Establishment of triple real- time PCR detection method for Vibrio cholera,Vibrio Parahaemolyticus and Listeria monocytogenes in marine food products
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摘要: 目的:建立一种同时定量检测霍乱弧菌、副溶血性弧菌、单增李斯特菌的三重PCR方法。方法:依据霍乱弧菌CTX遗传单元中zot毒力基因;副溶血性弧菌TDH中tl基因;单增李斯特菌hly基因,设计特异性引物探针,建立和评价了检测试剂盒性能。结果:霍乱弧菌、副溶血性弧菌、单增李斯特菌浓度在10~2~10~5cfu/m L标准曲线线性相关系数分别为0.9998、0.999、0.9963。三种致病菌10~5cfu/m L与10~3cfu/m L两种检测浓度对数值,批内变异系数分别为0.40%、0.83%、0.49%与0.75%、0.81%、0.64%;0.76%、1.03%、0.62%与1.06%、0.72%、1.26%;0.62%、0.51%、0.81%和1.38%、0.76%、2.34%;批间变异系数分别为0.58%与0.76%;0.83%与1.01%;0.66%与1.58%。检测灵敏度分别为:23、16、35 cfu/m L。结论:本文所建立的检测试剂盒适合于海产品等产品中三种致病菌的快速筛检。Abstract: Objective: In order to establish a effective method to detect Vibrio cholera,Vibrio Parahaemolyticus and Listeria monocytogenes by triple real- time PCR.Methods: Specific primers and probes were designed,which based on the zot toxin gene of CTX in Vibrio cholera,tl gene of TDH in Vibrio Parahaemolyticus and hly gene of Listeria monocytogenes. What is more,the performance of the test kit was established and evaluated. Results: The correlation coefficients of standard curve linears for Vibrio cholera,Vibrio Parahaemolyticus and Listeria monocytogenes were 0.9998,0.999 and 0.9963. The intra- assay coefficient variables of the three pathpgens at 10~5 cfu / m L and 103 cfu / m L concentration logarithm were 0.40%,0.83%,0.49% and0.75%,0.81%,0.64%; 0.76%,1.03%,0.62% and 1.06%,0.72%,1.26%; 0.62%,0.51%,0.81% and 1.38%,0.76%,2.34%; inter- assay coefficient variables were 0.58% and 0.76%,0.83% and 1.01%,0.66% and 1.58%. The sensitivity indicated that the lowest accurate quantitative concentrations of Vibrio cholerae,Vibrio parahaemolyticus,Listeria monocytogenes were 23,16,35 cfu / m L. Conclusion: A rapid detection method for testing the three pathogens of aquatic products was established.
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[1] 滕勇勇,王琪,吴雷,等.致病性弧菌的生物学特性和致病因子研究进展[J].热带医学杂志,2014,14(10):1396-1398. [2] Liu D.Identification,subtyping and virulence determination of Listeria monocytogenes,an omprotant food-borne pathogen[J].Journal of Medical Microbiology,2006,55(6):645-659.
[3] Marija Z,Konrad J D,Wolfgang K.Practical relevance of methodologies for detecting and tracing of Listeria monocytogenes in ready to eat foods and manufacture environments[J].Food Science and Technology,2011,44(2):351-362.
[4] Chstterjee S,Ghosh K,Raychoudhuri A,et al.Phenotypic and genotypic traits and epidemiological implication of Vibrio Cholerae O1 and O139 strains in India during 2003[J].J Med Microbiol,2007,56(6):828-832.
[5] 中华人民共和国卫生部疾病预防控制司.霍乱防治手册[M].第六版.北京:人民卫生出版社,2012. [6] Jio R,Si H P,Yeon SY.Simultaneous detection of Listeria species isolated from meat processed food s using multiplex PCR[J].Food Control,2013,32(2):659-664.
[7] 江迎鸿,谭贵良,陈亚波,等.多重PCR方法检测食品中霍乱弧菌、副溶血性弧菌和单核细胞增生李斯特氏菌[J].广东农业科学,2011(11):135-137. [8] 段强德,王录军,付宏岐,等.多重PCR快速检测食品中单核细胞增生性李斯特菌检测分析[J].山西农业科学,2015,61(3):38-40. [9] 蒋蔚,易力,陈永军,等.水产品中霍乱弧菌、副溶血弧菌和创伤弧菌多重PCR检测方法的建立[J].中国动物传染病学报,2016,24(1):44-51. [10] 陈丽,张红河,曹海芬,等.荧光聚合酶链反应检测副溶血弧菌TLH与TDH[J].检验医学;2007,22(4):450-454 [11] Fykse E M,Nilsen T,Nielsen A D,et al.Real-time PCR and NASBA for rapid and sensitive detection of vibrio cholera in ballast water[J].Mar Pollut Bull,2012,64(2):200-206.
[12] 李宏,杨大伟,刘云国,等.多重荧光定量PCR同时检测霍乱弧菌、副溶血性弧菌和创伤弧菌的方法研究[J].中国卫生检验杂志,2011,21(5):1180-1182. [13] 胡兴娟,沈飚,余辉,等.多重荧光定量PCR法检测海产品中副溶血性弧菌、沙门氏菌和单增李斯特菌[J].中国食品卫生杂志,2016,28(4):440-444. [14] Edwards KA,March JC.GM(1)-Functionalized Liposomes in a Microtiter Plate Assay for Cholera Toxin in Vibrio Cholerae Culture Samples[J].Anal Biochem,2007,368(1):39-48.
[15] 王环宇,李亚鹏,杨军,等.食品中单核细胞增生性李斯特氏菌PCR快速检测方法[J].中国食品卫生杂志,2004,16:10-13.
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