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

Chen, Rujing; Chen, Qiu-Yong; Wu, Xuemin; YongLiang, Che; Wang, Chenyan; LongBai, Wang; Shu, Yan; Lun-jiang, Zhou

doi:10.1155/2020/5673145

Cited by 2 publications

(4 citation statements)

References 34 publications

(37 reference statements)

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“…During the development of the ddPCR method, we chose the gB gene to design the primers and probe. The gB gene is highly conserved among members of the Herpesviridae family and is frequently used as the target gene to detect BoHV-1 [ 32 , 33 ]. The primers and probe used for ddPCR were the same as those used for qPCR, and we also developed a qPCR assay to validate the design.…”

Section: Discussionmentioning

confidence: 99%

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

Zhao

Chen

et al. 2022

BMC Vet Res

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Background Infectious bovine rhinotracheitis (IBR) caused by bovine alphaherpesvirus 1 (BoHV-1) is one of the most important contagious diseases in bovine. This is one of the most common infectious disease of cattle. This has led to high economic losses in the cattle farming industry. BoHV-1 can potentially be transmitted via semen during natural or artificial insemination (AI). Therefore, testing methods for the early diagnosis of BoHV-1 infection are urgently needed for international trade of ruminant semen. In this study, we developed a novel droplet digital PCR (ddPCR) assay for the detection of BoHV-1 DNA in semen samples. Results The ddPCR results showed that the detection limit was 4.45 copies per reaction with high reproducibility. The established method was highly specific for BoHV-1 and did not show cross-reactivity with specify the organisms (BTV, BVDV, Brucella, M . bovis). The results of clinical sample testing showed that the positivity rate of ddPCR (87.8%) was higher than that of qPCR (84.1%). Conclusions The ddPCR assay showed good accuracy for mixed samples and could be a new added diagnostic tool for detecting BoHV-1.

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Section: Discussionmentioning

confidence: 99%

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

Zhao

Chen

et al. 2022

BMC Vet Res

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“…In order to alleviate the shortage of human donor organs available for allograft, xenotransplantation using pig cells, tissues, or organs have been proposed (1)(2)(3). This may be related to the transmission of porcine mediated disease, so the maintenance of designated pathogen free pigs is required for xenotransplantation (4)(5)(6).…”

Section: Introductionmentioning

confidence: 99%

“…Porcine cytomegalovirus (PCMV), also known as Suid herpesvirus 2 (SuHV2), is an enveloped virus with a double-stranded linear DNA genome. PCMV can cause fever, reduced general condition, loss of appetite, numbness, neurological signs, and respiratory symptoms (e.g., sneezing, coughing, and dyspnea), acute to subacute disease, and high mortality and morbidity in piglets (3,7). In a previous studies, the virus have been reported to be immunosuppressive pathogen primarily affecting the immune function of the macrophages and T lymphocytes, which can cause preclinical infection in adult pigs and reproductive failure in pregnant sows (7,8).…”

Section: Introductionmentioning

confidence: 99%

“…In addition, companies such as Novateinbio and MyBioSource have ELISA products that detect antibody or antigen of PCMV, there are no commercialized products using molecular diagnostic methods until now. Real-time fluorescent quantitative PCR technology has become a powerful alternative platform for detection and differentiation of pathogenic viruses (3,17,18).…”

Section: Introductionmentioning

confidence: 99%

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One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples

Wang

Song²,

Shin³

et al. 2020

Front. Vet. Sci.

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Porcine cytomegalovirus (PCMV) is a pathogen that must be removed from pigs for use as organ donors in xenotransplantation. Recently, it has been found that when donor pigs are infected with PCMV, a pig-to-non-human-primate xenotransplantation lower transplant survival by 2-3 times. Therefore, highly sensitive methods are needed to maintain designated pathogen free (DPF) pig and screen for xenografts. The purpose of this study was to evaluate the performance of commercially available method with one-tube nested real-time PCR assay to quickly detect PCMV infection in clinical samples and compare the results with those of sequence analysis. Molecular diagnostic methods were used to evaluate 127 samples, including tissues and blood samples from pigs suspected of PCMV infection. The detection rate for positive PCMV was 38.6% (n = 49), 23.6% (n = 30), and 12.6% (n = 16) in one-tube nested real-time PCR, nested PCR, and conventional PCR methods, respectively. All PCMV-positive samples in conventional PCR or nested PCR methods were also positive in the one-tube nested real-time PCR assay. All the PCR products in the three methods were checked for amplification of PCMV gene by PCR and subsequent direct sequencing. The results of one-tube nested real-time PCR were found to be consistent with those of sequence analysis for all the samples and showed good agreement (κ = 1). Our study found that the one-tube nested real-time PCR assay is more sensitive than the other two methods. This assay required approximately 1.5 h for completion. Therefore, we concluded that one-tube nested real-time PCR assay is a fast and reliable method for the characterizing pathogen responsible for PCMV infection.

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

Cited by 2 publications

References 34 publications

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

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

One-Tube Nested Real-Time PCR Assay for Rapid Screening of Porcine Cytomegalovirus in Clinical Samples

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