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?:abstract
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In order to establish a specific and sensitive TaqMan- based real- time RT- PCR assay for detecting Bovine coronavirus (BCoV) and its RNA in calf diarrhea clinical samples, a pair of primers and one probe targeting to nsp10 polymerase gene of BcoV were designed After optimizing the reaction conditions, a TaqMan-based real-time RT-PCR assay was successfully established The assay showed a good linear relationship within the range of 3 36 x 101 copies/L- 3 36 x 109 copies/L, with a correlation coefficient of R2=0 9996, and the amplification efficiency was 109% This assay only specifically detected BCoV with a detection limit of 33 6 copies/L, whereas no other common unrelated pathogens could be detecteed Compared to other methods, this assay has a slightly higher sensitivity;both the intra- and the inter- assay coefficients of variation were less than 5%, indicating its good reproducibility The detection rate of BCoV for diarrhea samples was higher than that of a previously reported TaqManbased real-time RT-PCR assay Moreover, 153 diarrhea samples of dairy calves were collected from Xinjiang and Liaoning regions, 71 23% of the samples were detected as BCoV positive, and the farm positive rate was 100%, suggesting that BCoV infection in cattle is common in these regions In conclusion, a real-time RT-PCR assay targeting the BCoV polymerase gene was successfully established for the first time This assay has high sensitivity, specificity, and reproducibility, which would contribute to the detection and epidemiological investigation of BCoV
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