2012年的分子检测分析鉴定出新型人类冠状病毒（HCoV）以及用锁核酸（LNA）进行的探针修饰。

[Molecular detection assays for 2012 identified novel human coronavirus (HCoV) and probe modification with locked nucleic acid (LNA)].

作者信息

Zhou Wei-Min, Lu Rou-Jian, Gong He-Yuan, Li Hui, Duan Xi-Jie, Yang Yang, Tan Wen-Jie

机构信息

National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, China.

出版信息

Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2012 Dec;26(6):401-4.

PMID:23627013

Abstract

OBJECTIVE

To develop and optimize the molecular detection assays for recently identified human coronavirus (HCoV) infection.

METHODS

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.

RESULTS

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.

CONCLUSION

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.

摘要

目的

开发并优化针对最近鉴定出的人类冠状病毒（HCoV）感染的分子检测方法。

方法

基于英国健康保护局（HPA）报道的新型HCoV的208碱基对（bp）序列，我们设计并获得了几对用于检测新型HCoV的引物（F-1，R-1；F-2，R-2）和Taqman探针（TZ1，TZ2）。其中两个探针用锁核酸（LNA）进行了修饰（LNA-TZ1，LNA-TZ2）。然后，在本研究中开发并优化了逆转录聚合酶链反应（RT-PCR）和各种实时RT-PCR检测方法。我们还将我们的检测方法与欧洲团队最近基于upE或开放阅读框1b（ORF1b）靶点报道的实时RT-PCR检测方法进行了比较。

结果

使用临床样本组开发了针对新型HCoV的RT-PCR或实时RT-PCR检测方法，该方法与其他HCoV和几种常见呼吸道病毒无交叉反应。检测方法的分析灵敏度为每个反应少于50 - 500个拷贝，与实时RT-PCR检测方法中未用LNA标记相比，用LNA标记的Taqman探针时检测效果有所提高。基于upE和LNA-TZ1的检测方法优于其他方法。