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目的基于副溶血性弧菌tdh1 DNA序列中特异性变异位点,建立新的大流行株鉴别方法。方法通过对多种型别菌株的tdh序列进行比对,查找大流行株特异性变异位点,并基于该位点设计位点特异性PCR体系和检测方法。以GS-PCR和tdh基因联合检测为参照方法,用已知大流行株、非大流行株和2014年收集的1 067株副溶血性弧菌对新方法进行验证。结果 3株菌株的6条tdh全序列存在26个多态性位点,其中tdh1基因的第368位碱基的变异[G/A]能用于鉴别大流行株,本研究基于此位点建立了tdh1_368位点特异性PCR。该变异能将1996年后的大流行O3∶K6型菌株与1996前的进行区分,也能将大流行株其他血清型变种与非流行菌株进行区分。对2014年的1 067株菌株的检测结果显示,tdh1_368位点特异性PCR与参考方法检测结果完全一致。结论本研究以副溶血性弧菌tdh基因的多态性位点建立检测大流行菌型的试验方法,从对1 067株菌株的实测结果来看,tdh1_368位点特异性PCR与既往报道的方法具有高度的一致性,且指标更为直接。
Objective To establish a new method for identifying pandemic strains based on the specific mutation sites in DNA sequence of Vibrio parahaemolyticus tdh1. Methods The tdh sequences of various strains were compared to find out the specific mutation sites of the pandemic strain. Based on this site, a site-specific PCR system and detection method were designed. The new method was validated by the combined detection of GS-PCR and tdh genes, using known pandemic strains, non-pandemic strains and 1 067 strains of Vibrio parahaemolyticus collected in 2014. Results There were 26 polymorphic loci in the six tdh sequences of the three isolates. The variation of base 368 of the tdh1 gene [G / A] could be used to identify the pandemic strain. Based on this locus, Tdh1_368 site-specific PCR. This variant distinguishes between the pandemic O3: K6 strain after 1996 and the pre-1996 strain and can also distinguish other serotypes of the pandemic from non-endemic strains. The results of 1 067 strains in 2014 showed that tdh1_368 site-specific PCR was completely consistent with the reference method. Conclusion In this study, we established a test method for the detection of the pandemic strain based on the polymorphic loci of tdh gene of Vibrio parahaemolyticus. Based on the measured results of 1 067 strains, tdh1_368 site-specific PCR and previously reported methods With a high degree of consistency, and indicators more direct.