College of Veterinary Medicine, Shandong Agricultural University, Tai'an, Shandong 271018, China.
College of Veterinary Medicine, Shandong Agricultural University, Tai'an, Shandong 271018, China.
J Virol Methods. 2023 Dec;322:114810. doi: 10.1016/j.jviromet.2023.114810. Epub 2023 Sep 9.
Duck Tembusu virus disease, caused by duck Tembusu virus (DTMUV), brings great harm to duck industry. Early diagnosis is of great significance for the prevention and control of this disease. In order to develop a specific and sensitive method for rapid diagnosis of DTMUV, reverse-transcriptase recombinase aided amplification combined with lateral flow dipstick (RT-RAA-LFD) method for detection of DTMUV was established. Firstly, downstream primer was labeled with biotin and probe was labeled with FAM, and primer concentration, reaction time, and reaction temperature were optimized. Then, the specificity and sensitivity of this method was investigated. The results of specificity test showed that it had no cross reaction with other common pathogens such as low pathogenic avian influenza virus (AIV), Newcastle disease virus (NDV), duck hepatitis A virus (DHV), and duck Reovirus. The results of sensitivity test showed that the minimum detection limit of this method was 10 copies/μL, which was 1000 times than conventional RT-PCR (10 copies/μL), and equivalent to that of fluorescent quantitative PCR. Furthermore, this RT-RAA-LFD method demonstrated excellent intragroup and intergroup consistency. Finally, the RT-RAA-LFD assay and real-time PCR were both utilized to examine 58 clinical samples concurrently. The results showed that the RT-RAA-LFD method (5/58) was more sensitive than the fluorescence quantitative PCR method (4/58). In summary, RT-RAA-LFD method established in this study had a strong specificity and high sensitivity, which provided technical support for clinical detection of DTMUV.
鸭坦布苏病毒病是由鸭坦布苏病毒（DTMUV）引起的，给养鸭业带来了巨大危害。早期诊断对该病的防控具有重要意义。为了开发一种用于快速诊断 DTMUV 的特异性和灵敏性方法，建立了逆转录重组酶辅助扩增结合侧流层析试纸条（RT-RAA-LFD）法检测 DTMUV。首先，在下游引物上标记生物素，在探针上标记 FAM，并优化引物浓度、反应时间和反应温度。然后，对该方法的特异性和灵敏度进行了研究。特异性试验结果表明，该方法与低致病性禽流感病毒（AIV）、新城疫病毒（NDV）、鸭肝炎 A 病毒（DHV）和鸭呼肠孤病毒等常见病原体无交叉反应。灵敏度试验结果表明，该方法的最低检测限为 10 拷贝/μL，比常规 RT-PCR（10 拷贝/μL）灵敏 1000 倍，与荧光定量 PCR 相当。此外，该 RT-RAA-LFD 方法在组内和组间具有良好的一致性。最后，同时使用 RT-RAA-LFD 检测和实时荧光定量 PCR 对 58 份临床样本进行检测。结果表明，RT-RAA-LFD 法（5/58）比荧光定量 PCR 法（4/58）更灵敏。综上所述，本研究建立的 RT-RAA-LFD 方法具有较强的特异性和较高的灵敏度，为 DTMUV 的临床检测提供了技术支持。
