| The burden of infectious disease is high due to delayed disease detection, especially in developing countries and rural areas. Point-of-care testing (POCT) promises to revolutionize diagnostics by enabling rapid clinical tests at near-patient settings and in resource-poor environments. The objective of this research has been to develop a rapid, reliable, low-cost, disposable microfluidic diagnostic device for POC nucleic acid testing (POC-NAT). The microfluidic device was fabricated from thermoplastic material using hot embossing and thermal bonding techniques and the external fluidic and thermal controls were simplified to make them cost-efficient for disposable applications. A continuous flow polymerase chain reaction (CF-PCR) device for nucleic acid amplification was first developed and optimized by thermal and fluidic modeling. In order to overcome surface inhibition of PCR, a quantitative study was performed using response surface methodology (RSM) to analyze the effects of the related factors. The optimized PCR device was integrated with upstream sample preparation for the POC-NAT, detecting C.Difficile deoxyribonucleic acid (DNA) extracted from patient stool samples with a lower detection limit of 1copy/50mul reaction. The new microfluidic diagnostic device was also applied to test influenza A virus in crude clinical samples. The microfluidic test was found to be more sensitive and specific than the rapid immunoassays currently used, exhibiting 100% specificity and 95.9% sensitivity based on tests of n = 146 specimens (positive predictive value=100% and negative predictive value=96% compared to a bench top quantitative PCR reaction). The microfluidic test detected influenza A in specimens with viral loads as low as 10 3 cDNA copies/ml and was able to detect influenza A ribonucleic acid (RNA) directly from clinical specimens in 3 hours or less. The new test represents a major improvement over viral culture in terms of turnaround time and sensitivity, over rapid immunoassay tests in terms of sensitivity and specificity, and over bench top RT-PCR in terms of ease of use and portability. |
