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1
Lemasova, L. V., G. A. Tkachenko, E. V. Prokhvatilova, L. I. Belitskaya, D. V. Viktorov, and A. V. Toporkov. "ASSESSMENT OF THE POSSIBILITY OF APPLICATION IN LABORATORY PRACTICE OF REAGENT KIT FOR DIAGNOSIS OF GLANDERS AND MELIOIDOSIS BY REAL-TIME POLYMERASE CHAIN REACTION." Russian Clinical Laboratory Diagnostics 64, no. 11 (November 15, 2019): 700–704. http://dx.doi.org/10.18821/0869-2084-2019-64-11-700-704.
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The reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR is designed for detecting in vitro diagnostics and differentiate the DNA of glanders and melioidosis pathogens by real-time multiplex PCR in biological (clinical) material and cultures of microorganisms, as well as environmental objects and solid food products (rice). During clinical testing diagnostic value of reagent kit AmpligenBurk-mallei/pseudomallei-RT PCR has been studied. Based on the results obtained, a high analytical sensitivity (1×103 microbe cells/ml) and specificity (100%) of PCR-RT with the developed reagent kit were established, regardless of the type of material being studied. The diagnostic sensitivity of PCR-RT using a set of reagents was at least 98.0% and specificity at least 99%. The stages of state examination have been completed, a registration certificate has been obtained at Roszdravnadzor, production, sale and use of reagent kit in medical laboratory practice have been permitted.
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Goncharova, E. V., A. E. Donnikov, V. V. Kadochnikova, S. A. Morozova, M. N. Boldyreva, I. S. Galkina, and D. V. Blinov. "Real-time RT-PCR diagnostics of virus causing COVID-19." FARMAKOEKONOMIKA. Modern Pharmacoeconomic and Pharmacoepidemiology 13, no. 1 (April 24, 2020): 52–63. http://dx.doi.org/10.17749/2070-4909.2020.13.1.52-63.
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Aim: the study was aimed to develop a reagent kit for the real-time RT-PCR diagnostics of virus causing COVID-19.Materials and Methods. Three target sites were chosen in the genome SARS-CoV-2. The testing included 220 samples, 48 artificially created positive samples (made from patients’ biomaterial) and 172 clinical samples (scrapes from nasal and pharyngeal cavities, bronchoalveolar lavage, expectoration, endotracheal/nasopharyngeal aspirate, feces, post-mortem material), obtained from two medical centers. Preliminary, the obtained biomaterial was analyzed with a reagent kit of comparison. The evaluation was performed with a confidential interval CI 95%. The calculation of CI for the sensitivity and specificity was made based on the distribution of χ2.Results. The authors developed a technology of novel coronavirus infection (COVID-19) real-time RT-PCR diagnostics for the application in practical healthcare and proposed the variants of testing at all the stages (preanalytical, analytical, and post-analytical, including automated results processing). The proposed reagent kit meets the requirements of the World Health Organization and the Ministry of Healthcare of the Russian Federation. The study results demonstrated high sensitivity and specificity. The sensitivity was 100% (95% CI) 95.6–100%; the specificity was 100% (95% CI) 96.7–100%.Conclusion. The proposed reagent kit was registered in the RF as a medical product; the registration certificate No. RZN 2020/9948 dated 01.04.2020. The application of the reagent kit in network laboratories will provide patients with access to testing for the virus causing COVID-19 and contribute to quick differential diagnostics, improvement of pandemic control, and accurate statistics on the spread of the virus.
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Hecht, Leonie-Sophie, Angeles Jurado-Jimenez, Markus Hess, Hussein El Halas, Gregor Bochenek, Hala Mohammed, Fahd Alzahrani, et al. "Verification and diagnostic evaluation of the RealStar® Middle East respiratory syndrome coronavirus (N gene) reverse transcription-PCR kit 1.0." Future Microbiology 14, no. 11 (July 2019): 941–48. http://dx.doi.org/10.2217/fmb-2019-0067.
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Aim: We report the diagnostic evaluation of a confirmatory reverse transcription-PCR (RT-PCR) kit targeting the Middle East respiratory syndrome coronavirus (MERS-CoV) N gene. Material & methods: 33 patient samples from two collections sites in Riyadh, Saudi Arabia, which were pre-characterized via real-time RT-PCR targeting MERS-CoV orf1a and upE, and were tested using the MERS-CoV N gene, as a confirmatory assay. This diagnostic procedure follows a two-step diagnostics scheme, recommended by the WHO. Results: 18/33 samples tested positive, 11/33 tested negative for MERS-CoV RNA and 2/33 showed uncertain results. Conclusion: The results suggest, that the RealStar® MERS-CoV (N gene) RT-PCR kit 1.0 can be considered a suitable and reliable confirmatory assay in combination with the RealStar MERS-CoV RT-PCR kit 1.0 according to the diagnostic scheme recommended by WHO.
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Hu, ZB, W. Ma, CC Uphoff, H. Quentmeier, and HG Drexler. "c-kit expression in human megakaryoblastic leukemia cell lines." Blood 83, no. 8 (April 15, 1994): 2133–44. http://dx.doi.org/10.1182/blood.v83.8.2133.2133.
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Abstract A panel of 164 continuous human leukemia-lymphoma cell lines was analyzed for expression of c-kit using Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). The c-kit transcripts were detectable in cell lines assigned to the myeloid (in 7 of 29 by Northern blotting and in 4 of 8 by RT-PCR), monocytic (in 1 of 24 by Northern blotting and in 3 of 6 by RT-PCR), erythroid (in 6 of 8 by Northern blotting and in 5 of 5 by RT-PCR), and megakaryoblastic (in 10 of 10 by Northern blotting) lineages, c-kit expression was not seen by Northern blotting or RT-PCR analysis in any of the 93 lymphoid leukemia, myeloma, or lymphoma cell lines. Treatment of four megakaryoblastic cell lines with protein kinase C activators (phorbol ester 12-O-tetradecanoylphorbol 13-acetate and Bryostatin 1) led to terminal differentiation as assessed by morphologic alterations, changes in the surface marker profile, and growth arrest. These effects were associated with enhanced c-kit mRNA expression. Exposure to all- trans retinoic acid down-regulated c-kit mRNA levels, while simultaneously causing morphologic alterations in all four cell lines. Stimulation with growth factors (interleukin-3, granulocyte macrophage- colony stimulating factor, and insulin-like growth factors I and II), used to assess any role of c-kit in proliferative processes, did not lead to significant upregulation or downregulation of c-kit expression. The finding of constitutive and high expression of c-kit mRNA in all megakaryoblastic leukemia cell lines and its modulation by various reagents might further contribute to the understanding of megakaryopoietic proliferation, differentiation, and leukemogenesis.
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Hu, ZB, W. Ma, CC Uphoff, H. Quentmeier, and HG Drexler. "c-kit expression in human megakaryoblastic leukemia cell lines." Blood 83, no. 8 (April 15, 1994): 2133–44. http://dx.doi.org/10.1182/blood.v83.8.2133.bloodjournal8382133.
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A panel of 164 continuous human leukemia-lymphoma cell lines was analyzed for expression of c-kit using Northern blotting and reverse transcriptase-polymerase chain reaction (RT-PCR). The c-kit transcripts were detectable in cell lines assigned to the myeloid (in 7 of 29 by Northern blotting and in 4 of 8 by RT-PCR), monocytic (in 1 of 24 by Northern blotting and in 3 of 6 by RT-PCR), erythroid (in 6 of 8 by Northern blotting and in 5 of 5 by RT-PCR), and megakaryoblastic (in 10 of 10 by Northern blotting) lineages, c-kit expression was not seen by Northern blotting or RT-PCR analysis in any of the 93 lymphoid leukemia, myeloma, or lymphoma cell lines. Treatment of four megakaryoblastic cell lines with protein kinase C activators (phorbol ester 12-O-tetradecanoylphorbol 13-acetate and Bryostatin 1) led to terminal differentiation as assessed by morphologic alterations, changes in the surface marker profile, and growth arrest. These effects were associated with enhanced c-kit mRNA expression. Exposure to all- trans retinoic acid down-regulated c-kit mRNA levels, while simultaneously causing morphologic alterations in all four cell lines. Stimulation with growth factors (interleukin-3, granulocyte macrophage- colony stimulating factor, and insulin-like growth factors I and II), used to assess any role of c-kit in proliferative processes, did not lead to significant upregulation or downregulation of c-kit expression. The finding of constitutive and high expression of c-kit mRNA in all megakaryoblastic leukemia cell lines and its modulation by various reagents might further contribute to the understanding of megakaryopoietic proliferation, differentiation, and leukemogenesis.
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Deng, Ming Y., He Wang, Gordon B. Ward, Tammy R. Beckham, and Thomas S. McKenna. "Comparison of Six RNA Extraction Methods for the Detection of Classical Swine Fever Virus by Real-Time and Conventional Reverse Transcription–PCR." Journal of Veterinary Diagnostic Investigation 17, no. 6 (November 2005): 574–78. http://dx.doi.org/10.1177/104063870501700609.
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Six RNA extraction methods, i.e., RNAqueous kit, Micro-to-midi total RNA purification system, NucleoSpin RNA II, GenElute mammalian total RNA kit, RNeasy mini kit, and TRIzol LS reagent, were evaluated on blood and 7 tissues from pig infected with classical swine fever virus (CSFV). Each of the 6 extraction methods yielded sufficient RNA for positive results in a real-time reverse transcription–PCR (RT-PCR) for CSFV, and all RNA, except the one extracted from blood by TRIzol LS reagent, yielded positive results in both a conventional RT-PCR for CSFV and a conventional RT-PCR for an endogenous gene encoding β-actin. The RNA extracted from blood by TRIzol LS reagent became positive in both conventional RT-PCR assays when it was diluted to 1:2, 1:4, or up to 1:64 in nuclease-free water. It is concluded that all 6 methods are more or less useful for the detection of CSFV by real-time and conventional RT-PCR in swine blood and tissues. However, some of the 6 reagents offer certain advantages not common to all 6 extraction procedures. For example, RNA extracted by the TRIzol LS reagent constantly had the highest yield; that by the RNAqueous kit had the highest A260/A280 ratio for almost all samples; and that by the NucleoSpin RNA II and the GenElute mammalian total RNA kit was most likely to be free of contaminations with genomic DNA.
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Ningrum, Esti Prasetya, Sedyo Hartono, Sri Sulandari, and Susamto Somowiyarjo. "Multiplex RT-PCR Assay for Crinivirus Detection Using RNA Prepared from Three Extraction Methods on Tomato Plant." Jurnal Perlindungan Tanaman Indonesia 23, no. 2 (December 3, 2019): 250. http://dx.doi.org/10.22146/jpti.36558.
