A modified single-tube one-step product-enhanced reverse transcriptase (mSTOS-PERT) assay with heparin as DNA polymerase inhibitor for specific detection of RTase activity

2006 ◽  
Vol 37 (4) ◽  
pp. 305-312 ◽  
Author(s):  
Xiao-Yong Fan ◽  
Guo-Zhen Lü ◽  
Li-Na Wu ◽  
Jing-Hua Chen ◽  
Wen-Qing Xu ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Specific Detection ◽  
Single Tube ◽  
One Step ◽  
Polymerase Inhibitor

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

PLoS ONE ◽  
2021 ◽  
Vol 16 (6) ◽  
pp. e0252789
Author(s):  
Yukiko Nakura ◽  
Heng Ning Wu ◽  
Yuya Okamoto ◽  
Muneyuki Takeuchi ◽  
Koichiro Suzuki ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Reverse Transcriptase ◽  
Real Time ◽  
Dna Polymerase ◽  
Internal Control ◽  
Leukemia Virus ◽  
Rt Pcr ◽  
Thermotoga Petrophila ◽  
One Step ◽  
Thermostable Dna Polymerase

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.

scholarly journals One-step RNA pathogen detection with reverse transcriptase activity of a mutated thermostable Thermus aquaticus DNA polymerase

2010 ◽  
Vol 5 (2) ◽  
pp. 224-231 ◽  
Author(s):  
Ramon Kranaster ◽  
Matthias Drum ◽  
Nicole Engel ◽  
Manfred Weidmann ◽  
Frank T. Hufert ◽  
...  
Keyword(s):  
Reverse Transcriptase ◽  
Dna Polymerase ◽  
Pathogen Detection ◽  
Reverse Transcriptase Activity ◽  
Thermus Aquaticus ◽  
Transcriptase Activity ◽  
One Step

scholarly journals Detection of Viable Listeria monocytogenes with a 5′ Nuclease PCR Assay

1999 ◽  
Vol 65 (5) ◽  
pp. 2122-2127 ◽  
Author(s):  
Dawn-Marie Norton ◽  
Carl A. Batt
Keyword(s):  
Listeria Monocytogenes ◽  
Reverse Transcriptase ◽  
Cell Viability ◽  
Dna Polymerase ◽  
Pcr Assay ◽  
Single Tube ◽  
Fluorogenic Probe ◽  
Accurate Indicator ◽  
Nuclease Assay

ABSTRACT A 5′ nuclease assay has been developed to detect viableListeria monocytogenes. The assay targets the hemolysin A (hlyA) transcript, which is found only in L. monocytogenes. The single-tube, reverse transcriptase (RT), fluorogenic probe-based assay was formatted by using TthDNA polymerase whose activity was modulated by using the manganese-chelating morpholinepropanesulfonic acid (MOPS) buffer. This assay was quantitative over a 3-log-unit range of template concentrations when tested with an in vitro-transcribedhlyA mRNA. The viability of L. monocytogeneswas reduced by heating at various temperatures and times up to a maximum of a 9-D inactivation. The location of the primer had a pronounced effect on the utility of the assay, and primers located in the most distal regions of the hlyA transcript appeared to correlate with the number of CFU while primers located more internal on the amplicon overestimated the cell viability. The assay with primers that included the 3′ end of the transcript was an accurate indicator of viability as measured by CFU determination or staining with 5-sulfofluorescein diacetate.

One-step icELISA developed with novel antibody for rapid and specific detection of diclazuril residue in animal-origin foods

2020 ◽  
Vol 37 (10) ◽  
pp. 1633-1639
Author(s):  
Yanfang Zhang ◽  
Shufang Li ◽  
Tao Peng ◽  
Pimiao Zheng ◽  
Zile Wang ◽  
...  
Keyword(s):  
Animal Origin ◽  
Specific Detection ◽  
One Step ◽  
Animal Origin Foods
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