检测创伤弧菌两种毒力基因的环介导等温扩增检测方法的建立
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摘要
为建立快速检测致病性创伤弧菌两种毒素的环介导等温扩增(loop-mediated isothermal amplification,LAMP)方法,分别以创伤弧菌溶血素A基因(hemolysin gene A,HA)和多重毒素(repeats in toxin,RTX)毒力基因的保守序列作为靶序列设计内、外引物。通过肉眼观察白色沉淀初步判断检测结果,通过琼脂糖电泳最终确定检测结果。LAMP检测创伤弧菌的两种基因的灵敏度都达到10 CFU/mL。特异性结果表明致病性创伤弧菌的结果为阳性,而其他几种常见弧菌和食源菌检测结果为阴性。在人工模拟样品检测中,LAMP结果显示,采用水煮法提取DNA,从样品处理到报告结果耗时1.5 h,鱼肉中创伤弧菌的检出限为1×10~2 CFU/g(RTX)和1×10~4 CFU/g(HA);采用同样方法提取DNA进行普通PCR,其检出限为1×10~3 CFU/g(RTX)和1×10~4 CFU/g(HA),耗时4 h。结果表明,本研究建立的LAMP方法检测创伤弧菌两种毒力基因时具有灵敏度高、特异性强、方法简便等优点,适用于食品和养殖业中的现场快速检测。
In the present study, we developed and validated a loop-mediated isothermal amplification(LAMP) assay for detection of two toxins genes of Vibrio vulnificus. The inner and outer primers of LAMP were designed based on the conserved sequences of V. vulnificus hemolysin gene A(HA) and repeats-in-toxin(RTX) virulent gene. The detection method was preliminary evaluated by formation of the white deposition and confirmed through agarose gel electrophoresis. The sensitivity of this detection method was 10 CFU/mL for two LAMP genes of V. vulnificus. This method showed good specificity to V. vulnificus as it did not react with any other bacterial species. The bacterial DNA was extracted by water-boiling method and it took 1.5 h from sample processing to obtaining results. The detection limits were 10~2 CFU/g for RTX and 10~4 CFU/g for HA. On the contrast, the detection limit of common PCR method was 10~3 CFU/g for RTX and 10~4 CFU/g for HA and it took 4 h to complete test. This LAMP method may be used for on-site diagnostics of V. vulnificus in the food and agricultural industries.
In the present study, we developed and validated a loop-mediated isothermal amplification(LAMP) assay for detection of two toxins genes of Vibrio vulnificus. The inner and outer primers of LAMP were designed based on the conserved sequences of V. vulnificus hemolysin gene A(HA) and repeats-in-toxin(RTX) virulent gene. The detection method was preliminary evaluated by formation of the white deposition and confirmed through agarose gel electrophoresis. The sensitivity of this detection method was 10 CFU/mL for two LAMP genes of V. vulnificus. This method showed good specificity to V. vulnificus as it did not react with any other bacterial species. The bacterial DNA was extracted by water-boiling method and it took 1.5 h from sample processing to obtaining results. The detection limits were 10~2 CFU/g for RTX and 10~4 CFU/g for HA. On the contrast, the detection limit of common PCR method was 10~3 CFU/g for RTX and 10~4 CFU/g for HA and it took 4 h to complete test. This LAMP method may be used for on-site diagnostics of V. vulnificus in the food and agricultural industries.
引文
[1]Han F F,Wang F,Ge B.Detecting potentially virulent Vibrio vulnificus strains in raw oysters by quantitative loop-mediated isothermal amplification[J].Appl Environ Microbiol,2011,77(8):2589-2595.
[2]Surasilp T,Longyant S,Rukpratanporn S,et al.Rapid and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification combined with lateral flow dipstick targeted to rpo S gene[J].Mol Cell Probes,2011,25(4):158-163.
[3]Han F F,Ge B.Quantitative detection of Vibrio vulnificusin raw oysters by real-time loop-mediated isothermal amplification[J].Int J Food Microbiol,2010,142(1-2):60-66.
[4]Ren C H,Hu C Q,Luo P,et al.Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification[J].Microbiol Res,2008,164(5):514-521.
[5]Han F F,Ge B.Evaluation of a loop-mediated isothermal amplification assay for detecting Vibrio vulnificus in raw oysters[J].Foodborne Pathog Dis,2008,5(3):311-320.
[6]Nemoto J,Kojima T,Kusaba K,et al.Rapid detection of Vibrio vulnificus using a loop-mediated isothermal amplification method[J].Kansenshogaku Zasshi,2008,82(5):407-413.
[7]Fall J,Chakraborty G,Kono T,et al.Establishment of loop-mediated isothermal amplification method(LAMP)for the detection of Vibrio nigripulchritudo in shrimp[J].FEMS Microbiol Lett,2008,288(2):171-177.
[8]孙敏.创伤弧菌LAMP检测方法的建立6[A]//中国畜牧兽医学会.动物检疫学分会2010年学术年会论文集[C].中国畜牧兽医学会,2010,5:12-17.
[9]徐义刚,李苏龙,杨君宏,等.水产品中创伤弧菌DNA环介导恒温扩增快速检测方法的建立及初步应用[J].中国生物工程杂志,2010(6):96-102.
[10]张静,施慧,谢建军,等.利用LAMP法快速检测致病性哈维氏弧菌[J].上海海洋大学学报,2010,19(5):601-607.
