用于牛精液中牛病毒性腹泻病毒1型（BVDV-1）和蓝舌病病毒（BTV）灵敏检测的逆转录酶液滴数字PCR技术的开发与应用

Development and application of reverse transcriptase droplet digital PCR technology for sensitive detection of BVDV-1 and BTV in bovine semen.

作者信息

Yu Zhichao, Chen Linjun, Cui Qiang, Yan Han, Li Junyan, Luo Xiaoping, Li Yingying, Ju Xianghong, Yong Yanhong, Zhao Namula, Zhao Zhiguo

机构信息

Department of Veterinary Medicine, College of Coastal Agricultural Sciences, Guangdong Ocean University, Zhanjiang, 524088, Guangdong, China.

Technology Center, Hohhot Customs District, Hohhot, 010020, Inner Mongolia, China.

出版信息

BMC Vet Res. 2025 Jan 30;21(1):44. doi: 10.1186/s12917-025-04506-4.

DOI:10.1186/s12917-025-04506-4

PMID:39885446

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11783913/

Abstract

BACKGROUND

Bovine viral diarrhoea virus genotype 1 (BVDV-1) and bluetongue virus (BTV) are potent viral pathogens that may be transmitted through semen, resulting in the spread of diseases via artificial insemination. Thus, establishing an early detection method for BVDV-1 and BTV infection is important for the trading of semen. In this study, we developed two RT‒ddPCR methods to detect BVDV-1 and BTV, and each method was evaluated for repeatability, limit of detection and specificity. The sensitivity of these methods was compared with that of RT‒qPCR (WOAH) by analysing clinical samples.

RESULTS

The RT‒ddPCR results revealed that both methods exhibited good repeatability at low concentrations, with detection limits of 1.05 copies/µL and 0.662 copies/µL per reaction for BVDV-1 and BTV, respectively; additionally, both methods exhibited high specificity and did not exhibit cross-reaction with other important semen-transmitted pathogens. Eighty bovine semen samples and twenty mixed semen samples were tested. The results revealed that the positivity rates of BVDV-1 and BTV RT‒ddPCR (25% and 23%, respectively) were greater than those of RT‒qPCR (19% and 18%, respectively).

CONCLUSIONS

RT‒ddPCR was highly sensitive for detecting low concentrations of BVDV-1 and BTV in clinical samples and could be a good supplement for qPCR testing.

摘要

背景

牛病毒性腹泻病毒1型（BVDV-1）和蓝舌病病毒（BTV）是强效病毒病原体，可通过精液传播，导致疾病通过人工授精传播。因此，建立BVDV-1和BTV感染的早期检测方法对于精液交易很重要。在本研究中，我们开发了两种RT-ddPCR方法来检测BVDV-1和BTV，并对每种方法的重复性、检测限和特异性进行了评估。通过分析临床样本，将这些方法的灵敏度与RT-qPCR（世界动物卫生组织）的灵敏度进行了比较。

结果

RT-ddPCR结果显示，两种方法在低浓度下均具有良好的重复性，BVDV-1和BTV每个反应的检测限分别为1.05拷贝/微升和0.662拷贝/微升；此外，两种方法均具有高特异性，且与其他重要的精液传播病原体未出现交叉反应。对80份牛精液样本和20份混合精液样本进行了检测。结果显示，BVDV-1和BTV RT-ddPCR的阳性率（分别为25%和23%）高于RT-qPCR的阳性率（分别为19%和18%）。