A Prototype Biomarker Detector Combining Biomarker Extraction and Fixed Temperature PCR

Abstract

PCR is the most sensitive molecular diagnostic available for infectious diseases. The goal for low-resource settings is a simple, inexpensive instrument. Toward this goal, we previously published a self-contained sample preparation instrument that uses magnetics and prearrayed reagents in thin tubing to extract nucleic acids and perform isothermal amplification and detection of extracted biomarkers. To incorporate PCR thermal cycling, after biomarker is magnetically extracted from a patient sample, the section of tubing containing the extracted biomarker and PCR reagents is alternately positioned within two constant temperature blocks. This instrument was evaluated initially by extracting and amplifying a 140 bp fragment of the IS6110 sequence of tuberculosis from TE buffer. The mean cycle threshold for 5 × 10 6 copies of IS6110 was 25.5 ± 1.5 cycles (n = 4), which was significantly different from negative control samples (34.0 ± 2.6 cycles; n = 3). Using a more clinically relevant sample, we extracted and amplified Plasmodium falciparum DNA from malaria-infected human blood cultures. The average cycle threshold for 1% parasitemia samples was 24.7 ± 1.5 cycles (n = 3) and significantly different from negatives (31.5 ± 2.1 cycles; n = 3). This approach integrates biomarker extraction, PCR amplification, and detection in a simple, linear tubing design with potential for use as a low-resource instrument.

Adaptive PCR Based on Hybridization Sensing of Mirror-Image l-DNA

“…6–8 Achieving these conditions at the programmed temperature set points, however, is often compromised by variations in prepared DNA samples, 3,9,10 error in the volumes of pipetted reagents, 3,6,11 or inaccuracies in thermal cycler temperature calibrations. 6,12,13 These challenges are particularly acute in applications seeking to make PCR accessible outside of well-controlled laboratory settings, such as in point-of-care settings where the contents of prepared samples and reaction temperatures are more difficult to control. 10,13–16 Methods that more directly monitor the reaction using fluorescence probes have shown promise for overcoming some of the challenges with indirect thermal sensing, 17–22 yet these methods still rely on temperature calibrations to estimate primer annealing and target melting and do not compensate for variation in reaction contents that impact primer and target hybridization.…”

“…6,12,13 These challenges are particularly acute in applications seeking to make PCR accessible outside of well-controlled laboratory settings, such as in point-of-care settings where the contents of prepared samples and reaction temperatures are more difficult to control. 10,13–16 Methods that more directly monitor the reaction using fluorescence probes have shown promise for overcoming some of the challenges with indirect thermal sensing, 17–22 yet these methods still rely on temperature calibrations to estimate primer annealing and target melting and do not compensate for variation in reaction contents that impact primer and target hybridization. To enable PCR-based nucleic acid amplification in settings that have insufficient resources to precisely maintain sample contents and reaction temperatures, new PCR designs are needed that more directly monitor the key hybridization events during the reaction, rather than relying on rigid temperature cycling programs and consistent sample preparation.…”

“…Because efficient and specific amplification only occurs within a narrow range of reaction conditions, − discrepancies between the estimated and actual hybridization states of the targets and primers cause PCR failure. Consequently, temperature set points, cycle timing, and reaction contents must be precisely maintained to ensure complete target melting, stringent primer annealing, and efficient polymerase activity during each cycle. − Achieving these conditions at the programmed temperature set points, however, is often compromised by variations in prepared DNA samples, ,, error in the volumes of pipetted reagents, ,, or inaccuracies in thermal cycler temperature calibrations. ,, These challenges are particularly acute in applications seeking to make PCR accessible outside of well-controlled laboratory settings, such as in point-of-care settings where the contents of prepared samples and reaction temperatures are more difficult to control. ,− Methods that more directly monitor the reaction using fluorescence probes have shown promise for overcoming some of the challenges with indirect thermal sensing, − yet these methods still rely on temperature calibrations to estimate primer annealing and target melting and do not compensate for variation in reaction contents that impact primer and target hybridization. To enable PCR-based nucleic acid amplification in settings that have insufficient resources to precisely maintain sample contents and reaction temperatures, new PCR designs are needed that more directly monitor the key hybridization events during the reaction, rather than relying on rigid temperature cycling programs and consistent sample preparation.…”

Development of an Automated, Non-Enzymatic Nucleic Acid Amplification Test

“…The microfluidic tubing is held between the gears, which are controlled by a stepper motor. Rotation of the gears therefore moves the microfluidic tubing which contains the various reaction fluids, as previously described for one-directional sample prep devices [28,29]. The reaction cassette was moved at a speed of 0.2 cm/s to transport the magnetic beads, and 7.5 cm/s to break the beads out of the magnetic field.…”

Stimuli-Responsive Nanodiamond-Based Biosensor for Enhanced Metastatic Tumor Site Detection

“…While there are a number of methods available for the detection of cancer biomarkers, these biomarkers are most often identified as genes, microRNA, or proteins that are uniquely expressed or silenced in metastatic cancer. 5–12 Such biomarkers include heat shock protein 70 (Hsp70), epithelial cadherin (E-cadherin), and death-associated protein kinase (DAPK). 13–15 Many of these biomarkers were identified through increased understanding of the underlying molecular mechanisms of metastasis progression.…”