Artykuły w czasopismach na temat „Real-Time PCR technique”
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RAZA, ABIDA, i NAUREEN A KHATTAK. "REAL TIME PCR;". Professional Medical Journal 19, nr 06 (3.11.2012): 751–59. http://dx.doi.org/10.29309/tpmj/2012.19.06.2455.
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In recent years, real-time PCR has come forward as a robust and widely used molecular technique in clinical and biologicalsettings. Although it can detect very minute quantities of target nucleic acid, but quantification of specific nucleic acids is not an easy task.Accurate and precise quantification is hampered by a number of factors that may include assay development and validation, fluorophoresselection, handling during sample preparation, storage, reaction procedures, and batch analysis conditions. Even minor variations aresignificantly magnified by the exponential nature of this technique. Current review gives an insight of the advantages, limitations, assaychemistries, quantitation parameters, and quality control issues related to this technology. Moreover it will also highlight the utilization of Realtime PCR in clinical oncology, virology, microbiology, and gene expression studies.
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Saienko, Artem, Mykyta Peka, Viktor Balatsky, Sergii Korinnyi i Oleksandr Tsereniuk. "GMO analysis technique based on PCR and real-time PCR methods". Pig breeding the interdepartmental subject scientific digest, nr 75-76 (7.12.2021): 58–67. http://dx.doi.org/10.37143/0371-4365-2021-75-76-06.
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The use of genetically modified organisms (GMOs) is promising for overcoming the shortage of food products in the world and solving the problem of hunger arising in various regions. At the same time, the use of GMOs has become a cause of debate, as the safety of consuming GMO products for human health remains unproven. The risks associated with GMOs cause public concern, which has led to the restriction of the use of GMOs and their products in many countries and the need for constant control of their content in food products. This study describes methods based on the polymerase chain reaction (PCR), such as Real-time PCR and PCR with electrophoretic separation of amplificates, which are generally accepted in GMO analysis. In order to control the presence of GMOs in food raw materials, feed and finished products of plant and animal origin, screening is carried out for the presence of the most common genetic engineering structures used during the creation of GMOs: CaMV 35S promoter and NOS terminator. Both Real-time PCR and PCR with electrophoretic separation of amplificates allow to establish the presence of GMOs with high accuracy, and Real-time PCR is also used to determine the concentration of GMOs in the studied samples. The work presents a typical electrophoresis with visualization of the obtained PCR fragments that required electrophoretic separation and fragments that were synthesized in the Real-time PCR reaction, and determined the approximate sizes of the obtained fragments relative to the pBR322 DNA-MspI molecular weight marker. The approach described in this study, based on the use of PCR techniques, can be successfully used for GMO analysis of all groups of raw materials and finished products of animal and plant origin, and is also well adapted for the detection of various genetic engineering structures, not limited to the CaMV 35S promoter and terminator NOS, which makes it possible to increase the efficiency of such analysis and continue its application in relation to new genetic engineering structures used during the creation of GMOs. Keywords: genetically modified organisms (GMOs), PCR methods, GMO analysis, raw materials and finished products of plant and animal origin
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Skitovich, G. S., N. B. Shadrova, O. V. Pruntova i K. V. Serova. "REAL-TIME PCR OPTIMIZATION FOR LISTERIA MONOCYTOGENES GENOME DETECTION". Veterinary Science Today, nr 3 (3.10.2018): 63–68. http://dx.doi.org/10.29326/2304-196x-2018-3-26-63-68.
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Listeria monocytogenes is one of the major food contaminants causing the illness, called Listeriosis. Listeriosis incidence is much less, than the number salmonellosis and campylobacteriosis cases, but the clinical disease is significantly more severe and has a higher mortality. That’s why the development of species-specific PCR techniques to detect L. monocytogenes genome is a topical task. L. monocytogenes bacteria genome detection technique using real-time polymerase chain reaction (qRT-PCR) was improved. The amplification target was a highly specific and suitable for qualification of all strains iap gen, coding L. monocytogenes р60 surface protein. Optimum magnesium concentration (6 mM) and primer annealing temperature (57 °С) were selected. The sensitivity and specificity of the technique were identified. Detection threshold was 120 target molecules. The results obtained demonstrate that optimized qRT-PCR version, based on iap gen amplification, enables to detect L. monocytogenes in animal product and food samples. Optimized qRT-PCR-based screening tests ensure rapid and reliable results.
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Burggraf, Siegfried, i Bernhard Olgemöller. "Simple Technique for Internal Control of Real-Time Amplification Assays". Clinical Chemistry 50, nr 5 (1.05.2004): 819–25. http://dx.doi.org/10.1373/clinchem.2003.027961.
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Abstract Background: In real-time PCR assays, the most accurate way to identify false-negative results, e.g., those caused by PCR inhibitors, is to add to samples an internal control that will be coamplified with the target (e.g., pathogen) DNA. Current internal control procedures, however, which usually involve the introduction of a DNA fragment, are complex, time-consuming, and expensive. Methods: Single-stranded oligonucleotides, which contain little more than primer and probe binding sites, were used as internal controls in real-time PCR assays. Mismatches were included in the probe-binding region of the internal control oligonucleotide (ICO) to prevent probe–control hybridization during the fluorescence acquisition step of the PCR. Amplified ICOs were detected by melting point analysis. ICOs could be added directly to the sample material before DNA extraction. Results: To demonstrate the feasibility of the new approach, we designed ICOs for the LightCycler hybridization probe assays for Mycobacterium tuberculosis complex, hepatitis B virus, herpes simplex virus, and varicella zoster virus. In each case, the controls did not interfere with detection of the pathogen, but were clearly detectable during a subsequent melting point analysis. Conclusions: A single-stranded oligonucleotide that mimics the target region of the pathogen but is clearly distinguishable from the target during melting point analysis can serve as a simple, cost-effective internal control for real-time amplification assays. Such control oligonucleotides are easy to design and inexpensive. A costly second probe system is not necessary. Moreover, the internally controlled assay uses only one fluorescence detection channel of the instrument, leaving the second channel free for multiplex applications.
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Bębnowska, Dominika, Rafał Hrynkiewicz i Paulina Niedźwiedzka-Rystwej. "Real-Time PCR Confirms Infection with Lagovirus europaeus". Applied Sciences 11, nr 2 (11.01.2021): 656. http://dx.doi.org/10.3390/app11020656.
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Lagovirus europaeus GI.1/GI.2 is an etiological agent causing the highly dangerous rabbit hemorrhagic disease (RHD). Molecular research is the basic tool today that can help solve epidemic problems related to the expansion of pathogens in the world. By using the real-time polymerase chain reaction technique (PCR), we detected three different strains of Lagovirus europaeus/GI.1, which is an RNA virus infecting mainly rabbits. The results showed that the method used was fast, very specific, and effective.
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Bębnowska, Dominika, Rafał Hrynkiewicz i Paulina Niedźwiedzka-Rystwej. "Real-Time PCR Confirms Infection with Lagovirus europaeus". Applied Sciences 11, nr 2 (11.01.2021): 656. http://dx.doi.org/10.3390/app11020656.
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Lagovirus europaeus GI.1/GI.2 is an etiological agent causing the highly dangerous rabbit hemorrhagic disease (RHD). Molecular research is the basic tool today that can help solve epidemic problems related to the expansion of pathogens in the world. By using the real-time polymerase chain reaction technique (PCR), we detected three different strains of Lagovirus europaeus/GI.1, which is an RNA virus infecting mainly rabbits. The results showed that the method used was fast, very specific, and effective.
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Christopher-Hennings, Jane, Matthew A. Dammen, Shelleen R. Weeks, William B. Epperson, Shri N. Singh, Gina L. Steinlicht, Ying Fang i in. "Comparison of Two DNA Extractions and Nested PCR, Real-Time PCR, a New Commercial PCR Assay, and Bacterial Culture for Detection of Mycobacterium Avium Subsp. Paratuberculosis in Bovine Feces". Journal of Veterinary Diagnostic Investigation 15, nr 2 (marzec 2003): 87–93. http://dx.doi.org/10.1177/104063870301500201.
