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

Lunjiang Zhou,Shan Yan,Xuemin Wu,Rujing Chen,Yongliang Che,Chenyan Wang,Longbai Wang,Qiuyong Chen

doi:10.1155/2020/5673145

Lunjiang Zhou, Shan Yan + Show 6 more

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https://doi.org/10.1155/2020/5673145

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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 y = −3.249x + 38.958 corresponding to the amplification efficiency at 99.8%. The limit of the qPCR method was 51.9 copies/μl. 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.

Highlights

Xenotransplantation was proposed to alleviate the current shortage in human donor organs available for allotransplantation [1, 2]
Xenotransplantation may be associated with the transmission of porcine zoonotic microorganisms, including porcine endogenous retroviruses (PERVs), porcine cytomegalovirus (PCMV), porcine hepatitis E virus (HEV), and other porcine lymphotropic herpesviruses (PLHV, containing PLHV-1, PLHV-2, and PLHV-3) [3,4,5]
DPOL Gene Analysis. e virus was identified by us previously [21], and the virus was isolated using the same strategy described by Gu et al [6]. e complete DNA polymerase (DPOL) gene of PCMV was amplified by using the primers with overlapped three fragments (DPOL-1, DPOL-2, and DPOL-3, Table 1), which encompassed the complete DPOL gene of PCMV. e overlapped DPOL gene fragments were harvested with Gel Extraction Kit D2500 (Omega Bio-Tek, Guangzhou, China), TM T-A cloned using pMD 18-T Vector Cloning Kit (Takara Biomedical Technology, Beijing, China). e positive recombinant plasmids were sequenced using the Sanger method by a commercial company (Sangon Biotech, Shanghai, China) in both directions

Summary

Introduction

Xenotransplantation was proposed to alleviate the current shortage in human donor organs available for allotransplantation [1, 2]. Several diagnostic methods, including virus isolation, polymerase chain reaction (PCR) methods [8,9,10], loopmediated isothermal amplification assay (LAMP) [11], enzyme-linked immunosorbent assay (ELISA) [12,13,14], and Western-blot analysis [15] had been reported for detection of PCMV infection. These assays are either laborintensive, less sensitive, require agarose gel analysis for the amplification products or had a risk of contamination, which may lead to false results. A TaqMan based real-time fluorescent qPCR method, targeting the highly conserved

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Conclusion