实时定量PCR法快速检测水产品中的副溶血性弧菌

doi:10.3969/j.issn.1004-1524.2019.05.19

浙江农业学报 ›› 2019, Vol. 31 ›› Issue (5): 823-828.DOI: 10.3969/j.issn.1004-1524.2019.05.19

实时定量PCR法快速检测水产品中的副溶血性弧菌

陈琳^1,2, 周青青¹, 顾青^1,*, 郦萍¹

1.浙江工商大学浙江省食品微生物技术研究重点实验室,浙江杭州 310018;
2.杭州娃哈哈集团有限公司,浙江杭州 310018

收稿日期:2018-12-07 出版日期:2019-05-25 发布日期:2019-05-23
通讯作者: *顾青,E-mail: guqing2002@hotmail.com
作者简介:陈琳(1985—),女,黑龙江牡丹江人,博士研究生,研究方向为食品生物技术。E-mail: chenlinlucky@163.com
基金资助:
浙江省重大专项(2015C02039,2015C02022);国家自然科学基金(31606449,31871775);浙江省自然科学基金(LY16C200002,LQ18C200004)

Detection of Vibrio parahaemolyticus in seafood products by real-time quantitative PCR assay

CHEN Lin^1,2, ZHOU Qingqing¹, GU Qing^1,*, LI Ping¹

1. Key Laboratory for Food Microbial Technology of Zhejiang Province, Zhejiang Gongshang University, Hangzhou 310018, China;
2. Hangzhou Wahaha Group Co., Ltd., Hangzhou 310018, China

Received:2018-12-07 Online:2019-05-25 Published:2019-05-23

摘要/Abstract

摘要： 副溶血性弧菌是一种广泛存在于水产品中的食源性致病菌,可污染食物,引起严重的食物中毒。利用实时定量PCR方法建立一种快速检测水产品中副溶血性弧菌的方法,根据随机扩增多态性分析(RAPD)鉴定特异性片段,设计特异性引物。采用建立的实时定量PCR方法检测市售水产品20份,检出阳性食品6份,最小检测灵敏度为50 fg DNA,与传统微生物检测结果相比无显著差异。该检测方法特异性强,灵敏度高。

关键词: 副溶血性弧菌, 随机扩增多态性分析, 实时荧光定量PCR

Abstract: Vibrio parahaemolyticus is a food-borne pathogen, which can contaminate food and cause serious and acute gastroenteritis to human beings. In this study, a real-time polymerase chain reaction (PCR) assay was developed for the quantitative detection of Vibrio parahaemolyticus in aquatic products. Randomly amplified polymorphic DNA (RAPD) method was introduced to characterize species-specific DNA fragment of V. parahaemolyticus, and specific primers were designed. The proposed method was used to detect V. parahaemolyticus in 20 aquatic products on sale. It was shown that 6 samples out of 20 were carrying V. parahaemolyticus. The minimum detection sensitivity was 50 fg DNA. Compared with the methods recommended in national standard, the proposed method was swifter and more convenient to detect V. parahaemolyticus in auqatic products.

Key words: Vibrio parahaemolyticus, randomly amplified polymorphic DNA, real-time polymerase chain reaction

中图分类号:

陈琳, 周青青, 顾青, 郦萍. 实时定量PCR法快速检测水产品中的副溶血性弧菌[J]. 浙江农业学报, 2019, 31(5): 823-828.

CHEN Lin, ZHOU Qingqing, GU Qing, LI Ping. Detection of Vibrio parahaemolyticus in seafood products by real-time quantitative PCR assay[J]. , 2019, 31(5): 823-828.

