REP-PCR及ERIC-PCR法对分离自海产品副溶血性弧菌分型分析

REP-PCR and ERIC-PCR Analysis for the Typing of Vibrio parahaemolyticus Isolated from Sea Products Marketed in Shanghai

目的:采用细菌基因外重复回文序列扩增(REP-PCR)和肠道细菌基因间重复序列扩增(ERIC-PCR)两种方法对副溶血性弧菌2株标准株,13株实验室保存菌株和73株分离菌株共88株进行分子分型。方法:通过REP和ERIC-PCR指纹图谱扩增,利用NTsys-pc软件采用Dice系数对指纹图谱进行聚类结果分析。结果:所有供试菌株可通过此两种方法进行分型得到清晰的指纹图谱,并反映出副溶血性弧菌具有丰富的遗传多样性,REP-PCR扩增出5～9条分布在609～4200bp之间的条带,可将副溶血性弧菌分为5个群12类型,分辨指数达0.93,其中tdh+株在相似系数0.86时可聚类在第Ⅰ群;ERIC-PCR扩增出5～11条分布在400～7593bp之间的条带,将副溶血性弧菌分为7个群11个类型,分辨指数为0.94,其中tdh+株在相似系数0.76时可聚类在第Ⅰ群。结论:两种方法均显现出很好的分型能力,能够很好地将环境分离tdh+菌株和标准菌株聚类在一起,其中REP-PCR较ERIC-PCR重复性更好。

Abstract： Objective： The enterobacterial repetitive intergenic consensus sequence-polymerase chain reaction （ERIC-PCR） and repetitive extragenic palindromic element-polymerase chain reaction （REP-PCR） were used to analyze the types of 88 l, Tbrio parahaemolyticus including 2 standard strains, 13 laboratory strains and 73 isolated strains. Methods： REP and ERIC primers were used to amplify the repeated sequences. NTsys-pc soft-ware was used to calculate the discriminative index of REP- and ERIC-PCR with Dice coefficients. Results： All 88 tested V. parahamolyticus could be typed by REP- and ERIC- PCR clearly and revealed abundant genetic diversity in V. parahamolyticus. According to the results of REP-PCR-amplified 5-9 bands within 609-4200 bp, E parahamolyticus was typed into 5 groups and 12 types with discrimination index of 0.93, and the tdh~ strains with resolving index of 0.86 could be classified in Group I. According to ERIC-PCR-amplified 5-11 bands within 400-7593 bp, V. parahamolyticus was typed into 7 groups and 11 types with discrimination index of 0.94, and the tdh strains with discrimination index of 0.76 could be classified in group I. Conclusion： The isolated tdh strains are clustered together with standard strains using the two methods. Although both methods exhibit good typing capability, REP- PCR is more repeatable and stable than ERIC-PCR.