Using quantitative real-time polymerase chain reaction (qRT-PCR) for detection microcystin producing cyanobacteria

Introduction: Cyanobacterial blooms (CBs) have become a growing concern worldwide. In the natural environment, potentially toxic (can produce toxins) and non-toxic (can not produce toxins) colonies often co-exist within a bloom. Methods: The present study aimed to quantify toxic and non-toxic cells of cyanobacteria in the Tri An Reservoir (TAR) using a quantitative real-time polymerase chain reaction (qRT-PCR). Results: Results showed that the Microcystis genus dominated the cyanobacterial communities in the TAR. Microcystis was also the primary microcystins (MC) producing cyanobacteria in the water. Total cyanobacteria and Microcystis cells ranged from 152103 to 27106 copy/L and from 105103 to 19106 copy/L, respectively. The cell number of potentially MC-producing cyanobacteria (corresponding to the Microcystis mcyD gene) varied from 27103 to 13106 copy/L. MC concentrations often present in raw water with a concentration of up to 4.8 mg/L. Our results showed that the MC concentration in raw water was positively correlated with the mcyD copy number, suggesting that Microcystis spp. are the main toxin producers in the TAR's surface water. Conclusion: Our study suggested that qRT-PCR techniques and traditional count are comparable and could be used to quantify cyanobacteria. In addition, the qRT-PCR techniques can determine the toxic cyanobacterial cells and could be used as a tool for early monitoring of toxic cyanobacteria in lakes and reservoirs.

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Detection of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in clinical samples using Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

IOP Conference Series Earth and Environmental Science ◽

10.1088/1755-1315/762/1/012026 ◽

2021 ◽

Vol 762 (1) ◽

pp. 012026

Author(s):

A Atikana ◽

L Sukmarini ◽

Hariyatun ◽

A M Ridwanulah ◽

H A Nugroho ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Severe Acute Respiratory Syndrome ◽

Real Time ◽

Reverse Transcription ◽

Clinical Samples ◽

Chain Reaction ◽

Qrt Pcr ◽

Polymerase Chain

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Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

Virology Journal ◽

10.1186/s12985-020-01467-y ◽

2020 ◽

Vol 17 (1) ◽

Author(s):

Yang Zhang ◽

Chunyang Dai ◽

Huiyan Wang ◽

Yong Gao ◽

Tuantuan Li ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Clinical Samples ◽

Chain Reaction ◽

Pcr Assay ◽

Qrt Pcr ◽

Highly Sensitive ◽

One Step ◽

Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30 to 60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The limit of detection (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI: 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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Quantitation of 35S Promoter in Maize DNA Extracts from Genetically Modified Organisms Using Real-Time Polymerase Chain Reaction, Part 2: Interlaboratory Study

Journal of AOAC International ◽

10.1093/jaoac/88.2.558 ◽

2005 ◽

Vol 88 (2) ◽

pp. 558-573 ◽

Cited By ~ 18

Author(s):

Max Feinberg ◽

Sophie Fernandez ◽

Sylvanie Cassard ◽

Chrystèle Charles-Delobel ◽

Yves Bertheau ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Genetically Modified Organisms ◽

