Integrated Extreme Real-Time PCR and High-Speed Melting Analysis in 52 to 87 Seconds.

BACKGROUND: Extreme PCR in <30 s and high-speed melting of PCR products in <5 s are recent advances in the turnaround time of DNA analysis. Previously, these steps had been performed on different specialized instruments. Integration of both extreme PCR and high-speed melting with real-time fluorescence monitoring for detection and genotyping is presented here. METHODS: A microfluidic platform was enhanced for speed using cycle times as fast as 1.05 s between 66.4 °C and 93.7 °C, with end point melting rates of 8 °C/s. Primer and polymerase concentrations were increased to allow short cycle times. Synthetic sequences were used to amplify fragments of hepatitis B virus (70 bp) and Clostridium difficile (83 bp) by real-time PCR and high-speed melting on the same instrument. A blinded genotyping study of 30 human genomic samples at F2 c.*97, F5 c.1601, MTHFR c.665, and MTHFR c.1286 was also performed. RESULTS: Standard rapid-cycle PCR chemistry did not produce any product when total cycling times were reduced to <1 min. However, efficient amplification was possible with increased primer (5 µmol/L) and polymerase (0.45 U/µL) concentrations. Infectious targets were amplified and identified in 52 to 71 s. Real-time PCR and genotyping of single-nucleotide variants from human DNA was achieved in 75 to 87 s and was 100% concordant to known genotypes. CONCLUSIONS: Extreme PCR with high-speed melting can be performed in about 1 min. The integration of extreme PCR and high-speed melting shows that future molecular assays at the point of care for identification, quantification, and variant typing are feasible.

Near-Infrared Optical Imaging for Diagnosis of Maxillary Sinusitis.

Computed tomography (CT) is the current gold standard imaging for chronic rhinosinusitis (CRS) but is limited by cost, risk of radiation, and difficulty of being performed in the typical outpatient primary care setting. We describe the novel use of a low-cost, handheld technology to deliver an intraoral near-infrared (NIR) wavelength light to optically image the maxillary sinuses. Digital images were collected for subjects presenting with sinus disease using an intraoral NIR light source for transillumination of the maxillary sinuses, captured by a modified digital single-lens reflex camera. Light intensity contrasts were enhanced using computer analysis and subsequently compared to CT findings. NIR illumination produced unique patterns reflecting different disease states: normal sinus anatomy, mild sinus disease and/or mucosal thickening, and complete opacification of the sinus. Current results suggest that NIR imaging may facilitate the diagnosis of sinusitis in the outpatient setting with minimal cost and no radiation exposure.