scholarly journals Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a fourplex real-time quantitative reverse transcription-PCR assays

2021 ◽  
Author(s):  
Mathieu Durand ◽  
Philippe Thibault ◽  
Simon Lévesque ◽  
Ariane Brault ◽  
Alex Carignan ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Internal Control ◽  
Reverse Transcription ◽  
Selective Advantage ◽  
Locked Nucleic Acid ◽  
Quantitative Detection ◽  
Respiratory Pathogens ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

AbstractThe early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of the coronavirus disease 2019 (COVID-19). In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give SARS-CoV-2 a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human β2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected ≤ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of 9 logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to detect multiple viral target sequences containing typical mutations of SARS-CoV-2 variants in a single reaction, allowing quicker detection of circulating relevant variants.

scholarly journals Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its first variants in fourplex real-time quantitative reverse transcription-PCR assays

2022 ◽  
Vol 9 (1) ◽  
pp. 1-20
Author(s):  
Mathieu Durand ◽  
Philippe Thibault ◽  
Simon Lévesque ◽  
Ariane Brault ◽  
Alex Carignan ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Real Time ◽  
Internal Control ◽  
Reverse Transcription ◽  
Locked Nucleic Acid ◽  
Clinical Samples ◽  
Quantitative Detection ◽  
Respiratory Pathogens ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human β2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected £ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.

scholarly journals Rapid and Quantitative Detection of Hepatitis A Virus from Green Onion and Strawberry Rinses by Use of Real-Time Reverse Transcription-PCR

2005 ◽  
Vol 71 (9) ◽  
pp. 5624-5626 ◽  
Author(s):  
X. C. Shan ◽  
P. Wolffs ◽  
M. W. Griffiths
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Hepatitis A ◽  
Hepatitis A Virus ◽  
Quantitative Detection ◽  
Pcr Assay ◽  
Reverse Transcription Pcr ◽  
Immunomagnetic Capture ◽  
Capture Method

ABSTRACT In this study, an immunomagnetic capture method and a real-time reverse transcription-PCR assay were used to quantify hepatitis A virus (HAV) in green onion and strawberry rinses. This combined protocol detected as low as 0.5 PFU HAV in produce rinses and concentrated HAV levels up to 20-fold.

scholarly journals Implementation of an in-house real-time reverse transcription-PCR assay to detect the emerging SARS-CoV-2 N501Y variants

2021 ◽  
pp. 104868
Author(s):  
Marielle BEDOTTO ◽  
Pierre-Edouard FOURNIER ◽  
Linda HOUHAMDI ◽  
Philippe COLSON ◽  
Didier RAOULT
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

scholarly journals Real-Time Reverse Transcription-PCR Assay for Comprehensive Detection of Human Rhinoviruses

2007 ◽  
Vol 46 (2) ◽  
pp. 533-539 ◽  
Author(s):  
X. Lu ◽  
B. Holloway ◽  
R. K. Dare ◽  
J. Kuypers ◽  
S. Yagi ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

Development of a real-time reverse transcription-PCR assay to detect and quantify group A rotavirus equine-like G3 strains

2021 ◽  
Author(s):  
Eric M. Katz ◽  
Mathew D. Esona ◽  
Rashi Gautam ◽  
Michael D. Bowen
Keyword(s):  
Viral Load ◽  
Real Time ◽  
Reverse Transcription ◽  
Dynamic Range ◽  
Limit Of Detection ◽  
Human Populations ◽  
Pcr Assay ◽  
Group A Rotavirus ◽  
Reverse Transcription Pcr ◽  
Group A

Since 2013, group A rotavirus strains characterized as novel DS-1-like inter-genogroup reassortant ‘equine-like G3’ strains have emerged and spread across five continents among human populations in at least 14 countries. Here we report a novel one-step TaqMan quantitative real-time reverse transcription-PCR assay developed to genotype and quantify the viral load for samples containing rotavirus equine-like G3 strains. Using a universal G forward primer and a newly designed reverse primer and TaqMan probe, we developed and validated an assay with a linear dynamic range of 2.3 × 10 9 – 227 copies per reaction and a limit of detection of 227 copies. The percent positive agreement, percent negative agreement, and precision of our assay were 100.00%, 99.63%, and 100.00%, respectively. This assay can simultaneously detect and quantify the viral load for samples containing DS-1-like inter-genogroup reassortant equine-like G3 strains with high sensitivity and specificity, faster turnaround time, and decreased cost and will be valuable for high-throughput screening of stool samples collected to monitor equine-like G3 strain prevalence and circulation among human populations throughout the world.

scholarly journals Quantitative Reverse Transcription-PCR Assay with an Internal Standard for the Detection of Prostate-specific Antigen mRNA

1999 ◽  
Vol 45 (9) ◽  
pp. 1397-1407 ◽  
Author(s):  
Alice Ylikoski ◽  
Minna Sjöroos ◽  
Åke Lundwall ◽  
Matti Karp ◽  
Timo Lövgren ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Detection Limit ◽  
Prostate Specific Antigen ◽  
Reverse Transcription ◽  
Specific Antigen ◽  
Internal Standard ◽  
Quantitative Detection ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Reverse Transcription Pcr

Abstract Background: Circulating prostate cells can be detected with a reverse transcription-PCR (RT-PCR) assay for prostate-specific antigen (PSA) mRNA. We have developed a new quantitative RT-PCR method for measuring PSA mRNA. Methods: The method uses a PSA-like internal standard (IS) mRNA that is added into the sample at the beginning of the RNA extraction and coamplified by RT-PCR with the PSA in the sample. After PCR amplification, the IS and PSA products are selectively detected by hybridization in a microtitration plate using probes labeled with fluorescent europium chelates. Results: The method was validated with PSA and IS mRNAs and PSA-expressing cells to obtain a detection limit of 50 PSA mRNA copies (i.e., signal 2 times the mean of zero signal), linearity up to 106 copies, and detection of a single PSA-expressing cell. In preliminary evaluations, 60% (n = 10) of the prostate cancer patients with skeletal metastases gave results above the detection limit (500 PSA mRNA copies in 5 mL of blood). The total number of PSA copies ranged from 900 ± 200 to 44 100 ± 4900 (mean ± SD) in the samples, corresponding to ∼1–100 PSA-expressing cells in 5 mL of blood. In the controls (n = 34), none of the healthy females and 2 of 19 healthy males had detectable PSA mRNA [700 ± 100 and 2000 ± 900 (mean ± SD) PSA mRNA copies in 5 mL of blood for the 2 males]. Conclusions: The assay provides sensitive and quantitative detection of PSA mRNA expression from blood samples and can be used to establish the clinically significant number of PSA mRNA copies in prostate cancer.

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