Microscale sample preparation for PCR of C. difficile infected stool

Gillers, Sara; Atkinson, Christopher D.; Bartoo, Aaron C.; Mahalanabis, Madhumita; Boylan, Michael O.; Schwartz, John H.; Klapperich, Catherine M.; Singh, Satish K.

doi:10.1016/j.mimet.2009.05.020

Cited by 13 publications

(11 citation statements)

References 15 publications

(12 reference statements)

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“…In order to demonstrate the concept of minimally instrumented nucleic acid extraction, we also utilized a portable version of the microfluidic nucleic acid extraction instrument and protocols developed previously in our laboratory [41], [42], [43], [44]. The portable version of this extraction system is called SNAP-System for Nucleic Acid Preparation.…”

Section: Methodsmentioning

confidence: 99%

“…The PPM columns ( Figure 2 ) were housed in unaltered FinnTip Universal 250 µl filter-less pipette tips (Thermo Fisher Scientific, Waltham, MA). These PPM columns were fabricated as previously described [41], [45] with some modification. Briefly, the pre-polymer monomer solution was prepared by mixing 125 µl Ethylene dimethacrylate (98%, EDMA), 125 µl Butyl methacrylate (99%, BUMA) (both Sigma-Aldrich, St. Louis, MO), 750 µl 1-dodecanol, and 8.0 mg azobisisobutyronitrile (AIBN).…”

Section: Methodsmentioning

confidence: 99%

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Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis

Huang

Mahalanabis

et al. 2013

PLoS ONE

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In order to counter the common perception that molecular diagnostics are too complicated to work in low resource settings, we have performed a difficult sample preparation and DNA amplification protocol using instrumentation designed to be operated without wall or battery power. In this work we have combined a nearly electricity-free nucleic acid extraction process with an electricity-free isothermal amplification assay to detect the presence of Clostridium difficile (C. difficile) DNA in the stool of infected patients. We used helicase-dependent isothermal amplification (HDA) to amplify the DNA in a low-cost, thermoplastic reaction chip heated with a pair of commercially available toe warmers, while using a simple Styrofoam insulator. DNA was extracted from known positive and negative stool samples. The DNA extraction protocol utilized an air pressure driven solid phase extraction device run using a standard bicycle pump. The simple heater setup required no electricity or battery and was capable of maintaining the temperature at 65°C±2°C for 55 min, suitable for repeatable HDA amplification. Experiments were performed to explore the adaptability of the system for use in a range of ambient conditions. When compared to a traditional centrifuge extraction protocol and a laboratory thermocycler, this disposable, no power platform achieved approximately the same lower limit of detection (1.25×10−2 pg of C. difficile DNA) while requiring much less raw material and a fraction of the lab infrastructure and cost. This proof of concept study could greatly impact the accessibility of molecular assays for applications in global health.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis

Huang

Mahalanabis

et al. 2013

PLoS ONE

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“…The pH values of 3, 5.2, and 8 were chosen on the basis of the pK a values of the silica surface groups, the depurination of DNA (below pH 2), and the dissolution of silica (above pH 8) [30]. DNA adsorption and elution from the silica surface was quantified under conditions that mimic those commonly used in commercial kits and integrated POC diagnostic devices that isolate NAs from human samples [31–33]. However, we incubated the DNA and silica particles significantly longer than most test protocols call for to make sure that equilibrium was reached.…”

Section: Introductionmentioning

confidence: 99%

Low concentration DNA extraction and recovery using a silica solid phase

2017

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DNA extraction from clinical samples is commonly achieved with a silica solid phase extraction column in the presence of a chaotrope. Versions of these protocols have been adapted for point of care (POC) diagnostic devices in miniaturized platforms, but commercial kits require a high amount of input DNA. Thus, when the input clinical sample contains less than 1 μg of total DNA, the target-specific DNA recovery from most of these protocols is low without supplementing the sample with exogenous carrier DNA. In fact, many clinical samples used in the development of POC diagnostics often exhibit target DNA concentrations as low as 3 ng/mL. With the broader goal of improving the yield and efficiency of nucleic acid-based POC devices for dilute samples, we investigated both DNA adsorption and recovery from silica particles by using 1 pg– 1 μg of DNA with a set of adsorption and elution buffers ranging in pH and chaotropic presence. In terms of adsorption, we found that low pH and the presence of chaotropic guanidinium thiocyanate (GuSCN) enhanced DNA-silica adsorption. When eluting with a standard low-salt, high-pH buffer, > 70% of DNA was unrecoverable, except when DNA was initially adsorbed with 5 M GuSCN at pH 5.2. Unrecovered DNA was either not initially adsorbed or irreversibly bound on the silica surface. Recovery was improved when eluting with 95°C formamide and 1 M NaOH, which suggested that DNA-silica-chaotrope interactions are dominated by hydrophobic interactions and hydrogen bonding. While heated formamide and NaOH are non-ideal elution buffers for practical POC devices, the salient results are important for engineering a set of optimized reagents that could maximize nucleic acid recovery from a microfluidic DNA-silica-chaotrope system.

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“…Other improvements to consider will include on-board storage of primers and HDA buffer components, multiplexing to detect multiple target pathogens or strains and decreasing HDA reaction volumes on-chip. Although we used E. coli in broth medium for our proof-of-concept study, the same method of extraction with our μSPE chips has been shown previously to work in the background of complex biological samples of blood, urine, and stool for gram-negative bacteria such as E. coli and gram-positive bacteria such as Enterococcus faecalis , Bacillus subtilis and Clostridium difficile , and for RNA isolation from viruses and mammalian cells (Bhattacharyya and Klapperich 2006, 2008; Gillers et al 2009; Kulinski et al 2009; Mahalanabis et al 2009). Thus we anticipate that this integrated device may be applied for a wide variety of point-of-care diagnostic applications.…”

Section: Discussionmentioning

confidence: 99%

“…Detailed methods on the manufacture of these channels and the μSPE columns are found in our previous reports of DNA extraction from bacteria in urine, blood and stool (Gillers et al 2009; Kulinski et al 2009; Mahalanabis et al 2009). The HDA was first performed in microtubes to establish that μSPE-extracted DNA was suitable for HDA and to determine the optimal concentrations and conditions for the reactions.…”

Section: Methodsmentioning

confidence: 99%

An integrated disposable device for DNA extraction and helicase dependent amplification

Mahalanabis

ALMuayad

et al. 2010

Biomed Microdevices

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112

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Here we report the demonstration of an integrated microfluidic chip that performs helicase dependent amplification (HDA) on samples containing live bacteria. Combined chip-based sample preparation and isothermal amplification are attractive for world health applications, since the need for instrumentation to control flow rate and temperature changes are reduced or eliminated. Bacteria lysis, nucleic acid extraction, and DNA amplification with a fluorescent reporter are incorporated into a disposable polymer cartridge format. Smart passive fluidic control using a flap valve and a hydrophobic vent (with a nanoporous PTFE membrane) with a simple on-chip mixer eliminates multiple user operations. The device is able to detect as few as ten colony forming units (CFU) of E. coli in growth medium.

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Microscale sample preparation for PCR of C. difficile infected stool

Cited by 13 publications

References 15 publications

Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis

Low Cost Extraction and Isothermal Amplification of DNA for Infectious Diarrhea Diagnosis

Low concentration DNA extraction and recovery using a silica solid phase

An integrated disposable device for DNA extraction and helicase dependent amplification

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