scholarly journals Effect of Heat inactivation on Real-Time Reverse Transcription PCR of the SARS-COV-2 Detection

2020 ◽  
Author(s):  
yanxia liu ◽  
zhengan cao ◽  
mei chen ◽  
Yan zhong ◽  
yuhao luo ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Reverse Transcription ◽  
Statistical Difference ◽  
Rna Extraction ◽  
Heat Inactivation ◽  
Nasopharyngeal Swab ◽  
Reverse Transcription Pcr ◽  
Nucleic Acid Isolation ◽  
Heat Treated

Background: Real-time reverse transcription PCR (rRT-PCR) is commonly used to diagnose SARS-CoV-2 infection. Heat inactivation prior to nucleic acid isolation may allow safe testing, while the effects of heat inactivation on SARS-CoV-2 rRT-PCR detection result need to be determined. Methods: 14 positive nasopharyngeal swab specimens were inactivated at 56°C for 30min, 56°C for 60min, 60°C for 30min, 60°C for 75min, and 100°C for 10min, and another 2 positive nasopharyngeal swab specimens were also inactivated at 100°C for 10min, 100°C for 30min, 100°C for 60min, after which the samples were isolated and detected by rRT-PCR. Results: All 14 heat treated samples remained positive. The range of threshold cycle (Ct) values observed when detecting ORF1a/b was 27.228-34.011 in heat-treated samples, while 25.281-34.861 in unheated samples, and the range of threshold cycle (Ct) values observed at the time of detecting N was 25.777-33.351 in heat-treated samples, while 24.1615-35.433 in unheated samples, on basis of which it showed no statistical difference otherwise a good correlation of Ct values between the heat-inactivated samples and the untreated samples. However, the 2 samples inactivated at 100°C 30min, 100°C 60min turned into negative. Conclusions: Heat inactivation at 56°C for 30min, 56°C for 60min, 60°C for 30min, 60°C for 75min, and 100°C for 10min shall not affect the detection results of Real-Time Reverse Transcription PCR of the SARS-COV2. Furthermore, it is recommended to inactive nasopharyngeal swab specimens 10min at 100°C before RNA extraction in consideration of efficiency and reliable results. Key Words: SARS-CoV-2, Heat Inactivation, rRT-PCR, Comparison

scholarly journals Extraction-free protocol combining proteinase K and heat inactivation for detection of SARS-CoV-2 by RT-qPCR

PLoS ONE ◽  
2021 ◽  
Vol 16 (2) ◽  
pp. e0247792
Author(s):  
Valeria Genoud ◽  
Martin Stortz ◽  
Ariel Waisman ◽  
Bruno G. Berardino ◽  
Paula Verneri ◽  
...  
Keyword(s):  
Rna Extraction ◽  
Standard Technique ◽  
Proteinase K ◽  
Clinical Samples ◽  
Heat Inactivation ◽  
Nasopharyngeal Swab ◽  
Reverse Transcription Pcr ◽  
Extraction Step ◽  
Commercial Kits ◽  
Inexpensive Procedure

Real-time reverse transcription PCR (RT-qPCR) is the gold-standard technique for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection in nasopharyngeal swabs specimens. The analysis by RT-qPCR usually requires a previous extraction step to obtain the purified viral RNA. Unfortunately, RNA extraction constitutes a bottleneck for early detection in many countries since it is expensive, time-consuming and depends on the availability of commercial kits. Here, we describe an extraction-free protocol for SARS-CoV-2 detection by RT-qPCR from nasopharyngeal swab clinical samples in saline solution. The method includes a treatment with proteinase K followed by heat inactivation (PK+HID method). We demonstrate that PK+HID improves the RT-qPCR performance in comparison to the heat-inactivation procedure. Moreover, we show that this extraction-free protocol can be combined with a variety of multiplexing RT-qPCR kits. The method combined with a multiplexing detection kit targeting N and ORF1ab viral genes showed a sensitivity of 0.99 and a specificity of 0.99 from the analysis of 106 positive and 106 negative clinical samples. In conclusion, PK+HID is a robust, fast and inexpensive procedure for extraction-free RT-qPCR determinations of SARS-CoV-2. The National Administration of Drugs, Foods and Medical Devices of Argentina has recently authorized the use of this method.

