二重PCR快速检测牛伊氏锥虫和牛巴贝斯虫的研究

Rapid and sensitive detection of Trypanosoma evansi and Babesia bovis in bovine by double PCR

根据伊氏锥虫ITS序列(FJ416612)和GenBank报道的牛巴贝斯虫棒状结合蛋白1序列(FJ588013),设计合成2对特异性诊断引物,建立了牛伊氏锥虫和牛巴贝斯虫的二重PCR诊断方法。结果表明,建立的二重PCR诊断方法可扩增出牛伊氏锥虫和牛巴贝斯虫的特异性条带,大小分别为213bp和360bp,最低检出量分别为4.00pg和0.38pg。但对牛双芽巴贝斯虫、分歧巴贝斯虫、环形泰勒虫、附红细胞体、巴氏杆菌不敏感。用该二重PCR方法对95份水牛样品和134份奶牛样品进行检测,结果发现,水牛伊氏锥虫的感染率为13.7%(13/95)、牛巴贝斯虫的感染率为7.4%(7/95),无混合感染;奶牛伊氏锥虫的感染率为3.7%(5/134)、牛巴贝斯虫的感染率为0(0/134)。说明该二重PCR方法具有敏感、快速、特异的特点,适用于牛伊氏锥虫和牛巴贝斯虫的流行病学调查。

Two pairs of species-specific primers （ST1 and [T2, B1 and B2） for Trypanosoma evansi and Babesia bovis were designed using the ITS sequence （FJ416612） of T. evansi and elavate binain sequences （FJ588013） of B. boris （Obtained from GenBank）, and a double PCR amplification method for the diagnosis of T. evansi and B. boris infection was developed. The amplification products of 2136 and 360 bp corresponded to the T. evansi and B. bovis, respectively, have been resolved successfully with the lowest detection limit of 4.00 pg （for T. evansi） and 0.38 pg （for B. boris）. These two pairs of primers could not amplify the specific bands in case of Babeisia bigemina, Babeisia divergens, Theileda annulata, Wenyonli eperythrozoon, Pasteurella multoeida. The blood samples of 95 buffalos and 134 dairy cows was collected from Guangxi and analyzed using double PCR amplification method. The infection rate of T. evansi and B. boris in buffalos was found to be 13.7% （13/95） and 7.4% （7/95）, respectively. A very little or no mixed infection has been observed in dairy cows, it was 3.7% （5/134） and 0 （0/134）, respectively. Due to the high sensitivity, specificity and rapidness of this procedure, it can be suitably used for the large-scale epidemiological study of T. evansi and B. bovis in bovine.