Quadriplex PCR assay for diagnosis of haemoprotozoan diseases in bovines

Abstract

The present study was conducted to standardize a quadriplex PCR assay for rapid, sensitive, specific and simultaenous diagnosis of major haemoprotozoan diseases of bovines. Primers targeting cytochrome b gene, repetitive nucleotide sequences, gene encoding carbomyl phosphate synthetase II and small subunit rRNA were used for detection of Trypanosoma evansi, Theileria annulata, Babesia bovis and Babesia bigemina generating amplicon of 257bp, 312bp, 446bp and 689bp, respectively. Multiplex PCR assay was optimized in a final volume of 25 µl containing 12.5 µl dream taq green master mix (2X), 0.5 µl of each cytob1 primer, 0.4 µl of each TR3/TR4 primer, 0.4 µl of Bb1/Bb2 primer and 0.6 µl of Bg3/Bg4 primer, 4µl of template DNA and rest NFW. Twenty DNA samples extracted from field blood samples were screened by quadriplex PCR assay. Primers were found specific as did not produce amplicon with non-target DNA. Limit of detection was determined using tenfold serial dilution of parasitic DNA. Quadriplex PCR assay could able to amplify DNA of Theileria annulata and Babesia bigemina at copy number 1000 whereas of Babesia bovis and Trypanosoma evansi at copy number 100, respectively.  Out of 20 samples, T. annulata was seen in 15% sample, B.bigemina and B.bovis in 5 % sample and B. bigemina + T. annulata in 10% samples, rest sample were negative. Assay was found to be highly specific and can be used for simultaneous diagnosis of major haemoprotozoan diseases.