Establishment And Preliminary Application Of Taq Man Fluorescence Quantitative PCR For Detection Of Goose Parvovirus

Goose parvovirusis(GP)is a highly infectious disease that is known to be susceptible to infection by goose parvovirus(GPV),also known as goose plague,Is characterized by rapid transmission speed,infection rate,morbidity and mortality,to bring the goose industry has brought heavy damage,seriously affected the development of China's goose industry.Prevention and control of GP,early diagnosis is the key,the current stage of GPV detection using the common PCR method.However,compared with the Taq Man fluorescence quantitative PCR method,the common PCR method cannot quantitatively detect the viral load of GPV,and can only qualify the diagnosis of GP,and cannot make accurate judgment on the severity of the disease and cannot provide the prevention and treatment of the disease Accurate basis.Therefore,this study is to establish a rapid,accurate and absolutely quantitative method for the detection of goose microarray Taq Man fluorescence quantitative PCR.According to the published GPV-VP3 gene sequence(JN836326)published in GenBank,a pair of specific primers and probes were designed and synthesized in the conserved region.The VP3 gene(120bp)was amplified by TA cloning to construct a standard positive plasmid as the internal standard,and then the probe concentration,primer concentration and annealing temperature and other reaction conditions to optimize the screening,and sensitivity,specificity and repeatability and other tests.The results showed that the standard curve was obtained by Taq Man fluorescence quantitative PCR.From the curve,it was found that the amount of template DNA in the reaction was linear with the Ct value of Taq Man fluorescence quantitative PCR,the correlation coefficient was 0.999.This method has a minimum detection rate of 109 times the dilution of GPV.Compared with ordinary PCR,the sensitivity is 100 times higher.(DEV),canine parvovirus(CPV),Newcastle disease virus(NDV)and goose paramyxovirus(GPMV)did not cross-react;the difference between the group and the group was small,the coefficient of variation was less than 2%.The Taq Man fluorescence quantitative PCR was used to detect 20 cases of suspected GPV infection.The positive rate was 90%.The detection rate was 80%in 16 cases.The results showed that the Taq Man fluorescence quantitative PCR method was simple,specific and reproducible,and could be used for pathogen detection and quantitative analysis of GP.