Development of an efficient one-step real-time reverse transcription polymerase chain reaction method for severe acute respiratory syndrome-coronavirus-2 detection

The general methods to detect the RNA of severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) in clinical diagnostic testing involve reverse transcriptases and thermostable DNA polymerases. In this study, we compared the detection of SARS-CoV-2 by a one-step real-time RT-PCR method using a heat-resistant reverse transcriptase variant MM4 from Moloney murine leukemia virus, two thermostable DNA polymerase variants with reverse transcriptase activity from Thermotoga petrophila K4 and Thermococcus kodakarensis KOD1, or a wild-type DNA polymerase from Thermus thermophilus M1. The highest performance was achieved by combining MM4 with the thermostable DNA polymerase from T. thermophilus M1. These enzymes efficiently amplified specific RNA using uracil-DNA glycosylase (UNG) to remove contamination and human RNase P RNA amplification as an internal control. The standard curve was obtained from 5 to 105 copies of synthetic RNA. The one-step real-time RT-PCR method’s sensitivity and specificity were 99.44% and 100%, respectively (n = 213), compared to those of a commercially available diagnostic kit. Therefore, our method will be useful for the accurate detection and quantification of SARS-CoV-2.

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Escherichia coli DNA polymerase III ε subunit increases Moloney murine leukemia virus reverse transcriptase fidelity and accuracy of RT–PCR procedures

Analytical Biochemistry ◽

10.1016/j.ab.2006.10.009 ◽

2007 ◽

Vol 360 (1) ◽

pp. 84-91 ◽

Cited By ~ 34

Author(s):

Bahram Arezi ◽

Holly H. Hogrefe

Keyword(s):

Escherichia Coli ◽

Reverse Transcriptase ◽

Dna Polymerase ◽

Murine Leukemia Virus ◽

Leukemia Virus ◽

Moloney Murine Leukemia Virus ◽

Dna Polymerase Iii ◽

Rt Pcr ◽

Polymerase Iii ◽

Murine Leukemia

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High sensitive RNA detection by one-step RT-PCR using the genetically engineered variant of DNA polymerase with reverse transcriptase activity from hyperthermophilies

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2017.10.004 ◽

2018 ◽

Vol 125 (3) ◽

pp. 275-281 ◽

Cited By ~ 5

Author(s):

Hiroyuki Okano ◽

Misato Baba ◽

Katsuhiro Kawato ◽

Ryota Hidese ◽

Itaru Yanagihara ◽

...

Keyword(s):

Reverse Transcriptase ◽

Dna Polymerase ◽

Reverse Transcriptase Activity ◽

Genetically Engineered ◽

Rt Pcr ◽

Rna Detection ◽

Transcriptase Activity ◽

One Step

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Double-Quencher Probes Improved the Detection Sensitivity of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) by One-Step RT-PCR

10.1101/2020.03.17.20037903 ◽

2020 ◽

Cited By ~ 6

Author(s):

Yosuke Hirotsu ◽

Hitoshi Mochizuki ◽

Masao Omata

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Real Time ◽

Clinical Laboratory ◽

Human Life ◽

Positive Control ◽

Detection Sensitivity ◽

Rt Pcr ◽

Fluorescent Signal ◽

The Usa ◽

One Step

AbstractBackgroundSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerges in Wuhan City, Hubei Province, spreads worldwide, and threats the human life. The detection of SARS-CoV-2 is important for the prevention of the outbreak and management of patients. Real-time reverse-transcription polymerase chain reaction (RT-PCR) assay detected the virus in clinical laboratory.MethodsThis study utilized primers and single-quencher probes in accordance with the Centers for Disease Control and Prevention (CDC) in the USA and the National Institute of Infectious Diseases (NIID) in Japan. Moreover, we designed the double-quencher probes (YCH assay) according to the oligonucleotide sequence established by NIID. Using these assays, we conducted a one-step real-time RT-PCR with serial DNA positive control to assess the detection sensitivity.ResultsThe threshold cycle (Ct) value of RT-PCR was relatively low in CDC and YCH assays compared to NIID assay. Serial dilution assay showed that both CDC and YCH assays could detect a low-copy number of DNA positive control. The background fluorescent signal at the baseline was lower in YCH than that of NIID.ConclusionDouble-quencher probes decreased background fluorescent signal and improved detection sensitivity of SARS-CoV-2.

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Development and Validation of a Quantitative, One-Step, Multiplex, Real-Time Reverse Transcriptase PCR Assay for Detection of Dengue and Chikungunya Viruses

Journal of Clinical Microbiology ◽

10.1128/jcm.00299-16 ◽

2016 ◽

Vol 54 (7) ◽

pp. 1766-1773 ◽

Cited By ~ 20

Author(s):

Monika Simmons ◽

Todd Myers ◽

Carolina Guevara ◽

Donald Jungkind ◽

Maya Williams ◽

...

Keyword(s):

Reverse Transcriptase ◽

Sensitivity And Specificity ◽

Internal Control ◽

Chikungunya Virus ◽

Clinical Samples ◽

Rt Pcr ◽

Pcr Assay ◽

Denv Serotypes ◽

Reverse Transcriptase Pcr ◽

One Step

Dengue virus (DENV) and chikungunya virus (CHIKV) are important human pathogens with common transmission vectors and similar clinical presentations. Patient care may be impacted by the misdiagnosis of DENV and CHIKV in areas where both viruses cocirculate. In this study, we have developed and validated a one-step multiplex reverse transcriptase PCR (RT-PCR) to simultaneously detect, quantify, and differentiate between four DENV serotypes (pan-DENV) and chikungunya virus. The assay uses TaqMan technology, employing two forward primers, three reverse primers, and four fluorophore-labeled probes in a single-reaction format. Coextracted and coamplified RNA was used as an internal control (IC), andin vitro-transcribed DENV and CHIKV RNAs were used to generate standard curves for absolute quantification. The diagnostic 95% limits of detection (LOD) within the linear range were 50 and 60 RNA copies/reaction for DENV (serotypes 1 to 4) and CHIKV, respectively. Our assay was able to detect 53 different strains of DENV, representing four serotypes, and six strains of CHIKV. No cross-reactivity was observed with related flaviviruses and alphaviruses, To evaluate diagnostic sensitivity and specificity, 89 clinical samples positive or negative for DENV (serotypes 1 to 4) and CHIKV by the standard virus isolation method were tested in our assay. The multiplex RT-PCR assay showed 95% sensitivity and 100% specificity for DENV and 100% sensitivity and specificity for CHIKV. With an assay turnaround time of less than 2 h, including extraction of RNA, the multiplex quantitative RT-PCR assay provides rapid diagnosis for the differential detection of two clinically indistinguishable diseases, whose geographical occurrence is increasingly overlapping.

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