Wang Lihua, Li Yuzhen, Zhang Xirui, Madera Rachel, Pantua Homer, Craig Aidan, Muro Nina, Li Danqin, Retallick Jamie, Ferreyra Franco Matias, Truong Quang Lam, Nguyen Lan Thi, Shi Jishu
Center on Biologics Development and Evaluation, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.
Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS 66506, USA.
Microorganisms. 2025 Mar 7;13(3):615. doi: 10.3390/microorganisms13030615.
African swine fever (ASF), a highly contagious and lethal viral disease, continues to devastate the global swine industry. The emergence of ASF virus (ASFV) variants with varying genomic deletions poses significant challenges for ASF control. This study presents a novel, sensitive, and reliable quadplex real-time PCR assay for detecting ASFV variants lacking key genes (, , and ), either individually or in combination. The assay targets conserved regions within these genes, ensuring broad coverage of diverse ASFV genotypes. A porcine () internal control was incorporated to minimize false-negative results. Optimization and evaluation using spike-in tests demonstrated high sensitivity, with a limit of detection (LOD) ranging from 1 to 10 plasmid copies or 0.1 TCID of ASFV isolates per reaction. No cross-reactivity was observed when testing serum samples from pigs infected with other common swine viruses. Further validation across a diverse panel of samples, including those from naturally ASFV-infected field pigs ( = 54), experimentally ASFV-infected pigs ( = 50), PBS-inoculated pigs ( = 50), ASFV-free field pigs ( = 100), and feral pigs ( = 6), confirmed 100% specificity. This robust assay provides a valuable tool for rapid and accurate ASF surveillance and control efforts, facilitating the timely detection and mitigation of outbreaks caused by emerging ASFV variants.
非洲猪瘟（ASF）是一种极具传染性和致死性的病毒性疾病，持续对全球养猪业造成严重破坏。具有不同基因组缺失的非洲猪瘟病毒（ASFV）变体的出现，给非洲猪瘟的防控带来了重大挑战。本研究提出了一种新颖、灵敏且可靠的四重实时荧光定量PCR检测方法，用于单独或联合检测缺失关键基因（、和）的ASFV变体。该检测方法针对这些基因内的保守区域，确保广泛覆盖多种ASFV基因型。引入了猪（）内对照以尽量减少假阴性结果。通过加标试验进行的优化和评估显示出高灵敏度，每个反应的检测限（LOD）为1至10个质粒拷贝或0.1 TCID的ASFV分离株。检测感染其他常见猪病毒的猪的血清样本时未观察到交叉反应。在包括自然感染ASFV的田间猪（ = 54）、实验感染ASFV的猪（ = 50）、接种PBS的猪（ = 50）、无ASFV的田间猪（ = 100）和野猪（ = 6）在内的各种样本中进行的进一步验证，证实特异性为100%。这种强大的检测方法为快速准确的非洲猪瘟监测和防控工作提供了有价值的工具，有助于及时发现和缓解由新兴ASFV变体引起的疫情。
