布鲁氏菌PCR检测方法的应用与分子分型研究
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摘要
为弥补微生物学和血清学方法在布鲁氏菌检测中的不足,以及探索、建立一种快速、准确的布鲁氏菌基因分型方法,本研究开展了以下三个方面的研究:
首先,通过对已发表的布鲁氏菌属、种、型基因序列进行分析,寻找出布鲁氏菌种、型特异性基因分布规律,设计引物,运用VirB8-PCR,AMOS-PCR,B1-PCR方法对布鲁氏菌属、种、型进行鉴定。与此同时,依据聚合酶链反应-单链构象多态性(PCR-SSCP)原理,建立Ery-PCR-SSCP方法,用于牛种A19疫苗株与野毒株的区分鉴定。
其次,以布鲁氏菌致病力因子VirB7下游至VirB9上游基因序列为目的扩增片段,设计上、下游引物,优化血样、奶样中布鲁氏菌基因组DNA的提取方法,建立布鲁氏菌内参PCR(IR-PCR)检测方法,用于血液、奶液样本中布鲁氏菌的检测,以达到降低普通PCR法在检测布鲁氏菌中的假阴性率。
最后,对本实验室前期研制的布鲁氏菌VirB8基因PCR试剂盒进行组装,并对试剂盒各项特性(特异性、敏感性、保存期、可重复性)进行评测。同时应用该试剂盒对新疆、山西的2批65份血样进行了检测,评估该试剂盒的临床稳定性。
通过试验研究,获得以下结果:(1)VirB8-PCR,AMOS-PCR,B1-PCR方法能准确检测出布鲁氏菌,并能对其种、型进行鉴定;PCR-SSCP方法可将A19疫苗株与野毒株区分开来。(2)内参PCR方法在与细菌分离鉴定、血清学诊断的对比中,呈现出较高的阳性符合率(与细菌分离的阳性符合率为100%,与iELISA的阳性符合率为62.5%),其对血样、奶样的检出量分别为35CFU/ml、350CFU/ml,该方法适合于血样、奶样中布鲁氏菌的检测。(3)VirB8-PCR诊断试剂盒在对布鲁氏菌标准株、疫苗株及65份送检血样进行检测中,能从SAT阴性血样中检测出2份阳性,表现其敏感性高于SAT,同时实验数据表明,当每毫升血样中含有布鲁氏菌数量为35时即可被本试剂盒检测出阳性结果,体现出本试剂盒较高的敏感性。实验结果证实:本试剂盒的可重复性良好、特异性高,-20℃下可有效保存至少6个月。
In order to mark up for a dificiency in the appoach of microbiology and serological detection of Brucella. To explore and establish a rapid method of molecular typing of Brucella. The study is started by following three aspects of research.
First of all, To analyze a great number of partition Brucella of types sequence which have been issued as specific genotype, Searching for the distributing role of genotype- specific nucleotides, designing the specific primers, applying PCR method to identify specific type of Brucella. Established PCR-SSCP method distinguishes vaccine strain A19 from other specific types of Brucella.
Secondly, The downstream sequence of VirB7 to upstream sequence of VirB9 gene from type IV secretion system(TTSS)of brucella was used as template and the primers were designed. The extraction methods of DNA were optimized. Established IR-PCR (Internally Reference PCR) method of assay were used for detection of Brucella in anticoagulated blood and milk.
Finally, The PCR kit for detecting VirB8 gene of Brucella in blood was assembled and tested for its specificity, sensitivity, storage life and stability. Use the PCR kit, the blood samples from Xinjiang and Shanxi provinces of which were detected.
The results show that the VirB8-PCR,AMOS-PCR,B1-PCR method can be used to diagnosis of Brucellosis and identify specific type of Brucella; PCR-SSCP assay can be detected vaccine strain A19. 2. Established IR-PCR (Internally Reference PCR) method of assay is more effective to avoid operational errors of process of DNA extraction, of which the positive coincident rate of the IR-PCR detecting result with purification identification was 100 % and the positive coincident rate of the IR-PCR detecting result with iELISA was 62.5%; The detection limit was as low as 35CFU/ml for blood and 350CFU/ml for milk. The IR-PCR are very suitable for detecting brucella infection of anticoagulated blood and milk in laboratory. 3.The 2 samples showing negative by SAT were proved Brucella positive by the kit , the sensitivity of the kit being higher than that of the SAT. The detection limit was as low as 35CFU/ml for PCR kit, which revealed that the specificity of the kit is very reliable in -20℃for 6 months.
