牛乳中蜡样芽孢杆菌荧光定量PCR检测方法的建立及验证
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摘要
目的建立一种快速检测牛乳中蜡样芽孢杆菌的荧光定量PCR方法,并进行验证。方法根据GenBank中登录的蜡样芽孢杆菌gyrB基因序列及荧光定量PCR要求,设计合适的特异性引物,提取菌体DNA,对目的基因进行扩增及克隆;以重组质粒为模板进行荧光定量PCR扩增,绘制标准曲线;以蜡样芽孢杆菌DNA为模板进行荧光定量PCR扩增,确定其扩增条件。对建立的方法进行特异性及灵敏性验证。结果建立的方法检测其他3种牛乳中常见致病菌(大肠埃希菌、志贺菌、沙门菌)未见特异性扩增,即无交叉反应;最低检出限为1×10~(-7) ng/μL。以掺入蜡样芽孢杆菌的牛乳为样品,可检测到牛乳中10. 4 CFU/mL的蜡样芽孢杆菌,优于国标方法(GB 4789. 14-2014)检测含有相同菌落数的蜡样芽孢杆菌(仅能检测到1. 04×10~2 CFU/mL)。结论建立的方法特异性强,灵敏性高,适用于快速、准确检测牛乳中的蜡样芽孢杆菌,为实验室快速检测致病菌提供了参考。
Objective To establish and verify a fluorescence quantitative PCR assay for rapid detection of Bacillus cereus in milk. Methods Specific primers were designed according to the gyr B gene sequence of B. cereus in GenBank and the requirement of fluorescence quantitative PCR,based on which the bacterial DNA was extracted,and the target gene was cloned. Fluorescence quantitative PCR assay was performed using recombinant plasmid as a template,and a standard curve was plotted. Meanwhile,the fluorescence quantitative PCR assay was performed using DNA template of B. cereus,and the condition for amplification was determined. The established method was verified for specificity and sensitivity.Results The established fluorescence quantitative PCR assay showed no cross reactions with three common pathogenic bacteria in B. cereus,E. coli,Shigella shigae and salmonella. The minimum detection limit of the developed method was1 × 10-7 ng/L. The B. cereus at a concentration of 10. 4 CFU/mL in milk was detected by the method. However,by the method in GB 4789. 14-2014,only the B. cereus at a concentration of 1. 04 × 102 CFU/mL was detected.Conclusion The developed fluorescence quantitative PCR assay showed high specificity and sensitivity,which was suitable for rapid and accurate detection of B. cereus in milk. It provided a reference for rapid detection of pathogenic bacteria in laboratory.
Objective To establish and verify a fluorescence quantitative PCR assay for rapid detection of Bacillus cereus in milk. Methods Specific primers were designed according to the gyr B gene sequence of B. cereus in GenBank and the requirement of fluorescence quantitative PCR,based on which the bacterial DNA was extracted,and the target gene was cloned. Fluorescence quantitative PCR assay was performed using recombinant plasmid as a template,and a standard curve was plotted. Meanwhile,the fluorescence quantitative PCR assay was performed using DNA template of B. cereus,and the condition for amplification was determined. The established method was verified for specificity and sensitivity.Results The established fluorescence quantitative PCR assay showed no cross reactions with three common pathogenic bacteria in B. cereus,E. coli,Shigella shigae and salmonella. The minimum detection limit of the developed method was1 × 10-7 ng/L. The B. cereus at a concentration of 10. 4 CFU/mL in milk was detected by the method. However,by the method in GB 4789. 14-2014,only the B. cereus at a concentration of 1. 04 × 102 CFU/mL was detected.Conclusion The developed fluorescence quantitative PCR assay showed high specificity and sensitivity,which was suitable for rapid and accurate detection of B. cereus in milk. It provided a reference for rapid detection of pathogenic bacteria in laboratory.
