Enhanced nucleic acid amplification with blood in situ by wire-guided droplet manipulation (WDM)

Abstract

There are many challenges facing the use of molecular biology to provide pertinent information in a timely, cost effective manner. Wire-guided droplet manipulation (WDM) is an emerging format for conducting molecular biology with unique characteristics to address these challenges. To demonstrate the use of WDM, an apparatus was designed and assembled to automate polymerase chain reaction (PCR) on a reprogrammable platform. WDM minimizes thermal resistance by convective heat transfer to a constantly moving droplet in direct contact with heated silicone oil. PCR amplification of the GAPDH gene was demonstrated at a speed of 8.67 sec/cycle. Conventional PCR was shown to be inhibited by the presence of blood. WDM PCR utilizes molecular partitioning of nucleic acids and other PCR reagents from blood components, within the water-in-oil droplet, to increase PCR reaction efficiency with blood in situ. The ability to amplify nucleic acids in the presence of blood simplifies pre-treatment protocols towards true point-of-care diagnostic use. The 16s rRNA hypervariable regions V3 and V6 were amplified from Klebsiella pneumoniae genomic DNA with blood in situ. The detection limit of WDM PCR was 1 ng/µL or 105 genomes/µL with blood in situ. The application of WDM for rapid, automated detection of bacterial DNA from whole blood may have an enormous impact on the clinical diagnosis of infections in bloodstream or chronic wound/ulcer, and patient safety and morbidity.

A versatile-deployable bacterial detection system for food and environmental safety based on LabTube-automated DNA purification, LabReader-integrated amplification, readout and analysis

“…LabReader for DNA amplification , readout and analysis For DNA amplification, readout and analysis we used a 20 multichannel, multisample, UV−vis spectrophotometer/fluorometer that employs two frequencies of light simultaneously to interrogate the sample. T he LabReader is base d on a round geometry 37 , in which two LEDs serve as UV and fluorescence light sources to readout four different 25 wavelengths simultaneously. Figure 2 C sho ws a schematic overview of the LabReader optics.…”

“…a thermocycler), a stable laboratory environment, a continuous refrigeration chain for reagents or antibodies, and/or specially trained staff to 20 perform numerous manual steps [15][16][17][18][19][20] , all of which are expensive and generally preclude their use at the production site or in the field. 19,21 Automated sample preparation robots are commercially available, but they are expensive and often still require a specialized laboratory to perform manual pipetting steps for the 25 downstream diagnostic reaction. 22 They are therefore not practical for low-throughput testing laboratories or sales locations, who cannot afford buying expensive automation equipment or employing specialized staff.…”

“…21 Any automated diagnostic test for small-scale use should rely 30 on an easier-to-use, cheaper and more robust technology. 12 , 14, 19, 22 T hus, there has been an effort to develop low-cost, automated diagnostic biosensors, including nanotechnology-based sensors or microfluidic systems, such as droplet based [23][24][25] , centrifugal 26,27 , capillary 12, 19 , 28 , pneumatic 14, 28, 29 , paper-based 30-32 devices, and 35 lateral flow assays. 12,19,20,33 For the past decade, this has led to numerous publications about promising, early-stage technologies.…”

“…A heated brass piece 20 was fitted into the LabReader and heated with a Minco foil (10Ω, HK5565R10.0L12F) and a negative temperature coefficient, NT C (EPCOS, NT C B57540G1103F), was employed as temperature sensor. The NT C was connected to a serial resistor of 1.2kΩ, whose voltage was picked off from the temperature regulation 25 module (Carel, IR33DIN). For LAMP amplification an Isoplex VT EC screening kit (Mast Diagnostica) was used.…”

“…Here, different concentrations of E. coli in 25 water, milk and apple juice were extracted using the LabT ube. The DNA was collected in a removable PCR tube containing prestored PCR reagents and it was then automatically amplified, readout and analyzed inside the LabReader.…”