Detection Of Chicken Infectious Bronchitis Virus With Real-Time Fluorescent PCR

Infectious bronchitis virus(IBV)is a major cause of economic losses in the poultry industry and can be involved in respiratory disease,nephritis,and poor egg production and quality.In general,IBV infections can be diagnosed by detection of (parts of)IBV virus itself or the specific antibody response.There are a number of assays for diagnosing acute IBV infections.The most common for routine use are virus isolation(VI),immunofluorescence assay(IFA),immunoperoxidase assay(IPA) Tests commonly used are the haemagglutination inhibition(HI)test,the agar gel precipitation test(AGFT),and the enzyme-linked immunosorbent assay(ELISA).The virus neutralization test(VNT)is rarely used for routine diagnosis because it is relatively expensive and laborious.Choosing between tests and subsequent interpretation of the results can be very difficult and confusing,and is quite often thwarted by the poor documentation of the performance of tests after IBV infections in the field.These methods are time-consuming,low efficiency,low accuracy and sensitivity,which can not meet the diognostic expectations.Depending on the demands and local circumstances,a best choice or best combination of techniques for that situation can be made.PCR was applied to detect the animal infectious disease virus widely in recently years,which is more rapid and efficient.However,the gel electrophoresis is needed to determine the sizes of amplified product after normal PCR and the contamination,safety and time-consuming problems have been noted as significant issues.So a safer,more accurate,sensitive and rapid PCR method is required for these virus detection.The series of primers and Taqman probe were designed based on the region of S1 gene to detect IBV with normal PCR and Real-Time fluorescent PCR(RTF PCR)in this experiment.The specific primers and probes were found that they could detect all the IBV isolates collected from different sources.The protocol was approximately 100 times more sensitive than normal PCR and the amplified products were confirmed by specific fluorescent probe used during amplification.Strong fluorescent signal could be collected in the reaction when the RNA was directly extracted from the inoculated chick embryo kidney cell and infectious tissues which had no symptom.Furthermore,the pathogen isolation is not required and contamination is controlled because the whole detection process is finished in the contained tubes.This method can provide a specific,sensitive,rapid detection of IBV for chicken diseases preventive and control.