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Mandyhra, S. S., L. M. Muzykina, L. M. Ishchenko, G. A. Kovalenko, I. V. Halka, V. G. Spyrydonov, and S. A. Nychyk. "Approbation of RT-qPCR test kit for differential diagnosis of African and Classical swine fever." Scientific Messenger of LNU of Veterinary Medicine and Biotechnologies 20, no. 83 (March 2, 2018): 221–25. http://dx.doi.org/10.15421/nvlvet8343.
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The first Ukrainian real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) based test kit for the differential diagnosis of African (AFS) and Classical swine fever (CSF) has been developed in the Institute of Veterinary Medicine of NAAS. The proposed test kit allows simultaneous detection of three targets: ASFV DNA, CSFV cDNA and an internal control sample. The goal of this work was to provide an expert evaluation of the RT-qPCR kit for differential diagnosis of ASF and CSF according to appearance, analytical sensitivity, specificity and repeatability. Interdepartmental evaluation of the kit was conducted in the State Scientific and Research Institute of Laboratory Diagnostics and Veterinary and Sanitary Expertise (DNDILDEVSE) in accordance with the approved methodology. The RT-qPCR kit sensitivity was determined by testing 10-fold serial dilutions of the ASFV DNA and CSFV cDNA (concentration range was 103–100 copies/μl). For specificity determination reference samples of ASFV DNA different genotypes, ASF and CSF positive and negative field samples, as well as pathogens which cause similar to ASF and CSF clinical syndromes were used. Sample preparation and amplification were performed according to the test kit instructions. The amplification was accomplished on QuantStudio™ 5 System (Applied Biosystems). As a result of accomplished interdepartmental evaluation high sensitivity, specificity and repeatability of RT-qPCR kit were confirmed. In particular, it was determined that the limit of detection of the RT-qPCR kit was 5 copies of the ASFV and CSFV genomes per one reaction. The high specificity of the assay to ASFV (I, II, V, VIII, IX and X genotypes) and CSFV was confirmed. It was showп no cross-reactions with closely related pigs viruses (porcine circovirus type 2, porcine reproductive and respiratory syndrome virus and virus of Aujeszky's disease). The high enough repeatability of results was also confirmed. In conclusion, the obtained results are in compliance with the requirements of the RT-qPCR kit normative and technical documentation. This RT-qPCR kit will be recommended for use in veterinary medicine laboratories after its registration would be done.
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Buttner, Mark P., Patricia Cruz-Perez, and Linda D. Stetzenbach. "Enhanced Detection of Surface-Associated Bacteria in Indoor Environments by Quantitative PCR." Applied and Environmental Microbiology 67, no. 6 (June 1, 2001): 2564–70. http://dx.doi.org/10.1128/aem.67.6.2564-2570.2001.
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ABSTRACT Methods for detecting microorganisms on surfaces are needed to locate biocontamination sources and to relate surface and airborne concentrations. Research was conducted in an experimental room to evaluate surface sampling methods and quantitative PCR (QPCR) for enhanced detection of a target biocontaminant present on flooring materials. QPCR and culture analyses were used to quantitateBacillus subtilis (Bacillus globigii) endospores on vinyl tile, commercial carpet, and new and soiled residential carpet with samples obtained by four surface sampling methods: a swab kit, a sponge swipe, a cotton swab, and a bulk method. The initial data showed that greater overall sensitivity was obtained with the QPCR than with culture analysis; however, the QPCR results for bulk samples from residential carpet were negative. The swab kit and the sponge swipe methods were then tested with two levels of background biological contamination consisting of Penicillium chrysogenum spores. The B. subtilis values obtained by the QPCR method were greater than those obtained by culture analysis. The differences between the QPCR and culture data were significant for the samples obtained with the swab kit for all flooring materials except soiled residential carpet and with the sponge swipe for commercial carpet. The QPCR data showed that there were no significant differences between the swab kit and sponge swipe sampling methods for any of the flooring materials. Inhibition of QPCR due solely to biological contamination of flooring materials was not evident. However, some degree of inhibition was observed with the soiled residential carpet, which may have been caused by the presence of abiotic contaminants, alone or in combination with biological contaminants. The results of this research demonstrate the ability of QPCR to enhance detection and enumeration of biocontaminants on surface materials and provide information concerning the comparability of currently available surface sampling methods.
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Tuncel, Gulten, Mahmut Cerkez Ergoren, Buket Baddal, Pinar Tulay, Ayse Arikan, Emrah Guler, Cenk Serhan Ozverel, H. Kaya Suer, Murat Sayan, and Tamer Sanlidag. "Comparison of RT-qPCR results of different gene targets for SARS-CoV-2 in asymptomatic individuals during COVID-19 pandemic." EuroBiotech Journal 5, s1 (June 1, 2021): 26–31. http://dx.doi.org/10.2478/ebtj-2021-0018.
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Abstract A reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) is regarded as the most sensitive method available and is being used for screening procedure for all incoming passengers to Northern Cyprus for SARS-CoV-2. This study investigated the compatibility of two different RT-qPCR methodologies Diagnovital® and Bio-Speedy® by re-analyzing the previously confirmed positive samples. A total of 43 previously confirmed positive samples were re-analyzed by two different commercially available SARS-CoV-2 RT-qPCR kits. Only 23.5% of positive samples detected by Diagnovital® RT-qPCR kit were detected by Bio-Speedy® detection kit. In conclusion, adoption of Diagnovital® RT-qPCR kit detecting two regions of SARS-CoV-2 genome in our laboratories enabled the detection of SARS-CoV-2 in asymptomatic cases with higher sensitivity and contributed to the prevention of viral transmission within the country. The timely detection of infection in asymptomatic individuals may be the key to a successful fight against the COVID- 19 pandemic.
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Al-Saud, Haya, Khaldoun Al-Romaih, Razan Bakheet, Lina Mahmoud, Najla Al-Harbi, Ibtihaj Alshareef, Sara Bin Judia, et al. "Automated SARS-COV-2 RNA extraction from patient nasopharyngeal samples using a modified DNA extraction kit for high throughput testing." Annals of Saudi Medicine 40, no. 5 (September 2020): 373–81. http://dx.doi.org/10.5144/0256-4947.2020.373.
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ABSTRACT BACKGROUND: The pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) has prompted a need for mass testing to identify patients with viral infection. The high demand has created a global bottleneck in testing capacity, which prompted us to modify available resources to extract viral RNA and perform reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to detect SARS-COV-2. OBJECTIVES: Report on the use of a DNA extraction kit, after modifications, to extract viral RNA that could then be detected using an FDA-approved SARS-COV-2 RT-qPCR assay. MATERIALS AND METHODS: Initially, automated RNA extraction was performed using a modified DNA kit on samples from control subjects, a bacteriophage, and an RNA virus. We then verified the automated extraction using the modified kit to detect in-lab propagated SARSCOV-2 titrations using an FDA approved commercial kit (S, N, and ORF1b genes) and an in-house primer-probe based assay (E, RdRp2 and RdRp4 genes). RESULTS: Automated RNA extraction on serial dilutions SARS-COV-2 achieved successful one-step RT-qPCR detection down to 60 copies using the commercial kit assay and less than 30 copies using the in-house primer-probe assay. Moreover, RT-qPCR detection was successful after automated RNA extraction using this modified protocol on 12 patient samples of SARS-COV-2 collected by nasopharyngeal swabs and stored in viral transport media. CONCLUSIONS: We demonstrated the capacity of a modified DNA extraction kit for automated viral RNA extraction and detection using a platform that is suitable for mass testing. LIMITATIONS: Small patient sample size. CONFLICT OF INTEREST: None.
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Hennebique, Aurélie, Fabienne Gas, Hélène Batina, Cécilia De Araujo, Karine Bizet, and Max Maurin. "Evaluation of the Biotoxis qPCR Detection Kit for Francisella tularensis Detection in Clinical and Environmental Samples." Journal of Clinical Microbiology 59, no. 1 (October 28, 2020): e01434-20. http://dx.doi.org/10.1128/jcm.01434-20.
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ABSTRACTRapid and reliable detection and identification of Francisella tularensis (a tier 1 select agent) are of primary interest for both medical and biological threat surveillance purposes. The Biotoxis qPCR detection kit is a real-time quantitative PCR (qPCR) assay designed for the detection of Bacillus anthracis, Yersinia pestis, and F. tularensis in environmental or biological samples. Here, we evaluated its performance for detecting F. tularensis in comparison to previously validated qPCR assays. The Biotoxis qPCR was positive for 87/87 F. tularensis subsp. holarctica (type B) strains but also for F. tularensis subsp. novicida. It was negative for Francisella philomiragia and 24/24 strains belonging to other bacterial species. For 31 tularemia clinical specimens, the Biotoxis qPCR displayed a sensitivity between 90.32% and 96.55%, compared to qPCR tests targeting ISFtu2 or a type B-specific DNA sequence, respectively. All 30 nontularemia clinical specimens were Biotoxis qPCR negative. For water samples, the Biotoxis qPCR limit of detection was 1,000 CFU/liter of F. tularensis. For 57 environmental water samples collected in France, the Biotoxis qPCR was positive for 6/15 samples positive for ISFtu2 qPCR and 4/4 positive for type B qPCR. In conclusion, the Biotoxis qPCR detection kit demonstrated good performances for F. tularensis detection in various biological and environmental samples, although cross-amplification of F. tularensis subsp. novicida must be considered. This plate format assay could be useful to test a large number of clinical or environmental specimens, especially in the context of natural or intentional tularemia outbreaks.
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ASSURIAN, ANGELA, HELEN MURPHY, ALICIA SHIPLEY, HEDIYE NESE CINAR, ALEXANDRE DA SILVA, and SONIA ALMERIA. "Assessment of Commercial DNA Cleanup Kits for Elimination of Real-Time PCR Inhibitors in the Detection of Cyclospora cayetanensis in Cilantro." Journal of Food Protection 83, no. 11 (June 9, 2020): 1863–70. http://dx.doi.org/10.4315/jfp-20-139.
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ABSTRACT Inhibited reactions have occasionally been observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using quantitative real-time PCR (qPCR). Partial or total inhibition of PCR reactions, including qPCR, can occur, leading to decreased sensitivity or false-negative results. If inhibition occurs, this implies the need for additional purification or cleanup treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA cleanup kits (QIAquick purification kit from Qiagen [kit 1], OneStep PCR inhibitor removal by Zymo Research [kit 2], NucleoSpin genomic DNA cleanup XS from Macherey-Nagel [kit 3], DNA IQ system by Promega [kit 4], and DNeasy PowerPlant pro kit from Qiagen [5]) to minimize qPCR inhibition using the U.S. Food and Drug Administration–validated Bacteriological Analytical Manual (BAM) Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA cleanup kits evaluated was able to reduce the qPCR internal amplification control cycle threshold values to those considered to be normal for noninhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both a high oocyst level (200 oocysts) and a low oocyst level (10 oocysts). Of the five kits compared, kits 1, 2, and 3 did not show significant differences in the detection of C. cayetanensis, while significantly higher cycle threshold values, indicating lower recovery of the target DNA, were observed from kits 4 and/or 5 in samples seeded with 200 and 10 oocysts (P < 0.05). This comparative study provides recommendations on the use of commercial cleanup kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using the BAM Chapter 19b method. HIGHLIGHTS
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Dankova, Zuzana, Elena Novakova, Maria Skerenova, Veronika Holubekova, Vincent Lucansky, Dana Dvorska, Dusan Brany, et al. "Comparison of SARS-CoV-2 Detection by Rapid Antigen and by Three Commercial RT-qPCR Tests: A Study from Martin University Hospital in Slovakia." International Journal of Environmental Research and Public Health 18, no. 13 (July 1, 2021): 7037. http://dx.doi.org/10.3390/ijerph18137037.
