A Novel Technique Of Simultaneous Detecting Avian Poxvirus, Marek’s Disease Virus, And Egg Drop Syndrome Virus With Multiplex PCR

With the rapid development of economy, the scale of the poultry industry which has a long history is growing quickly in China. The disease is the major challenge of poultry, especially viral diseases. All of marek’s disease virus (MDV), avian poxvirus (APV) and egg drop syndrome virus (EDSV) are class B infectious viruses. Currently, the main methods for the detection of viruses include isolation and identification of viruses, serology, PCR etc. As sensitive as conventional PCR, the multiplex PCR is a method with high speed, high efficiency and high flux. The multiplex PCR which has the same basic principle as the conventional PCR could be used to simultaneously detect the several viruses.In this assay, MDV, APV, and EDSV were maintained in DF-1chicken embryo fibroblast culture (CEF), and DNA was isolated from cell culture-adapted viruses by guanidine isothiocyanate. Three pairs of specific primers were designed according to the DNA sequences of MDV, APV, and EDSV. Then the amplified fragments were cloned in pMD18-T vector to form pMD-MDV, pMD-APV and pMD-EDSV. Thus the multiplex PCR was established to detect three viruses after optimizing the annealing temperature, concentration of primers, PCR buffer, and the concentration of Mg2+. Therefore,the multiplex PCR reaction system was determined to be20μL:0.2μL GoTaq Flexi DNA polymerase(5U/μL),4μL5xGoTaq Flexi buffer,0.4μL dNTP(lOmM),2.4μL MgC12(25mM), APV-F/R0.4μL, MDV-F/R0.4μL, EDSV-F/R0.2μL, each template lμL, ddH2O8.0μL. The reaction conditions were as follows:initial denaturation at94℃for5min,40cycles ofdenaturation at94℃for30s, annealing at54℃for45s, and extension at72℃for30s, and final extension at72℃for10min.The multiplex PCR indicates the presence of MDV, APV, and EDSV through the production ofvirus-specific amplicons each having a different length.The sizes of the amplified products were175bp for MDV,118bp for APV and305bp for EDSV. No amplicons were produced with thenegative controls, which included newcastle disease virus(NDV), avian influenza virus(AIV), avian leukemia virus(ALV), infectious bursal disease virus(IBDV), and infectious bronchitis virus(IBV).With the10-fold dilution series, the lowest concentrationswith positive multiplex PCR results were103copies/μL for APV,104copies/μL for MDV, and EDSV. In contrast, the lowest concentrations with positive single PCR results were103copies/μL for APV andMDV,104copies/μL forEDSV. Thus, multiplex PCRwas slightly less sensitive than the single PCR. A total of30avian fecal samples were collected randomly from markets and MDV, APV, EDSV were all not detected by using multiplex PCR. But the viruses were detected from the artificially contaminated fecal samples. Moreover, MDV was detected from penguin fecal samples by multiplex PCR. Therefore, the multiplex PCR which has been established will play an important role in detecting viruses and controlling the epidemics.