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In this study, 5 combinations of 2 DNA extractions and 3 polymerase chain reaction (PCR) techniques were compared with culture for the detection of Mycobacterium paratuberculosis directly from bovine feces. These combinations included a new commercial extraction technique combined with a commercial PCR/Southern blot technique, nested PCR (nPCR), or real-time PCR, and a university-developed extraction combined with nPCR or real-time PCR. Four of the 5 combinations had statistically similar sensitivities between 93% and 100% and specificity between 95% and 100%, when compared with culture results from 63 bovine fecal samples. These results indicated that using a commercial extraction with a commercial PCR/Southern blot, nPCR, or real-time PCR, or a university-developed extraction with real-time PCR would result in similar sensitivities to culture for the identification of M. paratuberculosis from bovine feces and are valid alternatives to culture.
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Artika, I. Made, Yora Permata Dewi, Ita Margaretha Nainggolan, Josephine Elizabeth Siregar i Ungke Antonjaya. "Real-Time Polymerase Chain Reaction: Current Techniques, Applications, and Role in COVID-19 Diagnosis". Genes 13, nr 12 (16.12.2022): 2387. http://dx.doi.org/10.3390/genes13122387.
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Successful detection of the first SARS-CoV-2 cases using the real-time polymerase chain reaction (real-time PCR) method reflects the power and usefulness of this technique. Real-time PCR is a variation of the PCR assay to allow monitoring of the PCR progress in actual time. PCR itself is a molecular process used to enzymatically synthesize copies in multiple amounts of a selected DNA region for various purposes. Real-time PCR is currently one of the most powerful molecular approaches and is widely used in biological sciences and medicine because it is quantitative, accurate, sensitive, and rapid. Current applications of real-time PCR include gene expression analysis, mutation detection, detection and quantification of pathogens, detection of genetically modified organisms, detection of allergens, monitoring of microbial degradation, species identification, and determination of parasite fitness. The technique has been used as a gold standard for COVID-19 diagnosis. Modifications of the standard real-time PCR methods have also been developed for particular applications. This review aims to provide an overview of the current applications of the real-time PCR technique, including its role in detecting emerging viruses such as SARS-CoV-2.
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Yang, Wenli, H. D. Alan Lindquist, Vitaliano Cama, Frank W. Schaefer, Eric Villegas, Ronald Fayer, Earl J. Lewis, Yaoyu Feng i Lihua Xiao. "Detection of Toxoplasma gondii Oocysts in Water Sample Concentrates by Real-Time PCR". Applied and Environmental Microbiology 75, nr 11 (10.04.2009): 3477–83. http://dx.doi.org/10.1128/aem.00285-09.
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ABSTRACT PCR techniques in combination with conventional parasite concentration procedures have potential for the sensitive and specific detection of Toxoplasma gondii oocysts in water. Three real-time PCR assays based on the B1 gene and a 529-bp repetitive element were analyzed for the detection of T. gondii tachyzoites and oocysts. Lower sensitivity and specificity were obtained with the B1 gene-based PCR than with the 529-bp repeat-based PCR. New procedures for the real-time PCR detection of T. gondii oocysts in concentrates of surface water were developed and tested in conjunction with a method for the direct extraction of inhibitor-free DNA from water. This technique detected as few as one oocyst seeded to 0.5 ml of packed pellets from water samples concentrated by Envirocheck filters. Thus, this real-time PCR may provide a detection method alternative to the traditional mouse assay and microscopy.
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Valero, Clara, Laura de la Cruz-Villar, Óscar Zaragoza i María José Buitrago. "New Panfungal Real-Time PCR Assay for Diagnosis of Invasive Fungal Infections". Journal of Clinical Microbiology 54, nr 12 (14.09.2016): 2910–18. http://dx.doi.org/10.1128/jcm.01580-16.
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The diagnosis of invasive fungal infections (IFIs) is usually based on the isolation of the fungus in culture and histopathological techniques. However, these methods have many limitations often delaying the definitive diagnosis. In recent years, molecular diagnostics methods have emerged as a suitable alternative for IFI diagnosis. When there is not a clear suspicion of the fungus involved in the IFI, panfungal real-time PCR assays have been used, allowing amplification of any fungal DNA. However, this approach requires subsequent amplicon sequencing to identify the fungal species involved, increasing response time. In this work, a new panfungal real-time PCR assay using the combination of an intercalating dye and sequence-specific probes was developed. After DNA amplification, a melting curve analysis was also performed. The technique was standardized by using 11 different fungal species and validated in 60 clinical samples from patients with proven and probable IFI. A melting curve database was constructed by collecting those melting curves obtained from fungal species included in the standardization assay. Results showed high reproducibility (coefficient of variation [CV] < 5%; r > 0.95) and specificity (100%). The overall sensitivity of the technique was 83.3%, with the group of fungi involved in the infection detected in 77.8% of the positive samples with IFIs covered by molecular beacon probes. Moreover, sequencing was avoided in 67.8% of these “probe-positive” results, enabling report of a positive result in 24 h. This technique is fast, sensitive, and specific and promises to be useful for improving early diagnosis of IFIs.
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Kesmen, Zülal, i Hakiye Aslan. "Isıl İşlem Görmüş Sütlerde Salmonella Typhimurium Canlı Hücrelerinin PMA/Real-Time PCR ile Belirlenmesi". Turkish Journal of Agriculture - Food Science and Technology 5, nr 5 (7.06.2017): 518. http://dx.doi.org/10.24925/turjaf.v5i5.518-524.1094.
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Applying different technological processes during the production of food has a lethal effect on the bacteria but DNA of these bacterial strains may cause false positive results when detected by real time PCR technique because they preserve their existence for a certain period of time. To overcome this shortcoming of the real time PCR technique, a new method has been developed in recent years, based on the removal of dead cell DNA from the medium by treatment with Propodium Monoazide (PMA) before DNA extraction. In this study, real-time PCR method was combined with PMA application for the detection of live cells of Salmonella Typhimurium in heat treated milk samples. For this purpose, milk samples inoculated with S. Tyhimurium were heat treated at different temperatures (60, 65, 70 and 75°C) and times (15, 60, 300, 900 sec) and number of live bacteria was determined comparatively by direct real-time PCR, PMA/real-time PCR and conventional cultural method. As a result, unlike the direct real time PCR technique, PMA/real-time PCR method prevents to a certain extent of false positive results from dead cells at all tested temperatures and times but higher results were obtained from PMA/real-time PCR method when compared to conventional cultural results. Therefore, further studies should be carried out to optimize the conditions of the PMA application in order to eliminate the high positive results detected by the PMA / real-time PCR method
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Rezasoltani, Sama, Hossein Dabiri, Hamid Asadzadeh Aghdaei, Abbas Akhavan Sepahi, Mohammad Hossein Modarressi i Ehsan Nazemalhosseini Mojaradd. "An improved real-time qPCR technique for quantification of intestinal bacteria in human fecal samples". South Asian Journal of Experimental Biology 7, nr 5 (9.07.2018): 201–9. http://dx.doi.org/10.38150/sajeb.7(5).p201-209.
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The easy, cost-effective and rapid techniques for investigating the role of gut microbiota have become imperative owing to their importance in various diseases such as colon cancer and inflammatory bowel disease. Here we developed an absolute real-time qPCR technique for quantification of intestinal bacteria in human fecal samples for further investigation of gut microbiota composition in patients. At first, regarding bacterial species present in human fecal samples, species were cultured in anaerobic culture media and incubated at 37Cͦ in an anaerobic chamber. The number of CFU was counted. Eight fold serial dilutions of the bacterial suspension were prepared. To generate a standard curve for bacterial species in real-time PCR, DNA was extracted from different serial dilutions. Subsequently, DNA from human fecal samples was extracted. Now bacterial strains were applied as standard curve in absolute q PCR procedure for quantification of bacterial population in test samples. Based on absolute real-time analysis, intestinal bacteria were precisely quantified. The specificity of the primer pairs was checked by conventional PCR and real-time PCR regarding dissociation curve analysis. The amplification was linear in the range of 101-108. Dissociation curves had correlation coefficient values between 0.98 and 101/888. The efficiencies were between 95/073 and 99/804%. The absolute real-time PCR technique described here may be used for evaluating intestinal bacteria regarding diseases prevention.