参考文献

[1] AARTS H J M, JOOSTEN R G, HENKENS M H C, et al. Rapid duplex PCR assay for the detection of pathogenic Yersinia enterocolitica strains[J]. Journal of Microbiological Methods, 2001, 47(2): 209-217.
[2] YIN J F, WANG M Y, CHEN Y J, et al.Direct detection of Vibrio Vulnificus, Vibrio Parahaemolyticus, and Vibrio alginolyticus from clinical and environmental samples by a multiplex touchdown polymerase chain reaction assay[J]. Surgical Infections, 2018, 19(1): 48-53.
[3] ELMAHDI S, DASILVA L V, PARVEEN S.Antibiotic resistance of Vibrio parahaemolyticus and Vibrio vulnificus in various countries: a review[J]. Food Microbiology, 2016, 57: 128-134.
[4] JOTHIKUMAR N, GRIFFITHS M W.Rapid detection of Escherichia coli O157: H7 with multiplex real-time PCR assays[J]. Applied and Environmental Microbiology, 2002, 68(6): 3169-3171.
[5] LO C L H, LEUNG P H M, YIP S P, et al. Rapid detection of pathogenic Vibrio parahaemolyticus by a sensitive and specific duplex PCR-hybridization probes assay using LightCycler[J]. Journal of Applied Microbiology, 2008, 105(2): 575-584.
[6] MALINEN E.Comparison of real-time PCR with SYBR Green I or 5'-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria[J]. Microbiology, 2003, 149(1): 269-277.
[7] DOYLE T J.Completeness of notifiable infectious disease reporting in the United States: an analytical literature review[J]. American Journal of Epidemiology, 2002, 155(9): 866-874.
[8] 梅玲玲, 潘雪霞, 朱敏, 等. 浙江省副溶血性弧菌污染水平及贝类海产品风险评估[J]. 中国人兽共患病学报, 2012, 28(7): 700-704, 717.
MEI L L, PAN X X, ZHU M, et al.Contamination of Vibrio parahaemolyticus in Zhejiang Province and its risk assessment in shellfish[J]. Chinese Journal of Zoonoses, 2012, 28(7): 700-704, 717.(in Chinese with English abstract)
[9] KAUFMAN G E, BLACKSTONE G M, VICKERY M C L, et al. Real-time PCR quantification of Vibrio parahaemolyticus in oysters using an alternative matrix[J]. Journal of Food Protection, 2004, 67(11): 2424-2429.
[10] SU Y C, LIU C C.Vibrio parahaemolyticus: a concern of seafood safety[J]. Food Microbiology, 2007, 24(6): 549-558.
[11] 周微, 张伟钦, 付宇, 等. 荧光定量PCR方法快速检测原料乳中的大肠杆菌[J]. 中国乳品工业, 2009, 37(11): 39-42.
ZHOU W, ZHANG W Q, FU Y, et al.SYBR GreenⅠ fluorescence quantitative PCR detection of Escherichia coli in raw milk[J]. China Dairy Industry, 2009, 37(11): 39-42.(in Chinese with English abstract)
[12] 郭蔷, 冮洁, 武晓松. 采用荧光定量PCR方法快速检测蔬菜中沙门氏菌[J]. 食品研究与开发, 2016, 37(7): 123-127.
GUO Q, GANG J, WU X S.Real time fluorescence quantitative PCR method for rapid detection salmonella in vegetables[J]. Food Research and Development, 2016, 37(7): 123-127.(in Chinese with English abstract)
[13] SKORUPSKI K, TAYLOR R K.Control of the ToxR virulence regulon in Vibrio cholera by environmental stimuli[J]. Molecular Microbiology, 1997, 25(6): 1003-1009.
[14] GUTIERREZ WEST C K, KLEIN S L, LOVELL C R. High frequency of virulence factor genes tdh, trh, and tlh in Vibrio parahaemolyticus strains isolated from a pristine estuary[J]. Applied and Environmental Microbiology, 2013, 79(7): 2247-2252.
[15] TAKAHASHI H, IWADE Y, KONUMA H, et al.Development of a quantitative real-time PCR method for estimation of the total number of Vibrio parahaemolyticus in contaminated shellfish and seawater[J]. Journal of Food Protection, 2005, 68(5):1083-1088.

实时定量PCR法快速检测水产品中的副溶血性弧菌

Detection of Vibrio parahaemolyticus in seafood products by real-time quantitative PCR assay

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