Abstract
Clinical tests based on primer-initiated amplification of specific nucleic acid sequences achieve high levels of sensitivity and specificity. Despite these desirable characteristics, these tests have not reached their full potential because their complexity and expense limit their usefulness to centralized laboratories. This paper describes a device that integrates sample preparation and loop-mediated isothermal amplification (LAMP) with end point detection using a hand-held UV source and camera phone. The prototype device integrates paper microfluidics (to enable fluid handling) and a multilayer structure, or a "paper machine", that allows a central patterned paper strip to slide in and out of fluidic path and thus allows introduction of sample, wash buffers, amplification master mix, and detection reagents with minimal pipetting, in a hand-held, disposable device intended for point-of-care use in resource-limited environments. This device creates a dynamic seal that prevents evaporation during incubation at 65 °C for 1 h. This interval is sufficient to allow a LAMP reaction for the Escherichia coli malB gene to proceed with an analytical sensitivity of 1 double-stranded DNA target copy. Starting with human plasma spiked with whole, live E. coli cells, this paper demonstrates full integration of sample preparation with LAMP amplification and end point detection with a limit of detection of 5 cells. Further, it shows that the method used to prepare sample enables concentration of DNA from sample volumes commonly available from fingerstick blood draw.
Highlights
The unparalleled sensitivity and specificity of nucleic acid amplification tests (NAATs), led by those based on polymerase chain reaction (PCR), have become increasingly available in the developed world for the diagnosis of infectious, genetic, and other diseases
Starting with human plasma spiked with whole, ACS Paragon Plus Environment live E. coli cells, this paper demonstrates full integration of sample preparation with Loop-mediated Isothermal Amplification (LAMP)
Prior to amplification NAATs usually require sample preparation involving a series of steps to release the DNA, and to remove the many inhibitory compounds found in clinical samples[1,2]
Summary
The unparalleled sensitivity and specificity of nucleic acid amplification tests (NAATs), led by those based on polymerase chain reaction (PCR), have become increasingly available in the developed world for the diagnosis of infectious, genetic, and other diseases. In developing countries these tests are much less used because of their cost, complexity and requirements for expensive equipment and reagents. Prior to amplification NAATs usually require sample preparation involving a series of steps to release the DNA, and to remove the many inhibitory compounds found in clinical samples[1,2] These steps often require the use of complex laboratory equipment and, highly trained personnel. Because transporting patients and/or samples to these central facilities can be difficult, slow, and expensive in resource-limited environments with under-developed transportation systems, local healthcare outposts and traveling healthcare workers are the often the primary, or only, providers of healthcare[3,4,5,6]
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