RPB0385

Pathogen Description

Target Pathogen Pathogen Name NCBI Taxonomy ID Order Family Genus Species Pathogen type
Influenza A virus (H1N1) Influenza A virus (H1N1) 114727 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
F CTCACCGTGCCCAGTGAGCGAGGACTGCAG 30 \ 66.6 72.31 9218.02 \
R CCATCCTATTGTATATGAGGCCCATGCAAC 30 \ 46.67 60.86 9126 \

Gene Description

Target Gene GenBank ID
M gene \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
Fast, sensitive, and portable diagnostic methods are essential for efficient avian influenza control. RPA-CRISPR\Cas13a \ 20 min 39°C CRISPR\Cas13a 69 copies/μL \ \

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2023 CRISPR-Cas13a-based detection method for avian influenza virus Yuhan Wu,Jiaxing Zhan,Zhaomeng Shan,Yanbing Li,Yining Liu,Yan Li,Yixin Wang,Zhe Liu,Xuexia Wen,Xiurong Wang Frontiers in Microbiology 37886067 10.3389/fmicb.2023.1288951

CRISPR-Cas13a-based detection method for avian influenza virus

Author(s):

Yuhan Wu,Jiaxing Zhan,Zhaomeng Shan,Yanbing Li,Yining Liu,Yan Li,Yixin Wang,Zhe Liu,Xuexia Wen,Xiurong Wang

Journal:

Frontiers in Microbiology

Year:

2023

Abstract:

Avian influenza virus (AIV) causes huge losses to the global poultry industry and poses a threat to humans and other mammals. Fast, sensitive, and portable diagnostic methods are essential for efficient avian influenza control. Here, a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a based platform was developed to detect AIV. This novel method was developed to specifically detect H1-H16 subtypes of AIV with fluorescence and lateral flow-based readouts and exhibited no cross-reactivity with Newcastle disease virus, avian infectious bronchitis virus, or infectious bursal disease virus. The limit of detection was determined to be 69 and 690 copies/μL using fluorescence and lateral flow as readouts, respectively. The developed assay exhibited 100% consistency with quantitative real-time polymerase chain reaction in detecting clinical samples. The heating of unextracted diagnostic samples to obliterate nuclease treatment was introduced to detect viral RNA without nucleic acid extraction. Single-step optimization was used to perform reverse transcription, recombinase polymerase amplification, and CRISPR-Cas13a detection in a tube. These advances resulted in an optimized assay that could specifically detect AIV with simplified procedures and reduced contamination risk, highlighting the potential to be used in point-of-care testing.