在序列比對分析的基礎(chǔ)上，設(shè)計(jì)2對引物和2條MGB探針，經(jīng)體系優(yōu)化建立了可快速鑒別檢測兩種馬梨形蟲病病原（馬泰勒蟲和駑巴貝斯蟲）的雙重?zé)晒釶CR檢測方法。該方法可分別檢出22和31基因拷貝的蟲體DNA，且不與其他馬病病原發(fā)生交叉反應(yīng)。應(yīng)用建立的熒光PCR檢測方法，對采自進(jìn)出口馬匹的10份馬全血和20份馬血清進(jìn)行檢測，結(jié)果從馬全血中檢出2份馬泰勒蟲陽性樣品。使用一對馬泰勒蟲EMA1全基因擴(kuò)增引物，從2份陽性樣品中擴(kuò)增了EMA1基因?？寺y序后，對該基因全序列進(jìn)行分析，證實(shí)其為馬泰勒蟲特異基因序列；2份樣品的基因序列相似性為98.9%，存在6個(gè)氨基酸變異；2個(gè)樣品中的EMA1基因不完全一致，表明2個(gè)感染樣品中的蟲體可能為不同的馬泰勒蟲蟲株。
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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