Simple System for Isothermal DNA Amplification Coupled to Lateral Flow Detection

Abstract

Infectious disease diagnosis in point-of-care settings can be greatly improved through integrated, automated nucleic acid testing devices. We have developed an early prototype for a low-cost system which executes isothermal DNA amplification coupled to nucleic acid lateral flow (NALF) detection in a mesofluidic cartridge attached to a portable instrument. Fluid handling inside the cartridge is facilitated through one-way passive valves, flexible pouches, and electrolysis-driven pumps, which promotes a compact and inexpensive instrument design. The closed-system disposable prevents workspace amplicon contamination. The cartridge design is based on standard scalable manufacturing techniques such as injection molding. Nucleic acid amplification occurs in a two-layer pouch that enables efficient heat transfer. We have demonstrated as proof of principle the amplification and detection of Mycobacterium tuberculosis (M.tb) genomic DNA in the cartridge, using either Loop Mediated Amplification (LAMP) or the Exponential Amplification Reaction (EXPAR), both coupled to NALF detection. We envision that a refined version of this cartridge, including upstream sample preparation coupled to amplification and detection, will enable fully-automated sample-in to answer-out infectious disease diagnosis in primary care settings of low-resource countries with high disease burden.

Current Trends in the detection of Acute HIV Infection among Blood Donors: Reliability of Pooled Nucleic Acid Amplification Technology and the Need for Population Specific Algorithms: A Systematic Review

“…In contrast, of those anti-HIV repeat reactive samples that tested NAAT nonreactive and Western blot negative or indeterminate, 98.5% had an Abbott ELISA signal-cutoff ratio less than 15 [15]. The need for an optimized and realistic algorithm and pool size that is cost effective, for different settings and incidences via-as-vis methodology (such as hard spinning), taking into account HIV 1 genetic diversity, primerprobe mismatch and platforms as summarized in Table 6 below, cannot be over emphasized [16][17][18][19][20][21][22][23]26,27,[36][37][38][39][40][41][42][43][44][45]. Cannillo et al recently, reported that CAD as the most frequent cardiac complication in HIV patients (particularly those with low CD4 and high viral load) treated with HAART.…”

“…One of these universal platforms has tested 19 pools of 10 plasma samples from West Africa and recorded a high sensitivity. This simple, rapid, and robust test format at reduced cost is valuable for the screening of blood samples by laboratory workers in blood banks or diagnostic laboratories in developing countries [20,36]. These assays are aimed at increasing analytical sensitivity breaking the antigen-antibody complex and reducing the window period at low cost.…”

“…Studies of isothermal nucleic acid amplification system coupled to a lateral flow device have been presented by a group and will be useful in the acute detection various pathogen. The heating block will aid in antigenantibody complex dissociation [20,36].…”

Pouch-Chip Immunoassays and Nucleic Acid Amplification Tests

“…Pouch chips are an attractive design option for autonomous microfluidic devices, providing a means for fluid storage (reservoirs), fluid actuation (pumping), and flow control (valving) [7][8][9][10]. By attaching or laminating flexible polymer sheet materials (e.g.…”

“…Flexible sheet can also be used to realize diaphragm valves that constrict flow in channels or seal chambers for processing. Chip design, materials, fabrication, and accompanying instrumentation and actuators are presented in detail [8][9][10].…”