用DIG标记的寡核苷酸基因探针杂交技术进行人轮状病毒VP7G血清型鉴定

目的：建立用地高辛（Ｄｉｇｏｘｉｇｅｎｉｎ，ＤＩＧ）标记的寡核苷酸基因探针鉴定Ａ群轮状病毒ＶＰ７Ｇ血清型的分子杂交技术，并应用该技术研究河南Ａ群轮状病毒的Ｇ血清型分布。方法：根据轮状病毒编码糖蛋白ＶＰ７的基因核苷酸序列，选择该基因高保守区序列中与ＶＰ７Ｇ血清型高度相关的核苷酸片段，人工合成，并用ＤＩＧ标记。在优选的杂交条件下进行杂交：结果：①Ｇ１、Ｇ２、Ｇ３和Ｇ４四种Ｇ血清型特异性寡核苷酸探针只与同血清型的轮状病毒参考毒株杂交，无交叉杂交现象。灵敏度和特异性达到放射性同位素３２Ｐ标记的水平。②１８８份经ＰＡＧＥ确定的轮状病毒阳性标本中，７９份（４２．０％）、３５份（１８．６％）、１５份（７．９％）分别与Ｇ１、Ｇ２或Ｇ３型探针杂交；未检出Ｇ４型；５６份（２９．８％）未能分型；３份分型特殊。结论：该Ｇ血清型分型技术具有高度特异性和灵敏度

Typing of Human Rotavirus VP7 G Serotypes with Digoxigenin-Labeled Oligonucleotide Probes

Objective: To establish typing method of Human group A rotavirus VP7 G serotypes with digoxigenin-labeled oligonucleotide probes and to find out the distribution of G serotypes in Henan Province.Methods: The oligonucleotide sequences were selected from the conservative region of VP7 genes in human group A region knows to cord for a major serotype-specific neutralization epitope of G1～4 serotypes,respectively.We synthesized and labeled it with DIG .The hybridization were performed under the conditions by our selection.Results: ①The G1,G2,G3 and G4 serotype -specific oligonucleotide probes only hybridized with VP7 genes of reference rotavirus and no cross-hybridization.The sensitivity and specificity achieved the level of the probe labeled with 32 P .②Of 188 PAGE positive rotavirus samples,79(42.0%),35(18,.6%),15(7.9%) and zero samples hybridized with G1,G2,G3,and G4 probe,respectively,and 56(29.8%) samples were unserotyped.3(1.6%) rotavirus strains were found that serotypes were unusual. Conclusion: The rotavirus G serotyping method was highly specificity and sensitivity.The test were credibility and easy to perform,inespensive,nonradioactive,suitable for the situation in China,especially for epidemiology investigation and vaccine trails involved large amounts of samples.