由于SARS-CoV-2是一种高度传染性的病毒,替代可靠,快,仍然需要具有成本效益的方法来防止病毒传播,这些方法可以在实验室和即时检测中应用。逆转录实时荧光定量PCR(RT-qPCR)是目前检测RNA病毒的金标准,这需要逆转录酶将病毒RNA逆转录成cDNA,随后进行荧光定量PCR检测。经常使用的逆转录酶是不耐热的;检测过程由两个步骤组成:在相对较低的温度下进行逆转录反应,qPCR在相对较高的温度下进行,此外,如果要检测的RNA具有高级结构,则需要对其进行预处理。这里,我们基于超快速RTX-PCR和热球菌Argonaute介导的核酸检测(PAND)技术(URPAND),开发了快速,灵敏的单管SARS-CoV-2检测平台。URPAND是基于具有逆转录酶和DNA聚合酶活性的热稳定RTX(exo-)实现的超快速RTX-PCR过程。URPAND可以在30分钟内完成RT-PCR和PAND以检测一个管中的核酸。该方法可特异性检测SARS-CoV-2,检测限低至100拷贝/mL。单管URPAND临床样品的诊断结果与RT-qPCR测试显示100%一致。此外,由于其单核苷酸特异性,URPAND还用于鉴定SARS-CoV-2D614G突变体。URPAND平台是快速的,准确,管关闭,一管,易于操作,无大型仪器,这为SARS-CoV-2等RNA病毒的检测提供了新的策略。
Since SARS-CoV-2 is a highly transmissible virus, alternative reliable, fast, and cost-effective methods are still needed to prevent virus spread that can be applied in the laboratory and for point-of-care testing. Reverse transcription real-time fluorescence quantitative PCR (RT-qPCR) is currently the gold criteria for detecting RNA viruses, which requires reverse transcriptase to reverse transcribe viral RNA into cDNA, and fluorescence quantitative PCR detection was subsequently performed. The frequently used reverse transcriptase is thermolabile; the detection process is composed of two steps: the reverse transcription reaction at a relatively low temperature, and the qPCR performed at a relatively high temperature, moreover, the RNA to be detected needs to pretreated if they had advanced structure. Here, we develop a fast and sensitive one-tube SARS-CoV-2 detection platform based on Ultra-fast RTX-PCR and Pyrococcus furiosus Argonaute-mediated Nucleic acid Detection (PAND) technology (URPAND). URPAND was achieved ultra-fast RTX-PCR process based on a thermostable RTX (exo-) with both reverse transcriptase and DNA polymerase activity. The URPAND can be completed RT-PCR and PAND to detect nucleic acid in one tube within 30 min. This method can specifically detect SARS-CoV-2 with a low detection limit of 100 copies/mL. The diagnostic results of clinical samples with one-tube URPAND displayed 100% consistence with RT-qPCR test. Moreover, URPAND was also applied to identify SARS-CoV-2 D614G mutant due to its single-nucleotide specificity. The URPAND platform is rapid, accurate, tube closed, one-tube, easy-to-operate and free of large instruments, which provides a new strategy to the detection of SARS-CoV-2 and other RNA viruses.