首先,通过对已发表的布鲁氏菌属、种、型基因序列进行分析,寻找出布鲁氏菌种、型特异性基因分布规律,设计引物,运用VirB8-PCR,AMOS-PCR,B1-PCR方法对布鲁氏菌属、种、型进行鉴定。与此同时,依据聚合酶链反应-单链构象多态性(PCR-SSCP)原理,建立Ery-PCR-SSCP方法,用于牛种A19疫苗株与野毒株的区分鉴定。
其次,以布鲁氏菌致病力因子VirB7下游至VirB9上游基因序列为目的扩增片段,设计上、下游引物,优化血样、奶样中布鲁氏菌基因组DNA的提取方法,建立布鲁氏菌内参PCR(IR-PCR)检测方法,用于血液、奶液样本中布鲁氏菌的检测,以达到降低普通PCR法在检测布鲁氏菌中的假阴性率。
最后,对本实验室前期研制的布鲁氏菌VirB8基因PCR试剂盒进行组装,并对试剂盒各项特性(特异性、敏感性、保存期、可重复性)进行评测。同时应用该试剂盒对新疆、山西的2批65份血样进行了检测,评估该试剂盒的临床稳定性。
通过试验研究,获得以下结果:(1)VirB8-PCR,AMOS-PCR,B1-PCR方法能准确检测出布鲁氏菌,并能对其种、型进行鉴定;PCR-SSCP方法可将A19疫苗株与野毒株区分开来。(2)内参PCR方法在与细菌分离鉴定、血清学诊断的对比中,呈现出较高的阳性符合率(与细菌分离的阳性符合率为100%,与iELISA的阳性符合率为62.5%),其对血样、奶样的检出量分别为35CFU/ml、350CFU/ml,该方法适合于血样、奶样中布鲁氏菌的检测。(3)VirB8-PCR诊断试剂盒在对布鲁氏菌标准株、疫苗株及65份送检血样进行检测中,能从SAT阴性血样中检测出2份阳性,表现其敏感性高于SAT,同时实验数据表明,当每毫升血样中含有布鲁氏菌数量为35时即可被本试剂盒检测出阳性结果,体现出本试剂盒较高的敏感性。实验结果证实:本试剂盒的可重复性良好、特异性高,-20℃下可有效保存至少6个月。
In order to mark up for a dificiency in the appoach of microbiology and serological detection of Brucella. To explore and establish a rapid method of molecular typing of Brucella. The study is started by following three aspects of research.
First of all, To analyze a great number of partition Brucella of types sequence which have been issued as specific genotype, Searching for the distributing role of genotype- specific nucleotides, designing the specific primers, applying PCR method to identify specific type of Brucella. Established PCR-SSCP method distinguishes vaccine strain A19 from other specific types of Brucella.
Secondly, The downstream sequence of VirB7 to upstream sequence of VirB9 gene from type IV secretion system(TTSS)of brucella was used as template and the primers were designed. The extraction methods of DNA were optimized. Established IR-PCR (Internally Reference PCR) method of assay were used for detection of Brucella in anticoagulated blood and milk.
Finally, The PCR kit for detecting VirB8 gene of Brucella in blood was assembled and tested for its specificity, sensitivity, storage life and stability. Use the PCR kit, the blood samples from Xinjiang and Shanxi provinces of which were detected.
The results show that the VirB8-PCR,AMOS-PCR,B1-PCR method can be used to diagnosis of Brucellosis and identify specific type of Brucella; PCR-SSCP assay can be detected vaccine strain A19. 2. Established IR-PCR (Internally Reference PCR) method of assay is more effective to avoid operational errors of process of DNA extraction, of which the positive coincident rate of the IR-PCR detecting result with purification identification was 100 % and the positive coincident rate of the IR-PCR detecting result with iELISA was 62.5%; The detection limit was as low as 35CFU/ml for blood and 350CFU/ml for milk. The IR-PCR are very suitable for detecting brucella infection of anticoagulated blood and milk in laboratory. 3.The 2 samples showing negative by SAT were proved Brucella positive by the kit , the sensitivity of the kit being higher than that of the SAT. The detection limit was as low as 35CFU/ml for PCR kit, which revealed that the specificity of the kit is very reliable in -20℃for 6 months.
引文
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