Rapid Detection of SARS-CoV-2 Using Duplex Reverse Transcription-Multienzyme Isothermal Rapid Amplification in a Point-of-Care Testing

Hui Chen; Chang Sun; Yang Wang; Xiaojiao Gao; Jinwei You; Wanwan Yu; Ning Sun; Yang Yang; Xiaojun Li

doi:10.3389/fcimb.2021.678703

Rapid Detection of SARS-CoV-2 Using Duplex Reverse Transcription-Multienzyme Isothermal Rapid Amplification in a Point-of-Care Testing

Front Cell Infect Microbiol. 2021 Oct 22:11:678703. doi: 10.3389/fcimb.2021.678703. eCollection 2021.

Authors

Hui Chen^{1

2}, Chang Sun³, Yang Wang³, Xiaojiao Gao⁴, Jinwei You⁵, Wanwan Yu⁶, Ning Sun², Yang Yang², Xiaojun Li^{2

7}

Affiliations

¹ Department of Medicine, JiangSu University, Zhenjiang, China.
² Department of Basic Medical Laboratory, Institute of Clinical Laboratory Science, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing, China.
³ Department of Orthopaedics, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing, China.
⁴ Department of Clinical Laboratory, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing, China.
⁵ Department of Laboratory Animal, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing, China.
⁶ Department of Emergency, Jinling Hospital, Clinical School of Medical College, Nanjing University, Nanjing, China.
⁷ State Key Laboratory of Analytical Chemistry for Life Science, Department of Chemistry, Nanjing University, Nanjing, China.

Abstract

In December 2019, a severe acute respiratory syndrome caused by SARS-CoV-2 spread rapidly worldwide. Portable nucleic acid tests of SARS-CoV-2 are critically important for diagnostics. In this study, we used an isothermal amplification method-Multienzyme Isothermal Rapid Amplification (MIRA)-for rapid detection of SARS-CoV-2. We designed the primers and probes in ORF1ab and N gene of SARS-CoV-2. The amplicons could be monitored by lateral flow dipsticks (LFDs). The reaction temperature, time, concentrations of primers and probes, and working volume were optimized. Four commercial swab collection buffers were used to test the amplification efficacy of our assay without RNA extraction. Our assay was able to amplify duplex targets of SARS-CoV-2 in one single reaction using one-step RT-MIRA. The assay worked well in a low volume of 10 μl at 38°C for 20 min. Using three collection buffers without guanidinium, our assay was able to amplify efficaciously without RNA extraction. The 95% limit of detection (LoD) of the RT-MIRA assay was 49.5 (95% CI, 46.8-52.7) copies/ml for ORF1ab gene and 48.8 (95% CI, 46.5-52.6) copies/ml for N gene. There is no cross-reaction with other human respiratory pathogens, such as SARS-CoV, MERS-CoV, influenza A virus, influenza B virus, human adenovirus, respiratory syncytial virus, human parainfluenza virus, and coronavirus 229E in our assay. The precision evaluation revealed that the C50-20% to C50+20% range bounds the C5-C95 interval. This assay also showed high anti-interference ability. The extraction-free RT-MIRA and qPCR detection results of 243 nucleic acid specimens from suspected patients or national references showed a 100.0% (95% confidence interval, 94.2%-100.0%) positive predictive value and a 100.0% (95% confidence interval, 92.7%-100.0%) negative predictive value. Compared with qPCR, the kappa value of the two assays was 1.00 (P < 0.0001). In conclusion, we provide a portable and visualized method for detection of SARS-CoV-2 without RNA extraction, allowing its application in SARS-CoV-2 on-site detection.

Keywords: SARS-CoV-2; a low volume; duplex multienzyme isothermal rapid amplification; lateral flow dipstick; nucleic acid tests; point-of-care testing; without RNA extraction.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

COVID-19*
Humans
Nucleic Acid Amplification Techniques
Point-of-Care Testing
RNA, Viral / genetics
Reverse Transcription
SARS-CoV-2*

Substances

RNA, Viral