建立逆转录重组酶辅助扩增-侧向流动纸条和实时荧光逆转录重组酶辅助扩增方法检测鸡新城疫病毒。

Establishment of reverse transcription recombinase-aided amplification-lateral-flow dipstick and real-time fluorescence-based reverse transcription recombinase-aided amplification methods for detection of the Newcastle disease virus in chickens.

机构信息

College of Veterinary Medicine, Hebei Agricultural University, Baoding 071001, China.

出版信息

Poult Sci. 2020 Jul;99(7):3393-3401. doi: 10.1016/j.psj.2020.03.018. Epub 2020 Apr 15.

Newcastle disease is an acute and highly contagious disease of poultry caused by Newcastle disease virus infection, which does great harm to the poultry industry all over the world. To diagnose the disease simply and quickly, 2 detection methods were established based on reverse transcription recombinase-aided amplification (RT-RAA) technology. One is reverse transcription recombinase-aided amplification-lateral flow dipstick (RT-RAA-LFD) that is to combine RT-RAA with lateral flow dipstick; the other is real-time fluorescence-based reverse transcription recombinase-aided amplification (RF-RT-RAA) that is the combination of RT-RAA and exo probe. In this study, the reaction conditions such as reaction temperature and reaction time of the 2 methods were optimized, and their specificity and sensitivity were tested. The results showed that the RT-RAA-LFD method could be used to complete reaction within 23 min, and its lowest detectable limit was 10 copies/μL, 10 times higher than that of the conventional PCR method (10 copies/μL); the RF-RT-RAA method could be used to complete reaction within 26 min, and its lowest detectable limit was 10 copies/μL, 100 times higher than that of conventional PCR method (10 copies/μL), and it was as sensitive as real-time fluorescence-based quantitative PCR (10 copies/μL). The 2 methods had no cross reaction to the nucleic acid of other avian pathogens and showed good specificity. A total of 86 clinical samples suspected of the Newcastle disease virus were tested by conventional PCR, real-time fluorescence-based quantitative PCR, RT-RAA-LFD, and RF-RT-RAA. Based on the commonly used conventional PCR method, the other 3 detection methods had a coincidence rate of higher than 93%. In summary, RT-RAA-LFD and RF-RT-RAA had high specificity, sensitivity, and efficiency, which were suitable for clinical and laboratory diagnosis, respectively, and provided technical support for the prevention and control of Newcastle disease.

新城疫是由新城疫病毒感染引起的家禽急性、高度接触性传染病，对全球家禽业危害极大。为了简单、快速地诊断该病，建立了基于逆转录重组酶辅助扩增（RT-RAA）技术的 2 种检测方法。一种是将 RT-RAA 与侧向流试纸条相结合的逆转录重组酶辅助扩增-侧向流试纸条（RT-RAA-LFD）；另一种是将 RT-RAA 与外切探针相结合的实时荧光逆转录重组酶辅助扩增（RF-RT-RAA）。本研究优化了 2 种方法的反应温度和时间等反应条件，并对其特异性和敏感性进行了测试。结果表明，RT-RAA-LFD 方法可在 23 min 内完成反应，其最低检测限为 10 拷贝/μL，比常规 PCR 方法（10 拷贝/μL）高 10 倍；RF-RT-RAA 方法可在 26 min 内完成反应，其最低检测限为 10 拷贝/μL，比常规 PCR 方法（10 拷贝/μL）高 100 倍，与实时荧光定量 PCR 相当（10 拷贝/μL）。2 种方法与其他禽源性病原体的核酸无交叉反应，特异性良好。用常规 PCR、实时荧光定量 PCR、RT-RAA-LFD 和 RF-RT-RAA 检测 86 份疑似新城疫病毒的临床样本，以常规 PCR 法为对照，其余 3 种检测方法的符合率均在 93%以上。综上所述，RT-RAA-LFD 和 RF-RT-RAA 具有较高的特异性、敏感性和效率，分别适用于临床和实验室诊断，为新城疫的防控提供了技术支持。