Development of a highly specific LAMP assay for detection of Sarcocystis tenella and Sarcocystis gigantea in sheep.

ABSTRACT

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 × 105 copies/L for S. tenella and 6 × 104 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.