实时荧光PCR方法检测副溶血性弧菌的研究

建立了一种快速检测副溶血性弧菌（vibrio parahaemolyticus,VP）的荧光PCR（Real time polymerase chain re-action）方法。首先,从GenBank中获得副溶血性弧菌种特异性基因不耐热溶血毒素基因tl和直接耐热溶血素毒素基因tdh,用Primer Express2.0设计引物和Taqman荧光探针,在Roche荧光PCR上进行荧光PCR扩增,荧光曲线表明该荧光PCR可特异性地检测副溶血性弧菌,而大肠杆菌等其他14种细菌和空白对照都是阴性;检测方法灵敏度达10cfu/reaction。本研究建立的荧光PCR检测副溶血性弧菌的方法快速、特异性强,灵敏度高,稳定性好,可检测出总的和带tdh毒力基因的副溶血性弧菌,适合于大批量样品的检验,可广泛用于出入境检疫和动物防疫监督部门的疫情监测。

STUDY ON THE DETECTION OF VIBRIO PARAHAEMOLYTICUS USING REAL-TIME PCR METHOD

A rapid real time polymerase chain reaction(PCR) method to detect vibrio parahaemolyticus(VP) was established in this study. Primers and taqman probes were designed according to the two genes , the tl (the thermolabile hemolysin gene, a species-specific marker) and the tdh (the thermostable direct hemolysin gene) genes, with Primer Express 2.0. The assay was tested for specificity against 14 different strains of different species such as E.coli, Salmonella, Staphylococcus aureu, Shigella, Listeria monocytoge and so on. Only VP stains with the appropriate target genes could amplify target genes and generat fluorescent signal. The assay was proved to be able to detect 10 cfu (colony forming unit) per reaction. The newly-built real time PCR has high sensitivity, good specificity, reliable stability, so it has the potential to detect total and potentially virulent VP from environment sources and seafood.