禽腺病毒血清4型纳米PCR检测方法的建立与初步应用

Establishment and application of Nano PCR assay for detection on Fowl aviadenovirus serotype 4

为建立一种快速有效检测禽腺病毒血清4型(FAdV-4)的方法,本研究根据FAdV-4五邻体(Penton)基因序列,设计合成1对特异性引物,经过条件优化,建立了检测FAdV-4的纳米PCR方法(Nano PCR)。特异性试验结果显示,该方法对鸡新城疫病毒等12种家禽常见病原及国内报导的FAdV-1、FAdV-2、FAdV-8a、FAdV-8b、FAdV-11等5个主要血清型病毒的扩增结果均为阴性,仅FAdV-4检测结果为阳性,特异性较强。敏感性试验结果显示,该方法对FAdV-4的最低核酸检出量为54.0拷贝/μL,较常规PCR方法高10倍,敏感性较高。应用该方法和病毒分离培养法同时对87份临床样品进行检测,二者均检出14份阳性样品,总体符合率为100%。以上结果表明本研究建立的Nano PCR方法特异性好、敏感性高,可以用于FAdV-4的临床检测和分子流行病学调查。

To establish a rapid,specific and sensitive Nano PCR detecting method for Fowl aviadenovirus serotype 4(FAdV-4)diagnosis,a pair of primers were designed based on Penton gene of FAdV-4.The Nano PCR method was developed after the optimization of amplification conditions.The specific detection results of 12 common poultry pathogens and the FAdV-1,FAdV-2,FAdV-8a,FAdV-8b,FAdV-11 were all negative except for FAdV-4.The detection limit of the viral nucleic acid was 54.0 copies/μL.The Nano PCR method was approximately 10-fold more sensitive than regular PCR assay.Among 87 clinical samples,14 positive samples were identified by virus isolation and the Nano PCR method.The overall coincidence rate of two methods was 100%.These results indicated that the Nano PCR has good specificity,sensitivity and repeatability,which can be applied widely in clinical diagnosis and field surveillance of FAdV-4 infection.