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Koo, Seul-Bit-Na, Hyeon-Gyu Chi, Jong-Dae Kim, Yu-Seop Kim, Ji-Sung Park, Chan-Young Park i Deuk-Ju Lee. "Multiple Compact Camera Fluorescence Detector for Real-Time PCR Devices". Sensors 21, nr 21 (22.10.2021): 7013. http://dx.doi.org/10.3390/s21217013.
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The polymerase chain reaction is an important technique in biological research because it tests for diseases with a small amount of DNA. However, this process is time consuming and can lead to sample contamination. Recently, real-time PCR techniques have emerged which make it possible to monitor the amplification process for each cycle in real time. Existing camera-based systems that measure fluorescence after DNA amplification simultaneously process fluorescence excitation and emission for dozens of tubes. Therefore, there is a limit to the size, cost, and assembly of the optical element. In recent years, imaging devices for high-performance, open platforms have benefitted from significant innovations. In this paper, we propose a fluorescence detector for real-time PCR devices using an open platform camera. This system can reduce the cost, and can be miniaturized. To simplify the optical system, four low-cost, compact cameras were used. In addition, the field of view of the entire tube was minimized by dividing it into quadrants. An effective image processing method was used to compensate for the reduction in the signal-to-noise ratio. Using a reference fluorescence material, it was confirmed that the proposed system enables stable fluorescence detection according to the amount of DNA.
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Taha, Aza Bahadeen, Katan Sabir Ali i Furat Tahseen Sabeer. "Quantitation assay of hepatitis C virus RNA using real-time PCR technique". Cihan University-Erbil Scientific Journal 2017, Special-2 (2017): 242–52. http://dx.doi.org/10.24086/cuesj.si.2017.n2a22.
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Skoblov, M. Yu, E. D. Shibanova, E. V. Kovaleva, D. I. Bairamashvilli, Yu S. Skoblov i A. I. Miroshnikov. "DNA assay for recombinant pharmaceutical substances using the real-time PCR technique". Russian Journal of Bioorganic Chemistry 36, nr 1 (styczeń 2010): 104–8. http://dx.doi.org/10.1134/s1068162010010115.
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Hu, Yali, Mingming Zheng, Zhengfeng Xu, Xinru Wang i Hengmi Cui. "Quantitative real-time PCR technique for rapid prenatal diagnosis of Down syndrome". Prenatal Diagnosis 24, nr 9 (wrzesień 2004): 704–7. http://dx.doi.org/10.1002/pd.968.
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Nair, Smita, O. M. Roshna, V. P. Soumya, Vinayaka Hegde, M. Suresh Kumar, Ramaswamy Manimekalai i George V. Thomas. "Real-time PCR technique for detection of arecanut yellow leaf disease phytoplasma". Australasian Plant Pathology 43, nr 5 (13.02.2014): 527–29. http://dx.doi.org/10.1007/s13313-014-0278-7.
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Kaboré, Jacques, Hamidou Ilboudo, Charlie FA Compaoré, Oumou Camara, Mohamed Bamba, Hassane Sakandé, Mamadou Camara i in. "PO 8441 EXPERIMENTAL COMPARISON OF SENSITIVITY OF LAMP AND REAL-TIME PCR". BMJ Global Health 4, Suppl 3 (kwiecień 2019): A37.3—A38. http://dx.doi.org/10.1136/bmjgh-2019-edc.98.
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BackgroundHuman African trypanosomiasis, or sleeping sickness, remains a serious problem in tropical Africa. Timely diagnosis of this disease requires systematic population screening, particularly for Trypanosoma brucei gambiense, which has a long asymptomatic period.The lack of sensitivity and specificity of conventional diagnostic tests has led in recent years to the use of molecular tools. Amplification of parasite-specific DNA sequences significantly improved diagnosis of infection. However, these molecular tools still have some limitations especially in the case of low parasitaemia. Furthermore, research is still needed to make molecular detection a real control tool for the fight against sleeping sickness. The purpose of this study is to determine the threshold of sensitivity of real-time PCR using the 18S and TgsGp primers and of the LAMP technique, applied in the DiTECT-HAT project as molecular reference tests.MethodsWe used serial dilutions containing 0, 1, 10, 100, 103, 104, 105, 106 parasites per ml of blood. Samples were extracted, and DNA was amplified.ResultsThe analytical sensitivity of the 18S real-time PCR with the Taqman probe of the filter paper samples is 100 parasites/ml and that of the TgsGp real-time PCR with the Taqman probe of filter paper samples is 104 parasites/ml. For Lamp technique, the analytical sensitivity is 103 parasites/ml.ConclusionThis study shows that a ‘negative PCR’ would not mean ‘no parasite’. It suggests that DNA detection techniques should still be improved.
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Lin, Mei-Hui, Tse-Ching Chen, Tseng-tong Kuo, Ching-Chung Tseng i Ching-Ping Tseng. "Real-Time PCR for Quantitative Detection ofToxoplasma gondii". Journal of Clinical Microbiology 38, nr 11 (2000): 4121–25. http://dx.doi.org/10.1128/jcm.38.11.4121-4125.2000.
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The protozoan Toxoplasma gondii is one of the most common infectious pathogenic parasites and can cause severe medical complications in infants and immunocompromised individuals. We report here the development of a real-time PCR-based assay for the detection of T. gondii. Oligonucleotide primers and a fluorescence-labeled TaqMan probe were designed to amplify the T. gondii B1 gene. After 40 PCR cycles, the cycle threshold values (CT) indicative of the quantity of the target gene were determined. Typically, a CT of 25.09 was obtained with DNA from 500 tachyzoites of the T. gondii RH strain. The intra-assay coefficients of variation (CV) were 0.4, 0.16, 0.24, and 0.79% for the four sets of quadruplicate assays, with a mean interassay CV of 0.4%. These values indicate the reproducibility of this assay. Upon optimization of assay conditions, we were able to obtain a standard curve with a linear range (correlation coefficient = 0.9988) across at least 6 logs of DNA concentration. Hence, we were able to quantitatively detect as little as 0.05 T. gondii tachyzoite in an assay. When tested with 30 paraffin-embedded fetal tissue sections, 10 sections (33%) showed a CT of <40 and were scored as positive for this test. These results were consistent with those obtained through our nested-PCR control experiments. We have developed a rapid, sensitive, and quantitative real-time PCR for detection of T. gondii. The advantages of this technique for the diagnosis of toxoplasmosis in a clinical laboratory are discussed.
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Flori, Pierre, Bahrie Bellete, Fabrice Durand, Hélène Raberin, Céline Cazorla, Jamal Hafid, Frédéric Lucht i Roger Tran Manh Sung. "Comparison between real-time PCR, conventional PCR and different staining techniques for diagnosing Pneumocystis jiroveci pneumonia from bronchoalveolar lavage specimens". Journal of Medical Microbiology 53, nr 7 (1.07.2004): 603–7. http://dx.doi.org/10.1099/jmm.0.45528-0.
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Between January 2002 and July 2003, 173 bronchoalveolar lavage (BAL) specimens from 150 patients (19 HIV-infected and 131 non-HIV-infected patients) were evaluated for identification of Pneumocystis jiroveci (formerly known as Pneumocystis carinii f. sp. hominis) using staining techniques, conventional PCR (mtLSUrRNA gene) and real-time PCR (MSG gene). Test results were compared to Pneumocystis pneumonia (PCP) confirmed by typical clinical findings and response to treatment. Sensitivity and specificity of the techniques were 60 and 100 % for staining (where either one or both techniques were positive), 100 and 87.0 % for conventional PCR and 100 and 84.9 % for real-time PCR, respectively. The use of a concentration of 103 copies of DNA per capillary of BAL as a cut-off (determined by real-time PCR) increased specificity from 84.9 to 98.6 % without reducing the sensitivity of the technique. This technique is rapid (<3 h) and therefore of major interest in differentiating between asymptomatic carriage and PCP. A BAL specimen with <103 copies per capillary of Pneumocystis-specific DNA is more likely to indicate a chronic carrier state, but in such cases follow-up is required to ensure that the patient is not in the early stage of an active PCP.
