Rapid detection of Theileria sergenti by the polymerase chain reaction in Korean cattle

중합효소연쇄반응을 이용한 한우에 감염된 Theileria sergenti의 신속한 검출

To make the genomic DNA probe of Theileria sergenti, the merozoites were purified from erythrocytes of Korean cattle, The previous studies on the probe of T sergenti had resulted in two probes as KTS1 and KTS3 DNA fragment. Nucleotide sequence of both ends of the KTS1 and KST3 were determined in order to design primers for polymerase chain reaction. A pair of an uper primer(5'-CCTCTTGAAGTCATCCATGT-3'; nucleotide position 48) and a lower primer(5'-CACTGAGCTG GAAAGAGCTA-3'; nucleotide position 156) in pKTS1 were synthesized. The anticipated PCR product was 128bp in length. To examine the sensitivity of the PCR, KTS1 DNA and purified T sergenti DNA were serially diluted by tenfolds with distilled water. The primers were sensitive enough to detect 4ag of the authentic template DNA and 4fg of the purified T sergenti DNA by PCR. Furthermore, when the blood was serially diluted by two-folds with 0.9% saline, the pair could detect up to 0.00029%(about 164 parasites in $10{\mu}l$ of blood) of T sergenti infection in bovine erythrocytes by PCR. In a comparison of microscopic and PCR detection of T sergenti in the same samples from Chonbuk area, 47 and 51 out of 70 sample(67.1%) were positive by the former and by the latter method, respectively.