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Tomato infectious chlorosis virus (TICV) and Tomato chlorosis virus (ToCV) are members of the Crinivirus genus that induces yellowing symptoms in tomato plants. Detection of both viruses is generally carried out singly, thus it is necessary to develop a fast, accurate and efficient detection method to detect multiple viruses simultaneously in an effort to determine the suitable disease management strategies. This study was aimed to detect both viruses using the multiplex RT-PCR method and evaluate three methods of total RNA preparation used from tomato plants as RT-PCR templates. The methods evaluated were simple direct tube (SDT), simple dsRNA, and commercial kit (RNeasy Plant Mini Kit) as a comparison. The total source of RNA came from Crinivirus symptomatic tomato leaves from Kopeng, and Ketep (Central Java); Pakem (Yogyakarta); Malang (East Java); and Bogor (West Java). Single RT-PCR and multiplex RT-PCR using specific primers CPd I/CPd II and ToCV CF/ToCV CR with DNA band targets of 760 bp and 360 bp. The SDT and dsRNA methods have been successful in obtaining total RNA and viral RNA from tomato leaf samples. Total RNA RT-PCR with simple SDT and dsRNA methods followed by multiplex RT-PCR produces specific DNA band intensities that are comparable to Kit. RNA preparation with SDT and simple dsRNA methods is a simple, fast, easy and affordable method in providing templates for RT-PCR. Multiplex RT-PCR technique using two pairs of specific primers CPd I/CPd II and ToCV CF/ToCV CR is suitable for simultaneous testing of TICV and ToCV.
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Demeke, Tigst, Jemima Malabanan, Michelle Holigroski, and Monika Eng. "Effect of Source of DNA on the Quantitative Analysis of Genetically Engineered Traits Using Digital PCR and Real-Time PCR." Journal of AOAC INTERNATIONAL 100, no. 2 (March 1, 2017): 492–98. http://dx.doi.org/10.5740/jaoacint.16-0284.
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Abstract Seven commercially available DNA extraction kits were compared with a cetyltrimethylammonium bromide (CTAB)method to determine the suitability of the extracted DNA for RainDrop digital PCR (dPCR) and real-time PCR (RT-PCR) quantification of OXY235 canola, FP967 flax, and DP305423 soybean (spiked at the 0.1% level). For the kits, the highest amount of DNA extracted from a 0.2 g sample was obtained using OmniPrep for Plant for flax andDNeasy mericon Food for canola and soybean. For canola, DNA extracted with the Fast ID Genomic DNA Extraction Kit, FastDNA Spin Kit, GM Quicker 2, NucleoSpin Food, and DNeasy mericon Food was suitable for dPCR and RT-PCR. For flax, DNA extracted with Fast ID, FastDNA Spin Kit, OmniPrep for Plant, and NucleoSpin Food was suitable for RT-PCR. However, only Fast ID yielded DNA suitable for dPCR. For soybean, DNA extracted with five and six of the seven DNA extraction kits was suitable for dPCR and RT-PCR, respectively. Overall, Fast ID provided reliable results regardless of species or analysis method used. Canola, flax, and soybean DNA extracted with the CTAB method and then purified were suitable for both dPCR and RT-PCR. This is the first report showing the effect of different DNA extraction methods on the absolute quantification of genetically engineered traits using dPCR.
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Otter, Jonathan A., Nancy L. Havill, and John M. Boyce. "Evaluation of Real-Time Polymerase Chain Reaction for the Detection of Methicillin-ResistantStaphylococcus aureuson Environmental Surfaces." Infection Control & Hospital Epidemiology 28, no. 8 (August 2007): 1003–5. http://dx.doi.org/10.1086/519207.
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We compared real-time polymerase chain reaction (RT-PCR) with in vitro culture for detecting methicillin-resistantStaphylococcus aureusin samples from environmental surfaces. The sensitivity of RT-PCR, compared with culture, was 92.5%, and the specificity was 51.4%. Because of poor specificity, the RT-PCR kit tested is not suitable for the detection of MRSA on hospital surfaces.
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Stephenson, Patrick, David Crabtree, Katharine Evans, Heikki Salavirta, Pius Brzoska, Ana-Maria Leonte, Amanda Manolis, and Daniele Sohier. "Validation of the Thermo Scientific™ SARS-CoV-2 RT-PCR Detection Workflow for the Detection of SARS-CoV-2 from Stainless-Steel Environmental Surface Swabs: AOAC Performance Tested MethodSM 012103." Journal of AOAC INTERNATIONAL 104, no. 4 (April 5, 2021): 935–47. http://dx.doi.org/10.1093/jaoacint/qsab050.
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Abstract Background The Thermo Scientific™ SARS-CoV-2 reverse transcription–polymerase chain reaction (RT-PCR) Detection Workflow, packaged with Applied Biosystems™ TaqMan™ 2019-nCoV Assay Kit v1 targets three different SARS-CoV-2 genomic regions in a single RT-PCR reaction. Objective To validate the Thermo Scientific SARS-CoV-2 RT-PCR Workflow, for the detection of SARS-CoV-2 virus on stainless-steel surfaces as part of the AOAC Performance Tested MethodSM Emergency Response Validation program. Method The Applied Biosystems TaqMan 2019-nCoV Assay Kit v1, as part of the Thermo Scientific SARS-CoV-2 RT-PCR Workflow, was evaluated for specificity using in silico analysis of 15 764 SARS-CoV-2 sequences and 65 exclusivity organisms. The Thermo Scientific SARS-CoV-2 RT-PCR Workflow was evaluated in an unpaired study for one environmental surface (stainless steel) and compared to the U.S. Centers for Disease Control and Prevention 2019-Novel Coronavirus RT-PCR Diagnostic Panel, Instructions for Use (Revision 4, Effective 6/12/2020). Results In silico analysis showed that, of the 15 756 target SARS-CoV-2 genomes analyzed, 99% of the strains/isolates are perfectly matched to at least two of the three assays, and more than 90% have 100% homology to all three assays (ORF1ab, N-gene, S-gene) in the SARS-CoV-2 Kit. None of the 65 non-target strain genomes analyzed showed matching sequences. In the matrix study, the Thermo Scientific SARS-CoV-2 workflow showed comparable detection to the centers of disease control and prevention (CDC) method. Conclusions The Thermo Scientific SARS-CoV-2 RT-PCR Workflow is an effective procedure for detection of RNA from SARS-CoV-2 virus from stainless steel. Highlights The workflow provides equivalent performance results with the two tested RNA extraction platforms and the two tested RT-PCR instruments.
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Dinh, Phat X. "Multiplex RT-PCR assay to differentiate genotypes of porcine reproductive and respiratory syndrome virus in swine." Journal of Agriculture and Development 18, no. 06 (December 27, 2019): 8–13. http://dx.doi.org/10.52997/jad.2.06.2019.
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Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important diseases to swine industry worldwide. Due to the heterogeneity of field isolates, accurate detection of the PRRS virus is a diagnostic challenge. Recently, co-infection with NA-PRRSV, EU-PRRSV and HP-PRRSV isolates continuously increases in many countries, resulting in a significant impact on PRRSV diagnostics and disease control on farms. To facilitate rapid diagnosis and reliable discrimination of NA-PRRSV, EU-PRRSV and HP-PRRSV, a multiplex RT-PCR assay was established with three pairs of primers targeting highly conservative regions of nsp2 gene with predicted multiplex RT-PCR products of 364 bp, 161 bp and 259 bp, respectively. The primer pairs were optimized to be highly specific for PRRSV genotypes and were able to detect the target gene at the limit of 102 copies/μL for each gene. Clinical samples were used to evaluate this multiplex RT-PCR in parallel with a commercial real-time RT-PCR kit. Results showed over 95.2% (20/21 samples) agreement between the mRT-PCR and the real-time RT-PCR kit. Hence, it indicated that this multiplex RT-PCR could be useful for rapid and deferential diagnosis of NA-PRRSV, EU-PRRSV and HP-PRRSV in swine farms.
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Endarsih, Wiwik, Sedyo Hartono, and Sri Sulandari. "Perbaikan Metode Ekstraksi dsRNA Virus secara Sederhana untuk RT-PCR Tiga Virus Tumbuhan." Jurnal Perlindungan Tanaman Indonesia 21, no. 2 (January 8, 2018): 106. http://dx.doi.org/10.22146/jpti.26026.
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Replicative form (RF) of RNA viruses are dsRNA structured nucleic acid, always found in plants infected by RNA virus. The principle of dsRNA extraction is based on the different affinity of nucleic acids for the cellulose powder and the specific adsorption in 16.6% ethanol buffer. The study aims to develop the simple dsRNA extraction method for the preparation of RT-PCR detection for Rehmannia mosaic virus (ReMV), Cucumber mosaic virus (CMV), Tomato chlorosis virus (ToCV), and compared with commercial kit. The analysis was performed by quantification of nucleic acid with spectrophotometer, efficiency of method (level of complexity, time, cost per reaction) and sequencing. The RNA concentration with simple methode of dsRNA extraction was lower than kit extraction method but the both methods have same pure RNA result. The PCR and sequencing result showed that viral pathogen of pepper, tobacco, and tomato leaf was CMV, ReMV, and ToCV, respectively with amplicon size at 500, 568, and 360 bp. This method is quite cheap and the RNA quantity is proportional to the commercial kit. The simple method of dsRNA extraction can be proposed for the preparation of RT-PCR detection for CMV, ReMV, ToCV. IntisariReplicative form (RF) virus RNA merupakan asam nukleat berstruktur dsRNA, selalu ditemukan pada tumbuhan terinfeksi oleh virus RNA. Prinsip kerja ekstraksi dsRNA berdasarkan afinitas serbuk selulosa terhadap asam nukleat dan adsorbsi spesifik dsRNA pada konsentrasi etanol 16,6 %. Penelitian ini bertujuan untuk mengembangkan metode ekstraksi dsRNA secara sederhana untuk preparasi deteksi RT-PCR terhadap Rehmannia mosaic virus (ReMV), Cucumber mosaic virus (CMV), Tomato chlorosis virus (ToCV) dan dibandingkan dengan kit komersil. Data yang dibandingkan adalah kuantitas asam nukleat, analisa efisiensi metode (tingkat kerumitan, waktu, biaya per reaksi) serta sekuensing. Konsentrasi RNA hasil ekstraksi metode dsRNA secara sederhana lebih rendah dibanding dengan metode kit, namun kedua metode menghasilkan RNA yang murni. Berdasarkan hasil PCR dan sekuensing disimpulkan bahwa virus penyebab mosaik daun lada dan tembakau serta klorosis daun tomat berturut-turut adalah CMV, ReMV, dan ToCV dengan ukuran amplikon berturut turut 500, 568 dan 360 pb. Metode ini cukup murah dan kuantitas RNA yang dihasilkan sebanding dengan kit komersil. Ekstraksi RNA menggunakan metode dsRNA secara sederhana dapat dikembangkan untuk preparasi deteksi RT-PCR terhadap CMV, ReMV, ToCV.