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Cheng, Joanna, Ian Carter, Liping Wang i Peter Taylor. "Real-time PCR for laboratory diagnosis of Acanthamoeba keratitis". Microbiology Australia 32, nr 2 (2011): 111. http://dx.doi.org/10.1071/ma11111.
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Acanthamoeba keratitis is a painful vision-threatening disease of the human cornea. It is characterised by severe ocular pain or partial paracentral stromal ring infiltrate, which can be frequently misdiagnosed as herpes simplex virus keratitis. If the infection is not treated promptly, it may progress to ulceration of the cornea, loss of visual acuity, possibly blindness and even require enucleation. Acanthamoeba sp are found commonly in freshwater, tap water, seawater, hot springs and swimming pools. An epidemiologic case study revealed that major risk factors were the use of contact lenses, predominantly extended-wear soft lenses, the use of homemade rinsing saline and users who wear their lenses while swimming. The conventional method of detecting the formation of oocysts of Acanthamoeba by a culture technique takes an average three?five days. DNA amplification by PCR can improve turnaround time for the diagnosis. A study was carried out in this laboratory to compare the traditional culture method with a real-time PCR assay.
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Łagowski, Dominik, Sebastian Gnat, Aneta Nowakiewicz i Aleksandra Trościańczyk. "Real-Time PCR as an Alternative Technique for Detection of Dermatophytes in Cattle Herds". Animals 11, nr 6 (2.06.2021): 1662. http://dx.doi.org/10.3390/ani11061662.
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Dermatophytes are filamentous fungi with the ability to digest and grow on keratinized substrates. The ongoing improvements in fungal detection techniques give new scope for clinical implementations in laboratories and veterinary clinics, including the monitoring of the disease and carrier status. The technologically advanced methods for dermatophyte detection include molecular methods based on PCR. In this context, the aim of this study was to carry out tests on the occurrence of dermatophytes in cattle herds using qPCR methods and a comparative analysis with conventional methods. Each sample collected from ringworm cases and from asymptomatic cattle was divided into three parts and subjected to the real-time PCR technique, direct light microscopy analysis, and culture-based methods. The use of the real-time PCR technique with pan-dermatophyte primers detected the presence of dermatophytes in the sample with a 10.84% (45% vs. 34.17%) higher efficiency than direct analysis with light microscopy. Moreover, a dermatophyte culture was obtained from all samples with a positive qPCR result. In conclusion, it seems that this method can be used with success to detect dermatophytes and monitor cowsheds in ringworm cases and carriers in cattle.
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Tran, Linh Thi Nhut, My Thi Huynh Nguyen, Linh Nguy Hoang Le, Khoa Dang Le, Minh Hoang Nhat Nguyen, Tri Quang Le i Chuong Hoang Nguyen. "Building a molecular typing protocol for rs1801133 based on real-time PCR HRM technique". Science and Technology Development Journal - Natural Sciences 2, nr 3 (23.05.2019): 5–13. http://dx.doi.org/10.32508/stdjns.v2i3.747.
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rs1801133 is a single nucleotide polymorphism (SNP) located in the sequence of MTHFR on human chromosome 1. The alleles of this SNP affect the activity of the MTHFR enzyme. People bearing C/T genotype have 66% activity of MTHFR while people with T/T genotype have only 25% activity. These reduced activities of MTHFR cause homocysteinemia. There are several publications on the relationship between homocysteinemia and human diseases such as cardiovascular disease, neurological diseases, abnormal fetus, infertility and cancer. In this study, we built a molecular protocol for genotyping rs1801133 using real-time PCR HRM technique. This protocol could be used for diagnosis of molecular mechanism of homocysteinemia causing the mentoned above diseases as well as for the study of the relationship between rs1801133 and other human diseases. We successfully designed the primer pairs for genotyping and nucleotide sequencing rs1801133 by real-time PCR HRM and Sanger sequencing method. We also examined the optimal MgCl2 concentration for clear differentiation of three rs1801133 genotypes. Performance characteristics of the real-time PCR HRM protocol included of specificity, repeatability, reproducibility was evaluated and it showed good results. Comparison of genotyping results of rs1801133 between the realtime PCR HRM method and the Sanger nucleotide sequencing method showed good concordances. Finally, this real-time PCR HRM protocol for rs1801133 genotyping was applied on 100 human DNA samples to evaluate the clinical utility of the protocol.
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Esman, Anna, Anna Cherkashina, Konstantin Mironov, Dmitry Dubodelov, Svetlana Salamaikina, Anna Golubeva, Gasan Gasanov i in. "SARS-CoV-2 Variants Monitoring Using Real-Time PCR". Diagnostics 12, nr 10 (1.10.2022): 2388. http://dx.doi.org/10.3390/diagnostics12102388.
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According to the temporary recommendations of the 2021 World Health Organization (WHO), in addition to whole-genome sequencing, laboratories in various countries can also screen for known mutations utilizing targeted RT-PCR-based mutation detection assays. The aim of this work was to generate a laboratory technique to differentiate the main circulating SARS-CoV-2 variants in 2021–2022, when a sharp increase in morbidity was observed with the appearance of the Omicron variant. Real-time PCR methodology is available for use in the majority of scientific and diagnostic institutions in Russia, which makes it possible to increase the coverage of monitoring of variants in the territories of all 85 regions in order to accumulate information for the Central Services and make epidemiological decisions. With the methodology developed by the Central Research Institute of Epidemiology of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing (FSSCRP Human Wellbeing) (CRIE), more than 6000 biological samples have been typed, and 7% of samples with the Delta variant and 92% of samples with the Omicron variant have been identified as of 25 August 2022. Reagents for 140,000 definitions have been supplied to regional organizations.
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Pereira, Mariana R., Fabiana Rocha-Silva, Cidiane Graciele-Melo, Camila R. Lafuente, Telcia Magalhães i Rachel B. Caligiorne. "Comparison between Conventional and Real-Time PCR Assays for Diagnosis of Visceral Leishmaniasis". BioMed Research International 2014 (2014): 1–4. http://dx.doi.org/10.1155/2014/639310.
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The diagnosis of visceral leishmaniasis (VL) is a challenging issue and several studies worldwide have evaluated the different tools to reach a diagnostic solution. The polymerase chain reaction (PCR) has proven to be effective in detecting the genome ofLeishmaniaspecies in different biological samples. In this study, we compared the conventional PCR and real-time PCR using the Sybr Green system and their application in molecular diagnosis of visceral leishmaniasis in peripheral blood as a biological sample. The genus-specific conserved region of kinetoplast DNA (kDNA) was the target of amplification. We studied 30 samples from patients with suspect of visceral leishmaniasis who were treated by the Medical Clinic of Santa Casa de Belo Horizonte Hospital, Brazil. Among the samples studied, 19 had a confirmed diagnosis for VL by serology and/or by clinical findings. Among these 19 samples, 63% (n=12) presented positive results for serology and 79% (n=15) positive results in both PCR methodologies. This fact suggests that the PCR technique can assist in the diagnosis of visceral leishmaniasis in patients who do not have detectable antibodies by serology but can present the genome of the parasite circulating in whole blood. Also, it was possible to observe that there was conformity between the results of the techniques of cPCR and qPCR using the Sybr Green system in 100% of samples analyzed. These data suggest that both PCR techniques were equally effective for detection of the genome of the parasite in the patient’s blood.
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Voroshilina, ES, DL Zornikov i EA Panacheva. "Evaluation of the ejaculate microbiota by real-time PCR and culture-based technique". Laboratory diagnostics, nr 1 (11.03.2019): 41–45. http://dx.doi.org/10.24075/brsmu.2019.009.
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Among other things male sterility can be caused by inflammatory diseases of the urogenital tract, often associated with opportunistic microorganisms. Thus, it is necessary to implement modern methods for the detection and identification of opportunistic microorganisms in the urogenital tract. The aim of the work was to conduct comparative analysis of the ejaculate microbiota from men of the reproductive age and studied using quantitative polymerase chain reaction (PCR) and culture method. 86 samples of ejaculate collected from men aged 18–57 years after observing sexual abstinence for 3–5 days were examined. With culture study in 50% of samples we observed growth of gram positive facultative anaerobic bacteria in the amount less than 103 CFU/ml; in 16.3% of samples — the growth of bacteria was not observed. With real-time PCR in each sample 8–15 groups of microorganisms were detected (including the prevailing groups) in the amount of 102–106 GE/ml. In all 86 samples obligate anaerobes that cannot not be cultured in vitro were detected. The predominant groups of microorganisms, as determined by real-time PCR, were detected by the culture method only in 24.4% of cases.
