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

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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Clinical evaluation of periodontal pathogen levels by real-time polymerase chain reaction in peri-implantitis patients

International Journal of Implant Dentistry ◽

10.1186/s40729-021-00385-0 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Taichi Ito ◽

Gentaro Mori ◽

Yukari Oda ◽

Tomoki Hirano ◽

Hodaka Sasaki ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Real Time ◽

Bacterial Flora ◽

Periodontal Pathogen ◽

Periodontal Pathogens ◽

Chain Reaction ◽

Polymerase Chain ◽

Average Copy ◽

Number Of Bacteria ◽

Total Bacteria

Abstract Objective The mechanisms underlying the onset and progression of peri-implantitis are similar to those of periodontitis, and the causative bacteria are believed to similar. Previous studies support an association between peri-implantitis and periodontal pathogen. Thus, we investigated the bacterial flora of peri-implantitis patients in comparison to those of healthy implant and periodontitis patients. Materials and methods In total, 70 patients visiting Tokyo Dental College Chiba Hospital were divided into four groups: healthy, periodontitis, healthy implant, and peri-implantitis. For each group, the following five periodontal pathogens were detected using real-time polymerase chain reaction: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia, Treponema denticola, and Prevotella intermedia. Results The average copy number of total bacteria was significantly higher in the periodontitis group than in the other groups. P. gingivalis was detected in the periodontitis and peri-implantitis groups at levels as high as 18.92% and 12.29%, respectively, and P. intermedia was found in the peri-implantitis group at a rate of 2.06%. Nevertheless, periodontal pathogens were generally detected at lower levels in the peri-implantitis group than in the periodontitis group. Conclusion We found lower bacterial counts in the peri-implantitis group relative to the periodontitis group. Our results suggest that the peri-implant tissue is less resistant to bacteria, so even a small number of bacteria can be a risk factor for peri-implantitis and the causative agent of peri-implantitis can be bacteria other than periodontal pathogen.

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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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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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