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Rapid Detection of SARS-CoV-2 Virus Using Dual Reverse Transcriptional Colorimetric Loop-Mediated Isothermal Amplification.

ABSTRACT: The outbreak and pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a public health emergency of international concern. The rapid and accurate detection of the virus is a critical means to prevent and control the disease. Herein, we provide a novel, rapid, and simple approach, named dual reverse transcriptional colorimetric loop-mediated isothermal amplification (dRT-cLAMP) assay, to accelerate the detection of the SARS-CoV-2 virus without using expensive equipment. The result of this assay is shown by color change and is easily detected by the naked eye. To improve the detection accuracy, we included two primer sets that specifically target the viral orf1ab and N genes in the same reaction mixture. Our assay can detect the synthesized SARS-CoV-2 N and orf1ab genes at a low level of 100 copies/μL. Sequence alignment analysis of the two synthesized genes and those of 9968 published SARS-CoV-2 genomes and 17 genomes of other pathogens from the same infection site or similar symptoms as COVID-19 revealed that the primers for the dRT-cLAMP assay are highly specific. Our assay of 27 clinical samples of SARS-CoV-2 virus and 27 standard-added environmental simulation samples demonstrated that compared to the commercial kits, the consistency of the positive, negative, and probable clinical samples was 100, 92.31, and 44.44%, respectively. Moreover, our results showed that the positive, but not negative, standard-added samples displayed a naked-eye-detectable color change. Together, our results demonstrate that the dRT-cLAMP assay is a feasible detection assay for SARS-CoV-2 virus and is of great significance since rapid onsite detection of the virus is urgently needed at the ports of entry, health care centers, and for internationally traded goods.

SUBMITTER: Ji C

PROVIDER: S-EPMC8008787 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a major threat to public health. Rapid molecular testing for convenient and timely diagnosis of SARS-CoV-2 infections represents a challenge that could help to control the current pandemic and prevent future outbreaks. We aimed to develop and validate a multiplex and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using lyophilized LAMP reagents for sensitive and rapid detection of SARS-CoV-2. LAMP primers were designed for a set of gene targets identified by a genome-wide comparison of viruses. Primer sets that showed optimal features were combined into a multiplex RT-LAMP assay. Analytical validation included assessment of the limit of detection (LoD), intra- and inter-assay precision, and cross-reaction with other respiratory pathogens. Clinical performance compared to that of real-time reverse transcriptase-polymerase chain reaction (RT-qPCR) was assessed using 278 clinical RNA samples isolated from swabs collected from individuals tested for COVID-19. The RT-LAMP assay targeting the RNA-dependent RNA polymerase (RdRp), membrane (M), and ORF1ab genes achieved a comparable LoD (0.65 PFU/mL, CT=34.12) to RT-qPCR and was 10-fold more sensitive than RT-qPCR at detecting viral RNA in clinical samples. Cross-reactivity to other respiratory pathogens was not observed. The multiplex RT-LAMP assay demonstrated a strong robustness and acceptable intra- and inter-assay precision (mean coefficient of variation, 4.75% and 8.30%). Diagnostic sensitivity and specificity values were 100.0% (95% CI: 97.4-100.0%) and 98.6% (95% CI: 94.9-99.8%), respectively, showing high consistency (Cohen's kappa, 0.986; 95% CI: 0.966-1.000; p<0.0001) compared to RT-qPCR. The novel one-step multiplex RT-LAMP assay is storable at room temperature and showed similar diagnostic accuracy to conventional RT-qPCR, while being faster (<45 min), simpler, and cheaper. The new assay could allow reliable and early diagnosis of SARS-CoV-2 infections in primary health care. It may aid large-scale testing in resource-limited settings, especially if it is integrated into a point-of-care diagnostic device.

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Rapid Detection of SARS-CoV-2 Virus Using Dual Reverse Transcriptional Colorimetric Loop-Mediated Isothermal Amplification.

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