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| Establishment and Application of RT-PCR for Identification and Diagnosis of Duck reovirus Genotype Ⅰ and Ⅱ |
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Abstract There are two genotypes (ⅠandⅡ) of Duck reovirus (DRV) in clinical infection ducks (Anas platyrhynchos domestica). To establish a multiplex one-step RT-PCR method for the simultaneous detection and differentiation of DRV genotypeⅠ(classical DRV, C-DRV) and genotype Ⅱ (novel DRV, N-DRV) from clinical samples, two sets of primers were designed for a RT-PCR assay system according to S1 and S4 non-structural protein encoding sequences of two genotypes viruses. The amplification conditions, including primer concentration, annealing temperature and cycle times, were optimized. The results showed that the most effective and precise reaction system was as follow: PrimeScript 1 Step Enzyme Mix 1 μL, 2×1 Step Buffer 12.5 μL, each primer (20 μmol/L) 1 μL, RNA 1.5 μL, adding RNase Free dH2O to a final volume of 25 μL; The optimal reaction procedure was as follow: Reverse transcription for 30 min at 50 ℃; Pre-denaturation for 2 min at 94 ℃; Denaturation temperature for 2 min at 94 ℃, annealing temperature for 30 s at 55 ℃, extension for 30 s at 72 ℃, total of 30 cycles. These genotype-specific primer pairs could amplify 249 and 505 bp PCR products corresponding to C-DRV and N-DRV, respectively, and 2 fragments of 249 and 505 bp were simultaneously amplified from the mixed RNA sample of C-DRV and N-DRV. The assay exhibited high specificity as all negative controls and other duck pathogens, such as the Duck tembusu virus (DTMUV), Duck plague virus (DPV), Newcastle disease virus (NDV), Muscovy duck parvovirus (MDPV), Duck hepatitis A virus (DHAV-1) and H9N2 Avian influenza virus (AIV). The sensitivity of this method was determined to be 0.47 50% tissue culture infective dose (TCID50) for C-DRV and 0.62 TCID50 for N-DRV. Virus RNAs extracted from different generations and different times were detected by the developed multiplex one-step RT-PCR. The result showed that the method was found to be highly reproducible. In addition, RT-PCR assays of DRV diseased fowl samples (total 239) collected from different regions of Zhejiang province during 2011~2015 were taken. The detected results showed that the positive rates of C-DRV and N-DRV were 2.5% (6/239) and 31.4% (75/239), respectively. Six C-DRV positive samples were all Muscovy duck. In 75 N-DRV positive samples, there were 54 Muscovy duck (Cairna moschata) samples (positive rate 72%), 5 goose (Anser anser) samples (positive rate 6.7%) and 16 other breed duck samples (positive rate 21.3%). The results indicated that N-DRV was the currently prevalence genotype in Zhejiang province. P10 and P18 sequence of positive tissues were analyzed to confirm the accuracy of the test results. In addition, the multiplex one-step RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for C-DRV and N-DRV infections in duck, and the assay can provide effective technical support for molecular epidemiological survey of DRV.
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Received: 03 June 2016
Published: 07 November 2016
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