Genetically Modified ◽

Interlaboratory Study ◽

Performance Criteria ◽

Tolerance Interval ◽

Chain Reaction ◽

Qrt Pcr ◽

Polymerase Chain

Abstract The European Committee for Standardization (CEN) and the European Network of GMO Working Laboratories have proposed development of a modular strategy for stepwise validation of complex analytical techniques. When applied to the quantitation of genetically modified organisms (GMOs) in food products, the instrumental quantitation step of the technique is separately validated from the DNA extraction step to better control the sources of uncertainty and facilitate the validation of GMO-specific polymerase chain reaction (PCR) tests. This paper presents the results of an interlaboratory study on the quantitation step of the method standardized by CEN for the detection of a regulatory element commonly inserted in GMO maize-based foods. This is focused on the quantitation of P35S promoter through using the quantitative real-time PCR (QRT-PCR). Fifteen French laboratories participated in the interlaboratory study of the P35S quantitation operating procedure on DNA extract samples using either the thermal cycler ABI Prism® 7700 (Applied Biosystems, Foster City, CA) or Light Cycler® (Roche Diagnostics, Indianapolis, IN). Attention was focused on DNA extract samples used to calibrate the method and unknown extract samples. Data were processed according to the recommendations of ISO 5725 standard. Performance criteria, obtained using the robust algorithm, were compared to the classic data processing after rejection of outliers by the Cochran and Grubbs tests. Two laboratories were detected as outliers by the Grubbs test. The robust precision criteria gave values between the classical values estimated before and after rejection of the outliers. Using the robust method, the relative expanded uncertainty by the quantitation method is about 20% for a 1% Bt176 content, whereas it can reach 40% for a 0.1% Bt176. The performances of the quantitation assay are relevant to the application of the European regulation, which has an accepted tolerance interval of about ±50%. These data were fitted to a power model (r2 = 0.96). Thanks to this model, it is possible to propose an estimation of uncertainty of the QRT-PCR quantitation step and an uncertainty budget depending on the analytical conditions.

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Quantitative reverse transcriptase real-time polymerase chain reaction (qRT-PCR) in translational oncology: Lung cancer perspective

Lung Cancer ◽

10.1016/j.lungcan.2007.11.008 ◽

2008 ◽

Vol 59 (2) ◽

pp. 147-154 ◽

Cited By ~ 23

Author(s):

Marcin Skrzypski

Keyword(s):

Lung Cancer ◽

Polymerase Chain Reaction ◽

Reverse Transcriptase ◽

Real Time ◽

Chain Reaction ◽

Qrt Pcr ◽

Translational Oncology ◽

Polymerase Chain

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Quantitative expression analysis of Bodhesin genes in the buck (Capra hircus) reproductive tract by real-time polymerase chain reaction (qRT-PCR)

Animal Reproduction Science ◽

10.1016/j.anireprosci.2008.01.013 ◽

2009 ◽

Vol 110 (3-4) ◽

pp. 245-255 ◽

Cited By ~ 6

Author(s):

Luciana M. Melo ◽

Antônia S.F. Nascimento ◽

Felipe G. Silveira ◽

Rodrigo M.S. Cunha ◽

Nathália A.C. Tavares ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Expression Analysis ◽

Reproductive Tract ◽

Capra Hircus ◽

Chain Reaction ◽

Quantitative Expression ◽

Qrt Pcr ◽

Polymerase Chain

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Monitoring of invasive Aspergillosis antifungal therapy by quantitative reverse transcriptase real-time polymerase chain reaction (qRT- PCR)

10.26226/morressier.5ac39993d462b8028d899b37 ◽

2018 ◽

Author(s):

Hossein Khodadadi

Keyword(s):

Polymerase Chain Reaction ◽

Reverse Transcriptase ◽

Invasive Aspergillosis ◽

Real Time ◽

Antifungal Therapy ◽

Chain Reaction ◽

Qrt Pcr ◽

Polymerase Chain

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Analysis and validation of a highly sensitive one-step nested quantitative real-time polymerase chain reaction assay for specific detection of severe acute respiratory syndrome coronavirus 2

10.21203/rs.3.rs-74067/v1 ◽

2020 ◽

Author(s):

Yang Zhang ◽

Chunyang Dai ◽

Huiyan Wang ◽

Yong Gao ◽

Tuantuan Li ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Quantitative Polymerase Chain Reaction ◽

