Development of test-system for detection of H5 and H7 avian influenza virus RNA by multiplex real-time RT-PCR assay using internal control

• DOI: 10.29326/2304-196x-2025-14-1-40-46
• Publication Date: 2025-03-23T09:59:05Z
• Authors: A. D. Grekhneva; N. G Zinyakov; A. V. Andriyasov; A. A. Kozlov; E. V. Ovchinnikova; D. B. Andreychuk; P. D. Zhestkov; I. A. Chvala

Introduction. High pathogenicity avian influenza is a dangerous highly contagious viral infection of domestic and wild birds that recently has become widespread in Europe, Asia, Africa and Americas. The causative agent of the disease is type A influenza virus of subtypes H5 and H7. Real-time RT-PCR is one of the most rapid and effective techniques for avian influenza virus identification and typing, so development of the test system based on this technique with internal control to be used for control of the reaction main stages is of current importance. At the same time, the multiplex format of RT-PCR allows for simultaneous identification of several targets that reduces the consumption of reagents and the reaction time.Objective. Development of test-system for detection of H5 and H7 avian influenza virus RNA with multiplex real-time RT-PCR in biological samples and its characterization.Materials and methods. H5, H7, H3, H4, H10, H16 avian influenza virus, Newcastle disease virus, infectious bursal disease virus, infectious bronchitis virus, Marek’s disease virus, avian adenovirus isolates were used. MS2 bacteriophage was used as internal control.Results. Optimal primer-probe combinations were selected, test-system characteristics were determined: specificity for homologous and heterologous avian disease viruses, analytical sensitivity, reaction amplification efficiency, repeatability and reproducibility.Conclusion. Determination of the developed test system validation parameters has shown that it is specific only for H5 and H7 avian influenza virus, its analytical sensitivity for each subtype was 1.5 lg EID50/cm3 , and the amplification efficiency was 92 and 97%, respectively. The test system was validated through its use for testing biological samples submitted to the laboratory, the test results were consistent with the results of tests with standard diagnostic methods used in the Reference Laboratory for Avian Viral Diseases of the Federal Centre for Animal Health.