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The global pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is having a tremendous impact on the global economy, health care systems and the lives of almost all people in the world. The Central European country of Slovakia reached one of the highest daily mortality rates per 100,000 inhabitants in the first 3 months of 2021, despite implementing strong prophylactic measures, lockdowns and repeated nationwide antigen testing. The present study reports a comparison of the performance of the Standard Q COVID-19 antigen test (SD Biosensor) with three commercial RT-qPCR kits (vDetect COVID-19-MultiplexDX, gb SARS-CoV-2 Multiplex-GENERI BIOTECH Ltd. and Genvinset COVID-19 [E]-BDR Diagnostics) in the detection of infected individuals among employees of the Martin University Hospital in Slovakia. Health care providers, such as doctors and nurses, are classified as “critical infrastructure”, and there is no doubt about the huge impact that incorrect results could have on patients. Out of 1231 samples, 14 were evaluated as positive for SARS-CoV-2 antigen presence, and all of them were confirmed by RT-qPCR kit 1 and kit 2. As another 26 samples had a signal in the E gene, these 40 samples were re-isolated and subsequently re-analysed using the three kits, which detected the virus in 22, 23 and 12 cases, respectively. The results point to a divergence not only between antigen and RT-qPCR tests, but also within the “gold standard” RT-qPCR testing. Performance analysis of the diagnostic antigen test showed the positive predictive value (PPV) to be 100% and negative predictive value (NPV) to be 98.10%, indicating that 1.90% of individuals with a negative result were, in fact, positive. If these data are extrapolated to the national level, where the mean daily number of antigen tests was 250,000 in April 2021, it points to over 4700 people per day being misinterpreted and posing a risk of virus shedding. While mean Ct values of the samples that were both antigen and RT-qPCR positive were about 20 (kit 1: 20.47 and 20.16 for Sarbeco E and RdRP, kit 2: 19.37 and 19.99 for Sarbeco E and RdRP and kit 3: 17.47 for ORF1b/RdRP), mean Ct values of the samples that were antigen-negative but RT-qPCR-positive were about 30 (kit 1: 30.67 and 30.00 for Sarbeco E and RdRP, kit 2: 29.86 and 31.01 for Sarbeco E and RdRP and kit 3: 27.47 for ORF1b/RdRP). It confirms the advantage of antigen test in detecting the most infectious individuals with a higher viral load. However, the reporting of Ct values is still a matter of ongoing debates and should not be conducted without normalisation to standardised controls of known concentration.
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Curti, Lucía Ana, Ivana Primost, Sofia Valla, Daiana Ibañez Alegre, Cecilia Olguin Perglione, Guillermo Daniel Repizo, Julia Lara, et al. "Evaluation of a Lyophilized CRISPR-Cas12 Assay for a Sensitive, Specific, and Rapid Detection of SARS-CoV-2." Viruses 13, no. 3 (March 5, 2021): 420. http://dx.doi.org/10.3390/v13030420.
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We evaluated a lyophilized CRISPR-Cas12 assay for SARS-CoV-2 detection (Lyo-CRISPR SARS-CoV-2 kit) based on reverse transcription, isothermal amplification, and CRISPR-Cas12 reaction. From a total of 210 RNA samples extracted from nasopharyngeal swabs using spin columns, the Lyo-CRISPR SARS-CoV-2 kit detected 105/105 (100%; 95% confidence interval (CI): 96.55–100) positive samples and 104/105 (99.05%; 95% CI: 94.81–99.97) negative samples that were previously tested using commercial RT-qPCR. The estimated overall Kappa index was 0.991, reflecting an almost perfect concordance level between the two diagnostic tests. An initial validation test was also performed on 30 nasopharyngeal samples collected in lysis buffer, in which the Lyo-CRISPR SARS-CoV-2 kit detected 20/21 (95.24%; 95% CI: 76.18–99.88) positive samples and 9/9 (100%; 95% CI: 66.37–100) negative samples. The estimated Kappa index was 0.923, indicating a strong concordance between the test procedures. The Lyo-CRISPR SARS-CoV-2 kit was suitable for detecting a wide range of RT-qPCR-positive samples (cycle threshold range: 11.45–36.90) and dilutions of heat-inactivated virus (range: 2.5–100 copies/µL); no cross-reaction was observed with the other respiratory pathogens tested. We demonstrated that the performance of the Lyo-CRISPR SARS-CoV-2 kit was similar to that of commercial RT-qPCR, as the former was highly sensitive and specific, timesaving (1.5 h), inexpensive, and did not require sophisticated equipment. The use of this kit would reduce the time taken for diagnosis and facilitate molecular diagnosis in low-resource laboratories.
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Liu, Jason Yingjie. "Direct qPCR quantification using the Quantifiler® Trio DNA quantification kit." Forensic Science International: Genetics 13 (November 2014): 10–19. http://dx.doi.org/10.1016/j.fsigen.2014.06.016.
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Borghetti, Ivo Alberto, Miriam Ribas Zambenedetti, Luciana Requião, Deusilene Souza Vieira, Marco Aurélio Krieger, and Rita de Cássia Pontello Rampazzo. "External Control Viral-Like Particle Construction for Detection of Emergent Arboviruses by Real-Time Reverse-Transcription PCR." BioMed Research International 2019 (October 7, 2019): 1–4. http://dx.doi.org/10.1155/2019/2560401.
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Arboviruses have been emerging and reemerging worldwide, predominantly in tropical and subtropical areas. As many arbovirus infections, including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV), have similar signs and symptoms, clinical diagnosis of arbovirus infections is challenging. Therefore, reliable laboratory tests are necessary to improve the clinical management of patients with suspected arbovirus infections. Real-time reverse-transcription PCR (RT-qPCR) is among the more effective methods to distinguish these viruses. The aim of this study was to construct a unique positive external control derived from a unique plasmid using genetic engineering for specific use in RT-qPCR assays to detect Zika, dengue (1–4), and chikungunya. An external control derived from the MS2 bacteriophage was constructed using sequences from arbovirus and human genomes. Laboratories were asked to test the control in the ZDC Biomol kit, a RT-qPCR kit which is able to detect Zika, dengue serotypes 1–4, chikungunya, and an internal human control. RNA extracted from the external control was able to be amplified and detected in RT-qPCR assays for each virus detected by using the ZDC Biomol kit. The external control, samples from viral culture, and infected patient samples display similar amplification using this assay. The pET47b(+)MS2-ZDC vector is a viable expression system for the production of external control viral-like particles (MS2-ZDC). The RNA from the recombinant particles can be easily extracted and can function as a tool to validate all steps of process from the extraction to the amplification of all targets in specific reaction. Thus, the MS2-ZDC particles are laboratory-safe in order to avoid risk for operators, and the phages are effective as positive control for use in the ZDC Biomol kit amplifying all kit targets making them effective for commercial profile.
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Saeidi, Nazanin, Xiaoqiong Gu, Shin Giek Goh, Claire Lim Yi Xin, and Karina Yew-Hoong Gin. "Evaluating the efficacy of commercial kits for viral DNA/RNA extraction." Water Practice and Technology 12, no. 1 (March 1, 2017): 80–86. http://dx.doi.org/10.2166/wpt.2017.015.
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Extraction of viral DNA/RNA from environmental samples as part of the analytical procedure in quantifying waterborne viruses, is of great importance. In this study, two commercially available kits were compared to assess their performance, the MO BIO PowerViral Environmental DNA/RNA Isolation kit and the Qiagen QIAamp Viral RNA Mini kit. A performance assessment of extraction kits for detecting and quantifying six human enteric viruses as the commonest waterborne pathogens and one plant virus as an alternative fecal indicator has been carried out using quantitative PCR (qPCR). Water samples were collected from seven sites in Singapore during March and April 2015. In general, a strong association was observed between two different viral DNA/RNA extraction kits and detection frequency of targets (P = 0.017). The Qiagen kit showed higher extraction efficiency than the MoBio kit. However, in terms of quantification, a significant difference was only observed in the occurrences of NoV GI and PMMoV between two different kits (P < 0.05), although the kits showed similar efficiency removing qPCR inhibitors. The Qiagen kit was preferred for routine water quality monitoring.
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Tiam, Sandra Kim, Vincent Laderriere, Carole-Anne Gillis, Claude Fortin, and Isabelle Lavoie. "qPCR detection versus microscopy observations for assessing presence–absence of Didymosphenia geminata in Quebec rivers (Canada)." Water Quality Research Journal 52, no. 2 (May 24, 2015): 109–20. http://dx.doi.org/10.2166/wqrj.2017.030.
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Microscopic versus qPCR (quantitative polymerase chain reaction) analyses were compared for the detection of Didymosphenia geminata in biofilms and water filtrates from seven Gaspésie rivers (Canada). For the qPCR approach, two DNA extraction kits (QIAamp DNA Micro Kit, Qiagen and PowerSoil DNA Isolation Kit, Mo Bio Laboratories) and two pairs of primers were considered. The pair of primers D602F/D753Rext did not amplify D. geminata DNA whereas the pair of primers D602F/D753R was specific for D. geminata. Presence-absence diagnosis based on qPCR and microscopic analyses were consistent: D. geminata was detected in six of the seven rivers, both in the biofilm and filtrate samples. However, technical replications were needed at certain sites to observe the presence of D. geminata cells by microscopy. This underscores the necessity of replicate analyses, which is cost-effective to achieve when using qPCR due to the capacity to process tens of samples in a single PCR run in the context of a large scale assessment.
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Freire-Paspuel, Byron, Alfredo Bruno, Alberto Orlando, and Miguel Angel Garcia-Bereguiain. "Analytical and Clinical Evaluation of Two RT-qPCR SARS-CoV-2 Diagnostic Tests with Emergency Use Authorization in Ecuador." American Journal of Tropical Medicine and Hygiene 104, no. 5 (May 5, 2021): 1672–75. http://dx.doi.org/10.4269/ajtmh.20-1439.
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ABSTRACTDozens of RT-qPCR kits are available in the market for SARS-CoV-2 diagnosis, some of them with Emergency Use Authorization (EUA) by the Food and Drug Administration (FDA) or at least by a responsible agency of their country of origin, but many of them lack proper evaluation studies because of COVID-19 pandemic emergency. We evaluated the clinical performance of two commercially available kits in South America, the 2019-nCoV kit (Da An Gene, Guangzhou, China) and GenomeCoV19 kit (ABM, Richmond, Canada), for RT-qPCR SARS-CoV-2 diagnosis using the FDA EUA 2019-nCoV CDC kit (IDT, Coralville, IA) as gold standard. We found striking differences among clinical performance and analytical sensitivity in both kits; whereas the 2019-nCoV kit (Da An Gene) has a limit of detection of 2,000 copies/mL and 100% of sensitivity, the GenomeCoV19 kit (ABM) has a poor sensitivity of 75% and a limit of detection estimated to be over 8.000 copies/mL. The GenomeCoV19 kit (ABM) lacks clinical use authorization in Canada; however, the 2019-nCoV kit (Da An Gene) is authorized by the Chinese CDC. Our results support that only SARS-CoV-2 diagnosis kits with clinical use authorization from their country of origin should be exported to developing countries lacking proper evaluation agencies to avoid a deep impact of the COVID-19 pandemic due to unreliable diagnosis.
