在序列比对分析的基础上，设计2对引物和2条MGB探针，经体系优化建立了可快速鉴别检测两种马梨形虫病病原（马泰勒虫和驽巴贝斯虫）的双重荧光PCR检测方法。该方法可分别检出22和31基因拷贝的虫体DNA，且不与其他马病病原发生交叉反应。应用建立的荧光PCR检测方法，对采自进出口马匹的10份马全血和20份马血清进行检测，结果从马全血中检出2份马泰勒虫阳性样品。使用一对马泰勒虫EMA1全基因扩增引物，从2份阳性样品中扩增了EMA1基因。克隆测序后，对该基因全序列进行分析，证实其为马泰勒虫特异基因序列；2份样品的基因序列相似性为98.9%，存在6个氨基酸变异；2个样品中的EMA1基因不完全一致，表明2个感染样品中的虫体可能为不同的马泰勒虫虫株。

Establishmentand Application of a Duplex Real-time PCR Method for Detectionof Piroplasmosis Disease

Two pairs ofprimers and two MGB probes were designed based on sequence alignment，after system optimization，a duplex real-time PCR method foridentification and detection of two pathogens（Theileria equiand Babesia caballi）of piroplasmosisdisease was established. The detection limits were 22 and 31 gene copiesrespectively，and no cross reaction with otherpathogens of horse diseases was observed. By the established real-time PCR，10 whole blood and 20 serum samplescollected from imported and exported horses were tested，and 2 positive samples（against Theileria equi）were detected；then EMA1 gene from the two positivesamples were amplified through a pair of amplification primers of EMA1 gene ofTheileria equi. After being cloned and sequenced，the wholesequence of the genes were analyzed，and theamplified genes were confirmed to be the specific sequences of Theileria equi；The similarity of EMA1 sequence of thetwo samples was 98.9%，with six aminoacid mutations，indicating that the worms in the twosamples might belong to different strains of Theileria equi.

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