Detection of anatid herpesvirus 1 gC gene by TaqMan™ fluorescent quantitative real-time PCR with specific primers and probe

Final concentrations of primers each of 0.5 μmol/L and probe of 0.25 μmol/L were selected, and the optimized annealing temperature was 53°C. The combination of primers, probe and annealing temperature was used for subsequent experiments.

The amplification curves (Figure 1.a.) and standard curve (Figure 1.b.) of the TaqMan™ FQ-PCR were generated by using the 10-fold dilutions of pcDNA3.1-gC, which has already known its copies to undertake FQ-PCR reaction under optimum conditions with the iCycler IQ Detection System. The curve covered a dynamic range of eight log units of concentration and displayed a clear linear relationship with a correlation coefficient of 1.000 and high amplification efficiency (100%). By using the following formula, we were able to quantify the amount of unknown samples: Y = -3.321X + 45.822 (Y = threshold cycle, X = log starting quantity).

Ten-fold dilution series of pcDNA3.1-gC standard DNA (from 1.0 × 10⁵ to 1.0 × 10⁰ copies/reaction) were tested by the established FQ-PCR assay to evaluate the sensitivity of the system, the mean threshold cycle (Ct) values were 29.60, 33.10, 36.43, 39.30, 42.57 and N/A respectively. The results showed that the assay could detect down to 1.0 × 10¹ copies per reaction (Figure 2.). All liver samples were retested positive for AHV-1 from infected ducks with the established FQ-PCR assay, it indicated that this method was sensitive for clinical cases. Comparisons were made between the established FQ-PCR method and conventional PCR method by using 10-fold dilutions of viral DNA from infected allantoic fluid to calculate the end-point sensitivity of each assay. The results showed that the established FQ-PCR could detect viral DNA down to dilutions of 2.730 × 10¹, while the dilutions of only 2.730 × 10⁴ for conventional PCR.

The specificity test showed that pcDNA3.1-gC, AHV-1 attenuated vaccine (AHV-1 Cha) strain virus and AHV-1 virulent (AHV-1 Chv) strain virus were found positive for AHV-1 by the established FQ-PCR assay, while the bacteria, remaining viruses including negative control (liver sample of the healthy duck) were negative (Figure 3.). The results were confirmed by gel electrophoresis, there was a band of the expected size (78 bp) observed exclusively from samples of pcDNA3.1-gC, AHV-1 Cha and AHV-1 Chv. It indicated that the established FQ-PCR assay was highly specific.

In the intra-assay and inter-assay, the mean Ct values and standard deviations (SD) values were calculated. As shown in Table 1, the coefficient of variation (CV) values ranged from 0.44% to 2.03%, indicating that this assay was highly repeatable and reproducible.

AHV-1 gC gene and viral load quantification demonstrated that the AHV-1 gC copies of each sample could be calculated by using the Ct value determined from the standard curve. As shown in Table 2, AHV-1 gC can be detected in all analyzed tissues at 1 hour postinoculation. gC copies of all tissues reached a peak at 1 hour postinoculation in gC DNA vaccine-inoculated ducks, while the copies of most tissues (other than kidney) reached a peak at 4 hours postinoculation in AHV-1 Cha strain-infected ducks. The concentration of nucleic acid in DNA vaccine-inoculated ducks maintained 10⁷ copies/g level at 4 weeks postinoculation. The copies of the liver, spleen and thymus were more than other tissues in gC DNA vaccine-inoculated ducks, while the copies of the duodenum and rectum were relatively low in AHV-1 Cha strain-infected ducks.

AHV-1 Cha strain and Escherichia coli JM109 were obtained from Key Laboratory of Animal Diseases and Human Health of Sichuan Province. According to the gene libraries of AHV-1 constructed by the Avian Disease Research Center of Sichuan Agricultural University[56], the pMD18-gC plasmid was obtained through a 1296 bp fragment (gC gene) of PCR amplification was cloned into the pMD18-T vector (Takara, Japan), and then the result of sequencing compared with the sequences of AHV-1 in GenBank. Sequence was submitted to GenBank [GenBank: EU076811] by the Avian Disease Research Center of Sichuan Agricultural University [57].

Gosling new type viral enteritis virus, duck hepatitis virus type1, duck adenovirus, goose parvovirus, Marek's disease virus, AHV-1 Chv strain virus, Pasteurella multocida (5: A), Escherichia coli (O78) and Salmonella enteritidis (No. 50338) were provided by Key Laboratory of Animal Diseases and Human Health of Sichuan Province. They were propagated and the nucleic acid was extracted [58‐60].

The purified gC gene was obtained from pMD18-gC by using restriction enzymes (Eco R I and Xho I) (Takara, Japan), and was inserted into the eukaryotic expression vector pcDNA3.1(+) (Invitrogen, USA) according to the manufacturer's protocol. The constructed pcDNA3.1-gC plasmid was transformed into Escherichia coli JM109 cells. pcDNA3.1-gC plasmid was extracted by TIANprep plasmid extraction kit (Tiangen, China) according to manufacturer's protocol. The presence of target DNA was confirmed by PCR amplification with primers P1 and P2 (generated by Takara, Japan) targeting the gC gene on a Mycycler™ thermo cycler system (Bio-Rad, USA), their sequences were listed in Table 3. The product size was 1296 bp. DNA sequencing showed that pcDNA3.1-gC is real.

The FQ-PCR assay primers and TaqMan™ probe (named P3, P4 and P respectively, generated by Genecore Corporation, China) design was carried out by using the Primer Express™ software supplied by Applied Biosystems according to the sequence of gC gene [GenBank: EU076811] and their sequences were listed in Table 3. The forward and reverse primers amplified a 78 bp fragment of AHV-1 gC gene. The fluorogenic probe was labelled at 5' with FAM (6-carboxyfluorescein) dye as reporter and labelled at 3' with TAMRA (tetra-methylcarboxyrhodamine) as quencher.

