MHV、MPV和Reo-3三重TaqMan定量PCR检测方法的建立

doi:10.12300/j.issn.1674-5817.2025.130

实验动物与比较医学 ›› 2026, Vol. 46 ›› Issue (3): 408-415.DOI: 10.12300/j.issn.1674-5817.2025.130

MHV、MPV和Reo-3三重TaqMan定量PCR检测方法的建立

周忆旻¹(), 张昕宇¹, 杨佳诺¹, 刘梦佳¹, 司灿灿¹, 叶海绿², 孙文超¹()(), 兰添¹()()

^1.温州大学实验动物中心, 温州 325035
^2.温州肯恩大学理工学院实验中心, 温州 325060

收稿日期:2025-08-06 修回日期:2026-03-09 出版日期:2026-06-25 发布日期:2026-06-19
通讯作者:
孙文超（1987—），男，博士，副研究员，研究方向：实验动物传染病学的研究。E-mail：sunwenchao131@163.com。ORCID：0000-0002-8845-6242

兰添（1990—），女，硕士，实验师。研究方向：实验动物传染病学的研究。E-mail：827002151@qq.com。ORCID：0009-0006-4063-7817
作者简介:周忆旻（2001—），女，硕士研究生，研究方向：动物传染病学的研究。E-mail：1463203761@qq.com。ORCID：0009-0000-4259-3906
基金资助:
温州市基础性医疗卫生科技项目“基于数字PCR技术的SPF级实验动物病毒病原体快速检测关键技术研究与应用”(Y20220136);浙江省高校实验室工作研究项目“中外合作办学背景下高校SPF级动物实验室建设与管理机制初探—以温州肯恩大学为例”(YB202431)

Development of a Triplex TaqMan Quantitative PCR Method for Simultaneous Detection of MHV, MPV, and Reo-3

ZHOU Yimin¹(), ZHANG Xinyu¹, YANG Jianuo¹, LIU Mengjia¹, SI Cancan¹, YE Hailü², SUN Wenchao¹()(), LAN Tian¹()()

^1.Laboratory Animal Center, Wenzhou University, Wenzhou 325035
^2.Laboratory Center, College of Science, Mathematics and Technology, Wenzhou-Kean University, Wenzhou 325060

Received:2025-08-06 Revised:2026-03-09 Published:2026-06-25 Online:2026-06-19
Contact: SUN Wenchao (ORCID: 0000-0002-8845-6242), E-mail: sunwenchao131@163.com
LAN Tian (ORCID: 0009-0006-4063-7817), E-mail: 827002151@qq.com

摘要/Abstract

摘要：

目的针对实验小鼠中常见的小鼠肝炎病毒（mouse hepatitis virus，MHV）、小鼠细小病毒（mouse parvovirus，MPV）及呼肠孤病毒Ⅲ型（reovirus type 3，Reo-3），建立一种高特异性、高灵敏性的三重TaqMan定量PCR检测方法。方法选取3种病毒的保守基因序列构建重组质粒标准品，分别设计特异性引物和探针，通过预实验优化三重TaqMan定量PCR检测体系。通过10倍梯度稀释（10¹～10⁷拷贝/μL）对检测体系的灵敏度进行评价；并通过实验对批内和批间的重复性进行评价。结果在标准品浓度为10²～10⁷拷贝/μL范围内，拷贝数与Ct值之间呈良好线性相关关系（R²＞0.99），批内和批间的变异系数（coefficient of variation，CV）均小于5%，与小鼠巨细胞病毒（mouse cytomegalovirus，MCMV）、水疱性口炎病毒（vesicular stomatitis virus，VSV）、仙台病毒（Sendai virus，SeV）、小鼠肺炎病毒（pneumonia virus of mice，PVM）等均无交叉反应。结论本研究建立的三重TaqMan定量PCR检测方法具有较高的灵敏度、良好的重复性和较强的特异性，能够快速、特异、灵敏地检测MHV、MPV和Reo-3，实现单管多重病原体的同步检测，可应用于实验动物临床样本的检测以及流行病学的研究调查，为病毒感染防控及阻断传播提供有效的科学技术支撑。

关键词: 小鼠肝炎病毒, 小鼠细小病毒, 呼肠孤病毒Ⅲ型, 三重TaqMan定量PCR

Abstract:

Objective To establish a highly specific and sensitive triplex TaqMan quantitative PCR method for the detection of mouse hepatitis virus (MHV), mouse parvovirus (MPV), and reovirus type 3 (Reo-3) in laboratory mice. Methods Recombinant plasmid standards were constructed using conserved genomic regions of the three target viruses. Specific primers and probes were designed, and a triplex TaqMan quantitative PCR (qPCR) system was optimized through preliminary experiments. Sensitivity was evaluated using 10-fold serial dilutions (10¹-10⁷ copies/μL). Intra- and inter-assay repeatability were assessed via reproducibility experiments. Results A strong linear correlation (R²>0.99) was observed between copy number and Ct value at template concentrations of 10²-10⁷ copies/μL. Intra-assay and inter-assay coefficients of variation (CV) were both below 5%. No cross-reactivity was observed with mouse cytomegalovirus (MCMV), vesicular stomatitis virus (VSV), Sendai virus (SeV), or pneumonia virus of mice (PVM). Conclusion The triplex TaqMan quantitative PCR method established in this study has high sensitivity, good repeatability, and strong specificity. It enables the rapid, specific, and sensitive detection of MHV, MPV, and Reo-3, allows the simultaneous detection of multiple pathogens in a single tube, and can be applied to the detection of clinical samples from laboratory animals and to epidemiological investigations, thereby providing effective scientific and technological support for the prevention and control of viral infection and the interruption of transmission.

