Periódicos Brasileiros em Medicina Veterinária e Zootecnia

Specific detection of bovine coronavirus N protein with TaqMan probe qRT-PCR

Geng, Jin-jingGong, Zhuan-diLi, Qiong-yiShen, Xiao-yunWei, Suo-cheng

Background: Bovine Coronavirus (BCoV) can cause acute diarrhea in newborn calves and adult cattle. BCoV infectionmay cause losses to production by reduced weight gain and milk yield of the infected animals. Several methods have beenapplied to detect and diagnose BCoV. However, each assay has its deficiency. Currently, real-time quantitative PCR (qRTPCR) has been utilized to identify and quantify many viral pathogens since it is a highly sensitive. However, the technicalassay varies due to normalization control of the signal with an internal standard, typically a housekeeping gene.Materials, Methods & Results: The present study was aimed to establish a novel TaqMan probe real-time PCR (qRT-PCR)for detecting bovine coronaviruses (BCoV), and also to develop a diagnostic protocol which simplifies sample collectionand processing. One pair of specific primers, one pair of universal primers and a TaqMan probe were designed from theknown sequences of conserved nucleocapsid (N) protein of BCoV. Reaction systems of TaqMan qRT-PCR were optimizedincluding concentrations of the primers and probe as well as annealing temperatures. Prior to optimizing the assay, therecombinant plasmids of pMD18-T-BCoV-N were successfully constructed to make standard curves. The sensitivity, specificity and reproducibility were evaluated on the TaqMan qRT-PCR, respectively. A total of 321 feces specimens collectedfrom diarrheic calves were detected with this assay. The results showed the optimized reaction conditions for qRT-PCRwere 14.5 μM/L primers, 19.5 μM/L probes and 45.0°C annealing temperatures. The established TaqMan qRT-PCR assaycould specially detect BCoV without detecting any other viruses. Its minimum detection limit was 4.72 × 101 copies/μL.However, universal PCR could detect only 4.72 × 103 copies/μL...(AU)

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