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

doi:10.12300/j.issn.1674-5817.2025.130

Abstract

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

CLC Number:

Q95-33

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.

Figures/Tables 5

References 30

[1]	BARIC R S, SIMS A C. Development of mouse hepatitis virus and SARS-CoV infectious cDNA constructs[M]//Coronavirus Replication and Reverse Genetics. Berlin, Heidelberg: Springer, 2005: 229-252. DOI: 10.1007/3-540-26765-4_8 .
[2]	艾东旭, 孙菲, 李钰, 等. 嗜肠型小鼠肝炎病毒RT-PCR诊断方法的建立及其应用[J]. 实验动物与比较医学, 2018, 38(1): 36-39. DOI: 10.3969/j.issn.1674-5817.2018.01.006 .
	AI D X, SUN F, LI Y, et al. Establishment and application of RT-PCR diagnostic method for enterotropic mouse hepatitis virus[J]. Lab Anim Comp Med, 2018, 38(1): 36-39. DOI: 10.3969/j.issn.1674-5817.2018.01.006 .
[3]	王吉, 卫礼, 付瑞, 等. 长爪沙鼠小鼠肝炎病毒(MHV)RT-PCR检测方法的建立及初步应用[J]. 中国比较医学杂志, 2013, 23(2): 58-63. DOI: 10.3969/j.issn.1671-7856.2013.002.015 .
	WANG J, WEI L, FU R, et al. Establishment and preliminary application of a RT-PCR detection technique for mouse hepatitis virus in mice and Mongolian gerbils[J]. Chin J Comp Med, 2013, 23(2): 58-63. DOI: 10.3969/j.issn.1671-7856.2013.002.015 .
[4]	ASANO A, TORIGOE D, SASAKI N, et al. Identification of antigenic peptides derived from B-cell epitopes of nucleocapsid protein of mouse hepatitis virus for serological diagnosis[J]. J Virol Meth, 2011, 177(1): 107-111. DOI: 10.1016/j.jviromet.2011.07.006 .
[5]	赖国旗, 何明忠, 谭毅, 等. 小鼠肝炎病毒RT-PCR检测方法的研究[J]. 西南农业大学学报(自然科学版), 2004, 26(6): 762-763, 772. DOI: 10.3969/j.issn.1673-9868.2004.06.027 .
	LAI G Q, HE M Z, TAN Y, et al. Study of rt-pcr methods for MHV (mouse hepatitis virus) determination[J]. J Southwest Agric Univ (Nat Sci Ed), 2004, 26(6): 762-763, 772. DOI: 10.3969/j.issn.1673-9868.2004.06.027 .
[6]	国家市场监督管理总局, 国家标准化管理委员会. 实验动物微生物、寄生虫学等级及监测: [S/OL]. (2022-12-29)[2025-08-06]. .
	State Administration for Market Regulation, National Standardization Administration. Laboratory animal—Microbiological and parasitological standards and monitoring: [S/OL]. (2022-12-29)[2025-08-06]. .
[7]	JOH J, PROCTOR M L, DITSLEAR J L, et al. Epidemiological and phylogenetic analysis of institutional mouse parvoviruses[J]. Exp Mol Pathol, 2013, 95(1):32-37. DOI: 10.1016/j.yexmp.2013.03.009 .
[8]	BESSELSEN D G, ROMERO M J, WAGNER A M, et al. Identification of novel murine parvovirus strains by epidemiological analysis of naturally infected mice[J]. J Gen Virol, 2006, 87(6):1543-1556. DOI: 10.1099/vir.0.81547-0 .
[9]	JANUS L M, BLEICH A. Coping with parvovirus infections in mice: health surveillance and control[J]. Lab Anim, 2012, 46(1):14-23. DOI: 10.1258/la.2011.011025 .
