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Molecular detection assays for 2012 identified novel human coronavirus (HCoV) and probe modification with locked nucleic acid (LNA) / 中华实验和临床病毒学杂志
Article em Zh | WPRIM | ID: wpr-305026
Biblioteca responsável: WPRO
ABSTRACT
<p><b>OBJECTIVE</b>To develop and optimize the molecular detection assays for recently identified human coronavirus (HCoV) infection.</p><p><b>METHODS</b>Based on the 208 base pair(bp) sequence of novel HCoV reported by HPA of UK, we designed and obtained several pairs of primer (F-1, R-1; F-2, R-2) and Taqman probes (TZ1,TZ2) for detection of novel HCoV. Two of probes were modified with LNA (LNA-TZ1, LNA-TZ2). Then, RT-PCR and various real time RT-PCR assays were developed and optimized in this study. We also compared our assays with the real time RT-PCR assays reported recently by Europe team based on upE or ORF1b target.</p><p><b>RESULTS</b>The RT-PCR or real time RT-PCR assays for novel HCoV were developed without cross-reactivity with other HCoV and several common respiratory viruses using clinical specimen panel. The analytical sensitivity of assays were less than 50-500 copies per reaction and the detection was improved when Taqman probe modified with LNA-tagged, compared to no LNA-tagged in real time RT-PCR assays. The upE and LNA-TZ1 based assays were better than others.</p><p><b>CONCLUSION</b>The molecular detection sensitivity and specificity of TaqMan-based real time PCR assay could be improved when probe tagged with LNA. The upE or LNA-TZ1 based real time RT-PCR assay was recommend for detection of novel HCoV. This study laid a foundation for improving the performance of novel HCoV detection.</p>
Assuntos
Texto completo: 1 Base de dados: WPRIM Assunto principal: Oligonucleotídeos / RNA Viral / Sensibilidade e Especificidade / Classificação / Primers do DNA / Coronavirus / Reação em Cadeia da Polimerase Via Transcriptase Reversa / Genética / Métodos Tipo de estudo: Diagnostic_studies Limite: Humans Idioma: Zh Revista: Chinese Journal of Experimental and Clinical Virology Ano de publicação: 2012 Tipo de documento: Article
Texto completo: 1 Base de dados: WPRIM Assunto principal: Oligonucleotídeos / RNA Viral / Sensibilidade e Especificidade / Classificação / Primers do DNA / Coronavirus / Reação em Cadeia da Polimerase Via Transcriptase Reversa / Genética / Métodos Tipo de estudo: Diagnostic_studies Limite: Humans Idioma: Zh Revista: Chinese Journal of Experimental and Clinical Virology Ano de publicação: 2012 Tipo de documento: Article