Pathogen
DTMUV (live attenuated vaccine strain WF100 from QiLu Animal Health Products Co., Ltd.; Cat. no. 1502522), DHAV (live attenuated vaccine strain A66 from Chengdu Tecbond Biological Products Co., Ltd.; Cat. no. 220012214), and DEV (live attenuated vaccine strain C-KCE from Guangxi Liyuan Biological Co., Ltd.; Cat. no. 200352023) were isolated from the vaccine strain, respectively. The F gene of APMV-8 was synthesized by Sangon Biotech (Shanghai, China). AIV (H9N2), NDV, goose parvovirus (GPV) and FAdV (serotype 4) were isolated from clinical samples. Avian paramyxoviruses (APMV-4), Escherichia coli (E.coli, wild-type, serotype O2), Salmonella (SE, Typhimurium wild-type strain), Riemerella anatipestifer (SA, wild-type strain, serotype 10), Pasteurella multocida (serotype ST129), Clostridium perfringens (type A), and AIV (H3N8, H5N6, H6N5, H7N3) were maintained in our laboratory.
RNA extraction and cDNA synthesis
The nucleic acid of AIV, NDV, and DTMUV was extracted by Viral RNA Extraction Kit (Sangon Biotech, China), and then reverse transcribed into cDNA by Reverse Transcription Kit (Thermo Scientific, USA), according to the manufacturer’s instructions. Finally, the concentration and purity of each genome were determined by spectrophotometry (Thermo Scientific, USA) and preserved at −20 °C.
Primer and probe design
The complete gene sequences of AIV, NDV and DTMUV strains were downloaded from GenBank, and the viral gene sequences were aligned using DNAMAN (LynnonBiosoft, USA) to find gene conserved regions. The target genes including Matrix (M) gene for AIV, Fusion (F) gene for NDV and Envelope (E) gene for DTMUV were highly conserved (Table 5). We designed three pairs of specific primers and probes for each virus using Primer Premier 5 (Premier, Canada) in accordance with the results of sequence alignment. The fluorescent reporter dyes FAM, JOE and ROX were labeled at the 5’ end of the three viruses, and BHQ1, BHQ1, and BHQ2 were linked at the 3’ end for the simultaneous detection of the three genes in a single reaction(Table 5) .The primers were synthesized by Nanjing Kingsley Biotechnology Co. Ltd.
Standard plasmid preparation
Specific target fragments were amplified with the primers (Table 4), and then cloned into the pMD-18T vector (TaKaRa, China) to obtain the recombinant plasmids pMD-AIV, pMD-NDV, and pMD-DTMUV. The copy number of the recombinant plasmids was calculated using the following formula: copy number (copies/μL) = NA (copies/mol) × concentration (g/μL)/MW (g/mol), where NA is Avogadro’s number and MW is the base number times 340[24].
Experimental design for the Real-time PCR method
The Real-time PCR method was optimized using a D-optimal design consisting of 16 experiments. Three factors with three levels each were considered. These factors include annealing temperature (from 53 °C to 60 °C), primer concentration for each target gene (from 0.2 μM to 0.6 μM), and probes for each target gene (from 0.05 μM to 0.2 μM). The Ct value was used in statistical analysis. All analyses were performed using MODDE 12.1 software (Umetrics, Sweden). The relationship between the response Y and the variables Xi, Xj was expressed as Y = β0 + βiXi + βjXj + βijXiXj + βiiXi2 + βjjXj2 + . . . ε, where βs were the regression coefficients and ε was the experimental error. The linear coefficients βi and βj were the quantitative effect of the respective variables. The cross coefficient βij measured the interaction between the variables, and the square terms of βiiXi2 and βjjXj2 described the non-linear effects on the response [21]. In addition, we also evaluated the amplification efficiency, correlation coefficient (R2) and amplification curve.
Real-time PCR method
The real-time PCR was determined in a 20.0 μL reaction system with a LightCycler 96 real-time PCR system (Roche, Switzerland). The ingredients were 10.0 μL of TaKaRa Premix Ex Taq™ (Probe real-time PCR), 0.4 μM of the primers (AIV-F, AIV-R, NDV-F, NDV-R, DTMUV-F, and DTMUV-R) for AIV, NDV and DTMUV genes, 0.05 μM of the probes, and 1.0 μL of template. The PCR program was set as follows: pre-denaturation at 95 °C for 2 min and 40 amplification cycles of 95 °C for 10 s and 54 °C for 30 s. Multiple fluorescent signals were obtained once per cycle upon the completion of the extension step. The standard plasmid pMD18T-AIV, pMD18T-NDV and pMD18T-DTMUV were used as the positive control, ddH2O as the negative control. The assay was repeated at least three times within the study.
Conventional triplex PCR method
Conventional PCR assay was conducted in a 20.0 μL reaction system, which containing 10.0 μL of 2× Taq Master Mix (Vazyme Biotech, USA), 0.24 μM of the primer for AIV, 0.28 μM of the primer for NDV, 0.28 μM of the primer for DTMUV, 1.0 μL of template and ddH2O to a final volume of 20.0 μL. The amplification programme was that, pre-denaturation at 95 °C for 5 min, followed by 40 cycles of sequentially denaturation at 95 °C for 30 s, annealing at 54 °C for 30 s and extension at 72 °C for 20 s, a final extension at 72 °C for 10 min. The PCR products were analysed by 1.5% agarose gel electrophoresis. ddH2O was used as negative control.
Validation of the real-time PCR method
The specificity of the triplex real-time PCR was evaluated by cross-reactivity with other duck viruses and bacteria (including APMV-4, APMV-8, DHAV, DEV, GPV, FAdV, Escherichia coli, SE, RA, Pasteurella multocida and Clostridium perfringens). The standard plasmid DNAs were 10-fold serially diluted from 1×107 copies/μL to 1×101 copies/μL for each virus to determine the detection limit of the triplex real-time PCR method. In addition, standard plasmids in three different concentrations (1×107, 1×105 and 1×103 copies/μL) were used as templates to evaluate the repeatability and reproducibility of the real-time PCR method. The coefficients of variation (CV) for the Ct values of the intra- and inter-assay comparisons were determined by testing three replicates of each concentration in a single round of real-time PCR or by repeating three rounds of real-time PCR.
Pilot study of the real-time PCR method
A co-infection model was first designed to determine the detection efficiency of the developed method. Then, with the permission of the animal owner, 120 clinical samples were collected from duck farms and live poultry markets in Jiangsu Province and tested using the established PCR methods. The results are further validated by the published PCR methods [25-27].