Clinical Samples ◽

Chain Reaction ◽

Pcr Assay ◽

Qrt Pcr ◽

Highly Sensitive ◽

One Step ◽

Polymerase Chain

Abstract Background Coronavirus disease 2019 (COVID-19), caused by SARS-CoV-2, is posing a serious threat to global public health. Reverse transcriptase real-time quantitative polymerase chain reaction (qRT-PCR) is widely used as the gold standard for clinical detection of SARS-CoV-2. Due to technical limitations, the reported positive rates of qRT-PCR assay of throat swab samples vary from 30–60%. Therefore, the evaluation of alternative strategies to overcome the limitations of qRT-PCR is required. A previous study reported that one-step nested (OSN)-qRT-PCR revealed better suitability for detecting SARS-CoV-2. However, information on the analytical performance of OSN-qRT-PCR is insufficient. Method: In this study, we aimed to analyze OSN-qRT-PCR by comparing it with droplet digital PCR (ddPCR) and qRT-PCR by using a dilution series of SARS-CoV-2 pseudoviral RNA and a quality assessment panel. The clinical performance of OSN-qRT-PCR was also validated and compared with ddPCR and qRT-PCR using specimens from COVID-19 patients. Result The LoD (copies/ml) of qRT-PCR, ddPCR, and OSN-qRT-PCR were 520.1 (95% CI): 363.23–1145.69) for ORF1ab and 528.1 (95% CI: 347.7–1248.7) for N, 401.8 (95% CI: 284.8–938.3) for ORF1ab and 336.8 (95% CI: 244.6–792.5) for N, and 194.74 (95% CI: 139.7–430.9) for ORF1ab and 189.1 (95% CI: 130.9–433.9) for N, respectively. Of the 34 clinical samples from COVID-19 patients, the positive rates of OSN-qRT-PCR, ddPCR, and qRT-PCR were 82.35% (28/34), 67.65% (23/34), and 58.82% (20/34), respectively. Conclusion In conclusion, the highly sensitive and specific OSN-qRT-PCR assay is superior to ddPCR and qRT-PCR assays, showing great potential as a technique for detection of SARS-CoV-2 in patients with low viral loads.

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The use of quantitative real-time polymerase chain reaction (qRT-PCR) technologies to screen for the presence of total bacteria in a liquid sample

Pharmaceutical Regulatory Affairs Open Access ◽

10.4172/2167-7689.c1.022 ◽

2016 ◽

Vol 05 (02) ◽

Author(s):

Andy Moreno

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Liquid Sample ◽

Chain Reaction ◽

Qrt Pcr ◽

Polymerase Chain ◽

Total Bacteria

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Quantitative Real-Time Polymerase Chain Reaction for Bacterial Enumeration and Allelic Discrimination to Differentiate Xanthomonas Strains on Citrus

Phytopathology ◽

10.1094/phyto-95-1333 ◽

2005 ◽

Vol 95 (11) ◽

pp. 1333-1340 ◽

Cited By ~ 24

Author(s):

J. Cubero ◽

J. H. Graham

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Regulatory Protein ◽

Strain Identification ◽

Target Sequence ◽

Chain Reaction ◽

Allelic Discrimination ◽

Qrt Pcr ◽

Plant Samples ◽

Polymerase Chain

Quantitative real-time polymerase chain reaction (QRT-PCR) was developed for identification and enumeration of bacteria in citrus plant samples infected with Xanthomonas axonopodis pvs. citri and citrumelo, the cause of citrus bacterial canker (CBC) and citrus bacterial spot (CBS), respectively. Three sets of primers based on the pathogenicity gene (pth) in X. axonopodis pv. citri, a ribosomal gene in X. axonopodis pv. citrumelo, and the leucine-responsive regulatory protein (lrp) in both pathovars were combined with TaqMan probes and applied for specific strain detection and quantification. Calibration curves for bacterial abundance in plant samples obtained with the three primer-probe combinations were congruent with colony counts on plates of semiselective medium in most of the cases. However, apparent overestimation of bacterial cells by QRT-PCR indicated the presence of nonculturable or nonviable cells in some samples. In addition to quantification, the lrp primers and probes permitted differentiation by allelic discrimination of Xanthomonas strains infecting citrus tissues. This technique is based on the utilization of two probes that detect a single nucleotide difference in the target sequence between different strains and was validated with a collection of cultured Xanthomonas strains as well as tissue with CBC and CBS lesions. Allelic discrimination is demonstrated to be a more specific and sensitive protocol than previously developed PCR-based methods for strain identification and quantification.

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