RPB0290

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
RPA3-F TAATACGACTCACTATAGGGCAGGAGTCGGAATGCGTTTG 40 100μM 47.5 66.4 12400.1 \
RPA3-R TGGGCTAATATTTATAATTTTCCCCTCTCT 30 100μM 33.33 54.71 9087.97 \

Gene Description

Target Gene GenBank ID
HA gene KM245331.1

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
a diagnostic method that is quick in detection, capable of identifying very small amounts of pathogenic DNA\RNA, cost-effective, favorable for field diagnosis and easy to use. RPA-CRISPR\Cas13a primer 5.0 1.5h 37°C CRISPR\Cas13a system 10° copies/μL 10° copies/μL \

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2024 CRISPR\Cas13a-based genome editing for establishing the detection method of H9N2 subtype avian influenza virus Sha-Sha Chen,Yong-Lei Yang,Hong-Yun Wang,Tian-Kui Guo,Riaz-M Azeem,Chun-Wei Shi,Gui-Lian Yang,Hai-Bin Huang,Yan-Long Jiang,Jian-Zhong Wang,Xin Cao,Nan Wang,Yan Zeng,Wen-Tao Yang,Chun-Feng Wang Poultry Science 39096825 10.1016/j.psj.2024.104068

CRISPR\Cas13a-based genome editing for establishing the detection method of H9N2 subtype avian influenza virus

Author(s):

Sha-Sha Chen,Yong-Lei Yang,Hong-Yun Wang,Tian-Kui Guo,Riaz-M Azeem,Chun-Wei Shi,Gui-Lian Yang,Hai-Bin Huang,Yan-Long Jiang,Jian-Zhong Wang,Xin Cao,Nan Wang,Yan Zeng,Wen-Tao Yang,Chun-Feng Wang

Journal:

Poultry Science

Year:

2024

Abstract:

Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA (recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimize the constructed assay. The sensitivity of the optimized detection system is excellent, with a minimum detection limit of 10° copies/μL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV.