建立检测18种(型/亚型)呼吸道病毒的两种循环参数的实时荧光定量PCR  

Development of two panels of real-time PCR assay with different cycling parameters for detection of 18 type/subtypes of respiratory viruses

作　　者：陈钰静 丁细霞[1] 郭勇晖[1] 余楠[1] 潘玉先[1] 余志武[1] 陈满君 王压娣[1] 杜晓棠[1] 车小燕[1] 

机构地区：[1]南方医科大学珠江医院检验医学部,广州510282

出　　处：《临床检验杂志》2014年第5期340-344,共5页Chinese Journal of Clinical Laboratory Science

基　　金：国家重大科技专项(2012ZX10004-213;2013ZX10004805-004);广东省高等学校珠江学者岗位计划(2009GDUPS);广东省普通高校工程技术研究(开发)中心立项建设项目(GCZX-A1106)

摘　　要：目的建立两种不同循环参数的实时荧光定量PCR,检测包括流感病毒、冠状病毒等18种（型/亚型）呼吸道病毒。方法引用文献报道的18种（型/亚型）呼吸道病毒引物和探针,建立两种不同循环参数的实时荧光定量PCR法,考核方法的敏感性、特异性和准确性,并用临床标本进行验证。结果成功建立了可检测18种（型/亚型）呼吸道病毒的两种不同循环参数的实时荧光定量PCR法;该法特异性较好,最低检测限为10拷贝数/μL,变异系数（CV）为1.26%~3.43%,重复性良好;408份临床标本中,用该法检测鼻拭子标本阳性率为63.2%（225/356）,肺泡灌洗液标本阳性率为25.0%（13/52）。结论建立的实时荧光定量PCR法简便、特异、敏感、稳定,适用于临床常见呼吸道病毒感染的早期诊断。Objective To develop two panels of real-time PCR assays with different cycling parameters for detection of 18 types /subtypes of respiratory viruses,including influenza virus,coronavirus,etc. Methods The real-time PCR assays with two cycling parameters for detection of 18 types / subtypes of respiratory viruses was developed by using the primers and probes previously reported. The sensitivity,specificity and accuracy of the method were evaluated followed by detection of clinical specimens. Results The two panels of real-time PCR assays with different cycling parameters were successfully established which showed fine specificity. The lowest limit of detection of the assays was 10 copies / μL. The reproducibility of the assays was reliable with coefficient of variation from 1. 26% to 3. 43%. Among 408 clinical specimens,the detectable rates of nasal swabs and bronchoalveolar lavage fluid were 63. 2%（ 225 /356） and 25. 0%（ 13 /52）,respectively. Conclusion The two panels of real-time PCR assays for detection of 18 types / subtypes respiratory viruses with different cycling parameters were developed. The assays should be simple,specific and sensitive,thus applicable for early diagnosis of the infections of respiratory viruses in clinical laboratory.

关 键 词：呼吸道病毒 实时荧光定量聚合酶链反应 特异性 敏感性 

分 类 号：R373.1[医药卫生—病原生物学]