Design of oligonucleotide primers and a probe for the detection of toxocara canis by real-time PCR

Abstract

Toxocariasis is a dangerous zoonotic parasitic disease. Infection occurs when eggs of the parasite Toxocara enter the body; these eggs may be present in contaminated soil or transmitted through contact with infected animals. Once inside the body, larvae migrate to various organs and affect the liver, lungs, eyes, and other tissues. It causes visceral and ocular larva migrans syndrome, neurotoxocariasis, and covert or common toxocariasis. Toxocara canis is of significant importance for public health and the economy. The relevance of toxocariasis diagnosis is associated with its wide distribution, zoonotic nature, and manifestation of severe clinical forms in humans.

The aim of this study was to develop oligonucleotides for the detection of T. canis using real-time PCR. The efficiency of three primer pairs designed for T. canis detection was evaluated in order to identify the best option for use in real-time PCR. The studied oligonucleotides are located within the ITS2 gene and provide amplification of specific fragments of the expected size, confirming their high specificity and proper design. The highest amplification efficiency was observed for the primer pair T_can F2/R2 (105 bp), as confirmed by the intensity of the obtained band. The weaker PCR products obtained for T_can F1/R1 and T_can F3/R3 were probably associated with особенностями their binding to template DNA or the formation of secondary structures.

The primers T_can F2, T_can R2, and the probe T_can Probe2 demonstrated efficient and specific performance in real-time PCR, ensuring reliable detection of T. canis. The proposed oligonucleotides are recommended for the development of diagnostic test systems based on real-time PCR. This is important for early prevention of parasite spread, thereby reducing the risk of complications and increasing epidemiological safety.