New Molecular Detection Tools Adapted to Emerging Rhinoviruses and Enteroviruses
New Molecular Detection Tools Adapted to Emerging Rhinoviruses and Enteroviruses
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DOI：
10.1128/jcm.02339-08
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发表时间：
2009-06-01
影响因子：
9.4
通讯作者：
Kaiser, Laurent
中科院分区：
文献类型：
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作者：
Tapparel, Caroline;Cordey, Samuel;Kaiser, Laurent
人鼻病毒(HRV)和人类肠道病毒(HEV)是重要的呼吸道病原体。像其他RNA病毒一样，这些小核糖核酸病毒有一种内在的变异倾向。这导致了大量不同的血清型以及不断发现新的基因型别。这种巨大且不断增长的多样性不仅使实时PCR分析的设计复杂化，而且使免疫荧光无法在细胞中进行广泛的HRV和HEV检测或定量。在这项研究中，我们以基因组中最保守的5‘非翻译区为靶点，根据公开的序列设计了实时聚合酶链式反应(Panenterhino/GE/08)和基于肽核酸的杂交寡核苷酸探针(Panenterhino/GE/08 PNA探针)，以检测所有HRV和HEV物种成员。逆转录-聚合酶链式反应分析已经得到验证，不仅使用了质粒和病毒样本，而且还使用了量化的RNA转录本和大约1000个临床样本。这些新的通用检测聚合酶链式反应方法克服了循环毒株的变异性，降低了漏诊、出现和发散的HRV和HEV的风险。还专门设计了另一种实时聚合酶链式反应检测方法(Entero/GE/08)，以提供对脑脊液中HEV的灵敏和有针对性的检测。除了通用的探针外，我们还开发了检测细胞中HRV-A和HRV-B的特异性探针。这项研究为准确鉴定在人类体内传播的不同HEV和HRV提供了一个全面的工具箱。
Human rhinoviruses (HRV), and to a lesser extent human enteroviruses (HEV), are important respiratory pathogens. Like other RNA viruses, these picornaviruses have an intrinsic propensity to variability. This results in a large number of different serotypes as well as the incessant discovery of new genotypes. This large and growing diversity not only complicates the design of real-time PCR assays but also renders immunofluorescence unfeasible for broad HRV and HEV detection or quantification in cells. In this study, we used the 5' untranslated region, the most conserved part of the genome, as a target for the development of both a real-time PCR assay (Panenterhino/Ge/08) and a peptide nucleic acid-based hybridization oligoprobe (Panenterhino/Ge/08 PNA probe) designed to detect all HRV and HEV species members according to publicly available sequences. The reverse transcription-PCR assay has been validated, using not only plasmid and viral stocks but also quantified RNA transcripts and around 1,000 clinical specimens. These new generic detection PCR assays overcame the variability of circulating strains and lowered the risk of missing emerging and divergent HRV and HEV. An additional real-time PCR assay (Entero/Ge/08) was also designed specifically to provide sensitive and targeted detection of HEV in cerebrospinal fluid. In addition to the generic probe, we developed specific probes for the detection of HRV-A and HRV-B in cells. This investigation provides a comprehensive toolbox for accurate molecular identification of the different HEV and HRV circulating in humans.