科研動(dòng)態(tài)

為建立適合犬布魯氏菌病臨床檢測(cè)的單重PCR方法，通過(guò)對(duì)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(jì)1對(duì)引物并優(yōu)化反應(yīng)條件，建立了可初步鑒別犬4種布魯氏菌的PCR方法，然后對(duì)該方法的特異性和靈敏度進(jìn)行評(píng)價(jià)，并對(duì)20份犬布魯氏菌血清學(xué)陽(yáng)性的血液樣品進(jìn)行臨床檢測(cè)。結(jié)果顯示，利用建立的PCR反應(yīng)體系對(duì)牛種、羊種和豬種布魯氏菌均能擴(kuò)增出717 bp的目的條帶，對(duì)犬種布魯氏菌能擴(kuò)增出366 bp的目的條帶；最低可檢測(cè)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(cè)到3份犬種布魯氏菌陽(yáng)性樣品，與使用GB/T 18646—2018引物的PCR檢測(cè)結(jié)果一致。結(jié)果表明，本試驗(yàn)建立的單重PCR方法簡(jiǎn)捷、特異、敏感，適合基層獸醫(yī)實(shí)驗(yàn)室對(duì)犬布魯氏菌病的檢測(cè)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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