National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, 100193, People's Republic of China.
Beijing General Station of Animal Husbandry, Beijing, 100110, People's Republic of China.
Parasitol Res. 2024 Sep 14;123(9):324. doi: 10.1007/s00436-024-08349-0.
Sarcocystis infection in sheep has caused significant economic losses in the livestock industry, and the genetic similarity among Sarcocystis species highlights the need for precise diagnostic methods in sheep. This study developed a loop-mediated isothermal amplification (LAMP) method targeting COX-1 and 28S rRNA genes to detect Sarcocystis tenella and Sarcocystis gigantea, respectively. The LAMP method exhibited high specificity, selectively amplifying target DNA sequences without cross-reactivity with closely related protozoa, such as Toxoplasma gondii and Neospora caninum. Detection limits were determined as 3 × 10 copies/L for S. tenella and 6 × 10 copies/L for S. gigantea, enabling sensitive identification of low-level infections. Comparative analysis with conventional PCR on sheep cardiac tissues demonstrated a higher LAMP detection rate (80.0% vs 66.7%). In conclusion, the LAMP method offers superior sensitivity to conventional PCR, allows visual confirmation of results, and provides a rapid diagnostic tool for identifying S. tenella and S. gigantea infection in sheep. However, due to the limitation of sample availability, we were unable to assess all Sarcocystis species that use sheep as intermediate hosts, which warrants further research.
绵羊的肉孢子虫感染给畜牧业造成了重大经济损失，肉孢子虫种间的遗传相似性凸显出在绵羊中需要精确的诊断方法。本研究开发了一种针对 COX-1 和 28S rRNA 基因的环介导等温扩增（LAMP）方法，分别用于检测柔嫩艾美耳球虫和巨形艾美耳球虫。LAMP 方法具有高度特异性，仅对靶 DNA 序列进行选择性扩增，与密切相关的原生动物（如刚地弓形虫和新孢子虫）无交叉反应。检测限分别确定为 3×10 拷贝/L 的柔嫩艾美耳球虫和 6×10 拷贝/L 的巨形艾美耳球虫，能够灵敏地识别低水平感染。与绵羊心肌组织的常规 PCR 比较分析表明，LAMP 检测率更高（80.0%比 66.7%）。总之，LAMP 方法比常规 PCR 具有更高的灵敏度，允许对结果进行可视化确认，并为识别绵羊中的柔嫩艾美耳球虫和巨形艾美耳球虫感染提供了一种快速诊断工具。然而，由于样本可用性的限制，我们无法评估所有以绵羊为中间宿主的肉孢子虫种，这需要进一步研究。