[11]王耀焕,王瑞娜,周前进,等.环介导等温扩增联合横向流动试纸条快速检测创伤弧菌检测方法的建立[J].生物技术通报,2014(6):81-87.
[12]薛超波,许镇坚,黄朱梁,等.环介导等温扩增技术检测创伤弧菌方法的建立与应用[J].食品科学,2012,33(20):262-264.
[13]李桂满,侯敏,颜军,等.环介导等温扩增技术检测副溶血性弧菌的优势[J].职业与健康,2013,29(5):573-574+579.
[14]薛超波,黄朱梁,孙瑛,等.海产品中创伤弧菌实时浊度LAMP检测方法的建立[J].中国预防兽医学报,2013,35(11):912-915.
[15]张丽娜,王明义,杨小蕾,等.海洋创伤弧菌LAMP快速诊断方法的建立与评价[J].国际检验医学杂志,2016,37(5):577-579+582.
[16]周顺,高志鑫,张敏.创伤弧菌和副溶血弧菌双重LAMP检测方法的建立及初步应用[J].中国兽医学报,2016,36(11):1875-1881.
[17]S Zhou,Z X Gao,M Zhang,et al.Development of a quadruplex loop-mediated isothermal amplification assay for field detection of four Vibrio species associated with fish disease[J].Springer Plus,2016,5(1):1104.
[18]Zhou Q J,Wang L,Chen J,et al.Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip(on-chip LAMP)for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals[J].J Microbiol Methods,2014,104:26-35.
[19]Jones J L,Hara-Kudo Y,Krantz J A,et al.Comparison of molecular detection methods for Vibrio parahaemolyticus and Vibrio vulnificus[J].Food Microbiol,2012,30(1):105-111.
[2]Surasilp T,Longyant S,Rukpratanporn S,et al.Rapid and sensitive detection of Vibrio vulnificus by loop-mediated isothermal amplification combined with lateral flow dipstick targeted to rpo S gene[J].Mol Cell Probes,2011,25(4):158-163.
[3]Han F F,Ge B.Quantitative detection of Vibrio vulnificusin raw oysters by real-time loop-mediated isothermal amplification[J].Int J Food Microbiol,2010,142(1-2):60-66.
[4]Ren C H,Hu C Q,Luo P,et al.Sensitive and rapid identification of Vibrio vulnificus by loop-mediated isothermal amplification[J].Microbiol Res,2008,164(5):514-521.
[5]Han F F,Ge B.Evaluation of a loop-mediated isothermal amplification assay for detecting Vibrio vulnificus in raw oysters[J].Foodborne Pathog Dis,2008,5(3):311-320.
[6]Nemoto J,Kojima T,Kusaba K,et al.Rapid detection of Vibrio vulnificus using a loop-mediated isothermal amplification method[J].Kansenshogaku Zasshi,2008,82(5):407-413.
[7]Fall J,Chakraborty G,Kono T,et al.Establishment of loop-mediated isothermal amplification method(LAMP)for the detection of Vibrio nigripulchritudo in shrimp[J].FEMS Microbiol Lett,2008,288(2):171-177.
[8]孙敏.创伤弧菌LAMP检测方法的建立6[A]//中国畜牧兽医学会.动物检疫学分会2010年学术年会论文集[C].中国畜牧兽医学会,2010,5:12-17.
[9]徐义刚,李苏龙,杨君宏,等.水产品中创伤弧菌DNA环介导恒温扩增快速检测方法的建立及初步应用[J].中国生物工程杂志,2010(6):96-102.
[10]张静,施慧,谢建军,等.利用LAMP法快速检测致病性哈维氏弧菌[J].上海海洋大学学报,2010,19(5):601-607.
[11]王耀焕,王瑞娜,周前进,等.环介导等温扩增联合横向流动试纸条快速检测创伤弧菌检测方法的建立[J].生物技术通报,2014(6):81-87.
[12]薛超波,许镇坚,黄朱梁,等.环介导等温扩增技术检测创伤弧菌方法的建立与应用[J].食品科学,2012,33(20):262-264.
[13]李桂满,侯敏,颜军,等.环介导等温扩增技术检测副溶血性弧菌的优势[J].职业与健康,2013,29(5):573-574+579.
[14]薛超波,黄朱梁,孙瑛,等.海产品中创伤弧菌实时浊度LAMP检测方法的建立[J].中国预防兽医学报,2013,35(11):912-915.
[15]张丽娜,王明义,杨小蕾,等.海洋创伤弧菌LAMP快速诊断方法的建立与评价[J].国际检验医学杂志,2016,37(5):577-579+582.
[16]周顺,高志鑫,张敏.创伤弧菌和副溶血弧菌双重LAMP检测方法的建立及初步应用[J].中国兽医学报,2016,36(11):1875-1881.
[17]S Zhou,Z X Gao,M Zhang,et al.Development of a quadruplex loop-mediated isothermal amplification assay for field detection of four Vibrio species associated with fish disease[J].Springer Plus,2016,5(1):1104.
[18]Zhou Q J,Wang L,Chen J,et al.Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip(on-chip LAMP)for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals[J].J Microbiol Methods,2014,104:26-35.
[19]Jones J L,Hara-Kudo Y,Krantz J A,et al.Comparison of molecular detection methods for Vibrio parahaemolyticus and Vibrio vulnificus[J].Food Microbiol,2012,30(1):105-111.