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Raymond, Philippe, Sylvianne Paul, André Perron, and Louise Deschênes. "Norovirus Extraction from Frozen Raspberries Using Magnetic Silica Beads." Food and Environmental Virology 13, no. 2 (March 2, 2021): 248–58. http://dx.doi.org/10.1007/s12560-021-09466-0.
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AbstractHuman noroviruses (HuNoV) are among the main causes of acute gastroenteritis worldwide. Frozen raspberries have been linked to several HuNoV food-related outbreaks. However, the extraction of HuNoV RNA from frozen raspberries remains challenging. Recovery yields are low, and real-time quantitative reverse transcriptase PCR (RT-qPCR) inhibitors limit the sensitivity of the detection methodologies. A new approach using fine magnetic silica beads was developed for the extraction of HuNoV spiked on frozen raspberries. Relatively low recovery yields were observed with both the magnetic silica bead and the reference ISO 15216-1:2017 methods. High RT-qPCR inhibition was observed with the ISO 15216-1:2017 recommended amplification kit but could be reduced by using an alternative kit. Reducing RT-qPCR inhibition is important to limit the number of inconclusive HuNoV assays thus increasing the capacity to assess the HuNoV prevalence in frozen raspberries.
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Tozato, Claudia de Camargo, Vívian Ferreira Zadra, Caroline Rodrigues Basso, and João Pessoa Araújo Junior. "Canine distemper virus detection by different methods of One-Step RT-qPCR." Ciência Rural 46, no. 9 (May 17, 2016): 1601–6. http://dx.doi.org/10.1590/0103-8478cr20150932.
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ABSTRACT: Three commercial kits of One-Step RT-qPCR were evaluated for the molecular diagnosis of Canine Distemper Virus. Using the kit that showed better performance, two systems of Real-time RT-PCR (RT-qPCR) assays were tested and compared for analytical sensitivity to Canine Distemper Virus RNA detection: a One-Step RT-qPCR (system A) and a One-Step RT-qPCR combined with NESTED-qPCR (system B). Limits of detection for both systems were determined using a serial dilution of Canine Distemper Virus synthetic RNA or a positive urine sample. In addition, the same urine sample was tested using samples with prior centrifugation or ultracentrifugation. Commercial kits of One-Step RT-qPCR assays detected canine distemper virus RNA in 10 (100%) urine samples from symptomatic animals tested. The One-Step RT-qPCR kit that showed better results was used to evaluate the analytical sensitivity of the A and B systems. Limit of detection using synthetic RNA for the system A was 11 RNA copies µL-1 and 110 RNA copies µl-1 for first round System B. The second round of the NESTED-qPCR for System B had a limit of detection of 11 copies µl-1. Relationship between Ct values and RNA concentration was linear. The RNA extracted from the urine dilutions was detected in dilutions of 10-3 and10-2 by System A and B respectively. Urine centrifugation increased the analytical sensitivity of the test and proved to be useful for routine diagnostics. The One-Step RT-qPCR is a fast, sensitive and specific method for canine distemper routine diagnosis and research projects that require sensitive and quantitative methodology.
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Greiner, Georg, Michael Gurbisz, Franz Ratzinger, Nadine Witzeneder, Ingrid Simonitsch-Klupp, Gerlinde Mitterbauer-Hohendanner, Matthias Mayerhofer, et al. "Digital PCR: A Sensitive and Precise Method for KIT D816V Quantification in Mastocytosis." Clinical Chemistry 64, no. 3 (March 1, 2018): 547–55. http://dx.doi.org/10.1373/clinchem.2017.277897.
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Abstract BACKGROUND The analytically sensitive detection of KIT D816V in blood and bone marrow is important for diagnosing systemic mastocytosis (SM). Additionally, precise quantification of the KIT D816V variant allele fraction (VAF) is relevant clinically because it helps to predict multilineage involvement and prognosis in cases of advanced SM. Digital PCR (dPCR) is a promising new method for sensitive detection and accurate quantification of somatic mutations. METHODS We performed a validation study of dPCR for KIT D816V on 302 peripheral blood and bone marrow samples from 156 patients with mastocytosis for comparison with melting curve analysis after peptide nucleic acid-mediated PCR clamping (clamp-PCR) and allele-specific quantitative real-time PCR (qPCR). RESULTS dPCR showed a limit of detection of 0.01% VAF with a mean CV of 8.5% and identified the mutation in 90% of patients compared with 70% for clamp-PCR (P < 0.001). Moreover, dPCR for KIT D816V was highly concordant with qPCR without systematic deviation of results, and confirmed the clinical value of KIT D816V VAF measurements. Thus, patients with advanced SM showed a significantly higher KIT D816V VAF (median, 2.43%) compared with patients with indolent SM (median, 0.14%; P < 0.001). Moreover, dPCR confirmed the prognostic significance of a high KIT D816V VAF regarding survival (P < 0.001). CONCLUSIONS dPCR for KIT D816V provides a high degree of precision and sensitivity combined with the potential for interlaboratory standardization, which is crucial for the implementation of KIT D816V allele burden measurement. Thus, dPCR is suitable as a new method for KIT D816V testing in patients with mastocytosis.
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Feng, Qin. "A sensitive QPCR assay for EGFR mutation in plasma samples of NSCLC patients." Journal of Clinical Oncology 35, no. 15_suppl (May 20, 2017): 2595. http://dx.doi.org/10.1200/jco.2017.35.15_suppl.2595.
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2595 Background: Tyrosine kinase inhibitors (TKI) have improved the overall outlook and quality of life for most of EGFR mutation positive NSCLC patients. However, tumor tissues are often absent or insufficient for testing EGFR mutations to guide EGFR TKIs treatment of patients with nonsmall cell lung cancer (NSCLC). An assay that detects EGFR mutations in the plasma would provide a noninvasive technique to assess suitability for TK inhibitor therapy. The ACCB new EGFR Mutation Kit is a ARMS-PCR test for the qualitative detection of 45 mutations in exons 18, 19, 20, and 21 of the EGFR gene in DNA derived from human plasma from NSCLC patients. Methods: 10 mL tubes of blood were collected from patients who never had been treated by EGFR TKI, and plasma circulating tumor DNA were extracted from plasma by Biomark Circulating DNA Kit. Qubit 2.0 Fluorometer was used to make plasma circulating DNA tumor quantitation. The concentration of final DNA sample is ≦2ng/μl. Total, 272 plasma DNA samples from 246 lung adenocarcinoma, 23 lung squamous carcinoma and 3 lung adenosquamous carcinoma patients were collected. 104 paired tissue EGFR mutations were detected by the same kit. Results: EGFR mutation was detected in 88 from 272 plasma DNA samples, 86 lung adenocarcinoma, 1 lung squamous carcinoma, 1 lung adenosquamous carcinoma, with EGFR mutation rate were 35.0% (86/246), 4.3% (1/23), 33.3% (1/3) respectively. In 61 Ⅰ~Ⅲ and 211 Ⅳ stage NSCLC patients, EGFR mutation rate were 21.3% (13/61) and 35.5% (75/211) respectively. Conclusions: The ACCB new EGFR Mutation Kit is a sensitive, accurate, rapid, and reproducible assay capable of testing DNA extracted from human plasma from NSCLC patients.
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Freire-Paspuel, Byron, and Miguel Angel Garcia-Bereguiain. "Clinical Performance and Analytical Sensitivity of Three SARS-CoV-2 Nucleic Acid Diagnostic Tests." American Journal of Tropical Medicine and Hygiene 104, no. 4 (April 7, 2021): 1516–18. http://dx.doi.org/10.4269/ajtmh.20-1484.
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ABSTRACTHundreds of RT-qPCR kits are available in the market for SARS-CoV-2 diagnosis, some of them with emergency use authorization (EUA) by the Food Drug Administration (FDA) or their country of origin agency, but also many of them without any independent clinical performance evaluation. We performed a clinical evaluation for two Chinese SARS-CoV-2 RT-PCR kits available in South America, COVID-19 Nucleic Acid Test Kit (eDiagnosis Biomedicine, Wuhan, China) and 2019-nCoV Nucleic Acid Diagnostic Kit (Sansure Biotech, Changsha, China), for RT-qPCR SARS-CoV-2 diagnosis using the FDA EUA 2019-nCoV CDC kit (IDT, Coralville, IA) as gold standard. We found an excellent clinical performance and analytical sensitivity for both kits with sensitivity values of 100% and 95.3% and estimated limit of detection of 500 copies/mL and 1,000 copies/mL, for eDiagnosis and Sansure Biotech kits, respectively. COVID-19 Nucleic Acid Test Kit (eDiagnosis) and 2019-nCoV Nucleic Acid Diagnostic Kit (Sansure Biotech) are both made in China and hold EUA by the Chinese CDC. Also, Sansure Biotech kit has EUA by the FDA. In conclusion, our results endorse the use of these two commercially available kits imported to Ecuador for SARS-CoV-2 diagnosis, as they had the similar clinical performance as the gold standard from the CDC.
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Xie, Winny, and Yusmiati Yusmiati. "Optimization and Validation of a Real Time Reverse Transcriptase Polymerase Chain Reaction with RNA Internal Control to Detect Rubella RNA." Indonesian Biomedical Journal 5, no. 3 (December 1, 2013): 185. http://dx.doi.org/10.18585/inabj.v5i3.70.
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BACKGROUND: According to a report from WHO, cases of rubella infection in Indonesia has increased up to 10-fold from 2007 to 2011. Despite no data of congenital rubella syndrome in the report, there are approximately 45,000 cases of babies born with heart failure and 0.1-0.3% live births with congenital deafness in Indonesia. Allegedly, rubella infection during pregnancy may play a role in this condition. This study aimed to optimize and validate a real-time reverse transcriptase polymerase chain reaction (RT-qPCR) method to detect rubella virus RNA as an aid for the diagnosis of congenital rubella infection.METHODS: Method optimization was conducted using nucleic acids extracted from Trimovax Merieux vaccine with the High Pure Viral Nucleic Acid Kit. One step RT-qPCR was performed with Quantifast Multiplex RTPCR+R Kit. Target synthetic DNA was designed and used to determine the sensitivity of the method. RNA internal control was synthesized to control the process of extraction and amplification.RESULTS: The analytical sensitivity of this method was as low as 5 copies target synthetic DNA/μl. The mean Coefficient of Variation (CV) % of the critical threshold (Ct) obtained were 2.71%, 1.20%, 1.62%, and 1.59% for within run, between run, between kit lots, and between operators, respectively. Recovery of the target synthetic DNA from amniotic fluid was 100.51% (by the log copies/μl) at the concentration of 1,000,000 copies/μl.CONCLUSION: RT-qPCR is successfully used for the detection of rubella virus RNA in vaccine and synthetic nucleic acid. With its high sensitivity, good precision and recovery, this method offers a means to improve the diagnosis of congenital rubella infection in developing countries like Indonesia.KEYWORDS: congenital rubella, RT-qPCR, prenatal diagnosis, amniotic fluid
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Lista, Maria Jose, Pedro M. Matos, Thomas J. A. Maguire, Kate Poulton, Elena Ortiz-Zapater, Robert Page, Helin Sertkaya, et al. "Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation." PLOS ONE 16, no. 9 (September 15, 2021): e0256813. http://dx.doi.org/10.1371/journal.pone.0256813.
