A Multiplex RT-PCR Assay For Several Respiratory Viruses In Chicken Pathogens

Chicken respiratory tract infections in chicken production practice is a very common disease. Infectious pathogen including viruses, bacteria, mycoplasma, and other risk factors. Most cases is a mixture of several pathogenic infections or different pathogens has secondary infection, resulting in chick growth retardation, adult chicken, egg drop and cause the death of a variety of day-old chickens. Newcastle disease(Newcastle disease, ND), avian infectious bronchitis (Infectious laryngotrache, IB), as well as avian influenza (Avian influenza, AI) H5and H9subtypes is a kind of viral infectious diseases with similar respiratory symptoms in the clinical.And they are RNA viruses.Accurate and timely to make a diagnosis based solely on clinical symptoms and pathological findings is difficult, and different virus infection and its subsequent disposal program is not the same delay time to diagnosis or misdiagnosis can cause significant economic losses to the chicken business. In this experiment, therefore, the proposed ND-IB-AIH5-AIH94kinds of poultry respiratory pathogen multiplex RT-PCR differential diagnosis method, and its specificity and sensitivity of detection, clinical specificity, high sensitivity and time-saving laboratory differential diagnosis, its main results are as follows:1.4kinds of poultry respiratory pathogen amplification primer design and amplification of the establishmentFour pairs primers is designed according F gene fragment of the NDV, N gene fragment of the IBV and the HA fragment of H5,H9subtypes of the AIV, by gradient RT-PCR amplification of four pairs of primers were selected on the basis of the appropriate multiplex RT-PCR annealing temperature of four pairs of primers were the optimization of primer concentration, determined by ordinary RT-PCR annealing temperature of58℃, the concentration of the primers, for0.32pmol/μl (NDV),0.32pmol/μl (of IBV),0.24pmol/μl (H5),0.24pmol/μl (H9). Final design of the four pairs of primers and optimization of annealing temperature and primer concentration by ordinary RT-PCR technique specific amplification of NDV, IBV, of AIV the H5and H9subtypes of gene fragments, amplified fragment length for the other:520bp (NDV),748bp (IBV),151bp (H5).266bp (H9).2.4kinds of poultry respiratory pathogen multiplex RT-PCR conditions of the optimizationOrdinary RT-PCR based on the optimization of the multiplex RT-PCR, respectively, from the primer concentration, Taq enzyme concentration and Mg2+concentration, dNTP concentration, annealing temperature in these five areas to be optimized. Optimal results for the Mg2+concentration of3.0mM, dNTP concentration of0.09U/μl, annealing temperature of58℃, primer concentrations were0.32pmol/μl (NDV),0.32pmol/μl (IBV),0.24pmol/μl (H5),0.24pmol/μl (H9), optimization results can be a good differential diagnosis of these four viruses and the four virus mixed infection.3.4kinds of poultry respiratory pathogen multiplex RT-PCR method and its preliminary applicationEstablished for each primer can be amplified from the positive material corresponding bands from other pathogens, and the negative material and poultry other respiratory pathogens (infectious laryngotracheitis, Mycoplasma gallisepticum) nonspecific amplification with; established by multiplex RT-PCR method can detect10pgNDV, lngIBV, the H5subtype of10pgAIV,10pgAIV H9subtype RNA that established by the method of high specificity and sensitivity. That can be used to flock clinical outbreak rapid differential diagnosis of respiratory tract infection.