脑膜炎奈瑟菌基因组中可变数目串联重复序列与血清分群之间关系的研究

Research on the Relationship between Variable Number of Tandem Repeats Sequences and Serogrouping of Neisseria Meningitis

目的研究脑膜炎奈瑟菌(Neisseria Meningitides,Nm)基因组中串联重复序列(Variable Number of Tandem Repeats Sequences,VNTRs)与血清分群(A和C群)之间的关系。方法采用Nm血清抗体玻片凝集法对Nm菌株进行血清学鉴定;选择4对引物,应用聚合酶链反应(Polymerase Chain Reaction,PCR)扩增Nm基因组中4个位点的VNTR01～04,使用非加权配对算术平均法,应用统计学软件分析VNTRs拷贝(Copy)数与血清群之间的关系;以编码半乳糖转移酶基因中的VNTR01位点之后一段核苷酸序列为特异引物,对Nm进行PCR扩增。结果118株Nm菌株中,38株为A群,80株为C群,聚类分析可将118株菌分为5个组(I、II、III、IV、V)。95.8%(113/118)的菌株位于Ⅰ、Ⅱ组内,Ⅰ组中97.3%(36/37)的菌株为A群,Ⅱ组中98.7%(75/76)的菌株为C群。用两对特异引物对118株菌株进行PCR扩增,以PCR产物有或无做为判断结果,结果与血清分群有96.6%(114/118)的一致性。结论VNTRs拷贝数与血清群之间有较好的相关性,编码半乳糖转移酶基因中变异的核苷酸序列可以做为鉴别A、C群菌株的一种核酸标识。

Objective To research the relationship between variable number of tandem repeats sequences （VNTRs） in genome and serogrouping of Neisseria meningitides （N.meningitidis）. Methods 118 meningococcal strains which were isolated from invasive patients and carriers in China were serogrouped by antiseria agglutination testing. 38 strains were serogroup A. 80 were serogroup C. And then four variable number of tandem repeats sequences （VNTRs） loci in the DNA of 118 meningococcal strains were amplified by PCR. A dendrogram was constructed on the basis of DNA length of PCR productions by the Unweighted Pair Group Method with Arithmetic Averages （UPGMA）. The two pair primers-specific were designed based on the nucleotide variant afterward the VNTR01 locus （T01-A and T01-AC,T01-C and T01-AC） to amplify 118 meningococcal strains. Results 118 strains were classified into five VNTR groups. 98.3% （116/118） strains were identical between serogrouping and VNTRs genotyping. 97.3% （36/37） strains of serogr.oup A were categorized into groupⅠof VNTRs genotyping. 98.7% （75/76） strains of serogroup C were categorized into groupⅡof VNTRs genotyping. According to the presence or absence of PCR products using two pair primers-specific,118 strains were classif ied into two groups which correspond to serogroup A and C. The sensitivity of primers-specific T01-A and T01-AC was 89.5% （34/38）. The specificity of primers-specific T01-A and T01-AC was 100% （38/38）. The sensitivity of primers-specif ic T01-C and T01-AC was 100% （80/80）. The specif icity of primers-specif ic T01-C and T01-AC was 95.2% （80/84）. Conclusion VNTRs genotyping has high relation to serogrouping A and C. The nucleotide sequence variations in the galactosyl-transferase generelated to the serogrouping A and C.