肠炎血清型沙门菌多重PCR检测方法的建立及应用

Development of a multiplex PCR assay for the identification of Salmonellaenterica serovarenteritidis

目的为临床确诊及基层实验室快速准确鉴定肠炎血清型沙门菌提供实验室支持,同时为沙门菌传统血清学鉴定方法提供技术补充。方法根据沙门菌菌体抗原A/D1群基因、鞭毛抗原基因(fliC-HG)及肠炎沙门菌特异性基因片段(sdfI)设计引物,建立多重PCR体系并进行反应条件优化。对所建立的体系的特异性进行检测,并将该体系应用于浙江省2009-2010年分离的共计53株样本的检测。结果建立并优化了肠炎沙门菌检测的多重PCR体系,该体系具有高特异性,可准确快速鉴定肠炎血清型沙门菌,也可区分A群及D1群血清群的沙门菌,并能检测鞭毛抗原为HG的沙门菌,对实际样本检测符合率达100%。结论该体系能正确鉴定肠炎血清型沙门菌,可作为沙门菌传统血清学分型的辅助方法,可弥补商品化血清质量参差不齐的缺陷。并为肠炎沙门菌的监测与实验室诊断提供了一种简单快速、重复性好的新方法。

In order to develop a rapid method for the detection of the prevalent Salmonella enterica serovar enteritidis,a multiplex PCR method was developed and validated.Portions of the gene sequences of somatic antigen for Salmonella serogroups A/D1,flagellar antigens for fliC-HG and Salmonella difference fragment（sdfI） were targeted for amplification using four primer pairs.The multiplex PCR was developed and optimized.To valid the assay,genomic DNA from 14 Salmonella strains representing 14 serotypes and 18 non-salmonella strains was subjected to the PCR.The method was applied to the detection of 53 samples isolated from 2009 to 2010 in Zhejiang Province.The results showed the four targeted genes were correctly amplified only from Salmonella enteritidis.The assay could differentiate Salmonella serogroup A from Salmonella serogroup D1,and it also could identify the Salmonella with fliC-HG.The coincidence rate of the actual sample detection was up to 100%.It is concluded that this multiplex PCR method can identify Salmonella enteritidis enterica serovars directly.It can make up for deficiencies of uneven quality of commerical serum.It should be an assistant method to the traditional serotyping method.It is a simple,rapid,reliable and reproducible method of Salmonella enteritidis detection that will aid in surveillance,prevention and control of this pathogen.