Development And Application Of A One-step Real-time TaqMan RT-PCR Assay To Detect Duck Hepatitis Virus Type 1

The dissertation revolves around the development and application of a one-step real-time TaqMan RT-PCR assay to detect duck hepatitis virus type 1(DHV-1):â‘ Development of a one-step real-time TaqMan RT-PCR assay based on the 3D gene of DHV-1 genome;â‘¡Using the new assay,the distribution and amount of virulent strain in duck embryos were detected as well as the attenuated vaccine strain in chicken embryos;â‘¢Detection of DHV-1 in the tissues of artificially infected ducklings;â‘£Detection of DHV-1 in the stool of tolerated ducklings which were artificially infected with virulent strain and a molecular epidemiology survey of DHV-1 in ducks and conditions of Sichuan Province.The results are as follows:A one-step real-time reverse-transcription polymerase chain reaction assay(rRT-PCR) was developed for efficient detection of DHV-1.A pair of specific primers which showed 87bp fragment was designed against the conserved region in the 3D gene with a single conserved TaqMan^TM probe.It could obtain excellent linear when the DHV-1 RNA concentration between 1×10¹⁰ and 1×10⁵ copies.The detection limit of this assay was 10 viral genomic copies per reaction and it was highly specific to DHV-1 genera,whereas DPV,MPV,GPV,NGVEV,AIV(H5N2),DSHDV,Duck origin Oasturella multocida(5:A), Duck origin salmonella enteritidis,duck origin salmonella typhimurium,duck origin Escherichia coli(O78),allantoic fluid of normal duck embryos and liver of health duck showed negative results.Then,the rRT-PCR assay was used to determine the distribution and concentration of DHV-1 virulent strain in duck embryos as well as the DHV-1 attenuated vaccine strain in chicken embryos.The results revealed that the copy number of DHV-1 reached a peak in duck embryos and chicken embryos at 28-40 h,44-56 h post inoculation respectively,maintaining 10¹¹ copies/g level in embryoid bodies and 10⁸ -10¹⁰ copies/ml level in allantoic fluid.The RNA copy number in chorioallantoic membrane and muscle was higher than that in allantoic fluid,heart,liver,spleen,brain,intestine from dead duck and chicken embryos inoculated with DHV-1.Using this method and neutralization test to detect 10 liver samples taking from ducklings after artificially infected and 40 clinical liver samples taking from suspected ducklings showed that the positive results of artificially infected samples were the same,while the rRT-PCR method was more sensitive than neutralization test for clinical samples detection,and the positive rate was significant difference(P＜0.01).In conclusion,the rRT-PCR assay is rapid, sensitive,specific,reliable and quantitive.It will be a powerful tool for DHV-1 suspected case detection,distribution pattern of DHV-1 in vivo and molecular epidemiological screening.We used this rRT-PCR assay to detect DHV-1 of the artificially infected ducklings and the results showed that:after inoculated intramuscularly,the liver and Harder's glands were positive at 1 h post inoculation;and the muscle,heart,spleen,lung,kidney, duodenum and thymus were positive at 2 h.The positive results of jejunum,ileum and cecum came out at 4 h,and all the samples were positive at 6 h.All the tissues were positive at 6-72 h and the copy number of DHV-1 RNA in each tissue reached a peak at 24-48 h post inoculation,with the liver,thymus,Harder's glands,cecum,kidney and muscle containing high concentrations of DHV-1.All the samples were still positive until 2 w post inoculation except muscle,heart,lung,thymus,Harder's glands and brain.After inoculated orally,the liver and Harder's glands were positive at 2 h post inoculation;and the muscle,heart,spleen,kidney,duodenum and thymus were positive at 4 h.All the samples were positive at 6 h except lung,pancreas,cecum and brain.All the tissues were positive at 36-120 h and the copy number of DHV-1 RNA in each tissue reached a peak at 48-72 h post inoculation,with the liver,thymus,Harder's glands,rectum and heart containing high concentrations of DHV-1.All the samples were still positive until 2 w post inoculation except muscle,lung,Harder's glands and brain.We detected DHV-1 in the stool of tolerated ducklings which were artificially infected with virulent strain by oral and intramuscular challenge,and the results showed virus excretion pattern:in the oral challenge group,there are 2 of 5 ducks detected negative with stool within 60 days post inoculation,and all the stool of 5 ducks were negative at 120 d post inoculation.However,in the intramuscular challenge group,there was only 1 of 5 ducks detected negative with stool within 90 days post inoculation,and 2 of 5 ducks were still positive at 120 d post inoculation.1 of 10 cohabited ducks was started to be detected positive at 5 d post cohabitation,and there were 4 ducks detected positive until 14 d.The detection of samples collected from duck rising farms in Sichuan Province showed that, DHV-1 could be detected in the drinking water,natatorium,condition swab and anus swab with natatorium and anus swab containing high concentrations of DHV-1,drinking water containing low concentrations of it.The duck rising farms in Sichuan Province could be under the threat of DHV-1 infection,with the virus growth in vivo of ducks as well as in the illiquid natatorium.