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Young, Pampee P., Richard S. Buller, and Gregory A. Storch. "Evaluation of a Commercial DNA Enzyme Immunoassay for Detection of Enterovirus Reverse Transcription-PCR Products Amplified from Cerebrospinal Fluid Specimens." Journal of Clinical Microbiology 38, no. 11 (2000): 4260–61. http://dx.doi.org/10.1128/jcm.38.11.4260-4261.2000.
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We evaluated the DiaSorin DNA enzyme immunoassay (DEIA) kit for detection of enteroviral reverse transcription-PCR (RT-PCR) products amplified from cerebrospinal fluid. By use of an optical density of 0.05 as the absorbance cutoff, 35% of 198 specimens were PCR positive, whereas 16% were culture positive. DEIA was rapid and sensitive and can help implement enterovirus RT-PCR in clinical laboratories.
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Hymas, Weston C., Wade K. Aldous, Edward W. Taggart, Jeffery B. Stevenson, and David R. Hillyard. "Description and Validation of a Novel Real-Time RT-PCR Enterovirus Assay." Clinical Chemistry 54, no. 2 (February 1, 2008): 406–13. http://dx.doi.org/10.1373/clinchem.2007.095414.
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Abstract Background: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5′ nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry. Methods: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay–like plate end detection. Results: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid. Conclusions: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.
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Maulani, Tuti Rostianti, Hadi Susilo, Marlinda Indriati, and A. Suhaemi. "Deteksi Cemaran DNA Babi Dengan RT-PCR Pada Sosis Tanpa Logo Halal MUI Dari Empat Kecamatan di Kabupaten Pandeglang Banten." Gorontalo Agriculture Technology Journal 3, no. 2 (October 24, 2020): 72. http://dx.doi.org/10.32662/gatj.v3i2.1171.
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Sosis merupakan produk olahan daging yang cukup digemari di masyarakat khususnya di Kabupaten Pandeglang Provinsi Banten. Pentingnya sertifikat halal atau adanya logo halal MUI (Majelis Ulama Indonesia) untuk produk olahan daging membuat masyarakat muslim yakin untuk mengkonsumsinya. Tujuan dari penelitian ini adalah identifikasi cemaran DNA babi pada produk sosis yang beredar di 4 kecamatan di Wilayah Kabupaten Pandeglang tanpa logo halal MUI menggunakan RT-PCR (Real Time- Polymerase Chain Reaction) dan Pork Detection Kit. Metode dalam penelitan ini diawali dengan menggunakan Pork Detection Kit untuk pengujian antigen babi sebagai kontrol positif dan daging sapi sebagai kontrol negatif yang akan digunakan untuk running RT- PCR. Hasil running RT- PCR terhadap sampel DNA sosis di wilayah 4 (empat) kecamatan di Kabupaten Pandeglang (Majasari, Picung, Munjul, Cimanuk) tanpa label halal MUI menunjukkan trend DNA kontrol negatif pada kurva amplifaksi RT- PCR. Hal ini teridentifikasi bahwa sampel DNA sosis tanpa label halal MUI di 4 kecamatan di Kabupaten Pandeglang tidak tercemar DNA babi
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Said, Khadija, Zaydah R. de Laurent, Donwilliams O. Omuoyo, Clement Lewa, Elijah Gicheru, Robinson Cheruiyot, Brian Bartilol, et al. "An optimisation of four SARS-CoV-2 qRT-PCR assays in a Kenyan laboratory to support the national COVID-19 rapid response teams." Wellcome Open Research 5 (July 7, 2020): 162. http://dx.doi.org/10.12688/wellcomeopenres.16063.1.
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Background: The global COVID-19 outbreak relies on a quantitative real-time polymerase chain reaction (qRT-PCR) for the detection of severe acute respiratory syndrome coronavirus (SARS-CoV-2), to facilitate the roll-out of patient care and infection control measures. There are several qRT-PCR assays with little evidence on their comparability. We report alterations to the developers’ recommendations to sustain the testing capability in our setting, where the supply of testing reagents is limited. Methods: Standards generated from a serially-diluted positive control and previously identified positive/negative samples were used to determine the optimal volumes of the qRT-PCR reagents and to evaluate the validity and performance of four assays: Charité Berlin and European Virus Archive – GLOBAL (EVAg) primer-probe sets, and DAAN and Beijing Genomics Institute (BGI) premixed commercial kits. A multiplex and singleplex RT-PCR kit was used with the two primer-probe sets and the recommended assay volumes of the two premixed kits were altered. Results: In comparison to the multiplex RT-PCR kit, the singleplex RT-PCR kit combined with the primer-probe sets yielded consistent cycle threshold (Ct) values across the different titrations tested. The DAAN premixed kit produced comparable Ct values across the titrations, while the BGI kit showed incomparable Ct values and inconsistent results between batches using the manufacturer’s recommended volumes. Conclusion: We achieved a 2.5-fold and 4-fold increase in the number of tests/kit for the premixed kits and the primer-probe sets, respectively. The primer-probe set assays were reliable and consistent, and we preferred a combination of an EVAg and a Berlin target. Any inconclusive result was repeated by different individuals following the same protocol. DAAN was a consistent and reliable assay even at lower concentrations from the stated recommendations. BGI in contrast, required dilution to improve its performance and was hence an assay that was used in combination with EVAg or Berlin targets.
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Sarıgül, Figen, Osman Doluca, Sıla Akhan, and Murat Sayan. "Investigation of compatibility of severe acute respiratory syndrome coronavirus 2 reverse transcriptase-PCR kits containing different gene targets during coronavirus disease 2019 pandemic." Future Virology 15, no. 8 (August 2020): 515–24. http://dx.doi.org/10.2217/fvl-2020-0169.
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Aim: In the diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), reverse transcriptase-PCR (RT-PCR) technique is often used. We evaluated the compatibility of SARS-CoV-2 RT-PCR kits containing different gene targets during the pandemic. Materials & methods: Samples were tested by Bio-Speddy® ( RdRp gene) and Diagnovital® ( RdRp + E genes). The correlation between two assays were determined by Deming regression analysis and chi-square analyses. Results: Diagnovital PCR kit showed amplification in a narrow Ct range and conveniently sharper exponential amplification curves than Bio-Speedy PCR kit. While the correlation between the findings of the two kits was apparent even with single gene target, this correlation increased when a secondary biomarker was added to the correlation calculations. Conclusion: We have observed high correlation between different PCR kits, however, using different PCR kits during the pandemic may provide a more accurate diagnosis of SARS-CoV-2, since despite correlation there are a number of patients showing contradicting diagnosis.
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Diningsih, Erniawati, Gede Suastika, Tri Asmira Damayanti, and Slamet Susanto. "Deteksi Cepat Carnation mottle virus pada Tanaman Anyelir (Dianthus caryophyllus L.)." Jurnal Hortikultura 27, no. 1 (June 22, 2017): 95. http://dx.doi.org/10.21082/jhort.v27n1.2017.p95-104.
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<em>Carnation mottle virus </em>(CarMV) merupakan virus penting pada tanaman anyelir di Indonesia maupun di dunia. Deteksi virus yang mudah dan cepat, diperlukan untuk memantau sumber induk anyelir bebas virus. Tujuan penelitian adalah mengevaluasi tiga metode preparasi RNA total yang mudah dan cepat dari tanaman anyelir sebagai templat <em>one step </em>RT-PCR. Sumber RNA total adalah dari daun dan batang anyelir terinfeksi CarMV. Metode yang dievaluasi yaitu SDT, SEM, dan kit komersial sebagai pembanding. Optimasi dilakukan terhadap konsentrasi akhir primer (0.4 – 1.0 µM) dan MgCl<sub>2 </sub>(1.5 dan 2.0 mM). Metode SDT dan SEM berhasil mendapat RNA total dari tanaman anyelir baik dari sampel daun maupun batang. Keberhasilan yang didapat dengan metode SDT dan SEM sebanding dengan kit komersial. <em>One step</em> RT-PCR RNA total yang digabungkan dengan metode SDT dan SEM menghasilkan intensitas DNA yang sebanding dengan kit komersial. RNA total dari daun sebagai sumber templat <em>one step</em> RT-PCR terbaik dibandingkan batang. Preparasi RNA total dengan metode SDT dan SEM adalah metode cepat, mudah, dan murah dalam menyediakan templat <em>one step</em> RT-PCR. Konsentrasi primer 0.4 µM dan MgCl<sub>2 </sub>2 mM merupakan konsentrasi optimum dan menghasilkan hasil amplifikasi terbaik
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Pandya, Shridhar, Chetan Savaliya, and Kamlesh Thummar. "Role of Coroprotect Kit for Management of Covid 19: A Prophylaxis Case Study Report." Journal of Education, Teaching and Social Studies 4, no. 2 (May 4, 2022): p14. http://dx.doi.org/10.22158/jetss.v4n2p14.
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Globally Sars-Cov2 virus but with variety of Mutation like from Gamma to Delta, Delta to Omicron and at present in few country mixed Delta + Omicron cases found. At present pandemic condition needs a prophylaxis treatment against COVID-19. Drugs discoveries from Ayurvedic and phytoconstituents are working on interventions to combat COVID-19 infection. The purpose of the study was to evaluate preventive action (prophylaxis) of proposed intervention in around 500 healthy subjects with negative RT-PCR test, who have been administered with Coroprotect kit to explore the preventive potential of the intervention. Considering evidences from past research on Coroprotect kit together with the incidence of Positive RT-PCR rate in 500 subjects it can be concluded that Coroprotect kit could be a primary option to boost immunity against covid-19 and fight against infection. Surely the phytoconstituents based composition of Coroprotect kit holds promise to patients and healthcare stakeholders as safe and efficacious intervention in Covid 19 as prophylaxis and therapeutic as well.
