Development of a TaqMan Based Real-Time Fluorescent Quantitative PCR Assay for Detection of Porcine Cytomegalovirus in Semen

Overview

Journal Biomed Res Int

Publisher Wiley

Specialties Biomedical Engineering
Biotechnology
General Medicine

Date 2020 May 19

PMID 32420350

Citations 2

Authors

Rujing Chen

Qiuyong Chen

Xuemin Wu

Yongliang Che

Chenyan Wang

Longbai Wang

Shan Yan

Lunjiang Zhou

Affiliations

Soon will be listed here.

Abstract

This study described a TaqMan based real-time fluorescent quantitative PCR (qPCR) method to detect porcine cytomegalovirus (PCMV) infection, targeting the conserved region of the DNA polymerase (DPOL) gene. The standard curve showed a linear regression relationship with a coefficient of 0.999 and a slope of = -3.249 + 38.958 corresponding to the amplification efficiency at 99.8%. The limit of the qPCR method was 51.9 copies/l. The established qPCR method showed excellent specificity, with no cross-reaction observed with common porcine pathogens. The coefficient of variation for intra-assay and interassay variability ranged up to 1.51% and 2.24%, respectively. PCMV positive signals can be found in semen using this qPCR method, which suggested that we should pay more attention to PCMV contamination in semen in order to eliminate PCMV infection in artificial insemination and xenotransplantation.

Citing Articles

Development of a droplet digital PCR assay to detect bovine alphaherpesvirus 1 in bovine semen.

Yu Z, Zhao Z, Chen L, Yan H, Cui Q, Ju X BMC Vet Res. 2022; 18(1):125.

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References

Tang J, Frascaroli G, Lebbink R, Ostermann E, Brune W . Human cytomegalovirus glycoprotein B variants affect viral entry, cell fusion, and genome stability. Proc Natl Acad Sci U S A. 2019; 116(36):18021-18030. PMC: 6731671. DOI: 10.1073/pnas.1907447116. View

Sun S, Ahmad K, Wu X, Chen J, Fu H, Huang M . Development of Quantitative Real-Time PCR Assays for Rapid and Sensitive Detection of Two Species in Sugarcane. Biomed Res Int. 2018; 2018:8678242. PMC: 6106854. DOI: 10.1155/2018/8678242. View

Maes D, Van Soom A, Appeltant R, Arsenakis I, Nauwynck H . Porcine semen as a vector for transmission of viral pathogens. Theriogenology. 2015; 85(1):27-38. DOI: 10.1016/j.theriogenology.2015.09.046. View

Gu W, Zeng N, Zhou L, Ge X, Guo X, Yang H . Genomic organization and molecular characterization of porcine cytomegalovirus. Virology. 2014; 460-461:165-72. DOI: 10.1016/j.virol.2014.05.014. View

Garcia-Vazquez F, Mellagi A, Ulguim R, Hernandez-Caravaca I, Llamas-Lopez P, Bortolozzo F . Post-cervical artificial insemination in porcine: The technique that came to stay. Theriogenology. 2019; 129:37-45. DOI: 10.1016/j.theriogenology.2019.02.004. View

Burland T . DNASTAR's Lasergene sequence analysis software. Methods Mol Biol. 1999; 132:71-91. DOI: 10.1385/1-59259-192-2:71. View

Blomqvist G, Persson M, Wallgren M, Wallgren P, Morrell J . Removal of virus from boar semen spiked with porcine circovirus type 2. Anim Reprod Sci. 2011; 126(1-2):108-14. DOI: 10.1016/j.anireprosci.2011.04.014. View

Tamura K, Stecher G, Peterson D, Filipski A, Kumar S . MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol Biol Evol. 2013; 30(12):2725-9. PMC: 3840312. DOI: 10.1093/molbev/mst197. View

Morozov V, Morozov A, Denner J . New PCR diagnostic systems for the detection and quantification of porcine cytomegalovirus (PCMV). Arch Virol. 2016; 161(5):1159-68. DOI: 10.1007/s00705-016-2765-3. View

10.

Shirai J, Narita M, Iijima Y, Kawamura H . A cytomegalovirus isolated from swine testicle cell culture. Nihon Juigaku Zasshi. 1985; 47(5):697-703. DOI: 10.1292/jvms1939.47.697. View

11.

Denner J . The porcine virome and xenotransplantation. Virol J. 2017; 14(1):171. PMC: 5585927. DOI: 10.1186/s12985-017-0836-z. View

12.

Widen F, Goltz M, Wittenbrink N, Ehlers B, Banks M, Belak S . Identification and sequence analysis of the glycoprotein B gene of porcine cytomegalovirus. Virus Genes. 2002; 23(3):339-46. DOI: 10.1023/a:1012581508733. View

13.

Liu X, Zhu L, Shi X, Xu Z, Mei M, Xu W . Indirect-blocking ELISA for detecting antibodies against glycoprotein B (gB) of porcine cytomegalovirus (PCMV). J Virol Methods. 2012; 186(1-2):30-5. DOI: 10.1016/j.jviromet.2012.08.024. View

14.

Salam A, Horby P . The Breadth of Viruses in Human Semen. Emerg Infect Dis. 2017; 23(11):1922-1924. PMC: 5652425. DOI: 10.3201/eid2311.171049. View

15.

Shi W, Wang Y, Ren X, Gao S, Hua X, Guo M . EvaGreen-based real-time PCR assay for sensitive detection of salmonid alphavirus. Mol Cell Probes. 2018; 39:7-13. DOI: 10.1016/j.mcp.2018.03.002. View

16.

Liu X, Liao S, Zhu L, Xu Z, Zhou Y . Molecular epidemiology of porcine Cytomegalovirus (PCMV) in Sichuan Province, China: 2010-2012. PLoS One. 2013; 8(6):e64648. PMC: 3675093. DOI: 10.1371/journal.pone.0064648. View

17.

Su Y, Liu Y, Chen Y, Xing G, Hao H, Wei Q . A novel duplex TaqMan probe-based real-time RT-qPCR for detecting and differentiating classical and variant porcine epidemic diarrhea viruses. Mol Cell Probes. 2017; 37:6-11. DOI: 10.1016/j.mcp.2017.10.003. View

18.

Plotzki E, Keller M, Ivanusic D, Denner J . A new Western blot assay for the detection of porcine cytomegalovirus (PCMV). J Immunol Methods. 2016; 437:37-42. DOI: 10.1016/j.jim.2016.08.001. View

19.

Tajima T, HIRONAO T, Kajikawa T, Kawamura H . Application of enzyme-linked immunosorbent assay for the seroepizootiological survey of antibodies against porcine cytomegalovirus. J Vet Med Sci. 1993; 55(3):421-4. DOI: 10.1292/jvms.55.421. View

20.

Morozov V, Heinrichs G, Denner J . Effective Detection of Porcine Cytomegalovirus Using Non-Invasively Taken Samples from Piglets. Viruses. 2017; 9(1). PMC: 5294978. DOI: 10.3390/v9010009. View