Development and validation of a real-time PCR assay for the diagnosis of rabies virus Philippine strain in non-brain samples

Rabies is a fatal neurotropic and zoonotic disease responsible for thousands of deaths yearly. Direct fluorescent antibody test (dFAT), the gold standard in routine rabies diagnosis, requires dog brain samples, and takes 5-7 hours to obtain results. Brain specimen degradation due to inappropriate transport and storage conditions most of the time leads to false negative results, hence the need for an alternative diagnostic method that can also utilize non-brain specimens. In this study, an RT-qPCR assay was developed to specifically target the N gene of the Philippine rabies isolate. The assay was optimized using RNA from dFAT-positive dog brain tissues as templates. In-silico and in vitro evaluations both showed 100% specificity to rabies RNA, with a detection limit of 1 copy per microliter. Validation of the assay was done using dFAT-tested brain samples and potential brain specimen-alternates, specifically the dog nasal planum (NP) and follicle sinus complex (FSC). One hundred percent of the NP and FSC samples showed concordance with the respective dFAT-positive brain samples. Only 97% concordance was observed with the dFAT-negative brain samples. These results collectively validate the efficiency, sensitivity and specificity of the assay developed, indicating its potential utilization for in rabies diagnosis using clinical samples besides the brain tissues.