Establishment and Application of Diagnostic Test for Marek’s Disease Virus Based on TaqMan Probe Double Fluorescent Quantitative Polymerase-Chain-Reaction(FQ-PCR)

Marek’s disease (MD) is a highly contagious, malignant T-lymphomatosis of chickens. MD has a worldwide distribution and is thought to cause enormous loss to the poultry industry. The etiological agent Marek’s disease virus (MDV) are typical herpes viruses and belong to the family Herpesviridae, sub-family Alpha herpesvirinae. The MDV group consists of three closely related viruses with many antigens in common, and their division into individual serotypes, namely serotype 1, 2, and 3. Mixed infection, intervention from live vaccines, subclinical infection brings a great many difficulties on diagnosis, antidiastole, monitoring. Today MD is the only one in all tumorous disease that can be prevented by vaccine. Using live vaccines administered is the main measure. The quality of vaccine and immune effect is the key to control the disease triumphantly. In this study, we developed a Real-time PCR Based on TaqMan Probe Double Fluorescent Quantitative Polymerase-Chain- Reaction (FQ-PCR) for detection of MDV, studying and researching triple live vaccines in order to provide accurate laboratory data for early diagnosis and inspection of immunizing effect.Published sequences of the selected genes (Meq for MDV1, DNA-Polymerase for MDV2, SORF1 for HVT, and Ovotransferrin for chickens) were obtained from GenBank. Four pairs of primers and four internal dual-labeled fluorogenic probes were designed and synthesized. The fragments were cloned into T vector and transformed into DH5α. The positive recombinant plasmids were used as standard quantitative template to develop FQ-PCR, including optimization of PCR system, establishment of standard curve and evaluation to sensitivity, specificity and reproducibility. When compared with AGP and standard PCR , the FQ- PCR has a higher rate of positive (100﹪). FQ-PCR was 10-100 fold more sensitive than standard PCR, and could detect 2.01-2.78 copies for the virus sensitively. These methods were applied for studying triple live vaccines and monitoring the vaccine. Disciplinarian of three serotypes virus growth, valid range of inoculability, optimal dosage and the content of single patient forms unit were gained.The methods can differentiate field strains and vaccine strains on quantitative standard with the character of high isomerism and sensitivity. The detection can be rapidly completed in 1.5- 2.0 hours. The successful development of four separate FQ-PCR assays and three duplex assays for the quantitation and differentiation of MDV