Key words: Mouse hepatitis virus, Mouse parvovirus, Reovirus type 3, Triplex TaqMan quantitative PCR

中图分类号:

Q95-33

周忆旻,张昕宇,杨佳诺,等. MHV、MPV和Reo-3三重TaqMan定量PCR检测方法的建立[J]. 实验动物与比较医学, 2026, 46(3): 408-415. DOI: 10.12300/j.issn.1674-5817.2025.130.

ZHOU Yimin,ZHANG Xinyu,YANG Jianuo,et al. Development of a Triplex TaqMan Quantitative PCR Method for Simultaneous Detection of MHV, MPV, and Reo-3[J]. Laboratory Animal and Comparative Medicine, 2026, 46(3): 408-415. DOI: 10.12300/j.issn.1674-5817.2025.130.

图/表 5

参考文献 30

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引物/探针名称 Primer/probe name	引物/探针序列（5'→3'） Primer/probes sequence (5'→3')	扩增片段长度/bp Amplification fragment length/bp
MHV-F	CTTGGAACTAGTGATCCA	145
MHV-R	ACTGCAATTCATACACATC
MHV-P	CY5-TCATCAGCACCACCAGA GTTCTT-BHQ3
MPV-F	CTCTGGACTCTAACAACATA	104
MPV-R	GTACCTGTAAGGACTTGG
MPV-P	HEX-CACACCAGCAACAGACA ACCAA-BHQ1
Reo-3-F	CCAACGAATATGACACAG	125
Reo-3-R	CTACCTATCAAGTCTGCA
Reo-3-P	FAM-ACTACTTCACTCACTAC CGCACC-BHQ1

引物/探针名称 Primer/probe name	引物/探针序列（5'→3'） Primer/probes sequence (5'→3')	扩增片段长度/bp Amplification fragment length/bp
MHV-F	CTTGGAACTAGTGATCCA	145
MHV-R	ACTGCAATTCATACACATC
MHV-P	CY5-TCATCAGCACCACCAGA GTTCTT-BHQ3
MPV-F	CTCTGGACTCTAACAACATA	104
MPV-R	GTACCTGTAAGGACTTGG
MPV-P	HEX-CACACCAGCAACAGACA ACCAA-BHQ1
Reo-3-F	CCAACGAATATGACACAG	125
Reo-3-R	CTACCTATCAAGTCTGCA
Reo-3-P	FAM-ACTACTTCACTCACTAC CGCACC-BHQ1

病毒种类 Type of virus	拷贝数/（拷贝·μL^-1） Copy number/(copies·μL^-1）	组内重复实验 Intra-group repeatability experiments		组间重复实验 Inter-group repeatability experiments
病毒种类 Type of virus	拷贝数/（拷贝·μL^-1） Copy number/(copies·μL^-1）	CV/%	Ct （x̄±s）	CV/%	Ct （x̄±s）
小鼠肝炎病毒 Mouse hepatitis virus	0.87×10⁶	2.22	20.71±0.46	1.82	19.75±0.36
	0.87×10⁵	5.52	25.02±1.38	2.83	23.36±0.66
	0.87×10⁴	0.00	27.95±0.10	1.53	27.37±0.42
小鼠细小病毒 Mouse parvovirus	1.27×10⁶	1.69	20.76±0.35	1.28	19.47±0.25
	1.27×10⁵	0.93	24.74±0.23	1.78	23.64±0.42
	1.27×10⁴	2.95	29.17±0.86	1.01	28.82±0.29
呼肠孤病毒Ⅲ型 Reovirus type 3	1.21×10⁶	2.75	18.90±0.52	4.58	19.36±0.88
	1.21×10⁵	0.74	23.09±0.17	6.48	22.30±1.39
	1.21×10⁴	0.45	26.53±0.12	1.30	26.87±0.35

病毒种类 Type of virus	拷贝数/（拷贝·μL^-1） Copy number/(copies·μL^-1）	组内重复实验 Intra-group repeatability experiments		组间重复实验 Inter-group repeatability experiments
病毒种类 Type of virus	拷贝数/（拷贝·μL^-1） Copy number/(copies·μL^-1）	CV/%	Ct （x̄±s）	CV/%	Ct （x̄±s）
小鼠肝炎病毒 Mouse hepatitis virus	0.87×10⁶	2.22	20.71±0.46	1.82	19.75±0.36
	0.87×10⁵	5.52	25.02±1.38	2.83	23.36±0.66
	0.87×10⁴	0.00	27.95±0.10	1.53	27.37±0.42
小鼠细小病毒 Mouse parvovirus	1.27×10⁶	1.69	20.76±0.35	1.28	19.47±0.25
	1.27×10⁵	0.93	24.74±0.23	1.78	23.64±0.42
	1.27×10⁴	2.95	29.17±0.86	1.01	28.82±0.29
呼肠孤病毒Ⅲ型 Reovirus type 3	1.21×10⁶	2.75	18.90±0.52	4.58	19.36±0.88
	1.21×10⁵	0.74	23.09±0.17	6.48	22.30±1.39
	1.21×10⁴	0.45	26.53±0.12	1.30	26.87±0.35

MHV、MPV和Reo-3三重TaqMan定量PCR检测方法的建立

Development of a Triplex TaqMan Quantitative PCR Method for Simultaneous Detection of MHV, MPV, and Reo-3

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