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当前位置: 首页 > 文章 > 副溶血弧菌和霍乱弧菌双重荧光定量PCR快速检测方法的建立与应用 畜牧与兽医 2018 (4) 93-99
Position: Home > Articles > Establishment and application of theduplex real-time PCR of Vibrio parahaemolyticus and Vibrio cholera Animal Husbandry & Veterinary Medicine 2018 (4) 93-99

副溶血弧菌和霍乱弧菌双重荧光定量PCR快速检测方法的建立与应用

作  者：
凌娇;王权;万莹;白雪瑞;蒋蔚;潘翠玲
单  位：
南京农业大学动物医学院;中国农业科学院上海兽医研究所
关键词：
副溶血弧菌;霍乱弧菌;toxR;ompW;双重荧光定量PCR
摘  要：
建立一种基于TaqMan探针法同步定量检测副溶血弧菌和霍乱弧菌的双重荧光定量PCR方法。根据副溶血弧菌toxR基因和霍乱弧菌ompW基因设计特异性引物与TaqMan探针。toxR和ompW探针5'端分别标记FAM、CY5荧光报告基团,3'端均标记BHQ1荧光淬灭基团。结果显示:该方法的副溶血弧菌和霍乱弧菌最低检测限均达到10 CFU/m L,灵敏度高;特异性试验表明这两种细菌与其他病原菌(大肠杆菌O157、沙门氏菌、拟态弧菌、创伤弧菌)无交叉反应;批间和批内重复性实验表明变异系数均小于1.5%,说明重复性好。采用人工染菌虾肉样品,副溶血弧菌和霍乱弧菌的最低检测限分别为100 CFU/m L和50 CFU/m L,人工染菌贝类样品的最低检测限分别为50 CFU/m L和50 CFU/m L。两种细菌在虾肉中富集2 h后的最低检出限均为1 CFU/m L。结论:本研究所建立的双重荧光PCR检测方法具有灵敏度高、特异性强和重复性好的特点,包括DNA提取的整个检测过程可在1至3 h内完成,是同时快速检测副溶血弧菌和霍乱弧菌的有效手段。
译  名：
Establishment and application of theduplex real-time PCR of Vibrio parahaemolyticus and Vibrio cholera
作  者：
LING Jiao;WANG Quan;WAN Ying;BAI Xuerui;JIANG Wei;PAN Cuiling;College of Veterinary Medicine,Nanjing Agricultural University;Shanghai Veterinary Research Institute,CAAS;
关键词：
V. parahaemolyticus;;V. cholerae;;toxR;;ompW;;duplex real-time PCR
摘  要：
In this study,a Taq Man-based duplex real-time PCR assay was established for the detection of Vibrio parahaemolyticus(VP)and Vibrio cholera(VC). The specific primers and probes were designed based on the toxR gene of V. Parahaemolyticus and the omp W gene of V. cholera. The 5'-ends of probes for toxR and omp W were labeled with FAM and CY5 respectively,and their 3'-ends were all labeled with quencher BHQ1. The results showed that this PCR assay could unambiguously detect V. parahaemolyticus and V. cholera with a minimum detection limit of 10 CFU/m L indicating high sensitivity. The specificity of the assay showed that the two bacteria had no cross reaction with other pathogens(Such as Escherichia coli O157,Salmonella,Vibrio mimicus and Vibrio vulnificus). In addition,it had good repeatability between the batch and within the group,and the coefficient of variation was less than 1. 5%. When the developed method was applied to assaying food samples artificially contaminated by VP or VC,the detection limit was found to be 100 CFU/m L(VP) and 50 CFU/m L(VC)in shrimp samples or 50 CFU/m L(VP) and 50 CFU/m L(VC) in shellfish samples per reaction without an enrichment step. The minimum detection limit of the method was 1 CFU/m L in shrimps contaminated by VP or VC enriching for 2 hours. A conclusion could be drawn that the duplex real-time PCR assay established here was characteristic of high sensitivity,strong specificity,and good repeatability; and the entire detection procedure including DNAextracted by this method could be completed within 1 to 3 hours. This duplex real-time PCR assay was effective in rapid detection of V. parahaemolyticus and V. cholera.

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