环介导等温扩增法检测空肠弯曲菌
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摘要
目的空肠弯曲菌是重要的食源性疾病病例标本的病原菌检测项目,用环介导等温扩增(Loop-mediated isothermal amplification,LAMP)的方法,对食源性致病菌空肠弯曲菌进行快速检测。方法以空肠弯曲菌map A基因序列为检测靶基因,建立了空肠弯曲菌的LAMP快速检测方法。空肠弯曲菌的LAMP反应由DNA扩增试剂盒提供的反应体系加入引物和模板进行。做了空肠弯曲菌DNA模板灵敏度的检测、菌液灵敏度的检测和特异性检测。结果我们方法对空肠弯曲菌DNA的检测灵敏度为84. 5fg/反应管;对空肠弯曲菌菌液检测灵敏度为0. 8 CFU/反应管;特异性100%。结论对食源性疾病腹泻样本的空肠弯曲菌检测项目,可以采用map A-LAMP方法进行等温扩增初筛,初筛阳性的样品再有针对性地进行后续的传统培养。
Objective To detect the susceptibility of food borne pathogenic C. jejuni to Loop-mediated isothermal amplification( LAMP). Methods Taking the MAPA gene sequence of C. jejuni as the target gene,the LAMP method for rapid detection of C. jejuni was introduced,and then carried out by the addition of specific primers and templates for C. jejuni to the reaction mixture provided by the DNA amplification kit. The sensitivity testing for DNA template density,C. jejuni culture suspension density and the specificity procedures were conducted. Results In this study,the sensitivity of LAMP assay for detecting genomic DNA and culture suspension was 84. 5 fg/reaction tube and 0. 8 CFU/reaction tube,respectively. The specificity was100%. Conclusion The LAMP screening established in this study can be conducted to investigate the presence of C. jejuni in fecal specimens of foodborne diseases. For LAMP-positive specimens,they will be cultured immediately from the stored stool specimens through routine procedures.
Objective To detect the susceptibility of food borne pathogenic C. jejuni to Loop-mediated isothermal amplification( LAMP). Methods Taking the MAPA gene sequence of C. jejuni as the target gene,the LAMP method for rapid detection of C. jejuni was introduced,and then carried out by the addition of specific primers and templates for C. jejuni to the reaction mixture provided by the DNA amplification kit. The sensitivity testing for DNA template density,C. jejuni culture suspension density and the specificity procedures were conducted. Results In this study,the sensitivity of LAMP assay for detecting genomic DNA and culture suspension was 84. 5 fg/reaction tube and 0. 8 CFU/reaction tube,respectively. The specificity was100%. Conclusion The LAMP screening established in this study can be conducted to investigate the presence of C. jejuni in fecal specimens of foodborne diseases. For LAMP-positive specimens,they will be cultured immediately from the stored stool specimens through routine procedures.
引文
[1] Kaakoush NO,Casta? o-Rodríguez N,Mitchell HM,et al. Global epidemiology of campylobacter infection[J]. Clinical Microbiology Reviews,2015,28(3):687-720.
[2] Zhang H,Morrison S,Tang YW. Multiplex polymerase chain reaction tests for detection of pathogens associated with gastroenteritis[J]. Clinics in Laboratory Medicine,2015,35(2):461-486.
[3] Notomi T,Okayama H,Masubuchi HA,et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research,2000,28(12):E63.
[4] Mori Y,Kitao M,Tomita N,et al. Real-time turbidimetry of LAMP reaction for quantifying template DNA[J]. Journal of Biochemical and Biophysical Methods,2004,59(2):145-157.
[5] Mori Y,Nagamine K,Tomita N,et al. Detection of loop-mediated isothermal amplification reaction by turbidity derived from Magnesium pyrophosphate formation[J]. Biochemical and Biophysical Research Communications,2001,289(1):150-154.
[6] Iwamoto T,Sonobe T,Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex,M-avium,and M-intracellulare in sputum samples[J].Journal of Clinical Microbiology,2003,41(6):2616-2622.
[7] Lynch OA,Cagney C,Mcdowell DA,et al. Occurrence of fastidious Campylobacter spp. in fresh meat and poultry using an adapted cultural protocol[J]. International Journal of Food Microbiology,2011,150(2/3):171-177.
[8] Epps SV,Harvey RB,Hume ME,et al. Foodborne campylobacter:infections,metabolism,pathogenesis and reservoirs[J]. International Journal of Environmental Research and Public Health,2013,10(12):6292-6304.
[9] Centers for Disease Control and Prevention(CDC). Vital signs:incidence and trends of infection with pathogens transmitted commonly through food——Foodborne Diseases Active Surveillance Network,10 U. S. sites,1996-2010[J]. MMWR. Morbidity and Mortality Weekly Report,2011,60(22):749-755.