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There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/.
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Cuevas-Ferrando, Enric, Antonio Martínez-Murcia, Alba Pérez-Cataluña, Gloria Sánchez, and Walter Randazzo. "Assessment of ISO Method 15216 to Quantify Hepatitis E Virus in Bottled Water." Microorganisms 8, no. 5 (May 13, 2020): 730. http://dx.doi.org/10.3390/microorganisms8050730.
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Hepatitis E virus (HEV) is one of the causative agents of water-borne human viral hepatitis and considered in Europe an emerging zoonotic pathogen. Analysis of bottled water through a standard method validated for HEV can contribute towards the risk management of this hazard. Putting some recent reports by the European Food Safety Authority in place, this study aimed to assess the performance of the concentration and extraction procedures described in ISO 15216-1:2017 for norovirus and hepatitis A virus on HEV detection. Following the ISO recommendation, the bottled water samples were spiked using serially diluted HEV fecal suspensions together with mengovirus as process control and concentrated by filtration via positively charged nylon membranes. In order to extract viral RNA from the resulting concentrates, two different methods were compared in this study: The one recommended in the ISO norm, NucliSens® MiniMag® system (NS), and an alternative commercially available kit NucleoSpin®RNA virus kit (MN). Finally, three reverse transcription quantitative PCR (RT-qPCR) assays were used to quantify HEV titers. The evaluated procedures resulted in average HEV recoveries of 14.08 ± 4.90% and 3.58 ± 0.30% for the MN and NS methods, respectively. The limit of detection (LoD95%) was 1.25 × 104 IU/L for both extraction methods combined with the three RT-qPCR assays tested, with the exception of NS extraction coupled with RT-qPCR1 that showed a LoD95% of 4.26 × 103 IU/L. The method characteristics generated in this study support the limited suitability of the ISO 15216-1:2017 concentration procedure coupled with the evaluated RT-qPCR assays for detecting HEV in bottled water.
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Pinto, Gabriel Godinho, José Antonio Tesser Poloni, Diego D'Avila Paskulin, Fabio Spuldaro, Fernanda de Paris, Afonso Luís Barth, Roberto Ceratti Manfro, Elizete Keitel, and Alessandro C. Pasqualotto. "Quantitative detection of BK virus in kidney transplant recipients: a prospective validation study." Brazilian Journal of Nephrology 40, no. 1 (April 19, 2018): 59–65. http://dx.doi.org/10.1590/1678-4685-jbn-3776.
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Abstract Introduction: BK virus (BKV) infection in renal transplant patients may cause kidney allograft dysfunction and graft loss. Accurate determination of BKV viral load is critical to prevent BKV-associated nephropathy (BKVAN) but the cut-off that best predicts BKVAN remains controversial. Objective: To evaluate the performance of a commercial and an in-house qPCR test for quantitative detection of BK virus in kidney transplant recipients. Methods: This was a prospective study with kidney transplant recipients from two large university hospitals in Brazil. Patients were screened for BKV infection every 3 months in the first year post-transplant with a commercial and an in-house real time polymerase chain reaction (qPCR) test. BKVAN was confirmed based on histopathology. The area under the curve for plasma qPCR was determined from receiver operating characteristic analysis. Results: A total of 200 patients were enrolled. Fifty-eight percent were male, 19.5% had diabetes mellitus, and 82% had the kidney transplanted from a deceased donor. BKV viremia was detected in 32.5% and BKVAN was diagnosed in 8 patients (4%). BKVAN was associated with viremia of 4.1 log copies/mL, using a commercial kit. The cut-off for the in-house assay was 6.1 log copies/mL. The linearity between the commercial kit and the in-house assay was R2=0.83. Conclusion: Our study shows that marked variability occurs in BKV viral load when different qPCR methodologies are used. The in-house qPCR assay proved clinically useful, a cheaper option in comparison to commercial qPCR kits. There is an urgent need to make BKV standards available to the international community.
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Muñoz-Calderón, Arturo A., Susana A. Besuschio, Season Wong, Marisa Fernández, Lady J. García Cáceres, Patricia Giorgio, Laura A. Barcan, et al. "Loop-Mediated Isothermal Amplification of Trypanosoma cruzi DNA for Point-of-Care Follow-Up of Anti-Parasitic Treatment of Chagas Disease." Microorganisms 10, no. 5 (April 26, 2022): 909. http://dx.doi.org/10.3390/microorganisms10050909.
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A loop-mediated isothermal amplification assay was evaluated as a surrogate marker of treatment failure in Chagas disease (CD). A convenience series of 18 acute or reactivated CD patients who received anti-parasitic treatment with benznidazole was selected—namely, nine orally infected patients: three people living with HIV and CD reactivation, five chronic CD recipients with reactivation after organ transplantation and one seronegative recipient of a kidney and liver transplant from a CD donor. Fifty-four archival samples (venous blood treated with EDTA or guanidinium hydrochloride-EDTA buffer and cerebrospinal fluid) were extracted using a Spin-column manual kit and tested by T. cruzi Loopamp kit (Tc-LAMP, index test) and standardized real-time PCR (qPCR, comparator test). Of them, 23 samples were also extracted using a novel repurposed 3D printer designed for point-of-care DNA extraction (PrintrLab). The agreement between methods was estimated by Cohen’s kappa index and Bland–Altman plot analysis. The T. cruzi Loopamp kit was as sensitive as qPCR for detecting parasite DNA in samples with parasite loads higher than 0.5 parasite equivalents/mL and infected with different discrete typing units. The agreement between qPCR and Tc-LAMP (Spin-column) or Tc-LAMP (PrintrLab) was excellent, with a mean difference of 0.02 [CI = −0.58–0.62] and −0.04 [CI = −0.45–0.37] and a Cohen’s kappa coefficient of 0.78 [CI = 0.60–0.96] and 0.90 [CI = 0.71 to 1.00], respectively. These findings encourage prospective field studies to validate the use of LAMP as a surrogate marker of treatment failure in CD.
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Muñoz-Calderón, Arturo A., Susana A. Besuschio, Season Wong, Marisa Fernández, Lady J. García Cáceres, Patricia Giorgio, Laura A. Barcan, et al. "Loop-Mediated Isothermal Amplification of Trypanosoma cruzi DNA for Point-of-Care Follow-Up of Anti-Parasitic Treatment of Chagas Disease." Microorganisms 10, no. 5 (April 26, 2022): 909. http://dx.doi.org/10.3390/microorganisms10050909.
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A loop-mediated isothermal amplification assay was evaluated as a surrogate marker of treatment failure in Chagas disease (CD). A convenience series of 18 acute or reactivated CD patients who received anti-parasitic treatment with benznidazole was selected—namely, nine orally infected patients: three people living with HIV and CD reactivation, five chronic CD recipients with reactivation after organ transplantation and one seronegative recipient of a kidney and liver transplant from a CD donor. Fifty-four archival samples (venous blood treated with EDTA or guanidinium hydrochloride-EDTA buffer and cerebrospinal fluid) were extracted using a Spin-column manual kit and tested by T. cruzi Loopamp kit (Tc-LAMP, index test) and standardized real-time PCR (qPCR, comparator test). Of them, 23 samples were also extracted using a novel repurposed 3D printer designed for point-of-care DNA extraction (PrintrLab). The agreement between methods was estimated by Cohen’s kappa index and Bland–Altman plot analysis. The T. cruzi Loopamp kit was as sensitive as qPCR for detecting parasite DNA in samples with parasite loads higher than 0.5 parasite equivalents/mL and infected with different discrete typing units. The agreement between qPCR and Tc-LAMP (Spin-column) or Tc-LAMP (PrintrLab) was excellent, with a mean difference of 0.02 [CI = −0.58–0.62] and −0.04 [CI = −0.45–0.37] and a Cohen’s kappa coefficient of 0.78 [CI = 0.60–0.96] and 0.90 [CI = 0.71 to 1.00], respectively. These findings encourage prospective field studies to validate the use of LAMP as a surrogate marker of treatment failure in CD.
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Hoermann, Gregor, Karoline V. Gleixner, Graziella E. Dinu, Georg Greiner, Friedrich Wimazal, Emir Hadzijusufovic, Gerlinde Mitterbauer, Christine Mannhalter, Peter Valent, and Wolfgang R. Sperr. "KIT D816V Mutation Burden Predicts Prognosis and Survival In Patients With Mastocytosis and Correlates With The WHO Type Of The Disease." Blood 122, no. 21 (November 15, 2013): 4052. http://dx.doi.org/10.1182/blood.v122.21.4052.4052.
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Abstract Mastocytosis is characterized by abnormal expansion and accumulation of neoplastic mast cells in one or more organ systems. Traditionally, mastocytosis is divided into cutaneous mastocytosis (CM) and systemic mastocytosis (SM). In most patients with SM, the transforming somatic mutation KIT D816V is detected. However, only few studies have quantified the KIT D816V allele burden in CM and SM. The aim of the present study was to quantify KIT D816V in various forms of mastocytosis and to correlate the allele burden of KIT D816V with the disease category, serum tryptase levels and clinical outcomes. KIT D816V was quantified in bone marrow (BM) and peripheral blood (PB) cells by a real-time PCR (qPCR) assay based on allele-specific primers. In addition, BM and PB cells were also examined for the presence of KIT D816V by melting curve analysis after PCR clamping in all patients. Overall, 225 DNA samples (BM, n=112; PB, n=113) from 107 patients with mastocytosis (females: n=57; males, n=50; median age 49 years; range 18-73 years) were analyzed. Based on WHO criteria, 14 patients had CM, 3 the provisional diagnosis of mastocytosis in the skin (MIS), 66 indolent SM (ISM), 6 smouldering SM (SSM), 7 aggressive SM (ASM), one mast cell leukemia (MCL) and 10 patients SM with an associated hematologic non-mast cell lineage disorder (SM-AHNMD). KIT D816V was found in in 76/107 patients (71%) by melting curve analysis after PCR clamping, and in 92/107 patients (86%) by qPCR (p<0.005). In paired BM and PB samples of 43 patients an excellent correlation of the KIT D816V burden with almost identical results was found (r=0.98, p<0.001). When examining the KIT D816V allele burden in KIT D816V+ patients (n=92) in various categories of the disease, significant differences were found between CM (median KIT D816V allele fraction: 0.042%), MIS (median: 0.084%), ISM (median: 0.286%), SSM (median: 3.012%), ASM (median: 9.346%) and SM-AHNMD (median: 3.761%) (p<0.001). Moreover, we found that the KIT D816V allele burden correlates significantly with the serum tryptase level in our patients (r=0.50, p<0.005). Consecutive studies revealed that the KIT D816V allele fraction is of prognostic significance concerning survival as determined by Cox regression (p=0.015). As assessed by Kaplan Meier estimates and log rank testing, patients with a KIT D816V allele burden of ≥2% were found to have a significantly shorter survival than those with an allele burden of less than 2% (p=0.001) (Figure 1). Thirty patients were evaluated at diagnosis and during the follow up. In untreated patients with stable disease, the KIT D816V allele burden remained within a constant range. By contrast, in patients with disease progression, an increase in the KIT D816V burden over time was detectable. In patients responding to cytoreductive agents (cladribine n=4; hydroxyurea n=1) a significant decrease in the median KIT D816V allele burden (by 91.6%) after therapy compared to pre-therapeutic samples was observed (p=0.027). In summary, our data show that qPCR is a highly sensitive approach for the detection and quantification of KIT D816V in patients with mastocytosis and that the KIT D816V mutation burden differs significantly among patients in different WHO subtypes. Moreover, the KIT D816V allele burden correlates with serum tryptase levels and is of prognostic significance concerning survival in patients with mastocytosis. Finally, quantification of KIT D816V may serve as follow up parameter useful for determining the natural course and treatment responses in patients with mastocytosis. We recommend that the KIT D816V mutation burden is included as a novel parameter in daily practice and clinical trials in advanced SM.Figure 1Overall survival of patients with KIT D816V+ mastocytosis. Patients were split into those with a KIT D816V allele burden of<2% and those with an allelic burden of ≥2%. Survival was estimated by the method of Kaplan and Meier (p=0.001).Figure 1. Overall survival of patients with KIT D816V+ mastocytosis. Patients were split into those with a KIT D816V allele burden of<2% and those with an allelic burden of ≥2%. Survival was estimated by the method of Kaplan and Meier (p=0.001). Disclosures: Valent: Novartis: Consultancy, Honoraria, Research Funding.