scholarly journals Direct Saliva versus Conventional Nasopharyngeal Swab qRT-PCR to Diagnose SARS – CoV2: Validity Study

2021 ◽  
pp. 37-46
Author(s):  
Michael L. Tee ◽  
Paulyn Jean R. Ubial ◽  
Diana Rose E. Ranoa ◽  
Cherica A. Tee ◽  
Aedrian A. Abrilla ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Rna Extraction ◽  
Nasopharyngeal Swab ◽  
Diagnostic Validity ◽  
Real Time Quantitative Pcr ◽  
Qrt Pcr ◽  
Paired Samples ◽  
Feasible Alternative ◽  
Novel Coronavirus

Background: Saliva has been demonstrated as a feasible alternative specimen to nasopharyngeal swab for the detection of SARS-CoV-2 using real-time or quantitative reverse transcription polymerase chain reaction (qRT-PCR) method that bypasses the need for explicit viral ribonucleic acid (RNA) extraction. Aim: To assess the diagnostic validity of direct saliva-to-qRT-PCR in the detection of SARS-CoV-2 compared to conventional nasopharyngeal swab qRT-PCR. Methodology: Self-collected saliva samples were processed by heating at 95oC for 30 minutes followed by addition of buffer and detergent while viral RNA from nasopharyngeal swabs were extracted using the Sansure Biotech sample release reagent.  Paired samples were used as templates for qRT-PCR using the Sansure Novel Coronavirus (COVID-19) Nucleic Acid Diagnostic Kit and Sansure Biotech MA6000 Real-Time Quantitative PCR System. Direct saliva-to-qRT-PCR was compared to nasopharyngeal swab qRT-PCR in terms of diagnostic validity and agreement parameters, and both platforms were compared separately in terms of similar parameters with a composite reference standard (CRS) wherein the criteria for a positive result is SARS-CoV-2 detection in at least either nasopharyngeal swab or saliva. Results:  Of the 238 nasopharyngeal swab-saliva pairs tested, 20 (8.4%) nasopharyngeal swab and 24 (10.1%) saliva specimens tested positive. We documented a sensitivity of 85.0% (95% CI: 62.1%, 96.8%), specificity of 96.8% (95% CI: 93.5%, 98.7%), accuracy of 95.8% (95% CI: 92.4%, 98.0%) and Cohen Kappa of 0.75 (95% CI: 0.60, 0.90) when direct saliva-to-qRT-PCR was compared to the conventional platform. When the two platforms were individually compared to the CRS, numerically higher but not statistically significant sensitivity and accuracy were noted for direct saliva-to-qRT-PCR than for nasopharyngeal swab qRT-PCR. Conclusion: Direct saliva-to-qRT-PCR is non-inferior to nasopharyngeal swab qRT-PCR for detecting SARS-CoV-2 using the Sansure Novel Coronavirus Nucleic Acid Diagnostic Kit.

scholarly journals Development and Evaluation of Novel Real-Time Reverse Transcription-PCR Assays with Locked Nucleic Acid Probes Targeting Leader Sequences of Human-Pathogenic Coronaviruses

2015 ◽  
Vol 53 (8) ◽  
pp. 2722-2726 ◽  
Author(s):  
Jasper Fuk-Woo Chan ◽  
Garnet Kwan-Yue Choi ◽  
Alan Ka-Lun Tsang ◽  
Kah-Meng Tee ◽  
Ho-Yin Lam ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Real Time ◽  
Reverse Transcription ◽  
Locked Nucleic Acid ◽  
Small Rna Sequencing ◽  
Nucleic Acid Probes ◽  
Reverse Transcription Pcr ◽  
Highly Sensitive ◽  
Pcr Assays ◽  
Human Coronaviruses

