实时定量PCR法快速检测水产品中的副溶血性弧菌
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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。
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.
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.
引文
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[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.
[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.