Abstract:

Objective  To develop a duplex PCR assay for detection of Babesia caballi and Theileria equi.  Methods  Two pairs of primers were designed according to the BC48 gene of B. caballi and 18 s rRNA gene of T. equi， and a duplex PCR assay was developed by the optimization of reaction conditions. The specificity， sensitivity and reliability of the method were tested. The horse blood samples of suspected cases were collected from Yili region， and detected by the duplex PCR， microspopy， conventional PCR， and fluorescence quantitative PCR， and the results were compared.  Results  Using the duplex PCR assay， the specific fragments of 155 bp and 280 bp were amplified from DNA samples of B. caballi and T. equi， respectively. No specific fragment was amplified from DNA samples of B. bigemina、 Theilerdia annulata， Theilerdia sergenti， Toxoplasma gondii， Neospora caninum， and Trypanosoma evansi. The limit of detection was 4.85×105 copies/μl for B. caballi DNA and 4.85×104 copies/μl for T. equi DNA， respectively. Among the 24 blood samples， 11 were found B. caballi-positive by the duplex PCR assay， and 18 were T. equi-positive. The coincidence rate of microscopy， conventional PCR， and fluorescence quantitative PCR with duplex PCR was 91.7%（22/24）， 95.8%（23/24）， and 95.8%（23/24）， respectively.  Conclusion  A duplex PCR assay for simultaneous detection of B. caballi and T. equi is established.

Key words: Babesia caballi, Theileria equi, Duplex PCR