Development Of Indirect N-ELISA Antibody Test Kit And Detection Method Of Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) For Infectious Bronchitis Virus

Avian infectious bronchitis is one of the highly contagious respiratory and acute diseases in chickens and causes by infectious bronchitis virus(IBV),which can affect different days old chicken and ofen leads to co-infection with mycoplasm and Escherichia colibacillus etc.It can increase the motality of chicken flocks with huge economic loss in poultry.Nowadays,IB is widely epidemic and still one of the infectious diseases threatening poultry industry all over the world.IBV has numerous serotypes mainly because of its high potential mutations in genome,and the cross protection between different serotypes is very poor.Therefore it is a big chanllege to protect from IB.Therefore,it is urgent and essential to develop a rapid,sensitive,specific antibody and pathogen detection method.ELISA has been widely used and adopted to be the standard method by the OIE(OIE)because of the advantages of rapid,convenience,high sensitivity and specificity among all current methods of detection of IBV antibodies.At present,the whole virus is coated in the commercial IBV antibody test kit,which leads to cross reactivity among different serotype strains,and the whole virus purification is difficult and likely to have high risk of spreading as well as the virus testing cost is huge,which makes commercialization application of IBV antibody detection kit be limited.In spite of the traditional methods of detecting IBV are accurate,their operation is complex,time-consuming and laborious.Though DNA hybridization,RT-PCR and Real-time fluorescent quantitative RT-PCR have high sensitivity and specificity,time-consuming,the environment pollution of EB and expensive equipment and reagents can not be neglected.Therefore,it is very important and essential to develop a ELISA kit coated with the N gene protein of conservative and strong immunogenicity and a rapid,sensitive,simple and pratical method for IBV detection.Firstly,the presnet study was to develop indirect N-ELISA kits for clinical IBV antibody detection on the base of the indirect N-ELISA detection method established by our previous research team.The indirect N-ELISA antibody detection kit was developed via the optimal reaction conditions,repeatability intra and inter-assay reproducibility test,37℃ accelerated aging test,4℃ and-200 ℃ storage period of comparison,conformance testing and blocking test.The results showed that the intra-assay variation coefficient was 3.07%～8.34%,interassay variation coefficient was 2.33%～6.36%,respectively.The ELISA antibody detection kit could be stable at-20℃at least for one year.296 serum samples had been tested via the commercialized kit and indirect N-ELISA antibody detection kit.The negative coincidence rate is 86.39%,the positive coincidence rate is 97.3%,respectively.1368 serum samples from A,B,C,D and E chicken farms in Guangxi were detected by the developed IBV indirect N-ELISA kit.The results showed that the developed indirect N-ELISA kit could reflect the immue effect.In addition,it also could show increasing antibodies level trend.540 clinical samples detected by the indirect N-ELISA kits(their storage period was 2,4 and 6 months,respectively).The results showed that the anastomosis was very good,except that the antibody levels decreased slightly,Secondly,in order to prevent effectively and diminish mortality rate of IB and develop a rapid and practical method for the detection of IBV,a reverse transcription-loop-mediated isothermal amplification(RT-LAMP)with primers based on N gene sequence was established in the present study.The viral cDNA generated by reverse transcription was amplificated with Bst DNA polymera at 61～63℃,and the products could be visualized with naked-eye.The results showed that the method were sensitive(below in 900fg),specific as well as stable,therefore,it can be adopted by primary sector and poultry farm.Therefore,IBV indirect N-ELISA antibody test kit developed in the present study was specific,sensitive,safe,stable and cheap and the reverse transcription RT-LAMP was specific,sensitive,reproducible,simple and rapid.RT-LAMP can be carried our without special equipment.The present study was very valuable in the clinical application.