Standardization of Reverse Transcriptase – Polymerase Chain Reaction for the Detection of Paraflagellar Rod Gene of Trypanosoma evansi in Cattle
S. Sivajothi V. C. Rayulu P. M. Kondaiah D. Sreenivasulu Ch. Srilatha B. Sudhakara Reddy
Vol 8(4), 94-100
Paraflagellar rod is the major structural component of Trypanosoma evansi flagellum and is identified as a complex lattice of filaments which runs parallel to the axoneme throughout most of the length of the flagellum of Trypanosomatids. In the kinetoplastid species paraflagellar rod 2 gene is highly conserved. Therefore paraflagellar rod 2 gene was suggested as vaccine candidate as well as diagnostic antigen. Reverse transcriptase - polymerase chain reaction was standardized to investigate the existence of paraflagellar rod 2 gene in the local strain of Trypanosoma evansi. The conserved 5’ PFR gene of Trypanosoma evansi was amplified by using variable RNA concentrations and cycling conditions. The RNA amount was set in a volume of 3 µl at 20 ng/µl and the optimal reverse transcriptase - polymerase chain reaction cycling condition was established at 94ºC for 3 min, followed by 35 cycles of denaturation at 94°C for 30 sec, primer annealing at 60°C for 45 sec, extension at 72°C for 1 min with a final extension at 72°C for 15 min. The optimized amplification resulted in 1800 bp band of PFR 2 gene of Trypanosoma evansi parasites. Successful RT-PCR amplification, using cDNA generated out of the template RNA by reverse transcription yielded an 1800 bp specific product of the expected size from the host cell free Trypanosoma evansi parasites.
Keywords : Paraflagellar Rod Gene RT-PCR Trypanosoma evansi
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