鸭腺病毒3型荧光RPA恒温快速检测方法的建立与应用

Rapid Detection of Duck Adenovirus Type 3 Based on Isothermal Recombinase Polymerase Amplification Assay

关键词:  Dadv-3  Hexon基因  RPA  恒温快速检测

Key words:  Dadv-3  Hexon gene  RPA  constant temperature rapid detection.

摘要:

鸭腺病毒3型(Duck adenovirus type 3，DAdV-3)是我国近年来新发的禽腺病毒，致病力较强，已经严重影响到鸭养殖业。为实现对DAdV-3的快速检测，以DAdV-3的Hexon基因为靶基因，设计特异性重组酶聚合酶扩增(Recombinase polymerase amplification，RPA)引物和探针，并构建重组标准质粒命名为DAdV-H-18T，优化RPA反应条件后，建立DAdV-3的荧光RPA恒温快速检测方法。结果显示，该检测方法最佳反应条件为38 ℃、18 min；特异性强，与其他常见鸭病病原如鸭肝炎病毒、鸭坦不苏病毒等均无交叉反应。对重组质粒DAdV-H-18T的最低检测限为1 copy/μL；采用建立的RPA检测方法和qPCR对55份鸭组织临床样本进行检测，阳性率分别为65.4%、50.9%，检测符合率为92.7%。研究建立的DAdV-3荧光RPA方法，检测速度快，敏感性高，特异性强，为该病临床快速检测提供了有力工具。

Abstract:

Duck adenovirus type 3(DAdV-3)is a newly discovered poultry adenovirus in my country in recent years. It has a strong pathogenicity and has seriously affected the duck breeding industry. In order to realize the rapid detection of DAdV-3, specific Recombinase polymerase amplification(RPA) primers and probes were designed with the Hexon gene of DAdV-3 as the target gene, and the recombinant standard plasmid was constructed and named DAdV-H-18T. After optimizing the RPA reaction conditions, the establishment Fluorescent RPA isothermal rapid detection method for DAdV-3. The results showed that the optimal reaction condition of the detection method was 38 ℃、18 minutes; the specificity was strong, and there was no cross-reaction with other common duck disease pathogens such as duck hepatitis virus and duck tambusu virus. The minimum detection limit of the recombinant plasmid DAdV-H-18T was 1 copy/μL; the established RPA detection method and qPCR were tested on 55 duck tissue clinical samples, and the results showed that the positive rates of RPA and qPCR were 65.4%, 50.9%, the detection coincidence rate was 92.7%. This study successfully established the fluorescent RPA method of DAdV-3, which has the advantages of fast speed, high sensitivity and strong specificity, and provides a powerful tool for the rapid clinical detection of the disease.