Fikatas A, Dimitriou T G, Kyriakopoulou Z, Moschonas G D, Amoutzias G D, Mossialos D, Gartzonika C, Levidiotou-Stefanou S, Markoulatos P
Microbiology - Virology Laboratory, Department of Biochemistry & Biotechnology, School of Health Sciences, University of Thessaly, Larissa, Greece.
Bioinformatics Laboratory, Department of Biochemistry & Biotechnology, School of Health Sciences, University of Thessaly, Larissa, Greece.
Lett Appl Microbiol. 2017 Sep;65(3):234-240. doi: 10.1111/lam.12766. Epub 2017 Jul 25.
In this report a strand specific RT-PCR was established for the detection of the replicative negative RNA strand of poliovirus sabin 1 (Sabin1) and Echovirus 19 (E19) strains. The key for the successful conduction of the assay was the use of a specific reverse transcription primer targeting the 5'-UTR of enteroviruses that consisted of a stem-loop structure at the 5'-end and an enteroviral-specific sequence at the 3'-end. The stem loop RT-PCR was found to be an accurate and sensitive method, detecting even 10 CCID of poliovirus sabin 1 (Sabin1) and E19 strains 6 h postinfection (p.i.), while CPE appeared 3 days later. This assay was also validated in SiHa and Caski cell lines that are not used for the detection of enteroviruses. The negative RNA strand was detected 6 h and 12 h p.i. in SiHa and Caski cells, when these cell lines were inoculated with 10 and 1 CCID respectively, whereas CPE was observed 5 days p.i for SiHa cells and 8 days p.i for Caski cells and that only at 10 CCID . The results show that this approach may be used for replacing the time-consuming cell cultures in order to detect the active replication of enteroviruses.
Enteroviruses are positive stranded RNA viruses that may cause severe diseases. The conventional method for detection of active viral replication involves virus isolation in sensitive cell cultures followed by titration and seroneutralization. In this report, we describe the use of a stem-loop secondary structured oligonucleotide in RT-PCR assay for the detection of the replicative negative strand of the positive-stranded RNA of poliovirus sabin 1 and E19 strains. This approach proved to be a useful tool that may be used for replacing the time-consuming cell culture assays in order to detect the active replication of enteroviruses.
在本报告中，建立了一种链特异性逆转录聚合酶链反应（RT-PCR），用于检测脊髓灰质炎病毒1型减毒株（Sabin1）和埃可病毒19型（E19）毒株的复制性负链RNA。该检测方法成功实施的关键在于使用一种针对肠道病毒5'-非翻译区（5'-UTR）的特异性逆转录引物，该引物在5'-端具有茎环结构，在3'-端具有肠道病毒特异性序列。发现茎环RT-PCR是一种准确且灵敏的方法，在感染后6小时（p.i.）就能检测到脊髓灰质炎病毒1型减毒株（Sabin1）和E19毒株的10个组织培养感染剂量（CCID），而细胞病变效应（CPE）在3天后才出现。该检测方法也在未用于检测肠道病毒的SiHa和Caski细胞系中得到验证。当分别用10个和1个CCID接种SiHa和Caski细胞时，在感染后6小时和12小时检测到负链RNA，而SiHa细胞在感染后5天观察到CPE，Caski细胞在感染后8天观察到CPE，且仅在接种10个CCID时出现。结果表明，这种方法可用于替代耗时的细胞培养，以检测肠道病毒的活跃复制。
肠道病毒是正链RNA病毒，可引起严重疾病。检测病毒活跃复制的传统方法包括在敏感细胞培养物中进行病毒分离，随后进行滴定和血清中和。在本报告中，我们描述了在RT-PCR检测中使用茎环二级结构寡核苷酸来检测脊髓灰质炎病毒1型减毒株和E19毒株正链RNA的复制性负链。这种方法被证明是一种有用的工具，可用于替代耗时的细胞培养检测，以检测肠道病毒的活跃复制。
