Advertisement

Microchimica Acta

, Volume 183, Issue 3, pp 1195–1202 | Cite as

Parallel solid-phase isothermal amplification and detection of multiple DNA targets in microliter-sized wells of a digital versatile disc

  • Sara Santiago-Felipe
  • Luis Antonio Tortajada-Genaro
  • Rosa Puchades
  • Ángel Maquieira
Original Paper

Abstract

An integrated method for the parallelized detection of multiple DNA target sequences is presented by using microstructures in a digital versatile disc (DVD). Samples and reagents were managed by using both the capillary and centrifugal forces induced by disc rotation. Recombinase polymerase amplification (RPA), in a bridge solid phase format, took place in separate wells, which thereby modified their optical properties. Then the DVD drive reader recorded the modifications of the transmitted laser beam. The strategy allowed tens of genetic determinations to be made simultaneously within <2 h, with small sample volumes (3 μL), low manipulation and at low cost. The method was applied to high-throughput screening of relevant safety threats (allergens, GMOs and pathogenic bacteria) in food samples. Satisfactory results were obtained in terms of sensitivity (48.7 fg of DNA) and reproducibility (below 18 %). This scheme warrants cost-effective multiplex amplification and detection and is perceived to represent a viable tool for screening of nucleic acid targets.

Graphical Abstract

A low-cost method is presented for high-throughput screening of multiple DNA sequences. Parallelized isothermal amplification takes place in microwells integrated into a digital versatile disc. Optical detection is performed by a disc drive.

Keywords

Multiplex PCR DNA Recombinase polymerase amplification (RPA) Compact disc High-throughput screening Food safety 

Notes

Acknowledgments

This research has been funded through projects FEDER PrometeoII/2014/040 (GVA), and CTQ/2013/45875-R (MINECO). The Spanish Ministry of Education and Science provided S.S.F. with a grant for PhD studies.

Supplementary material

604_2016_1745_MOESM1_ESM.docx (829 kb)
ESM 1 (DOCX 828 kb)

