基于 bla CARB-17 基因建立水产品中副溶血弧菌的环介导等温扩增技术检测方法  

bla CARB-17-based LAMP assay for detecting Vibrio parahaemolyticus in aquatic products

作　　者：胡元庆 沈子晨 李凤霞 吕琳雪 周赞虎 HU Yuanqing;SHEN Zichen;LI Fengxia;LYU Linxue;ZHOU Zanhu(School of Biological Science and Biotechnology,Minnan Normal University,Zhangzhou 363000,China;Jiangsu Key Laboratory of Zoonosis,Yangzhou University,Yangzhou 225000,China;Comprehensive Technical Service Center,Zhangzhou Customs,Zhangzhou 363000,China)

机构地区：[1]闽南师范大学生物科学与技术学院,福建漳州363000 [2]扬州大学,江苏省人兽共患病学重点实验室,江苏扬州225000 [3]漳州海关综合技术服务中心,福建漳州363000

出　　处：《食品与发酵工业》2020年第23期198-206,共9页Food and Fermentation Industries

基　　金：福建省自然科学基金面上项目(2017J01453);江苏省人兽共患病学重点实验室资助项目(R1402)。

摘　　要：以β内酰胺酶编码基因-17(class A carbenicillin-hydrolyzingβ-lactamase family-17,bla CARB-17)为靶标,建立一种检测水产品中副溶血弧菌的高特异性环介导等温扩增(loop-mediated isothermal amplification,LAMP)技术。用Bacillus stearothermophilus(Bst)DNA聚合酶于63℃反应60 min,扩增副溶血弧菌ATCC17802的bla CARB-17基因,产物用质量浓度20 g/L琼脂糖凝胶电泳和SYBR Green I染色鉴定,优化体系的主要参数。用5株食源性病原菌作对照,验证体系的特异性;将基因组倍比稀释确定其灵敏度;稀释菌液涂抹新鲜虾样品做污染模拟实验,并对实际样品检测分析其可靠性。优化后体系中Mg 2+最适浓度为2.4 mmol/L,dNTPs最佳反应浓度是0.96 mmol/L,Bst DNA聚合酶最适用量是4.8 U,内外引物浓度比是8∶1,最佳反应条件是65℃、60 min。特异性实验显示仅副溶血弧菌为阳性;体系最低检测限为3.64×102 ng/μL;模拟实验的最低检测限为10 CFU/mL;实际样品LAMP检测与PCR符合率100%。该研究建立的LAMP方法操作简便、特异性强、灵敏性高,可用于现场快速检测副溶血弧菌。A highly specific loop-mediated isothermal amplification(LAMP)assay targeting the bla CARB-17 gene was developed for rapid and sensitive detection of Vibrio parahaemolyticus in aquatic products.The assay was optimized and conducted at 63℃for 60 min using Bacillus stearothermophilus(Bst)DNA polymerase.Amplification was analyzed via 20 g/L agarose gel electrophoresis followed by SYBR GreenⅠstaining.The specificity was determined by detecting V.parahaemolyticus ATCC 17802 and other 5 foodborne pathogens.The sensitivity was evaluated by detecting diluted genome DNA samples.The reliability was proved in both simulation experiment using experimentally contaminated shrimp samples and detection of aquatic samples from fish markets.The optimum detection condition was:2.4 mmol/L Mg 2+,0.96 mmol/L dNTPs,4.8 U Bst DNA polymerase,the ratio of inner and outer primer was 8∶1,and react at 65℃for 60 min.The result of specificity showed that V.parahaemolyticus ATCC 17802 was positive,and other 5 control strains were negative.The detection limit of LAMP assay was 3.64×102 ng/μL,and the detection limit in the simulation experiment was 10 CFU/mL.The LAMP assay showed 100%consistency with conventional PCR for detecting practical samples.The LAMP assay established in this experiment is convenient,sensitive and specific,and is suitable for rapid in-field detection of V.parahaemolyticus.

关 键 词：β内酰胺酶基因(bla CARB-17) 环介导等温扩增技术 副溶血弧菌 水产品 快速检测 

分 类 号：TS254.7[轻工技术与工程—水产品加工及贮藏工程]