Rapid and Sensitive Detection of RNA Viruses Based on Reverse Transcription Loop-Mediated Isothermal Amplification, Magnetic Nanoparticles, and Chemiluminescence
Rapid and Sensitive Detection of RNA Viruses Based on Reverse Transcription Loop-Mediated Isothermal Amplification, Magnetic Nanoparticles, and Chemiluminescence
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基于逆转录环介导的等温扩增、磁性纳米颗粒和化学发光的 RNA 病毒快速、灵敏检测
DOI：
10.1166/jbn.2016.2244
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发表时间：
2016
影响因子：
2.9
通讯作者：
He Nongyue
中科院分区：
文献类型：
--
作者：
Wang Jiuhai;Lu Peng;Yan Jieni;Zhang Yufan;Huang Lanye;Ali Zeeshan;Liu Bin;Li Zhiyang;He Nongyue
RNA病毒，特别是高致病性禽流感（HPAI）病毒，在世界范围内引起严重的健康问题，并造成巨大的经济损失。为了实施治疗和疾病控制战略，迫切需要早期检测这些致命RNA病毒的诊断工具。常规的基于逆转录聚合酶链反应（RT-PCR）的荧光（RT-PCR-CL）检测常用于病毒感染的诊断。然而，对昂贵的PCR机器和更长的热循环时间的要求是显着的缺点。本研究提出了一种基于逆转录环介导等温扩增（RT-LAMP）结合化学发光（CL）检测H7N9病毒的方法。该方法不需要任何昂贵的仪器，与RT-PCR-CL相比，处理时间显著缩短。由于包括RT-LAMP温度、探针浓度、杂交温度和杂交持续时间在内的几个因素可能会影响CL信号，因此对这些参数中的每一个进行了研究和优化。使用优化的RT-LAMP-CL方法可检测到1000拷贝/mL的H7N9 RNA。与传统的RT-PCR-CL相比，RT-LAMP的检测时间显著缩短。该技术对于病毒检测和诊断，特别是对于禽流感病毒，具有很大的前景。
RNA viruses, particularly, the highly pathogenic avian influenza (HPAI) virus, pose serious health concerns, and cause huge economic losses worldwide. Diagnostic tools for the early detection of these deadly RNA viruses are urgently needed to implement treatment and disease control strategies. Conventional reverse transcription polymerase chain reaction (RT-PCR)-based chemiluminescent (RT-PCR-CL) detection is frequently used for the diagnosis of viral infections. However, the requirements for expensive PCR machines and longer thermocycling times are significant drawbacks. In this study, we propose a method based on reverse transcription loop-mediated isothermal amplification (RT-LAMP) combined with chemiluminescence (CL) to detect H7N9 virus. The proposed method does not require any expensive instruments, and processing time is remarkably shortened compared to that of RT-PCR-CL. Since several factors including RT-LAMP temperature, probe concentration, hybridization temperature, and hybridization duration might affect the CL signal, each of these parameters was investigated and optimized. One thousand copies/mL of H7N9 RNA were detectable using the optimized RT-LAMP-CL method. The detection time was significantly reduced by using RT-LAMP, in comparison with conventional RT-PCR-CL. This technique holds great promise for viral detection and diagnosis, especially with regard to avian influenza virus.