山东省畜禽源性魏氏梭菌毒素型Multi-PCR调查
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摘要
为搞清楚山东省魏氏梭菌毒素型真实分布情况,建立了Multi—PCR检测方法,对近5年内本省流行过该病的德州、泰安、枣庄、青岛、临沂、烟台等地的24个养殖场采集的692个样品进行了检测,共分离到124株魏氏梭菌,检出率为17.9%:经Multi—PCR检测均为A犁。检测结果确实可靠,为准确的预防该菌感染提供了理论指导。同时本课题还建立了魏氏梭菌的快速PCR鉴定方法,具有较高应用价值。
本研究共分四部分:
第一部分:魏氏梭菌的分离和初步鉴定 将采集的粪便样品分别无菌称取5g稀释到50ml营养肉汤中,置于厌氧培养箱中43℃增菌18h,然后取出按“三线分离法”接种到血培养基上,43℃厌氧培养12h,可见圆形、突起、边缘整齐、表面湿润、光滑、双溶血环微绿的菌落。然后将菌落涂片,染色,镜检,同时对照在有氧环境中培养无菌落生长。镜检为典型革兰氏阳性的两端钝圆、粗大杆菌通过API-20A生化鉴定系统、牛乳暴烈发酵实验进行鉴定。共分离鉴定124株魏氏梭菌。
第二部分:魏氏梭菌的PCR快速诊断方法的建立及应用 建立了聚合酶链氏反应魏氏梭菌α毒素基因的检测方法,通过不断摸索条件优化了试剂使用剂量,缩短了反应时间,另外用该法对沙门氏菌、大肠杆菌、肠球菌和葡萄球菌扩增。结果表明,仅魏氏梭菌扩增出了特异性条带,最低能检测到1CFU,说明该方法具有很高的特异性和敏感性。用该法对山东齐河两个猪场、一个牛场、一个羊场和,一个兔场进行了样品检测,共检测到11株牛源(11/60),检出率为18.3%;4株猪源魏氏梭菌(4/20),检出率为20%;3株羊源魏氏梭菌(3/10),检出率为30%;3株兔源魏氏梭菌(3/12),检出率为25%,其PCR总检出率为20.6%,与常规细菌分离鉴定结果一致。应用显示本方法是魏氏梭菌的一种快速、简便的鉴定方法,具有很高的推广应用价值。
第三部分:魏氏梭菌Multi-PCR检测方法建立及在空气样品检测上的应用 根据GenBank上发表的序列设计了四对引物,通过不断优化反
In order to confirm the real Epidemical situation of Clostridium perfringens toxin types in Shandong Province, multiplex-PCR was established, by which we investigated 692 samples isolated from Dezhou,Taian, Zaozhuang,Qingdao,Linyi and Yantai. We detect 124 Clostridium perfringens positive,and all are type A.The survey result is absolutely true,which provide theory guide for previntion. The study established a fast PCR detect method to Clostridium perfringens,which has high specifity and possesses latent application prospect.The study consists of four parts.Part Ⅰ:Isolation and Toxin-type Identification of Clostridium perfringens of samples. 124 strains of Clostridium perfringens were isolated from animals feces. The study result showed isolation strains were toxin type A by API-20A biochemical test method.Part Ⅱ:A pair of primer was succeeded in designing and synthesizing according to the published sequence of the α -toxin of Clostridium perfringens.The PCR can amplify 325bp specific α -toxin fragments.It accords with the predicted amplification segament, however,it has no positive result with system to amplify Salmonella 、 E-coli 、 Enterococcus and Staphylococcus,so ,it can demonstrate the PCR amplification system has good specificity.In addition,it also possesses very high sensitivity because the system can detect the 1CFU/ml dilute template. The PCR technique was applied tor the detection of Clostridium perfringens in localized areas of Shandong province.The result showed that 21 of 102 samples were positive which is basically identical with other bacteria isolation methods.According to the above results ,the study has developed a Clostridium perfringens quick detection method which possesses latent application prospect. Part Ⅲ:For detection of Clostridium perfringens toxin types four pairs of primer for alpha, beta, epsilon and jota toxin gene were succeeded in designing and synthesizing according to the published sequence.It can
distinguish type A,B,C,D and E of Clostridium perfringens.The specific of PCR is high,only Clostridium perfringens show positive,other strains such as Salmonella > E-coli^ Enterococcus and Staphylococcus all show negative. The multiplex PCR was applied for the identification of toxin types of Clostridium perfringens from samples which were collected from cattle stable and had been identified by ELISA.The PCR result accord with the ELISA method.So the muliplex-PCR can be applied to epidemiology survey. Part IV: The multiplex PCR was applied for the identification of toxin types of Clostridium perfringens from samples which were collected from earlier epidemic districts of Shandong province. The results showed that all isolated 124 samples (17.9%) were Clostridium perfringens toxin type A, that was alpha toxin positive which is different from abroad reports.
