A colorimetric microplate assay for determination of Staphylococcus aureus DNA is described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H2O2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450 nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.μL−1. The potential application of the method was verified by analyzing spiked food samples.
This is a preview of subscription content, log in to check access.
Buy single article
Instant unlimited access to the full article PDF.
Price includes VAT for USA
Subscribe to journal
Immediate online access to all issues from 2019. Subscription will auto renew annually.
This is the net price. Taxes to be calculated in checkout.
Kadariya J, Smith TC, Thapaliya D (2014) Staphylococcus aureus and staphylococcal food-borne disease: an ongoing challenge in public health. Biomed Res Int 2014:9. https://doi.org/10.1155/2014/827965
Wei C, Zhong J, Hu T, Zhao X (2018) Simultaneous detection of Escherichia coli O157:H7, Staphylococcus aureus and Salmonella by multiplex PCR in milk. 3. Biotech 8(1):76. https://doi.org/10.1007/s13205-018-1086-5
Zhao X, Zhao F, Wang J, Zhong N (2017) Biofilm formation and control strategies of foodborne pathogens: food safety perspectives. RSC Adv 7(58):36670–36683. https://doi.org/10.1039/C7RA02497E
Zhao X, Wei C, Zhong J, Jin S (2016) Research advance in rapid detection of foodborne Staphylococcus aureus. Biotechnol Biotechnol Equip 30(5):827–833. https://doi.org/10.1080/13102818.2016.1209433
Gill AAS, Singh S, Thapliyal N, Karpoormath R (2019) Nanomaterial-based optical and electrochemical techniques for detection of methicillin-resistant Staphylococcus aureus: a review. Microchim Acta 186(2):114. https://doi.org/10.1007/s00604-018-3186-7
Gomez A, Miller NS, Smolina I (2014) Visual detection of bacterial pathogens via PNA-based padlock probe assembly and isothermal amplification of DNAzymes. Anal Chem 86(24):11992–11998. https://doi.org/10.1021/ac5018748
Mohsen MG, Kool ET (2016) The discovery of rolling circle amplification and rolling circle transcription. Acc Chem Res 49(11):2540–2550. https://doi.org/10.1021/acs.accounts.6b00417
Dar SA, Kuenen JG, Muyzer G (2005) Nested PCR-denaturing gradient gel electrophoresis approach to determine the diversity of sulfate-reducing bacteria in complex microbial communities. Appl Environ Microbiol 71(5):2325–2330. https://doi.org/10.1128/AEM.71.5.2325-2330.2005
Fang TH, Ramalingam N, Xian-Dui D, Ngin TS, Xianting Z, Lai Kuan AT, Peng Huat EY, Hai-Qing G (2009) Real-time PCR microfluidic devices with concurrent electrochemical detection. Biosens Bioelectron 24(7):2131–2136. https://doi.org/10.1016/j.bios.2008.11.009
Cook N (2003) The use of NASBA for the detection of microbial pathogens in food and environmental samples. J Microbiol Methods 53(2):165–174. https://doi.org/10.1016/S0167-7012(03)00022-8
Sun J, Najafzadeh M, Zhang J, Vicente V, Xi L, Hoog S (2011) Molecular identification of Penicillium marneffei using rolling circle amplification. Mycoses 54:e751–e759. https://doi.org/10.1111/j.1439-0507.2011.02017.x
Xu J, Guo J, Maina SW, Yang Y, Hu Y, Li X, Qiu J, Xin Z (2018) An aptasensor for staphylococcus aureus based on nicking enzyme amplification reaction and rolling circle amplification. AnBio 549:136–142. https://doi.org/10.1016/j.ab.2018.03.013
