鼠肝炎病毒(MHV)是一种高度传染性的鼠冠状病毒,具有对宿主动物造成伤害的高潜力。本研究旨在建立实时逆转录重组酶聚合酶扩增(RT-RPA)快速检测实验小鼠MHV的方法。
UNASSIGNED:设计了针对MHV参考菌株M基因中保守区域的RT-RPA测定的特异性引物和探针(登录号:FJ6647223)根据TwistDx手册说明。特异性,灵敏度,评估RT-RPA方法的可重复性,并与标准RT-qPCR方法进行比较。使用68个现场样品评估该测定的临床适用性。
UNASSIGNED:使用新开发的RT-RPA测定法在37°C下在20分钟内完成扩增,而使用RT-qPCR方法需要近60分钟。RT-RPA方法具有明显的省时优势。RT-RPA和RT-PCR方法的检测限相同,为4.45×101拷贝/μL。特异性表现为缺乏与MHV的交叉反应,小鼠肺炎病毒,仙台病毒,汉坦病毒,微小的小鼠病毒,和III型呼肠孤病毒。RT-RPA检测的MHV检出率为13.63%(9/66),RT-qPCR检测为15.15%(10/66)。Cohen的“kappa”(κ)分析结果在两种方法之间表现出非常好的一致性,κ≥0.750(因为κ=0.939)和p<0.0005(因为p=0.000)。
UASSIGNED:RT-RPA检测提供了一种简单的替代工具,快速,在实验室小鼠中可靠地检测MHV,具有在实验室中应用的巨大潜力。
Murine hepatitis virus (MHV) is a highly infectious murine coronavirus that has a high potential for causing harm to host animals. This study aimed to develop a real-time reverse transcription recombinase polymerase amplification (RT-RPA) method for rapid detection of MHV in laboratory mice.
UNASSIGNED: Specific primers and probes for RT-RPA assay were designed targeting the conserved region in the M gene of the MHV reference strain (accession no. FJ6647223) according to the TwistDx manual instructions. The specificity, sensitivity, and reproducibility of the RT-RPA method were evaluated and compared with those of the standard RT-qPCR method. The clinical applicability of this assay was evaluated using 68 field samples.
UNASSIGNED: Amplification using the newly developed RT-RPA assay was completed within 20 min at 37°C, while that using the RT-qPCR method required nearly 60 min. The RT-RPA method exhibited an obvious time-saving advantage. Both RT-RPA and RT-PCR methods had the same limit of detection, which was 4.45 × 101 copies/μL. The specificity was indicated by a lack of cross-reaction with MHV, pneumonia virus of mice, Sendai virus, hantavirus, minute virus of mice, and reovirus type III. The MHV detection rate of RT-RPA assays was 13.63% (9/66) and RT-qPCR assays was 15.15% (10/66). Cohen\'s \"kappa\" (κ) analysis results exhibited a very good agreement between two methods with the value of κ ≥ 0.750(since κ = 0.939) and p < 0.0005 (since p = 0.000).
UNASSIGNED: The RT-RPA assay offers an alternative tool for simple, rapid, and reliable detection of MHV in laboratory mice and has significant potential for application in laboratories.