Development and application of multiplex real-time PCR assay for diagnosis of Brucella abortus, Campylobacter fetus subspecies venerealis and Tritrichomonas fetus
Abstract
Brucellosis, Bovine campylobacteriosis and trichomoniasis are the diseases of major concern which are responsible for huge economic losses to livestock industry. Isolation and serological tests are considered as the gold standard assays for diagnosis of these causative agents but they are time-consuming and lack specificity. To control and eradicate a disease, a confirmatory diagnostic method which is sensitive, quick and specific is the foremost requirement. Therefore, in the present study a TaqMan probe based multiplex real time PCR assay was developed for simultaneous detection of Brucella abortus, Campylobacter fetus subsp. venerealis and Tritrichomonas fetus. The reproducible detection limit of mqPCR was found to be ~60 copies for Campylobacter fetus subsp. venerealis, ~ 3 copies for Brucella abortus while ~6 copies for Tritrichomonas fetus with high efficiency i.e. 93.64% for Campylobacter fetus subsp. venerealis, 104.18% for Tritrichomonas fetus and 95.88% for Brucella abortus. In terms of amount of template, limit of detection of mqPCR assay was as low as 0.02fg for Tritrichomonas fetus, 0.2fg for Campylobacter fetus subsp. venerealis and 1fg for Brucella abortus. Analytical sensitivity of initial single assay and multiplex assay were estimated using standard curve method. The R2 and slope of standard curve for mqPCR assay was 0.995, 0.992 and 0.993 and -3.220,-3.480 and -3.422 for Tritrichomonas fetus, Campylobacter fetus subsp. venerealis and Brucella abortus respectively. The mqPCR assay did not produce any nonspecific amplification when tested with related pathogens. The mqPCR assay was also compared with conventional PCR and it was found 100 times more sensitive than conventional PCR. Field samples (aborted foetuses, fetal parts, blood samples, discharge from repeat beeding animals, preputial washings and semen samples) collected from aborted animals, repeat breeders and bulls from different parts of Haryana were also tested with the developed mqPCR assay. Only one sample (uterine discharge) was found positive for Brucella abortus while none of the sample was found positive for Tritrichomonas fetus, Campylobacter fetus subsp. venerealis. In conclusion, the present study showed that the developed mqPCR assay is more sensitive, specific, have high reproducibility and repeatability and is faster than serological and conventional PCR methods in detecting the Brucella abortus, Campylobacter fetus subspecies venerealis and Tritrichomonas fetus from diverse group of biological samples. However, more number of samples should be tested for proper validation and before implementation of the developed mqPCR in routine diagnosis of these pathogens.
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