小鼠肺炎病毒双重荧光RT-PCR检测方法的建立及应用

doi:10.12300/j.issn.1674-5817.2026.024

摘要/Abstract

摘要：

目的为了满足进出境啮齿类实验动物、野生动物、生物材料和生物制品中小鼠肺炎病毒（pneumonia virus of mice，PVM）快速通关检疫的需要，建立PVM双重荧光RT-PCR检测方法。方法分别针对PVM编码F蛋白基因和M蛋白基因，设计引物和不同荧光探针标记的探针，建立双重荧光RT-PCR检测方法，并对方法的特异性、敏感性和重复性进行验证。结果建立的双重荧光RT-PCR方法能特异性的识别PVM，而与其它病毒无交叉反应；针对PVM两个基因的特异性引物和探针在同一检测体系中的扩增效率相近，两个荧光通道的检测下限均能达到10 copies/μL；经多个质粒标准品的批内重复和批间重复测试，双通道荧光检测的Ct值变异系数均小于2%；将其应用于PVM野鼠不同组织和体表样本的检测，其适用性广，可用于口鼻液、粪便等体表及环境样本的检测，可从口岸监测的鼠类样本中高效地检出PVM阳性样本。结论建立了PVM双重荧光RT-PCR方法，可为PVM疫情的早期诊断提供更加可靠的技术手段。

关键词: 小鼠肺炎病毒, 双重荧光RT-PCR方法, 融合蛋白基因, 基质蛋白基因

Abstract:

Objective In order to meet the needs for rapid entry and exit quarantine of rodent laboratory animals, wild animals, biological materials, and biological products for pneumonia virus of mice (PVM), a dual-fluorescence RT-PCR detection method for PVM has been established. Methods Primers and probes labeled with different fluorescent markers were designed specifically for the PVM fusion protein gene and the matrix protein gene to establish a duplex fluorescent RT-PCR detection method, and the specificity, sensitivity, and reproducibility of the method were validated. Results The established dual fluorescent RT-PCR method can specifically identify PVM without cross-reacting with other viruses. The specific primers and probes for the two PVM genes have similar amplification efficiency in the same detection system, and the detection limit under two fluorescent channels can both reach 10 copies/μL. Through intra-batch and inter-batch testing of multiple plasmid standards, the coefficient of variation of the Ct values in its dual-channel fluorescence detection was all less than 2%. Applying it to the detection of different tissues and body surface samples of PVM wild mice, it is highly versatile and can be used for testing body surface and environmental samples such as oral and nasal fluids and feces, allowing for efficient detection of PVM-positive samples from rodent samples monitored at ports. Conclusion A PVM dual-fluorescence RT-PCR method has been established, which can provide a more reliable technical approach for the early diagnosis of PVM outbreaks.

Key words: Pneumonia virus of mice, Dual fluorescent RT-PCR method, Fusion protein gene, Matrix protein gene

中图分类号:

Q95-3

熊炜,王奕婷,卢宇豪,等.小鼠肺炎病毒双重荧光RT-PCR检测方法的建立及应用[J]. 实验动物与比较医学.. DOI: 10.12300/j.issn.1674-5817.2026.024.

XIONG Wei,WANG Yiting,LU Yuhao,et al. Establishment and Application of a Dual-Fluorescence RT-PCR Method for Detecting Mouse Pneumonia Virus[J]. Laboratory Animal and Comparative Medicine. DOI: 10.12300/j.issn.1674-5817.2026.024.

图/表 6

表1 PVM双重荧光RT-PCR方法的引物和探针序列

Table 1 Primer and Probe Sequences for the PVM Dual-Fluorescence RT-PCR Method

名称 Name	序列 Sequence	扩增长度 Length
PVM-M正向引物PVM-M forward primer	5'- TCTTGATAAATGCTATCATAGCT-3'	96 bp
PVM-M反向引物PVM-M reverse primer	5'-GAATTGTCACACAGAAGCTC-3'
PVM-M荧光探针PVM-M fluorescent probe	5'- FAM- CTTCGTAATCCATGTTGCCCCAGTC -BHQ1-3'
PVM-F正向引物PVM-F forward primer	5'- TGGCTAGAGCTGATAATGGC -3'	93 bp
PVM-F反向引物PVM-F reverse primer	5'- CATACCCATTGTGGATCTCA -3'
PVM-F荧光探针PVM-F fluorescent probe	5'-VIC- TGGCTCATTATCATACTTCCCGTC -BHQ1-3'

注：1-1：PVM阳性样本的FAM荧光通道扩增曲线（设置复孔）；1-2：PVM阳性样本的VIC荧光通道扩增曲线（设置复孔）；2~12：MHV、Reo-3、MVM、SeV、BRSV、MV、NDV、CDV、PPRV、CPIV和阴性对照的两个荧光检测通道均未出现扩增。
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图1 PVM双重荧光RT-PCR方法的特异性试验
注：1-1：PVM阳性样本的FAM荧光通道扩增曲线（设置复孔）；1-2：PVM阳性样本的VIC荧光通道扩增曲线（设置复孔）；2~12：MHV、Reo-3、MVM、SeV、BRSV、MV、NDV、CDV、PPRV、CPIV和阴性对照的两个荧光检测通道均未出现扩增。

Figure 1 Specificity test of PVM duplex fluorescent RT-PCR method
Note： 1-1：FAM fluorescence channel amplification curves of PVM-positive samples （replicates were set）；1-2：VIC fluorescence channel amplification curves of PVM-positive samples （replicates were set）；2~12：No amplification was observed in the two fluorescence detection channels for MHV， Reo-3， MVM， SeV， BRSV， MV， NDV， CDV， PPRV， CPIV， and the negative control.

