| Duck Viral Hepatitis (DVH) is an acute, highly contact deadly infectious diseases caused by Duck hepatitis virus (DHV). It causes enormous losses to duck industry of our country even the world. In this study, we established one-step rRT-PCR and one-step duplex rRT-PCR methods which based on DHAV-1 VPO and DHAV-3 VP3 to detect DHAV-1 and DHAV-3.1. One-step rRT-PCR for DHAV-1Blasted nucleic acid sequences of DHAV-1, DHAV-2 and DHAV-3 in the NCBI, we designed primers and probe based on gene VPO of DHAV-1, then established a one-step rRT-PCR assay for DHAV-1. The one-step rRT-PCR assay for DHAV-1 could obtain excellent linear and repeatability when the DHAV-1 RNA concentration between 4.88×1010 copies/?L-4.88×101 copies/?L, expression:Y=-3.367X+42.688, R2=1.000, amplification efficiency is 98.1%, close rolerance, high reliability and the limit of the assay is 49copies/?L. This assay is specific that can only detect DHAV-1 and there was no amplification with non-DHAV-1 pathogen such as DPV, RA, E. coli, DH5a, SE, Sty, PM, NDV, MDPV, DSHDV, AIV, IBD, NGVEV and blank controls.2. One-step rRT-PCR for DHAV-3Blasted nucleic acid sequences of DHAV-1, DHAV-2 and DHAV-3 in the NCBI, we designed primers and probe based on gene VP3 of DHAV-3, then built standard plasmid, prepared cRNA-VP3 and established a one-step rRT-PCR method for DHAV-3. The one-step rRT-PCR assay for DHAV-3 could obtain excellent linear and repeatability when the DHAV-1 RNA concentration between 4.72×1010 copies/?L-4.72×101 copies/?L, expression:Y=-3.373X+44.261, R2=0.996, amplification efficiency is 97.9%, close rolerance, high reliability and the limit of the assay is 48copies/?L. This assay is specific that can only detect DHAV-3 and there was no amplification with non-DHAV-3 pathogen such as DPV, RA, E. coli, DH5a, SE, Sty, PM, NDV, MDPV, DSHDV. AIV, IBD, NGVEV and blank controls.3. One-step duplex rRT-PCR for DHAV-1 and DHAV-3Prepared cRNA-VPO and cRNA-VP3 as templates by ten-times step dilution and established a one-step duplex rRT-PCR method for DHAV-1 and DHAV-3. It could obtain excellent linear and repeatability when the DHAV-1 (DHAV-3) RNA concentration between 4,88×1010 copies/?L-4.88×101 copies/?L (4.72×1010 copies/?L-4.72×100 copies/?L), expression:Y=-3.371X+42.679 (Y=-3.398X+42.858), R2=0.999 (R2=0.998), amplification efficiency is 98.0%(96.9%), close rolerance, high reliability and the limit of the assay is 49 copies/?L (5 copies/?L). The one-step duplex rRT-PCR assay for DHAV-1 and DHAV-3 with high specificity and there was no amplification with non-DHAV-3 pathogen such as DPV, RA. E. coli. DH5a, SE, Sty, PM, NDV, MDPV. DSHDV, AIV.IBD, NGVEV and blank controls.4. DHAV-3 and its coinfection with DHAV-1 in duck embryosDetecting the distribution and content of DHAV-3 in duck embryos by one-step rRT-PCR method for DHAV-3, result showed that brain, muscle, heart, liver, gizzard, enteron, allantoic fluid, amniotic fluid, chorioallantoic membrane of duck embryos were infected virus (104-105 copies/g) after post inoculation about 24 h. Duck embryos died in a certain period of 56 to 60 h post inoculation and the viral copies of tissues in dead duck embryos was from 107 to 109 copies/g, and chorioallantoic membrane. Thirty-eight clinical samples were detected by the one-step single or duplex rRT-PCR assay, results showed that twenty-seven were DHAV-1, seven were DHAV-3 and the others were mixed infection.Detected the DHAV-1 and DHAV-3 in the tissues of duck embryos at different times post inoculation, the viral copies of DHAV-1 and DHAV-3 in the allantoic fluid, brain, muscle, heart, liver, gizzard and enteron of duck embryos reached their maximum about 108 to 1010 copies/g sixty hours post inoculation. The maximum viral copies of DHAV-1 in muscle, heart, liver, gizzard and enteron were about 1010 copies/g, and the virus content of brain and allantoic fluid was 109 copies/g. The maximum viral copies of DHAV-3 in brain, muscle, heart, liver, gizzard and enteron were about 10 to 10 copies/g and the virus content of allantoic fluid was 1010 copies/g.Detecting the DHAV-1 and DHAV-3 in allantoic fluid and soma of dead duck embryos by the one-step duplex rRT-PCR assay, results showed that when remained the amount of DHAV-1 inoculation (108 copies), the viral copies of DHAV-3 in the allantoic fluid was reduced from 107 to 104 copies/g and viral copies of DHAV-3 in the soma reduced from 107 to 106 copies/g with the reduction of DHAV-3 inoculation (108 to 104 copies). When remained the amount of DHAV-3 inoculation (108 copies), the viral copies of DHAV-1 in the allantoic fluid and soma were remain about 109 copies/g (copies/mL) with the reduction of DHAV-3 inoculation (108 to 104 copies). It showed that, DHAV-1 has a better proliferation ability than DHAV-3 and inhibited the proliferation of DHAV-3 on the other hand. |
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