Dataset Information

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

ABSTRACT:

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.

SUBMITTER: Yu Z

PROVIDER: S-EPMC8976375 | biostudies-literature | 2022 Apr

REPOSITORIES: biostudies-literature

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Publications

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

Yu Zhichao Z Zhao Zhiguo Z Chen Linjun L Yan Han H Cui Qiang Q Ju Xianghong X Yong Yanhong Y Liu Xiaoxi X Ma Xingbin X Zhang Guanhua G

BMC veterinary research 20220402 1

<h4>Background</h4>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 t ...[more]

PMID: 35366879

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Project description:The objective of this study was to establish a more sensitive and specific diagnostic method for detecting plasma BK polyomavirus (BKPyV) DNA load in patients after renal transplantation using droplet digital polymerase chain reaction (ddPCR) and to validate the methodology. The linear range, lower limit of detection, accuracy, precision, and specificity of the detection system were evaluated by using the WHO BKPyV standard (7.2 log10 IU/mL) as a reference, in accordance with the relevant documents of the Clinical and Laboratory Standards Institute. Plasma samples were collected from 74 renal transplantation patients with urinary BKPyV-DNA levels exceeding 7 log10 copies/mL. Quantitative PCR (qPCR) and ddPCR were performed, and their diagnostic efficacy for BKPyV-DNA in the diagnosis of BK polyomavirus-associated nephropathy was evaluated using a receiver operating characteristic (ROC) curve. The coefficients of variation for the repeated detection of BKPyV standard DNA were 2.55 and 4.71 at concentrations of 6.2 and 3.2 log10 IU/mL, respectively. The linear range was 2.2-6.2 log10 IU/mL, and the lowest detection limit was 100 IU/mL. By utilizing histopathological examination of renal biopsy as the gold standard for BKPyV diagnosis, the area under the ROC curve of 74 post-transplantation plasma samples detected by the ddPCR system was found to be 0.875 (95% CI: 0.797-0.953, P < 0.01). The optimal threshold was 512.86 copies/mL, with a sensitivity of 90.0% and a specificity of 67.6%. In comparison, the area under the ROC curve for qPCR was 0.668 (95% CI: 0.583-0.752, P < 0.01), with an optimal threshold of 11,481.54 copies/mL, a sensitivity of 35.0%, and a specificity of 100.0%. Pairwise comparison (Delong test) of the ROC curves of the two systems showed a significant difference in the area under the curve, with a difference of 0.207 and a P-value <0.01. The BKPyV nucleic acid detection system, based on ddPCR, is appropriate for the regular monitoring of the BK polyomavirus, specifically in plasma samples containing low viral DNA loads while it provides the benefits of both absolute quantification and high sensitivity.IMPORTANCEIt was previously believed that droplet digital polymerase chain reaction had limitations, including high cost, limited throughput, and cumbersome operation, which hindered its widespread application in clinical practice. However, the current fully automated digital PCR platform, combined with streamlined operations, can detect 96 samples at once, and the entire process can be completed within an hour, laying a solid foundation for its extensive use.

Dataset Information

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

Background

Results

Conclusions

Publications

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

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