反转录-环介导等温扩增技术快速检测志贺氏菌
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摘要
建立了反转录-环介导等温扩增技术的志贺氏菌快速检测方法。针对志贺氏菌特异保守基因ipa H设计多套引物,建立优化了的反应体系,并评价其准确性、特异性、灵敏度。以人工污染脱脂乳样品比较RT-LAMP与RT-PCR的灵敏度。结果表明,在65℃等温条件下,RT-LAMP反应可在30 min内完成。在活菌/损伤菌模型中,该方法表现出比国标法更好的准确性。在23株细菌标准菌株中仅对4株志贺氏菌有特异性检出。志贺氏菌RNA模板的检测灵敏度为7 fg/μL。人工污染脱脂乳样品检测灵敏度达到40 CFU/g,比RT-PCR方法高出2个数量级。表明所建立的志贺氏菌RT-LAMP扩增方法可以准确的检测志贺氏菌,同时具有快速、特异、灵敏的优势,可用于志贺氏菌的快速筛查和现场监控。
This study aimed to establish a method using reverse transcriptase loop-mediated isothermal amplification( RT-LAMP) for rapid detection of Shigella sp.. Multiple sets of primers were designed according to the specific conserved gene ipaH of genus Shigella. An optimized reaction system was established,and its accuracy,specificity,as well as sensitivity were evaluated using artificial contaminated samples. The results showed that RT-LAMP assay successfully detected ipaH within 30 min under isothermal conditions at 65 °C. In the viable/damage bacteria model,this method even showed suitable accuracy better than that of the national standard method. Only four strains from Shigella sp. were specifically detected from a mixture of 23 different bacterial standard strains. The detection sensitivity of Shigella RNA was 7 fg/μL. The detection sensitivity of artificially contaminated skimmed milk samples reached40 CFU/g,which was two orders of magnitude higher than the RT-PCR method. This RT-LAMP assay could accurately detect genus Shigella. This assay was rapid,specific and sensitive,which could be used for rapid screening and onsite monitoring of Shigella sp..
This study aimed to establish a method using reverse transcriptase loop-mediated isothermal amplification( RT-LAMP) for rapid detection of Shigella sp.. Multiple sets of primers were designed according to the specific conserved gene ipaH of genus Shigella. An optimized reaction system was established,and its accuracy,specificity,as well as sensitivity were evaluated using artificial contaminated samples. The results showed that RT-LAMP assay successfully detected ipaH within 30 min under isothermal conditions at 65 °C. In the viable/damage bacteria model,this method even showed suitable accuracy better than that of the national standard method. Only four strains from Shigella sp. were specifically detected from a mixture of 23 different bacterial standard strains. The detection sensitivity of Shigella RNA was 7 fg/μL. The detection sensitivity of artificially contaminated skimmed milk samples reached40 CFU/g,which was two orders of magnitude higher than the RT-PCR method. This RT-LAMP assay could accurately detect genus Shigella. This assay was rapid,specific and sensitive,which could be used for rapid screening and onsite monitoring of Shigella sp..
引文
[1]张帅,齐颖颖,张红星,等.快速检测生鲜肉中三种食源性致病菌[J].江苏农业学报,2016,32(4):939-945.
[2]LAURI A,CASTIGLIONI B,MARIANI P.Comprehensive analysis of Salmonella,sequence polymorphisms and development of a LDR-UA assay for the detection and characterization of selected serotypes[J].Applied Microbiology&Biotechnology,2011,91(1):189-210.
[3]KOTLOFF K L,WINICKOFF J P,IVANOFF B,et al.Global burden of Shigella infections:Implications for vaccine development and implementation of control strategies.[J].Bulletin of the World Health Organization,1999,77(8):651-666.
[4]崔庆刚,杨志远,杜永新,等.志贺氏菌的生化特性及检测方法的研究进展[J].上海畜牧兽医通讯,2017(1):23-25.
[5]NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loopmediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):E63.
[6]李月华,付博宇,马晓燕,等.原料乳中志贺氏菌的实时荧光环介导等温扩增技术研究[J].食品科技,2016(1):315-320.
[7]刘旭.三种食源性致病菌的LAMP检测方法研究[D].杭州:中国计量学院,2012.
[8]YAN M,XU L,JIANG H,et al.PMA-LAMP for rapid detection of Escherichia coli and shiga toxins from viable but non-culturable state[J].Microb Pathog,2017,105:245-250.
[9]李月,王丽,孙远明,等.DNA染料结合环介导等温扩增技术检测志贺氏菌死活细胞[J].食品工业科技,2011,32(8):216-219.
[10]KOBAYASHI H,OETHINGER M,TUOHY M J,et al.Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide[J].Letters in Applied Microbiology,2010,48(5):633-638.
[11]邹晓蕾,刘礼崔,罗立新.细菌总RNA提取方法的比较[J].现代食品科技,2013(8):1 948-195 4.
