Development and validation of the látex agglutination test (LAT) for diagnosis of canine distemper virus or preparation of RNA for direct use in RT-qPCR

Abstract

Canine distemper (CD) is a enzootic worldwide carnivorous disease with greater economic importance related to infection in dogs. Considering the contagious nature and high mortality rates of CD infected dogs, becomes necessary to develop affordable tools for diagnosis of the disease. Currently, RT-PCR and RT-qPCR followed by NESTED-PCR from urine samples are used for the diagnosis of disease, but these techniques have limitations such as cost, time and risk of contamination. The latex agglutination test (LAT) is a diagnostic method that has been applied in a wide variety of analytical tests for diagnosis of infections. Is a simple, rapid, specific and cost-effective and to date has not been developed for the diagnosis of canine distemper, probably by the low detection threshold. Although, several procedures may be used to optimize the LAT in order to increase the sensitivity of the test. For this purpose, antibodies from different animals will be tested under various conditions of antibody-binding latex (latex and antibody concentration, pH and binding time). Additionally, the latex will be previously conjugated with protein A and then conjugated to mammals antibodies. However, the result of LAT may be dependent on the viral load of the sample. The samples that fail to show visual agglutination will be centrifuged and used for concentration and extraction of viral nucleic acid, as well as other techniques already described using particle-bound for this purpose, aiming to increase the sensitivity of RT-qPCR reaction. This technology probably will eliminate the step of viral RNA extraction and qPCR-NESTED, reducing costs, optimizing reaction time and allowing greater safety concerning the laboratory contamination. The objective of this project is to develop, standardize and validate a new method of detection and capture by agglutination for rapid diagnosis of canine distemper in samples with high viral load or viral RNA preparation for use in RT-qPCR. (AU)