| Goose parvovirus (GPV) is a member of Parvoviridae depend virus. GPV mainly infects the3weeks-old goslings and Muscovy duck. Goslings infected by GPV often show bleeding, cellulose and exudative enteritis and finally develop into enteral embolism. Gosling plague caused by the virus spread fastly, highly mortality and the goslings and Muscovy duck survived show growth retardation, which bring great harm to the waterfowl breeding bring. Chinese scholar Fang Dingyi was the first to find the disease in1956, which after above a decade also found in many European countries and was reported as Derzsy’s disease, afterwards finally whose pathogens were confirmed as goose parvovirus. Many countries have reported the occurrence and prevalence of goose parvovirus disease since that and the disease was perennially popular in our country after its discovery, resulting in seriously economic loss. The major reason of goose parvovirus disease’s outbreak is the subclinical infected or latent infected goose whose feces contaminate egg and environment, so as to the virus access to the susceptible geese. Therefore, prevention and control to GPV need a fast and accurate diagnosis which can give the treatment time for infected goslings.At present,because of mixed infections in clinical disease, leading to clinical symptoms are not typical, it is difficult that diagnosis GPV infection with conventional methods of epidemiolo gy, clinical symptoms observed, pathological examination and so on. The previously established laboratory instruments, such as isolation and identification of viruses, virus neutralization test, a gar diffusion test, ELISA test and PCR, etc., although there are high specificity, sensitivity and t imeliness but have some shortcomings. In recent years, in order to avoid all the limitations of tra ditional detection methods, we began to apply genetic testing technology, analysis the complex microbial of clinical and environmental samples, which is extremely important for diagnosis and treatment of major infectious disease, and other work. And denaturing gradient gel electrophore sis and other traditional detection methods require complex manual, and difficult to grasp the tec hnical, the sensitivity is lowly, and the disadvantages of DNA microarray technology and other t echnologies is false positive, cost high and so on. Thus it is need an automated gene polymerase chain reaction technique in microbial mononuclear Cape acid polymorphism analysis, microbial drug resistance mutation detection, genotyping and other work identified. Denaturing high perfo rmance liquid chromatography (DHPLC) technology is a new tools in gene mutation testing, an d it has automated, high sensitivity, economic and other advantages, it can greatly save time, im prove the detection efficiency, and has been played an important effect on test and diagnostics.A large number of deaths of10day-old Jilin white geese were found at a goose farm in Tai Pingchi Nong’an County of Jilin province in2011May. Goose liver from death geese whose symptoms were typical made into emulsion and inoculates in allantoises cavity of12day-old goose embryo without the gosling plague maternal antibody. It resulted that the goose embryo began to die48h after inoculated the emulsion and the parvovirus-like particles were observed after negative staining under electron microscope in allantoises fluid. Geese showed gosling plague clinical symptoms and pathological characteristics in the test group in animal regression test. The virulence of the isolated virus was at10-65/0.2mL of ELD50which showed its toxicity was trong.Liver emulsion and allantoises fluid could be detected the specific fragment using PCR which sequencing was compared through genbank afterwards and phylogenetic tree was made, which showed that goose parvovirus was isolated, named as JLTP strain, in the first experiment and its homology was up to99%compared with Goose parvovirus strain GPV/CH/HLJ02/08capsid protein (VP3) gene.In the second experiment we use the established PCR combinated denaturing high performance liquid chromatography to establish a new method to detecte the GPV and the reaction conditions weret hat chromatographic column:PS.DVB&C18DNASep column (4.6mm×50mm, particle size3mm);column temperature:50℃; mobile phase:volume fraction50%A buffer solution,50%volume fraction of buffer solution B; flow rate:l mL/min. Specificity analysis showed a high specificity of the method; sensitivity analysis showed that the sensitivity of this method is100times higher than the conventional PCR method. The positive detection rate of clinical samples was up to100%with the PCR-DHPLC method established in this study, which proved that the PCR-DHPLC could using as a new detection method. The new method was capable of specifically detecting goose parvovirus exsting lesions, which could be used as an effective means for clinical diagnosis of GPV and which could put the works of diagnosis, surveillance and control of goose parvovirus disease in Jilin at a new stage. |
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