Multiplex one-step Real-time PCR by Taqman-MGB method for rapid detection of pan and H5 subtype avian influenza viruses

<div><p>Avian influenza virus (AIV) can infect a variety of avian species and mammals, leading to severe economic losses in poultry industry and posing a substantial threat to public health. Currently, traditional virus isolation and identification is inadequate for the early diagnosis because of its labor-intensive and time-consuming features. Real-time RT-PCR (RRT-PCR) is an ideal method for the detection of AIV since it is highly specific, sensitive and rapid. In addition, as the new quencher MGB is used in RRT-PCR, it only needs shorter probe and helps the binding of target gene and probe. In this study, a pan-AIV RRT-PCR for the detection of all AIVs and H5-AIV RRT-PCR for detection of H5 AIV based on NP gene of AIV and HA gene of H5 AIV were successfully established using Taqman-MGB method. We tested 14 AIV strains in total and the results showed that the pan-AIV RRT-PCR can detect AIV of various HA subtypes and the H5-AIV RRT-PCR can detect H5 AIV circulating in poultry in China in recent three years, including H5 viruses of clade 7.2, clade 2.3.4.4 and clade 2.3.2.1. Furthermore, the multiplex detection limit for pan-AIV and H5-AIV RRT-PCR was 5 copies per reaction. When this multiplex method was applied in the detection of experimental and live poultry market samples, the detection rates of pan-AIV and H5 AIV in RRT-PCR were both higher than the routine virus isolation method with embryonated chicken eggs. The multiplex RRT-PCR method established in our study showed high sensitivity, reproducibility and specificity, suggesting the promising application of our method for surveillance of both pan AIV and prevalent H5 AIV in live poultry markets and clinical samples.</p></div

AIV strains used in this study and the detection limit of RRT-PCR method.

<p>AIV strains used in this study and the detection limit of RRT-PCR method.</p

Primers and probes used in this study.

<p>Primers and probes used in this study.</p

Detection of H5 AIV in swabs from infected SPF chickens with virus isolation and RRT-PCR.

<p>Oral-pharyngeal swabs and cloacal swabs of 10 SPF chickens were collected at 24 h, 36 h and 48 h. The infection dose of SPF chicken was 0.2mL 10³ EID₅₀ of H5 AIV. At 24 h and 36 h, no H5 AIV was detected in oral-pharyngeal samples in traditional virus isolation method while the number of positive trachea swabs through RRT-PCR detection at 24 h and 36 h were 5 and 2 respectively. The number of positive samples in RRT-PCR matched that in virus isolation at 48 h.</p

Amplification plots and standard curves drew by ABI 7500 software in multiplex detection with standard plasmids.

<p>The threshold lines in amplification plots and linear curves in standard curves were draw automatically by ABI 7500 software. (A) NP standard curve between 5x10⁰ and 5x10⁵ copies (y = -3.346x+39.272, R² = 0.997, top), linear amplification plot (middle), and log amplification plot (bottom) in multiplex detection. (B) HA standard curve between 5x10⁰ and 5x10⁵ copies (y = -3.43x+40.183, R² = 0.998, top), linear amplification plot (middle), and log amplification plot (bottom) in multiplex detection.</p

Inter-assay in multiplex detection of H5 AIV and pan-AIV.

<p>Inter-assay in multiplex detection of H5 AIV and pan-AIV.</p

Positive percentage by virus isolation and RRT-PCR for live-poultry market samples from May 2016 to June 2016.

<p>110 oral-pharyngeal or cloacal samples were collected from live poultry market in May and June respectively in 2016. Columns from left to right are 10%, 16.4%, 4.5%, 12.7%, 17.3%, 26.4%, 15.5%, 22.7%, which showed RRT-PCR method was of higher positive percentage than virus isolation.</p

Intra-assay in multiplex detection of H5 AIV and pan-AIV.

<p>Intra-assay in multiplex detection of H5 AIV and pan-AIV.</p