OLIGONUCLEOTIDE PRIMER SEQUENCES FOR THE SPECIFIC AND SENSITIVE DETECTION OF BEBESIA BIGEMINA BY POLYMERASE CHAIN REACTION

摘要

Bovine babesiosis is an economically important tick-borne disease causing considerable mortality and morbidity in purebred and cross-bred cattle while the indigenous and the recovered animals act as reservoir of infection to the susceptible population. For improved management of the disease, refinement of the currently available diagnostic tools is an urgent need. The parasitological and serological techniques in use today suffer from sensitivity and specificity limitations, ideally required for detection of low grade reservoirs of the disease. The polymerase chain reaction (PCR) method offers an exciting prospect of meeting the above requirements. The present invention deals with discovery of pair of oligonucleotide primer sequences of 23 base pairs length: 5" TTCCCACACGGTAGCGCGCAAAA 3"(Forward primer) and 5" GGGCGGTGAAAAGAGCAACGACA 3" (Reverse primer) for the sensitive and specific detection of B. bigemina by PCR method. The primer set was designed and custom synthesi/ed to amplify a single DNA band of 40()bp. The dc novo primers used in the PCR assay were designed using the nucleotide sequence information obtained from a monomorphic DNA fragment amplified by arbitrary primer- polymerase chain reaction from Indian isolates of Bahesia bigemina. The nucleotide sequence data comparison of this product revealed no homology with any known nucleotide sequence information of proto/oan parasites or the common mammalian host species lor the target parasite. The sensitivity limit of the primer set was 5 picograms of template DNA and higher than the previously described primer sequence (Figueroa el c;/.1992). No cross-amplification was evident with heterologous DNAs such as, bovine / hubaline leucocyte DNA and genomic DNAs obtained from common protozoan parasites of bovids like, Theileria annulata, Tryptinosoma cvansi. and Toxoplasma gondii.