Test-system for detection of peste des petits ruminants virus genome by reverse transcription real-time PCR

Abstract

Peste des petits ruminants (PPR) is a highly contagious transboundary infection disease of small ruminants, characterized by fever, anorexia, ocular and nasal discharge, erosions and ulcers in digestive mucosa, diarrhoea and marked leucopoenia with immunosuppression. Because of the complexity of the PPR epizootic situation in neighboring countries (Tajikistan, China, Mongolia, Kazakhstan, Afghanistan) the risk of occurrence of this disease on the territories of Siberian, Ural, Far Eastern, North Caucasian and Southern Federal Districts of Russian Federation is very high. So, the development of molecular-genetic methods for the diagnosis of PPR and methods for stabilizing of biological samples represents scientific relevance. This article presents data on the development of a test system for detecting the PPR virus genome by reverse transcription Real-Time PCR. This technique is based on the amplification of a fragment (148 bp) of hemagglutinin gene of PPR virus using the original oligonucleotide primers and fluorescent-labeled hybridization probe. Analytical specificity of the developed test system was evaluated by testing the strains of PPR virus, rinderpest virus, bluetongue virus, as well as biological samples from clinically healthy animals and intact cell cultures. Positive results were obtained only with samples containing PPR virus (strains Epizootichesky, Nigeria 75/1 and 45G37/35-K). The analytical sensitivity of the developed test-system, determined using tenfold serial dilutions of cultural virus-containing material, is 0.83±0.22 lg TCID50/ml. To create a positive amplification control, a fragment of hemagglutinin gene synthesized usign Real-Time PCR was cloned in plasmid pTZ57 R/T in Escherichia coli. It was established that the plasmid DNA concentration for amplification linearity ranges from 2.4½107 to 24 molecules/μl. To assess the practical suitability of the developed test system for the diagnosis of PPR, the blood samples from sheep experimentally infected with PPR virus (strain Epizootichesky) were tested. As a result, the PPR virus genome was detected in blood samples from day 5 to day 12 after infection. Since transportation of biological samples over long distances may require during survey, we have developed “dry” blood method to collect and store blood samples. It has been shown that “dry” blood drops are stable at room temperature for a month and can be used in Real-Time PCR testing.