开发一种用于检测来自阿曼的 SARS-COV-2 的比色 RT-LAMP 检测方法。

Development of a colorimetric RT-LAMP assay for the detection of SARS-COV-2 isolated from Oman.

机构信息

Department of Animal and Veterinary Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, Muscat, Oman.

Department of Plant Sciences, College of Agricultural and Marine Sciences, Sultan Qaboos University, Muscat, Oman.

出版信息

J Infect Dev Ctries. 2022 Jun 30;16(6):952-958. doi: 10.3855/jidc.15377.

DOI:10.3855/jidc.15377

PMID:35797288

Abstract

INTRODUCTION

A rapid and sensitive COVID-19 diagnostic test is required to aid in the prevention and control of the current COVID-19 pandemic spread. We developed a colorimetric, rapid, and sensitive RT-LAMP assay for the diagnosis of COVID-19 viral infection.

METHODOLOGY

Complete genome sequences of 41 SARS-CoV-2 isolates from Oman were used in this study. Three primer sets (CoV_S1, CoV_S2, CoV_M1) were developed from all Omani SARS-CoV-2 genome sequences available at the time, targeting the spike protein gene and the M gene. The primer set (CoV_S1) was found to be the most sensitive and specific among the three designed sets. The sensitivity and specificity of the assay were compared to that of qRT-PCR. Direct testing of SARS-CoV-2 spiked saliva with the developed assay was evaluated. Lyophilized colorimetric assays were stored at room temperature and 4 °C and their ability to detect positive samples were tested for a period of 8 weeks.

RESULTS

The RT-LAMP assay was validated by testing 145 COVID-19 clinical samples with a sensitivity of 96.9% and specificity of 94.7% when compared to the validated qRT-PCR assay. The assay specificity was tested against SARS-CoV Frankfurt 1 RNA virus and avian coronaviruses as they tested negative with the developed assay. The assay was lyophilized and managed to detect the positive samples colorimetrically when stored at 4 °C for up to 8 weeks.

CONCLUSIONS

The assay can be utilized in its current form as a screening assay with the advantages of being simpler, quicker, and cheaper than the qRT-PCR.

摘要

简介

为了帮助预防和控制当前 COVID-19 疫情的传播，需要一种快速且灵敏的 COVID-19 诊断测试。我们开发了一种比色、快速且灵敏的 RT-LAMP 检测方法，用于诊断 COVID-19 病毒感染。

方法

本研究使用了来自阿曼的 41 种 SARS-CoV-2 分离株的完整基因组序列。根据当时可用的所有阿曼 SARS-CoV-2 基因组序列，设计了三组引物（CoV_S1、CoV_S2、CoV_M1），靶向刺突蛋白基因和 M 基因。在这三组设计的引物中，CoV_S1 组被发现是最敏感和特异的。比较了该检测方法与 qRT-PCR 的敏感性和特异性。评估了该方法对已开发的检测方法进行直接检测 SARS-CoV-2 加标唾液的效果。冻干比色检测在室温下和 4°C 下储存，并在 8 周的时间内测试其检测阳性样本的能力。

结果

通过对 145 份 COVID-19 临床样本进行检测，该 RT-LAMP 检测方法得到了验证，与已验证的 qRT-PCR 检测方法相比，其灵敏度为 96.9%，特异性为 94.7%。该检测方法的特异性已针对 SARS-CoV Frankfurt 1 RNA 病毒和禽冠状病毒进行了测试，因为它们用已开发的检测方法测试呈阴性。该检测方法经过冻干处理，在 4°C 下储存长达 8 周时，仍能够通过比色法检测阳性样本。