Based on findings in small RNA-sequencing (Seq) data analysis, we developed highly sensitive and specific real-time reverse transcription (RT)-PCR assays with locked nucleic acid probes targeting the abundantly expressed leader sequences of Middle East respiratory syndrome coronavirus (MERS-CoV) and other human coronaviruses. Analytical and clinical evaluations showed their noninferiority to a commercial multiplex PCR test for the detection of these coronaviruses.

scholarly journals Detection of Murine Norovirus 1 by Using Plaque Assay, Transfection Assay, and Real-Time Reverse Transcription-PCR before and after Heat Exposure

2007 ◽  
Vol 74 (2) ◽  
pp. 543-546 ◽  
Author(s):  
Leen Baert ◽  
Christiane E. Wobus ◽  
Els Van Coillie ◽  
Larissa B. Thackray ◽  
Johan Debevere ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Detrimental Effect ◽  
Heat Exposure ◽  
Plaque Assay ◽  
Heat Inactivation ◽  
Virus Infectivity ◽  
Murine Norovirus ◽  
Reverse Transcription Pcr ◽  
Before And After

ABSTRACT The correlation between the detection of murine norovirus 1 RNA by real-time reverse transcription-PCR and the infectivity by plaque assay before and after heat exposure (80°C) was examined. No correlation was found in the current study. Moreover, heat inactivation had a much stronger detrimental effect on virus infectivity than on the integrity of the viral genome.

scholarly journals Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257563
Author(s):  
Les Jones ◽  
Abhijeet Bakre ◽  
Hemant Naikare ◽  
Ravindra Kolhe ◽  
Susan Sanchez ◽  
...  
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Fluorescent Detection ◽  
Nasopharyngeal Swab ◽  
Live Virus ◽  
Reverse Transcription Pcr ◽  
Variant Virus ◽  
Alpha Variant

The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity.

scholarly journals Genogroup I and II Noroviruses Detected in Stool Samples by Real-Time Reverse Transcription-PCR Using Highly Degenerate Universal Primers

2004 ◽  
Vol 70 (12) ◽  
pp. 7179-7184 ◽  
Author(s):  
Gary P. Richards ◽  
Michael A. Watson ◽  
Rebecca L. Fankhauser ◽  
Stephan S. Monroe
Keyword(s):  
Real Time ◽  
Reverse Transcription ◽  
Rna Extraction ◽  
Universal Primers ◽  
Rt Pcr ◽  
Degenerate Primers ◽  
Reverse Transcription Pcr ◽  
Wide Range ◽  
Genetic Clusters ◽  
Stool Samples

ABSTRACT Genogroup I noroviruses from five genetic clusters and genogroup II noroviruses from eight genetic clusters were detected in stool extracts using degenerate primers and single-tube, real-time reverse transcription-PCR (RT-PCR) with SYBR Green detection. Two degenerate primer sets, designated MON 431-433 and MON 432-434, were designed from consensus sequences from the major clusters of norovirus based on the RNA-dependent RNA polymerase region of the norovirus genome. Viruses were extracted from stool samples within 20 min using a viral RNA extraction kit. Real-time RT-PCR for noroviruses generated semiquantitative results by means of the cycle threshold data and dilution endpoint standard curves. Presumptive product verification was achieved by evaluation of first-derivative melt graphs. Multiple clusters of noroviruses were identified simultaneously in a multiplex fashion by virtue of slight differences in melting temperature. The detection of 13 different genetic clusters suggests that the MON primers may serve as universal primers for most, if not all, of the noroviruses in a multiplex assay. Our technique provides a framework for broad application of real-time RT-PCR in clinical, environmental, and food testing laboratories for a wide range of noroviruses.

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