多杀性巴氏杆菌TaqMan荧光定量PCR检测方法的建立与应用
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摘要
为建立一种多杀性巴氏杆菌(Pm)快速敏感的检测方法,本实验根据Pm70株kmt1基因保守区设计特异性引物和TaqMan探针,建立了该菌的TaqMan荧光定量PCR检测方法,并优化了检测反应条件。本实验建立的荧光定量PCR方法可以特异性检测Pm,而对鸭疫里默氏杆菌、支气管败血波氏杆菌等菌株检测结果均为阴性,表明该方法具有良好的特异性。该方法对C48-1株标准品的检测灵敏度为1.75×10~1拷贝/μL,优于常规PCR检测方法(1.75×10~3拷贝/μL) 100倍;组内和组间重复性试验的变异系数均小于2%,具有良好的重复性。对20份疑似感染的临床病料样品(鸡肝脏、牛肺脏)进行增菌培养和分离鉴定,结果分离到8株多杀性巴氏杆菌;以建立的荧光定量PCR方法与常规PCR方法对增菌培养液进行检测,阳性率分别为40%(8/20)和25%(5/20),且荧光定量PCR方法与常规细菌分离鉴定的符合率为100%。对C48-1株感染致死的20份小鼠肝脏和脾脏组织研磨液进行检测,阳性率均为100%。本研究建立的方法能够快速、敏感的检测Pm,对于其临床和实验室的快速诊断具有重要意义。
To develop a rapid and sensitive method for Pasteurella multocida detection, the Taq Man real-time PCR was established with a pair of primers and probe targeting the conserved region of kmt1 gene of P.multocida Pm70 strain. The assay was highly specific for amplification from P.multocida, but no amplification from Riemerella anatipestifer, Brodetella bronchiseptica and so on. The detection limitation of the protocol was 1.75 ×10 copies/μL initial template, which was 100-fold more sensitive than that of conventional PCR. Moreover, the method was highly reproducible and a coefficient of variation was less than 2% for both intra-and inter-assay. Tweenty tissue samples collected from chicken(livers) and bovine(lungs) were detected, and 8 samples were positive, while the positive rate of detection was 45%(8/20) by the real-time PCR and 25%(5/20)by conventional PCR, respectively. Positive rates of 20 samples from livers and spleens of mice infected with P.multocida C48-1 strain were 100% by both real-time PCR and conventional PCR. Therefore, the real-time PCR assay provided a sensitive and rapid method for detection of P.multocida in the laboratory and clinical infection.
To develop a rapid and sensitive method for Pasteurella multocida detection, the Taq Man real-time PCR was established with a pair of primers and probe targeting the conserved region of kmt1 gene of P.multocida Pm70 strain. The assay was highly specific for amplification from P.multocida, but no amplification from Riemerella anatipestifer, Brodetella bronchiseptica and so on. The detection limitation of the protocol was 1.75 ×10 copies/μL initial template, which was 100-fold more sensitive than that of conventional PCR. Moreover, the method was highly reproducible and a coefficient of variation was less than 2% for both intra-and inter-assay. Tweenty tissue samples collected from chicken(livers) and bovine(lungs) were detected, and 8 samples were positive, while the positive rate of detection was 45%(8/20) by the real-time PCR and 25%(5/20)by conventional PCR, respectively. Positive rates of 20 samples from livers and spleens of mice infected with P.multocida C48-1 strain were 100% by both real-time PCR and conventional PCR. Therefore, the real-time PCR assay provided a sensitive and rapid method for detection of P.multocida in the laboratory and clinical infection.
引文
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[11]Blackall P J,Miflin J K.Identification and typing of Pasteurella multocida:A review[J].Avian Pathol,2000,29(4):271-287.
[12]汤细彪.猪源多杀性巴氏杆菌的分子流行病学与致病性研究[D].武汉:华中农业大学,2010.
[13]Liu Jian-jun,Yang Fan,He Jian-fan,et al.Study on the rapid detection of Dengue viruses by Taq Man MGB real-time fluorescent PCR[J].J Hygiene Res,2006,35(6):736-738.
[14]赵静,张红见,马梅琴,等.青藏高原牦牛多杀性巴氏杆菌荧光定量RQ-PCR检测方法的建立[J].中国兽医杂志,2013,(10):18-20.
[15]程苏云,梅玲玲,朱敏,等.沙门菌Taq Man-MGB探针实时荧光PCR检测技术研究[J].中国卫生检验杂志,2009,(06):1320-1323.
[2]Talan D A,Citron D M,Abrahamian F M,et al.Bacteriologic analysis of infected dog and cat bites[J].N Engl J Med,1999,340(2):85-92.
[3]Wilkie I W,Harper M,Boyce J D,et al.Pasteurella multocida:diseases and pathogenesis[J].Curr Top Microbiol Immunol,2012,361:1-22.
[4]唐先春,吴斌,索绪峰,等.猪多杀性巴氏杆菌的分离鉴定及生物学特性研究[J].畜牧兽医学报,2005,(06):590-595.
[5]张娜,李书光,陈金龙,等.兔F型多杀性巴氏杆菌的分离鉴定及其致病性[J].中国兽医科学,2012,(7):685-689.
[6]高明燕,徐步,赵宝华,等.多杀性巴氏杆菌检测、鉴定和分型研究进展[J].动物医学进展,2010,31(1):67-72.
[7]Bhimani M,Bhanderi B,Roy A.Loop-mediated isothermal amplification assay(LAMP)based detection of Pasteurella multocida in cases of haemorrhagic septicaemia and fowl cholera[J].Vet Italiana,2015,51(2):115.
[8]Townsend K M,Frost A J,Lee C W,et al.Development of PCR assays for species-and type-specific identification of Pasteurella multocida isolates[J].J Clin Microbiol,1998,36(4):1096.
[9]Davies R L,Maccorquodale R,Reilly S.Characterisation of bovine strains of Pasteurella multocida and comparison with isolates of avian,ovine and porcine origin[J].Vet Microbiol,2004,99(2):145-158.
[10]罗青平,杨峻,温国元,等.禽巴氏杆菌病疫苗研究进展[J].湖北畜牧兽医,2012,(12):14-16.
[11]Blackall P J,Miflin J K.Identification and typing of Pasteurella multocida:A review[J].Avian Pathol,2000,29(4):271-287.
[12]汤细彪.猪源多杀性巴氏杆菌的分子流行病学与致病性研究[D].武汉:华中农业大学,2010.
[13]Liu Jian-jun,Yang Fan,He Jian-fan,et al.Study on the rapid detection of Dengue viruses by Taq Man MGB real-time fluorescent PCR[J].J Hygiene Res,2006,35(6):736-738.
[14]赵静,张红见,马梅琴,等.青藏高原牦牛多杀性巴氏杆菌荧光定量RQ-PCR检测方法的建立[J].中国兽医杂志,2013,(10):18-20.
[15]程苏云,梅玲玲,朱敏,等.沙门菌Taq Man-MGB探针实时荧光PCR检测技术研究[J].中国卫生检验杂志,2009,(06):1320-1323.