[12]GB 4789.5-2012食品安全国家标准食品微生物检验志贺氏菌检验[S].北京:中国标准出版社,2012.
[13]FAN F X,WANG S J,LOU J,et al.Establishment of RT-LAMP assay to detect Salmonella Typhi in blood[J].Disease Surveillance,2012.
[14]梁玉林,刘秀,周振森,等.基于反转录-环介导等温扩增技术检测金黄色葡萄球菌[J].食品与发酵工业,2018,44(1):217-223.
[15]WANG Y,LI J,QIAO P,et al.Screening and application of a new aptamer for the rapid detection of Sudan dye III[J].European Journal of Lipid Science&Technology,2018:1 700 112.
[16]ZHANG L,WEI Q,HAN Q,et al.Detection of shigellain milk and clinical samples by magnetic immunocaptured-loop-mediated isothermal amplification assay[J].Frontiers in Microbiology,2018,9:94.
[17]周杨,万强,蔡芷荷,等.环介导等温扩增技术在食品中痢疾志贺氏菌快速检测[J].微生物学通报,2017,44(9):2 247-2 254.
[18]曾桂芬,龙北国,姜容,等.LAMP和PCR法检测痢疾志贺菌的特异性和灵敏性比较[J].热带医学杂志,2012,12(5):547-549,592.
[19]王建昌,胡连霞,段永生,等.志贺氏菌实时荧光单引物等温扩增方法的建立及应用[J].食品科学技术学报,2015,33(6):40-45.
[2]LAURI A,CASTIGLIONI B,MARIANI P.Comprehensive analysis of Salmonella,sequence polymorphisms and development of a LDR-UA assay for the detection and characterization of selected serotypes[J].Applied Microbiology&Biotechnology,2011,91(1):189-210.
[3]KOTLOFF K L,WINICKOFF J P,IVANOFF B,et al.Global burden of Shigella infections:Implications for vaccine development and implementation of control strategies.[J].Bulletin of the World Health Organization,1999,77(8):651-666.
[4]崔庆刚,杨志远,杜永新,等.志贺氏菌的生化特性及检测方法的研究进展[J].上海畜牧兽医通讯,2017(1):23-25.
[5]NOTOMI T,OKAYAMA H,MASUBUCHI H,et al.Loopmediated isothermal amplification of DNA[J].Nucleic Acids Research,2000,28(12):E63.
[6]李月华,付博宇,马晓燕,等.原料乳中志贺氏菌的实时荧光环介导等温扩增技术研究[J].食品科技,2016(1):315-320.
[7]刘旭.三种食源性致病菌的LAMP检测方法研究[D].杭州:中国计量学院,2012.
[8]YAN M,XU L,JIANG H,et al.PMA-LAMP for rapid detection of Escherichia coli and shiga toxins from viable but non-culturable state[J].Microb Pathog,2017,105:245-250.
[9]李月,王丽,孙远明,等.DNA染料结合环介导等温扩增技术检测志贺氏菌死活细胞[J].食品工业科技,2011,32(8):216-219.
[10]KOBAYASHI H,OETHINGER M,TUOHY M J,et al.Unsuitable distinction between viable and dead Staphylococcus aureus and Staphylococcus epidermidis by ethidium bromide monoazide[J].Letters in Applied Microbiology,2010,48(5):633-638.
[11]邹晓蕾,刘礼崔,罗立新.细菌总RNA提取方法的比较[J].现代食品科技,2013(8):1 948-195 4.
[12]GB 4789.5-2012食品安全国家标准食品微生物检验志贺氏菌检验[S].北京:中国标准出版社,2012.
[13]FAN F X,WANG S J,LOU J,et al.Establishment of RT-LAMP assay to detect Salmonella Typhi in blood[J].Disease Surveillance,2012.
[14]梁玉林,刘秀,周振森,等.基于反转录-环介导等温扩增技术检测金黄色葡萄球菌[J].食品与发酵工业,2018,44(1):217-223.
[15]WANG Y,LI J,QIAO P,et al.Screening and application of a new aptamer for the rapid detection of Sudan dye III[J].European Journal of Lipid Science&Technology,2018:1 700 112.
[16]ZHANG L,WEI Q,HAN Q,et al.Detection of shigellain milk and clinical samples by magnetic immunocaptured-loop-mediated isothermal amplification assay[J].Frontiers in Microbiology,2018,9:94.
[17]周杨,万强,蔡芷荷,等.环介导等温扩增技术在食品中痢疾志贺氏菌快速检测[J].微生物学通报,2017,44(9):2 247-2 254.
[18]曾桂芬,龙北国,姜容,等.LAMP和PCR法检测痢疾志贺菌的特异性和灵敏性比较[J].热带医学杂志,2012,12(5):547-549,592.
[19]王建昌,胡连霞,段永生,等.志贺氏菌实时荧光单引物等温扩增方法的建立及应用[J].食品科学技术学报,2015,33(6):40-45.