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. 2022 Apr 2;18(1):125.
doi: 10.1186/s12917-022-03235-2.

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

Zhichao Yu  1   2 , Zhiguo Zhao  3 , Linjun Chen  2 , Han Yan  2 , Qiang Cui  2 , Xianghong Ju  1 , Yanhong Yong  1 , Xiaoxi Liu  1 , Xingbin Ma  1 , Guanhua Zhang  4
Affiliations

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

Zhichao Yu et al. BMC Vet Res. .

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.

Keywords: Bovine alphaherpesvirus 1; Bovine semen; Detection method; Droplet digital PCR; Real-time PCR.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Optimization of the annealing temperature for the ddPCR method
Fig. 2
Fig. 2
qPCR standard curve from a 10-fold dilution series of plasmid templates. The slope of the fitted line is − 2.85, R2 = 0.944
Fig. 3
Fig. 3
Sensitivity analysis of the amplification curve of the qPCR method. Limit of detection of qPCR from serial dilutions (4.45 × 109 to 4.45 × 102) of plasmid templates. 1-10: 4.45 × 109-4.45 × 100 copies/μL dilution standard. 11: Negative control
Fig. 4
Fig. 4
Sensitivity analysis of the ddPCR assay with different DNA dilutions. 1-6: 4.45 × 104-4.45 × 10− 1 copies/μL dilution standard. NC: negative control
Fig. 5
Fig. 5
Sensitivity analysis of the qPCR assay with different DNA dilutions. 1-10: 4.45 × 109-4.45 × 100 copies/μL dilution standard
Fig. 6
Fig. 6
Repeatability analysis of the DNA concentration in each diluted sample determined by the ddPCR assay. 1-5: 4.45 × 104-4.45 × 100 copies/μL dilution standard
Fig. 7
Fig. 7
Specificity of the ddPCR assay. Lanes 1-6: BoHV-1 plasmid, BTV, BVDV, Brucella and Mycobacterium bovis. Lane NC: negative control
Fig. 8
Fig. 8
Specificity of the ddPCR assay. 1-6: BoHV-1 plasmid, BTV, BVDV, Brucella and Mycobacterium bovis. NC: negative control
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