Comparative study of the performance of two methods of RNA extraction by real-time RT-PCR (RT-qPCR) from central nervous system samples of cattle naturally infected with the rabies virus (RABV)

• Grant number: 17/13459-2
• Support type: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): September 01, 2017
• Effective date (End): December 31, 2018
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Preventive Veterinary Medicine
• Principal researcher: Enio Mori
• Grantee: Maria Eduarda Rodrigues Chierato
• Home Institution: Instituto Pasteur (IP). Coordenadoria de Controle de Doenças (CCD). Secretaria da Saúde (São Paulo - Estado). São Paulo , SP, Brazil

Abstract

Direct fluorescent antibody test (DFAT) is considered a rapid screening and the gold standard test for the rabies diagnosis by WHO and OIE due to high sensitivity and specificity. As the rabies virus (RABV) does not infect all central nervous system (CNS) structures uniformly, the detection of this agent by DAFT may have variable results. There is disagreement about which fragments would be most suitable for diagnosis purpose in cattle. Some situations require the development and implementation of alternative methodologies, such as reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR (RT-qPCR). However, there is a decline in accuracy in the analysis of results that cause the decrease efficiency in the amplification of nucleic acids associated with inhibitory factors in the TRIzol reagent. In this way, to infer which these inhibitory factors could cause low viral detection by RT-PCR and RT-qPCR, this study aims at comparing the amplification of two RNA extraction methods: TRIzol and kit by silica membrane columns. For this purpose, we will use CNS samples [brain (thalamus, cortex, hippocampus, cerebellum and brainstem) and spinal cord] diagnosed for rabies of 127 cattle from Rabies Diagnosis Section of Instituto Pasteur, previously collected during March 2015 to April 2016. (AU)