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Microfluidics and Nanofluidics

, Volume 14, Issue 3–4, pp 731–741 | Cite as

Sub-microliter scale in-gel loop-mediated isothermal amplification (LAMP) for detection of Mycobacterium tuberculosis

  • Dammika P. Manage
  • Linda Chui
  • Linda M. PilarskiEmail author
Research Paper

Abstract

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a common human disease that is prevalent in resource-deprived areas of the world. Current detection techniques for TB require expensive conventional instruments in a laboratory setting, preventing accessible and low cost diagnosis of the disease. Using a loop-mediated isothermal amplification (LAMP) assay, we have amplified and detected TB in a 6 × 8 semisolid polyacrylamide gel post array using an inexpensive prototype instrument. Each post contains 670 nL of volume, minimizing the need for large quantities of reagents. Amplified DNA is detected via fluorescence of the dye LCGreen Plus+, which is polymerized into the gel along with other reagents. The prototype device contains a Peltier element for heating, a diode laser as an excitation source, and a CCD camera for detecting fluorescence in real-time. About 12 Mycobacterium tuberculosis genomes per gel post can be detected within 75 min of amplification. This sensitivity is similar to that obtained by conventional methods using a commercial thermocycler. We achieved comparable LAMP amplification when the template is added externally or when the template is polymerized in the gel. This rapid isothermal amplification technology, with its simple thermal requirements, has the potential to be integrated into micro-devices and serves as a model for implementing future low-cost point of care diagnostics.

Keywords

Miniaturization Point-of-care Tuberculosis In-gel DNA amplification Isothermal 

Abbreviations

LAMP

Loop-mediated isothermal amplification

R-LAMP

Real-time LAMP

R-PCR

Real-time-PCR

MCA

Melting curve analysis

LRE

Linear regression of efficiency

Notes

Acknowledgments

This work was funded by AHFMR Interdisciplinary Team Grants Program.

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Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Dammika P. Manage
    • 1
  • Linda Chui
    • 2
    • 3
  • Linda M. Pilarski
    • 1
    • 3
    Email author
  1. 1.Department of OncologyUniversity of Alberta and Cross Cancer InstituteEdmontonCanada
  2. 2.Provincial Laboratory for Public HealthEdmontonCanada
  3. 3.Department of Laboratory Medicine and PathologyUniversity of AlbertaEdmontonCanada

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