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Locatelli, Giuseppe, Fabio Santoro, Fabrizio Veglia, Alberto Gobbi, Paolo Lusso i Mauro S. Malnati. "Real-Time Quantitative PCR for Human Herpesvirus 6 DNA". Journal of Clinical Microbiology 38, nr 11 (2000): 4042–48. http://dx.doi.org/10.1128/jcm.38.11.4042-4048.2000.
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The diagnosis of human herpesvirus 6 (HHV-6) infection represents a complex issue because the most widely used diagnostic tools, such as immunoglobulin G antibody titer determination and qualitative DNA PCR with blood cells, are unable to distinguish between latent (clinically silent) and active (often clinically relevant) infection. We have developed a new, highly sensitive, quantitative PCR assay for the accurate measurement of HHV-6 DNA in tissue-derived cell suspensions and body fluids. The test uses a 5′ nuclease, fluorogenic assay combined with real-time detection of PCR amplification products with the ABI PRISM 7700 sequence detector system. The sensitivity of this method is equal to the sensitivity of a nested PCR protocol (lower detection limit, 1 viral genome equivalent/test) for both the A and the B HHV-6 subgroups and shows a wider dynamic range of detection (from 1 to 106 viral genome equivalents/test) and a higher degree of accuracy, repeatability, and reproducibility compared to those of a standard quantitative-competitive PCR assay developed with the same reference DNA molecule. The novel technique is versatile, showing the same sensitivity and dynamic range with viral DNA extracted from different fluids (i.e., culture medium or plasma) or from tissue-derived cell suspensions. Furthermore, by virtue of its high-throughput format, this method is well suited for large epidemiological surveys.
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Takahashi, T., i T. Nakayama. "Novel Technique of Quantitative Nested Real-Time PCR Assay for Mycobacterium tuberculosis DNA". Journal of Clinical Microbiology 44, nr 3 (1.03.2006): 1029–39. http://dx.doi.org/10.1128/jcm.44.3.1029-1039.2006.
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Marubini, E., P. Verderio, C. Casini Raggi, M. Pazzagli i C. Orlando. "Statistical Diagnostics Emerging from External Quality Control of Real-Time PCR". International Journal of Biological Markers 19, nr 2 (kwiecień 2004): 141–46. http://dx.doi.org/10.1177/172460080401900209.
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Besides the application of conventional qualitative PCR as a valuable tool to enrich or identify specific sequences of nucleic acids, a new revolutionary technique for quantitative PCR determination has been introduced recently. It is based on real-time detection of PCR products revealed as a homogeneous accumulating signal generated by specific dyes. However, as far as we know, the influence of the variability of this technique on the reliability of the quantitative assay has not been thoroughly investigated. A national program of external quality assurance (EQA) for real-time PCR determination involving 42 Italian laboratories has been developed to assess the analytical performance of real-time PCR procedures. Participants were asked to perform a conventional experiment based on the use of an external reference curve (standard curve) for real-time detection of three cDNA samples with different concentrations of a specific target. In this paper the main analytical features of the standard curve have been investigated in an attempt to produce statistical diagnostics emerging from external quality control. Specific control charts were drawn to help biochemists take technical decisions aimed at improving the performance of their laboratories. Overall, our results indicated a subset of seven laboratories whose performance appeared to be markedly outside the limits for at least one of the standard curve features investigated. Our findings suggest the usefulness of the approach presented here for monitoring the heterogeneity of results produced by different laboratories and for selecting those laboratories that need technical advice on their performance.
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Giglio, Steven, Paul T. Monis i Christopher P. Saint. "Legionella Confirmation Using Real-Time PCR and SYTO9 Is an Alternative to Current Methodology". Applied and Environmental Microbiology 71, nr 12 (grudzień 2005): 8944–48. http://dx.doi.org/10.1128/aem.71.12.8944-8948.2005.
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ABSTRACT The currently accepted culture techniques for the detection of Legionella spp. in water samples (AS/NZS 3896:1998 and ISO 11731 standard methods) are slow and laborious, requiring from 7 to 14 days for a result. We describe a fully validated rapid confirmation technique that uses real-time PCR incorporating the intercalating dye SYTO9 for the direct identification of primary cultures, significantly decreasing turnaround time and allowing faster remedial action to be taken by the industry.
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Loftie-Eaton, Wesley, Allison Tucker, Ann Norton i Eva M. Top. "Flow Cytometry and Real-Time Quantitative PCR as Tools for Assessing Plasmid Persistence". Applied and Environmental Microbiology 80, nr 17 (27.06.2014): 5439–46. http://dx.doi.org/10.1128/aem.00793-14.
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ABSTRACTThe maintenance of a plasmid in the absence of selection for plasmid-borne genes is not guaranteed. However, plasmid persistence can evolve under selective conditions. Studying the molecular mechanisms behind the evolution of plasmid persistence is key to understanding how plasmids are maintained under nonselective conditions. Given the current crisis of rapid antibiotic resistance spread by multidrug resistance plasmids, this insight is of high medical relevance. The conventional method for monitoring plasmid persistence (i.e., the fraction of plasmid-containing cells in a population over time) is based on cultivation and involves differentiating colonies of plasmid-containing and plasmid-free cells on agar plates. However, this technique is time-consuming and does not easily lend itself to high-throughput applications. Here, we present flow cytometry (FCM) and real-time quantitative PCR (qPCR) as alternative tools for monitoring plasmid persistence. For this, we measured the persistence of a model plasmid, pB10::gfp, in threePseudomonashosts and in known mixtures of plasmid-containing and -free cells. We also compared three performance criteria: dynamic range, resolution, and variance. Although not without exceptions, both techniques generated estimates of overall plasmid loss rates that were rather similar to those generated by the conventional plate count (PC) method. They also were able to resolve differences in loss rates between artificial plasmid persistence assays. Finally, we briefly discuss the advantages and disadvantages for each technique and conclude that, overall, both FCM and real-time qPCR are suitable alternatives to cultivation-based methods for routine measurement of plasmid persistence, thereby opening avenues for high-throughput analyses.
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Bao-Zheng, Luo, Bo Qing-Ru, Chen Jing-Fan, Xu Hai-Nie, Yang Su, Yang Jian-Yun, Sha Cai-Hua i Liao Xiu-Yun. "Establishment of real-time fluorescent PCR assay to detectStreptococcus suisserotype 2". Chinese Journal of Agricultural Biotechnology 4, nr 1 (kwiecień 2007): 69–74. http://dx.doi.org/10.1017/s1479236207001350.
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AbstractA real-time fluorescent polymerase chain reaction (PCR) assay was established to detectStreptococcus suisserotype 2. Primers and Taqman probe were designed according tocps2I(capsular polysaccharide 2I) gene using bio-software Primer Express2.0 and Oligo6.0. An 81 bp DNA fragment was amplified fromS. suisserotype 2 genomic DNA, and the PCR product was cloned into pMD18-T vector and confirmed by DNA sequencing. The real-time fluorescent PCR amplification curve on a Lightcycler® showed that the method is accurate and specific forS. suisserotype 2 amplification, whereas reference bacteriaS. suis,Escherichia coli,Salmonellasp.,Staphylococcus aureus,Shigellasp.,Listeria monocytogenesstrains and a blank control were all negative. Tenfold serial dilutions ofS. suisserotype 2 were used to measure the sensitivity of real-time fluorescent PCR: ten copies of bacteria could be detected in one PCR reaction and only 30 min were required for a single test. To examine the stability of the real-time fluorescent PCR, the positive control was detected at two different times. The threshold cycle (Ct) values showed no statistical differences (P>0.05). Thus, this method was stable and repeatable. These results indicate that this real-time fluorescent PCR technique could be applied for epidemic supervision in entry–exit inspection and quarantine.
