Microfluidic Chip for Molecular Amplification of Influenza A RNA in Human Respiratory Specimens

Abstract

A rapid, low cost, accurate point-of-care (POC) device to detect influenza virus is needed for effective treatment and control of both seasonal and pandemic strains. We developed a single-use microfluidic chip that integrates solid phase extraction (SPE) and molecular amplification via a reverse transcription polymerase chain reaction (RT-PCR) to amplify influenza virus type A RNA. We demonstrated the ability of the chip to amplify influenza A RNA in human nasopharyngeal aspirate (NPA) and nasopharyngeal swab (NPS) specimens collected at two clinical sites from 2008–2010. The microfluidic test was dramatically more sensitive than two currently used rapid immunoassays and had high specificity that was essentially equivalent to the rapid assays and direct fluorescent antigen (DFA) testing. We report 96% (CI 89%,99%) sensitivity and 100% (CI 95%,100%) specificity compared to conventional (bench top) RT-PCR based on the testing of n = 146 specimens (positive predictive value = 100% (CI 94%,100%) and negative predictive value = 96% (CI 88%,98%) ). These results compare well with DFA performed on samples taken during the same time period (98% (CI 91%,100%) sensitivity and 96% (CI 86%,99%) specificity compared to our gold standard testing). Rapid immunoassay tests on samples taken during the enrollment period were less reliable (49% (CI 38%,61%) sensitivity and 98% (CI 98%,100%) specificity). The microfluidic test extracted and amplified influenza A RNA directly from clinical specimens with viral loads down to 10 3 copies/ml in 3 h or less. The new test represents a major improvement over viral culture in terms of turn around time, over rapid immunoassay tests in terms of sensitivity, and over bench top RT-PCR and DFA in terms of ease of use and portability.

Loss of GSK-3 Causes Abnormal Astrogenesis and Behavior in Mice

“…Our data suggest that over-proliferation of astrocytes causes an enlarged brain in GSK-3 mutant mice. Our results are consistent with previous studies that have shown the role of GSK-3 in proliferation of multiple cell types including neural progenitors [11], hematopoietic stem cells [43], pancreatic mesenchymal stem cells [44], fibroblast [45], skin cancer cells [46], and mammary cancer cells [47]. Pharmacological inhibition or genetic elimination of GSK-3 promotes proliferation of these cells.…”

One-heater flow-through polymerase chain reaction device by heat pipes cooling

“…4 There have been some reports on performing flow-through PCR for two-temperature thermal cycling inside the microdevice. [5][6][7][8][9][10] Some of them combine the annealing and extension stages at the same temperature region, or the extension region is not created by direct heating but by lateral heat conduction from heaters for denaturation and annealing.…”

“…The denaturation and annealing regions were designed to be narrower than the extension region. 10 On conventional layouts with three heating regions, large sized chip modules are required for a suitable arrangement of the microchannel to complete the PCR process. The developed five-temperature-region thermocycler concept overcomes these limitations.…”

Thermally multiplexed polymerase chain reaction

“…3,4 Other detection techniques, such as immunoassays and direct fluorescent antigen testing, are being replaced in favor of the improved sensitivity and shorter turnaround time of PCR. 5 Still, PCR remains relatively expensive due to high reagent costs, and conventional PCR instruments, such as thermoelectric and convective thermocyclers, are limited by large reaction volumes (>5 ll) and thermal inaccuracies. [6][7][8] Further, despite the dozens to hundreds of sample capacities of modern PCR instruments, operation is typically limited to uniform thermal conditions and average reaction durations on the order of 1 h. Yet, different genetic targets have corresponding optimal annealing temperatures that depend on the length and GC content of their primers.…”