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Suh, In Bum, Jaegyun Lim, Hyo Seon Kim, Guil Rhim, Heebum Kim, Hana Kim, Sae-Mi Lee, et al. "Development and Evaluation of AccuPower COVID-19 Multiplex Real-Time RT-PCR Kit and AccuPower SARS-CoV-2 Multiplex Real-Time RT-PCR Kit for SARS-CoV-2 Detection in Sputum, NPS/OPS, Saliva and Pooled Samples." PLOS ONE 17, no. 2 (February 10, 2022): e0263341. http://dx.doi.org/10.1371/journal.pone.0263341.
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Rapid and accurate detection of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is essential for the successful control of the current global COVID-19 pandemic. The real-time reverse transcription polymerase chain reaction (Real-time RT-PCR) is the most widely used detection technique. This research describes the development of two novel multiplex real-time RT-PCR kits, AccuPower® COVID-19 Multiplex Real-Time RT-PCR Kit (NCVM) specifically designed for use with the ExiStation™48 system (comprised of ExiPrep™48 Dx and Exicycler™96 by BIONEER, Korea) for sample RNA extraction and PCR detection, and AccuPower® SARS-CoV-2 Multiplex Real-Time RT-PCR Kit (SCVM) designed to be compatible with manufacturers’ on-market PCR instruments. The limit of detection (LoD) of NCVM was 120 copies/mL and the LoD of the SCVM was 2 copies/μL for both the Pan-sarbecovirus gene and the SARS-CoV-2 gene. The AccuPower® kits demonstrated high precision with no cross reactivity to other respiratory-related microorganisms. The clinical performance of AccuPower® kits was evaluated using the following clinical samples: sputum and nasopharyngeal/oropharyngeal swab (NPS/OPS) samples. Overall agreement of the AccuPower® kits with a Food and Drug Administration (FDA) approved emergency use authorized commercial kit (STANDARD™ M nCoV Real-Time Detection kit, SD BIOSENSOR, Korea) was above 95% (Cohen’s kappa coefficient ≥ 0.95), with a sensitivity of over 95%. The NPS/OPS specimen pooling experiment was conducted to verify the usability of AccuPower® kits on pooled samples and the results showed greater than 90% agreement with individual NPS/OPS samples. The clinical performance of AccuPower® kits with saliva samples was also compared with NPS/OPS samples and demonstrated over 95% agreement (Cohen’s kappa coefficient > 0.95). This study shows the BIONEER NCVM and SCVM assays are comparable with the current standard confirmation assay and are suitable for effective clinical management and control of SARS-CoV-2.
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Domnich, Alexander, Andrea Orsi, Donatella Panatto, Vanessa De Pace, Valentina Ricucci, Patrizia Caligiuri, Giulia Guarona, et al. "Comparative Diagnostic Performance of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Kit for the Rapid Detection of SARS-CoV-2." Pathogens 10, no. 12 (December 15, 2021): 1629. http://dx.doi.org/10.3390/pathogens10121629.
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Although the reverse transcription-polymerase chain reaction (RT-PCR) is considered a standard-of-care assay for the laboratory diagnosis of SARS-CoV-2, several limitations of this method have been described. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative molecular assay and is potentially able to overcome some intrinsic shortcomings of RT-PCR. In this study, we evaluated the diagnostic performance of the novel HG COVID-19 RT-LAMP assay. In this retrospective analysis, a total of 400 routinely collected leftover nasopharyngeal samples with a known RT-PCR result were tested by means of the HG COVID-19 RT-LAMP assay. The overall sensitivity and specificity values of HG COVID-19 RT-LAMP versus RT-PCR were 97.0% (95% CI: 93.6–98.9%) and 98.5% (95% CI: 95.7–99.7%), respectively. Inter-assay agreement was almost perfect (κ = 0.96). Concordance was perfect in samples with high viral loads (cycle threshold < 30). The average time to a positive result on RT-LAMP was 17 min. HG COVID-19 RT-LAMP is a reliable molecular diagnostic kit for detecting SARS-CoV-2, and its performance is comparable to that of RT-PCR. Shorter turnaround times and the possibility of performing molecular diagnostics in the point-of-care setting make it a valuable option for facilities without sophisticated laboratory equipment.
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Ternovoi, V. A., Yu V. Kononova, A. V. Zaykovskaya, E. V. Chub, A. S. Volynkina, T. P. Mikryukova, E. S. Kotenev, O. V. Pyankov, A. O. Sementsova, and V. B. Loktev. "DEVELOPMENT AND ASSESSMENT OF A REAGENT KIT FOR RNA DETECTION OF CRIMEAN-CONGO HEMORRHAGIC FEVER VIRUS WITH USING REVERSE TRANSCRIPTION LOOP-MEDIATED ISOTHERMAL AMPLIFICATION METHOD." Russian Clinical Laboratory Diagnostics 64, no. 9 (September 15, 2019): 571–77. http://dx.doi.org/10.18821/0869-2084-2019-64-9-571-577.
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This study presents the results of laboratory trials of the reagent kit for the rapid detection of RNA of the Crimean-Congo hemorrhagic fever virus (CCHFV) using loop-mediated isothermal amplification with reverse transcription (RT-LAMP). The developed RT-LAMP reagent kit was used to detect the CCHFV and showed a sensitivity of 103 GE/ml of viral RNA, which is sufficient for detection of the CCHFV in the early stage of human infections. The kit showed high specificity and no cross-reactivity with viral panel from the State collection of viruses of the FBRI SRC VB «Vector» (arboviruses and hemorrhagic fever viruses). Laboratory trials of the RT-LAMP kit are showed a high analytical and diagnostic sensitivity and specificity for RNA detection of the CCHFV and high speed of the analysis (60-70 min with sample preparation) compared to real-time PCR. Approbation of the kit field version has showed the possibility of setting the RT-LAMP reaction and viral RNA detection without the using of analytical equipments.
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Bosevska, Golubinka, Nikola Panovski, Eleni Dokić, and Violeta Grunevska. "RT-PCR Detection of HIV in Republic of Macedonia." Bosnian Journal of Basic Medical Sciences 8, no. 4 (November 20, 2008): 350–55. http://dx.doi.org/10.17305/bjbms.2008.2896.
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The aim of the study was to detect HIV RNA in seropositive patients using RT-PCR method and thus, to establish PCR methodology in the routine laboratory works.The total of 33 examined persons were divided in two groups: 1) 13 persons seropositive for HIV; and 2) 20 healthy persons - randomly selected blood donors that made the case control group. The subjects age was between 25 and 52 years (average 38,5).ELFA test for combined detection of HIV p24 antigen and anti HIV-1 + 2 IgG and ELISA test for detection of antibodies against HIV-1 and HIV-2, were performed for each examined person. RNA from the whole blood was extracted using a commercial kit based on salt precipitation. Detection of HIV RNA was performed using RT-PCR kit. Following nested PCR, the product was separated by electrophoresis in 1,5 % agarose gel. The result was scored positive if the band of 210bp was visible regardless of intensity Measures of precaution were taken during all the steps of the work and HIV infected materials were disposed of accordingly.In the group of blood donors ELFA, ELISA and RT-PCR were negative. Assuming that prevalence of HIV infection is zero, the clinical specificity of RT-PCR is 100 %. The analytical specificity of RT-PCR method was tested against Hepatitis C and B, Human Papiloma Virus, Cytomegalovirus, Herpes Simplex Virus, Rubella Virus, Mycobacterium tuberculosis, Chlamydia trachomatis. None of these templates yielded amplicon. In the group of 13 seropositive persons, 33 samples were analyzed. HIV RNA was detected in 15 samples. ELISA and ELFA test were positive in all samples. Different aliquots of the samples were tested independently and showed the same results. After different periods of storing the RNA samples at -70°C, RT-PCR reaction was identical to the one performed initially. The obtained amplicons were maintained frozen at -20°C for a week and the subsequently performed electrophoresis was identical to the previous one. The reaction is fast, simple for manipulation; with low detection level of 60 IU/ml. RT-PCR needs a small amount of RNA, as well as a small volume of sample. HIV RNA was detected in different periods of time with different clinical presentations in patients, with or without antiretroviral therapy.RT-PCR method gives the opportunity for reliable determination of HIV-1 RNA with border of detection of 60 IU/ml. The test is reproducible and has high analytical and clinical specificity
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Shanmugam, Chandrakumar, Michael Behring, Vishwas Luthra, Sixto M. Leal, Sooryanarayana Varambally, George J. Netto, and Upender Manne. "Meta-analysis of the robustness of COVID-19 diagnostic kit performance during the early pandemic." BMJ Open 12, no. 4 (April 2022): e053912. http://dx.doi.org/10.1136/bmjopen-2021-053912.
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BackgroundAccurate detection of SARS-CoV-2 is necessary to mitigate the COVID-19 pandemic. However, the test reagents and assay platforms are varied and may not be sufficiently robust to diagnose COVID-19.MethodsWe reviewed 85 studies (21 530 patients), published from five regions of the world, to highlight issues involved in the diagnosis of COVID-19 in the early phase of the pandemic. All relevant articles, published up to 31 May 2020, in PubMed, BioRiXv, MedRiXv and Google Scholar, were included. We evaluated the qualitative (9749 patients) and quantitative (10 355 patients) performance of RT-PCR and serologic diagnostic tests for real-world samples, and assessed the concordance (5538 patients) between test performance in meta-analyses. Synthesis of results was done using random effects modelling and bias was evaluated according to QUADAS-2 guidelines.ResultsThe RT-PCR tests exhibited heterogeneity in the primers and reagents used. Of 1957 positive RT-PCR COVID-19 participants, 1585 had positive serum antibody (IgM±IgG) tests (sensitivity 0.81, 95% CI 0.66 to 0.90). While 3509 of 3581 participants RT-PCR negative for COVID-19 were found negative by serology testing (specificity 0.98, 95% CI 0.94 to 0.99). The chemiluminescent immunoassay exhibited the highest sensitivity, followed by ELISA and lateral flow immunoassays. Serology tests had higher sensitivity and specificity for laboratory approval than for real-world reporting data.DiscussionThe robustness of the assays/platforms is influenced by variability in sampling and reagents. Serological testing complements and may minimise false negative RT-PCR results. Lack of standardised assay protocols in the early phase of pandemic might have contributed to the spread of COVID-19.
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Shaker, Rouba, Ebla Abdalrahman, Zainab Ali, Lina Reslan, Houda Harastani, Amjad Haidar, Soha Ghanem, et al. "Wide Variability in the Sensitivity and Specificity of Rotavirus Immunoassay Diagnostic Kits in Practice." Journal of Infection in Developing Countries 15, no. 11 (November 30, 2021): 1701–7. http://dx.doi.org/10.3855/jidc.11922.
