多重实时荧光定量PCR技术检测儿童呼吸道新发病毒方法的建立与应用  

Detection and identification of respiratory emerging infectious viruses in children by multiplex real time PCR

作　　者：翁学军 张甦 杜英 沈霞萍 袁新华 程红玲 李刚 邢建明 

机构地区：[1]浙江省湖州市妇幼保健院检验科,313000

出　　处：《中华检验医学杂志》2013年第6期538-542,共5页Chinese Journal of Laboratory Medicine

基　　金：浙江省湖州市科技局科研资助项目（2009YS10）

摘　　要：目的建立儿童新发呼吸道病毒的多重实时荧光定量PCR检测方法，并利用该方法进行临床标本的检测。方法根据人类偏肺病毒、冠状病毒HKU1、冠状病毒NL63、人博卡病毒、KI多瘤病毒和wu多瘤病毒的保守区域设计并合成引物和探针，确定引物和探针的特异性，优化引物和探针的组合浓度，通过检测具有类似临床症状的其他病原体的标本评价该多重荧光定量PCR方法的特异性，构建质粒标准品用于分析检测该方法的灵敏度和重复性；并对2009至2012年湖州市妇幼保健院及湖州市中心医院收集的322例呼吸道感染患儿的痰液标本进行检测分析。结果成功建立了这6种新发呼吸道病毒的多重荧光定量PCR检测方法，能够同时或分别对其进行检测。体系中筛选的引物和探针具有很好的特异性，并优化出适合的引物探针组合浓度，灵敏度分析最低检测限为10拷贝数／μl，重复性分析中批内变异系数（CV）为1．2％～3．4％，批间CV为0．08％～0．85％，均小于5％。322份临床标本中，人类偏肺病毒阳性17份，阳性率5．28％，人博卡病毒9份，阳性率2．80％，未检到冠状病毒HKU1、冠状病毒NL63、KI多瘤病毒和WU多瘤病毒。结论多重荧光定量PCR方法具有简单快速，灵敏性和特异度高等特点，并且能实现多种新发病毒同时并行检测，可为儿童呼吸道新发病毒感染的临床诊治提供技术支持。Objective To develop a rapid, sensitive and specific multiplex real time fluorescence quantitative PCR for detecting and identifying respiratory emerging infectious viruses in children, and apply the detection for clinical specimens. Methods The conserved regions of six new discovery respiratory viruses, human metapneumovirs, coronavirus HKU1, coronavirus NL63, human bocavirus, Karolinska Institute polyomavirus and WU polyomavirus, were selected as target genes. The specific primers and TaqMan probes of the six viruses according to the principle of the multiplex real time fluorescence quantitative PCR were designed and synthesized. The combined concentration of these primers and probes were optimized to determine the specificity of these primers and probes. The specificity of the method was evaluated by detecting the other pathogen with similar clinical symptom. The standard preparations of the six viruses were constructed for analyzing the sensitivity and repeatability of the method. The method was applied for 322 sputum specimens in children with respiratory tract infection collected by Maternal and Child Care Hospital of Huzhou and Huzhou Centeral Hospital from 2009 to 2012. Results The six new discovery respiratory viruses were detected respectively and simuhaneously by using the multiplex real time PCR accurately and specifically, and the sensitivity was 10 copies/ixl. The coefficient of variation of intra-assay in repeatability performance analysis was 1.2% - 3.4% and the CV of inter-assay was 0. 08% - 0. 85% , which all less than 5%. Among those 322 clinical specimens, 17（5.28%） were positive for human metapneumovirns; 9 （2.80%） were positive for human bocavirus. The other four viruses were undetected. Conclusions Multiplex real time PCR was a rapid, specific and sensitive method, which candetect variety of emerging infectious viruses simultaneously. It can be used in screening large numbers of samples, and support technology for clinical diagnosis and epidemiology study.

关 键 词：呼吸系统 多瘤病毒属 变性肺病毒 冠状病毒属 荧光 儿童 

分 类 号：R457.12[医药卫生—治疗学]