Bessaud Maël, Autret Arnaud, Jegouic Sophie, Balanant Jean, Joffret Marie-Line, Delpeyroux Francis
Unité postulante de biologie des virus entériques, Institut Pasteur, 25 rue du Dr Roux, 75 015 Paris, France.
J Virol Methods. 2008 Nov;153(2):182-9. doi: 10.1016/j.jviromet.2008.07.010. Epub 2008 Aug 29.
Human enteroviruses are among the most common viruses infecting humans. These viruses are known to be able to infect a wide range of tissues and are believed to establish persistent infections. Enteroviruses are positive-sense single-stranded RNA viruses whose replication involves the synthesis of negative strand intermediates. Therefore, the specific detection of negatively stranded viral RNA in tissues or cells is a reliable marker of active enteroviral replication. The present report presents the development of a real-time RT-PCR allowing the specific detection and quantification of negatively stranded viral RNA. Since it was known that specific amplification of single-stranded RNA can be made difficult by false-priming events leading to false-positive or overestimated results, the assay was developed by using a tagged RT primer. This tagged RT-PCR was shown to be able to amplify specifically negative RNA of enteroviruses grown in cell cultures by preventing the amplification of cDNAs generated by false-priming.
人肠道病毒是感染人类的最常见病毒之一。已知这些病毒能够感染多种组织，并被认为会建立持续性感染。肠道病毒是正链单链RNA病毒，其复制涉及负链中间体的合成。因此，在组织或细胞中特异性检测负链病毒RNA是肠道病毒活跃复制的可靠标志物。本报告介绍了一种实时逆转录聚合酶链反应（RT-PCR）的开发，该方法可特异性检测和定量负链病毒RNA。由于已知单链RNA的特异性扩增可能会因错误引物事件而变得困难，从而导致假阳性或结果高估，因此该检测方法是通过使用带标签的逆转录引物开发的。这种带标签的RT-PCR被证明能够通过防止错误引物产生的cDNA扩增，特异性扩增细胞培养物中生长的肠道病毒的负链RNA。
