单核细胞增生李斯特氏菌实时荧光RPA检测方法的建立及应用
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摘要
本研究旨在建立一种快速检测单核细胞增生李斯特氏菌(Listeria monocytogenes,LM)的实时荧光重组酶聚合酶扩增(real-time RPA)方法。基于LMhly A基因保守序列,设计合成特异性RPA引物和exo探针,所建立的LM real-time RPA方法能够在37℃等温条件下20 min内完成扩增。该方法仅对LM基因组DNA为阳性扩增,对其它20种致病菌基因组DNA的扩增均为阴性;以LM纯培养菌液的基因组DNA进行10倍列梯度稀释,并以之作为模板,其灵敏性可达到5×10-1 pg,与实验室已建立的real-time PCR方法一致。人工污染实验中,对于LM接菌量为3 CFU/25 g的羊肉和生菜样品,增菌14 h即可通过建立的real-time RPA方法检出,与传统培养方法和real-time PCR检测结果均一致,但是前者所需时间明显少于后者。本文建立的LM real-time RPA方法特异性强,灵敏性高,操作简单,反应迅速,并能够摆脱对价格昂贵的荧光PCR仪和专业实验室的需求,可应用于食品突发事件中LM的现场快速检测,对保障我国食品安全具有重要意义。
The aim of this study was to establish a method for the rapid detection of Listeria monocytogenes(LM) by real-time recombinase polymerase amplification(real-time RPA). The primers and exo probe real-time RPA were designed basing on the conserved region of the hly A gene of LM, and the assay was performed successfully at 37 for 20 min. The real℃-time RPA assay could specifically amplify the genomic DNA of LM, but not other 20 bacteria. Using 10-fold serial dilutions of the genomic DNA of LM, the data showed that the limit of detection of the assay was 5×10-1 pg, which was the same as the real-time PCR. In the artificial contamination assays, the Listeria monocytogenes could be detected after 14 hours enrichment culture by the real-time RPA for the mutton and lettuce samples, which were inoculated initially with LM at the concentration of 3 CFU/25 g. The detection results of real-time RPA were the same as those of the traditional culture method and real-time PCR, while the reaction time was much less than the latter two assays. The developed real-time RPA assay is highly specific, highly sensitive, rapid, easy to perform, and is independent of the expensive fluorescence thermal cycler and good laboratory circumstance, which could be applied in the on-site detection of LM in the food safetyoutbreaksand is of great significance for ensuring the food safety.
The aim of this study was to establish a method for the rapid detection of Listeria monocytogenes(LM) by real-time recombinase polymerase amplification(real-time RPA). The primers and exo probe real-time RPA were designed basing on the conserved region of the hly A gene of LM, and the assay was performed successfully at 37 for 20 min. The real℃-time RPA assay could specifically amplify the genomic DNA of LM, but not other 20 bacteria. Using 10-fold serial dilutions of the genomic DNA of LM, the data showed that the limit of detection of the assay was 5×10-1 pg, which was the same as the real-time PCR. In the artificial contamination assays, the Listeria monocytogenes could be detected after 14 hours enrichment culture by the real-time RPA for the mutton and lettuce samples, which were inoculated initially with LM at the concentration of 3 CFU/25 g. The detection results of real-time RPA were the same as those of the traditional culture method and real-time PCR, while the reaction time was much less than the latter two assays. The developed real-time RPA assay is highly specific, highly sensitive, rapid, easy to perform, and is independent of the expensive fluorescence thermal cycler and good laboratory circumstance, which could be applied in the on-site detection of LM in the food safetyoutbreaksand is of great significance for ensuring the food safety.
