Establishment And Application Of Nested PCR And Loop-mediated Isothermal Amplification Detection Methods For Avian Adenovirus Type 4

Since June 2015,a new avian infectious disease,Hydropericardium hepatitis syndrome(HHS),caused by fowl adenovirus serotype 4(FAd V-4),has been prevalent in Shandong,Anhui,Jiangsu and Henan provinces.The disease is prone to be co-infection and mainly harms broilers aged 3-6 weeks,with a mortality rate of up to 90%,and brings great economic losses to poultry industry.Conventional laboratory testing methods need special instruments,which are expensive,time-consuming,laborious,and the testing site is limited.There are many inconveniences in clinic detection in the chicken production process.Therefore,it is necessary to establish a fast,sensitive and convenient testing method.In this study,nested PCR and loop-mediated isothermal amplification(LAMP)detection methods were established to detect FAd V-4.By aligning 27 published avian adenovirus genome sequences in Gen Bank,and selecting highly conserved regions,specific nested PCR primers for group I avian adenoviruses and FAd V-4 were designed,respectively.The detection method was established,and the reaction conditions were optimized.The optimal reaction conditions for nested PCR were as follows: the optimal annealing temperature of the first round reaction was 60.4?,the annealing time was 35 s,the cycle number was 30 times,the external primer concentration was 0.9 ?mol/L,the Mg2+ concentration was 2.5 mmol/L,and the r Taq DNA polymerase concentration was 1 U;the optimal annealing temperature of the second round reaction is 54.6?,the annealing time is 30 s,the cycle is 30 times,the internal primer concentration is 0.5 ?mol/L,the Mg2+ concentration is 1.0 mmol/L,and the r Taq DNA polymerase concentration is 1 U.The detection method has the highest efficiency for interested gene amplification,and has no cross-reaction with other chicken-derived viruses.At the same time,the detection method has high sensitivity,and the lowest detectable virus DNA is 5.2 pg/?L.Furthermore,specific LAMP primers were designed according to the FAd V-4 Hexon gene sequence,and a LAMP detection method for FAd V-4 was established.The reaction conditions and reaction system were optimized.After optimization,the reaction temperature was 62?,and the reaction time was 30 min.When the ratio of external primer to internal primer concentration was 1:2,the amplification effect was significantly improved.If the Mg2+ concentration was 6.0 ?mol/L,the current best amplification effect was shown.The sensitivity test result shows that the method is 10 times more sensitive and has better specificity compared with the established nested PCR detection method.Using two detection methods to test 102 clinical samples at the same time,it was found that the detection rates of the two methods were found to be 34.3% and 20.5%,respectively.For its higher specificiality and sensitivity,LAMP detectiong method has better application prospect to detect FAd V-4 infection in poultry production.