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Griffiths, Lisa J., Martin Anyim, Sarah R. Doffman, Mark Wilks, Michael R. Millar i Samir G. Agrawal. "Comparison of DNA extraction methods for Aspergillus fumigatus using real-time PCR". Journal of Medical Microbiology 55, nr 9 (1.09.2006): 1187–91. http://dx.doi.org/10.1099/jmm.0.46510-0.
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Newer methods such as PCR are being investigated in order to improve the diagnosis of invasive aspergillosis. One of the major obstacles to using PCR to diagnose aspergillosis is a reliable, simple method for extraction of the fungal DNA. The presence of a complex, sturdy cell wall that is resistant to lysis impairs extraction of the DNA by conventional methods employed for bacteria. Numerous fungal DNA extraction protocols have been described in the literature. However, these methods are time-consuming, require a high level of skill and may not be suitable for use as a routine diagnostic technique. Here, a number of extraction methods were compared: a freeze–thaw method, a freeze–boil method, enzyme extraction and a bead-beating method using Mini-BeadBeater-8. The quality and quantity of the DNA extracted was compared using real-time PCR. It was found that the use of a bead-beating method followed by extraction with AL buffer (Qiagen) was the most successful extraction technique, giving the greatest yield of DNA, and was also the least time-consuming method assessed.
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Deubelbeiss, A., M. L. Zahno, M. Zanoni, D. Bruegger i R. Zanoni. "Real-Time RT-PCR for the Detection of Lyssavirus Species". Journal of Veterinary Medicine 2014 (16.10.2014): 1–12. http://dx.doi.org/10.1155/2014/476091.
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The causative agents of rabies are single-stranded, negative-sense RNA viruses in the genus Lyssavirus of Rhabdoviridae, consisting of twelve classified and three as yet unclassified species including classical rabies virus (RABV). Highly neurotropic RABV causes rapidly progressive encephalomyelitis with nearly invariable fatal outcome. Rapid and reliable diagnosis of rabies is highly relevant for public and veterinary health. Due to growing variety of the genus Lyssavirus observed, the development of suitable molecular assays for diagnosis and differentiation is challenging. This work focused on the establishment of a suitable real-time RT-PCR technique for rabies diagnosis as a complement to fluorescent antibody test and rabies tissue culture infection test as gold standard for diagnosis and confirmation. The real-time RT-PCR was adapted with the goal to detect the whole spectrum of lyssavirus species, for nine of which synthesized DNA fragments were used. For the detection of species, seven probes were developed. Serial dilutions of the rabies virus strain CVS-11 showed a 100-fold higher sensitivity of real-time PCR compared to heminested RT-PCR. Using a panel of thirty-one lyssaviruses representing four species, the suitability of the protocol could be shown. Phylogenetic analysis of the sequences obtained by heminested PCR allowed correct classification of all viruses used.
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Hamad, M., A. Khashan i M. Sood. "Molecular detection of NDV by Real-Time PCR in Baghdad". Al-Anbar Journal of Veterinary Sciences 11, nr 1 (czerwiec 2018): 95–99. http://dx.doi.org/10.37940/ajvs.2018.11.1.11.
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Newcastle disease virus (NDV) is widely spread in Iraqi chicken that is associated with notable economic losses. Current study was carried out to identify NDV in the North of Baghdad by employing a Molecular methods based on the Real-Time PCR technique, the primers and the probe also were designed based on the conserved fragment coding matrix protein(M gene).These genes of virus show the highest level of conservation, and therefore the test is adaptable for detection of NDV. In the current study, 14 clinically suspicious birds believed to be infected with ND, had been chosen from 14 different commercial poultry farms in various areas of North Baghdad brought to the Aurok and Al-Emad Lab. Baghdad/ Iraq. The infected birds were examined and the tissue samples collected from ceca tonsils, spleens, trachea, lungs and brains of each farm. These samples were submitted to examination directly by Real-time PCR. The Results of the samples collected from 14 poultry flocks were showed positive results in ten samples with the SYBR Green I real-time PCR and the other 4 flocks failed to show any positive results with both gene primers.
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Krejmer-Rabalska, Martyna, Lukasz Rabalski, Michael Jukes, Marlinda Lobo de Souza, Sean Moore i Boguslaw Szewczyk. "New Method for Differentiation of Granuloviruses (Betabaculoviruses) Based on Real-Time Polymerase Chain Reaction (Real-Time PCR)". Viruses 11, nr 2 (29.01.2019): 115. http://dx.doi.org/10.3390/v11020115.
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Baculoviridae is a highly diverse family of rod-shaped viruses with double-stranded DNA. To date, almost 100 species have had their complete genomic sequences deposited in the GenBank database, a quarter of which comprises granuloviruses (GVs). Many of the genomes are sequenced using next-generation sequencing, which is currently considered the best method for characterizing new species, but it is time-consuming and expensive. Baculoviruses form a safe alternative to overused chemical pesticides and therefore there is a constant need for identifying new species that can be active components of novel biological insecticides. In this study, we have described a fast and reliable method for the detection of new and differentiation of previously analyzed granulovirus species based on a real-time polymerase chain reaction (PCR) technique with melting point curve analysis. The sequences of highly conserved baculovirus genes, such as granulin and late expression factors 8 and 9 (lef-8 and lef-9), derived from GVs available to date have been analyzed and used for degenerate primer design. The developed method was tested on a representative group of eight betabaculoviruses with comparisons of melting temperatures to allow for quick and preliminary granulovirus detection. The proposed real-time PCR procedure may be a very useful tool as an easily accessible screening method in a majority of laboratories.
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Wu, Yajun, Ying Chen, Bin Wang, Yunhua Gao, Liqun Bai i Haiyan Wang. "SYBR Green Real-Time PCR Used to Detect Celery in Food". Journal of AOAC INTERNATIONAL 93, nr 5 (1.09.2010): 1530–36. http://dx.doi.org/10.1093/jaoac/93.5.1530.
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Abstract Celery was found to provoke human allergenic response in some countries. Labeling of celery ingredients was required by the European Union, and the threshold set at 10 mg/kg (0.001%). In our study, a celery mannitol transporter (Mat3) gene-based detection method was established by means of SYBR Green real-time PCR technique. No cross-reactivity was found between celery and the other food materials. Absolute detection limit (LODa), relative detection limit (LODr), and practical detection limit (LODp) of the method were determined through experiments on pure celery DNA, DNA mix, and spiked food samples. The method was able to detect 0.001 raw food sample and 0.01 heated food sample. The utility of the method was confirmed by the investigation of 13 commercial foods.
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Fenicia, Lucia, Fabrizio Anniballi, Dario De Medici, Elisabetta Delibato i Paolo Aureli. "SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A". Applied and Environmental Microbiology 73, nr 9 (16.03.2007): 2891–96. http://dx.doi.org/10.1128/aem.02234-06.
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ABSTRACT Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 101 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%).
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Park, YoungBin, You-Hee Cho, YoungMee Jee i GwangPyo Ko. "Immunomagnetic Separation Combined with Real-Time Reverse Transcriptase PCR Assays for Detection of Norovirus in Contaminated Food". Applied and Environmental Microbiology 74, nr 13 (25.04.2008): 4226–30. http://dx.doi.org/10.1128/aem.00013-08.
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ABSTRACT We developed an immunomagnetic separation (IMS) technique combined with real-time TaqMan reverse transcriptase PCR (RT-PCR), which allowed detection of norovirus at a level as low as 3 to 7 RT-PCR units from artificially contaminated strawberries. The inoculum recovery rate ranged from 14 to 30%. The data demonstrate that IMS combined with real-time RT-PCR will be useful as a rapid and sensitive method for detecting food-borne microbial contaminants.
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Ma, Lei, Fanwen Zeng, Bihong Huang, Feng Cong, Ren Huang, Jingyun Ma i Pengju Guo. "Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay". BioMed Research International 2018 (6.11.2018): 1–7. http://dx.doi.org/10.1155/2018/5035139.