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Introduction: Most hospitals rely on rapid antigen-detection kits for the diagnosis of rotavirus infection. Several small studies reviewed the sensitivity and specificity of some of these kits. These studies showed discrepancy in results obtained for sensitivity and specificity that varied according to the type of kit used, area of study, and type of test used as standard for diagnosis of rotavirus infection. The objective of the study is to determine the sensitivity and specificity of five commonly used rotavirus immunoassay kits in comparison to RT-PCR as standard. Methodology: Stool samples (N = 1,414) collected from children under 5 years of age hospitalized with gastroenteritis were tested for rotavirus by immunoassay kits and RT-PCR in a prospective hospital-based surveillance study conducted at 7 centers in Lebanon. Concordance and discrepancy between the two methods was used to calculate sensitivity and specificity, using RT-PCR as the “gold standard”. Results: The sensitivity and specificity were respectively 95.08% and 86.62% for the SD Bioline® (Standard Diagnostics, Inc, South Korea) kit calculated on 645 samples, 65.86% and 45.90% for the VIROTECT® (Trinity Biotech, Ireland) kit calculated on 327 samples, 83.9% and 64.2% for the Rota-Strip (C-1001) (Coris Bioconcept, Belgium) calculated on 95 samples, 52.3% and 10.9% for the Acon® (Acon Laboratories, Inc, California, USA) kit calculated on 122 samples, 68.1% and 20% for the VIKIA® Rota-Adéno (Biomerieux, France) kit calculated on 32 samples. Conclusion: A wide discrepancy was detected between the calculated and advertised sensitivity and specificity for most of the kits.
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Boudet, Agathe, Robin Stephan, Sophie Bravo, Milène Sasso, and Jean-Philippe Lavigne. "Limitation of Screening of Different Variants of SARS-CoV-2 by RT-PCR." Diagnostics 11, no. 7 (July 12, 2021): 1241. http://dx.doi.org/10.3390/diagnostics11071241.
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Since January 2021, the diffusion of the most propagated SARS-CoV-2 variants in France (UK variant 20I/501Y.V1 (lineage B.1.1.7), 20H/H501Y.V2 (lineage B.1.351) and 20J/H501Y.V3 (lineage P.1)) were urgently screened, needing a surveillance with an RT-PCR screening assay. In this study, we evaluated one RT-PCR kit for this screening (ID SARS-CoV-2/UK/SA Variant Triplex®, ID Solutions, Grabels, France) on 2207 nasopharyngeal samples that were positive for SARS-CoV-2. Using ID Solutions kit, 4.1% (92/2207) of samples were suspected to belonged to B.1.351 or P.1 variants. Next-generation sequencing that was performed on 67.4% (62/92) of these samples confirmed the presence of a B.1.351 variant in only 75.8% of the samples (47/62). Thirteen samples belonged to the UK variant (B.1.1.7), and two to A.27 with N501Y mutation. The thirteen with the UK variant presented one mutation in the S-gene, near the ΔH69/ΔV70 deletion (S71F or A67S), which impacted the detection of ΔH69/ΔV70 deletion. Using another screening kit (PKampVariantDetect SARS-CoV-2 RT-PCR combination 1 and 3® PerkinElmer, Waltham, MA, USA) on the misidentified samples, we observed that the two mutations, S71F or A67S, did not impact the detection of the UK variant. In conclusion, this study highlights the limitations of the screening strategy based on the detection of few mutations/deletions as well as it not being able to follow the virus evolution.
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Dedkov, V. G., A. A. Deviatkin, E. M. Poleschuk, M. V. Safonova, M. L. Markelov, and G. A. Shipulin. "Development and evaluation of the RT-PCR kit for the rabies virus diagnosis." Problems of Virology, Russian journal 61, no. 5 (October 20, 2016): 235–40. http://dx.doi.org/10.18821/0507-4088-2016-61-4-235-240.
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Kim, K., H. Katayama, M. Kitajima, Y. Tohya, and S. Ohgaki. "Development of a real-time RT-PCR assay combined with ethidium monoazide treatment for RNA viruses and its application to detect viral RNA after heat exposure." Water Science and Technology 63, no. 3 (February 1, 2011): 502–7. http://dx.doi.org/10.2166/wst.2011.249.
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A method was developed for discriminating damaged viruses or naked viral RNA from intact viruses by ethidium monoazide (EMA) treatment before RT-PCR. The applied EMA treatment consisted of three steps: (1) EMA dose, (2) exposure to light, and (3) additional purification by spin-column gel filtration. Approximately 4-log reduction in viral RNA concentration was observed by adding a dose of 10 μg/mL-EMA with 300 s of light irradiation. Although residual EMA can be an inhibitor of RT-PCR, its effect was reduced by spin-column gel filtration or a QIAamp® Viral RNA Mini Kit. EMA-RT-PCR was applied to the thermally treated PV1. Results of EMA-RT-PCR were similar to the plaque assay when PV1 was thermally inactivated. Although this is a preliminary study investigating applicability of the EMA-RT-PCR method for RNA viruses, the results suggest that the method is potentially applicable for the selective detection of epidemiologically important enteric viruses in water such as enteroviruses and noroviruses.
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Sun, Hong, Chelsea Harrington, Nancy Gerloff, Mark Mandelbaum, Stacey Jeffries-Miles, Lea Necitas G. Apostol, Ma Anne-Lesley D. Valencia, et al. "Validation of a redesigned pan-poliovirus assay and real-time PCR platforms for the global poliovirus laboratory network." PLOS ONE 16, no. 8 (August 6, 2021): e0255795. http://dx.doi.org/10.1371/journal.pone.0255795.
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Surveillance and detection of polioviruses (PV) remain crucial to monitoring eradication progress. Intratypic differentiation (ITD) using the real-time RT-PCR kit is key to the surveillance workflow, where viruses are screened after cell culture isolation before a subset are verified by sequencing. The ITD kit is a series of real-time RT-PCR assays that screens cytopathic effect (CPE)-positive cell cultures using the standard WHO method for virus isolation. Because ITD screening is a critical procedure in the poliovirus identification workflow, validation of performance of real-time PCR platforms is a core requirement for the detection of poliovirus using the ITD kit. In addition, the continual update and improvement of the ITD assays to simplify interpretation in all platforms is necessary to ensure that all real-time machines are capable of detecting positive real-time signals. Four platforms (ABI7500 real-time systems, Bio-Rad CFX96, Stratagene MX3000P, and the Qiagen Rotor-Gene Q) were validated with the ITD kit and a redesigned poliovirus probe. The poliovirus probe in the real-time RT-PCR pan-poliovirus (PanPV) assay was re-designed with a double-quencher (Zen™) to reduce background fluorescence and potential false negatives. The updated PanPV probe was evaluated with a panel consisting of 184 polioviruses and non-polio enteroviruses. To further validate the updated PanPV probe, the new assay was pilot tested in five Global Polio Laboratory Network (GPLN) laboratories (Madagascar, India, Philippines, Pakistan, and Democratic Republic of Congo). The updated PanPV probe performance was shown to reduce background fluorescence and decrease the number of false positives compared to the standard PanPV probe.
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Dennis, Lacayo-Leñero, Erick Crespo-Solís, Patricia Guzmán-Uribe, María Isabel Tussie-Luna, Maria Teresa Tusie-Luna, Adriana Rosas-López, Mabel Cerrillo-Hinojosa, and Yayoi Segura-Kato. "Experience on the Application of a Commercial RT-PCR Kit on the Diagnosis of 28 Chromosomal Translocations Associated with Acute Leukemia in Adults." Blood 124, no. 21 (December 6, 2014): 5323. http://dx.doi.org/10.1182/blood.v124.21.5323.5323.
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Abstract BACKGROUND: The cytogenetic aspects of acute leukemia have been studied for over 30 years. Genomic sequencing has permitted the identification of recurrent mutations that have been associated with the heterogeneous behavior of acute leukemia. Currently, the genomic profile of patients with acute leukemia is important regarding risk classification and therapeutic approach. At this moment there are scarce studies of the genomic profile in adult patients with acute leukemia in Mexico. OBJECTIVES: To describe genomic alterations found in adult patients with acute leukemia in a cancer reference hospital in Mexico. Evaluate the efficacy of a commercial multiple PCR and nested qualitative PCR kit (HemaVision DNA Technology A/S, HV01-28N), for the detection of 28 translocations on acute leukemia. To assess by a quantitative method with RT-PCR the detection of the most frequently reported mutations in acute myeloid leukemia(38%) [t(15;17) PML-RARa, inv(16) CBF-MYH11, t(8;21) RUNX1-RUNX1T1 and acute lymphoid leukemia (32%) [t(9;22) BCR/ABL, t(4;11) MLL-AFF1, t(1;19) TCF3-PBX1]. Compare the results between the commercial kit, RT-PCR and conventional cytogenetic studies. METHODS: Patients with recent diagnosis of acute leukemia (myeloid and lymphoid) according to World Health Administration (WHO) 2008 criteria were included between the period of 03/25/2013 and 12/15/13. All patients were given informed consent form and approval of the local Ethics Committee We performed bone marrow smear and bone marrow biopsy of all patients. 5 mL of bone marrow were collected on heparin for cytogenetic study including karyotipe and FISH analysis. 5 mL of bone marrow were collected for molecular biology studies including PCR-kit and genome sequencing technique. 5mL of peripheral blood with heparin were obtained for karyotype in case of not finding metaphases for the analysis on bone marrow specimen. RESULTS: 24 patients were enrolled on the study. The mean age was 42 years (17-69). 8/24 were male (33.3%) and 16/24 were female (66.6%). Acute myeloid leukemia 11/24 (45.8%), acute lymphoblastic leukemia 8/24 (33.3%), acute promyeolocytic leukemia 3/24 (12.5%), acute biphenotypic leukemia 1/24 (4.2%) and one case of blastic phase of chronic myeloid leukemia (4.2%). The commercial PCR platform detected only two of the 28 translocations in 4/24 patients (16.6%). 2/24 (8.3%) BCR-ABL and 2/24 (8.3%) PML-RARa. In contrast to RT-PCR that detected 7/24 patients (29%). 4/24 (16%) BCR-ABL, 2/24 (8.3%) PML-RARa and 1/24 (4.1%) CBFB-MYH11. FISH analysis detected the 4/24 (16%) translocations of the commercial kit. Karyotype detected structural chromosomal anomalies in 9/24 patients (37.5%). After the analysis of the 24 samples by quantitative RT-PCR for the six most frequent rearrangements, we also identified 2 additional cases of BCR-ABL. CONCLUSIONS: The commercial qualitative PCR kit was inferior on detecting genomic alterations (16.6%) than quantitative PCR (29%) or conventional cytogenetic study (37.5%). The RT-PCR quantitative method with specific probe for the most frequent genomic alterations in acute leukemia in addition to cytogenetic analysis seems to be a better cost-effective strategy than the commercial multiple PCR platforms. Disclosures No relevant conflicts of interest to declare.