[10] Hara-Kudo Y,Takatori K. Contamination level and ingestion dose of foodborne pathogens associated with infections[J]. Epidemiology and Infection,2011,139(10,SI):1505-1510.
[11] Singh H,Rathore RS,Singh S. Comparative analysis of cultural isolation and PCR based assay for detection of campylobacter jejuni in food and faecal samples[J]. Brazilian Journal of Microbiology,2011,42(1):181-186.
[12]徐义刚,李丹丹,田长永,等.应用DNA环介导恒温扩增技术快速检测空肠弯曲菌[J].中国农业科学,2012,45(13):2751-2757.
[13] Yamazaki W,Taguchi M,Ishibashi M,et al. Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli[J]. Journal of Medical Microbiology,2008,57(Pt 4):444-451.
[14] Buchan BW,Ledeboer NA. Emerging technologies for the clinical microbiology laboratory[J]. Clinical Microbiology Reviews,2014,27(4):783-822.
[15] Johnson JG,Livny J,Dirita VJ. High-throughput sequencing of Campylobacter jejuni insertion mutant libraries reveals map A as a fitness factor for chicken colonization[J]. Journal of Bacteriology,2014,196(11):1958-1967.
[16] Begum S,Sekar M,Gunaseelan L,et al. Molecular identification of Campylobacter jejuni and coli from chicken,calves and dogs to determine its potential threat on human being[J]. Veterinary World,2015,8(12):1420-1423.
[2] Zhang H,Morrison S,Tang YW. Multiplex polymerase chain reaction tests for detection of pathogens associated with gastroenteritis[J]. Clinics in Laboratory Medicine,2015,35(2):461-486.
[3] Notomi T,Okayama H,Masubuchi HA,et al. Loop-mediated isothermal amplification of DNA[J]. Nucleic Acids Research,2000,28(12):E63.
[4] Mori Y,Kitao M,Tomita N,et al. Real-time turbidimetry of LAMP reaction for quantifying template DNA[J]. Journal of Biochemical and Biophysical Methods,2004,59(2):145-157.
[5] Mori Y,Nagamine K,Tomita N,et al. Detection of loop-mediated isothermal amplification reaction by turbidity derived from Magnesium pyrophosphate formation[J]. Biochemical and Biophysical Research Communications,2001,289(1):150-154.
[6] Iwamoto T,Sonobe T,Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex,M-avium,and M-intracellulare in sputum samples[J].Journal of Clinical Microbiology,2003,41(6):2616-2622.
[7] Lynch OA,Cagney C,Mcdowell DA,et al. Occurrence of fastidious Campylobacter spp. in fresh meat and poultry using an adapted cultural protocol[J]. International Journal of Food Microbiology,2011,150(2/3):171-177.
[8] Epps SV,Harvey RB,Hume ME,et al. Foodborne campylobacter:infections,metabolism,pathogenesis and reservoirs[J]. International Journal of Environmental Research and Public Health,2013,10(12):6292-6304.
[9] Centers for Disease Control and Prevention(CDC). Vital signs:incidence and trends of infection with pathogens transmitted commonly through food——Foodborne Diseases Active Surveillance Network,10 U. S. sites,1996-2010[J]. MMWR. Morbidity and Mortality Weekly Report,2011,60(22):749-755.
[10] Hara-Kudo Y,Takatori K. Contamination level and ingestion dose of foodborne pathogens associated with infections[J]. Epidemiology and Infection,2011,139(10,SI):1505-1510.
[11] Singh H,Rathore RS,Singh S. Comparative analysis of cultural isolation and PCR based assay for detection of campylobacter jejuni in food and faecal samples[J]. Brazilian Journal of Microbiology,2011,42(1):181-186.
[12]徐义刚,李丹丹,田长永,等.应用DNA环介导恒温扩增技术快速检测空肠弯曲菌[J].中国农业科学,2012,45(13):2751-2757.
[13] Yamazaki W,Taguchi M,Ishibashi M,et al. Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli[J]. Journal of Medical Microbiology,2008,57(Pt 4):444-451.
[14] Buchan BW,Ledeboer NA. Emerging technologies for the clinical microbiology laboratory[J]. Clinical Microbiology Reviews,2014,27(4):783-822.
[15] Johnson JG,Livny J,Dirita VJ. High-throughput sequencing of Campylobacter jejuni insertion mutant libraries reveals map A as a fitness factor for chicken colonization[J]. Journal of Bacteriology,2014,196(11):1958-1967.
[16] Begum S,Sekar M,Gunaseelan L,et al. Molecular identification of Campylobacter jejuni and coli from chicken,calves and dogs to determine its potential threat on human being[J]. Veterinary World,2015,8(12):1420-1423.