A Loop-Mediated Isothermal Amplification Integrated G-Quadruplex Molecular Beacon (LAMP-GMB) Method for the Detection of Staphylococcus aureus in Food
- 60 Downloads
Staphylococcus aureus is a common pathogen in foodstuffs, and an effective detection assay is therefore essential for ensuring food safety. In this study, we established a label-free loop-mediated isothermal amplification integrated G-quadruplex molecular beacon (LAMP-GMB) chemiluminescence method for S. aureus detection based on its nuc gene that encodes a thermostable nuclease. Target DNA was amplified using the LAMP technique; the result LAMP products were specially recognized by GMB detection probe, subsequently induced the release of G-rich sequence, and formed a complex spatial structure G-quadruplex. The structure can bind with hemin and catalyze the H2O2-mediated oxidation of luminol reagent to produce the chemiluminescence signal for chemiluminescent detection. Under the optimized conditions, the developed method showed a good linear relationship (R2 = 0.9882) in the range of 101-105 fg μL−1 and was able to detect as low as 10 fg μL−1 genomic DNA from S. aureus. Using spiked milk samples, the method exhibited a linear relationship (R2 = 0.9893) ranging from 5 × 101 to 5 × 107 CFU mL−1 with the limit of detection of 50 CFU mL−1. Importantly, the detection assay did not require the preparation and use of labeled primers for the LAMP reaction, increasing the reliability and efficiency of the assay. Therefore, the developed LAMP-GMB method has the potential to be widely applied to S. aureus monitoring work in food field.
KeywordsStaphylococcus aureus LAMP G-quadruplex Luminol Chemiluminescence
This study was funded by the Fund for Qing Lan Project of Jiangsu, the Fundamental Research Funds for the Central Universities (KYYJ201708), and Special Funds of Agro-Product Quality Safety Risk Assessment of Ministry of Agriculture of the People’s Republic of China (GJFP201701505).
Compliance with Ethical Standards
Conflict of Interest
Jingguo Xu declares that he has no conflict of interest. Yimin Hu declares that she has no conflict of interest. Jia Guo declares that she has no conflict of interest. Yumeng Yang declares that she has no conflict of interest. Jiarong Qiu declares that she has no conflict of interest. Xuanxuan Li declares that he has no conflict of interest. Zhihong Xin declares that he has no conflict of interest
This article does not contain any studies with human participants or animals performed by any of the authors.
- Chen Z, Liao Y, Ke X, Zhou J, Chen Y, Gao LL, Chen Q, Yu S (2011) Comparison of reverse transcription loop-mediated isothermal amplification, conventional PCR and real-time PCR assays for Japanese encephalitis virus. Mol Biol Rep 38:4063–4070. https://doi.org/10.1007/s11033-010-0525-0 CrossRefPubMedGoogle Scholar
- Feuillie C, Merheb MM, Gillet B, Montagnac G, Daniel I, Hänni C (2014) Detection of DNA sequences refractory to PCR amplification using a biophysical SERRS assay (surface enhanced resonant Raman spectroscopy). PLoS One 9:e114148. https://doi.org/10.1371/journal.pone.0114148 CrossRefPubMedPubMedCentralGoogle Scholar
- Gao F, Fan T, Wu J, Liu S, du Y, Yao Y, Zhou F, Zhang Y, Liao X, Geng D (2017) Proximity hybridization triggered hemin/G-quadruplex formation for construction a label-free and signal-on electrochemical DNA sensor. Biosens Bioelectron 96:62–67. https://doi.org/10.1016/j.bios.2017.04.024 CrossRefPubMedGoogle Scholar
- Hirayama H, Kageyama S, Moriyasu S, Sawai K, Onoe S, Takahashi Y, Katagiri S, Toen K, Watanabe K, Notomi T, Yamashina H, Matsuzaki S, Minamihashi A (2004) Rapid sexing of bovine preimplantation embryos using loop-mediated isothermal amplification. Theriogenology 62:887–896. https://doi.org/10.1016/j.theriogenology.2003.12.007 CrossRefPubMedGoogle Scholar
- Kumar D, Chauhan TKS, Agarwal RK, Dhama K, Goswami PP, Mariappan AK, Tiwari AK, Mishra BP (2017) A double-stranded probe coupled with isothermal amplification for qualitative and quantitative detection of avian reovirus. Arch Virol 162:979–985. https://doi.org/10.1007/s00705-016-3184-1 CrossRefPubMedGoogle Scholar
- Liu W, Huang S, Liu N, Dong D, Yang Z, Tang Y, Ma W, He X, Ao D, Xu Y, Zou D, Huang L (2017) Establishment of an accurate and fast detection method using molecular beacons in loop-mediated isothermal amplification assay. Sci Rep 7:40125. https://doi.org/10.1038/srep40125 CrossRefPubMedPubMedCentralGoogle Scholar
- Nurul Najian AB, Engku Nur Syafirah EA, Ismail N, Mohamed M, Yean CY (2016) Development of multiplex loop mediated isothermal amplification (m-LAMP) label-based gold nanoparticles lateral flow dipstick biosensor for detection of pathogenic Leptospira. Anal Chim Acta 903:142–148. https://doi.org/10.1016/j.aca.2015.11.015 CrossRefPubMedGoogle Scholar
- Wang Y, Li H, Wang Y, Zhang L, Xu J, Ye C (2017) Loop-mediated isothermal amplification label-based gold nanoparticles lateral flow biosensor for detection of enterococcus faecalis and Staphylococcus aureus. Front Microbiol 8:192. https://doi.org/10.3389/fmicb.2017.00192 CrossRefPubMedPubMedCentralGoogle Scholar
- Wolk DM, Struelens MJ, Pancholi P, Davis T, Della-Latta P, Fuller D, Picton E, Dickenson R, Denis O, Johnson D, Chapin K (2009) Rapid detection of Staphylococcus aureus and methicillin-resistant S. aureus (MRSA) in wound specimens and blood cultures: multicenter preclinical evaluation of the Cepheid Xpert MRSA/SA skin and soft tissue and blood culture assays. J Clin Microbiol 47:823–826. https://doi.org/10.1128/JCM.01884-08 CrossRefPubMedPubMedCentralGoogle Scholar
- Yang R, Zhang H, Li X, Ye L, Gong M, Yang J, Yu J, Bai J (2018b) A multiplex loop-mediated isothermal amplification assay for rapid screening of Acinetobacter baumannii and D carbapenemase OXA-23 gene. Biosci Rep 38:BSR20180425. https://doi.org/10.1042/bsr20180425 CrossRefPubMedPubMedCentralGoogle Scholar