Abstract / Summary
Plasmodium ovale ( P. ovale ) is one of five malaria parasites known to infect humans. Current research confirms the existence of two genetically distinct species, P. ovale wallikeri ( Pow ) and P. ovale curtisi ( Poc ), which differ in their pathogenicity and virulence in humans. Accurate discrimination between these species is crucial for appropriate patient management and epidemiological surveillance. Commonly employed methods such as qPCR and serological assays are limited by high cost, long processing times, and inadequate sensitivity or specificity. We designed five candidate crRNAs and selected two highly specific ones to construct separate detection systems: one for Pow and another for Poc . PCR-CRISPR/Cas12a detection system was then established and optimized. The limit of detection and specificity of the system were evaluated, and its detection performance was further validated using clinical samples. The Pow CRISPR/Cas12a system demonstrated the detection limit of 100 copies/μL, while the Poc system achieved the detection limit of 30 copies/μL, with no cross-reactivity observed between the two species, indicating high sensitivity and specificity. We further validated the assay using 137 clinical samples from imported malaria patients, yielding the sensitivity of 96.77% and specificity of 99.05% for Poc and sensitivity of 83.78% and specificity of 100% for Pow . This method represents a significant step forward in the molecular diagnosis of P. ovale malaria. Its combination of specificity, ease of use, and potential for adaptation makes it a highly promising tool for enhancing epidemiological surveillance and supporting malaria elimination efforts, particularly in the context of managing malaria cases.