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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.
References
Ai JW, Zhou X, Xu T, Yang M, Chen Y, He GQ, Pan N, Cai Y, Li Y, Wang X, Su H, Wang T, Zeng W, Zhang WH (2019) CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis. Emerg Microbes Infect 8:1361–1369. https://doi.org/10.1080/22221751.2019.1664939
Chen JS, Ma E, Harrington LB, Costa D, Tian M, Palefsky X, Doudna JA (2018) CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360:436–439. https://doi.org/10.1126/science.aar6245
Dai B, Xiang A, Qu D, Chen G, Wang L, Wang W, Zhai D, Wang L, Lu Z (2022) Rapid and Sensitive Assay of Helicobacter pylori with one-tube RPA-CRISPR/Cas12 by portable array detector for visible analysis of thermostatic nucleic acid amplification. Front Microbiol 13:858247. https://doi.org/10.3389/fmicb.2022.858247
Doudna JA, Charpentier E (2014) Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science 346:1258096. https://doi.org/10.1126/science.1258096
Gomes F, Henriques M (2016) Control of bovine Mastitis: Old and recent therapeutic approaches. Curr Microbiol 72:377–382. https://doi.org/10.1007/s00284-015-0958-8
Goto M, Hayashidani H, Takatori K, Hara-Kudo Y (2007) Rapid detection of enterotoxigenic Staphylococcus aureus harbouring genes for four classical enterotoxins, SEA, SEB, SEC and SED, by loop-mediated isothermal amplification assay. Lett Appl Microbiol 45:100–107. https://doi.org/10.1111/j.1472-765X.2007.02142.x
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337:816–821. https://doi.org/10.1126/science.1225829
Karimzadeh R, Ghassab RK (2022) Identification of nuc nuclease and sea enterotoxin genes in Staphylococcus aureus isolates from nasal mucosa of burn hospital staff: a cross-sectional study. New Microbes New Infect 47:100992. https://doi.org/10.1016/j.nmni.2022.100992
Katsuda K, Hata E, Kobayashi H, Kohmoto M, Kawashima K, Tsunemitsu H, Eguchi M (2005) Molecular typing of Staphylococcus aureus isolated from bovine mastitic milk on the basis of toxin genes and coagulase gene polymorphisms. Vet Microbiol 105:301–305. https://doi.org/10.1016/j.vetmic.2004.12.004
Kozhakhmetova S, Zholdybayeva E, Tarlykov P, Atavliyeva S, Syzdykov T, Daniyarov A, Mukhtarova K, Ramankulov Y (2021) Determinants of resistance in Bacteroides fragilis strain BFR_KZ01 isolated from a patient with peritonitis in Kazakhstan. J Glob Antimicrob Resist 25:1–4. https://doi.org/10.1016/j.jgar.2021.02.022
Lee SY, Oh SW (2022) Filtration-based LAMP-CRISPR/Cas12a system for the rapid, sensitive and visualized detection of Escherichia coli O157:H7. Talanta 241:123186. https://doi.org/10.1016/j.talanta.2021.123186
Leijon M, Atkins E, Persson Waller K, Artursson K (2021) Longitudinal study of Staphylococcus aureus genotypes isolated from bovine clinical mastitis. J Dairy Sci 104:11945–11954. https://doi.org/10.3168/jds.2021-20562
Liu X, Qiu X, Han L, Yue Y, Xu S, Li F, Yao J, Sun L, Li Z (2023) Three novel Cas12a orthologs with robust DNA cleavage activity suitable for nucleic acid detection. Gene 852:147055. https://doi.org/10.1016/j.gene.2022.147055
Mustafa MI, Makhawi AM (2021) SHERLOCK and DETECTR: CRISPR-Cas Systems as potential Rapid Diagnostic Tools for Emerging Infectious Diseases. J Clin Microbiol 59:e00745-20. https://doi.org/10.1128/jcm.00745-20
Normanno G, Corrente M, La Salandra G, Dambrosio A, Quaglia NC, Parisi A, Greco G, Bellacicco AL, Virgilio S, Celano GV (2007) Methicillin-resistant Staphylococcus aureus (MRSA) in foods of animal origin product in Italy. Int J Food Microbiol 117:219–222. https://doi.org/10.1016/j.ijfoodmicro.2007.04.006
Stella S, Mesa P, Thomsen J, Paul B, Alcón P, Jensen SB, Saligram B, Moses ME, Hatzakis NS, Montoya G (2018) Conformational activation promotes CRISPR-Cas12a catalysis and resetting of the endonuclease activity. Cell 175:1856-1871e21. https://doi.org/10.1016/j.cell.2018.10.045
Wang D, Chen G, Lyu Y, Feng E, Zhu L, Pan C, Zhang W, Liu X, Wang H (2022) A CRISPR/Cas12a-based DNAzyme visualization system for rapid, non-electrically dependent detection of Bacillus anthracis. Emerg Microbes Infect 11:428–437. https://doi.org/10.1080/22221751.2021.2012091
Xu JH, Kang L, Yuan B, Feng ZH, Li SQ, Wang J, Wang YR, Xin WW, Gao S, Li JX, Sun YS, Wang JL, Yuan Y (2022) Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis. Front Microbiol 13:901520. https://doi.org/10.3389/fmicb.2022.901520
You Y, Zhang P, Wu G, Tan Y, Zhao Y, Cao S, Song Y, Yang R, Du Z (2021) Highly specific and sensitive detection of Yersinia pestis by Portable Cas12a-UPTLFA platform. Front Microbiol 12:700016. https://doi.org/10.3389/fmicb.2021.700016
Zetsche B, Abudayyeh OO, Gootenberg JS, Scott DA, Zhang F (2020) A survey of genome editing activity for 16 Cas12a orthologs. Keio J Med 69:59–65. https://doi.org/10.2302/kjm.2019-0009-OA
Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, Van Der Oost J, Regev A, Koonin EV, Zhang F (2015) Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163:759–771. https://doi.org/10.1016/j.cell.2015.09.038
Zhang H, Yao S, Sheng R, Wang J, Li H, Fu Y, Li J, Zhang X, Zhao C (2022) A cascade amplification strategy for ultrasensitive Salmonella typhimurium detection based on DNA walker coupling with CRISPR-Cas12a. J Colloid Interface Sci 625:257–263. https://doi.org/10.1016/j.jcis.2022.06.027
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).
Author information
Authors and Affiliations
Contributions
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.
Corresponding author
Ethics declarations
Ethical approval
DNA samples were provided by Dr. G. Chuzhebayeva, and this was confirmed by the Local Bioethics Commission of A.Baitursynov Kostanay Regional University.
Competing interests
The authors declare no competing interests.
Conflict of interest
All authors declare no conflict of interest associated with this publication.
Additional information
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
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 48, 291–300 (2024). https://doi.org/10.1007/s11259-023-10212-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11259-023-10212-z