References

  1. 1.
    Li Y, Guo SJ, Shao N, Tu S, Xu M, Ren ZR, Ling X, Wang GQ, Lin ZX, Tao SC (2011) A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray. Lab Chip 11:3609–3618CrossRefGoogle Scholar
  2. 2.
    Ng JK, Chong S (2011) Multiplexing Capabilities of Biosensors for Clinical Diagnostics. INTECH Open Access Publisher, RijekaGoogle Scholar
  3. 3.
    Shrestha HK, Hwu KK, Chang MC (2010) Advances in detection of genetically engineered crops by multiplex polymerase chain reaction methods. Trends Food Sci Tech 21:442–454CrossRefGoogle Scholar
  4. 4.
    Shao N, Jiang SM, Zhang M, Wang J, Guo SJ, Li Y, Jiang HW, Liu CX, Zhang DB, Yang LT, Tao SC (2014) MACRO: a combined microchip-PCR and microarray system for high-throughput monitoring of genetically modified organisms. Anal Chem 86:1269–1276CrossRefGoogle Scholar
  5. 5.
    Baker M (2012) Digital PCR hits its stride. Nat Methods 9:541–544CrossRefGoogle Scholar
  6. 6.
    Hoffmann J, Trotter M, von Stetten F, Zengerle R, Roth G (2012) Solid-phase PCR in a picowell array for immobilizing and arraying 100,000 PCR products to a microscope slide. Lab Chip 12:3049–3054CrossRefGoogle Scholar
  7. 7.
    Gole J, Gore A, Richards A, Chiu YJ, Fung HL, Bushman D, Chiang HI, Chun J, Lo YH, Zhang K (2013) Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells. Nat Biotechnol 31:1126–1132CrossRefGoogle Scholar
  8. 8.
    McCalla SE, Tripathi A (2011) Microfluidic reactors for diagnostics applications. Annu Rev. Biomed Eng 13:321–343Google Scholar
  9. 9.
    Westin L, Xu X, Miller C, Wang L, Edman CF, Nerenberg M (2000) Anchored multiplex amplification on a microelectronic chip array. Nat Biotechnol 18:199–204CrossRefGoogle Scholar
  10. 10.
    Khan Z, Poetter K, Park DJ (2008) Enhanced solid phase PCR: mechanisms to increase priming by solid support primers. Anal Biochem 375:391–393CrossRefGoogle Scholar
  11. 11.
    Khodakov DA, Ellis AV (2014) Recent developments in nucleic acid identification using solid-phase enzymatic assays. Microchim Acta 181:1633–1646CrossRefGoogle Scholar
  12. 12.
    Shin Y, Perera AP, Kim KW, Park MK (2013) Real-time, label-free isothermal solid-phase amplification/detection (ISAD) device for rapid detection of genetic alteration in cancers. Lab Chip 13:2106–2114CrossRefGoogle Scholar
  13. 13.
    Kersting S, Rausch V, Bier FF, von Nickisch-Rosenegk M (2014) Rapid detection of Plasmodium falciparum with isothermal recombinase polymerase amplification and lateral flow analysis. Malaria J 13:99–107CrossRefGoogle Scholar
  14. 14.
    del Río JS, Yehia Adly N, Acero-Sánchez JL, Henry OY, O’Sullivan CK (2014) Electrochemical detection of Francisella tularensis genomic DNA using solid-phase recombinase polymerase amplification. Biosens Bioelectron 54: 674–678Google Scholar
  15. 15.
    Lutz S, Weber P, Focke M, Faltin B, Hoffmann J, Müller C, Mark D, Roth G, Munday P, Armes N, Piepenburg O, Zengerle R, von Stetten F (2010) Microfluidic lab-on-a-foil for nucleic acid analysis based on isothermal recombinase polymerase amplification (RPA). Lab Chip 10:887–893CrossRefGoogle Scholar
  16. 16.
    Santiago-Felipe S, Tortajada-Genaro LA, Morais S, Puchades R, Maquieira A (2014) One-pot isothermal DNA amplification–Hybridisation and detection by a disc-based method. Sensor Actuat B-Chem 204:273–281CrossRefGoogle Scholar
  17. 17.
    Tortajada-Genaro LA, Santiago-Felipe S, Amasia M, Russom A, Maquieira A (2015) Isothermal solid-phase recombinase polymerase amplification on microfluidic digital versatile discs (DVDs). RSC Adv 5:29,987–29,995CrossRefGoogle Scholar
  18. 18.
    Wu J, Zhu Y, Xue F, Mei Z, Yao L, Wang X, Zheng L, Liu J, Liu G, Peng C, Chen W (2014) Recent trends in SELEX technique and its application to food safety monitoring. Microchimica Acta 181:479–491CrossRefGoogle Scholar
  19. 19.
    Roda A, Mirasoli M, Roda B, Bonvicini F, Colliva C, Reschiglian P (2012) Recent developments in rapid multiplexed bioanalytical methods for foodborne pathogenic bacteria detection. Microchimica Acta 178:7–28CrossRefGoogle Scholar
  20. 20.
    Xu S (2012) Electromechanical biosensors for pathogen detection. Microchimica Acta 178:245–260CrossRefGoogle Scholar
  21. 21.
    Santiago-Felipe S, Tortajada-Genaro LA, Puchades R, Maquieira A (2014) Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis. Anal Chim Acta 811:81–87CrossRefGoogle Scholar
  22. 22.
    International Organization for Standardization, Geneva, Switzerland (2005a) ISO 21569:2005. Foodstuffs-methods of analysis for the detection of genetically modified organisms and derived products-Qualitative nucleic acid based methodsGoogle Scholar
  23. 23.
    International Organization for Standardization, Geneva, Switzerland (2005b) ISO 21570:2005. Foodstuffs-methods of analysis for the detection of genetically modified organisms and derived products-Quantitative nucleic acid based methodsGoogle Scholar
  24. 24.
    Tortajada-Genaro LA, Rodrigo A, Hevia E, Mena S, Niñoles R, Maquieira A (2015) Microarray on digital versatile disc for identification and genotyping of Salmonella and Campylobacter in meat products. Analytical and bioanalytical chemistry 407:7285–7294CrossRefGoogle Scholar
  25. 25.
    Adessi C, Matton G, Ayala G, Turcatti G, Mermod JJ, Mayer P, Kawashima E (2006) Solid phase DNA amplification: characterisation of primer attachment and amplification mechanisms. Nucleic Acids Res 28:e87CrossRefGoogle Scholar
  26. 26.
    Shin Y, Kim J, Lee TY (2014) A solid phase-bridge based DNA amplification technique with fluorescence signal enhancement for detection of cancer biomarkers. Sensor Actuat B-Chem 199:220–225CrossRefGoogle Scholar
  27. 27.
    Li G, Chen Q, Li J, Hu X, Zhao J (2010) A compact disk-like centrifugal microfluidic system for high-throughput nanoliter-scale protein crystallization screening. Anal Chem 82:4362–4369CrossRefGoogle Scholar
  28. 28.
    Zhou QJ, Wang L, Chen J, Wang RN, Shi YH, Li CH, Zhang DM, Yan XJ, Zhang YJ (2014) Development and evaluation of a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP) for rapid and simultaneous detection of ten pathogenic bacteria in aquatic animals. J Microbiol Meth 104:26–35CrossRefGoogle Scholar
  29. 29.
    Focke M, Stumpf F, Roth G, Zengerle R, von Stetten F (2010) Centrifugal microfluidic system for primary amplification and secondary real-time PCR. Lab Chip 10:3210–3212CrossRefGoogle Scholar
  30. 30.
    Gorkin R, Park J, Siegrist J, Amasia M, Lee BS, Park JM, Kim J, Kim H, Madou M, Cho YK (2010) Centrifugal microfluidics for biomedical applications. Lab Chip 10:1758–1773CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2016

Authors and Affiliations

  • Sara Santiago-Felipe
    • 1
  • Luis Antonio Tortajada-Genaro
    • 1
  • Rosa Puchades
    • 1
  • Ángel Maquieira
    • 1
  1. 1.Instituto Interuniversitario de Reconocimiento Molecular y Desarrollo Tecnológico (IDM) - Departamento de QuímicaUniversitat Politècnica de ValènciaValenciaSpain

Personalised recommendations