FQ-PCR was performed in an iCycler iQ Multicolor Real-Time PCR Detection System (Bio-Rad, USA) with a reaction mixture (20 μL) containing 10 μL 2 × Premix Ex Taq™ (Takara, Japan) and 2 μL standard template according to the manufacturer's protocol. Autoclaved double-filtered nanopure water was added to get the final volume to 20 μL. The reactions were optimized in triplicate based on primers (P3 and P4) and TaqMan™ probe (P) concentration selection criteria, which was performed according to 5 × 5 matrix of primers concentrations (0.2, 0.3, 0.4, 0.5 and 0.6 μmol/L) and probe concentrations (0.1, 0.2, 0.25, 0.3 and 0.35 μmol/L). The two-step PCR cycling condition as follows: initial denaturation and hot-start Taq DNA polymerase activation at 95°C for 5 min, 45 cycles of denaturation at 94°C for 5 s, primer annealing and extension at 53°C for 30 s with fluorescence acquisition during each annealing and extension stage. The tests were carried out by using the 0.2 mL PCR tubes (Axygen, USA).

The recombinant plasmid pcDNA3.1-gC was used to establish standard curve as standard DNA of FQ-PCR. pcDNA3.1-gC concentration was determined by taking the absorbance at 260 nm by using a Smartspec 3000 spectrophotometer (Bio-Rad, USA) and purity was confirmed by using the 260/280 nm ratio. The pcDNA3.1-gC copies/μL was calculated and the purified plasmid DNA was serially diluted 10-fold in TE buffer, pH 8.0, from 5.0 × 10⁷ to 5.0 × 10⁰ plasmid copies/μL. The Primers (P3 and P4) were used for this amplification, These dilutions were used as amplification standards to construct the standard curve by plotting the plasmid copy number logarithm against the Ct values under optimum conditions. The standard curve and its correlation coefficient were generated through the software of iCycler IQ Detection System (Bio-Rad, USA) according to the manufacturer's protocol.

The sensitivity of the assay was used as the limit extent of detection when testing 10-fold diluted DNA standards in triplicate. The dilution of plasmid pcDNA3.1-gC was ranging from 1.0 × 10⁵ to 1.0 × 10⁰ copies/reaction. This test was performed under optimum conditions.

The different 40 liver samples had been confirmed positive for AHV-1 by using the conventional PCR from infected ducks, these samples were retested with the established FQ-PCR method to evaluate the sensitivity of this method for clinical cases.

AHV-1 Cha strains was propagated in the allantoic cavity of 10-day-old SPF duck embryo. The allantoic fluid was harvested from dead embryo. Viral DNA from allantoic fluid was extracted by using TIANamp viral Genomic (DNA/RNA) extracting kit (Tiangen, China) according to the manufacture's instructions, then examined by the established FQ-PCR method and conventional PCR under same circumstance in triplicate after it was 10-fold diluted with sterile ultrapure water. The detection limit of the FQ-PCR was determined based on the highest dilution that resulted in the presence of Ct value in real-time PCR detection. The detection limit of the conventional PCR was determined through the highest dilution that resulted in the presence of clear amplified fragments (78 bp) on the agarose gel. The end-point sensitivity of both assays were calculated.

The specificity of the assay was evaluated by testing the different kinds of templates including pcDNA3.1-gC, AHV-1 Cha, AHV-1 Chv, gosling new type viral enteritis virus, duck hepatitis virus type1, duck adenovirus, goose parvovirus, Marek's disease virus, Pasteurella multocida (5: A), Escherichia coli (O78) and Salmonella enteritidis (No. 50338), then the liver DNA of the healthy duck should be added in this experiment as a negative control.

In order to assess intra-assay variability, eight dilutions of pcDNA3.1-gC (1.0 × 10⁸-1.0 × 10¹ copies/reaction) were prepared separately. These samples were assayed simultaneously in triplicate in a same experiment. Five experiments were performed on different days in order to assess inter-assay variability, using eight pcDNA3.1-gC dilutions (1.0 × 10⁸-1.0 × 10¹ copies/reaction). All tests were performed under optimum conditions. The mean Ct values, SD values and CV values were calculated independently for each DNA dilution.

This study was conducted with 90 AHV-1-free Peking ducks (28 days old) from a AHV-1-free farm which were certificated with qualitative PCR as described by Song[61]. 60 ducks were randomly divided into two equal groups in this study (30 in each group). Thirty non-immunized ducks in Groups 3 used as controls. Ducks in Groups 1-2 were inoculated with 200 μg pcDNA3.1-gC (2.714 × 10¹³ copies) as DNA vaccine by intramuscular route and with 0.2 mL AHV-1 Cha strain vaccine (6.692 × 10¹¹ copies) by subcutaneous route respectively. At each of ten sampling times, three vaccinated ducks of each immune group were chosen randomly for sampling. The liver, pancreas, spleen, kidney, lung, thymus, heart, brain, duodenum, rectum, Harderian gland and bursa of Fabricius were collected at 1 h, 4 h, 8 h, 12 h, 1 d, 3 d, 5 d, 7 d, 2 wk and 4 wk postinoculation respectively. DNA of all these samples were extracted by Animal cell/tissue DNA magnetic bead extraction kit (Bioeasy Technology, China) in Thermo Scientific KingFisher (mL) (Thermo, USA) from 15 mg tissues according to the manufacturer's protocol, followed by being dissolved in 50 μL sterile ultrapure water. Then, 2 μL DNA of each sample was prepared to detect AHV-1 gC accumulation in triplicate by TaqMan™ FQ-PCR.