RPB0276

Pathogen Description

Target Pathogen Pathogen Name NCBI Taxonomy ID Order Family Genus Species Pathogen type
Staphylococcus aureus Staphylococcus aureus, Micrococcus aureus, Staphylococcus pyogenes aureus 1280 Bacillales Staphylococcaceae Staphylococcus Staphylococcus aureus Bacterium

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
S.A -F1 ACTAAAAAATTACATAAAGAACCTGCGACA 30 250 nM 30 54.11 9193.11 \
S.A -R1 GTCTGAATGTCATTGGTTGACCTTTGTACA 30 250 nM 40 58.51 9218.04 \
Nuc-P FAM-CAAGTCTAAGTAGCTCAGCAAATGCATCACA-BHQ1 31 1 µM 41.94 60.13 9481.26 \

Gene Description

Target Gene GenBank ID
nuc gene \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
rapid, efficient, and ultrasensitive detection of pathogens in real food samples. RPA-CRISPR\Cas12a Primer Premier 5 and NCBI BLAST 40 min 37 °C CRISPR\Cas12a assay 1 copy/μL 5 copies \

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2024 Efficient magnetic enrichment cascade single-step RPA-CRISPR\Cas12a assay for rapid and ultrasensitive detection of Staphylococcus aureus in food samples Yujie Ma,Hongjuan Wei,Yunxiang Wang,Xiaodan Cheng,Hong Chen,Xingsheng Yang,Hongsheng Zhang,Zhen Rong,Shengqi Wang Journal Of Hazardous Materials 38228008 10.1016/j.jhazmat.2024.133494

Efficient magnetic enrichment cascade single-step RPA-CRISPR\Cas12a assay for rapid and ultrasensitive detection of Staphylococcus aureus in food samples

Author(s):

Yujie Ma,Hongjuan Wei,Yunxiang Wang,Xiaodan Cheng,Hong Chen,Xingsheng Yang,Hongsheng Zhang,Zhen Rong,Shengqi Wang

Journal:

Journal Of Hazardous Materials

Year:

2024

Abstract:

Staphylococcus aureus (S. aureus) is a prevalent foodborne pathogen that could cause severe food poisoning. Thus, rapid, efficient, and ultrasensitive detection of S. aureus in food samples is urgently needed. Here, we report an efficient magnetic enrichment cascade single-step recombinase polymerase amplification (RPA)-CRISPR/Cas12a assay for the ultrasensitive detection of S. aureus. Magnetic beads (MBs) functionalized with S. aureus-specific antibodies were initially used for S. aureus enrichment from the complex matrix, with 98% capture efficiency in 5 min and 100-fold sensitivity improvement compared with unenriched S. aureus. Next, a single-step RPA-CRISPR/Cas12a-based diagnostic system with optimized extraction-free bacteria lysis was constructed. This assay could detect as low as 1 copy/μL (five copies/reaction) of extracted DNA template and 10 CFU/mL of S. aureus within 40 min. Furthermore, the assay could effectively detect S. aureus in real food samples such as lake water, orange juice, pork, and lettuce, with concordant results to qPCR assays. The proposed cascade signal-amplification assay eliminates the need for lengthy bacterial culture and complex sample preparation steps. Hence, the proposed assay shows great application potential for rapid, efficient, and ultrasensitive detection of pathogens in real food samples.