為建立適合犬布魯氏菌病臨床檢測的單重PCR方法，通過對比粗糙型（犬種）與光滑型（羊種、牛種和豬種）布魯氏菌基因組DNA的序列差異，設(shè)計1對引物并優(yōu)化反應條件，建立了可初步鑒別犬4種布魯氏菌的PCR方法，然后對該方法的特異性和靈敏度進行評價，并對20份犬布魯氏菌血清學陽性的血液樣品進行臨床檢測。結(jié)果顯示，利用建立的PCR反應體系對牛種、羊種和豬種布魯氏菌均能擴增出717 bp的目的條帶，對犬種布魯氏菌能擴增出366 bp的目的條帶；最低可檢測到犬種、羊種、豬種和牛種布魯氏菌基因組DNA濃度分別為5.07×10-2、6.20×10-2、7.80×10-3和5.50×10-2 ng/μL；20份血液樣品中共檢測到3份犬種布魯氏菌陽性樣品，與使用GB/T 18646—2018引物的PCR檢測結(jié)果一致。結(jié)果表明，本試驗建立的單重PCR方法簡捷、特異、敏感，適合基層獸醫(yī)實驗室對犬布魯氏菌病的檢測與鑒定。
Development of a Single PCR Assay for Detection of Four Kinds of Brucella in Dogs
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.
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