Development and Validation of Heminested RT-PCR and qRT-PCR Assays for Comprehensive Detection of Rabies Virus in the Suspected Rabid Brain and Saliva Samples

Background: Rabies virus (RV) is one of the most dangerous zoonotic disease and major public health problems in most of the world, especially underdeveloped countries. Rabies is preventable by proper vaccination, even shortly after exposure. Today, it seems a fast, sensitive, and reliable rabies diagnostic method is required, which might reduce the financial burden of inappropriate diagnosis. Objectives: The aim of this study was to develop and validate two molecular techniques, including heminested RT-PCR and qRT-PCR assays, for comprehensive detection of rabies virus in the suspected rabid brain and saliva samples. Methods: In this study, we developed qRT-PCR as a fast, sensitive, and specific method for rapid detection of rabies virus in brain and saliva samples. Also, the sensitivity and specificity of the method were compared with heminested RT-PCR test and direct fluorescent antibody (dFA) as a serologic gold standard method of World Health Organization (WHO) and MIT (mouse inoculation test) as a confirmatory test. Results: A combination of compatible primers based on RNA-dependent RNA polymerase (L) gene of the Pasteur virus fixed strain (PV) (accession number. M13215) was used for developing the qRT-PCR assay. Primer and probes were designed according to other Iran circulating viruses genomes that were available in public databases (GenBank). The clinical sensitivities of qRT-PCR and heminested RT-PCR methods were determined 97.14% and 94.3%, respectively. In addition, the clinical specificities of qRT-PCR and heminested RT-PCR methods were determined 93.75% and 88.24%, respectively. Also, the analytical sensitivities of qRT-PCR and heminested RT-PCR methods were about 5 × 102 and 5 × 103 FFU/mL, respectively. Conclusions: In this study, qRT-PCR assay as a diagnostic molecular method with high sensitivity and specificity was developed for the detection of the rabies virus genome in both brain and saliva samples. Therefore, this rapid, accurate, and cost-effective detection and quantification method may be used as an investigative tool, which can be valid for detection of target viral genome in the research and diagnosis field.