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Rublenko, N. "Identification of Salmonella spp and serovars Typhimurium, Enteritidis by qPCR." Naukovij vìsnik veterinarnoï medicini, no. 1(154) (May 21, 2020): 21–31. http://dx.doi.org/10.33245/2310-4902-2020-154-1-21-31.
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This article presents the results of the identification of the Salmonella genus as well as serovars Enteritidis and Typhimurium by a real-time polymerase chain reaction. We constructed three pairs of primers and fluorescent probes to simultaneously identify the Salmonella genus, serovars Enteritidis and Typhimurium in a qPCR. The specificity of the primers was evaluated on Salmonella strains of different serovars from the National Center for Strains of Microorganisms (UNCMS) strains of the State Scientific Control Institute of Biotechnology and Strains of Microorganisms (SSCIBSM) and 46 Salmonella strains isolated from poultry. E. coli ATCC 25922, Bacillus cereus ATCC 11778, Listeria monocytogenes ATCC 19112 from UNCMS collection were used to check the specificity of the primers as heterologous samples. Bacterial DNA was extracted using a DNA Sorb B (Amplisens) kit, and realtime PCR was accomplished with the "Real-time PCR kit" (Syntol) on Bio-rad CFX. A series of 10-fold S. Typhimurium and S. Enteritidis DNA dilutions were studied to evaluate the sensitivity of the primers: 10-1-10-5. The analytical sensitivity of primers for detection of the genus Salmonella is: for S. Typhimurium - 0.25 ng/sample (Typhimurium) and S. Enteritidis - 0.27 ng/ sample (Enteritidis). The results of the studies confirmed the specificity of the primer set and the high sensitivity. No hybridization of primers with DNA samples of other bacteria found, in particular, the nonspecific reaction products were absent. The primer sets for the detection of DNA of Enteritidis and Typhimurium serovars also has high specificity. If necessary, this set of primers can be used to perform a multiplex qPCR, that can simultaneously identify bacteria of the Salmonella genus and differentiate Enteritidis and Typhimurium serovars. Keywords: Salmonella, bacteria, polymerasechainreaction, DNA, qPCR.
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Medeiros, S. O., B. J. A. Silva, A. L. Carneiro, O. C. Ferreira Júnior, and A. Tanuri. "Avaliação de dois testes sorológicos comerciais para diagnóstico das infecções pelo FIV e pelo FeLV." Arquivo Brasileiro de Medicina Veterinária e Zootecnia 71, no. 2 (April 2019): 447–54. http://dx.doi.org/10.1590/1678-4162-10111.
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RESUMO FIV e FeLV são retrovírus associados principalmente com neoplasias. Dois testes rápidos são disponibilizados no Brasil para o diagnóstico dessas infecções: um kit de imunocromatografia de fluxo bidirecional (SNAP® Combo IDEXX) e um kit de imunocromatografia de fluxo lateral unidirecional (ALERE/BIONOTE Anigen Rapid). O objetivo deste estudo foi comparar o teste SNAP® com o teste ALERE. Amostras de sangue de 178 gatos foram testadas utilizando-se ambos os kits. A reação em cadeia de polimerase em tempo real (qPCR) foi empregada como método confirmatório para todos os resultados. O teste SNAP® apresentou sensibilidade e especificidade de 100% para FIV; a sensibilidade e a especificidade do teste ALERE foram de 96,15% e 98,68%, respectivamente. A sensibilidade e a especificidade para o FeLV foram de 93,02% e 96,30% para o teste SNAP® e de 90,70% e 97,78% para o teste ALERE. Ainda em relação ao FeLV, três amostras com resultado positivo na qPCR obtiveram resultado falso-negativo em ambos os testes. Não houve diferença estatisticamente significante entre os métodos. Considerando a qPCR como padrão-ouro, o teste SNAP® apresentou maior sensibilidade e especificidade para o FIV, e o teste ALERE apresentou maior especificidade para o FeLV. Os resultados mostraram uma boa correlação entre os testes.
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Yang, Yujie, Jie Cheng, Yongni Zhang, Jiabao Guo, Bin Xie, Wenyi Zhang, Zhaojin Zhu, and Yi Zhu. "Electroacupuncture at Zusanli (ST36) Repairs Interstitial Cells of Cajal and Upregulates c-Kit Expression in Rats with SCI-Induced Neurogenic Bowel Dysfunction." Evidence-Based Complementary and Alternative Medicine 2020 (November 27, 2020): 1–9. http://dx.doi.org/10.1155/2020/8896123.
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Background. Electroacupuncture (EA) could improve colonic transit activity in rats with neurogenic bowel dysfunction (NBD) caused by spinal cord injury (SCI). The function of interstitial cells of Cajal (ICCs) and c-Kit expression may play essential roles in this process. Material and Methods. Thirty-six Sprague Dawley rats were randomized to the sham group, the SCI group, or the SCI + EA group (bilateral Zusanli, 30 min/day, 14 days). Changes in the ultrastructural morphology of ICCs were observed. The c-Kit expression on different levels was analyzed by immunohistochemistry, Western blotting, and RT-qPCR, respectively. Results. Abnormal morphology of ICCs and downregulation of the c-Kit expression occurred after SCI. While the number of ICCs was increased, the ultrastructural morphology was improved significantly in EA rats. They also showed better improvement in c-Kit expression at both protein and gene levels. Conclusion. Abnormal ICCs in colon tissues and the downregulated expression of c-Kit could be observed after SCI. EA at Zusanli (ST36) could improve the colon function by repairing the morphology and increasing the number of ICCs and upregulating c-Kit expression.
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Folta, Adam, Tomas Jurcek, Blanka Kubesova, Daniela Zackova, Jiri Mayer, and Ivana Jeziskova. "A Comparison of Two Standardized Quantitative RT-PCRs with CE IVD Kit for Digital PCR in CML Patients with Different Level of BCR-ABL1 Transcripts." Blood 134, Supplement_1 (November 13, 2019): 1646. http://dx.doi.org/10.1182/blood-2019-125551.
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The detection and quantification of BCR-ABL1 transcripts play a key role in therapy management of chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors (TKIs). Reverse transcription quantitative PCR (RT-qPCR) is considered the gold standard strategy for monitoring of minimal residual disease (MRD). Recently, digital PCR (dPCR) was introduced as a new quantification method based on determination of absolute target sequence quantity. In connection with the deep molecular response (MR) assessment and the option of TKIs treatment cessation, the dPCR has a potential of high sensitivity, robustness and accuracy of BCR-ABL1 transcripts detection. The aim of present work was to compare the results of two standardized RT-qPCR methods with RT-dPCR detection in clinical samples of CML patients with different BCR-ABL1 fusion transcripts level. In total, 62 peripheral blood samples of CML patients with the level of BCR-ABL1 transcripts (international scale, IS) between 100% and undetectable were enrolled. The samples were analyzed by three methods: (1) CE IVD Xpert BCR-ABL Ultra Kit (GeneXpert, Cepheid), (2) conventional RT-qPCR assay according to ELN guidelines and certified by EUTOS for the level of MR4.5 BCR-ABL1 detection (ABI7300, Applied Biosystems), and (3) RT-dPCR using CE IVD QXDx BCR-ABL %ID Kit (Bio-Rad) on QX200 Droplet Digital PCR System (Bio-Rad). All steps were performed according to manufacturer´s instructions. A linear regression analysis and an overall reporting bias analysis using the Bland-Altman test were used for comparison of the methods. Based on the results of GeneXpert as the determined routine method for BCR-ABL1 transcripts quantification, the samples were divided into two groups: 50/62 samples were scored as BCR-ABL1 positive (the first group), while 12/62 samples were scored as BCR-ABL1 undetectable (the second group). The first group of GeneXpert positive samples was analyzed using both conventional RT-qPCR and RT-dPCR (both in duplicates). The median of the sum of ABL copies per sample was 180,511 copies (range 56,466 - 536,453) by RT-qPCR vs. 39,960 (range 14,360 - 71,690) by RT-dPCR. Both conventional RT-qPCR and RT-dPCR did not detect any BCR-ABL1 transcript in 4/50 samples. In addition, RT-dPCR did not detect any BCR-ABL1 transcript in 5 other samples (Figure 1). Linear regression analysis showed good correlation between both sets of assays: GeneXpert vs. RT-dPCR (R2=0.965, N=41) and conventional RT-qPCR vs. RT-dPCR (R2=0.977, N=41). The slopes of regression curve were not significantly different from the value of 1 for both sets of the compared assays (1.02; 95% CI, range 0.95 - 1.08 and 1.02; 95% CI, range 0.97 - 1.08, respectively). In addition, an overall bias (Bland-Altman analyses) was -0.05 (GeneXpert vs. RT-dPCR) and -0.12 (conventional RT-qPCR vs. RT-dPCR) suggesting good concordance in % IS reporting between RT-dPCR and two standardized quantitative BCR-ABL1 assays. The evaluation of MR level was identical by all three methods in 26/50 samples. The comparison of two methods revealed consistent MR level by GeneXpert vs. RT-dPCR in 29/50 samples and by conventional RT-qPCR vs. RT-dPCR in 28/50 samples. The RT-dPCR scored 12/50 samples (GeneXpert vs. RT-dPCR) and 13/50 samples (conventional RT-qPCR vs. RT-qPCR) to the different level of MR compared to standardized RT-qPCR methods. The second group of samples (12/62) undetectable for BCR-ABL1 transcripts according to GeneXpert was analyzed using conventional RT-qPCR and RT-dPCR in tetraplicates to reach higher sensitivity. The BCR-ABL1 negativity was confirmed in 11/12 samples. One sample was BCR-ABL1 positive by both the conventional RT-qPCR (0.0008% IS) and RT-dPCR (0.0013% IS). We showed that the results of quantitative detection of BCR-ABL1 transcripts (% IS) obtained by the CE IVD RT-dPCR kit are in good correlation with the results of both standardized RT-qPCR methods and all three methods provide comparable sensitivity. RT-qPCR method yielded higher number of copies of control ABL gene per sample compared to RT-dPCR although the input amount of RNA into the RT reaction was the same. The MR level evaluation of RT-dPCR vs. both RT-qPCR methods was identical to the level MR3.0 (category >0.01% IS), while for samples in deep MR (category ≤ 0.01% IS, MR4.0 and below) revealed partial difference in categorization into the individual MR levels. Supported by MH CZ - DRO (FNBr, 65269705). Disclosures Zackova: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Angelini: Consultancy; Incyte: Consultancy. Mayer:AOP Orphan Pharmaceuticals AG: Research Funding.
