Development of a Multiplex RT-qPCR for the Detection of Different Clades of Avian Influenza in Poultry

Abstract

Since the initial detection of H5N1, a highly pathogenic avian influenza (HPAI) virus, in 1996 in China, numerous HPAI H5 lineages have been classified, and they continue to pose a threat to animal and human health. In this study, we developed a novel primer/probe set that can be employed to simultaneously detect pan-H5 HPAI and two clades, 2.3.2.1 and 2.3.4.4, of H5Nx viruses using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The sensitivity and specificity of these primer sets and probes were confirmed with a number of different subtypes of influenza virus and the H5-HA gene plasmid DNA. In particular, the multiplex RT-qPCR assay was successfully applied to the simultaneous detection of H5 HPAI and different virus clades in clinical field samples from a poultry farm. Therefore, this multiplex assay and a novel detection primer set and probes will be useful for the laboratory diagnosis and epidemiological field studies of different circulating H5 HPAI virus clades in poultry and migratory wild birds.

Avian Influenza in Wild Birds and Poultry: Dissemination Pathways, Monitoring Methods, and Virus Ecology

“…After subsequent incubation, allantoic fluids should be recovered and tested using a screening test, such as hemagglutination, or influenza A type-specific tests (agar gel immunodiffusion test, neuraminidase inhibition (NI) tests, and others). An alternative approach to the detection of AIV particles in the fluid is by use of molecular techniques to detect the presence of nucleic acid signatures specific to influenza A, such as real-time reverse transcription polymerase chain reaction (RT-PCR) testing [55], along with more specific assays developed for the simultaneous detection of different viral clades in field samples and in poultry [56,57]. It should be noted that, for large-scale screening experiments, the current strategy of viral reproduction in inoculated chicken embryos followed by a hemagglutination assay (Figure 1) is regarded as the primary method for viral detection, while RT-PCR and sequencing remain as means of final proof [54,58,59].…”

Diagnostic performance and clinical feasibility of a novel one-step RT-qPCR assay for simultaneous detection of multiple severe acute respiratory syndrome coronaviruses

“…In total, 19 different cultured viruses were evaluated. Four SARS-CoV-2 isolates from Korea (wild-type, alpha variant, beta variant, and delta variant), human coronavirus NL63 (Korea/CN0601/14), human coronavirus 229E (Korea/KUMC-9), human coronavirus OC43 (KBPV-VR-8), and MERS-CoV (MERS-CoV/KOR/KNIH/002_05_2015) were obtained from the Korea Centers for Disease Control and Prevention (KCDC, Republic of Korea), and other viruses, including porcine epidemic diarrhea virus (Korea/SM98), dengue virus type 2 (Korea/DENV-2/KBPV-VR-29), human H3N2 influenza A virus (Korea/37/2012), human H1N1 influenza A virus (California/04/09), human influenza B virus (B/Brisbane/60/2008), parainfluenza virus 1 (Korea/KUMC-44), human respiratory syncytial virus (HRSV-A/IC688/12), and adenovirus type 3 (KUMC-62), were obtained from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) or purchased from the Korea Bank for Pathogenic Viruses (Republic of Korea) [ 23 ]. RNA from SARS-CoV (HKU-39849) was provided by Dr. Seungtaek Kim (Institute Pasteur Korea) [ 24 ], and viral RNA samples from bat-derived SARS-related CoV (GenBank accession numbers: MK991935 and MK991936) from bat fecal samples, which were used in specificity assays (Table 2 ), were provided by Prof. Hye Kwon Kim (Chungbuk National University, South Korea) [ 25 ].…”

Reassortant Highly Pathogenic H5N6 Avian Influenza Virus Containing Low Pathogenic Viral Genes in a Local Live Poultry Market, Vietnam

“…Samples were then tested for AIVs using a matrix (M) gene-specific real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) method according to the WHO guideline for animal influenza virus detection [16]. Continuously, the samples containing influenza A virus were examined for HPAIV and clade detection based on an RT-qPCR assay following a previous report [17]. Positive AIV samples were isolated by inoculating 10-day-old embryonated chicken eggs in a biosafety level two plus facility at the College of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi, Vietnam.…”