A microfluidic chip integrating DNA extraction amplification and detection for the

A microfluidic chip integrating DNA extraction amplification and detection for the identification of bacteria in saliva is described. and strains of bacteria can be simultaneously identified in the same sample by varying the primers and probes used in each of the seven reaction wells. In initial tests as little as 30 fg (8-12 copies) of MSSA gDNA in buffer has been successfully amplified and detected with this device. 1 Introduction A point-of-care (POC) device able to rapidly identify bacteria in clinical samples would provide more immediate and accurate information for better treatment options at clinical or primary care facilities. Current methods of diagnosing bacterial infections using labor-intensive culture methods can take more than 24 IL-11 hours delaying effective treatment and limiting SH-4-54 potential options.1 2 Nucleic acid tests including techniques such as the polymerase chain reaction (PCR) are alternatives to culture for identifying bacteria. These tests can positively identify bacteria in a few hours from specific nucleic acid sequences. Traditional PCR methods require the use of specialized equipment expensive reagents and trained personnel to complete the assays.3-5 Thus PCR is generally still performed in centralized laboratories by trained technicians with results supplied in a similar time frame (~24 h) to many culture techniques.4 6 Microfluidic devices integrating SH-4-54 PCR can make this diagnostic tool available for POC testing. Microfluidics offers many advantages over current tube-based PCR procedures including lower reagent consumption faster cycling times lower cost per test and automated processing for use by minimally trained personnel.1 3 4 7 8 Microfluidic systems can be designed to be portable SH-4-54 with disposable chips that eliminate contamination concerns between samples. The small device footprints achievable can incorporate parallel processing units increasing throughput and thus detection of multiple pathogens simultaneously.3 8 To fully integrate a PCR assay onto a POC device for sample-in answer-out capability the following steps are required: cell lysis DNA extraction and removal of SH-4-54 PCR inhibitors amplification via thermocycling and amplicon detection.11-13 Several chip designs have been described that integrate cell lysis and DNA extraction with PCR by using silica-based separations or magnetic beads for SH-4-54 extraction.3 4 14 15 Since silica and some magnetic beads are PCR inhibitors the DNA must be eluted often with ethanol a strong PCR inhibitor before downstream amplification.16-19 Chip designs have been reported that performed cell lysis in the PCR chamber without DNA extraction or isolation of the targeted cells also called direct PCR.13 20 This is sufficient for samples that do not contain PCR inhibitors but many clinical samples contain a wide variety of inhibitors and require extraction for successful PCR. Cell lysis in the PCR chamber without DNA extraction has also been demonstrated with antibody-functionalized magnetic beads used to separate the target cells from the rest of the sample.21 If more than one species is targeted antibody-functionalized beads would be needed for each type making the addition of new targets difficult. Many chip designs incorporating cell lysis and DNA extraction are limited to only a few reaction chambers 3 4 13 21 reducing the potential for multiplexing reactions. A chip design containing 12 reaction chambers for easy multiplexing has been described22 but it does not integrate cell lysis or DNA extraction on-chip. AOMs have previously been used to extract DNA from samples with subsequent PCR amplification directly on the AOM.16 17 23 The amount of DNA extracted has been found to depend not only on the size of the AOM’s pores but also on pH and salt concentration with larger pores sometimes performing better than smaller pores.17 AOMs can also inhibit PCR to some degree but the basic pH of the master mix along with adding BSA and extra polymerase to the reaction mixture will release nucleic SH-4-54 acids bound to the membrane and minimize the inhibition.16 17 23 26 We have developed a chip that utilizes these properties of the AOM to integrate DNA extraction and PCR in multiple reaction chambers with on-chip detection in a simple and functional device. Figure 1 shows an image of the PDMS/AOM/glass hybrid chip (a) and a schematic of its cross-section (b). The device uses an AOM sandwiched between an array of seven parallel reaction wells and a microfluidic coating to control fluid.