注：扩增曲线从左到右PVM质粒模板的浓度分别为10⁵ copies/μL、10⁴ copies/μL、10³ copies/μL、10² copies/μL、10 copies/μL、1 copies/μL和阴性对照（即0 copies/μL）（设置复孔）。
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图2 PVM双重荧光RT-PCR方法的敏感性试验
注：扩增曲线从左到右PVM质粒模板的浓度分别为10⁵ copies/μL、10⁴ copies/μL、10³ copies/μL、10² copies/μL、10 copies/μL、1 copies/μL和阴性对照（即0 copies/μL）（设置复孔）。

Figure 2 Sensitivity test of PVM dual-fluorescence RT-PCR method
Note： The amplification curves from left to right correspond to PVM plasmid template concentrations of 10? copies/μL， 10? copies/μL， 103 copies/μL， 102 copies/μL， 10 copies/μL， 1 copy/μL， and the negative control （i.e.， 0 copies/μL）（replicates were set）.

表2 双重荧光RT-PCR方法检测PVM的重复性测试

Table 2 Repeatability test of PVM dual-fluorescence RT-PCR method

质粒浓度（拷贝/μL） Plasmid concentration（copies/μL）	M蛋白基因（FAM通道） M protein gene (FAM channel)		F蛋白基因（VIC通道） F protein gene (VIC channel)
质粒浓度（拷贝/μL） Plasmid concentration（copies/μL）	Ct值 Ct value	变异系数 Coefficient of Variation	Ct值 Ct value	变异系数 Coefficient of Variation
批内（n=5）
10⁵	21.75±0.14	0.66%	21.14±0.10	0.46%
10⁴	25.03±0.20	0.79%	24.26±0.18	0.76%
10³	28.65±0.27	0.93%	27.45±0.22	0.82%
10²	31.93±0.32	1.02%	30.92±0.32	1.03%
批间（n=12）
10⁵	21.89±0.19	0.89%	21.54±0.27	1.24%
10⁴	25.28±0.28	1.12%	24.50±0.34	1.40%
10³	28.82±0.28	0.98%	27.58±0.32	1.17%
10²	32.78±0.43	1.31%	31.87±0.46	1.44%

注：PVM阳性野鼠的血液、口鼻液、粪便、肝、脾、肺、肾等样本两个荧光通道均呈阳性扩增。
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图3 双重荧光RT-PCR方法检测PVM阳性鼠的组织和体液样本
注：PVM阳性野鼠的血液、口鼻液、粪便、肝、脾、肺、肾等样本两个荧光通道均呈阳性扩增。

Figure 3 Detection of tissue and body fluid samples from PVM-positive mice using the dual fluorescence RT-PCR method
Note： Blood， oral and nasal secretions， feces， liver， spleen， lungs， and kidney samples from PVM-positive wild mice showed positive amplification in both fluorescence channels.

注：1~2-1：2份PVM阳性盲样的FAM荧光通道扩增曲线；1~2-2：2份自制PVM阳性盲样的VIC荧光通道扩增曲线；3-1：#2023634号野鼠PVM阳性的FAM荧光通道扩增曲线；3-2：#2023634 号野鼠PVM阳性的VIC荧光通道扩增曲线；4~56：47份PVM检测呈阴性的野鼠样本及6份阴性盲样对照样本无扩增曲线。
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图4 双重荧光RT-PCR方法检测口岸野鼠中的PVM
注：1~2-1：2份PVM阳性盲样的FAM荧光通道扩增曲线；1~2-2：2份自制PVM阳性盲样的VIC荧光通道扩增曲线；3-1：#2023634号野鼠PVM阳性的FAM荧光通道扩增曲线；3-2：#2023634 号野鼠PVM阳性的VIC荧光通道扩增曲线；4~56：47份PVM检测呈阴性的野鼠样本及6份阴性盲样对照样本无扩增曲线。

Figure 4 Detection of PVM among port field murine using duplex fluorescent RT-PCR
Note： 1~2-1： Amplification curves in the FAM fluorescence channel for 2 PVM-positive blinded samples； 1~2-2： Amplification curves in the VIC fluorescence channel for 2 PVM-positive blinded samples； 3-1： Amplification curve of FAM fluorescent channel for PVM-positive field mouse #2023634； 3-2： Amplification curve of VIC fluorescent channel for PVM-positive field mouse #2023634； 4~56： No amplification curves were observed in 47 PVM-negative field mouse samples and 6 negative blind control samples.

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