Rapid Detection of SARS-CoV-2 by Low Volume Real-Time Single Tube Reverse Transcription Recombinase Polymerase Amplification Using an Exo Probe with an Internally Linked Quencher (Exo-IQ)
Rapid Detection of SARS-CoV-2 by Low Volume Real-Time Single Tube Reverse Transcription Recombinase Polymerase Amplification Using an Exo Probe with an Internally Linked Quencher (Exo-IQ)
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DOI：
10.1093/clinchem/hvaa116
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发表时间：
2020-08-01
影响因子：
9.3
通讯作者：
Hufert, Frank T.
中科院分区：
文献类型：
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作者：
Behrmann, Ole;Bachmann, Iris;Hufert, Frank T.
背景技术背景：目前的SARS-CoV-2疫情已蔓延到几乎每个国家，截至2020年5月26日，确诊病例超过500万例，死亡人数超过30万。快速的第一线测试协议需要爆发控制和surveillance.METHODS：我们使用计算机和手动设计，以产生一套合适的逆转录重组酶聚合酶扩增（RT-RPA）引物和核酸外切酶探针，内部淬灭（exo-IQ），序列靶向SARS冠状病毒-2 N基因。通过扩增体外转录的RNA标准品测定RT-RPA敏感性。使用来自常见呼吸道病毒的32份核酸样本的选择性样本组证明了试验选择性。为了验证针对全长SARS-CoV-2 RNA的测定，筛选了来自细胞培养上清液和19个鼻咽拭子样品（8个SARS-CoV-2阳性和11个阴性）的总病毒RNA。结果：RT-RPA检测的95%检测概率确定为7.74（95%CI：2.87-27.39）RNA拷贝/反应。该检测试剂盒与任何其他筛选的具有临床意义的冠状病毒或呼吸道病毒均无交叉反应性。当与RT-qPCR（n = 20）相比时，开发的RT-RPA测定产生100%的诊断灵敏度和特异性。运行时间为15至20分钟，对于高RNA浓度，在7分钟内可获得第一个结果，报道的检测方法构成了迄今为止最快的基于核酸的SARS-CoV-2检测方法之一，并可能提供一种简单易用的RT替代方法。qPCR用于需要时的一线筛查。
BACKGROUND: The current outbreak of SARS-CoV-2 has spread to almost every country with more than 5 million confirmed cases and over 300,000 deaths as of May 26, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance.METHODS: We used computational and manual designs to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ), sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated with a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays.RESULTS: The 95% detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87-27.39) RNA copies per reaction. The assay showed no cross-reactivity to any other screened coronaviruses or respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n = 20).CONCLUSIONS: With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple-to-use alternative to RT-qPCR for first-line screening at the point of need.