致病性副溶血性弧菌菌落杂交检测体系的优化
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摘要
对常见食源性致病菌副溶血性弧菌的杂交条件进行了优化。基于副溶血性弧菌的tdh,trh,toxR基因选用了3种探针序列,从菌落杂交样品的预处理方法、杂交时间和显色检测等方面对基于副溶血性弧菌毒力基因的原位杂交实验进行了条件优化。结果表明,采用80℃热固定2 h,37℃预杂交10 min后杂交8 h以及封闭1 h的改良方法可获得高特异性、低背景且着色清晰的实验结果。优化的菌落杂交技术检测副溶血性弧菌与传统检测方法相比,具有高效、准确、可区分致病性菌株的优点,为水产品中副溶血性弧菌致病菌株和非致病菌株的同步检测和筛选提供参考。
Based on selected 3 reported probe sequences of tdh,trh,and toxR,hybridization conditions of common foodborne pathogenic V. parahaemolyticus( Vp) have been optimized,including pretreatment of colony hybridization samples,time,and developing and other aspects were carried out conditions optimization based on gene hybridization experiment in situ of Vp virulence. The results showed that a high specific,low background,and distinctly colored results have been acquired adopting an improved methods of 80 ℃ heat fixation for 2 h,hybridization for 8 h after 37℃ pre-hybridization for 30 min as well as sealing for 1 h. As compared with traditional detection method the optimized colony hybridization technology to detect Vp possessing the advantages of high efficiency,accuracy,being able to distinguish. This study provides a technical support for the detection of pathogenic and nonpathogenic Vp in aquaproducts synchronistic detection and screening.
Based on selected 3 reported probe sequences of tdh,trh,and toxR,hybridization conditions of common foodborne pathogenic V. parahaemolyticus( Vp) have been optimized,including pretreatment of colony hybridization samples,time,and developing and other aspects were carried out conditions optimization based on gene hybridization experiment in situ of Vp virulence. The results showed that a high specific,low background,and distinctly colored results have been acquired adopting an improved methods of 80 ℃ heat fixation for 2 h,hybridization for 8 h after 37℃ pre-hybridization for 30 min as well as sealing for 1 h. As compared with traditional detection method the optimized colony hybridization technology to detect Vp possessing the advantages of high efficiency,accuracy,being able to distinguish. This study provides a technical support for the detection of pathogenic and nonpathogenic Vp in aquaproducts synchronistic detection and screening.
引文
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[13]Suffredini E,Cozzi L,Ciccaglioni G,et al.Development of a colony hybridization method for the enumeration of total and potentially enteropathogenic Vibrio parahaemolyticus in shellfish[J].International journal of food microbiology,2014,186:22-31.
[14]刘超兰,王海英,李文芳,等.菌落原位杂交法筛选特定菌种的方法初探[J].酿酒科技,2013,(8):15-18.
[15]吕欣,彭霞薇,呼庆,等.利用DGGE-菌落原位杂交法分离土壤中精喹禾灵降解菌[J].环境科学,2013,1(1):263-270.
[16]杨颖,张伟,区炳明,等.猪源大肠杆菌Nissle 1917菌株的分离鉴定[J].微生物学通报,2017,44(4):845-851.
[17]Saidi SM,Chowdhury N,Awasthi SP,et al.Prevalence of Vibrio cholerae O1 E1 Torvariant in a cholera-endemic zone of Kenya[J].Journal of medical microbiology,2014,63(3):415-420.
[18]丁海燕,冯伟,李凌智,等.菌落原位杂交技术在环境微生物检测中的应用[J].安徽农业科学,2015,43(14):25-26.
[19]Givens CE,Bowers JC,De Paola A,et al.Occurrence and distribution of Vibrio vulnificus and Vibrio parahaemolyticuspotential roles for fish,oyster,sediment and water[J].Letters in applied microbiology,2014,58(6):503-510.
