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

Abstract

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.

Development and evaluation of a TaqMan MGB RT-PCR assay for detection of H5 and N8 subtype influenza virus

“…In this study, DNA plasmids were used for analytical sensitivity testing instead of RNA run-off transcripts as described previously [28]. The detection limit of the assay was 10 copies per reaction for both H5 and N8 genes.…”

“…Regarding reproducibility, inter-assays and intra-assays were analysed using different concentrations of plasmids as described previously [ 29 ]. The results of intra-assays (Table 3 ) and inter-assays (Table 4 ) revealed that the coefficients of variation (CV%) were all < 2%, suggesting our RRT-PCR method is highly reproducible [ 30 ].…”

“…The sensitivity of the RRT-PCR assay was determined for each reaction using 10-fold serial dilutions of H5 and N8 plasmids, with DNA ranging from 1 to 10 9 copies per reaction [28]. To evaluate the clinical sensitivity and specificity of the RRT-PCR assay, sixweek-old female BALB/c mice (n = 24) were Note: Primers and probes were targeted to the conserved regions of the H5-HA and N8-NA genes, and the H5 gene-specific probe was labelled with FAM at the 5′ end, while the N8 gene-specific probe was labelled with HEX to allow specific detection of H5N8 AIVs in a single reaction.…”

“…The sensitivity of the RRT-PCR assay was determined for each reaction using 10-fold serial dilutions of H5 and N8 plasmids, with DNA ranging from 1 to 10 9 copies per reaction [ 28 ]. To evaluate the clinical sensitivity and specificity of the RRT-PCR assay, six-week-old female BALB/c mice ( n = 24) were anesthetized by isoflurane and inoculated intranasally with H5N8 virus in 0.05 mL phosphate buffered saline.…”

Replication of a Dog-Origin H6N1 Influenza Virus in Cell Culture and Mice

“…To quantify the viral RNA copies in the animal samples, qRT-PCR was performed using the modified primers and probe targeting nucleoprotein gene [ 24 ] and the RT-qPCR-probe 1-step Go No-ROX kit (Biosystems, London, UK). The reaction was conducted using qTOWER3G Real-time PCR Thermal Cycler (Analytik Jena AG, Jena, Germany).…”