Development Of Loop-Mediated Isothermal Amplification (Lamp)for The Detection Of Chicken Anemia Virus

Chicken infectious anemia is caused by chicken anemia virus, which induces immuno-uppressive in young chicken. One day old chicken is easiest infected by this virus and causes mortality ange from10%to20%. The reaction to vaccination is reduced, easy to infect other disease, and decrease in production, which causes huge economic loss.Chicken anemia virus is widely detected in flock of chicken in most districts for long time because rare obvious clinical features easy to be observed. The serological investigation indicated that almost all of the flocks of chicken were infected by this virus in China, which increased difficulty of control this disease. So it is necessary to detect the virus. Loop-mediated isothermal amplification (LAMP) was developed in2000, and was emphasized in detection in different areas. This method showed advantage in several aspects such as simplification in reaction condition and easy to judge by naked eyes under natural light, which facilitated the utilization in clinical detection. This study tried to develop a LAMP test to detect CAV, and test the potential as a commercial detection kit used in clinic. Based on the alignment of genomes of CAV and the characterization of CAV genome, the conserved VP2gene was chosen as primer target region. The online software of primerexplorer V4was used to design the LAMP primers. The real-time turbidity machine was used to select the LAMP primer set in large number. Several primer sets were tested the specificity and sensitivity, and the best set of primer was picked out.Results showed that there was no non-specific reaction to common avian disease viruses. It was the same or more sensitive in one hour reaction than conventional PCR in30cycles. The LAMP assay was referenced to the conventional PCR and sequencing. The correlation of LAMP in first22clinical samples was100%to PCR test.In the first pre-commercial kit, there was85.1%identity in the challenge chicken samples to PCR and83.6%correlation to the PCR in clinical samples. Besides, in24CAV isolates detection test, it defined24out of24. And it showed high reproducibility both in intra or inter reproduce test. After more than one year’s reservation, the first pre-commercial kit still sensitive to detect the103copies of standard plasmid.In the second pre-commercial kit, LAMP test was93.5%and83.7%correlated to PCR and sequencing in artificial challenge samples and clinical samples, respectively. Further tests were continued to evaluate the pre-commercial kit. In this study, the LAMP method was developed successfully to detect CAV, which was specific, sensitive and reproductive. It showed prospective to develop a commercial kit in future.