[2]Ma C,Deng X,Ke C,et al.Epidemiology and etiology characteristics of foodborne outbreaks caused by Vibrio parahaemolyticus during 2008-2010 in Guangdong province,China[J].Foodborne pathogens and disease,2014,11(1):21-29.
[3]Wang S,Duan H,Zhang W,et al.Analysis of bacterial foodborne disease outbreaks in China between 1994 and 2005[J].Pathogens and Disease,2007,51(1):8-13.
[4]Chiou CS,Hsu SY,Chiu SI,et al.Vibrio parahaemolyticus serovar O3:K6 as cause of unusually high incidence of foodborne disease outbreaks in Taiwan from 1996 to 1999[J].Journal of Clinical Microbiology,2000,38(12):4621-4625.
[5]Gooch JA,Depaola A,Bowers J,et al.Growth and survival of Vibrio parahaemolyticus in postharvest American oysters[J].Journal of food protection,2002,65(6):970-974.
[6]Okuda J,Ishibashi M,Hayakawa E,et al.Emergence of a unique O3:K6 clone of Vibrio parahaemolyticus in Calcutta,India,and isolation of strains from the same clonal group from Southeast Asian travelers arriving in Japan[J].Journal of Clinical Microbiology,1997,35(12):3150-3155.
[7]Yamamoto A,Iwahori J,Vuddhakul V,et al.Quantitative modeling for risk assessment of Vibrio parahaemolyticus in bloody clams in southern Thailand[J].International journal of food microbiology,2008,124(1):70-78.
[8]Daniels NA,Ray B,Easton A,et al.Emergence of a new Vibrio parahaemolyticus serotype in raw oysters:a prevention quandary[J].Jama,2000,284(12):1541-1545.
[9]Su YC,Liu C.Vibrio parahaemolyticus:a concern of seafood safety[J].Food microbiology,2007,24(6):549-558.
[10]赵永刚.副溶血弧菌tdh、trh和tlh基因的克隆、表达及基因敲除对其溶血活性的影响[D].青岛:中国海洋大学,2010:11.
[11]Shirai H,Ito H,Hirayama T,et al.Molecular epidemiologic evidence for association of thermostable direct hemolysin(TDH)and TDH-related hemolysin of Vibrio parahaemolyticus with gastroenteritis[J].Infection and immunity,1990,58(11):3568-3573.
[12]谢冰,徐亚同.环境微生物的分子生物学研究方法[J].世界科技研究与发展,2003,25(2):48-53.
[13]Suffredini E,Cozzi L,Ciccaglioni G,et al.Development of a colony hybridization method for the enumeration of total and potentially enteropathogenic Vibrio parahaemolyticus in shellfish[J].International journal of food microbiology,2014,186:22-31.
[14]刘超兰,王海英,李文芳,等.菌落原位杂交法筛选特定菌种的方法初探[J].酿酒科技,2013,(8):15-18.
[15]吕欣,彭霞薇,呼庆,等.利用DGGE-菌落原位杂交法分离土壤中精喹禾灵降解菌[J].环境科学,2013,1(1):263-270.
[16]杨颖,张伟,区炳明,等.猪源大肠杆菌Nissle 1917菌株的分离鉴定[J].微生物学通报,2017,44(4):845-851.
[17]Saidi SM,Chowdhury N,Awasthi SP,et al.Prevalence of Vibrio cholerae O1 E1 Torvariant in a cholera-endemic zone of Kenya[J].Journal of medical microbiology,2014,63(3):415-420.
[18]丁海燕,冯伟,李凌智,等.菌落原位杂交技术在环境微生物检测中的应用[J].安徽农业科学,2015,43(14):25-26.
[19]Givens CE,Bowers JC,De Paola A,et al.Occurrence and distribution of Vibrio vulnificus and Vibrio parahaemolyticuspotential roles for fish,oyster,sediment and water[J].Letters in applied microbiology,2014,58(6):503-510.