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Porcine deltacoronavirus (PDCoV) is a newly discovered coronavirus, which belongs to the family Coronaviridae. It causes watery diarrhea, vomiting, and dehydration in newborn piglets. A sensitive RT-PCR method is urgently required to detect PDCoV infection. In this study, we developed and evaluated a conventional RT-PCR assay and a SYBR green-based real-time RT-PCR assay that targeted the PDCoV n gene. Both assays are specific and have the same limit of detection at 2 × 101 copies of RNA molecules per reaction. Eighty-four clinical samples were subjected to both conventional RT-PCR and real-time RT-PCR, and the same positive rate (41.7%) was achieved, which was much higher than the positive rate (26.2%) using a previously described one-step RT-PCR technique. In summary, a conventional RT-PCR technique was successfully established for the detection of PDCoV with the same detection limit as a SYBR green-based real-time RT-PCR assay.
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Manivannan, Kavitha, Samar Mohamed Mahmoud, Malathi Ramasamy, Abeer A. E. Shehata, Hanaa Ahmed, Chandrasekar Solaimuthu i Kaviyarasan Dhandapani. "Molecular detection of brucellosis in dromedary camels of Qatar by real-time PCR technique". Comparative Immunology, Microbiology and Infectious Diseases 78 (październik 2021): 101690. http://dx.doi.org/10.1016/j.cimid.2021.101690.
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Aguayo-Patrón, Sandra, Reyna Castillo-Fimbres, Luis Quihui-Cota i Ana María Calderón de la Barca. "Use of real-time polymerase chain reaction to identify Entamoeba histolytica in schoolchildren from northwest Mexico". Journal of Infection in Developing Countries 11, nr 10 (31.10.2017): 800–805. http://dx.doi.org/10.3855/jidc.9350.
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Introduction: Entamoeba histolytica, E. dispar, and E. moshkovskii are morphologically identical, but intestinal amebiasis is caused only by E. histolytica. Mexico is among the countries with high amebae infection rates, although the contribution of pathogenic amoeba to the total detected cases remains unknown, especially in the northwestern dry region. Therefore, the aim of this study was to identify the actual prevalence of E. histolytica using real-time polymerase chain reaction (PCR) in schoolchildren of northwestern Mexico.
Methodology: Participants were children from five public elementary schools in the low-socioeconomic-level suburban areas of Hermosillo, Sonora, Mexico. One stool sample was collected from each child and analyzed by the Faust technique for Entamoeba spp. and by real-time PCR for E. histolytica.
Results: Analysis of stool samples from 273 children (9.0 ± 1.5 years of age) resulted in 25 (9.2%) positive for E. histolytica/E. dispar/E. moshkovskii by the Faust technique; of these, 3 were positive for E. histolytica by real-time PCR. In addition, 2 samples that were negative for E. histolytica/E. dispar/E. moshkovskii by the Faust technique were positive by real-time PCR.
Conclusions: The actual prevalence of E. histolytica in our study population was 1.8%, which is lower than those reported in previous studies in other Mexican regions.
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Cheng, Ching-Yang, Jing-Ruei Chi, Sin-Rong Lin, Chi-Chiang Chou i Chin-Cheng Huang. "Rapid quantification of Salmonella Typhimurium inoculated to meat products by real-time PCR". Acta Veterinaria Hungarica 57, nr 1 (1.03.2009): 25–38. http://dx.doi.org/10.1556/avet.57.2009.1.3.
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The objective of this study was to use a 5′-nuclease (TaqMan) real-time PCR method with primers and probe specific to thespaQgene as a rapid approach to quantitatively determineSalmonellaTyphimurium. The result showed that the correlation coefficient between real-time PCR estimates and bovine serum albumin (BSA) plate counts ofS. Typhimurium was 0.99, independently of 105-fold numbers of bystanderEscherichia coliO157:H7 or total viable counts. The sensitivity of the real-time quantitative PCR assay was 10 CFU/mL for pureS. Typhimurium culture without enrichment. A known number ofS. Typhimurium target cells were inoculated to dumpling fillings and chicken nuggets and DNA was extracted for real-time PCR analysis. The sensitivity was 60 CFU/g forS. Typhimurium inoculated to the food samples without any preceding procedure of enrichment. The duration of the entire experiment from DNA isolation and purification to PCR amplification was less than 12 h. This study demonstrated that realtime PCR is a rapid and reliable technique for quantifyingS. Typhimurium possessing thespaQgene in pure culture and in meat products.
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Romero-Soto, Fabian Oswaldo, Mohammad Mahdi Aeinehvand, Marc Madou i Sergio Omar Martinez-Chapa. "(Digital Presentation) An Electrochemical Sensor in a Disc for Real-Time PCR Assisted with Thermal Convective Flow". ECS Meeting Abstracts MA2022-02, nr 61 (9.10.2022): 2230. http://dx.doi.org/10.1149/ma2022-02612230mtgabs.
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Nowadays, the polymerase chain reaction (PCR) has extensively adapted to microfluidic devices for several features such as the low consumption of reagents and sample, portability, high-throughput, and low fabrication costs [1]. The reduction of the amplification time has been a relevant parameter since the invention of the PCR, where rapid microfluidic PCR is especially preferred for the detection of infectious diseases [1,2]. The continuous-flow-based PCR is a type of microfluidic PCR that proposes the mobilization of the PCR mixture through a microchannel with different fixed temperature zones to achieve the required thermal-cycling [3]. This approach has smaller thermal inertia because only the PCR mixture needs to be heated and cooled instead of the entire microfluidic device. The continuous-flow approach allows rapid-thermal cycling with lower power consumption and, therefore, reduces the overall amplification time. The Lab-on-a-disc, or centrifugal microfluidic platform, has adapted the continuous-flow PCR by utilizing the flow generated by thermal convection in a ring-structured microchannel [4]. During spinning, embedded heaters in the bottom of the CD heat specific section of the microchannel (see Figure 1) at a fixed temperature and, subsequently, create a natural convection flow in the PCR mixture and move through the microchannel. The centrifugal-assisted thermal convection (CATC) technique not only enables the recirculation pathway for the continuous-flow PCR but overcomes the unidirectional nature of centrifugation force on the CD. Despite the advantage of rapid PCR on Lab-on-a-Disc, the CATC technique lacks the implementation of a detection method with high sensitivity and resolution, aiming for a fully integrated microfluidic platform. Electrochemical detection can provide fast responses with minimum power consumption for miniaturized devices. This work proposes the integration of an electrochemical sensor to the CATC technique in a circular microchannel for the real-time monitoring of DNA amplification. The electrochemical sensor consists of interdigitated gold electrodes located above the ring microchannel to quantify the amplicon concentration after each cycle mediated by the intercalation of Methylene Blue between the newly-generated DNA (see figure 1A). The characterization of the sensor is performed through voltammetric techniques such as cyclic voltammetry (CV) and linear sweep voltammetry (LSV), where the latter is utilized for amplicon quantification. To optimize the CATC technique combined with the electrochemical sensor, a COMSOL simulation is performed to improve the amplification ratio and amplification time in the CD by analyzing the microchannel geometry and flow rate (see figure 1B). Both the thermal control for the thermal-assisted microchannel and the electrochemical sensor are powered and controlled via the “electrified Lab-on-a-Disc” (eLoaD) board [5] (see figure 1C). [1] Y. Zhang, P. Ozdemir, Anal. Chim. Acta 638 (2009) 115–125. [2] X. Dong, L. Liu, Y. Tu, J. Zhang, G. Miao, L. Zhang, S. Ge, N. Xia, D. Yu, X. Qiu, TrAC - Trends Anal. Chem. 143 (2021) 116377. [3] M.B. Kulkarni, S. Goel, Eng. Res. Express 2 (2020). [4] M. Saito, K. Takahashi, Y. Kiriyama, W.V. Espulgar, H. Aso, T. Sekiya, Y. Tanaka, T. Sawazumi, S. Furui, E. Tamiya, Anal. Chem. 89 (2017) 12797–12804. [5] S.M. Torres Delgado, J.G. Korvink, D. Mager, Biosens. Bioelectron. 117 (2018) 464–473. Figure 1
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Ворошилина, Е. С., Д. Л. Зорников i Е. А. Паначева. "Сравнительное исследование микробиоты эякулята методом количественной ПЦР и культуральным методом". Вестник Российского Государственного медицинского университета, nr 1 (11.03.2019): 44–49. http://dx.doi.org/10.24075/vrgmu.2019.009.
