Primers and probe design
Specific primers and a TaqMan probe to detect DTMUV were designed using Primer Express 2.0. The target sequence (115-bp long) was selected from a relatively conserved region on the E gene of DTMUV, Fengxian virus (FXV) strain (GenBank accession no. HQ833330). The fluorescent reporter dye, FAM, was located at the 5' end of the probe; the quencher, TAMRA, was located at the 3' end. The primers used were the EF primer (forward, 5'-TGTCTTATGCAGGTACCGATG-3') and the ER primer (reverse, 5'-CGTATGGGTTGACTGTTATCA-3'). The probe used was the TaqMan probe EP (FAM-AGTTCCCATATCCATGTC-TAMRA).
FXV, one of the DTMUV isolates, was propagated in specific pathogen-free embryonated chicken eggs. The embryos were carefully monitored, and those that died 24-120 h post-inoculation were pooled, homogenized, and centrifuged along with the chorioallantoic membranes. The supernatants were stored at -80°C until they were used for molecular analyses. The FXV titer was determined according to the 50% egg lethal dose (ELD50).
RNA extraction and reverse transcription
RNA was extracted from FXV by using the RNeasy mini kit (Qiagen Inc., Valencia, CA) according to the manufacturer's instructions. The total RNA was stored at -80°C until use.
First-strand cDNA was synthesized using AMV reverse transcriptase (TaKaRa Biotechnology, Dalian, China) according to the manufacturer's instructions. Briefly, the following reagents were added and mixed: 4 μL of 5× reverse transcriptase buffer, 2 μL of dNTP mixture (10 mmol/L), 1 μL of random primer (50 mmol/L), 2 μL of AMV reverse transcriptase, 0.5 μL of RNAase inhibitor (40 U/μL), 5 μL of total RNA, and 5.5 μL of RNAase-free water. The reaction mixture was sequentially incubated at 42°C for 60 min and then at 72°C for 15 min. The DNA was subsequently stored at -20°C.
Preparing the standard plasmid DNA
PCR was used to amplify the E gene in a reaction mixture consisting of 5 μL of 10× buffer, 4 μL of dNTP, 2 μL of each primer (10 μM of EF and ER), and 0.5 μL of Ex Taq Hot Start (TaKaRa Biotechnology, Dalian, China). The thermal conditions were as follows: initial denaturation at 94°C for 5 min; 35 cycles at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s; and a final extension at 72°C for 10 min.
The PCR product was then inserted into a pMD18-T vector, (TaKaRa Biotechnology, Dalian, China). After the recombinant plasmid was amplified in DH5α host bacteria, it was purified using a commercial test kit (Axygen Scientific Inc., Union, CA). The concentration of the plasmid was determined according to the OD260 value, and the number of copies of the plasmid was calculated . The plasmids were maintained at -20°C for use as standard DNA in subsequent experiments.
Establishing a standard curve for the real-time PCR
The real-time PCR for the E gene was conducted in a 25 μL reaction system containing the following ingredients: 2.5 μL of 10× buffer, 2.5 μL of dNTP, 1 μL of each primer (10 μM of EF and ER), 0.6 μL of the probe (10 μM of EP), 1 μL of recombinant plasmid, 0.2 μL of Ex Taq Hot Start, and 16.2 μL of sterile water.
Real-time PCR was performed on Mastercycler ep realplex (Eppendorf, Germany) by using the following thermal cycles: 95°C for 2 min, 40 cycles at 95°C for 20 s, and 54°C for 1 min. The assay was repeated at least 3 times with each template and the negative control.
Determining the specificity of the real-time PCR
To determine the specificity of the real-time PCR, the FXV and 5 well-known viruses causing infectious diseases in ducks, including duck plague virus (DPV), avian influenza virus (AIV), duck hepatitis virus (DHV), duck parvovirus (DPVV), and Newcastle disease virus (NDV), were tested under the conditions described above.
Sensitivity of the real-time PCR
To determine the lower detection limit, the plasmid stock was diluted with sterile water to 5 × 107 copies/μl, 5 × 106 copies/μl, 5 × 105 copies/μl, 5 × 104 copies/μl, 5 × 103 copies/μl, 5 × 102 copies/μl, 5 × 101 copies/μl, and 5 × 100 copies/μl. Four replicates of each dilution and 2 negative controls (blanks) were then tested by the real-time PCR and conventional PCR.
Reproducibility of the real-time PCR
To test the reproducibility of the real-time PCR, standard plasmids in 3 different concentrations (5 × 105 copies/μl, 5 × 104 copies/μl, and 5 × 103 copies/μL) were aliquoted and stored at -80°C for future use as templates for intra- and inter-assay comparisons. The variations in the inter-assay were assessed by testing 5 replicates of each concentration in a single round of real-time PCR, and the variations in the intra-assay were assessed by repeating 5 rounds of real-time PCR. The coefficients of variation (CV) for the Ct values of the intra- and inter-assay comparisons were determined.
Detecting samples from experimentally or naturally infected ducks
The shelducks were inoculated intranasally with 105 ELD50 of the virus in 0.2 mL of supernatant of homogenized embryos and chorio-allantoic membranes of eggs that died 24-120 h post-inoculation with FX2010. One day after they were inoculated, 2 contact ducks were introduced into the isolator and placed among the ducks that had been inoculated intranasally. Oropharyngeal and cloacal swabs were collected from all ducks on days 2, 3, and 5 post-inoculation. All swabs were suspended in 1 mL of phosphate-buffered saline (PBS) and used for RNA extraction with an RNeasy Mini kit (Qiagen) or virus isolation. Two ducks from the inoculated group were euthanized 4 and 7 days post-inoculation, respectively. Two contact ducks were euthanized 6 days post-contact, and 2 non-infected ducks were euthanized as negative controls. Samples of blood, liver, spleen, kidneys, heart, brain, pancreas, and lungs were collected from the euthanized ducks. The samples were weighed, homogenized, and diluted in PBS (pH 7.4) at a ratio of 1:1 (mL/g). A portion of each sample was used for total RNA extraction and applied to the reverse transcriptase real-time PCR assay. In addition, a portion of each sample was used to isolate the virus in embryonated chicken eggs.
Twenty-one clinical samples, including blood, spleen, liver, and brain samples, from sick farm-raised ducks suspected of being infected with DTMUV were tested using both real-time PCR and conventional PCR.