RPB0289

Pathogen Description

Target Pathogen Pathogen Name NCBI Taxonomy ID Order Family Genus Species Pathogen type
Influenza A virus (H9N2) Influenza A virus (A\chicken\Yunnan\Baoshan2\2007(H9N2)) 479038 Articulavirales Orthomyxoviridae Alphainfluenzavirus Influenza A virus Virus

Primer Description

Primer Name Sequence(5'-3') Length(bp) Primer Final Concentration(μM） GC Content（%） Predicted Melting Temperature(℃） Molecular Weight(g/moles) Positions in GenBank accession number
H9-F GGGGTAGCTGCGCAGTGCAATGTCAGACAGA 31 10 µM 58.06 69.46 9650.31 \
H9-R TTCTAGATCTAGTAGAGGACTATTCGGGGCCATAGC 36 10 µM 47.22 63.5 11115.27 \
H9-probe ACAACATTGCCCTTCCAAAATGTAAGTAAG(FAM-dT)(THF)(BHQ1-dT) GCATTTGGAAACTGC[C3-spacer] 45 10 μM 40 65.67 13821.06 \

Gene Description

Target Gene GenBank ID
HA \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
\ RT–RAA SnapGene software (version 4.3.6) 30 min 42°C \ 1759 copies per reaction at a 95% confidence 163 copies 1

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2024 Establishment of a Real-Time Fluorescence Isothermal Recombinase-Aided Amplification Method for the Detection of H9 Avian Influenza Virus Yuxin Zhang,Cheng Zhang,Jiaqi Li,Yejin Yang,Ligong Chen,Heng Wang,Zitong Yang,Mingda Zhang,Huan Cui,Shishan Dong Diagnosis, Prevention and Control in Avian Virus Infections 39330790 10.3390/vetsci11090411

Establishment of a Real-Time Fluorescence Isothermal Recombinase-Aided Amplification Method for the Detection of H9 Avian Influenza Virus

Author(s):

Yuxin Zhang,Cheng Zhang,Jiaqi Li,Yejin Yang,Ligong Chen,Heng Wang,Zitong Yang,Mingda Zhang,Huan Cui,Shishan Dong

Journal:

Diagnosis, Prevention and Control in Avian Virus Infections

Year:

2024

Abstract:

The H9 subtype of avian influenza virus (AIV) has been characterized by its rapid spread, wide range of prevalence, and continuous evolution in recent years, leading to an increasing ability for cross-species transmission. This not only severely impacts the economic benefits of the aquaculture industry, but also poses a significant threat to human health. Therefore, developing a rapid and sensitive detection method is crucial for the timely diagnosis and prevention of H9 AIVs. In this study, a real-time fluorescent reverse transcription recombinase-aided isothermal amplification (RT-RAA) technique targeting the hemagglutinin (HA) of H9 AIVs was established. This technique can be used for detection in just 30 min at a constant temperature of 42 °C, and it exhibits good specificity without cross-reactivity with other viruses. Sensitivity tests revealed that the detection limit of RT-RAA was 163 copies per reaction, and the visual detection limit was 1759 copies per reaction at a 95% confidence interval, both of which are capable of detecting low concentrations of standards. Furthermore, RT-RAA was applied to detect 155 clinical samples, and compared to real-time fluorescent quantitative PCR (RT-qPCR), RT-RAA demonstrated high accuracy, with a specificity of 100% and a kappa value of 0.96, indicating good correlation. Additionally, with the assistance of a portable blue imaging device, we can visually observe the amplification products, greatly facilitating rapid detection in resource-limited environments. The RT-RAA detection method developed in this study does not require expensive equipment or highly skilled staff, making it beneficial for the accurate and low-cost detection of H9 AIVs.