Development and validation of heminested rt-pcr and qrt-pcr techniques for detection of rabies virus genomes for the first time in iran

Azadeh Rasooli,1 Fereshteh amiri,2 Zohreh fadajan,3 Iman salahshourifar,4 Rouzbeh bashar,5 Maryam fazeli,6,*

1. Department of Biochemistry, Faculty of Sciences, Payame Noor University, Tehran, Iran
2. Department of Biology, College of Basic Science, Tehran Science and Research Branch, Islamic Azad University, Tehran, Ir
3. Department of Biology, College of Basic Science, Tehran Science and Research Branch, Islamic Azad University, Tehran, Ir
4. Department of Biology, College of Basic Science, Tehran Science and Research Branch, Islamic Azad University, Tehran, Ir
5. WHO Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran, Tehran, Iran
6. WHO Collaborating Center for Reference and Research on Rabies, Pasteur Institute of Iran, Tehran, Iran

Abstract

Introduction

Rabies virus (rv) is one of the most dangerous zoonotic disease and major public health problem in most of the world, especially underdeveloped countries. most carnivorous, domesticated animals and bats can be infected with the virus. they can transmit it to other mammals. 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 as well as physiological stress on patients because of the extremely high fatality rate of the disease. the aim of this study was to develop and validate two molecular techniques, heminested rt-pcr and qrt-pcr assays, for comprehensive detection of iran circulating rabies virus genomes in the suspected rabid brain and saliva samples.

Methods

In this study, we tried to develop 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 was determined compared to heminested rt-pcr test and direct fluorescent antibody (dfa) and mit (mouse inoculation test).

Results

A combination of 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. primers and probe designed were blasted using the ncbi primer-blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/) with other iran circulating virus genomes that were available in public databases (genbank). the clinical sensitivities of qrt-pcr and heminested rt-pcr methods were calculated 97.14% and 94.3%, respectively; while, the clinical specificities of qrt-pcr and heminested rt-pcr methods were calculated 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.

Conclusion

In this study, qrt-pcr and heminested rt-pcr assays as two diagnostic molecular methods with high sensitivity and specificity were developed for detection of rabies virus genome. therefore, these rapid, accurate, and cost-effective detection methods may perhaps be the investigative tools which can be valid for detection of target viral genome for use in the research and diagnosis field.

Keywords

Rabies virus; molecular diagnosis; reverse transcriptase polymerase chain reaction; real-time polyme