Genetic identification of Staphylococcus aureus isolates from cultured milk samples of bovine mastitis using isothermal amplification with CRISPR/Cas12a-based molecular assay
Amanzholova M. Shaizadinova A. Bulashev A. Abeldenov S.
February 2024Springer Science and Business Media B.V.
Veterinary Research Communications
2024#48Issue 1291 - 300 pp.
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.
Cas endonuclease , Cas12a , CRISPR , LAMP , RPA , Staphylococcus aureus
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National Center for Biotechnology, Astana, 010000, Kazakhstan
L.N. Gumilyov Eurasian National University, Astana, Kazakhstan
Al-Farabi Kazakh National University, Almaty, Kazakhstan
S. Seifullin Kazakh Agrotechnical Research University, Astana, Kazakhstan
National Center for Biotechnology
L.N. Gumilyov Eurasian National University
Al-Farabi Kazakh National University
S. Seifullin Kazakh Agrotechnical Research University
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