Development of oligonucleotide primers for identification of the spring carp viremia virus (Cyprinus carpio) by polymerase chain reaction

Spring carp viremia is a contagious disease affecting cyprinids, primarily the common carp (Cyprinus carpio). The disease is endemic in Europe, America and several Asian countries, where it causes significant morbidity and mortality in affected fish. The Office International des Epizooties (OIE) has included spring carp viremia (SCV) in the list of notifiable diseases. The virus is excreted mainly in the faeces and urine of clinically infected fish and by the carriers themselves. Diagnosis should be confirmed by virus neutralization or polymerase chain reaction (PCR) and nucleotide sequence analysis of the PCR products. There is no treatment for spring carp viremia. This article is devoted to the development of primers for real-time polymerase chain reaction to detect the genome of the spring carp viremia virus. The studies were conducted at the Kropotkin Regional Veterinary Laboratory in Kropotkin, Krasnodar krai. Carp samples were collected by taking tissue material and exudate from the abdominal cavity. Primers were selected at the Department of Biology, Ecology, Parasitology, Aquatic Bioresources and Aquaculture of the Ulyanovsk State Agrarian University. The study material included 4 organ samples from carp fish received by the Kropotkin Regional Veterinary Laboratory. Real-time polymerase chain reaction was performed in a reaction mixture volume of 25 μl per 1 RNA sample. The samples under study were detected using a CFX 96 Bio-Rad (USA) amplifier. Fluorescence was measured by channels at the 5` ends with FAM/green dye and at the 3` ends with BHQ1 quencher. As a result of the RTPCR, 6 fluorescence curves were identified, together with two positive controls, crossing the Threshold line. The values of the "Ct" indicator ranged from 13 to 35. The obtained results of the reaction indicate their good reproducibility and reliability.