威海地区两种海洋弧菌多重降落PCR体系的建立与应用

Establishment and application of multiplex touchdown PCR system for detection of two kinds of Marine vibrio in Weihai region

目的分析威海地区食源性疾病中,海洋性弧菌的检出率,并建立一种可以同时检测创伤弧菌及溶藻弧菌的多重降落PCR反应体系。方法收集怀疑食源性疾病患者粪便标本进行分离培养鉴定并分析,并且针对创伤弧菌的vvhA基因与溶藻弧菌的toxR基因分别设计两对特异性引物,应用Primer-BLAST检测引物特异性。应用该引物建立多重降落PCR,并检测93份粪便标本,其结果与细菌培养法进行比较结果。结果威海地区怀疑食源性疾病患者粪便标本中共检出海洋性弧菌45例,以副溶血性弧菌为主,随后为创伤性弧菌和溶藻弧菌。所建立的多重降落PCR对致病菌两种基因(vvhA、toxR)DNA的检测下限分别为104CFU/mL、103CFU/mL;与细菌培养法相比,灵敏度及总灵敏度均达100%,检测vvhA、toxR基因特异性分别为96.47%、95.45%,总特异性达95.95%。结论我们建立的多重降落PCR检测体系可用于由创伤弧菌和溶藻弧菌引起的食源性感染的快速诊断与流行病学调查。

Objective To develop a multiplex touchdown PCR system for simultaneous detection Vibrio vulnificus and Vibrio alginolyticus and analyze the detection rate of Marine vibrio in weihai foodborne diseases.Methods The fecal specimens of patients with suspected foodborne diseases were separated,cultured and analyzed,two pairs of specific primers were designed based on vvhA gene of Vibrio vulnificus and toxR gene of Vibrio alginolyticus and detected primer specificity with primerBLAST.The primers were used to establish multiplex touchdown PCR,and detected 93 fecal specimens,and the results were compared with the bacterial culture method.Results 45 cases of Marine vibrio were detected in fecal samples from patients with suspected foodborne diseases,mainly by the Vibrio parahaemolyticus,followed by Vibrio vulnificus and Vibrio alginolyticus.Multiplex touchdown PCR established two genes of pathogenic bacteria（vvhA,toxR）DNA detection limit were 104 CFU/mL,103 CFU/mL;Compared with the bacterial culture method,the sensitivity and total sensitivity reached 100% .The specificity of vvhA and toxR gene was 96.47% and 95.45% respectively,and the total specificity was 95.95% .Conclusion The system of multiplex touchdown PCR detection we established can be used in the rapid diagnosis and epidemiological investigation of foodborne infection caused by Vibrio vulnificus and Vibrio alginolyticus.