Zhan Z, Li H, Liu J, Xie G, Xiao F, Wu X, Aguilar ZP, Xu H (2020) A competitive enzyme linked aptasensor with rolling circle amplification (ELARCA) assay for colorimetric detection of listeria monocytogenes. Food Control 107:106806. https://doi.org/10.1016/j.foodcont.2019.106806
Shi D, Huang J, Chuai Z, Chen D, Zhu X, Wang H, Peng J, Wu H, Huang Q, Fu W (2014) Isothermal and rapid detection of pathogenic microorganisms using a nano-rolling circle amplification-surface plasmon resonance biosensor. Biosens Bioelectron 62:280–287. https://doi.org/10.1016/j.bios.2014.06.066
Najafzadeh MJ, Sun J, Vicente VA, Hoog GSD (2011) Rapid identification of fungal pathogens by rolling circle amplification using Fonsecaea as a model. Mycoses 54(5):e577. https://doi.org/10.1111/j.1439-0507.2010.01995.x
Russell C, Welch K, Jarvius J, Cai Y, Brucas R, Nikolajeff F, Svedlindh P, Nilsson M (2014) Gold nanowire based electrical DNA detection using rolling circle amplification. ACS Nano 8(2):1147–1153. https://doi.org/10.1021/nn4058825
Huang R, He L, Xia Y, Xu H, Liu C, Xie H, Wang S, Peng L, Liu Y, Liu Y, He N, Li Z (2019) A sensitive Aptasensor based on a Hemin/G-Quadruplex-assisted signal amplification strategy for electrochemical detection of gastric Cancer Exosomes. Small 15(19):1900735. https://doi.org/10.1002/smll.201900735
Teng J, Ye Y, Yao L, Yan C, Cheng K, Xue F, Pan D, Li B, Chen W (2017) Rolling circle amplification based amperometric aptamer/immuno hybrid biosensor for ultrasensitive detection of Vibrio parahaemolyticus. Microchim Acta 184(9):3477–3485. https://doi.org/10.1007/s00604-017-2383-0
Yao L, Ye Y, Teng J, Xue F, Pan D, Li B, Chen W (2017) In vitro isothermal nucleic acid amplification assisted surface-enhanced Raman spectroscopic for ultrasensitive detection of Vibrio parahaemolyticus. Anal Chem 89(18):9775–9780. https://doi.org/10.1021/acs.analchem.7b01717
Tian B, Han Y, Fock J, Strömberg M, Leifer K, Hansen MF (2019) Self-assembled magnetic nanoparticle–Graphene oxide Nanotag for Optomagnetic detection of DNA. ACS Appl Nano Mater 2(3):1683–1690. https://doi.org/10.1021/acsanm.9b00127
Gou D, Xie G, Li Y, Zhang X, Chen H (2018) Voltammetric immunoassay for Mycobacterium tuberculosis secretory protein MPT64 based on a synergistic amplification strategy using rolling circle amplification and a gold electrode modified with graphene oxide, Fe3O4 and Pt nanoparticles. Microchim Acta 185(9):436. https://doi.org/10.1007/s00604-018-2972-6
Yao C, Xiang Y, Deng K, Xia H, Fu W (2013) Sensitive and specific HBV genomic DNA detection using RCA-based QCM biosensor. Sensors Actuators B Chem 181:382–387. https://doi.org/10.1016/j.snb.2013.01.063
Yang X, Yang K, Zhao X, Lin Z, Liu Z, Luo S, Zhang Y, Wang Y, Fu W (2017) Terahertz spectroscopy for the isothermal detection of bacterial DNA by magnetic bead-based rolling circle amplification. Analyst 142:4661–4669. https://doi.org/10.1039/C7AN01438D
Wang J, Li H, Li T, Ling L (2018) Determination of bacterial DNA based on catalytic oxidation of cysteine by G-quadruplex DNAzyme generated from asymmetric PCR: application to the colorimetric detection of Staphylococcus aureus. Microchim Acta 185(9):410. https://doi.org/10.1007/s00604-018-2935-y
Bendayan M (2000) A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology. Biotech Histochem 75(5):203–242. https://doi.org/10.3109/10520290009068433
Liu L, Xia N, Liu H, Kang X, Liu X, Xue C, He X (2014) Highly sensitive and label-free electrochemical detection of microRNAs based on triple signal amplification of multifunctional gold nanoparticles, enzymes and redox-cycling reaction. Biosens Bioelectron 53:399–405. https://doi.org/10.1016/j.bios.2013.10.026
