Abstract：
Murine hepatitis virus (MHV) infection is one of the most prevalent types of mice infection in laboratory. MHV could cause death in mice and even interfere with the results in animal experiments. Herein, we developed two isothermal approaches based on the Multienzyme Isothermal Rapid Amplification (MIRA), for rapid detection of MHV in conserved M gene. We designed and screened several pairs of primers and probes and the isothermal fluorescence detector was applied for the exonuclease Ⅲ reverse transcription MIRA (exo-RT-MIRA) assay. To further simplify the workflow, the portable fluorescence visualization instrument, also as a palm-sized handheld system, was used for the naked-eye exo-RT-MIRA assay. The amplification temperature and time were optimized. The assay could be processed well at 42 °C 20 min for the exo-RT-MIRA and the naked-eye exo-RT-MIRA assay. The limit of detection (LoD) of the exo-RT-MIRA assay was 43.4 copies/μL. The LoD of the naked-eye exo-RT-MIRA assay was 68.2 copies/μL. No nonspecific amplifications were observed in the two assays. A total of 107 specimens were examined by qPCR and two assays developed. The experimental results statistical analysis demonstrated that the exo-RT-MIRA assay with the qPCR yielded sufficient agreement with a kappa value of 1.000 (p < 0.0001). The results also exhibited a good agreement (kappa value, 0.961) (p < 0.0001) between the naked-eye exo-RT-MIRA assay and the qPCR assay. In our study, the exo-RT-MIRA assay and the naked-eye exo-RT-MIRA assay presented the possibility of new methods in MHV point-of-testing diagnosis.
摘要：
小鼠肝炎病毒（MHV）感染是实验室中最常见的小鼠感染类型之一。MHV可能导致小鼠死亡，甚至干扰动物实验的结果。在这里，我们开发了两种基于多酶等温快速扩增（MIRA）的等温方法，用于保守M基因中MHV的快速检测。我们设计并筛选了几对引物和探针，并将等温荧光检测器用于外切核酸酶Ⅲ逆转录MIRA（exo-RT-MIRA）测定。为了进一步简化工作流程，便携式荧光可视化仪器，也作为一个手掌大小的手持系统，用于裸眼exo-RT-MIRA测定。优化扩增温度和时间。对于exo-RT-MIRA和裸眼exo-RT-MIRA测定,该测定可以在42°C下处理20分钟。外-RT-MIRA测定的检测限(LoD)为43.4拷贝/μL。裸眼exo-RT-MIRA测定的LoD为68.2拷贝/μL。在两个测定中未观察到非特异性扩增。通过qPCR检查了总共107个样本,并开发了两个测定。实验结果统计学分析表明,具有qPCR的exo-RT-MIRA测定产生与1.000的卡伯值足够一致(p<0.0001)。结果也表现出良好的一致性(卡伯值，0.961）（p<0.0001）在裸眼外切RT-MIRA测定和qPCR测定之间。在我们的研究中,exo-RT-MIRA测定和裸眼exo-RT-MIRA测定为MHV点诊断提供了新方法的可能性。
