镇江地区肠道病毒71型VP1基因的检测及系统进化分析

Detection and Phylogenetic Analysis of Enterovirus 71 in Zhenjiang

目的检测我国镇江地区2008年手足口病(HFMD)散发病例中的肠道病毒71型(EV71),并进行毒株的种系进化分析。方法采集临床HFMD患者粪便标本,抽提病毒RNA,通过巢式聚合酶链反应技术进行特异性扩增。经琼脂糖凝胶电泳鉴定为阳性的标本,采用EV71特异性引物对其VP1区的核苷酸序列进行扩增。扩增的VP1片段经纯化后与测序载体PET32a(+)连接,转化大肠埃希菌DH5α,并对阳性质粒进行酶切和测序鉴定。利用Clustalx和MEGA4.0分析软件,进行系统进化分析。结果通过聚合酶链反应扩增获得EV71VP1基因,根据遗传进化分析发现该病毒株与1999、2000年在美国亚特兰大以及2000年中国北京地区流行的EV71型遗传距离较近,均属于C型。结论镇江地区HFMD患者中分离的EV71病毒株属C基因型。

Objective To detect enterovirus 71 （ EV71 ） gene among patients with hand-foot-and-mouth disease（HFMD） in Zhengjiang in 2008 and to analyze the evolution by phylngenetic analysis. Methods The EV71 VPl genes were amplified by nested PCR. The amplicons were inserted into the vector PET32a（ ＋ ） and tranducted into the E. coli DH5et. The VP1 gene sequences（891 nucleotides）were determined and compared by phylogenetic analysis. Results The EV 71 VPI genes were successfully amplified by PCR. The EV71 isolates from Zhenjiang belonged to genotype C by phylogenetic analysis and this genotype was different from the strains isolated from other regions of China. Conclusion The EV71 strains from Zhenjiang belonged to genotype C.