鸭腺病毒3型PCR检测方法的建立

Specific Diagnosis for Duck Adenovirus Type 3 by PCR Method

鸭3型腺病毒(Duck adenovirus type 3,DAdV-3)为近年来新发的以引起鸭肝脏肿大出血、肾脏大面积出血点为特征的鸭群新发疫病。为建立特异性的检测DAdV-3的PCR方法,研究通过分析鸭群中鸭腺病毒A型(DAdV-A)、鸭源禽腺病毒4型(FAdV-4)、鸭2型腺病毒(DAdV-2)和DAdV-3的Fiber 2基因特征,建立特异性检测DAdV-3的PCR方法。结果显示:优化后的PCR方法最佳退火温度为54℃,对DAdV-3扩增片段大小为548 bp;敏感性强,最低检测限为36.4 pg;特异性好,对DAdV-A、FAdV-4、鸭病毒性肠炎(DEV)、番鸭细小病毒(MDPV)、鹅细小病毒(GPV)、鸭圆环病毒(DuCV)和鹅多瘤病毒(GHPV)均无特异性扩增。表明该方法可有效用于开展新发DAdV-3的流行病学调查。

Duck adenovirus type 3(DAdV-3),which is newly identified adenovirus in ducklings,with necrosis and hemorrhage in liver and spleen.In this study,the Fiber 2 gene sequences of duck adenoviruse A(DAdV-A),fowl adenovirus 4(FAdV-4),duck adenovirus 2(DAdV-2) and DAdV-3 occurred in ducks were retrieved from GenBank in order to develop a specific PCR tool for DAdV-3 infection.Primer design software Oligo 7.0 was used to design specific primers based on the Fiber 2 gene characterization after genetic comparison.Then PCR method was established after conditional optimized.The results showed the optimal annealing temperature was 54 ℃,which only could amplify DAdV-3infection with 548 bp in length.The PCR method showed high sensitivity,with low detection limit at 36.4 pg.The PCR method was demonstrated good specificity,with no cross-amplification from common adenoviruses occurred in ducks(i.e.DAdV-A,FAdV-4) and other common duck-origin pathogens(i.e.duck enteritis virus,Muscovy duck parvovirus,goose parvovirus,duck circovirus and goose hemorrhagic polyomavirus).It′s concluded that PCR method for specific diagnosis with DAdV-3 was developed in this study,which laid foundation for further DAdV-3 epidemiological investigation.