引文
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[8]YANG I C,SHIH D Y,HUANG T P,et al.Establishment of a novel multiplex PCR assay and detection of toxigenic strains of the species in the Bacillus cereus group[J].J Food Prot,2005,68(10):2123-2130.
[9]RAMESH A,PADMAPRIYA B P,CHRASHEKAR A.Application of a convenient DNA extraction method and multiplex PCR for the direct detection of Staphylococcus aureus and Yersinia enterocolitica in milk samples[J].Mol Cell Probes,2002,16(4):307-314.
[10]JI D,XIN S J.Development and data analysis of real-time fluorescent quantitative PCR[J].Lett Biotechnol,2009,20(4):598-600.(in Chinese)纪冬,辛绍杰.实时荧光定量PCR的发展和数据分析[J].生物技术通讯,2009,20(4):598-600.
[11]HAUGE S.Food poisoning caused by S.aureus,Cl.perfringens,B.cereus and Salmonella species[J].Tidsskrift for Den Norske Lgeforening Tidsskrift for Praktisk Medicin Ny Rkke,1972,92(8):589-591.
[12]WANG Y,ZHOU G P.Analysis on fatal cases caused by Bacillus cereus induced foodborne poisoning[J].Chin J Food Hyg,2011,23(2):191-194.(in Chinese)王洋,周帼萍.蜡样芽孢杆菌食物中毒死亡案例分析[J].中国食品卫生杂志,2011,23(2):191-194.
[13]DI PINTO A,BONERBA E,BOZZO G.Occurrence of potentially enterotoxigenic Bacillus cereus in infant milk powder[J].Eu Food Res Technol,2013,237(2):275-279.
[14]OWUSU-KWARTENG J,WUNI A,AKABANDA F,et al.Prevalence,virulence factor genes and antibiotic resistance of Bacillus cereus sensu lato,isolated from dairy farms and traditional dairy products[J].Bmc Microbiol,2017,17(1):65.
[15]BARTOSZEWICZ M,KROTEN M A,SWIECICKA I.Germination and proliferation of emetic Bacillus cereus sensu lato strains in milk[J].Folia Microbiologica,2013,58(6):529-535.
[16]LI C J,ZHANG Y B,LUO X C,et al.Application of fluorescent quantitative polymerase chain reaction technique in cetection of clinic microorganism[J].Biotechnol Bulle,2011,2:66-69.(in Chinese)李春娟,张一兵,罗喜钢.荧光定量PCR技术在临床微生物检测中的应用[J].生物技术通报,2011,2:66-69.
[17]YAN L,WANG X Y,GUO Y C,et al.Rapid detection of Listeria monocytogenes in pork samples by real-time PCR with Taqman probe[J].J Hyg Res,2014,43(2):177-182.(in Chinese)闫琳,王晓英,郭云昌.应用Taqman实时PCR法检测猪肉中单核细胞增生李斯特菌[J].卫生研究,2014,43(2):177-182.
[18]WEHRLE E,DIDIER A,MORAVEK M,et al.Detection of Bacillus cereus with enteropathogenic potential by multiplex real-time PCR based on SYBR green I[J].Mol Cell Probes,2010,24(3):124-130.
[2]ZHAO Y M,REN G P.Research improvement of Bacillus cereus in dairy products[J].China Dairy Industry,2014,42(4):46-49.(in Chinese)赵月明,任国谱.乳制品中蜡样芽孢杆菌的研究进展[J].中国乳品工业,2014,42(4):46-49.
[3]KANG B Y,MU G Q,CHEN L J,et al.Investigation and analysis of bacillus in yogurt production environment[J].China Dairy Cattle,2013(11):36-40.(in Chinese)康博燕,牟光庆,陈历俊,等.酸奶生产环境中芽孢杆菌的调查与分析[J].中国奶牛,2013(11):36-40.