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Okino, Cintia Hiromi, Rodrigo Giglioti, Pamella Cristini Silva, Henrique Nunes de Oliveira, and Márcia Cristina de Sena Oliveira. "Comparative evaluation of DNA extraction kit, matrix sample and qPCR assays for bovine babesiosis monitoring." Molecular Biology Reports 45, no. 6 (October 25, 2018): 2671–80. http://dx.doi.org/10.1007/s11033-018-4436-9.
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Bochicchio, Maria Teresa, Jessica Petiti, Paola Berchialla, Barbara Izzo, Emilia Giugliano, Emanuela Ottaviani, Santa Errichiello, et al. "Droplet Digital PCR for BCR–ABL1 Monitoring in Diagnostic Routine: Ready to Start?" Cancers 13, no. 21 (October 30, 2021): 5470. http://dx.doi.org/10.3390/cancers13215470.
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BCR–ABL1 mRNA levels represent the key molecular marker for the evaluation of minimal residual disease (MRD) in chronic myeloid leukemia (CML) patients and real-time quantitative PCR (RT-qPCR) is currently the standard method to monitor it. In the era of tyrosine kinase inhibitors (TKIs) discontinuation, droplet digital PCR (ddPCR) has emerged to provide a more precise detection of MRD. To hypothesize the use of ddPCR in clinical practice, we designed a multicentric study to evaluate the potential value of ddPCR in the diagnostic routine. Thirty-seven RNA samples from CML patients and five from healthy donors were analyzed using both ddPCR QXDxTMBCR-ABL %IS Kit and LabNet-approved RT-qPCR methodologies in three different Italian laboratories. Our results show that ddPCR has a good agreement with RT-qPCR, but it is more precise to quantify BCR–ABL1 transcript levels. Furthermore, we did not find differences between duplicate or quadruplicate analysis in terms of BCR–ABL1% IS values. Droplet digital PCR could be confidently introduced into the diagnostic routine as a complement to the RT-qPCR.
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Matsumura, Itaru, Hirohisa Nakamae, Chikashi Yoshida, Linda Fletcher, Susan Branford, Daisuke Koga, Takayuki Sogabe, Yuzuru Kanakura, and Tomoki Naoe. "Odk-1201, One-Step RT-qPCR Major BCR-ABL/ABL mRNA Kit for the International Scale, with High Sensitivity to Detect Deeper MR." Blood 124, no. 21 (December 6, 2014): 1805. http://dx.doi.org/10.1182/blood.v124.21.1805.1805.
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Abstract Background: Molecular monitoring by the measurement of major BCR-ABL mRNA levels is important for the evaluation of the therapeutic efficacy of TKI in CML patients. However, there is no commercial kit easily available in each laboratory or hospital, which can estimate deeper responses below MR4.5that is 4.5-log reduction by the international scale (IS) 0.0032%. Methods: An ODK-1201 kit (Otsuka Pharmaceutical Co. Japan) is based on a HawkZ05 Fast One-Step RT-PCR Kit (Roche, Indianapolis, IN, USA). In this kit, reverse transcription and quantitative PCR (RT-qPCR) are performed in one tube, of which products are measured by ABI 7500 fast Dx (Life Technologies Japan, Japan). To isolate enough amount of RNA for the evaluation of molecular responses below MR4.5in CML patients, 7 ml of peripheral blood is required for this kit. To get the conversion factor (CF) for the IS, the panel of WHO International Standard was obtained from NIBSC, which comprises four ampoules each containing freeze-dried BCR-ABL-positive and -negative cells at various ratios from 0.01% to 10%. RNAs were isolated from each ampoule with a QIAamp RNA Blood Mini Kit (QIAGEN) and were subjected to the measurement of major BCR-ABL and ABL mRNAs with an ODK-1201 kit. Since %BCR-ABL/ABL in the WHO standard panel was already defined, CF was calculated by comparing the values of %BCR-ABL/ABL obtained by ODK-1201 with those of WHO standard panel. To validate the results obtained by ODK-1201, we collected 267 peripheral blood samples from CML patients as a multicenter study, and 120 samples in these were measured by ODK-1201 and in the international reference laboratory (Adelaide Lab), respectively. In addition, we conducted another multicenter study to compare ODK-1201 with an Ipsogen BCR-ABL1 Mbcr IS-MMR DX Kit (QIAGEN) and a One-Step qRT-PCR BCR-ABL Kit (Molecular MD), in which we measured the samples from 201 CML patients, 25 patients with non-CML hematologic malignancies and 25 healthy volunteers by these methods. All of the samples were obtained after the written informed consent was given. Results: The CF of ODK-1201 determined by the WHO standard panel was 1.12. After conversion to the IS by this CF, %BCR-ABL/ABLIS determined by ODK-1201 ranged from 0.0007% to 144.6867% in CML patients. In contrast, all of the values were below 0.0007% in the samples from patients with non-CML hematologic malignancies and healthy volunteers. The overall correlation between ODK-1201 and Adelaide Lab system was very good (94% within 3-fold bias) and the evaluation of MMR was concordant in 84% of CML samples. As for 46 CML samples with %BCR-ABL/ABL IS between 0.0032% and 0.1%, IS values by ODK-1201 were similar to the ones by Adelaide Lab system (with MR4.5; 0.0032%) with 89% within 3-fold and high correlation coefficient (r=0.89). Also, the correlation between ODK-1201 and Ipsogen was r = 0.97 (n = 224) and that between ODK-1201 and Molecular MD kit was r = 0.97 (n = 221), indicating that ODK-1201 was as accurate as Ipsogen and Molecular MD kits. Together, these results indicate that ODK-1201 was capable of measuring at least MR4.5 with the lowest value detected as 0.0007% (calculated sensitivity of less than MR5.0) by the IS. Conclusions: We developed a major BCR-ABLmRNA kit, ODK-1201 with CF for the IS and confirmed its high sensitivity (0.0007%) and accuracy. The procedure is very simple and takes only 2.5 h, and it needs a small amount of blood because RT-qPCR is performed in a single tube. It will enable us to evaluate deeper molecular responses in any laboratories. Conflict of interest :received research fundinand and honoraria from Otsuka I.M. received honoraria from Otsuka during the conduct of the study. H.N. received research funding from Otsuka during the conduct of the study and received research funding and honoraria from otsuka outside the submitted work. C.Y. received research funding from Otsuka during the conduct of the study and honoraria from Novartis and Bristol Myers Squibb. S.B. received research funding and honoraria from Novartis, Bristol Myers Squibb, Otsuka and Ariad. L.F. has no conflict of interest. K.D. and T.S. are employee of Otsuka. Y.K. received honoraria from Otsuka during the conduct of the study. T.N. received grants and personal fees from Otsuka during the conduct of the study, and grants and personal fees from Otsuka outside the submitted work. Disclosures Matsumura: Otsuka: Consultancy, Honoraria. Nakamae:Otsuka : Research Funding. Yoshida:Otsuka: Research Funding. Branford:Otsuka: Research Funding. Koga:Otsuka: Employment. Sogabe:Otsuka: Employment. Kanakura:Otsuka: Consultancy. Naoe:Otsuka: Consultancy.
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Lim, Da Hye, Hyunseul Jee, Kyung Chul Moon, Chae Seung Lim, and Woong Sik Jang. "Development of a Simple DNA Extraction Method and Candida Pan Loop-Mediated Isothermal Amplification Assay for Diagnosis of Candidemia." Pathogens 11, no. 2 (January 18, 2022): 111. http://dx.doi.org/10.3390/pathogens11020111.
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To reduce the morbidity and mortality of candidemia patients through rapid treatment, the development of a simple, rapid molecular diagnostic method that is based on nucleic acid extraction and is superior to conventional methods for detecting Candida in the blood is necessary. We developed a multiplex Candida Pan/internal control (IC) loop-mediated isothermal amplification (LAMP) assay and a simple DNA extraction boiling protocol using Chelex-100 that could extract yeast DNA in blood within 20 min. The Chelex-100/boiling method for DNA extraction showed comparable efficiency to that of the commercial QIAamp UCP Pathogen Mini Kit using Candida albicans qPCR. In addition, the Candida Pan/IC LAMP assay showed superior sensitivity to that of general Candida Pan and species qPCRs against clinical DNA samples extracted with the QIAamp UCP Pathogen Mini Kit and Chelex-100/boiling method. The Candida Pan/IC LAMP assay followed by Chelex-100/boiling-mediated DNA extraction showed high sensitivity (100%) and specificity (100%) against clinical samples infected with Candida. These results suggest that the Candida Pan/IC LAMP assay could be used as a rapid molecular diagnostic test for candidemia.
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Alnahash, Abdullah, Young-Min Song, Sae-Kyung Min, Hyun-Jin Lee, Min-Ji Kim, Yoon-Hee Park, Je-Uk Park, and Jun-Beom Park. "Effects of Connective Tissue Growth Factor on the Cell Viability, Proliferation, Osteogenic Capacity and mRNA Expression of Stem Cell Spheroids." Applied Sciences 11, no. 14 (July 16, 2021): 6572. http://dx.doi.org/10.3390/app11146572.
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Background: Connective tissue growth factor (CTGF) is a cellular communication network factor family protein involved in many cellular functions. The purpose of this study was to determine the effects of CTGF on the proliferation, osteogenic capacity, and mRNA expression of spheroids composed of gingiva-derived mesenchymal stem cells (GMSCs). Methods: CTGF was applied at final concentrations of 0, 25, 50, 100, and 200 ng/mL. Qualitative cell viability was determined using Live/Dead kit assay. Metabolic viability was determined with a colorimetric assay kit. Osteogenic activity was analyzed with alkaline phosphatase activity and Alizarin Red S staining. Quantitative polymerase chain reaction (qPCR) was used to assess the expression levels of RUNX2, BSP, OCN, and COL1A1. Results: In general, there was no significant difference in cell viability between the groups on Days 1, 4, and 7. Addition of CTGF produced an increase in Alizarin Red S staining. qPCR results demonstrated that the mRNA expression levels of RUNX2, BSP, OCN, and COL1A1 were significantly increased with the addition of CTGF. Conclusions: Based on these findings, we conclude that CTGF can be applied for increased osteogenic differentiation of stem cell spheroids.
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Abdulkadir, Hani, Jennine Grootens, Matilda Kjellander, Eva Hellstrom Lindberg, Gunnar Nilsson, and Johanna Ungerstedt. "Histone Deacetylase Inhibitor SAHA Mediates Epigenetic Silencing of KIT D816V Mutated Systemic Mastocytosis Primary Mast Cells and Selective Apoptosis of Mutated Mast Cells." Blood 126, no. 23 (December 3, 2015): 2834. http://dx.doi.org/10.1182/blood.v126.23.2834.2834.