[10]	吴雪伶, 樊金萍, 冯建平, 等. 重组细胞中鼠细小病毒检测方法的建立及初步应用[J]. 中国生物制品学杂志, 2012, 25(8):1030-1034, 1042. DOI:10.13200/j.cjb.2012.08.103.wuxl.006 .
	WU X L, FAN J P, FENG J P, et al. Development and preliminary application of a method for detection of murine minute virus in recombinant cells[J]. Chin J Biol, 2012, 25(8):1030-1034, 1042. DOI:10.13200/j.cjb.2012.08.103.wuxl.006 .
[11]	潘金春, 罗银珠, 吴瑞可, 等. 小鼠感染细小病毒的临床特征分析[J]. 中国实验动物学报, 2017, 25(1): 64-69.DOI:10.3969/j.issn.1005-4847.2017.01.012 .
	PAN J C, LUO Y Z, WU R K, et al. Analysis of the clinical features of mice with minute virus infection[J]. Acta Lab Anim Sci Sin, 2017, 25(1): 64-69. DOI:10.3969/j.issn.1005-4847.2017.01.012 .
[12]	方勤, 朱作言. 呼肠孤病毒结构与功能研究进展[J]. 病毒学报, 2003, 19(4):381-384. DOI:10.13242/j.cnki.bingduxuebao.001515 .
	FANG Q, ZHU Z Y. Advances in research of reovirus structure and function[J]. Chin J Virol, 2003, 19(4):381-384. DOI:10.13242/j.cnki.bingduxuebao.001515 .
[13]	OUATTARA L A, BARIN F, BARTHEZ M A, et al. Novel human reovirus isolated from children with acute necrotizing encephalopathy[J]. Emerg Infect Dis, 2011, 17(8): 1436-1444. DOI: 10.3201/eid1708.101528 .
[14]	TYLER K L, BARTON E S, IBACH M L, et al. Isolation and molecular characterization of a novel type 3 reovirus from a child with meningitis[J]. J Infect Dis, 2004, 189(9): 1664-1675.
[15]	BROWNLEE R D, ARDESHIR A, BECKER M D, et al. A field strain of minute virus of mice (MVMm) exhibits age- and strain-specific pathogenesis[J]. J Gen Virol, 2018, 99(4): 558-566. DOI: 10.1099/jgv.0.001044 .
[16]	LIU J L, YAO L, ZHAI F F, et al. Development and application of a triplex real-time PCR assay for the simultaneous detection of avian influenza virus subtype H5, H7 and H9[J]. J Virol Meth, 2018, 252: 49-56. DOI: 10.1016/j.jviromet. 2017.11.005 .
[17]	CHAMBERLAIN J S, GIBBS R A, RAINER J E, et al. Deletion screening of the Duchenne muscular dystrophy locus via multiplex DNA amplification[J]. Nucl Acids Res, 1988, 16(23):11141-11156. DOI: 10.1093/nar/16.23.11141 .
[18]	赵迎峰, 马畅, 刘彪, 等. 三种小鼠肠道病毒多重PCR检测方法的建立和初步应用[J]. 实验动物科学, 2024, 41(5): 36-41. DOI: 10.3969/j.issn.1006-6179.2024.05.007 .
	ZHAO Y F, MA C, LIU B, et al. Establishment and preliminary application of a multiplex PCR method for detecting three types of enterovirus in mice[J]. Lab Anim Sci, 2024, 41(5): 36-41. DOI: 10.3969/j.issn.1006-6179.2024.05.007 .
[19]	庞然. 鼠痘病毒和小鼠肝炎病毒双重qPCR检测方法的建立[D]. 上海: 东华大学, 2023. DOI:10.27012/d.cnki.gdhuu.2023.001874 .
	PANG R. Establishment of a dual qPCR detection method for murine poxvirus and mouse hepatitis virus[D]. Shanghai: Donghua University, 2023. DOI:10.27012/d.cnki.gdhuu.2023.001874 .
[20]	王淑菁, 林欢, 付瑞, 等. 同时检测小鼠微小病毒(MVM)和小鼠细小病毒(MPV)的荧光定量PCR的建立及应用[J]. 实验动物科学, 2018, 35(4):29-32. DOI:10.3969/j.issn.1006-6179.2018.04.004 .
