实验犬布氏杆菌的多重PCR检测与分型鉴定

Detection of Brucellosis in Canine by Multiplex Polymerase Chain Reaction

目的建立实验犬及相关生物制品布氏杆菌的多重PCR检测与分型鉴定方法。方法选择布氏杆菌Omp2基因同源性较高的区域设计引物对布氏杆菌进行多重PCR扩增,扩增结果一致的样本进行酶切以区分不同型,同时进行序列测定,以确定该方法的准确性;然后验证该方法的特异性和敏感性。结果成功扩增得到目的条带,并通过酶切区分五种布氏杆菌;PCR产物与布氏杆菌DNA序列同源性达到99%,并验证了该方法的检测结果。实验结果证明该方法特异性较好,灵敏性为1.8×10-7μg/mL。结论成功建立布氏杆菌多重PCR检测与分型鉴定方法,所建立的方法特异性好,灵敏度高。本研究对保证实验犬群的质量,保护饲养人员、实验人员的身体健康具有重要意义。

ObjectiveTo establish multiplex PCR assay for detects Brucellosis in laboratory canine and related biological products.Method These primers of multiplex PCR were designed according to the Omp2 gene of Brucellosis,and the PCR reaction was optimum,and then some samples were digested by three restriction enzyme for differentiate Brucellas.We also cloned PCR products and measured sequence after multiplex PCR to determine the accuracy of the assay.Then the specificity and sensitivity of the assay was tested.Results The target bands were successfully amplified,and these samples were successfully distinguished by digestion of the PCR product;It was 99% homology between the target segment by multiplex PCR and Brucellosis DNA sequence from GeneBank.The specificity of this method is better,1.8 × 10^-7 μg /ml samples can be detect.Conclusion The Brucella PCR detection and typing of multiple identification methods was established successful,the assay have good specificity and high sensitivity.The assay is important to ensure the quality of laboratory canine and protect the health of the animal keepers and laboratory personnels.