摘要
目的建立一种更加简便的A组人轮状病毒(HRV)核酸斑点杂交VP7分型方法,以便用于对HRV流行情况进行调查。方法在HRVVP7编码基因各G基因型间高度变异而型内高度保守区域设计分型探针,在该区域的两侧相对保守区域设计一对通用引物,利用PCR分别将地高辛标记HRV5种常见型别(G1～4,G9型)的DNA探针,建立基于VP7的斑点杂交方法;选取经抗原检测和聚丙烯酰胺凝胶电泳(PAGE)检测均为HRV阳性的2006至2008年住院腹泻患儿粪便标本200份,RT-PCR扩增VP7全基因,并对扩增阳性产物应用斑点杂交方法进行G型别分析。结果建立的斑点杂交方法在5种型别探针间无交叉反应,各型探针的检测灵敏度可达到10pg。200份PAGE阳性标本中162份RT-RCR扩增VP7基因阳性,斑点杂交显示G1型41例(25.3%),G2型2例(1.2%),G3型63例(38.9%),G9型35例(21.6%),混合感染19例(11.7%),杂交未分出型2例(1.2%),未检测到G4型HRV。结论本研究所建立的斑点杂交方法敏感度和特异度强,适合在HRV大规模分子流行病学调查时应用。通过该方法的初步应用,发现北京地区婴幼儿HRV除了常见的G1、G2和G3型外,还有G9型感染。
Objective To set up a more convenient and effective dot-blot hybridization assay for surveiUance of HRV circulating in Beijing in recent years. Methods In order to show multiplescquence align and analyze the nucleotide sequence of HRV VP7 gene, the software of DNAStar, MegAlign and Primer Premier 5 were used for well-known G serotypes of human rotaviruses from GenBank database. A region which was highly divergent among genes from different serotypes and conserved within genes of VP7s from same serotypes （ nt 72-378 ） was selected as probe. One universal primer pair was designed based on the sequences on both sides of this highly divergent region （ nt 51-7 l and 379-399） for PCR amplified and digoxigenin labelled probes to be used for five common HRV genotypes （ G1-4, G9 ） in a dot-blot hybridization for VP7 genotyping. Full length VP7 genes of HRV from 200 fecal specimens which were collected from hospitalized children with diatThea who were detected HRV positive were identified polyacrylamide gel electrophoresis （PAGE） and rotavirus antigen was amplified by RT-PCR. The RT-PCR products were genotyped by the established dot-blot hybridization. VP7 genes of rotavirus from some specimens were genotyped by both dot-blot hybridization and nested PCR for comparison. Results The DNA dot-blot hybridization assay set up in this study was of high sensitivity and specificity by showing no cross reaction among probes from different genotypes. Each of these five probes could detect 10 pg of DNA for corresponding genotype. Out of 200 HRV positive stool specimens, 162 samples could be amplified VP7 gene products. Dot-blot hybridization showed that 41 （25. 3% ）, 2 （ 1.2% ）, 63 （58.9 % ） and 35 （21.6%） samples were G1, G2, G3 and G9 genotypes, respectively. Two of 162 cases could not be genotyped and no G4 HRV was detected. Among these genotyped samples, 19 showed that they were co-infected by 2 rotaviruses with different genotypes. The nucleotide sequence analysis of VP7 genes from some of the genotyped samples by DNA dot-blot hybridization demonstrated that the genotypes determined by these two assays were consistent. Of 60 rotavirus positive fecal specimens used for genotyping by nested PCR, 53 were genotyped and 7 were unable to be done, whereas all of these 60 samples were genotyped by this dot-blot hybridization. Conclusions The dot-blot hybridization assay established in this study was highly sensitive and specific and could be used for HRV surveys. Data indicated that in addition to the common G1, G2 and G3 genotypes, rotavirus G9 had been infected in Beijing in recent years.
出处
《中国循证儿科杂志》
CSCD
2009年第5期436-441,共6页
Chinese Journal of Evidence Based Pediatrics
关键词
A组人轮状病毒
地高辛
斑点杂交
G基因型
Human group A rotavirus
Digoxigenin
Dot-blot hybridization
G genotype
