| 刊名 | Animal Husbandry and Feed Science |
| 作者 | Wang Keke1*, Liang Xinxin1, Jiang Gangqiang1, Long Zhixin1, Hudusi Aierken1, Liu Zhiling2, Wang Yan3, Wu Xiaowei2, Xiao Yuanyuan1, Bai Meihua1 |
| 作者单位 | 1. Urumqi Customs Technology Centre, Urumqi 830063, China; 2. Guangzhou Customs Technology Centre, Guangzhou 510623, China; 3. Animal and Plant and Food Inspection and Quarantine Technology Center, Shanghai Customs, Shanghai 200120, China |
| DOI | 10.19578/j.cnki.ahfs.2024.01-03.004 |
| 年份 | 2024 |
| 刊期 | 1 |
| 页码 | 21-25 |
| 关键词 | Equine arteritis virus (EAV); ORF7 gene; Fluorescence quantitative RT-PCR |
| 摘要 | [Objective] The paper was to establish a fluorescence quantitative RT-PCR method for detection of equine arteritis virus (EAV). [Method] Primers and probes were developed for the EAV ORF7 gene sequence, and the reaction system was optimized. Standard curves were established, leading to the initial development of the EAV fluorescence quantitative RT-PCR assay. The accuracy, specificity, and sensitivity of this method were subsequently evaluated. [Result] The EAV fluorescence quantitative RT-PCR assay demonstrated optimal performance at an annealing temperature of 61°C, with a final concentration of primer and probe set at 0.6 μmol/L. The plasmid standard demonstrated a strong linear correlation with Ct values within the range of 1.6×107-1.6×102 copies/μL. The equation of the standard curve was determined to be y= -2.68x+32.88, with an R² value of 0.9927. Consequently, the EAV fluorescence quantitative RT-PCR assay was successfully established. The methodology employed was effective in detecting EAV, Theileria equi, equine herpesvirus-1 (EHV-1), equine herpesvirus-4 (EHV-4), and equine influenza virus (EIV). The findings indicated that the method was specifically capable of detecting EAV, while the other pathogens tested yielded negative results. The method demonstrated a high degree of specificity. It was employed to detect the standard plasmid cRNA synthesized through in vitro transcription following a 10-fold dilution. The results indicated that the minimum detection limit of the method was 1.6×10² copies/μL, and it exhibited high sensitivity. The coefficient of variation, both within and between groups, was maintained at 1.8%, indicating good reproducibility. In this study, the fluorescence quantitative RT-PCR assay developed was utilized alongside the EAV fluorescence quantitative RT-PCR assay established by previous researchers to analyze a total of 234 clinical samples. Both methods yielded a positive detection rate of 14.1%, and the coincidence rate between the two techniques was found to be 100%. [Conclusion] The fluorescence quantitative RT-PCR assay developed in this study offers a novel approach and concept for the prevention and control of equine viral arteritis (EVA). |
