Establishment And Application Of Fluorescence Quantitative PCR For Detecting Goose Parvovirus And Comparing And Analyzing Of The VP3 Sequence Of Goose Parvovirus

Goose parvovirus(GPV) is the causative agent of a disease of high mortality and morbidity in geese and Muscovy ducks, which causes great loss to geese cultivation. In the reseach , we established a method of Fluorescence Quantitative PCR(FQ-PCR) for detecting GPV. The study indicates that the LUXTM assay is rapid, reliable and sensitive and it has the potential for use as an alternative molecular method for GPV diagnosis , and the distribution and duration regularity of GPV SD2005 in artificially-infected goslings body was studied by FQ-PCR. The experiments provide essential datas for explaining pathogenic mechanism and controlling technology for GPV. VP3 genes of four strains of GPV in home were amplified,sequenced,compared and analyzed, which provides theory foundation for studying the character of immunogenicity and virulence in the molecule lever.The research divides four parts:Part One: Isolation and Identification of two Goose Parvovirus of Shandong ProvinceWith 12 days goose embryo , two wild strains of GPV of Shandong province were isolated. By challenge test, the goslings had oblivious clinic symptom and anatomical pathology of gosling plague, but the control group was normal. In the test of double agar-gel immunodiffusion, a line of precipitation between the anti-GPV serum and the pathogen was visible. With a pair of primers designed according to the sequence gene of VP1 of GPV in GenBank, a fragment was amplified by PCR as expectation. The amplified DNA was cloned into pMD18-T vector and sequenced. Sequence indicated that both DNAs of the amplified and the GPV sequence in GenBank displayed 98% identity. According to the results, we can determined that the virus is GPV, and named it SD2002 and SD2005. Part Two: Establishment and Primary Application of LUXTM Primer FQ-PCR for Detecting Goose parvovirusFQ-PCR assay, based on light upon extension (LUXTM) fluorogenic primer and LightCycle technology, was developed for rapid detection of Goose parvovirus. The assay exhibited high specificity as all the negative controls (samples from healthy sero-negative goose) and other viral DNA, such as DPV,NGVEV,were not detected. Up to 1000 copies of the GPV gene plasmid were detected by the assay, as few as 2.5 pg DNA of target sequences per reaction, thus revealing a high sensitivity. The study indicates that the LUXTM assay reported below is rapid, reliable and sensitive and it has the potential for use as an alternative molecular method for GPV diagnosis.Part Three: Study on the regularity of distribution of the virus in geese experimentally infected with GPV which isolated from Shandong by FQ- PCRGoose parvovirus(GPV) SD2005 was isolated from a 16-day-old dead goose. Fluorescence quantitative PCR(FQ-PCR) assay was developed for rapid detection of Goose parvovirus. The distribution and duration regularity of GPV SD2005 in artificially-infected gosling body was studied by FQ-PCR. The results were as follows: At hour 8 post-infection (PI), the GPV DNA can be detected from lingua,all enteric organs,feces,all digestive organs,blood,heart and liver. The GPV DNA reached the period of maximum quantity at 48 h PI, and the GPV DNA could be detected from all organs, The fastigium raised from 48 h PI to 168 h PI. Subsequently, the quantities of GPV DNA in gosling body declined quickly. At 720 h PI, however, the GPV DNA could be detected from feces,liver and spleen. The experiment provides essential essay datas for collecting samples and controlling technology for GPV. Part Four: Comparing and analyzing of the VP3 gene sequence of different GPV strainsA pair of primer corresponding to the gene of strain B of GPV , was used to amplify VP3 gene of four strains of GPV in home by the Polymerase Chain Reaction (PCR). The PCR product was cloned,transferred,identified,sequenced and analyzed. The result of sequence analysis showed that the VP3 gene is 1605bp and encode 534 amino acids. Sequence comparison of the VP3 gene from different virus strains showed that VP3 of these virus share a high homology from 96.4 percent to 99.8 percent in amino acid, the identity of partial VP3 gene of Shandong strains with China strain including Taiwan's is higher than with European's, and only 0.7% difference in amino acid was found between the two Shandong strains. There were no typical difference in amino acids between vaccine strains and others.