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目的对浙江省2004~2013年分离的54株脑膜炎奈瑟菌(Neisseria meningitidis,Nm)进行基因分型研究,了解菌株间的遗传进化关系。方法参照细菌分子分型和基因组差异公共数据库(Pubmlst)提供的引物与方法,获得多位点序列分型(Multilocus Sequence Typing,MLST)序列型(Sequence Type,ST)。采用进化分析软件(Splitstree)4、聚类分析软件(e BURST)V3等处理所得序列数据,并构建遗传进化图谱。结果 54株Nm得到21个STs,其中11个STs为新发现,主要序列型ST-7和ST-4821分别占29.63%和27.78%。不同血清群其STs/ST-克隆群(Complex)有明显不同,A群菌株78.95%(15/19)为ST-7/ST-5Complex(15/19);B群菌株STs分散,57.14%(8/14)呈单体存在;C群菌株82.35%(14/17)为ST-4821/ST-4821Complex。不同来源分离株其ST-Complex不同,来自病人及密切接触者分离株ST-5Complex分别占48.15%(13/27)及33.33%(4/12)、ST-4821Complex分别占40.74%(11/27)及25.00%(3/12),而来自健康带菌者分离株ST-4821Complex克隆群占40.00%(6/15)。7个管家基因的核苷酸多态性(Pi)范围为0.01298(adk)~0.11100(aro E)。Splitstree 4构建径向系统进化树显示,浙江省Nm菌株分属5个分枝,主要序列型ST-7及ST-4821分别分布于3分枝及2分枝中,4分枝中均为浙江省STs。e BURST V3聚类分析发现,浙江省STs/ST-5Complex基因型相对稳定,但浙江省STs/ST-4821Complex基因型已具遗传多样性变化。结论浙江省Nm以A群78.95%(15/19)ST-7/ST-5Complex和C群82.35%(14/17)ST-4821/ST-4821Complex两大克隆群占绝对优势,B群57.14%(8/14)呈单体分散。浙江省A、B、C群均存在ST-4821Complex相同基因型菌株,可能不同血清群之间已产生荚膜转换,其种群间已显示基因重组进化特征,这是中国首次从A群Nm中检出ST-4821/ST-4821Complex及ST-5798/ST-4821Complex菌株。
Objective To study the genotyping of 54 Neisseria meningitidis isolates from 2004 to 2013 in Zhejiang Province and to understand the genetic evolution among strains. Methods Multilocus Sequence Typing (MLST) Sequence Type (ST) was obtained with reference to the primers and methods provided by the PubMed database. The obtained sequence data were processed by using evolutionary analysis software (Splitstree) 4 and cluster analysis software (e BURST) V3, and the genetic evolution map was constructed. Results A total of 21 STs were found in 54 strains of Nm, 11 of which were newly discovered. The major sequences ST-7 and ST-4821 accounted for 29.63% and 27.78% respectively. The STs / ST-clone complex of different serogroups was significantly different. The group A strains were 78.95% (15/19) ST-7 / ST-5Complex (15/19) 8/14) were present as monomer; Group C strain 82.35% (14/17) was ST-4821 / ST-4821 Complex. The ST-Complex of different isolates was different from that of ST-5Complex from patients and close contacts, accounting for 48.15% (13/27) and 33.33% (4/12), respectively. ST-4821Complex accounted for 40.74% (11/27 ) And 25.00% (3/12), respectively, whereas the clone from the healthy carrier ST-4821 Complex accounted for 40.00% (6/15). Nucleotide polymorphisms (Pi) of seven housekeeping genes ranged from 0.01298 (adk) to 0.11100 (aro E). The construction of the radial phylogenetic tree by Splitstree 4 showed that the strains of Nm in Zhejiang Province belonged to 5 branches, the major sequences of ST-7 and ST-4821 were located in 3 branches and 2 branches, respectively, and all the 4 branches were in Zhejiang Province STs. e BURST V3 cluster analysis found that Zhejiang Province STs / ST-5Complex genotype is relatively stable, but Zhejiang province STs / ST-4821Complex genotype has a genetic diversity changes. Conclusion The two clones of 78.95% (15/19) ST-7 / ST-5Complex in group A and 82.35% (14/17) ST-4821 / ST-4821Complex in group C accounted for 57.1% (8/14) were monomer dispersed. The same genotype of ST-4821Complex exists in Zhejiang province A, B and C groups, and it is possible that capsular conversion has occurred among different serogroups and the genetic recombination characteristics have been shown among the populations. ST-4821 / ST-4821 Complex and ST-5798 / ST-4821 Complex strains.