单重PCR鉴别犬4种布鲁氏菌检测方法的建立

Development of a Single PCR Assay for Detection of Four Kinds of Brucella in Dogs

为建立适合犬布鲁氏菌病临床检测的单重PCR方法,通过对比粗糙型(犬种)与光滑型(羊种、牛种和猪种)布鲁氏菌基因组DNA的序列差异,设计1对引物并优化反应条件,建立了可初步鉴别犬4种布鲁氏菌的PCR方法,然后对该方法的特异性和灵敏度进行评价,并对20份犬布鲁氏菌血清学阳性的血液样品进行临床检测。结果显示,利用建立的PCR反应体系对牛种、羊种和猪种布鲁氏菌均能扩增出717 bp的目的条带,对犬种布鲁氏菌能扩增出366 bp的目的条带;最低可检测到犬种、羊种、猪种和牛种布鲁氏菌基因组DNA浓度分别为5.07×10^(-2)、6.20×10^(-2)、7.80×10^(-3)和5.50×10^(-2) ng/μL;20份血液样品中共检测到3份犬种布鲁氏菌阳性样品,与使用GB/T 18646—2018引物的PCR检测结果一致。结果表明,本试验建立的单重PCR方法简捷、特异、敏感,适合基层兽医实验室对犬布鲁氏菌病的检测与鉴定。

In order to establish a single PCR assay appropriate for detection of brucella in dogs,one pair of primers were designed through comparison of genomic DNA sequences between rough phenotype(B.canis)and smooth phenotype(B.melitensis,B.abortus and B.suis)strains,after optimization of reaction conditions,a PCR assay was established for preliminary detection of the four kinds of brucella,then its specificity and sensitivity were evaluated,and 20 Brucella-seropositive blood samples from dogs were tested clinically.The results showed that,for B.melitensis,B.abortus and B.suis,the fragment with the length of 717 bp could be amplified by the established method,and for B.canis,the fragment with the length of 366 bp could be amplified;the lowest genomic DNA concentrations of B.canis,B.melitensis,B.suis and B.abortus were 5.07×10-2,6.20×10-2,7.80×10-3 and 5.50×10-2 ng/μL,respectively;three positive samples for B.canis were detected out in 20 blood samples,which was consistent with the PCR results of using GB/T 18646—2018 primers.As a conclusion,the established assay was appropriate for detection of brucella in dogs in field veterinary laboratories due to its advantages of simplicity,specificity and sensitivity.