	WANG S J, LIN H, FU R, et al. Establishment and application of the real-time PCR for detecting minute virus of mouse (MVM) and mouse parvovirus (MPV) simultaneously in laboratory mice[J]. Lab Anim Sci, 2018, 35(4):29-32. DOI:10.3969/j.issn.1006-6179.2018.04.004 .
[21]	禹思宇, 周智君, 谭建锡, 等. 鼠呼肠孤病毒3型荧光定量RT-PCR检测方法的建立[J]. 湖南畜牧兽医, 2020(2):37-40. DOI:CNKI:SUN:HNCM.0.2020-02-019 .
	YU S Y, ZHOU Z J, TAN J X, et al. Establishment of fluorescent quantitative RT-PCR method for detection of mouse reovirus type 3[J]. Hunan J Anim Sci Vet Med, 2020(2):37-40. DOI:CNKI:SUN:HNCM.0.2020-02-019 .
[22]	JACOBY R O, LINDSEY J R. Risks of infection among laboratory rats and mice at major biomedical research institutions[J]. ILAR J, 1998, 39(4):266-271. DOI: 10.1093/ilar.39.4.266 .
[23]	MCINNES E F, RASMUSSEN L, FUNG P, et al. Prevalence of viral, bacterial and parasitological diseases in rats and mice used in research environments in Australasia over a 5-y period[J]. Lab Anim, 2011, 40(11):341-350. DOI: 10.1038/laban1111-341 .
[24]	PRITCHETT-CORNING K R, COSENTINO J, CLIFFORD C B. Contemporary prevalence of infectious agents in laboratory mice and rats[J]. Lab Anim, 2009, 43(2):165-173. DOI: 10.1258/la.2008.008009 .
[25]	WON Y S, JEONG E S, PARK H J, et al. Microbiological contamination of laboratory mice and rats in Korea from 1999 to 2003[J]. Exp Anim, 2006, 55(1):11-16. DOI: 10.1538/expanim.55.11 .
[26]	SMITH P C, NUCIFORA M, REUTER J D,et al. Reliability of soiled bedding transfer for detection of mouse parvovirus and mouse hepatitis virus[J]. Comp Med, 2007, 57(1):80-86.
[27]	李欣悦, 佟巍, 丛日旭, 等. 三种小鼠肠道病毒核酸检测技术在小鼠健康监测中的应用[J]. 中国比较医学杂志, 2018, 28(8): 38-42. DOI: 10.3969/j.issn.1671-7856.2018.08.008 .
	LI X Y, TONG W, CONG R X, et al. Application of nucleic acid detection techniques for three mouse enteroviruses in mouse health monitoring[J]. Chin J Comp Med, 2018, 28(8):38-42. DOI: 10.3969/j.issn.1671-7856.2018.08.008 .
[28]	YIN Y W, XIONG C Y, SHI K C, et al. Multiplex digital PCR: a superior technique to qPCR for the simultaneous detection of duck Tembusu virus, duck circovirus, and new duck reovirus[J]. Front Vet Sci, 2023, 10:1222789. DOI: 10.3389/fvets.2023.1222789 .
[29]	GOTO Y, FUKUNARI K, TADA S, et al. A multiplex real-time RT-PCR system to simultaneously diagnose 16 pathogens associated with swine respiratory disease[J]. J Appl Microbiol, 2023, 134(11):lxad263. DOI: 10.1093/jambio/lxad263 .
[30]	鲍显伟, 李昊, 李小龙, 等. BVDV、IBRV和AKAV多重PCR检测方法的建立与应用[J]. 中国兽医学报, 2024, 44(1):39-43, 87. DOI:10.16303/j.cnki.1005-4545.2024.01.06 .
	BAO X W, LI H, LI X L, et al. Establishment and application of multiplex PCR assays for BVDV, IBRV and AKAV[J]. Chin J Vet Sci, 2024, 44(1):39-43, 87. DOI:10.16303/j.cnki.1005-4545.2024.01.06 .

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