巢式PCR在腺病毒F亚属引起的致腹泻肠道感染检测中的应用  

Utilization of Nest PCR in Intestinal Infection Caused by F subgenus of Enteric Adenovirus

作　　者：张志华[1] 王华洪[2] 兰喆[1] 

机构地区：[1]湖南省华容县疾病预防控制中心,湖南华容414200 [2]岳阳市疾病预防控制中心

出　　处：《实用预防医学》2009年第6期1936-1938,共3页Practical Preventive Medicine

摘　　要：目的建立一种特异、敏感、快速地检测致腹泻肠道腺病毒F亚属的巢式PCR方法,了解引起肠道致腹泻感染中腺病毒的携带情况。方法根据腺病毒F亚属基因编码序列,设计一对通用引物扩增靶基因片段,扩增后片段长度为520 bp,另根据相对保守序列设计一对特异性的引物以第一次PCR扩增后的产物为模板,再进行PCR扩增,片段长度为300 bp。通过ABI3130基因检测仪来评价该方法的特异性;对腺病毒模板做10倍比系列稀释后进行巢式PCR检测,并对其敏感性进行分析,对PCR扩增产物进行电泳和测序鉴定。结果肠道致腹泻肠道感染中通过巢式PCR扩增反应可检出腺病毒F亚属,通过基因序列测定证实了PCR产物的特异性;在敏感性检测中PCR检测肠道腺病毒模板的下限为2.08×10-4μg/ml。结论本研究建立了一个快速、特异、敏感的肠道腺病毒PCR检测方法,适用于流行病学调查及临床检测。Objective To set up a nest polymerase chain reaction （PCR） assay for the rapid and sensitive detection of F subgenus of enteric adenovirus, and to know the prevalence of enteric adenovirus in diarrhea . Methods According to the sequences of F subgenus of enteric adenovirus gene, a couple of consensus primers were designed to amplify the target gene sequence, the size of the amplified fragment was 520 bp. In addition, a specific primer was designed to reamplify the target gene as the template was the first PCR product and the product was 300 bp. The specificity of the method was evaluated by ABI3130 gene sequence analysis, the PCR products were serially diluted by 10 times and amplified by nest PCR to verify the sensitivity and the detection limit, at last the PCR products were assayed by electrophoresis and DNA sequence analysis. Results In intestinal infection, we could detect F subgenus of enteric adenovirus through nest POR, the specificity was verified by gene analysis and in the sensibilitY test the lowest limit we had detected was 2.08 × 10^-4ug/ml. Conclusions The nest PCR assay is a rapid, specific and sensitive detection method for F subgenus of enteric adenovirus, and it can be used for epidemiological investigation and clinical detection.

关 键 词：巢式PCR 检测 肠道腺病毒 基因测序 

分 类 号：R373.2[医药卫生—病原生物学]