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Abstract Systemic mastocytosis (SM) is a myeloproliferative disease for which there is currently no specific therapy. Over 90% of the patients carry the D816V point mutation that renders the KIT receptor constitutively active. In the current study, we assessed the sensitivity of mast cell line HMC1.2 and primary SM patient mast cells to histone deacetylase inhibitors, and found that SAHA is most efficient. SAHA induced a rapid downregulation of KIT mRNA, with a subsequent reduction in total KIT protein as well as cell surface KIT. This was followed by major mast cell apoptosis. Primary SM patient mast cells cultured ex vivo were even more sensitive to SAHA than HMC1.2 cells, whereas healthy subject mast cells were unaffected. There was a correlation between cell death and SM disease severity, where cell death was more pronounced in the case of aggressive disease, with almost 100% cell death among mast cells from the mast cell leukemia patient. Using ChIP qPCR, we found that the level of active chromatin mark H3K18ac/totalH3 decreased significantly in the KIT region, due to an increase in H3 density. This epigenetic silencing was specific to the KIT region and not seen in control genes upstream and downstream of KIT. Primary analysis of ChIP-seq data for histone marks H3K4me3 and H3K27me3, demonstrates a downregulation of transcription factors involved in activation of KIT receptor, such as MAPK, for the SAHA treated samples. This indicates an indirect epigenetic silencing of KIT. Our results therefore demonstrate that SAHA epigenetically silences KIT, and work is ongoing to elucidate the exact mechanisms of KIT regulation. Altogether, SAHA maybe a specific treatment for SM. Disclosures No relevant conflicts of interest to declare.
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Genoud, Valeria, Martin Stortz, Ariel Waisman, Bruno G. Berardino, Paula Verneri, Virginia Dansey, Melina Salvatori, Federico Remes Lenicov, and Valeria Levi. "Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR." PLOS ONE 16, no. 2 (February 26, 2021): e0247792. http://dx.doi.org/10.1371/journal.pone.0247792.
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Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.
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Gaur, Ritu, Dipesh Kumar Verma, Ritin Mohindra, Kapil Goyal, Shipra Gupta, Vidhi Singla, Vaibhav Sahni, et al. "Buccal swabs as non-invasive specimens for detection of severe acute respiratory syndrome coronavirus-2." Journal of International Medical Research 49, no. 5 (May 2021): 030006052110169. http://dx.doi.org/10.1177/03000605211016996.
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Introduction The current gold standard for detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA involves subjecting nasopharyngeal or oropharyngeal swabs to reverse transcription quantitative PCR (RT-qPCR). However, both sample types need to be collected by trained professionals. Using self-collected buccal swabs as an alternative could simplify and accelerate diagnosis of coronavirus disease 2019 (COVID-19). Objective To assess self-collected buccal swab samples as an alternative method for SARS-CoV-2 detection in patients with COVID-19. Methods Buccal swab samples were self-collected by 73 patients with COVID-19. Total RNA was extracted using Qiagen kits. RNA encoding the SARS-CoV-2 Env protein and human RNase P as an internal control was amplified using the TRUPCR® SARS-CoV-2 RT-qPCR kit version 2.1 and a Bio-Rad CFX96 Real-Time Detection System. Result The sensitivity of RT-qPCR from buccal swabs was 58.9% (43/73; 95% confidence interval [CI] 46.77%–70.27%) and that of RT-qPCR from saliva was 62.90% (39/62; 95% CI 49.69%–74.84%) taking positive SARS-CoV-2 RT-qPCR from nasopharyngeal swabs as the gold standard. Conclusion Self-collected buccal swabs are promising alternatives to nasopharyngeal or oropharyngeal swabs for SARS CoV-2 detection.
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Ramos, Lucia, I. P. Shamini Pushparajah, M. Shahjahan Kabir, Bethan E. Parry, and Kerry R. Everett. "Propidium monoazide combined with qPCR to differentiate live and dead conidia of Neofabraea actinidiae." New Zealand Plant Protection 71 (July 30, 2018): 348. http://dx.doi.org/10.30843/nzpp.2018.71.218.
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Neofabraea actinidiae can occasionally cause post-harvest rot in kiwifruit. Quantitative polymerase chain reaction (qPCR) analysis represents a feasible and accurate option for identifying and quantifying this rot but is limited because qPCR results do not differentiate live and dead conidia. Propidium monoazide (PMA) is a photoreactive dye that penetrates into the damaged cell-wall membranes of dead conidia binding to the DNA and thus suppressing its amplification by qPCR. A commercial kit containing PMA was trialled for differentiating between live and dead N. actinidiae conidia. The most suitable conditions were 1 μM PMA with 10 min light emitting diode (LED) exposure, and could clearly distinguish high concentrations of live from similar concentrations of dead conidia when tested separately and as a mixture. Low concentrations of live N. actinidiae conidia could be distinguished from dead ones when tested separately, but not as a mixture. Additional work is needed to optimise the effectiveness of the PMA binding and apply this concept in the orchard.
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Lin, Shu-Hsien, Kun-Ta Wu, Chih-Chi Wang, Kuang-Tzu Huang, Kuang-Den Chen, Chih-Che Lin, Li-Wen Hsu, and King-Wah Chiu. "HCV RNA in serum and liver samples of patients undergoing living donor liver transplantation." Journal of International Medical Research 49, no. 8 (August 2021): 030006052110349. http://dx.doi.org/10.1177/03000605211034945.
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Objective To compare hepatitis C virus (HCV) RNA levels from serum and explanted native liver samples from patients undergoing living donor liver transplantation (LDLT). Methods This was a prospective observational study. Serum and liver samples were collected from consecutive serum anti-HCV-positive transplant recipients between February 2016 to August 2019. HCV RNA was extracted from liver samples and subjected to one-step reverse-transcription qPCR. using the TopScript One Step qRT-PCR Probe Kit with HCV qPCR probe assay and human GAPDH qPCR probe assay on a ViiA7 Real-Time PCR System. Results Among the 80 patients, 36% (29/80) were HCV RNA positive in serum and 85% (68/80) had positive hepatic HCV RNA. Post-liver transplantation, 4% (3/80) patients were serum positive. Conclusions Our study suggests that pre-transplant serum HCV RNA levels may give an underestimate of the number of positive HCV RNA cases and that hepatic HCV RNA data may be more accurate.
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Preißler, Kathleen, Alexander Dennis Watzal, Miguel Vences, and Sebastian Steinfartz. "Detection of elusive fire salamander larvae (Salamandra salamandra) in streams via environmental DNA." Amphibia-Reptilia 40, no. 1 (2019): 55–64. http://dx.doi.org/10.1163/15685381-18000007.
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Abstract In the face of the global biodiversity crisis, the monitoring of species richness and diversity is experiencing an increased demand entailing a raise in cost and time investment. The analysis of species-specific DNA fragments in environmental samples (eDNA) such as from water or soil, facilitate the molecular detection of species without the specific sampling of individuals. The invasive chytrid fungus Batrachochytrium salamandrivorans (Bsal) is infecting natural fire salamander populations (Salamandra salamandra) and causes chytridiomycosis resulting in infrequent regional extinctions of populations across Central Europe. With regard to the expanding distribution of Bsal over the last years, cost-effective monitoring of fire salamanders is important for the conservation of this species. Based on a real-time quantitative PCR (qPCR) assay, we developed a new protocol to detect S. salamandra larvae in streams via eDNA, using species-specific primers of the mitochondrial control region (D-loop). We tested the efficiency of qPCR primer sets for six combinations of DNA extraction kits coupled with subsequent PCR inhibitor removal kits for obtaining qPCR-detectable S. salamandra eDNA from water filters, that were taken both from natural streams and artificial water tanks in the laboratory as positive controls. We found that the DNeasy Blood & Tissue Kit in combination with the DNeasy PowerClean CleanUp Kit performed best for detecting salamander larvae from natural streams. Our experimental protocol paves the way for resource-saving approaches to monitor S. salamandra larvae, but also confirms the limits to this eDNA approach in that it requires optimized laboratory protocols.
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Grgicak, Catherine M., Zena M. Urban, and Robin W. Cotton. "Investigation of Reproducibility and Error Associated with qPCR Methods using Quantifiler® Duo DNA Quantification Kit*." Journal of Forensic Sciences 55, no. 5 (July 12, 2010): 1331–39. http://dx.doi.org/10.1111/j.1556-4029.2010.01460.x.
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Barra, Gustavo Barcelos, Ticiane Henriques Santa Rita, Ana Luisa Santa Cruz Almeida, Rafael Henriques Jácomo, and Lídia Freire Abdalla Nery. "Serum Has Higher Proportion of Janus Kinase 2 V617F Mutation Compared to Paired EDTA-Whole Blood Sample: A Model for Somatic Mutation Quantification Using qPCR and the 2-∆∆Cq Method." Diagnostics 10, no. 3 (March 12, 2020): 153. http://dx.doi.org/10.3390/diagnostics10030153.
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Detection of the Janus Kinase-2 (JAK2) V617F mutation is a diagnostic criterion for myeloproliferative neoplasms, and high levels of mutant alleles are associated with worse outcomes. This mutation is usually tested on blood DNA by allele-specific qPCR (AS-qPCR) and measured using absolute quantification. However, some automated DNA extractions co-extracts of PCR inhibitors from blood and qPCR absolute quantification need increased efforts in order to maintain standard curves. JAK2 V617F can also be detected in serum using droplet digital PCR (ddPCR), a specimen with less inhibitors and favorable to automated extractions, but ddPCR instruments are not wide available as qPCR thermocyclers. Here, we evaluate whether JAK2 V617F could be accurately quantified by AS-qPCR using the 2-∆∆Cq method on blood DNA and validate the assay using gold-standard molecular diagnostic protocols. Next, we apply the validated method to assess if the mutation could be reliably detected/quantified in serum. JAK2 V617F could be quantified by AS-qPCR using the 2-∆∆Cq method—the assay was highly accurate (bias of 1.91%) compared to a commercial kit, highly precise (total CV% of 0.40%, 1.92%, 11.12% for samples with 93%, 54%, and 2.5% of mutant allele), highly sensitive (limit of detection of 0.15%), and demonstrated a linear detection response from 1.1% to 99.9%. Serum presented a higher mutant allele burden compared to the paired whole blood (mean of 4%), which allows for an increased JAK2 mutant detection rate and favors increased JAK2 V617F high-throughput analysis.
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Senina, M. Е., Y. А. Savochkina, L. V. Skvortsov, T. I. Popova, and L. V. Dzhedzheia. "The qPCR analysis of vaginal microflora for the diagnosis of bacterial vaginosis." Biomedical Chemistry: Research and Methods 2, no. 2 (2019): e00084. http://dx.doi.org/10.18097/bmcrm00084.
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The correct information about of the vaginal microflora plays an important role in preventing the occurrence of urinary tract infections and sexually transmitted infections among women. Disbalance of obligate and facultative microflora causes disbacteriosis, a risk factor for emergence of infectious diseases. It is known that the cause of bacterial vaginosis (BV) is not a single pathogen but a impairments in of the general balance of the vaginal microflora, which manifests a decrease of the normal microflora (Lactobacillus spp) and intense increase of pathogenic aerobic and anaerobic bacteria. The development of molecular genetic analysis methods, in particular, approaches based on the use of polymerase chain reaction (PCR), significantly expanded understanding of the diversity of microbial biotopes, including identification of the key and new «players» in the development of BV. The aim of our study was to evaluate the performance of real-time PCR kit «Femoscreen» («Lytech», Russia) for comprehensive BV diagnosis.
