Development and Evaluation of a Test System for the Detection of Echinococcus Granulosus Using Real-Time PCR

Cystic echinococcosis remains an important zoonotic disease in Kazakhstan, requiring reliable molecular methods for the detection and monitoring of E. granulosus. The aim of this study was to develop and analytically evaluate a G1/G3 complex-oriented real-time PCR test system targeting the mitochondrial cox1 gene for the detection of Echinococcus granulosus sensu stricto. Sheep liver echinococcal cysts were used as biological material. Cyst fluid was examined by light microscopy, and DNA was extracted using a QIAamp DNA Mini Kit. The nucleotide sequences of cox1 from different genotypes of E. granulosus sensu lato were aligned, and a G1/G3 oligonucleotide complex system was developed. The expected amplicon sizes were 108 bp. In silico analysis revealed suitable oligonucleotide characteristics, including acceptable melting temperatures, GC content, and the absence of critical secondary structures. Classical PCR confirmed amplification of the expected fragments, and the systems were adapted for real-time PCR using FAM/BHQ1-labeled probes. The developed real-time PCR assay can serve as a basis for further improving the molecular diagnostics of echinococcosis, timely detection of E. granulosus, and increasing the effectiveness of epidemiological surveillance.