兔源肺炎克雷伯氏菌重组酶聚合酶扩增检测方法的建立
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摘要
为建立一种检测兔源肺炎克雷伯氏菌(Klebsiella pneumoniae)重组酶聚合酶扩增方法(recombinase polymerase amplification,RPA),根据肺炎克雷伯氏菌的phoE基因保守序列设计引物,扩增片段大小为277 bp,并对反应条件进一步优化,最终建立了适宜于快速准确检测兔源肺炎克雷伯氏菌的重组酶聚合酶等温扩增方法。该方法可特异性检测出兔源肺炎克雷伯氏菌,最低检出细菌量为8.3×10~1 CFU/mL,灵敏度比传统PCR高100倍。本研究建立的肺炎克雷伯氏菌RPA检测方法特异性强、操作简单,从而为生产中的兔源肺炎克雷伯氏菌现场快速检测提供了一种新方法。
In order to establish a recombinase polymerase amplification(RPA)assay for detecting Klebsiella pneumonia of rabbit origin,a pair of primers were designed based on the conservative sequence of PhoE gene of Klebsiella pneumoniae,and the fragment with the length of 277 bp was amplified,then the reaction conditions were further optimized,finally,the RPA assay was established,by which,the Klebsiella pneumoniae of rabbit origin could be specifically detected with the minimum bacteria of 8.3×10~1 CFU/mL,and its sensitivity was 100 times higher than that of traditional PCR. Therefore,the method established in the study had characteristics of strong specificity and simple operation,which would provide a new method for rapid detection of Klebsiella pneumoniae of rabbit origin.
In order to establish a recombinase polymerase amplification(RPA)assay for detecting Klebsiella pneumonia of rabbit origin,a pair of primers were designed based on the conservative sequence of PhoE gene of Klebsiella pneumoniae,and the fragment with the length of 277 bp was amplified,then the reaction conditions were further optimized,finally,the RPA assay was established,by which,the Klebsiella pneumoniae of rabbit origin could be specifically detected with the minimum bacteria of 8.3×10~1 CFU/mL,and its sensitivity was 100 times higher than that of traditional PCR. Therefore,the method established in the study had characteristics of strong specificity and simple operation,which would provide a new method for rapid detection of Klebsiella pneumoniae of rabbit origin.
引文
[1]王召朋,柴同杰,牟特,等.青岛地区兔肺炎克雷伯氏菌分离鉴定[J].泰安山东畜牧兽医,2012,33(5):16-18.
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[9]CHIU C H,SU L H,WU T L,et al.Liver abscess caused by Klebsiella pneumoniae in siblings[J].Journal of clinical microbiology,2001,39(6):2351-2353.
[10]李国军,王德毅,周玉龙,等.蓝狐肺炎克雷伯氏菌感染病例[J].中国兽医杂志,2007,43(2):56-57.
[11]蔡双双,刘晓丽,段圣洁,等.水禽源肺炎克雷伯菌的分离鉴定及致病性观察[J].中国兽医杂志,2018,54(7):79-81.
[12]胡冬梅,陈世平.肺炎克雷伯菌的耐药性及PFGE电泳核型[J].中华微生物学和免疫学杂志,2000,20(4):319-322.
[13]董萌萌,张伟涛,徐彦召,等.猪源肺炎克雷伯菌的分离鉴定及生物学分析[J].河南科技学院学报(自然科学版),2018,46(3):52-57.
[14]文菁,曾玲,孙玉林.肺炎克雷伯氏菌新型等温扩增检测方法的建立[J].岭南师范学院学报,2018,39(3):69-79.
[15]CHEN H T,ZHANG J,YANG S H,et al.Rapid detection of porcine parvovirus DNA by sensitive loop-mediated isothermal amplification[J].Journal of virological methods,2009,158(1/2):100-103.
[16]姜一曈,王昭华,鑫婷,等.基于重组酶聚合酶扩增技术的犬细小病毒快速检测方法的建立[J].中国预防兽医学报,2018,40(10):926-929.
[2]钟世勋.毛皮动物肺炎克雷伯氏菌分离菌株的系统发育分析及其微胶囊疫苗的研制[D].山东农业大学,2013.
[3]WANG J,ZHANG Y,ZHANG R,et al.Recombinase polymerase amplification assay for rapid detection of porcine circovirus 3[J].Molecular and cellular probes,2017,36:58-61.
[4]ZACHARY A C,MIGUEL M C,ALEJANDRO C G,et al.Recombinase polymerase amplification-based assay to diagnose giardia in stool samples[J].The American journal of tropical medicine and hygiene,2015,92(3):583-587.
[5]WANG J,WANG J,LIU L,et al.Development of a real-time recombinase polymerase amplification assay for rapid and sensitive detection of porcine circovirus 2[J].Archives of virology,2017,162(8):2293-2296.
[6]DIANCOURT L,PASSET V,VERHOEF J,et al.Multilocus sequence typing of Klebsiella pneumoniae nosocomial isolates[J].Journal of clinical microbiology,2005,43(8):4178-4182.
[7]刘俊凌,杨俊文.腹泻病儿致泻病原学及临床研究[J].中国妇幼保健,2004(14):113-114.
[8]WEI Z Q,CHEN Y G,YU Y S,et al.Nosocomial spread of multi-resistant Klebsiella pneumoniae containing a plasmid encoding multiple beta-lactamases[J].Journal of medical microbiology,2005,54(9):885-888.
[9]CHIU C H,SU L H,WU T L,et al.Liver abscess caused by Klebsiella pneumoniae in siblings[J].Journal of clinical microbiology,2001,39(6):2351-2353.
[10]李国军,王德毅,周玉龙,等.蓝狐肺炎克雷伯氏菌感染病例[J].中国兽医杂志,2007,43(2):56-57.
[11]蔡双双,刘晓丽,段圣洁,等.水禽源肺炎克雷伯菌的分离鉴定及致病性观察[J].中国兽医杂志,2018,54(7):79-81.
[12]胡冬梅,陈世平.肺炎克雷伯菌的耐药性及PFGE电泳核型[J].中华微生物学和免疫学杂志,2000,20(4):319-322.
[13]董萌萌,张伟涛,徐彦召,等.猪源肺炎克雷伯菌的分离鉴定及生物学分析[J].河南科技学院学报(自然科学版),2018,46(3):52-57.
[14]文菁,曾玲,孙玉林.肺炎克雷伯氏菌新型等温扩增检测方法的建立[J].岭南师范学院学报,2018,39(3):69-79.
[15]CHEN H T,ZHANG J,YANG S H,et al.Rapid detection of porcine parvovirus DNA by sensitive loop-mediated isothermal amplification[J].Journal of virological methods,2009,158(1/2):100-103.
[16]姜一曈,王昭华,鑫婷,等.基于重组酶聚合酶扩增技术的犬细小病毒快速检测方法的建立[J].中国预防兽医学报,2018,40(10):926-929.