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.

Spatio-temporal and trade export risk analysis of bluetongue disease in France: A case study of China

“…According to the United Nations International Trade Statistics Database ( 28 ), from 2014 to 2018, China imported 539 kg of bovine semen from France. Semen from viremia animals is infectious, and recipient cows can become infected when they receive infectious semen ( 29 – 31 ). Therefore, importing semen from France carries certain risks.…”

Development and evaluation of a time-resolved fluorescence labelled immunochromatographic strip assay for rapid and quantitative detection of bovine herpesvirus 1

“…Various test methods have been developed for the diagnosis of BoHV-1, including loop-mediated isothermal amplification assay, duplex real-time PCR and droplet digital PCR assays ( Marin et al, 2016 ; Peltzer et al, 2021 ; Ali et al, 2022 ; Yu B. et al, 2022 ; Yu Z. et al, 2022 ). Both droplet digital PCR assays and loop-mediated isothermal amplification are very sensitive methods for the diagnosis of BoHV-1 infections; however, these methods can only be performed by personnel with technical expertise in well-equipped laboratories.…”

Development of a droplet digital PCR assay for the sensitive detection of iridovirus in Andrias davidianus

“…Droplet digital PCR (ddPCR) is a new generation absolute quantitive PCR technique that offers high sensitivity, precision and reproducibility in molecular detection (Hou et al, 2023). Compared to the above PCR methods, ddPCR is less susceptible to PCR inhibitors (Guti'errez‐Aguirre et al, 2015; Li et al, 2018) and enables the absolute quantitation of nucleic acids without requiring a standard curve (Yu et al, 2022). The ddPCR method has been proven to be successful in detecting viral and bacterial infections in aquatic animals such as common carp (Wang et al, 2021), salmonid (Ammon et al, 2022; Jia et al, 2017; Orioles et al, 2022), tilapia (Zhao et al, 2023), largemouth bass (Jiang et al, 2023), shrimp (Zhang et al, 2022) and oysters (Tan et al, 2018).…”

“…Droplet digital PCR (ddPCR) is a new generation absolute quantitive PCR technique that offers high sensitivity, precision and reproducibility in molecular detection (Hou et al, 2023). Compared to the above PCR methods, ddPCR is less susceptible to PCR inhibitors (Guti'errez-Aguirre et al, 2015;Li et al, 2018) and enables the absolute quantitation of nucleic acids without requiring a standard curve (Yu et al, 2022).…”