两种恒河猴肠道腺病毒基因型分型方法的比较分析  

An analysis of two intestinal adenovirus genotyping methods in rhesus macaques

作　　者：卢建溪[1] 钱师宇[2] 王强[1] 阳莉[1] 李刚[3] 

机构地区：[1]中山大学附属第三医院疫苗研究所,广东广州510630 [2]暨南大学医学院,广东广州510632 [3]中山大学附属第三医院感染科,广东广州510630

出　　处：《海南医学》2011年第12期15-19,共5页Hainan Medical Journal

基　　金：国家高技术研究发展计划(编号:2007AA021002);广东省自然科学基金资助项目(编号:7001521);广东省科技计划资助项目(编号:2008B030301101)

摘　　要：目的通过对两种肠道腺病毒基因型分型方法进行比较,以寻找出简便快速的肠道腺病毒基因型分型方法。方法采集57例圈养的恒河猴粪便样本,方法一:将粪便悬液过滤上清感染BSC-1细胞,提取病变细胞的基因组DNA进行PCR扩增,然后克隆、测序,确定病毒基因型;方法二:从粪便悬液过滤上清中直接提取基因组DNA进行巢式PCR扩增,对PCR阳性者进行克隆、测序以及基因型分型。结果 57份样本中,两种方法均有12份样本检出腺病毒,阳性率为21.1%(12/57)。系统进化分析显示,两种方法检出的腺病毒型别一致。提示这些序列主要可分为两大组:类SAdV-6组(2个非重复序列)和类SAdV-7组(9个非重复序列)。此外,有三个克隆,其最相似序列分别为:SAdV-1、SAdV-3和HAdV-52。结论虽然两种方法的敏感性、特异性一样,但方法二是直接提取粪便悬液过滤上清基因组DNA进行巢式PCR扩增,而无需方法一进行3次体外细胞感染实验后从病变细胞中提取基因组DNA进行PCR扩增,节省了实验的时间及成本,特别是避免了体外细胞感染过程中可能出现的污染,因此值得推荐。Objective To search and classify genotyping method of gastrointestinal adenoviruses conveniently and quickly.Methods Stool samples were collected from a colony of captively bred rhesus macaques and two methods were used to figure out the presence and genotypes of adenovirus.Method 1：Stool samples were suspended in Herpes buffer and filtered.The filtration was used to infect BSC-1 cell.Genomic DNA was extracted from the infected cells and used as PCR template,and the PCR positive fragments were cloned for sequencing and phylogenetic analyses;method 2：DNA extracted directly from fecal samples was used as the template for nested PCR.The PCR product was also cloned and submitted for sequencing.Results 57 stool samples were detected with the two methods.The PCR positive rate was the same 21.1%（12/57）,as well as the genotypes identified by the subsequent sequencing analysis.The results suggested that the viral DNA clones were primarily segregated into two large groups：SAdV-6（2 non-redundant sequences） and SAdV-7（9 non-redundant sequences）.In addition,there were three clones with more similarity to SAdV-1,SAdV-3 and HAdV-52 respectively.Conclusion Although the sensitivity and specificity of two methods is the same,but method 2 saved time and cost,especially avoided the possible contamination that may occur during the process of in vitro infection,so method 2 is superior to method 1.

关 键 词：恒河猴 猴子腺病毒 基因型 

分 类 号：R-332[医药卫生]