RPB0152

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
RPA-NUC-FW TGGTCCTGAAGCAAGTGCTTTTACGAAAAA 30 0.42 40 60.32 9254.09 \
RPA-NUC-RV TCTCTTTTTTCGCTTGTGCTTCACTTTTTCTTA 33 0.42 33.3 58.01 9949.49 \

Gene Description

Target Gene GenBank ID
nuc \

Assay Description

Application Assay Primer Designing Software Reaction Time(min) Assay Temperature(℃) Readout System(s) Limit of Detection(LoD) Sensitivity(%) Specificity(%)
identification of Staphylococcus aureus RPA-CRISPR/Cas12a assay \ 40 39 CRISPR/Cas12a assay 10² copies per reaction \ \

Publication Description

Year of Publication Title Author(s) Journal PMID DOI
2023 Genetic identification of Staphylococcus aureus isolates from cultured milk samples of bovine mastitis using isothermal amplification with CRISPR/Cas12a-based molecular assay Meruyert Amanzholova,Aisha Shaizadinova,Aitbay Bulashev,Sailau Abeldenov Veterinary research communications 37673833 10.1007/s11259-023-10212-z

Genetic identification of Staphylococcus aureus isolates from cultured milk samples of bovine mastitis using isothermal amplification with CRISPR/Cas12a-based molecular assay

Author(s):

Meruyert Amanzholova,Aisha Shaizadinova,Aitbay Bulashev,Sailau Abeldenov

Journal:

Veterinary research communications

Year:

2023

Abstract:

Bovine mastitis, a common and costly disease in dairy cattle, is primarily caused by Staphylococcus aureus. Timely and accurate detection of this pathogen is crucial for effective disease management. In this study, we developed and validated a novel molecular diagnostic assay based on the CRISPR/Cas12a system coupled with Recombinase Polymerase Amplification (RPA) and Loop-Mediated Isothermal Amplification (LAMP). We utilized specific primers targeting the nucleotide sequences of the S.aureus genes of interest, such as nuc and sea. RPA/LAMP reactions were performed under optimized conditions, and the resulting products were subsequently subjected to CRISPR/Cas12a detection. The CRISPR/Cas12a assay successfully detected the target nuc and sea genes, with a limit of detection of 104 and 102 gene copies per reaction, respectively. All 13 S.aureus clinical isolates were identified by RPA-CRISPR/Cas12a assay. The total reaction time is approximately 1 h. The assay demonstrated high sensitivity for the detection of S.aureus in both laboratory and clinical samples.