Sui Xin, Zhang Xu, Fei Dongliang, Zhang Zhen, Ma Mingxiao
Institute of Biological Sciences, Jinzhou Medical University, Jinzhou, Liaoning, China.
The First Affiliated Hospital, Jinzhou Medical University, Jinzhou, Liaoning, China.
PeerJ. 2019 Jan 8;6:e6068. doi: 10.7717/peerj.6068. eCollection 2019.
Hemorrhagic fever with renal syndrome is in most cases caused by the Hantaan virus (HTNV) and Seoul virus (SEOV). To develop and apply reverse transcription loop-mediated isothermal amplification (RT-LAMP) to detect HTNV and SEOV simultaneously, which was faster, more cost effective, and easier to perform as the target gene amplified rapidly. In this article an assay based on LAMP is demonstrated, which only employs such apparatus as a water bath or a heat block.
A chromogenic method using the calcein/Mn complex and real-time turbidity monitoring method were used to assess reaction progress of the reaction, and the specificity of the RT-LAMP-based assay was assessed by detecting cDNAs/cRNAs generated from Coxsackievirus A16, Influenza virus, lymphocytic choriomeningitis virus, mouse poxvirus, rotavirus, mouse hepatitis virus. In addition, 23 clinical specimens were used to determine the agreement between the RT-LAMP assay with reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence (IFT) method.
The detection limit of RT-LAMP to HNTV and SEOV was as low as 10 copies/μL with optimized reaction conditions, which was much more sensitive than the RT-PCR method (100-1,000 copies/μL). At the same time, the detection results of 23 clinical specimens have also illustrated the agreement between this the RT-LAMP assay with RT-PCR and IFT.
This RT-LAMP assay could be used to perform simultaneous and rapid detection of HTNV and SEOV to the clinical specimens.
肾综合征出血热在大多数情况下由汉坦病毒（HTNV）和汉城病毒（SEOV）引起。为了开发并应用逆转录环介导等温扩增技术（RT-LAMP）同时检测HTNV和SEOV，该技术具有快速、成本效益高且操作简便的特点，因为目标基因能快速扩增。本文展示了一种基于LAMP的检测方法，该方法仅需水浴或热块等仪器。
使用钙黄绿素/锰复合物的显色法和实时浊度监测法评估反应进程，并通过检测柯萨奇病毒A16、流感病毒、淋巴细胞性脉络丛脑膜炎病毒、小鼠痘病毒、轮状病毒、小鼠肝炎病毒产生的cDNA/cRNA评估基于RT-LAMP检测方法的特异性。此外，使用23份临床标本确定RT-LAMP检测法与逆转录聚合酶链反应（RT-PCR）和免疫荧光法（IFT）之间的一致性。
在优化的反应条件下，RT-LAMP对HTNV和SEOV的检测限低至10拷贝/μL，比RT-PCR方法（100 - 1000拷贝/μL）灵敏得多。同时，23份临床标本的检测结果也表明了RT-LAMP检测法与RT-PCR和IFT之间的一致性。
这种RT-LAMP检测法可用于对临床标本同时进行快速检测HTNV和SEOV。
