Specificity of Primers and Probes for Molecular Diagnosis of Leishmania (Leishmania) chagasi in Dogs and Wild Animals

Molecular tools, especially real-time polymerase chain reaction (qPCR), are relevant tools for laboratory diagnosis due to their sensitivity, specificity, rapid results, and ability to quantify parasite load. This study evaluated the specificity of the LEISH-1/LEISH-2 primer pair with the TaqMan MGB probe in serum samples previously classified by indirect Enzyme-Linked Immunosorbent Assay (ELISA) (30 positive dogs, 30 negative dogs; 9 positive wild animals and 16 negative wild animals) using in silico analyses (Primer-BLAST, Multiple Alignment using Fast Fourier Transform-MAFFT^®, Geneious, RNAfold, and SnapGene) and Real-Time Polymerase Chain Reaction (qPCR) experimentation. Unexpected amplification occurred in all negative samples, revealing critical specificity failures mainly associated with the probe. In silico analyses confirmed these findings, indicating structural incompatibilities and low selectivity of the sequences. To address this limitation, a new set of oligonucleotides, named GIO, was designed. Computational analyses showed superior performance of GIO, with greater structural stability, absence of unfavorable secondary structures, and improved specificity. Although experimental validation is still required, the results suggest that GIO has strong potential for use in more robust and reliable diagnostic protocols for visceral leishmaniasis across different epidemiological contexts.