Detection of influenza A virus from live-bird market poultry swab samples in China by a pan-IAV, one-step reverse-transcription FRET-PCR

Abstract

The persistent public health threat of animal to human transmission of influenza A virus (IAV) has stimulated interest in rapid and accurate detection of all IAV subtypes in clinical specimens of animal origin. In this study, a new set of primers and probes was designed for one-step pan-IAV reverse-transcription fluorescence resonance energy transfer (FRET)-PCR. The detection limit of one-step pan-IAV RT FRET-PCR was 10 copies of the matrix gene per reaction, and proved to be equivalent or superior to virus isolation in detecting nine IAV subtypes. Application of the pan-IAV RT FRET-PCR to oral-pharyngeal and cloacal swab specimens collected from healthy poultry in 34 live bird markets in 24 provinces of China revealed that 9.2% of the animals (169/1,839) or 6.3% of their oral-pharyngeal or cloacal swabs (233/3,678) were positive for IAV, and 56.8% of IAV-positive samples were of the H9N2 subtype. Paralleling detection of IAV in H9N2-infected SPF chickens and chickens from LBM showed that pan-IAV FRET-PCR had a higher detection limit than virus isolation in eggs while the results by FRET-PCR and virus isolation overall matched. It is expected that this strategy can be useful for facile surveillance for IAV in clinical samples from a variety of sources.

In Vitro and In Vivo Antiviral Activity of Gingerenone A on Influenza A Virus Is Mediated by Targeting Janus Kinase 2

“…The 293T cells seeded in 6-well plates were infected with 0.01 MOI H5N1 virus and then incubated in the absence or presence of Gin A (0, 10, 25, 50 μM) for 16 h. Total RNA was extracted from H5N1 virus-infected cells by using a Qiagen RNA extraction kit. Viral RNA levels were measured by using a one-step reverse-transcription FRET-PCR to amplify the M gene according to our recent publication [ 22 ].…”

Development of a multiplex probe combination-based one-step real-time reverse transcription-PCR for NA subtype typing of avian influenza virus

“…Total RNAs were extracted from allantoic fluids, trachea, or cloacal swabs by using the High-Pure PCR Template Preparation Kit (Roche Molecular Biochemicals, Indianapolis, IN, USA) according to the manufacturer’s instructions 29 . Total RNA was eluted in 20 μl elution buffer for each sample.…”

“…Three-week-old specific-pathogen-free (SPF) White Leghorn chickens from Beijing Meiliyaweitong Experimental Animal Technology Co., Ltd, were inoculated intranasally with 10 6 EID 50 of AIV H9N2 in a 0.2 ml volume (n = 20). Trachea and cloacal swabs were collected from chickens at 3, 5, and 7 days post-infection (dpi), and resuspended in 1 ml PBS for rRT-PCR detection and virus isolation 29 . For virus isolation, the samples were inoculated into the allantoic cavities of 10-day-old embryonated chicken eggs, after 3 days of incubation at 35 °C, the presence of hemagglutinating agents was determined by performing hemagglutination assays using 1% chicken erythrocytes.…”

New frontiers in applied veterinary point‐of‐capture diagnostics: Toward early detection and control of zoonotic influenza

“…A lengthier, two‐step assay involving conserved influenza A target–matrix protein, for example—followed by priority or OIE‐reportable subtypes (H5, H7, and H9) is also feasible. On‐site detection of a novel influenza subtype outside of this framework remains a challenge until a rapid, portable pan‐influenza platform is developed mirroring the one‐step pan‐influenza A virus benchtop PCR primer and probe sets that have been described …”