引文
[1]吴蜀豫,李迎惠,冉陆,等.中国2001年11省(市)食品中李斯特菌污染状况的主动监测[J].中华流行病学杂志,2003,24(8):657-660WU Shu-yu,LI Ying-hui,RAN Lu,et al.Active surveillance on Listeria monocytogenes in seven kinds of food in 11provinces of China in 2001[J].Chin J Epiderniol,2003,24(8):657-660
[2]Liu D Y,Ainsworth A J,Austin F W,et al.Use of PCR primers derived from a putative transcriptional regulator gene for species-specific determination of Listeria monocytogenes[J].International Journal of Food Microbiology,2004,91:297-304
[3]朱静鸿,龚云伟,武艳力.食品中单核细胞增生李斯特菌污染状况调查[J].中国卫生工程学,2016,5:491-493ZHU Jing-hong,GONG Yun-wei,WU Yan-li.Contamination status of Listeria monocytogenes in food[J].Chin J Health Eng Oct,2016,5:491-493
[4]赵薇,刘桂华,王艳秋,等.食品中单核细胞增生李斯特菌污染及耐药状况调查[J].中国卫生检验杂志,2012,6:1394-1395ZHAO Wei,LIU Gui-hua,WANG Yan-qiu,et al.Contamination status and drug resistance of Listeria monocytogenes in food[J].Chinese Journal of Health Laboratory Technology,2012,6:1394-1395
[5]Th?nnings S,Knudsen J D,Sch?nheyder H C,et al.Antibiotic treatment and mortality in patients with Listeria monocytogenes meningitis or bacteraemia[J].Clinical Microbiology&Infection the Official Publication of the European Society of Clinical Microbiology&Infectious Diseases,2016,22(8):725-730
[6]Rietberg K,Lloyd J,Melius B,et al.Outbreak of Listeria monocytogenes infections linked to a pasteurized ice cream product served to hospitalized patients[J].Epidemiology&Infection,2016,144(13):2728-2731
[7]Melo J,Andrew P W,Faleiro M L.Listeria monocytogenes,in cheese and the dairy environment remains a food safety challenge:The role of stress responses[J].Food Research International,2015,67:75-90
[8]Scallan E,Hoekstra Rm,Angulo Fj,et al.Foodborne illnessacquired in the United States-major pathogens[J].Emerg Infect Dis,2011,17(1):7-15
[9]CDC.Recipe for Food Safety[EB/OL].[2013-10-29].http://www.cdc.gov/vitalsigns/listeria/index.html
[10]GB 4789.30-2016,食品安全国家标准食品微生物学检验单核细胞增生李斯特氏菌检验[S]GB 4789.30-2016,National food safety standard Food microbiological examination:Listeria moncytogenes[S]
[11]唐廷廷,韩国全,王利娜,等.分子生物学技术在食源性致病菌检测中的研究进展[J].食品安全质量检测学报,2016,9:3497-3502TANG Ting-ting,HAN Guo-quan,WANG Li-na,et al.Research progress of molecular biology techniques on detecting foodborne pathogens[J].Journal of Food Safety and Quality,2016,9:3497-3502
[12]刘万静,刘斌,李湘平,等.实时定量荧光PCR快速鉴定食品中单核细胞增生李斯特氏菌[J].食品安全质量检测学报,2017,8(1):252-255LIU Wan-jing,LIU Bin,LI Xiang-ping,et al.Rapid detection of Listeria monocytogenes in food by real-time PCR[J].Journal of Food Safety and Quality,2017,8(1):252-255
[13]黄朱梁,薛超波,孙瑛,等.食品中单核细胞增生李斯特氏菌实时浊度(LAMP)检测方法的建立[J].食品工业,2015,1:277-280HUANG Zhu-liang,XUE Chao-bo,SUN Ying,et al.Development of a real-time turbidimeter-based loop-mediated isothermal amplification(LAMP)method for detection of Listeria monocytogenes[J].Food Industry,2015,1:277-280
[14]徐潮,李亮,金芜军,等.荧光RPA技术检测转基因水稻科丰6号[J].分子植物育种,2014,12(5):875-880XU Chao,LI Liang,JIN Wu-jun,et al.Event-specific real-time RPA detection of transgenic rice Kefeng6[J].Molecular Plant Breeding,2014,12(5):875-880
[15]Wang J,Liu L,Li R,et al.Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplification[J].Journal of Veterinary Diagnostic Investigation Official Publication of the American Association of Veterinary Laboratory Diagnosticians Inc,2016,28(5):574
[16]Wang J,Wang J,Li R,et al.Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification[J].Bmc Veterinary Research,2017,13(1):241
[17]Boyle D S,Mcnerney R,Low H T,et al.Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification[J].Plos One,2014,9(8):e103091
[18]Euler M,Wang Y,Otto P,et al.Recombinase polymerase amplification assay for rapid detection of Francisella tularensis[J].Journal of Clinical Microbiology,2012,50(7):2234-2238
[19]Wu Y D,Zhou D H,Zhang L X,et al.Recombinase polymerase amplification(RPA)combined with lateral flow(LF)strip for equipment-free detection of cryptosporidium,spp.oocysts in dairy cattle feces[J].Parasitology Research,2016,115(9):3551
[20]顾思宇,张红星,金君华,等.LAMP法检测单核细胞增生性李斯特菌的研究[J].食品科技,2016,8:297-301GU Si-yu,ZHANG Hong-xing,JIN Jun-hua,et al.Detection of Listeramonoy ctogenes by LAMP[J].Food Science and Technology,2016,8:297-301