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Enin, Yaroslav S., Oleg I. Kit, Yuriy A. Gevorkyan, Natalya V. Soldatkina, Dmitry A. Kharagezov, Anton G. Milakin, Vyacheslav A. Sustretov, Andrey Dashkov, and Liubov Yu Vladimirova. "Detection of KRAS mutations in colon adenocarcinoma by droplet digital PCR." Journal of Clinical Oncology 37, no. 15_suppl (May 20, 2019): e15080-e15080. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e15080.
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e15080 Background: The screening of mutations in the KRAS gene is a traditional marker of the effectiveness of targeted therapy for colorectal cancer. Mutations in the 12 codon of the second exon of the KRAS gene are present in 40% of colorectal tumors, and monitoring of the mutational status together with the level of mutant DNA is of particular clinical interest. However, the sensitivity level of the traditionally used real-time polymerase chain reaction (RT-PCR) method is insufficient in some cases. Recently, Droplet Digital PCR (DD-PCR) has been considered as an alternative method, which is more sensitive. The purpose of this study was to compare the methods of detection of somatic mutations in the second exon of the KRAS gene using DD-PCR versus RT-PCR. Methods: This study included 134 patients with colon adenocarcinoma. The presence or absence of activating mutations in the second exon of the KRAS gene in samples of FFPE blocks was detected by RT-PCR using the “Real-time-PCR-KRAS-7M reagent kit” (Biolink, Russia) and Digital Droplet PCR using the “KRAS Screening Multiplex kit” (Bio-Rad, USA). Results: For all 134 samples, selected for the study, the WT status of the KRAS gene was identified by the RT-PCR method. According to the results of DD-PCR for 131 samples (96.2%), a positive amplification response of mutant DNA with more than 200 events was obtained. The number of amplicons varied from 312 to 117917 per sample, the median was 2940 copies. According to recent trials, a clinically significant level of mutational events must exceed 5% of the total number of amplicons in a sample. In our study, 12.9% of cases (17 patients) met this criterion. Conclusions: The DD-PCR method demonstrated a much higher analytical sensitivity for the detection of SNP mutations in comparison with RT-PCR that may be of critical importance in the therapeutic decision.
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Liang, Yang, Bo Jiao, Chuan-Feng Wu, Shu-Min Xiong, Long-Jun Gu, Zhu Chen, and Sai-Juan Chen. "AML1-ETO9a Correlated with C-KIT Overexpression/Mutation and Indicated Poor Disease Outcome in t(8;21) Acute Myeloid Leukemia." Blood 110, no. 11 (November 16, 2007): 1822. http://dx.doi.org/10.1182/blood.v110.11.1822.1822.
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Abstract AML1-ETO fusion gene is generated from chromosome translocation t(8;21) in acute myeloid leukemia (AML). It is recently reported that its spliced variant AML1-ETO9a induced leukemia rapidly in murine model. Here we detected AML1-ETO9a expression in 58 of 71 (81.7%) patients with t(8;21) AML by using qualitative RT-PCR. Quantitative RT-PCR revealed significantly higher C-KIT levels in the AML1-ETO9a positive group than in the negative group (P=0.0001). Among 29 cases harbored C-KIT mutations, 28 (96.6%) expressed AML1-ETO9a. Clinically, patients expressing AML1-ETO9a exhibited significantly elevated white blood cell counts and a short overall survival time (P=0.0455 and 0.0039, respectively). Morphologically, although there is no difference of leukemic blasts percentage between AML1-ETO9a positive and negative cases (P=0.1169), the latter possessed more aberrant myelocytes (P=0.0462). Taken together, AML1-ETO9a correlated with C-KIT overexpression/mutation and indicated poor disease outcome in t(8;21) AML.
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Blomqvist, S., A. Skyttä, M. Roivainen, and T. Hovi. "Rapid Detection of Human Rhinoviruses in Nasopharyngeal Aspirates by a Microwell Reverse Transcription-PCR–Hybridization Assay." Journal of Clinical Microbiology 37, no. 9 (1999): 2813–16. http://dx.doi.org/10.1128/jcm.37.9.2813-2816.1999.
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A rapid and sensitive microwell reverse transcription (RT)-PCR–hybridization assay was developed to detect human rhinoviruses in clinical specimens and cell culture suspensions. Two hundred three nasopharyngeal aspirates collected from children with symptoms of respiratory disease were analyzed by a classical rolling-tube cell culture method, microwell culture of HeLa Ohio cell monolayers, and RT-PCR with detection of the amplicons in a microwell hybridization assay. The RT-PCR was also done with harvests of the microwell cultures. RNA was extracted with a commercial kit, and the RT-PCR procedure was carried out with microtiter-format equipment. A confirmatory test that exploited a blocking oligonucleotide at the hybridization step was developed to reliably identify marginally positive specimens. Of the 203 nasopharyngeal aspirate specimens, rhinovirus or rhinoviral RNA was detected in 111 specimens (55%). Ninety-eight specimens (48%) were found to be positive by RT-PCR of the original nasopharyngeal aspirates, while the conventional rolling-tube cell culture method yielded 52 (26%) positive specimens. This RT-PCR method with solid-phase hybridization is easy to perform, sensitive, and specific and will be especially useful for analysis of large numbers of clinical specimens.
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Orłowska, Anna, Jan F. Żmudziński, Marcin Smreczak, Paweł Trębas, and Anna Marzec. "Diagnostic reliability of different RT-PCR protocols for the detection of bluetongue virus serotype 14 (BTV-14)." Journal of Veterinary Research 61, no. 4 (December 1, 2017): 391–95. http://dx.doi.org/10.1515/jvetres-2017-0065.
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AbstractIntroduction:The reverse transcription polymerase chain reaction (RT-PCR) is one of the most extensively used methods for identification of animals infected with bluetongue virus (BTV). There are several RT-PCR protocols published and several real-time RT-PCR (rtRT-PCR) commercial kits available on the market. Because Poland faced BTV-14 infection in 2012, different protocols were implemented in the country to confirm the RT-PCR results positive for this virus. The article presents a comparative study of several RT-PCR protocols and discusses their diagnostic reliability and applicability.Material and Methods:Six rtRT-PCR/RT-PCR protocols were compared for the laboratory diagnostic of fourteen BTV-14 isolates circulating in Poland in 2012–2014.Results:All 14 isolates were positive in the protocols of Shawet al.(18), a commercial LSI NS3 kit, and Eschbaumeret al.(5). Four out of fourteen BTV-14 isolates gave positive results in Hoffmann’s 2 and 6 protocols and none of the 14 isolates yielded positive results in Maanet al.(8) method. Phylogenetic study of a short fragment of 450 nt of BTV segment 2 (258–696 positions) revealed 100% identity within Polish variants and with Russian and Spanish isolates.Conclusion:The paper points to the possible false negative results in the diagnosis of BTV infections depending on the protocol used.
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Han, Yi, Jia-Dao Chen, Zu-Mei Liu, Yuan Zhou, Jia-Hong Xia, Xin-Ling Du, and Man-Wen Jin. "Functional ion channels in mouse cardiac c-kit+ cells." American Journal of Physiology-Cell Physiology 298, no. 5 (May 2010): C1109—C1117. http://dx.doi.org/10.1152/ajpcell.00207.2009.
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Cardiac c-kit+ cells are generally believed to be the major population of stem/progenitor cells in the heart and can be used as a cell source for cardiomyoplasty; however, the cellular electrophysiological properties are not understood in this type of cells. The present study was designed to investigate functional ion channels in undifferentiated mouse cardiac c-kit+ cells using approaches of whole cell patch voltage clamp, RT-PCR, and cell proliferation assay. It was found that three types of ionic currents were present in mouse cardiac c-kit+ cells, including a delayed rectifier K+ current (IKDR) inhibited by 4-aminopyridine (4-AP), an inward rectifier K+ current ( IKir) decreased by Ba2+, and a volume-sensitive chloride current ( ICl.vol) inhibited by 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB). RT-PCR revealed that the corresponding ion channel genes, Kv1.1, Kv1.2, and Kv1.6 (for IKDR), Kir.1.1, Kir2.1, and Kir2.2 (likely responsible for IKir), and Clcn3 (for ICl.vol), were significant in mouse cardiac c-kit+ cells. The inhibition of ICl.vol with NPPB and niflumic acid, but not IKDR with 4-AP and tetraethylammonium, reduced cell proliferation and accumulated the cell progression at G0/G1 phase in mouse cardiac c-kit+ cells. Our results demonstrate that three types of functional ion channel currents (i.e., IKDR, IKir, and ICl.vol) are present in mouse cardiac c-kit+ cells, and ICl.vol participates in regulating cell proliferation.
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A. Saleh, B. A. Jarullah, J. Aed Gati, and. "Prevalence of Bovine viral diarrhea virus in cattle herds from Basrah and Nassirya Provinces by direct and indirect Elisa and Real time qPCR." Al-Qadisiyah Journal of Veterinary Medicine Sciences 11, no. 2 (December 25, 2012): 1. http://dx.doi.org/10.29079/vol11iss2art184.
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The current study was conducted to investigate the prevalence of BVD virus in Basrah and Nassirya city by using ELISA and RT-PCR. Two hundreds and eighty two samples of non vaccinated cattle sera samples collected from two regions of Iraq (188 samples from Nassirya city and 92 samples from Basrah city). Samples tested by Enzyme Linked Immunosorbent Assay (ELISA) antigen capture. Positive results were 20 samples ( 8 sample in Thi-Qar and 12 positive samples from Basrah). All samples submitted to indirect ELISA(IDEXX HerdCheck ELISA )for detect BVDV antibodies .Genotyping of all 20 positive samples to antigen detection were tested by Real time PCR, using Cador BVDV ½ kit, after extraction of virus RNA by QIAamp mini kit. The results revealed that there were 20 positive sample according to direct ELISA(Ag detection), while 66 sample were positive to indirect ELISA, as well as, the result of RT-PCR showed that there were two sample positive to BVDV type-1 (one sample form each city).Key words: BVDV, Genotype, ELISA, Iraq, Real time PCR.
