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

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 103 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

Sequence alignment of microfluidic assay RT-PCR products to standard reference influenza strain A/PR/8/34 M1 gene (position number: 193–252).

<p>The sequence presented here excludes primer sequences.</p

Cumulative sensitivity and specificity of the microfluidic assay for influenza A for decreasing specimen viral loads.

<p>Cumulative sensitivity and specificity of the microfluidic assay for influenza A for decreasing specimen viral loads.</p

On-chip PCR product concentration vs. copy number showing NP swab and NP aspirate specimens.

<p>On-chip PCR product concentration vs. copy number showing NP swab and NP aspirate specimens.</p

The microfluidic assay flow.

<p>(A) Image of two microfluidic chips with attached thin film heaters and the two-barrel syringe pump. Glass syringes were connected to each chip with flexible tubing to load reagents and samples. Three ports were glued at the inlets of SPE channel and the waste port, and the outlet of the PCR channel. (B) Channel design with three sections: sample preparation (SPE), RT chamber and continuous flow PCR channel. Two fixed resistance heaters are attached via thermal tape to the bottom of the chip. Fluid flow between the channels was linear, and changes in fluid resistance allowed for valveless operation. The depth is 500 µm for SPE and RT channels, and the PCR channel is 100 µm deep. The widths are 500 µm for the SPE column, 1 mm for the RT chamber, and vary from 200 to 400 µm for the wide and narrow sections of the continuous flow PCR channel. The chip is 70 mm in length, 25 mm in width and 1.4 mm in height. (C) Microfluidic assay process flow. The nasopharyngeal sample is mixed with lysis buffer, applied to the chip, the chip is run, and the PCR products are read using a commercial capillary electrophoresis chip.</p

Summary statistics for DFA.

<p> <i>(95% CI).</i></p

Summary statistics for the microfluidic assay.

<p><i>(95% CI)</i>.</p

Representative on-chip RT-PCR products on 12% polyacrylamide gel.

<p>P: cultured influenza A virus (A/PR/8/34) microfluidic assay result. N: negative control (nuclease free water) in microfluidic chip result. Positive specimens: 26B, 53, 27B, 20B, 22B, 21B, 115, 117, 31B, 30B, 171. negative specimens: 149, 56, 24, 92, 45, 173, 302, 64, 272, 22, 24, 112, 150, 307, 173, 118, 91, 20, 32, 6, 8. L: GeneRuler™ Ultra Low Range DNA Ladder (Fermentas, Glen Burnie, Maryland).</p