Development And Application Of Fluorescent Quantitative RT-PCR For Detecting Duck Reovirus Virus

Avian reovirus (ARV) has spread widely in the world by infecting naturally or artificially chicken, turkey, muscovy duck, geese and other poultry. Reovirus was isolated from a variety of avain by many scholars. It had been discovered that there was a significant difference in terms of clinical symptoms and pathological changes of different poultry infected with reovirus, which brought great difficulties to diagnose in poultry. It was reported in the main breeding areas of duck in china the serious infection of duck reovirus had caused huge economic losses. Therefore, it is an inevitable trend to find a rapid, accurate and sensitive assay for the detection of duck reovirus. In this study, a pair of primers was designed according to the S2 gene sequences of duck reovirus, the fluorescent quantitative RT-PCR method for detecting duck reovirus was established, which provided tools for detecting distribution and concentration of the virus in different tissues. It is of great significance in elucidating the pathogenesis of this disease, also in diagnosis, prevention and epidemiological investigation. The research contents are as follows:1. Preparation of the plasmid standard A pair of specific primers was designed against the conserved region in the S2 gene for amplification of 285 bp fragment. The PCR products were cloned into pMD18-T vector and subsequently the recombinant was transformed into E.coli DH5α. The positive recombinant plamids identified by PCR and sequencing were obtained and then named pMD18-TS2. The concentration and purity of the constructed plasmid were determined by a BioPhotometer Plus in three replicates, followed by taking the average of optical dentisy readings for all replicates. The DNA copy number was calculated following the formula. After quantitation, standard plasmid was diluted serially in 10-fold dilutions ranging from 1.48×1010 to 1.48×100 copies/μL.2. Protocol optimization of fluorescent quantitative RT-PCR assay The concentration of primers and annealing temperature were optimized to achieve the optimal working conditions. In accordance with the reaction conditions of optimization, A series of 10-fold dilutions with plasmid concentration ranging from 1.48×109 to 1.48×100 copies/μL were used to amplify and establish a standard curve. The result showed a good gradient among amplification curves of plasmid standards. In addition, the standard curve revealed a linear relationship between cycle threshold (Ct) and plasmid concentration ranging from 1.48×109 to 1.48×101 opies/μL. The correlation coefficient for the associated standard curve was 0.998 and the amplification efficiency values of the real-time RT-PCR were calculated to be 95.16%. Using the equation as followed, the nucleic acid copy number of duck reovirus for unknown samples could be quantitated, Y=-3.444X+39.344(where Y is the threshold cycle, and X the log of the starting quantity). The assay exhibited high specificity, no crossing-reaction with Duck enteritis virus (DEV), Duck hepatitis virus (DHV), Riemerella anatipestifer (RA), Pasteurella, Salmonella. The intra-assay and inter-assay coefficient of variations of the assay were in the range of 0.94%-3.57% and 1.12%-5.61%, respectively, showing a good repeatability.3. Comparison of detecting DRV between using conventional RT-PCR and fluorescent quantitative RT-PCR assay All collected tissues from ducklings of experimental infection and 10 clinical samples were detected by both conventional RT-PCR and fluorescent quantitative RT-PCR. It can be observed that, amongst the 80 samples from experimental infection,42 samples (52.5%) were found to be positive by fluorescent quantitative RT-PCR assay and 36 samples (45%) by conventional RT-PCR. DRV could be detected in 8 kind of tissues collected from ducklings infected artificially. Detection rate of spleen, bursa of Fabricius, thymus and lung was high but there was a lower detection rate of liver and pancreas. The concentration of virus in spleen group was higher than that in other groups (P<0.05). Amongst the 10 clinical samples,6 samples (60%) were positive by fluorescent quantitative assay but 3 samples (30%) by conventional RT-PCR.SYBR Green I fluorescent quantitative RT-PCR is a accurate, sensitive and specific assay for detection and quantitation of duck reovirus. Spleen was the preferred organ for detection of this virus.