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Mascuch, Samantha J., Sara Fakhretaha-Aval, Jessica C. Bowman, Minh Thu H. Ma, Gwendell Thomas, Bettina Bommarius, Chieri Ito, et al. "A blueprint for academic laboratories to produce SARS-CoV-2 quantitative RT-PCR test kits." Journal of Biological Chemistry 295, no. 46 (September 3, 2020): 15438–53. http://dx.doi.org/10.1074/jbc.ra120.015434.
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Widespread testing for the presence of the novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in individuals remains vital for controlling the COVID-19 pandemic prior to the advent of an effective treatment. Challenges in testing can be traced to an initial shortage of supplies, expertise, and/or instrumentation necessary to detect the virus by quantitative RT-PCR (RT-qPCR), the most robust, sensitive, and specific assay currently available. Here we show that academic biochemistry and molecular biology laboratories equipped with appropriate expertise and infrastructure can replicate commercially available SARS-CoV-2 RT-qPCR test kits and backfill pipeline shortages. The Georgia Tech COVID-19 Test Kit Support Group, composed of faculty, staff, and trainees across the biotechnology quad at Georgia Institute of Technology, synthesized multiplexed primers and probes and formulated a master mix composed of enzymes and proteins produced in-house. Our in-house kit compares favorably with a commercial product used for diagnostic testing. We also developed an environmental testing protocol to readily monitor surfaces for the presence of SARS-CoV-2. Our blueprint should be readily reproducible by research teams at other institutions, and our protocols may be modified and adapted to enable SARS-CoV-2 detection in more resource-limited settings.
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Pappa, Styliani A., Panagiota I. Kontou, Pantelis G. Bagos, and Georgia G. Braliou. "Urine-Based Molecular Diagnostic Tests for Leishmaniasis Infection in Human and Canine Populations: A Meta-Analysis." Pathogens 10, no. 3 (February 27, 2021): 269. http://dx.doi.org/10.3390/pathogens10030269.
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Leishmaniasis is a neglected tropical disease affecting humans and domesticated animals with high mortality in endemic countries. The pleiotropy of symptoms and the complicated gold-standard methods make the need for non-invasive, highly sensitive diagnostic tests imperative. Individual studies on molecular-based Leishmania diagnosis in urine show high discrepancy; thus, a data-evidenced comparison of various techniques is necessary. We performed a systematic review and meta-analysis using the bivariate method of diagnostic methods to pool sensitivities and specificities. We investigated the impact of DNA-extraction method, PCR type, amplified locus, host species, leishmaniasis form, and geographical region. The pooled sensitivity was 69.2%. Tests performed with the kit-based DNA extraction method and qPCR outweighed in sensitivity the phenol-chloroform-based and PCR methods, while their combination showed a sensitivity of 79.3%. Amplified locus, human or canine as host and cutaneous or visceral leishmaniasis revealed similar sensitivities. Tests in European and Middle Eastern countries performed better than tests in other regions (sensitivity 81.7% vs. 43.7%). A combination of kit-based DNA extraction and qPCR could be a safer choice for molecular diagnosis for Leishmania infection in urine samples in European–Middle Eastern countries. For the rest of the world, more studies are needed to better characterize the endemic parasite species.
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Mehl, Martin, Patricia Meinhardt, Erika Lorenzen, Caroline Knoll, Patrizia Howaldt, Jennifer Geister, Steffen Mergemeier, and Markus Lacorn. "Detection of SARS-CoV-2 Virus on Stainless-Steel Surfaces: AOAC Performance Tested MethodSM 022102." Journal of AOAC INTERNATIONAL 104, no. 4 (April 8, 2021): 924–34. http://dx.doi.org/10.1093/jaoacint/qsab049.
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Abstract Background The SureFast® SARS-CoV-2 PLUS Test is a reverse transcription qPCR (RT-qPCR) assay for the direct, qualitative detection of novel coronavirus (SARS-CoV-2) RNA from stainless-steel environmental sample swabs. Objective To validate the SureFast SARS-CoV-2 PLUS Kit as part of the AOAC Research Institute’s Emergency Response Validation Performance Tested Method(s)SM program. Method The SureFast SARS-CoV-2 PLUS Kit was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms (both near neighbors and background organisms) using the ThermoBLAST program. The candidate method was evaluated in an unpaired study design for one environmental surface (stainless steel) and compared to the US Centers for Disease Control and Prevention 2019-Novel Coronavirus (2019-nCoV) Real-Time RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020). Results Results of the in silico analysis demonstrated 99.99% selectivity of the method in being able to detect target sequences of the known CoV-2 genomes and discriminate them from near neighbors. In the matrix study, the candidate method demonstrated statistically significant better recovery of the target analyte than the PCR detection reference method. Conclusions The SureFast SARS-CoV-2 PLUS Kit is a rapid and accurate method that can be utilized by food producers to detect the causative agent of COVID-19 on stainless-steel contact surfaces. Highlights SureFast SARS-CoV-2 PLUS test method is highly specific for primer/probe binding to the E target genome region for the SARS-CoV-2 virus, 99.99% binding specificity using in silico analysis.
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Azzopardi, Kristy I., Myra Hardy, Ciara Baker, Rhian Bonnici, Stacey Llewellyn, James S. McCarthy, Rebecca J. Traub, and Andrew C. Steer. "Detection of six soil-transmitted helminths in human stool by qPCR- a systematic workflow." PLOS ONE 16, no. 9 (September 30, 2021): e0258039. http://dx.doi.org/10.1371/journal.pone.0258039.
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Soil-transmitted helminths (STH) infect up to one-quarter of the global population, with a significant associated disease burden. The main human STH are: Ancylostoma spp. and Necator americanus (hookworms); Ascaris lumbricoides, Trichuris trichiura, and Strongyloides stercoralis. The aim of this study was to establish a scalable system for stool STH multiplex quantitative real-time polymerase chain reactions (qPCR). Stool samples collected in Fiji and preserved in potassium dichromate were transferred to Melbourne at ambient temperature. Samples were washed to remove potassium dichromate and DNA was extracted with the Mini-Beadbeater-24 and a column-based kit. A SYBR green qPCR to detect the vertebrate mitochondrial gene was used as a DNA extraction control. Samples were tested using a probe-based multiplex qPCR targeting A. lumbricoides, T. trichiura and S. stercoralis, and in a second multiplex reaction to detect hookworms to the species level (A. duodenale, A. ceylanicum, N. americanus). An internal amplification control in both multiplex assays was included to prevent false-negative results due to PCR inhibitors. Samples were homogenised for a single cycle of 40 seconds to release STH DNA and washed stool was stored for up to 15 weeks at -30°C without compromising DNA. Our multiplex qPCR detected multiple species of STH without reduced sensitivity compared to singleplex. qPCR data from 40 stools was validated against STH-positive stools determined by microscopy. We have developed and validated an efficient and staged system for detecting six clinically important STH affecting humans that could be easily implemented without advanced automation in any qPCR-capable laboratory.
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Sekikawa, Takahiro. "A new immunomagnetic bead separation–surfactant extraction treatment protocol for rapid and sensitive quantitative PCR detection of Cryptosporidium parvum DNA." Water Supply 17, no. 1 (July 27, 2016): 161–68. http://dx.doi.org/10.2166/ws.2016.125.
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The Cryptosporidium oocyst is encased in a robust wall that is extremely resistant to detrimental environmental factors such as chlorine used to disinfect potable water. Therefore, extracting oocyst DNA is not a trivial undertaking. Standard procedures used to extract DNA from oocysts, such as freeze–thaw (F/T) methods and DNA purification kits, are time-consuming and expensive and are difficult to implement in routine clinical practice. Therefore, we developed a surfactant extraction treatment (SET) that efficiently extracts DNA from the oocyst. Immunomagnetic separation (IMS) combined with quantitative real-time polymerase chain reaction (qPCR) detects pathogenic microorganisms with high sensitivity. The objective of the present study was to evaluate SET for its ability to simplify qPCR detection of 18S rDNA directly from immunomagnetic bead–oocyst conjugates. DNA extracted directly from the conjugates using SET did not affect DNA amplification in the qPCR assay. Further, the rate of DNA amplification using IMS–SET was greater than that using F/T combined with the DNA purification kit. The rate of recovery of oocysts from surface water samples spiked with oocysts did not differ significantly from previously published values. These data demonstrate that the new IMS–SET protocol using qPCR can simplify the recovery and detection of Cryptosporidium oocysts.
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Klajmon, Adrianna, Aldona Olechowska-Jarząb, Dominika Salamon, Agnieszka Sroka-Oleksiak, Monika Brzychczy-Włoch, and Tomasz Gosiewski. "Comparison of Antigen Tests and qPCR in Rapid Diagnostics of Infections Caused by SARS-CoV-2 Virus." Viruses 14, no. 1 (December 23, 2021): 17. http://dx.doi.org/10.3390/v14010017.
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Diagnostics of the coronavirus disease 2019 (COVID-19) using molecular techniques from the collected respiratory swab specimens requires well-equipped laboratory and qualified personnel, also it needs several hours of waiting for results and is expensive. Antigen tests appear to be faster and cheaper but their sensitivity and specificity are debatable. The aim of this study was to compare a selected antigen test with quantitative polymerase chain reaction (qPCR) tests results. Nasopharyngeal swabs were collected from 192 patients with COVID-19 symptoms. All samples were tested using Vitassay qPCR SARS-CoV-2 kit and the Humasis COVID-19 Ag Test (MedSun) antigen immunochromatographic test simultaneously. Ultimately, 189 samples were tested; 3 samples were excluded due to errors in taking swabs. The qPCR and antigen test results were as follows: 47 positive and 142 negative, and 45 positive and 144 negative, respectively. Calculated sensitivity of 91.5% and specificity of 98.6% for the antigen test shows differences which are not statistically significant in comparison to qPCR. Our study showed that effectiveness of the antigen tests in rapid laboratory diagnostics is high enough to be an alternative and support for nucleic acid amplification tests (NAAT) in the virus replication phase in the course of COVID-19.
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Moura, André, Mikael Arrais Hodon, Paulo Martins Soares Filho, Marina de Azevedo Issa, Ana Paula Ferreira de Oliveira, and Antônio Augusto Fonseca Júnior. "Comparison of nine DNA extraction methods for the diagnosis of bovine tuberculosis by real time PCR." Ciência Rural 46, no. 7 (July 2016): 1223–28. http://dx.doi.org/10.1590/0103-8478cr20151489.
Full textAbstract:
ABSTRACT: Bovine tuberculosis is an infectious disease with a high impact on the cattle industry, particularly in developing countries. PCR is a very sensitive method for detection of infectious agents, but the sensitivity of molecular diagnosis is largely dependent on the efficiency of the DNA extraction methods. The objective of this study was to evaluate DNA extraction methods for direct detection of Mycobacterium bovis in bovine tissue. Nine commercial kits for DNA extraction were evaluated when combined with two real time PCRs. The DNeasy Blood & Tissue Kit from QIAGEN showed better performance and sensitivity followed by the DNA Mini Kit RBC and FTA Elute Micro Card. Results suggested that, even when the analytical sensitivity of the qPCR is very high, the extraction method can influence the diagnostic sensitivity.