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Одной из причин мужского бесплодия могут быть воспалительные заболевания урогенитального тракта, развитие которых в ряде случаев ассоциировано с условно-патогенными микроорганизмами (УПМ). В связи с этим актуальна проблема внедрения современных методов выявления и идентификации УПМ в урогенитальном тракте. Целью работы было провести сравнительный анализ результатов исследования микробиоты эякулята мужчин репродуктивного возраста с помощью количественной полимеразной цепной реакции (ПЦР) (тест Андрофлор) и культурального метода. Исследовали 86 образцов эякулята, собранных у мужчин в возрасте 18–57 лет после соблюдения полового воздержания в течение 3–5 суток. При культуральном исследовании в 50% образцов наблюдали рост грамположительных факультативно анаэробных бактерий в количестве менее 103 КОЕ/мл; в 16,3% образцов — роста бактерий отмечено не было. При использовании ПЦР в реальном времени (ПЦР-РВ) в каждом образце выявляли 8–15 групп микроорганизмов (в том числе определяли преобладающую) в количестве 102–106 ГЭ/мл. Во всех 86 образцах были обнаружены облигатные анаэробы, которые не культивируются in vitro. Преобладающие группы микроорганизмов, определяемые в ПЦР-РВ, были выявлены культуральным методом только в 24,4% случаев.
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Jaton, Katia, Jacques Bille i Gilbert Greub. "A novel real-time PCR to detect Chlamydia trachomatis in first-void urine or genital swabs". Journal of Medical Microbiology 55, nr 12 (1.12.2006): 1667–74. http://dx.doi.org/10.1099/jmm.0.46675-0.
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Screening for Chlamydia trachomatis infections can be performed on urine samples and genital swabs using molecular techniques. A novel approach was developed that combined an automated extraction procedure, an automated liquid-handling system and real-time PCR to detect C. trachomatis from urine or swabs. This novel real-time PCR approach was compared to the commercial Cobas Amplicor system on 628 specimens. In a retrospective analysis, 51 samples that tested positive using the Cobas assay were also positive with the real-time PCR, whereas the 49 samples negative with Cobas were also negative with the real-time PCR, for an overall agreement of 100 %. Among 528 prospective samples consecutively received at the authors' laboratory with a request for C. trachomatis PCR, five PCR reactions were inhibited when tested with Cobas. These five inhibited samples were found negative with the real-time PCR. Among the remaining 523 samples, 45 (8.6 %) were positive with both methods, 476 (91 %) were negative with both methods, and 2 (0.4 %) were positive with Cobas but negative with the real-time PCR. Thus, when considering Cobas as the gold standard, the overall agreement was 99.6 %, the sensitivity of the real-time PCR was 95.7 % and the specificity was 100 %. The two discrepant samples were retested in parallel and were found negative with both methods. When testing a batch of 25 samples, both reagent costs and laboratory technician time were reduced with the new technique (7.30 euros per sample and 134 min) compared to Cobas (11.20 euros per sample and 232 min). Moreover, due to reduced organizational constraints, the median time from sample reception to result was only 24 h using the automated platform. Overall, this novel real-time PCR approach exhibited an excellent specificity and a sensitivity similar to that of Cobas Amplicor PCR for the detection of C. trachomatis. Given its high throughput potential and low costs/laboratory technician time requirement, it may be useful for future use in large C. trachomatis screening programs.
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Bustin, SA. "Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays". Journal of Molecular Endocrinology 25, nr 2 (1.10.2000): 169–93. http://dx.doi.org/10.1677/jme.0.0250169.
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The reverse transcription polymerase chain reaction (RT-PCR) is the most sensitive method for the detection of low-abundance mRNA, often obtained from limited tissue samples. However, it is a complex technique, there are substantial problems associated with its true sensitivity, reproducibility and specificity and, as a quantitative method, it suffers from the problems inherent in PCR. The recent introduction of fluorescence-based kinetic RT-PCR procedures significantly simplifies the process of producing reproducible quantification of mRNAs and promises to overcome these limitations. Nevertheless, their successful application depends on a clear understanding of the practical problems, and careful experimental design, application and validation remain essential for accurate quantitative measurements of transcription. This review discusses the technical aspects involved, contrasts conventional and kinetic RT-PCR methods for quantitating gene expression and compares the different kinetic RT-PCR systems. It illustrates the usefulness of these assays by demonstrating the significantly different levels of transcription between individuals of the housekeeping gene family, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH).
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Raoot, Amita, i Geeta Dev. "Assessment of Status ofrpoBGene in FNAC Samples of Tuberculous Lymphadenitis by Real-Time PCR". Tuberculosis Research and Treatment 2012 (2012): 1–5. http://dx.doi.org/10.1155/2012/834836.
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Introduction.Multidrug resistance tuberculosis (MDR TB), the combined resistance ofMycobacterium tuberculosisto isoniazid (INH) and rifampin (RFM) is a major public health problem in India as it ranks second among the MDR-TB high burden countries worldwide. WHO recommends RFM resistance as a “surrogate marker” for detecting MDR. FNAC is the most widely used noninvasive investigative technique for TB lymphadenitis. Real-time polymerase chain reaction, an extremely versatile technique can be used for the timely detection and treatment of MDR TB by assessing RFM resistance status in the FNAC samples of TB lymphadenitis.Aim.To assess the status ofrpoBgene by real-time PCR in FNAC samples of TB lymphadenitis.Materials and Methods.Thirty FNAC samples from patients with persistent LAP or appearance of new LAP after 5 months or more of Anti Tubercular Treatment were assessed for status ofrpoBgene by Real-Time PCR using probe covering the “hot spot resistance” region of therpoBgene.Result.By using probe covering codons 531 and 526 ofrpoBgene, we could detect 17 of 30 (56.7%) rifampin resistant isolate. The PCR could detectMtbDNA in 100% of cases.Conclusion.Use of molecular methods like Real-Time PCR for detection of MDR-TB in FNAC samples is time saving, logical and economical approach over the culture based method.
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Cao, Yiqi, Miao Yu, Guihua Dong, Bing Chen i Baiyu Zhang. "Digital PCR as an Emerging Tool for Monitoring of Microbial Biodegradation". Molecules 25, nr 3 (6.02.2020): 706. http://dx.doi.org/10.3390/molecules25030706.
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Biodegradation of contaminants is extremely complicated due to unpredictable microbial behaviors. Monitoring of microbial biodegradation drives us to determine (1) the amounts of specific degrading microbes, (2) the abundance, and (3) expression level of relevant functional genes. To this endeavor, the cultivation independent polymerase chain reaction (PCR)-based monitoring technique develops from endpoint PCR, real-time quantitative PCR, and then into novel digital PCR. In this review, we introduce these three categories of PCR techniques and summarize the timely applications of digital PCR and its superiorities than qPCR for biodegradation monitoring. Digital PCR technique, emerging as the most accurately absolute quantification method, can serve as the most promising and robust tool for monitoring of microbial biodegradation.
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Abbas, Adeeb, Majeed A. Sabbah, Abdul-salam Hatam, Luma A. Yasser i Baan Abdul-Latif. "Molecular diagnosis of Iraqi chronic myeloid leukemia patients using quantitative real-time PCR". Journal of Biotechnology Research Center 4, nr 2 (1.06.2010): 64–69. http://dx.doi.org/10.24126/jobrc.2010.4.2.125.
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hronic myeloid leukemia (CML), also known as chronic granulocytic leukemia, is a form of leukemia characterized by the increased and unregulated growth of myeloid cells in the bone marrow and the accumulation of these cells in peripheral blood. Total RNA extraction, cDNA and quantitative real-time PCR (Q-RT-PCR) were done for thirty CML Iraqi patients. Hematology tests (hemoglobin, platelets and WBCs counts) were done for the same samples in the same time. The results of this study show that some samples with normal hematology values have BCR-ABL (p210) fusion transcript with molecular analysis by Q-RT-PCR. This indicates the importance of this technique in the diagnosis and monitoring the therapy of CML patients.