[4]ZHOU G P,LIANG T G,DING S J.Analysis on 299 Bacillus cereus food poisoning cases in 1986-2007[J].Chin J Food Hyg,2009,21(5):450-454.(in Chinese)周帼萍,梁天光,丁淑娟.1986-2007年中国299起蜡样芽胞杆菌食物中毒案例分析[J].中国食品卫生杂志,2009,21(5):450-454.
[5]CHEN C H,DING H C.A colony blot immunoassay for the rapid identification of Bacillus cereus[J].J Food Prot,2004,67(2):387.
[6]HANSEN B M,LESER T D,HENDRIKSEN N B.Polymerase chain reaction assay for the detection of Bacillus cereus group cells[J].FEMS Microbiol Lett,2001,202(2):209-213.
[7]PENG H.Isolation and enumeration of Bacillus cereus from foods on novel chromogenic plating medium[J].Food Microbiol,2001,18(3):231-238.
[8]YANG I C,SHIH D Y,HUANG T P,et al.Establishment of a novel multiplex PCR assay and detection of toxigenic strains of the species in the Bacillus cereus group[J].J Food Prot,2005,68(10):2123-2130.
[9]RAMESH A,PADMAPRIYA B P,CHRASHEKAR A.Application of a convenient DNA extraction method and multiplex PCR for the direct detection of Staphylococcus aureus and Yersinia enterocolitica in milk samples[J].Mol Cell Probes,2002,16(4):307-314.
[10]JI D,XIN S J.Development and data analysis of real-time fluorescent quantitative PCR[J].Lett Biotechnol,2009,20(4):598-600.(in Chinese)纪冬,辛绍杰.实时荧光定量PCR的发展和数据分析[J].生物技术通讯,2009,20(4):598-600.
[11]HAUGE S.Food poisoning caused by S.aureus,Cl.perfringens,B.cereus and Salmonella species[J].Tidsskrift for Den Norske Lgeforening Tidsskrift for Praktisk Medicin Ny Rkke,1972,92(8):589-591.
[12]WANG Y,ZHOU G P.Analysis on fatal cases caused by Bacillus cereus induced foodborne poisoning[J].Chin J Food Hyg,2011,23(2):191-194.(in Chinese)王洋,周帼萍.蜡样芽孢杆菌食物中毒死亡案例分析[J].中国食品卫生杂志,2011,23(2):191-194.
[13]DI PINTO A,BONERBA E,BOZZO G.Occurrence of potentially enterotoxigenic Bacillus cereus in infant milk powder[J].Eu Food Res Technol,2013,237(2):275-279.
[14]OWUSU-KWARTENG J,WUNI A,AKABANDA F,et al.Prevalence,virulence factor genes and antibiotic resistance of Bacillus cereus sensu lato,isolated from dairy farms and traditional dairy products[J].Bmc Microbiol,2017,17(1):65.
[15]BARTOSZEWICZ M,KROTEN M A,SWIECICKA I.Germination and proliferation of emetic Bacillus cereus sensu lato strains in milk[J].Folia Microbiologica,2013,58(6):529-535.
[16]LI C J,ZHANG Y B,LUO X C,et al.Application of fluorescent quantitative polymerase chain reaction technique in cetection of clinic microorganism[J].Biotechnol Bulle,2011,2:66-69.(in Chinese)李春娟,张一兵,罗喜钢.荧光定量PCR技术在临床微生物检测中的应用[J].生物技术通报,2011,2:66-69.
[17]YAN L,WANG X Y,GUO Y C,et al.Rapid detection of Listeria monocytogenes in pork samples by real-time PCR with Taqman probe[J].J Hyg Res,2014,43(2):177-182.(in Chinese)闫琳,王晓英,郭云昌.应用Taqman实时PCR法检测猪肉中单核细胞增生李斯特菌[J].卫生研究,2014,43(2):177-182.
[18]WEHRLE E,DIDIER A,MORAVEK M,et al.Detection of Bacillus cereus with enteropathogenic potential by multiplex real-time PCR based on SYBR green I[J].Mol Cell Probes,2010,24(3):124-130.