Application of three duplex real-time PCR assays for simultaneous detection of human seasonal and avian influenza viruses

Abstract

This study was performed to develop real-time PCR (qPCR) for detection of human seasonal and avian influenza viruses in duplex format. First duplex qPCR detects haemagglutinin (HA) gene of influenza virus A(H1N1)pdm09 and HA gene of influenza virus A(H3N2), the second reaction detects neuraminidase (NA) gene of influenza virus A(H3N2) and NA gene of influenza virus A(H1N1)pdm09 and A(H5N1), and the third reaction detects HA gene of influenza A(H5N1) and nonstructural protein gene of influenza B virus. Primers and probes were designed using multiple alignments of target gene sequences of different reference strains. Assays were optimised for identical thermocycling conditions. Their specificity was confirmed by conventional PCR and monoplex qPCR with nucleic acids isolated from different influenza viruses and other respiratory pathogens. Plasmid constructs with a fragment of specific gene were used to assess sensitivity of the assay. The limit of detection ranged from 27 to 96 cDNA copies/reaction. Clinical specimens (n = 107) have been tested using new assays, immunofluorescence and monoplex qRT-PCR. It has been shown that developed assays have been capable of rapid and accurate simultaneous detection and differentiation of influenza viruses. They are more sensitive than immunofluorescence and at least as sensitive as monoplex qRT-PCR.

Natural Infection with Highly Pathogenic Avian Influenza A/H5N1 Virus in Pet Ferrets

“…One-step reverse transcription real-time PCR (RT-qPCR) was performed using the CFx96 system (BioRad, Hercules, CA, USA). Tests for SARS-CoV-2 were conducted with a commercially available Novel Coronavirus (COVID-19) Real-Time Multiplex RT-PCR Kit (LifeRiver, San Diego, CA USA), and tests for IAVs were performed with an in-house method described by Stefańska et al [ 16 ].…”

“…To determine the limit of detection (LOD) of the method and the range of its linearity, we used pBluescript plasmids with a cloned cDNA fragment (385 nucleotides) covering the HA gene region (Epoch LifeScience ®, Missouri City, TX, USA), for which primers and a probe were previously designed. LOD was determined as the lowest cDNA concentration that would be detected with 95% probability when testing at least 24 samples constituting replicates of 5 dilutions of plasmid DNA on a decimal logarithmic scale [ 16 ]. RT-qPCR results with a quantification cycle (Cq) of ≤35.00 were considered positive.…”

“…These cases resulted from infection with the highly pathogenic avian influenza (HPAI) virus A/H5N1 [1][2][3][4]. Although A/H5N1 virus infections have been occasionally observed in cats in the past [5], the scale of these outbreaks in Poland turned out to be unprecedented, both in the European and the global context, which was reflected in the WHO announcement of 16 July 2023 (https://www.who.int/emergencies/disease-outbreak-news/item/2023 -DON476 accessed on 5 June 2024). In total, the A/H5N1 virus infection was confirmed in at least 34 cats.…”

Upper Respiratory Tract Disease in a Dog Infected by a Highly Pathogenic Avian A/H5N1 Virus

“…One-step reverse transcription real-time PCR (RT-qPCR) was performed using the CFx96 system (BioRad, Hercules, CA, USA). Real-Time Multiplex RT-PCR Kit (LifeRiver, San Diego, CA, USA) for SARS-CoV-2 detection and with an in-house method described by Stefa ńska et al for IAVs [28] RT-qPCR results with a quantification cycle (Cq) of ≤35.00 were considered positive.…”