Osterback Riikka, Tevaluoto Tuire, Ylinen Tiina, Peltola Ville, Susi Petri, Hyypiä Timo, Waris Matti
Department of Virology, University of Turku, Turku, Finland.
J Clin Microbiol. 2013 Dec;51(12):3960-7. doi: 10.1128/JCM.01646-13. Epub 2013 Sep 18.
Human rhinoviruses (HRVs) and human enteroviruses (HEVs) are significant respiratory pathogens. While HRV infections are restricted to the respiratory tract, HEV infections may spread to secondary target organs. The method of choice for sensitive specific detection of these viruses is reverse transcription (RT)-PCR with primers targeting the conserved 5' noncoding region of the viral RNA. On the other hand, sequence similarities between HRVs and HEVs complicate their differential detection. In this study, we describe the use of locked nucleic acid (LNA) analogues in short double-dye probes which contained only two selectively HRV- or HEV-specific bases. The double-stranded DNA dye BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-methyl-quinolinium chloride) was used with the LNA probes in a tricolor real-time PCR assay to allow specific detection of HRVs (probes labeled with 6-carboxyfluorescein [FAM] [green]) and HEVs (Cy5 [red]) with additional melting curve analysis (BOXTO [yellow]). The functionality of the probes was validated in PCR and RT-PCR assays using plasmids containing viral cDNA, quantified viral RNA transcripts, cultivated rhino- and enterovirus prototypes, and clinical specimens. Of 100 HRV and 63 HEV prototypes, the probes correctly identified all HEVs except one that produced only a BOXTO signal. Among 118 clinical specimens with sequencing results, concordant results were obtained for 116 specimens. Two specimens were reactive with both probes, but sequencing yielded only a single virus. Real-time PCR with LNA probes allowed sensitive group-specific identification of HRVs and HEVs and would enable relative copy number determination. The assay is suitable for rapid and accurate differential detection of HRVs and HEVs in a diagnostic laboratory setting.
人鼻病毒（HRV）和人肠道病毒（HEV）是重要的呼吸道病原体。虽然HRV感染局限于呼吸道，但HEV感染可能扩散至继发靶器官。对这些病毒进行灵敏特异检测的首选方法是使用针对病毒RNA保守5'非编码区的引物进行逆转录（RT）-PCR。另一方面，HRV和HEV之间的序列相似性使它们的鉴别检测变得复杂。在本研究中，我们描述了在短双染料探针中使用锁核酸（LNA）类似物，这些探针仅包含两个选择性的HRV或HEV特异性碱基。双链DNA染料BOXTO（4-[6-(苯并恶唑-2-基-(3-甲基-)-2,3-二氢-(苯并-1,3-噻唑)-2-亚甲基)]-1-甲基-喹啉氯化铵）与LNA探针一起用于三色实时PCR检测，以通过额外的熔解曲线分析（BOXTO[黄色]）实现对HRV（用6-羧基荧光素[FAM][绿色]标记的探针）和HEV（Cy5[红色]）的特异性检测。使用含有病毒cDNA的质粒、定量的病毒RNA转录本、培养的鼻病毒和肠道病毒原型以及临床标本，在PCR和RT-PCR检测中验证了探针的功能。在100株HRV和63株HEV原型中，除了一株仅产生BOXTO信号的病毒外，探针正确鉴定了所有的HEV。在118份有测序结果的临床标本中，116份标本获得了一致的结果。两份标本与两种探针均有反应，但测序仅产生一种病毒。使用LNA探针的实时PCR能够灵敏地对HRV和HEV进行组特异性鉴定，并能够确定相对拷贝数。该检测方法适用于诊断实验室环境中对HRV和HEV进行快速准确的鉴别检测。