[2]Liu D Y,Ainsworth A J,Austin F W,et al.Use of PCR primers derived from a putative transcriptional regulator gene for species-specific determination of Listeria monocytogenes[J].International Journal of Food Microbiology,2004,91:297-304
[3]朱静鸿,龚云伟,武艳力.食品中单核细胞增生李斯特菌污染状况调查[J].中国卫生工程学,2016,5:491-493ZHU Jing-hong,GONG Yun-wei,WU Yan-li.Contamination status of Listeria monocytogenes in food[J].Chin J Health Eng Oct,2016,5:491-493
[4]赵薇,刘桂华,王艳秋,等.食品中单核细胞增生李斯特菌污染及耐药状况调查[J].中国卫生检验杂志,2012,6:1394-1395ZHAO Wei,LIU Gui-hua,WANG Yan-qiu,et al.Contamination status and drug resistance of Listeria monocytogenes in food[J].Chinese Journal of Health Laboratory Technology,2012,6:1394-1395
[5]Th?nnings S,Knudsen J D,Sch?nheyder H C,et al.Antibiotic treatment and mortality in patients with Listeria monocytogenes meningitis or bacteraemia[J].Clinical Microbiology&Infection the Official Publication of the European Society of Clinical Microbiology&Infectious Diseases,2016,22(8):725-730
[6]Rietberg K,Lloyd J,Melius B,et al.Outbreak of Listeria monocytogenes infections linked to a pasteurized ice cream product served to hospitalized patients[J].Epidemiology&Infection,2016,144(13):2728-2731
[7]Melo J,Andrew P W,Faleiro M L.Listeria monocytogenes,in cheese and the dairy environment remains a food safety challenge:The role of stress responses[J].Food Research International,2015,67:75-90
[8]Scallan E,Hoekstra Rm,Angulo Fj,et al.Foodborne illnessacquired in the United States-major pathogens[J].Emerg Infect Dis,2011,17(1):7-15
[9]CDC.Recipe for Food Safety[EB/OL].[2013-10-29].http://www.cdc.gov/vitalsigns/listeria/index.html
[10]GB 4789.30-2016,食品安全国家标准食品微生物学检验单核细胞增生李斯特氏菌检验[S]GB 4789.30-2016,National food safety standard Food microbiological examination:Listeria moncytogenes[S]
[11]唐廷廷,韩国全,王利娜,等.分子生物学技术在食源性致病菌检测中的研究进展[J].食品安全质量检测学报,2016,9:3497-3502TANG Ting-ting,HAN Guo-quan,WANG Li-na,et al.Research progress of molecular biology techniques on detecting foodborne pathogens[J].Journal of Food Safety and Quality,2016,9:3497-3502
[12]刘万静,刘斌,李湘平,等.实时定量荧光PCR快速鉴定食品中单核细胞增生李斯特氏菌[J].食品安全质量检测学报,2017,8(1):252-255LIU Wan-jing,LIU Bin,LI Xiang-ping,et al.Rapid detection of Listeria monocytogenes in food by real-time PCR[J].Journal of Food Safety and Quality,2017,8(1):252-255
[13]黄朱梁,薛超波,孙瑛,等.食品中单核细胞增生李斯特氏菌实时浊度(LAMP)检测方法的建立[J].食品工业,2015,1:277-280HUANG Zhu-liang,XUE Chao-bo,SUN Ying,et al.Development of a real-time turbidimeter-based loop-mediated isothermal amplification(LAMP)method for detection of Listeria monocytogenes[J].Food Industry,2015,1:277-280
[14]徐潮,李亮,金芜军,等.荧光RPA技术检测转基因水稻科丰6号[J].分子植物育种,2014,12(5):875-880XU Chao,LI Liang,JIN Wu-jun,et al.Event-specific real-time RPA detection of transgenic rice Kefeng6[J].Molecular Plant Breeding,2014,12(5):875-880
[15]Wang J,Liu L,Li R,et al.Rapid and sensitive detection of canine parvovirus type 2 by recombinase polymerase amplification[J].Journal of Veterinary Diagnostic Investigation Official Publication of the American Association of Veterinary Laboratory Diagnosticians Inc,2016,28(5):574
[16]Wang J,Wang J,Li R,et al.Rapid and sensitive detection of canine distemper virus by real-time reverse transcription recombinase polymerase amplification[J].Bmc Veterinary Research,2017,13(1):241
[17]Boyle D S,Mcnerney R,Low H T,et al.Rapid detection of Mycobacterium tuberculosis by recombinase polymerase amplification[J].Plos One,2014,9(8):e103091
[18]Euler M,Wang Y,Otto P,et al.Recombinase polymerase amplification assay for rapid detection of Francisella tularensis[J].Journal of Clinical Microbiology,2012,50(7):2234-2238
[19]Wu Y D,Zhou D H,Zhang L X,et al.Recombinase polymerase amplification(RPA)combined with lateral flow(LF)strip for equipment-free detection of cryptosporidium,spp.oocysts in dairy cattle feces[J].Parasitology Research,2016,115(9):3551
[20]顾思宇,张红星,金君华,等.LAMP法检测单核细胞增生性李斯特菌的研究[J].食品科技,2016,8:297-301GU Si-yu,ZHANG Hong-xing,JIN Jun-hua,et al.Detection of Listeramonoy ctogenes by LAMP[J].Food Science and Technology,2016,8:297-301