实时荧光跨越式滚环等温扩增结合PMA检测虾产品中的活副溶血性弧菌

doi:10.7506/spkx1002-6630-20200927-329

食品科学 ›› 2021, Vol. 42 ›› Issue (24): 289-295.doi: 10.7506/spkx1002-6630-20200927-329

• 安全检测 • 上一篇

实时荧光跨越式滚环等温扩增结合PMA检测虾产品中的活副溶血性弧菌

董晶，徐慧，郭威，卢鑫，张伟

（1.河北农业大学食品科技学院，河北保定 071000；2.河北农业大学理工学院，河北沧州 061100；3.河北农业大学生命科学学院，河北保定 071000）

发布日期:2021-12-30
基金资助:
国家自然科学基金面上项目（31371772）；河北省自然科学基金重点项目（C2019204342）；河北省重点研究开发计划项目（18275501D）；中央引导地方科技发展资金项目-基础研究项目（216Z5501G）；河北农业大学食品加工学科群经费资助项目（2021-06）

Detection of Vibrio parahaemolyticus in Edible Crustaceans by Real-time Fluorescence Saltatory Rolling Circle Amplification Combined with Propidium Monoazide

DONG Jing, XU Hui, GUO Wei, LU Xin, ZHANG Wei

(1. College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, China; 2. College of Science and Technology, Hebei Agricultural University, Cangzhou 061100, China; 3. College of Life Sciences, Hebei Agricultural University, Baoding 071000, China)

Published:2021-12-30

摘要/Abstract

摘要： 目的：为快速检测虾产品中的活副溶血性弧菌，建立一种实时荧光跨越式滚环等温扩增（real-time fluorescent saltatory rolling circle amplification，RF-SRCA）与叠氮溴化丙锭（propidium monoazide，PMA）染料相结合的检测方法。方法：依据副溶血性弧菌的特异性基因toxR设计并筛选引物，对PMA-RF-SRCA检测方法进行特异性、灵敏度及检出限分析，并与PMA-实时聚合酶链式反应（real-time polymerase chain reaction，real-time PCR）方法进行对比。对76 份样品进行检测，以GB 4789.7—2013方法为标准，对PMA-RF-SRCA方法的相对敏感性、相对特异性、相对符合率进行计算，以对本方法的实用性进行评估。结果：此方法的引物特异性良好，12?株副溶血性弧菌结果均呈阳性，18?株非副溶血性弧菌结果均呈阴性；PMA-RF-SRCA法灵敏度为3.2×100?CFU/mL，检出限为2.08×101?CFU/g，此方法的灵敏度是PMA-real-time PCR的100?倍。经过实际样品检测，PMA-RF-SRCA方法的敏感性、特异性、符合率分别为100.00%、96.00%和98.68%。结论：PMA-RF-SRCA方法特异性强、灵敏度高，可用于虾产品中的活副溶血性弧菌的检测与筛查。

关键词: 实时荧光跨越式滚环等温扩增；叠氮溴化丙锭；副溶血性弧菌；toxR基因；食品；检测

Abstract: Objective: A novel and rapid method for the detection of Vibrio parahaemolyticus in shrimp products was established by combined use of real-time fluorescent saltatory rolling circle amplification (SRCA) and propidium monoazide (PMA). Methods: Primers were designed and screened based on the V. parahaemolyticus-specific gene toxR. The specificity, sensitivity and detection limit of the PMA-RF-SRCA method were analyzed and compared with those of PMA combined with real-time polymerase chain reaction (PMA-real-time PCR). To evaluate its practicability, the relative sensitivity, relative specificity and relative accuracy of PMA-RF-SRCA were calculated using the national standard method GB 4789.7-2013 as a reference standard when it was used to detect 76 samples. Results: The specificity of the designed primers was excellent, giving positive results for 12 V. parahaemolyticus strains and negative results for 18 non-V. parahaemolytic strains. PMA-RF-SRCA was 100-fold more sensitive than PMA-RF-SRCA with a sensitivity of 3.2 × 100 CFU/mL and a detection limit of 2.08 × 101 CFU/g. Compared with the GB 4789.7-2013 method, the relative sensitivity, relative specificity and relative accuracy of PMA-RF-SRCA were 100.00%, 96.00% and 98.68%, respectively. Conclusion: The PMA-RF-SRCA method has the advantages of strong specificity and high sensitivity, and is suitable for the detection and screening of live V. parahaemolyticus in edible crustaceans.

Key words: real-time fluorescent saltatory rolling circle amplification; propidium monoazide; Vibrio parahaemolyticus; gene toxR; food; detection

中图分类号:

TS201

董晶，徐慧，郭威，卢鑫，张伟,. 实时荧光跨越式滚环等温扩增结合PMA检测虾产品中的活副溶血性弧菌[J]. 食品科学, 2021, 42(24): 289-295.

DONG Jing, XU Hui, GUO Wei, LU Xin, ZHANG Wei. Detection of Vibrio parahaemolyticus in Edible Crustaceans by Real-time Fluorescence Saltatory Rolling Circle Amplification Combined with Propidium Monoazide[J]. FOOD SCIENCE, 2021, 42(24): 289-295.

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实时荧光跨越式滚环等温扩增结合PMA检测虾产品中的活副溶血性弧菌

Detection of Vibrio parahaemolyticus in Edible Crustaceans by Real-time Fluorescence Saltatory Rolling Circle Amplification Combined with Propidium Monoazide

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