A Mismatch-Tolerant RT-Quantitative PCR: Application to Broad-Spectrum Detection of Respiratory Syncytial Virus

Li, Yingxue; Wan, Zhenzhou; Hu, Yihong; Zhou, Yi; Chen, Qin; Zhang, Chiyu

doi:10.2144/btn-2018-0184

Cited by 16 publications

(17 citation statements)

References 35 publications

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“…The reason for two samples testing positive by the RT-qPCR assay but negative by the RT-LAMP assay may be the very low copy numbers (having high CT values of >38 in the RT-qPCR assay). However, regarding the other two samples tested positive by the RT-LAMP assay (with a high time threshold of >45 min) but negative by the RT-qPCR assay, the reason is speculated to be the presence of viral variants, that have caused mismatches with the primers and/or probe [11,14].…”

Section: Discussionmentioning

confidence: 99%

“…Real-time PCR (qPCR) is the most robust and widely used technology for the qualitative and quantitative diagnosis of viruses, including coronaviruses [8][9][10][11]. Since the outbreak of COVID-19, 2 of 10 many real-time qPCR (RT-qPCR) assays had been developed, and have played an essential role in laboratory confirmation of SARS-CoV-2 infection [3,9,12].…”

Section: Introductionmentioning

confidence: 99%

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A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

Wan

et al. 2020

IJMS

Self Cite

209

249

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COVID-19 has become a major global public health burden, currently causing a rapidly growing number of infections and significant morbidity and mortality around the world. Early detection with fast and sensitive assays and timely intervention are crucial for interrupting the spread of the COVID-19 virus (SARS-CoV-2). Using a mismatch-tolerant amplification technique, we developed a simple, rapid, sensitive and visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for SARS-CoV-2 detection based on its N gene. The assay has a high specificity and sensitivity, and robust reproducibility, and its results can be monitored using a real-time PCR machine or visualized via colorimetric change from red to yellow. The limit of detection (LOD) of the assay is 118.6 copies of SARS-CoV-2 RNA per 25 µL reaction. The reaction can be completed within 30 min for real-time fluorescence monitoring, or 40 min for visual detection when the template input is more than 200 copies per 25 µL reaction. To evaluate the viability of the assay, a comparison between the RT-LAMP and a commercial RT-qPCR assay was made using 56 clinical samples. The SARS-CoV-2 RT-LAMP assay showed perfect agreement in detection with the RT-qPCR assay. The newly-developed SARS-CoV-2 RT-LAMP assay is a simple and rapid method for COVID-19 surveillance.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

Wan

et al. 2020

IJMS

Self Cite

209

249

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“…The inconsistency and FNR of RT-qPCR can be attributed to many different factors, including the variation that occurs in viral RNA sequences, which subsequently affects results that use primers in the ORF1a/b gene and N genes. The influence of variation in viral RNA sequences can be minimized by the mismatch-tolerant amplification methods (Li et al, 2019;Zhou et al, 2019) which would be very helpful for improving the sensitivity and reliability of RNA detection. Another factor that thwarts the accuracy and consistency of RT-qPCR tests is sampling procedures, since the viral loads varies in different anatomic sites.…”

Section: Rt-qpcr Dpcr Lamp and Crisprmentioning

confidence: 99%

Diagnostic methods and potential portable biosensors for coronavirus disease 2019

Cui

Zhou

2020

Biosensors and Bioelectronics

317

251

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Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre-including this research content-immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

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“…Since the COVID-19 outbreak [12], many real-time qPCR (RT-qPCR) detection reagents have been developed and used in the laboratory tests for the detection of SARS-CoV-2 [13]. However, RT-qPCR requires professional personnel, sophisticated experimental equipment, and a duration of 1.5 h [14][15][16].…”

Section: Introductionmentioning

confidence: 99%

Establishment and Application of Reverse Transcription Recombinase-Aided Amplification as A Rapid Detection Method for SARS-COV-2

Ai¹,

Liu

Ye³

et al. 2020

Preprint

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Background: By the end of August 2020, >23 million cases and 800,000 deaths were attributed to SARS-CoV-2 in >200 countries. The improvement of simple, rapid, and efficient detection methods is of great significance for the early detection, timely isolation, and protection of susceptible populations. This study aimed to provide an alternative method for the rapid detection of viral nucleic acid.Methods: This study provided a rapid nucleic acid detection method mediated by recombinant enzyme based on the novel coronavirus (SARS-CoV-2). Primers and probes were designed based on the N gene sequence of coronavirus. The method was performed at 39 °C, the detection time was short (<20 min), and the detection limit was up to 101 copies/mL.Results: The primer-probe did not show any cross-reaction with adenovirus, Zika virus, influenza B virus, and chikungunya virus, with good specificity. A total of 106 clinical throat swab samples were compared by reverse transcription recombinase-aided amplification (RT-RAA) and commercial reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR); the results were identical.Conclusions: The novel coronavirus RT-RAA method established in this study had high sensitivity, strong specificity, simple operation, and fast detection speed, and hence, is suitable for the rapid detection of novel coronavirus under the current epidemic situation.

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A Mismatch-Tolerant RT-Quantitative PCR: Application to Broad-Spectrum Detection of Respiratory Syncytial Virus

Cited by 16 publications

References 35 publications

A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

A Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

Diagnostic methods and potential portable biosensors for coronavirus disease 2019

Establishment and Application of Reverse Transcription Recombinase-Aided Amplification as A Rapid Detection Method for SARS-COV-2

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