添加有扩增内标的副溶血弧菌PCR检测方法

Detection of Vibrio parahaemolyticus by PCR method with internal amplification control

【目的】发掘副溶血弧菌特异性更强的检测靶点,并人工构建扩增内标,建立可以有效避免假阴性的新PCR检测体系。【方法】利用生物信息学方法,从副溶血弧菌(Vibrio parahaemolyticus)基因组DNA中发掘特异性很高的序列,并设计相应的特异性引物,人工构建扩增内标,建立PCR检测体系。【结果】本研究发掘得到的序列vp1332特异性很强,经检索,该序列是编码ABC转运子接合蛋白组分的基因片段,根据此序列设计一对特异检测引物(vp1332L/vp1332R),同时,构建了扩增内标,并建立了PCR检测体系。利用该体系对296株副溶血弧菌和33株非副溶血弧菌进行检测,结果显示,所有以副溶血弧菌为模板的PCR反应均可扩增到一条343bp的特异片段,而模板来源于非副溶血弧菌的则只能扩增到一条499bp的扩增内标片段。灵敏度实验表明,该PCR反应体系的检测灵敏度为1.6×102cfu/mL。人工污染实验表明,起始染菌量为1.24cfu/25g样品时经8h增菌,即可检测到副溶血弧菌。实际样品检测结果也证实该方法的有效性。【结论】本研究建立的PCR反应体系能特异地检测副溶血弧菌,并可有效地排除假阴性,提高检测准确率。

[Objective]The aim of the study was to explore a new detective target in Vibrio parahaemolyticus,which was more specific than others at present,to construct an internal amplification control （IAC）,and finally to develop a new PCR system which could effectively eliminate false-negative results. [Methods] Genomic comparison analysis was used to explore V. parahaemolyticus specific targets,which were then evaluated and used to design specific primers. An IAC was constructed by the compound primer technology. PCR parameters were optimized,and its reaction system was developed. [Results] A V. parahaemolyticus species-specific sequence （vp1332） which encodes probable binding protein component of ABC transporter was mined and selected as a detection target. A pair of specific primers （vp1332L/vp1332R） was designed based on this sequence for the development of a PCR assay. An internal amplification control was constructed and added into the PCR detection system,which was co-amplified with the target sequence so as to indicate the existence of inhibitors. Specificity of this PCR system was tested with 296 V. parahaemolyticus strains and 33 non-V. parahaemolyticus strains,and the results showed that there was a 343-bp amplicon resulted from all V. parahaemolyticus strains,while there was no this amplicon but only a 499-bp IAC amplicon appeared for all non- V. parahaemolyticus strains. The detection limit of this assay for purified V. parahaemolyticus genomic DNA was 1.6×10^2 cfu/mL. Artificial contamination assays showed that V. parahaemolyticus could be detected after eight hours enrichment when the original concentration of this bacterium was 1.24 cfu/25 g. The PCR method developed in this study was also evaluated with Seafood samples,and the results demonstrated that it worked effectively. [Conclusion] A novel PCR method was successfully developed in this study,which could effectively detect V. parahaemolyticus with high accuracy and could especially eliminate false-negative results.