Synchronous Detection of BPV and BVDV with Duplex Taqman qPCR Method

Background: Bovine parvovirus (BPV) and bovine viral diarrhea virus (BVDV) are commonly etiologies causing diarrhea in dairy herds. BPV is a member of bocaparvovirus genus with a non-enveloped capsid. BVDV, belonging to Pestivirus genus in Flaviviridae, possesses a single-stranded RNA, and is classified into BVDV-1 and BVDV-2 genotypes according to the 5’UTR sequence. 21 genetic groups of BVDV-1 and four groups of BVDV-2 have been found. Diagnosis of viral diarrhea is often relied on virus detection by isolation or detection of serum antibody. The main objective of the present study was to establish a duplex real time PCR (qPCR) based on Taqman probe to detect synchronously BPV and BVDV. Materials, Methods & Results: TaqMan probe and primers were designed and synthesized from the sequences of conserved 5′ - untranslated regions (5′ UTR) of Haden strain of BPV and NADL strain of BVDV. The cDNAs were transcribed in vitro to make standard curves before optimizing the assay. DNA/PCR products were ligated into pMD18-T vector, and then used to transfer BL-21 competent cells to acquire the recombinant plasmids of pMD18-T-BPV and pMD18-T-BVDV. Optimum reaction conditions were comparatively selected. The sensitivity, specificity and reproducibility of TaqMan probe qRT-PCR were evaluated respectively. The results showed the concentrations of pMD18-T-BPV or pMD18-T-BVDV were 2.0 × 1010 DNA copies/μL, respectively. A duplex Taqman qPCR method was developed by optimizing the amplification conditions to simultaneously detect BPV and BVDV. The assay targets at highly conserved VP2 gene of BPV and 5′ UTR gene of BVDV. This qPCR assay was assessed for specificity and sensitivity using DNA of BPV and cDNA of BVDV. For clinical validation, 308 samples were tested from clinically diarrhea calves. The results showed that optimum annealing temperature was achieved in 43.2 ℃ fro duplex BPV and BPIV. Dynamic curves and standard curves were created following amplification of recombinant plasmids using the optimized duplex Taqman BPV and BVDV, with an amplification efficiency of 95.69%. Duplex Taqman qPCR could only detect DNA of BPV and cDNA of BVDV with a strong specificity. The detection limitation was as low as 2.0 × 102 copies/μL of pMD18-T-BPV plasmid and 2.0 × 101 copies/μL for pMD18-T-BVDV plasmid, respectively. Sensitivity of detection was 100-fold higher than conventional PCR. Duplex Taqman qPCR had excellent repeatability or stability with less than 1.2% of intra-assay and inter-assay. 35 and 47 positive feces samples were identified using duplex Taqman qPCR in comparison to 30 and 42 positives for universal PCR, respectively. Discussion: The bovine viral diarrhea virus (BVDV) is a key pathogenic factor in bovine diarrhea. Currently, few effective measures are available for the treatment or prevention for BVDV and BPV infections in animals. The technique was proven to be repeatable and linear over a range of at least 5 magnitudes, from 101 to 105 RNA/DNA copies, thus ensuring an accurate measurement of BPV DNA and BVDV RNA loads in clinical samples. In conclusion, a duplex Taqman qPCR was established for detecting simultaneously BPV and BVDV. Taqman qPCR method was rapid and specific assay. This assay was 100-fold sensitive than conventional PCR. It will be propitious to rapidly and differentially diagnose pathogens of viral diarrhea of dairy farms. Taqman qPCR method was rapid and specific assay and had a sensitivity of 2.0 copies/μL

Specific Detection of Bovine Coronavirus N Protein with TaqMan Probe qRT-PCR

Background:  Bovine Coronavirus (BCoV) can cause acute diarrhea in newborn calves and adult cattle. BCoV infection may cause losses to production by reduced weight gain, reduced milk yield. Several methods have been applied to detect and diagnose BCoV. However, each assay has its deficiency. Currently, real-time quantitative PCR (qRT-PCR) has been utilized to identify and quantify many viral pathogens since it is a highly sensitive. However, the technical assay varies due to normalization control of the signal with an internal standard, typically a housekeeping gene. The main objective of the present study to establish a novel TaqMan probe real-time PCR (qRT-PCR) for detecting BCoV.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 collection and processing. One pair of specific primers, one pair of universal primers and a TaqMan probe were designed from the known sequences of conserved nucleocapsid (N) protein of BCoV. Reaction systems of TaqMan qRT-PCR were optimized including concentrations of the primers and probe as well as annealing temperatures. Prior to optimizing the assay, the recombinant 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 collected from diarrheic calves were detected with this assay. The results showed the optimized reaction conditions for qRT-PCR were 14.5 μM/L primers, 19.5 μM/L probes and 45.0°C annealing temperatures. The established TaqMan qRT-PCR assay could 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. Its sensitivity was 100-fold stronger than universal PCR. In conclusion, this TaqMan qRT-PCR had excellent specificity, sensitivity and stability with a 100-fold sensitivity stronger than universal PCR. Minimum detection limit was 4.72 × 101 copies/μL. This method was a cost-effective method to diagnose diarrhea and distinguish pathogens in dairy farms.Discussion:  In this study, the authors developed a quantitative real-time PCR (qRT-PCR) in this study based on the TaqMan probe of BCoV. This TaqMan qRT-PCR assay selected and used one pair of specific primers (BCoV-qF/BCoV-qR) and a specific TaqMan probe (BCoV-probe) targeting the conserved nucleocapsid (N) gene. The specificity of primers and probes was validated with Primer-BLAST. The specificity of the qRT-PCR was confirmed by the negative control and other six viruses. The findings demonstrated that TaqMan qRT-PCR could only detect BCoV. This verified the qRT-PCR had an excellent specificity. It is obvious that this TaqMan qRT-PCR assay can detect only BCoV with stronger sensitivity and reproducibility than other real-time PCR methods. The sensitivity test indicated the minimum detection limit of the TaqMan qRT-PCR was 4.72 × 101copies/μL, or 47.2 copies/μL. Sensitivity of the TaqMan qRT-PCR assay was increased by 100-fold as compared to universal PCR with a good inter-assay and intra-assay reproducibility. Thereby, based on the high sensitivity of the assay of this qRT-PCR assay it may be a cost-effective method to diagnose BCoV infections and indentify the etiologic agents of diarrhea syndrome in the dairy farms