可变数目串联重复序列技术用于中国C群脑膜炎奈瑟菌基因分型的研究

Study on the use of variable-number tandem repeats to examine genetic diversity of Neisseria menineitidis serogroup C isolates in China

目的利用脑膜炎奈瑟菌基因组中可变数目串联重复序列(VNTR)特征,对中国C群脑膜炎奈瑟菌菌株进行基因分型。方法中国C群脑膜炎奈瑟菌菌株109株,选择脑膜炎奈瑟菌DNA中4个VNTR位点,PCR扩增含有串联重复序列的DNA片段,选择每一个VNTR位点有差别的PCR产物进行测序,序列比对,测算串联重复序列的拷贝数。Bio-Rad Gel DocTM XR凝胶成像分析系统计算PCR产物DNA片段的碱基含量,换算成串联重复数;对109株菌株4个位点的串联重复序列拷贝数进行聚类分析,依据聚类分析结果进行基因分型,并将VNTR基因分型结果与脉冲场凝胶电泳基因分型(PFGE)结果进行比较。结果109株C群脑膜炎奈瑟菌菌株分为22个VNTR基因型,同一暴发来源的菌株具有相同VNTR特征;VNTR基因分型方法与PFGE基因分型具有相关关系。结论应用VNTR技术可以对中国C群脑膜炎奈瑟菌进行基因分型和分子流行病学方面的研究,VNTR基因分型可较好地应用于追溯流行性脑脊髓膜炎暴发传染源。

Objective To study the variable number of tandem repeats （VNTR） genotyping of serogroup C Neisseria meningitldis strains discovered in China. Methods We randomly selected 109 serogroup C meningococci strains. Four VNTR loci in ,Neisseria rneningitidis DNA genome were studied. The polymorphism numbers of copies in the four VNTR loci were studied by polymerase chain reaction （PCR）. The PCR products resulting from the amplification of the four VNTR for each isolate were electrophoresed and photographed under Bio-Rad Gel DocTM XR imagery equipments. PCR products were analyzed and the molecular weight of gel images were computed and analyzed by software. To reckon the repeat numbers in each locus of every isolate, different DNA segments in each VNTR locus were sequenced. Using UPGMA cluster analysis techniques to study the genotyping of copies numbers in the four VNTR loci in C meningococci strains genome and to compare the result of VNTR typing with pulsed-field gel electrophoresis（PFGE） typing from 105 isolates of the studied 109 isolaes. Results 109 serogroup C meningococci isolates were divided into 22 VNTR genotypes. Identical types were distributed in the strains which associated with the same outbreaks. Correlation was observed between VNTR clusters and PFGE clusters Conclusion As one of the molecular techniques, VNTR could be used for serogroup C rneningococci isolates genotyping,in China. VNTR analysis might be used for fine typing of meningococcal isolates which belonged to the same clonal complex.