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Aydogan, Sibel, Ziya Cibal Acikgoz, Aysegul Gozalan, Fisun Kirca, Tuba Muderris, and Elife Ozturk. "Comparison of a novel real-time PCR method (RTA) and Artus RG for the quantification of HBV DNA and HCV RNA." Journal of Infection in Developing Countries 11, no. 07 (July 31, 2017): 543–48. http://dx.doi.org/10.3855/jidc.8363.
Full textAbstract:
Introduction: The purpose of this study was to evaluate and compare two manual isolation and real-time (RT) polymerase chain reaction (PCR) kits (RTA RT-PCR with RTA isolation kit and Artus RG RT-PCR with QIAamp isolation kit) for molecular diagnosis of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections.
Methodology: The study was conducted on 121 and 54 clinical samples for the detection of HBV DNA and HCV RNA, respectively, with an additional 8 HCV RNA external quality control samples.
Results: Though a high correlation was observed between the two kits for the HBV DNA (r = 0.955, p = 0.001) and HCV RNA quantifications (r = 0.828, p = 0.001), discordant results were found in nine of the HBV DNA and in six of the HCV RNA samples. The mean difference between the two systems was found to be 0.4 log IU/mL in Quality Control for Molecular Diagnostics (QCMD) HCV RNA samples by Bland-Altman analysis.
Conclusions: Although there was a high correlation between HBV DNA and HCV RNA tests according to the results of the study, the RTA system requires improvement for the determination of HCV RNA.
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Bosevska, Golubinka, Elizabeta Janceska, Gordana Kuzmanovska, Vladimir Mikik, and Nikola Panovski. "Methods for Molecular Surveillance of Influenza Used in Macedonia." PRILOZI 35, no. 2 (December 1, 2014): 25–30. http://dx.doi.org/10.2478/prilozi-2014-0003.
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AbstractThe aim: To present and compare different Nucleic Acid Testing assays used for laboratory diagnosis of influenza virus infection in our country.Materials and methods: Respiratory samples used were nose and throat swabs. The RNA extraction was performed with a QIAamp viral RNA kit. During the season 2009–2010 the first 25 samples were tested with: conventional gel-based RT-PCR and CDC rtRT-PCR using published specific matrix and HA gene primers and probes for influenza virus typing and subtyping.Results: Of 25 samples tested with conventional RT-PCR7(28%) were positive for influenza A, but negative for A/H1seasonal and A/H3. Retested with rtRT-PCR 9(36%) were positive for influenza A, 8(32%) were positive for A/H1pdm and 1(4%) was A/H3. Two samples positive with rtRT-PCR for influenza A were negative with RT-PCR. The sensitivity of the RT-PCR in comparison with rtRT-PCR is 100% and the specificity is 88.89%. Positive predictive value for RT-PCR is 77.78%, and negative predictive value is 100%. RT-PCR is a four-step and rtRT-PCR a one-step procedure. The turn-around time of RT-PCR is 6 hours and for rtRT-PCR it is 2 hours.Discussion and conclusion: For surveillance purposes nose and throat swabs are the more easy and practical to collect. It was proved that RT-PCR is too laborious, multi-step and time-consuming. The sensitivity of both assays is equal. The specificity of rtRT-PCR is higher. NAT assays for detection of influenza viruses have become an integral component of the surveillance programme in our country. They provide a fast, accurate and sensitive detection of influenza.
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Srivastava, Shilpi, Amita Jain, Shally Awasthi, and Mastan Singh. "Comparitive performance of in-house RT-PCR for NSP4 gene with a commercial enzyme immunoassay for the detection of rotavirus in stool samples of children with diarrhoea." South Asian Journal of Experimental Biology 4, no. 6 (February 4, 2015): 336–38. http://dx.doi.org/10.38150/sajeb.4(6).p336-338.
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Rotaviruses are the most significant pathogen in the etiology of watery diarrhoea in infants and young children all over the world. Laboratory diagnosis is important both for clinical management and estimation of the disease bur-den. We collected faecal samples from 260 children under the age of five years hospitalized with acute diarrhoea and tested them for rotavirus anti-gen by Enzyme immuno assay (EIA) kit [RIDASCREEN]. RNA was also extract-ed from supernatant of 10% stool suspensions in Phosphate Buffered Saline (PBS) and amplified by Reverse transcriptase polymerase chain reaction (RT-PCR) for Non Structural Protein 4 (NSP4) gene. EIA was positive in 58(22.3%) samples while RT-PCR was positive in 45(17.3%) samples. Compared to EIA, RT-PCR was 77.5% sensitive and 100% specific. The Positive Predictive Value (PPV) and Negative Predictive Value (NPV) were 100% and 93.9% respective-ly. We infer that RT-PCR is a moderately sensitive but highly specific diagnos-tic test for rotavirus infection in this study.
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Heyming, Theodore, Kellie Bacon, Bryan Lara, Chloe Knudsen-Robbins, Aprille Tongol, and Terence Sanger. "SARS-CoV-2 Serology Testing in an Asymptomatic, At-Risk Population: Methods, Results, Pitfalls." Infectious Disease Reports 13, no. 4 (October 21, 2021): 910–16. http://dx.doi.org/10.3390/idr13040082.
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The primary aim of this study was to determine the seroprevalence of SARS-CoV-2 antibodies in a population of pediatric healthcare workers (HCWs). This study was conducted 14 May–13 July 2020. Study participants included pediatric HCWs at a pediatric hospital with either direct patient contact or close proximity to patient-care areas. SARS-CoV-2 antibodies were assessed via the Wytcote Superbio SARS-CoV-2 IgM/IgG Antibody Fast Detection Kit and the Abbott Architect SARS-CoV-2 IgG assay. Participants underwent RT-PCR testing upon entry to the study and following rapid IgM+/IgG+ results; respiratory panel PCR (RP-PCR) was performed following IgM+ results. A total of 57 of 289 (19.7%) of participants demonstrated positive serology as assessed by the Wytcote rapid kit (12 on Day 1 and 45 throughout the study). However, only one of these participants demonstrated IgG+ serology via the Abbott assay. Two participants tested SARS-CoV-2+ via RT-PCR testing. One individual was adenovirus+ and enterovirus/rhinovirus+. In our study population, we observed a seroprevalence of SARS-CoV-2 antibodies of 0.35%. The lack of concordance between antibody tests suggests that the Wytcote rapid test kit may not be of use as a screening tool. However, the feasibility of the overall process indicates that a similar methodology may have potential for future epidemiologic surveillance.
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Gorgievski-Hrisoho, Meri, Jean-Daniel Schumacher, Nevenka Vilimonovic, Daniel Germann, and Lukas Matter. "Detection by PCR of Enteroviruses in Cerebrospinal Fluid during a Summer Outbreak of Aseptic Meningitis in Switzerland." Journal of Clinical Microbiology 36, no. 9 (1998): 2408–12. http://dx.doi.org/10.1128/jcm.36.9.2408-2412.1998.
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Enteroviruses (EV) are among the most common causes of aseptic meningitis. Standard diagnostic techniques are often too slow and lack sensitivity to be of clinical relevance. EV RNA can be detected within 5 h by a commercially available reverse transcription-PCR (RT-PCR) test kit. Cerebrospinal fluid (CSF) samples from 68 patients presenting with aseptic meningitis during a summer outbreak in Switzerland were examined in parallel with cell culture and commercial RT-PCR. RT-PCR was positive in all 16 CSF specimens positive by cell culture (100%). In addition, 42 of 52 (80%) CSF samples negative by cell culture were PCR positive. In 26 of these 42 (62%) patients, viral culture from other sites (throat swab or stool) was also positive. The CSF virus culture took 3 to 7 days to become positive. Echovirus 30 was the type most often isolated in this outbreak. The sensitivity of CSF RT-PCR based on clinical diagnosis during this aseptic meningitis outbreak in patients with negative bacterial culture results was 85%, i.e., considerably higher than the sensitivity of CSF virus culture (24%). We conclude that this commercial RT-PCR assay allows a positive diagnosis with minimal delay and may thus influence clinical decisions.
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Chukwudi, Ijeoma Chekwube, Kenneth Ikejiofor Ogbu, Pam Dachung Luka, Refiloe Petunia Malesa, Livio Edward Heath, Emmanuel Ikenna Ugochukwu, and Kennedy Foinkfu Chah. "Comparison of colorimetric loop-mediated isothermal amplification kit and reverse transcription-polymerase chain reaction in the diagnosis of peste des petits ruminants in sheep and goats in Southeast Nigeria." November-2020 13, no. 11 (2020): 2358–63. http://dx.doi.org/10.14202/vetworld.2020.2358-2363.
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Background and Aim: Peste des petits ruminants (PPR) is an acute, extremely contagious transboundary viral disease of small ruminants with severe economic consequences, caused by PPR virus. Cost-effective and rapid diagnosis of the disease is essential for prompt management and control. This study aimed to compare the application of a commercial colorimetric loop-mediated isothermal amplification (cLAMP) kit and reverse transcriptase-polymerase chain reaction (RT-PCR) in the diagnosis of PPR in sheep and goats in Southeast Nigeria. Materials and Methods: Nasal swab samples were collected from West African Dwarf sheep and goats showing clinical signs suggestive of PPR (n=80) and those without any clinical signs (n=140) of the disease. The diagnosis was achieved through detection of PPR viral genome in the samples using a cLAMP kit and RT-PCR. cLAMP assay was done directly on nasal swab samples without ribosomal nucleic acid extraction. A set of six primers targeting the matrix gene protein was used for the cLAMP assay. Results: PPR viral genome was detected by both cLAMP and RT-PCR in 51 (63.8%) of the 80 samples from sheep and goats with signs suggestive of PPR while 14 (10%) of those without signs tested positive for PPR by both assay methods. There was a 100% agreement in the cLAMP and RT-PCR results. However, cLAMP was a faster, easier, and less expensive method compared to RT-PCR. Conclusion: The cLAMP assay demonstrates the potential for a point of care diagnosis in the field and a valuable diagnostic tool in areas with poor electricity supply as well as in a less equipped diagnostic laboratory. Since the reagents are affordable, cLAMP can be a diagnostic tool of choice in the detection and surveillance of PPR virus in countries with limited resources.
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Pećar, Dino, Ivana Čeko, Lana Salihefendić, and Rijad Konjhodžić. "Assessment of Ion S5 NGS protocol for SARS-CoV-2 genome sequencing." Genetics & Applications 5, no. 2 (December 27, 2021): 24. http://dx.doi.org/10.31383/ga.vol5iss2pp24-30.