Lai W, Zeng Q, Tang J, Zhang M, Tang D (2018) A conventional chemical reaction for use in an unconventional assay: a colorimetric immunoassay for aflatoxin B1 by using enzyme-responsive just-in-time generation of a MnO2 based nanocatalyst. Microchim Acta 185(2):92. https://doi.org/10.1007/s00604-017-2651-z
Ambrosi A, Castañeda MT, Killard AJ, Smyth MR, Alegret S, Merkoçi A (2007) Double-codified gold Nanolabels for enhanced Immunoanalysis. Anal Chem 79(14):5232–5240. https://doi.org/10.1021/ac070357m
Szemes M, Bonants P, de Weerdt M, Baner J, Landegren U, Schoen CD (2005) Diagnostic application of padlock probes—multiplex detection of plant pathogens using universal microarrays. NAR 33(8):e70–e70. https://doi.org/10.1093/nar/gni069
Li J, Chu X, Liu Y, Jiang J-H, He Z, Zhang Z, Shen G, Yu R-Q (2005) A colorimetric method for point mutation detection using high-fidelity DNA ligase. NAR 33:e168. https://doi.org/10.1093/nar/gni163
Hua D, Xu Z, Anil K, Guozang Z, Xiaoning Z, Xing-Jie L (2013) Long genomic DNA amplicons adsorption onto unmodified gold nanoparticles for colorimetric detection of bacillus anthracis. ChCom 49(1):51–53. https://doi.org/10.1039/c2cc37037a
Ning Y, Gao Q, Zhang X, Wei K, Chen L (2016) A Graphene oxide-based sensing platform for the determination of methicillin-resistant Staphylococcus aureus based on Strand-displacement polymerization recycling and synchronous fluorescent signal amplification. J Biomol Screen 21(8):851–857. https://doi.org/10.1177/1087057116653564
Zhang S, Wu Z, Shen G, Yu R (2009) A label-free strategy for SNP detection with high fidelity and sensitivity based on ligation-rolling circle amplification and intercalating of methylene blue. Biosens Bioelectron 24(11):3201–3207. https://doi.org/10.1016/j.bios.2009.03.012
Xiang Y, Zhu X, Huang Q, Zheng J, Fu W (2015) Real-time monitoring of mycobacterium genomic DNA with target-primed rolling circle amplification by a Au nanoparticle-embedded SPR biosensor. Biosens Bioelectron 66:512–519. https://doi.org/10.1016/j.bios.2014.11.021
Xiang Y, Deng K, Xia H, Yao C, Chen Q, Zhang L, Liu Z, Fu W (2013) Isothermal detection of multiple point mutations by a surface plasmon resonance biosensor with Au nanoparticles enhanced surface-anchored rolling circle amplification. Biosens Bioelectron 49:442–449. https://doi.org/10.1016/j.bios.2013.04.044
The authors are grateful for providing language help from the American Journal Experts.
This work was supported by “The National Key R&D Program of China” (No.2016YFD0401202), Special Project of Tianjin Innovation Platform (No.17PTGCCX00230), Tianjin science and technology planning project(No. 18PTSYJC00130).
Conflict of interest
The author(s) declare that they have no conflict of interest.
Human and Animal Rights
This article does not contain any studies with laboratory animals or human participants performed by any of the authors.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
About this article
Cite this article
Li, Y., Wang, J., Wang, S. et al. Rolling circle amplification based colorimetric determination of Staphylococcus aureus. Microchim Acta 187, 119 (2020). https://doi.org/10.1007/s00604-019-4082-5
- Rolling circle amplification (RCA)
- Colorimetric microplate assay
- Multifunctional gold nanoparticles
- Biotin–streptavidin system
- Staphylococcus aureus