本研究共分四部分:
第一部分:魏氏梭菌的分离和初步鉴定 将采集的粪便样品分别无菌称取5g稀释到50ml营养肉汤中,置于厌氧培养箱中43℃增菌18h,然后取出按“三线分离法”接种到血培养基上,43℃厌氧培养12h,可见圆形、突起、边缘整齐、表面湿润、光滑、双溶血环微绿的菌落。然后将菌落涂片,染色,镜检,同时对照在有氧环境中培养无菌落生长。镜检为典型革兰氏阳性的两端钝圆、粗大杆菌通过API-20A生化鉴定系统、牛乳暴烈发酵实验进行鉴定。共分离鉴定124株魏氏梭菌。
第二部分:魏氏梭菌的PCR快速诊断方法的建立及应用 建立了聚合酶链氏反应魏氏梭菌α毒素基因的检测方法,通过不断摸索条件优化了试剂使用剂量,缩短了反应时间,另外用该法对沙门氏菌、大肠杆菌、肠球菌和葡萄球菌扩增。结果表明,仅魏氏梭菌扩增出了特异性条带,最低能检测到1CFU,说明该方法具有很高的特异性和敏感性。用该法对山东齐河两个猪场、一个牛场、一个羊场和,一个兔场进行了样品检测,共检测到11株牛源(11/60),检出率为18.3%;4株猪源魏氏梭菌(4/20),检出率为20%;3株羊源魏氏梭菌(3/10),检出率为30%;3株兔源魏氏梭菌(3/12),检出率为25%,其PCR总检出率为20.6%,与常规细菌分离鉴定结果一致。应用显示本方法是魏氏梭菌的一种快速、简便的鉴定方法,具有很高的推广应用价值。
第三部分:魏氏梭菌Multi-PCR检测方法建立及在空气样品检测上的应用 根据GenBank上发表的序列设计了四对引物,通过不断优化反
In order to confirm the real Epidemical situation of Clostridium perfringens toxin types in Shandong Province, multiplex-PCR was established, by which we investigated 692 samples isolated from Dezhou,Taian, Zaozhuang,Qingdao,Linyi and Yantai. We detect 124 Clostridium perfringens positive,and all are type A.The survey result is absolutely true,which provide theory guide for previntion. The study established a fast PCR detect method to Clostridium perfringens,which has high specifity and possesses latent application prospect.The study consists of four parts.Part Ⅰ:Isolation and Toxin-type Identification of Clostridium perfringens of samples. 124 strains of Clostridium perfringens were isolated from animals feces. The study result showed isolation strains were toxin type A by API-20A biochemical test method.Part Ⅱ:A pair of primer was succeeded in designing and synthesizing according to the published sequence of the α -toxin of Clostridium perfringens.The PCR can amplify 325bp specific α -toxin fragments.It accords with the predicted amplification segament, however,it has no positive result with system to amplify Salmonella 、 E-coli 、 Enterococcus and Staphylococcus,so ,it can demonstrate the PCR amplification system has good specificity.In addition,it also possesses very high sensitivity because the system can detect the 1CFU/ml dilute template. The PCR technique was applied tor the detection of Clostridium perfringens in localized areas of Shandong province.The result showed that 21 of 102 samples were positive which is basically identical with other bacteria isolation methods.According to the above results ,the study has developed a Clostridium perfringens quick detection method which possesses latent application prospect. Part Ⅲ:For detection of Clostridium perfringens toxin types four pairs of primer for alpha, beta, epsilon and jota toxin gene were succeeded in designing and synthesizing according to the published sequence.It can
distinguish type A,B,C,D and E of Clostridium perfringens.The specific of PCR is high,only Clostridium perfringens show positive,other strains such as Salmonella > E-coli^ Enterococcus and Staphylococcus all show negative. The multiplex PCR was applied for the identification of toxin types of Clostridium perfringens from samples which were collected from cattle stable and had been identified by ELISA.The PCR result accord with the ELISA method.So the muliplex-PCR can be applied to epidemiology survey. Part IV: The multiplex PCR was applied for the identification of toxin types of Clostridium perfringens from samples which were collected from earlier epidemic districts of Shandong province. The results showed that all isolated 124 samples (17.9%) were Clostridium perfringens toxin type A, that was alpha toxin positive which is different from abroad reports.
引文
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