| Infectious bronchitis (IB), caused by infectious bronchitis virus (IBV), is an acute and highly contagious disease in chickens. The virus mainly harm the respiratory system、 digestive and reproductive system of chickenes, causing dyspnea, lesion in the kidney and declines in egg production. The morbidity and mortality are high in some field casese, affecting the healthy development of poultry industry. For development and application of fast IBV detection method, primers were designed after arrangement and analysis of IBV genetic sequence, and study on the RT-PCR and the loop-mediated isothermal amplification (LAMP) detection method were performed, providing a reliable detection method for quick clinical diagnosis.A pair of M gene PCR primers were desighed according to published IBV’s sequence in GenBank. IBV’s RT-PCR detection method was developed by constructed vector IBV-pMD19-T、optimized reaction options、sensitivity and specificity test of detection method, the method started to apply in the field of clinical detection. The results show that the segment around271bp was amplified by the developed RT-PCR detection method, which has good sensitivity and specificity. The sensitivity of detection method can be60copies, the result of MDV、NDV and mycoplasma were all the negative.The detection result of20samples showed that only4samples were positive, and the positive rate was20%.3pair of M gene LAMP primers were designed on the basis of published IBV’s sequence in GenBank. The LAMP detection method was preliminary studied by optimized reaction options、sensitivity and specificity test of detection method.The agarose gel electrophoresis showed that incubation at63℃for45min, the amplification segment of RT-LAMP detection method was above160bp. The Detection Limit is more than RT-PCR method, the sensitivity of RT-LAMP detection method could be6copies. The result of MDV、NDV and mycoplasma were all the negative, with very good specificity and sensitivity. |
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