Development of the Polymerase Chain Reaction Technique (PCR) for the Detection of Babesia canis (B. canis) Infection in Dogs
To develop the polymerase chain reaction technique for the detection of B.canis in the dogs and to identify the subspecies of B.canis in Thailand.
Materials & Methods
DNA from B. canis-infected canine blood samples was extracted and used to perform PCR using primer located at the intervening 5.8S coding region of rRNA gene. The PCR conditions were consisted of an initial 2 minutes denaturation at 94°C, then 35 cycles of denaturation at 94°C for 60 seconds, annealing at 55°C for 60 seconds, and extension at 72°C for 90 seconds. The PCR product was digested with the restriction enzyme AspS9 and analyzed by 2% agarose gel electrophoresis. The PCR product was also sequenced and compared with B. canis sequences published on GenBank.
All samples gave the PCR product similar in size, which was approximately 800 bp. For AspS9 restriction enzyme analysis, all of PCR product was cut into two fragments, which consisted of 600 bp and 200 bp DNA fragments. This result showed that all of B. canis samples were B.canis vogeli. The sequencing result was in accordance with the restriction enzyme analysis.
According to these results, the subspecies of B. canis in Thailand was B.canis vogeli or mainly B.canis vogeli. This PCR technique is fast and inexpensive which may be suitable for the typing of subspecies of B. canis.