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.
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Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
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Acknowledgements
We thank Dr. G. Chuzhebayeva (associate professor, Department of Veterinary Sanitation, A.Baitursynov Kostanay Regional University) for providing genomic DNA of S.aureus isolates.
Funding
This research was funded by the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (Grant No. AP09259771) and the Ministry of Agriculture of the Republic of Kazakhstan (BR10764944).
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M.A., A.S., A.B. and S.A. wrote the main manuscript text. M.A., A.S. and S.A. prepared all figures. All authors reviewed the manuscript.Conceptualization, [Sailau Abeldenov] S.A.; methodology, S.A., [Aisha Shaizadinova] A.S., [Meruyert Amanzholova] M.A.; software, S.A., A.S., M.A.; validation, S.A., A.S., M.A.; formal analysis, [Aitbay Bulashev] A.B., S.A.; investigation, S.A., A.S., M.A.; resources, S.A.; data curation, S.A., A.S., M.A.; writing—original draft preparation, S.A., A.S., M.A.; writing—review and editing, S.A., A.S., M.A., A.B.; visualization, S.A.; supervision, S.A.; project administration, S.A.; funding acquisition, S.A., A.B.
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DNA samples were provided by Dr. G. Chuzhebayeva, and this was confirmed by the Local Bioethics Commission of A.Baitursynov Kostanay Regional University.
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Amanzholova, M., Shaizadinova, A., Bulashev, A. et al. Genetic identification of Staphylococcus aureus isolates from cultured milk samples of bovine mastitis using isothermal amplification with CRISPR/Cas12a-based molecular assay. Vet Res Commun (2023). https://doi.org/10.1007/s11259-023-10212-z
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DOI: https://doi.org/10.1007/s11259-023-10212-z