目的 建立一种快速区分鉴别田鼠型与非田鼠型鼠疫耶尔森菌(鼠疫菌)的分子分型方法。方法 设计特异引物对93株不同来源、不同生物型鼠疫菌的天冬氨酸酶基因(aspA)进行PCR扩增,用限制性内切酶Hpy CH4Ⅳ对扩增产物进行限制性片段长度多态性(RFLP)分析;直接测序法检测aspA基因多态性。结果 aspA基因扩增产物经Hpy CH4Ⅳ酶切后呈现2种基因型:田鼠型鼠疫菌(38株)均为101、126 bp 2个条带;非田鼠型鼠疫菌(55株)均为227 bp单一条带。测序结果证实酶切识别位点发生突变导致带型差异。结论 初步建立了鼠疫菌PCR?RFLP(聚合酶链反应-限制性片段长度多态性)法aspA基因分型方案,通过Hpy CH4Ⅳ酶切带型差异区分高毒力的非田鼠型菌株与低毒力的田鼠型菌株,适用于疫源地流行病学调查。
Objective To develop a simple genotyping method for rapid differentiation of Yersinia pestis strains between Microtus and non-Microtus biotypes. Methods Specific primers were designed and applied to amplify the aspartase gene (aspA) through polymerase chain reaction (PCR) among 93 Y. pestis strains of diverse origins and biotypes. The PCR products were genotyped by restriction fragment length polymorphism (RFLP) analysis using the restriction enzyme Hpy CH4Ⅳ. Genetic polymorphisms of aspA were determined by direct DNA sequencing. Results Among the 93 test strains, the amplified products of aspA digested with Hpy CH4Ⅳ showed two genotypes: Microtus biotype strains (38) all had two RFLP bands in sizes of 101 and 126 bp, respectively; and non-Microtus biotype strains (58) consistently had one single RFLP band in size of 227 bp. Direct sequencing data confirmed that mutation at the recognition site for restriction enzyme led to differences in the RFLP banding patterns. Conclusion A new genotyping method (aspA-PCR-RFLP) was established for rapid differentiation of Y. pestis strains between Microtus biotype with low virulence and non-Microtus biotype with high virulence according to different RFLP banding patterns. The proposed method can be used for epidemiologic surveillance in plague foci.
[1] Bevins SN, Baroch JA, Nolte DL, et al. Yersinia pestis: examining wildlife plague surveillance in China and the USA[J]. Integr Zool, 2012,7(1):99-109.
[2] Li Y, Cui Y, Hauck Y, et al. Genotyping and phylogenetic analysis of Yersinia pestis by MLVA: insights into the worldwide expansion of Central Asia plague foci[J]. PLoS One,2009,4(6):e6000.
[3] Zhou D, Tong Z, Song Y, et al. Genetics of metabolic variations between Yersinia pestis biovars and the proposal of a new biovar, microtus[J]. J Bacteriol,2004,186(15):5147-5152.
[4] Morelli G, Song Y, Mazzoni CJ, et al. Yersinia pestis genome sequencing identifies patterns of global phylogenetic diversity[J]. Nat Genet,2010,42(12):1140-1143.
[5] Cui Y, Li Y, Gorge O, et al. Insight into microevolution of Yersinia pestis by clustered regularly interspaced short palindromic repeats[J]. PLoS One,2008,3(7):e2652.
[6] Li Y, Dai E, Cui Y, et al. Different region analysis for genotyping Yersinia pestis isolates from China[J]. PLoS One,2008,3(5):e2166.
[7] Bearden SW, Sexton C, Pare J, et al. Attenuated enzootic (pestoides) isolates of Yersinia pestis express active aspartase[J]. Microbiology,2009,155(Pt 1):198-209.
