新型冠状病毒一步法可视化恒温快速检测方法的建立

Establishment of a New Method for Rapid Detection of SARS-CoV-2 by Visual LAMP(Without Nucleic Acid Extraction)

新型冠状病毒(SARS-CoV-2)感染导致急性或致命性肺炎,该病毒是继严重急性呼吸系统综合征病毒(SARS-CoV)和中东呼吸系统综合征病毒(MERS-CoV)之后发现的又一由动物传染给人且能够人传人的冠状病毒。为快速确诊感染SARS-CoV-2的人和动物,急需建立一种快速高效的检测方法。本研究根据GenBank公布的SARSCoV-2全基因组序列,利用LAMP Desiner2.0软件筛选高效且特异性强的组合环引物和Bst 4.0 DNA聚合酶的扩增特性,即依赖RNA为模板进行DNA合成,建立一种高效、快速、灵敏检测SARS-CoV-2的方法。结果表明,该方法实现了5~20拷贝的病毒RNA检测目标,并可直接检测灭活病毒。因此,临床样本无需RNA提取,经灭活处理后即可进行样品检测。扩增后的核酸分子与OG(Orange-Green)染料结合,阳性样品为绿色,阴性样品为橙黄色。本研究建立的SARS-CoV-2一步法可视化恒温快速检测方法,实现了核酸快速检测,且具有高灵敏度,为SARS-CoV-2检测提供了新的方法选择。

A new type of coronavirus(SARS-CoV-2)infection caused acute or fatal pneumonia.The virus is another coronavirus that is transmitted from animal to human and capable of transmitted from human to human,following the severe acute respiratory syndrome coronavirus(SARS-CoV)and the Middle East respiratory syndrome coronavirus(MERS-CoV).In order to control the epidemic as soon as possible,there is an urgent need for rapid detection and confirmation of infected patients.In this study,according to the SARS-CoV-2 whole genome published in GenBank as target gene,LAMP Desiner 2.0 software was used to screen efficient and highly specific combinatorial loop primers.The amplification characteristics of Bst 4.0 DNA polymerase relys RNA as template for DNA synthesis.Viral RNA-positive test results showed that 5 to 20 copies of virus nucleic acid could be detected.The inactivated virus was directly used as amplification template for clinical detection.The amplified nucleic acid molecules are combined with OG(Orange-Green)dye.Positive samples are green and negative samples are orange-yellow.The established SARS-CoV-2 one-step visual constant temperature rapid detection method realizes rapid detection of nucleic acids with high sensitivity.This study provides a new method for SARS-CoV-2 detection.