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Monitoring of the lineages SARS-CoV-2 is equally important in a fight against COVID-19 epidemics, as is regular RT - PCR testing. Ion AmpliSeq Library kit plus is a robust and validated protocol for library preparation, but certain optimizations for better sequencing results were required. Clinical SARS-CoV-2 samples were transported in three different viral transport mediums (VTM), on arrival at the testing lab, samples were stored on -20OC. Viral RNA isolation was done on an automatic extractor using a magnetic beads-based protocol. Screening for positive SARS-CoV-2 samples was performed on RT–PCR with IVD certified detection kit. This study aims to present results as follows: impact of first PCR cycle variation on library quantity, comparison of VTMs with a quantified library, maximum storage time of virus and correlation between used cDNA synthesis kit with generated target base coverage. Our results confirmed the adequacy of the three tested VTMs for SARS-CoV-2 whole-genome sequencing. Tested cDNA synthesis kits are valid for NGS library preparation and all kits give good quality cDNA uniformed in viral sequence coverage. Results of this report are useful for applicative scientists who work on SARS-CoV-2 whole-genome sequencing to compare and apply good laboratory practice for optimal preparation of the NGS library.
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Chenchev, I., L. Polychronova, S. Chakarova, and I. Genova. "REAL-TIME PCR FOR DETECTION OF EQUINE ARTERITIS VIRUS." Archives of Veterinary Medicine 3, no. 1 (June 28, 2010): 3–11. http://dx.doi.org/10.46784/e-avm.v3i1.189.
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The aim of this study was to develop and validate the real-time RT-PCR method in detection of the equine arteritis virus in the nasal swabs, semen plasma and whole blood samples from horses with clinical signs of the disease. The 66 samples - 28 nasal swab samples from mares, 23 semen plasma samples from stallions, 6 whole blood samples from stallions and mares, 7 samples - 10% internal organ tissues suspensions from aborted foetus were used in investigation. Two reference strains „ARVAC” and “Bucyrus” were used as the positive controls. RNA was isolated from cell culture supernatant fluid which was infected with reference viruses and whole blood from infected animals by commercial kit Viral RNA Mini Kit (Qiagen, Germany). The real-time RT-PCR - one step protocol, developed by Balasuriya et al. (2002) was used for the amplification of the 204 bp ORF7 segment of the EAV genome. The 96% of samples showed the positive results by real time RT-PCR. The 3.5% we investigated again and only 0.5 % of the samples were negative. The controls tested positive and this contributed for precise interpretation of the obtained results. The samples which show high CT values were amplified again only with primers and the products were visualized in 2% agarose gel. The positive reaction products show the DNA fragments of about 200 bp.
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Tomeczkowski, J., A. Beilken, D. Frick, B. Wieland, A. Konig, MH Falk, A. Reiter, K. Welte, and KW Sykora. "Absence of c-kit receptor and absent proliferative response to stem cell factor in childhood Burkitt's lymphoma cells." Blood 86, no. 4 (August 15, 1995): 1469–80. http://dx.doi.org/10.1182/blood.v86.4.1469.bloodjournal8641469.
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The cytokine stem cell factor (SCF) synergizes with interleukin-7 (IL- 7) to enhance the proliferation of pre-B cells. To examine the role of SCF and its receptor, c-kit, in the pathogenesis of pediatric Burkitt's lymphomas (BL), we investigated the expression of SCF and c-kit in BL cells and the mitogenic activity of SCF on BL cells. A panel of 13 BL cell lines and 7 fresh biopsy tumors was investigated. BL cells were stimulated either by Epstein-Barr virus (EBV) infection or by different reagents and cytokines, and expression of SCF and c-kit was studied on the mRNA level by Northern blot analysis and reverse-transcriptase polymerase chain reaction (RT-PCR), followed by Southern blotting. c- kit expression was also studied by fluorescence-activated cell sorting and by crosslinking of digoxigenin-labeled recombinant human SCF to the cell surface. Proliferation of BL cell lines was measured by 3H- thymidine incorporation. Low-level expression of c-kit mRNA was detected in 2 of 13 unstimulated BL cell lines and in 1 fresh BL tumor. One cell line showed upregulation of c-kit mRNA with A23187 and downregulation with phorbol myristate acetate. Neither c-kit nor SCF could be detected in any other cell line under any condition of stimulation as analyzed by Northern blot analysis, RT-PCR followed by Southern blot analysis, crosslinking, and immunofluorescence. No response to SCF was seen in 3H-thymidine incorporation assays. We conclude that most BL cells express neither SCF nor c-kit and that the low-level expression of c-kit in some BL cells most likely has no biologic significance.
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Khandker, Elisha. "Detection of human papillomavirus by hybrid capture and real time PCR methods in patients with chronic cervicitis and cervical intraepithelial neoplasia." IMC Journal of Medical Science 10, no. 2 (January 12, 2017): 45–48. http://dx.doi.org/10.3329/imcjms.v10i2.31109.
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Background and objectives: Cervical cancer due to Human papillomavirus (HPV) is one of the leading causes of morbidity and mortality in women. Testing of HPV can identify women who are at risk of cervical cancer. Nowadays, molecular methods like real time polymerase chain reaction (PCR) and hybrid capture technique are applied for detecting HPV in cervical specimens. The objective of the present study was to determine the rate of HPV infection in women with chronic cervicitis and cervical intraepithelial neoplasia (CIN) by a commercial real time polymerase chain reaction test kit and by a hybrid capture HPV DNA test.Methods: Women aged between 20 to 55 years with chronic cervicitis and CIN were enrolled in the study after obtaining informed consent. Cervical specimen was collected by using cervical brush and stored in transport medium until used. HPV was detected by High Risk Screen Real-TM Quant 2x (Sacace, Biotechnologies SrI, Italy) real time PCR kit (HR RT-PCR) and by Hybrid Capture-2 High-Risk HPV DNA (Hc-2; Digene Corporation, USA) test.Results: Total 72 women with chronic cervicitis and CIN of different grades were included in the study. Out of this, HPV infection detected by HR RT-PCR was 31 (43%) and by Hc-2 was 14 (19.4%). Both the tests were able to detect HPV infection in all the CIN 3 cases and in most of the CIN 2 cases. However, HR RT-PCR detected higher number of HPV in chronic cervicitis and CIN1 cases.Conclusion: The study has shown that HR RT-PCR and Hc-2 tests are equally effective in detecting HPV infection in patients with CIN 2 and CIN 3 lesions. However, HR RT-PCR is more sensitive test for detecting HPV in chronic cervicitis and early CIN lesions and, therefore can be used in epidemiological study to detect presence of HPV in general population.IMC J Med Sci 2016; 10(2): 45-48
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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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Alfaro-Fernández, A., C. Córdoba-Sellés, M. C. Cebrián, J. A. Sánchez-Navarro, A. Espino, R. Martín, and C. Jordá. "First Report of Tomato torrado virus in Tomato in the Canary Islands, Spain." Plant Disease 91, no. 8 (August 2007): 1060. http://dx.doi.org/10.1094/pdis-91-8-1060b.
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In 2003, greenhouse-grown tomato crops (Lycopersicon esculentum Mill.) in the Canary Islands (Spain) were observed showing an initial yellowing in defined areas at the base of the leaflet that later developed into necrotic spots or an extensive necrotic area progressing from the base to tip. Fruits were also affected, showing necrotic areas and often developing cracking. Generally, the plants that were affected seemed to be burnt, their growth was reduced, and the production level was seriously damaged. Similar symptoms have been observed in Murcia (Spain) since 2001, which have been recently associated with Tomato torrado virus (ToTV) infection (2). Twenty-two tomato samples showing “torrado disease” symptoms were collected from different greenhouses between 2003 and 2006 in Las Palmas (Canary Islands, Spain). To verify the identity of the disease, double-antibody sandwich (DAS)-ELISA was performed on leaf and fruit extracts of symptomatic plants using polyclonal antibodies specific to Potato virus Y (PVY), Tomato mosaic virus (ToMV), Tomato spotted wilt virus (TSWV) (Loewe Biochemica, Sauerlach, Germany), and Pepino mosaic virus (PepMV) (DSMZ, Braunschweig, Germany). Total RNA was extracted from the 22 tomato samples with the RNAwiz Extraction kit (Ambion, Huntingdon, United Kingdom) and tested using one-step reverse-transcription (RT)-PCR with the SuperScript Platinum Taq kit (Invitrogen Life Technologies, Barcelona, Spain) with primers specific to PepMV (1) and ToTV (2). All analyses included healthy tomato plants as negative controls. Five of the twenty-two tomato samples were positive for PepMV and negative for the other viruses tested by serological analysis. However, all 22 samples were positive in RT-PCR performed with the primers specific to ToTV segment RNA2. The RT-PCR assay to detect ToTV produced an amplicon of the expected size (580 bp). No amplification product was observed when healthy plants or a water control were used as a template in the RT-PCR reaction. The ToTV RT-PCR product was purified (High Pure PCR Product Purification kit, Roche Diagnostics, Mannheim, Germany) and sequenced. BLAST analysis of one sequence (GenBank Accession No. EF436286) showed 99% identity to ToTV RNA2 sequence (GenBank Accession No. DQ388880). To our knowledge, this is the first report of ToTV in the Canary Islands. References: (1) I. Pagán et al. Phytopathology 96:274, 2006. (2) M. Verbeek et al. Online Publication. doi:10.1007/s00705-006-0917-6. Arch. Virol., 2007.
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Logoida, Mariia, Pavol Kristian, Andrea Schreiberova, Patrícia Denisa Lenártová, Veronika Bednárová, Elena Hatalová, Ivana Hockicková, et al. "Comparison of Two Diagnostic Methods for the Detection of Hepatitis B Virus Genotypes in the Slovak Republic." Pathogens 11, no. 1 (December 24, 2021): 20. http://dx.doi.org/10.3390/pathogens11010020.
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The hepatitis B virus (HBV), belonging to the Hepadnaviridae family, is responsible for a global health concern still in the 21st century. The virus is divided into 10 genotypes, which differ in geographical distribution and in their effect on disease progression and transmission, susceptibility to mutations, and response to treatment. There are many methods for diagnostics of HBV and differentiating its genotypes. Various commercial kits based on real-time polymerase chain reaction (RT PCR) and hybridization available, as well as whole genome sequencing or the sequencing of only individual parts of the genomes. We compared a commercial kit AmpliSens HBV-genotype-FRT, based on RT PCR, with an adapted method of amplification of the surface genomic region combined with Sanger sequencing. In the examined samples we identified the A, B, C, D, and E genotypes. By PCR with Sanger sequencing, the genotypes were determined in all 103 samples, while by using the commercial kit we successfully genotyped only 95 samples, including combined genotypes, which we could not detect by sequencing.
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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.