Sample preparation and assay refinements for pathogen detection platforms

Lim, Daniel V.; Kearns, Elizabeth A.; Leskinen, Stephaney D.; Magaña, Sonia; Stroot, Joyce M.; Hunter, Dawn M.; Schlemmer, Sarah M.

doi:10.1117/12.807763

Cited by 3 publications

(3 citation statements)

References 32 publications

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“…Humic acids, proteins and polysaccharides, indicated by low A 260 /A 230, adversely affect PCR amplification kinetics [7,8,33,34]. A majority of the extracts in this study had low A 260 /A 230 purity ratios.…”

Section: Discussionmentioning

confidence: 95%

DNA extract characterization process for microbial detection methods development and validation

Olson

Morrow

2012

BMC Res Notes

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BackgroundQuantitative polymerase chain reaction (qPCR) assays used in pathogen detection require rigorous methods development including characterizing DNA extraction products. A DNA extract characterization process is demonstrated using DNA extracted from five different cells types (two Gram-negatives: Escherichia coli, and Burkholderia thailandensis, spores and vegetative cells from the Gram-positive Bacillus cereus, and yeast Saccharomyces cerevisiae) with six different methods.ResultsDNA extract quantity (concentration and extraction efficiency) and quality (purity and intactness) varied by cell type and extraction method enabling the demonstration of different DNA characterization methods. DNA purity was measured using UV spectroscopy, where the A260/A280 and A260/A230 ratios are indicators of different contaminants. Reproducibility of UV spectroscopy measurements decreased for DNA concentrations less than 17.5 ng/μL. Forty-seven extracts had concentrations greater than 17.5 ng/μL, 25 had A260/A280 above 2.0, and 28 had A260/A230 ratios below 1.8 indicating RNA and polysaccharide contamination respectively. Based on a qPCR inhibition assay the contaminants did not inhibit PCR. Extract intactness was evaluated using microfluidic gel electrophoresis. Thirty-five samples had concentrations above the limit of quantification (LOQ, roughly 11 ng/ μL), 93.5% of the DNA was larger than 1kb and 1% was smaller than 300 bp. Extract concentrations ranged from 1502.2 ng/μL to below the LOQ when UV spectroscopy, fluorometry, and qPCR were used. LOQ for UV spectroscopic and fluorometric measurements were 3.5 ng/μL and 0.25 ng/μL respectively. The qPCR LOQ varied by cell type (5.72 × 10-3 ng/μL for E. coli, 2.66 × 10-3 ng/μL, for B. cereus, 3.78 × 10-3 ng/μL for B. thailandensis, and 7.67 × 10-4 ng/μL for S. cerevisiae). A number of samples were below the UV spectroscopy (n = 27), flurometry (n = 15), and qPCR (n = 3) LOQ.ConclusionThe presented DNA extract characterization process provides measures of DNA quantity and quality applicable to microbial detection methods development and validation studies. Evaluating DNA quality and quantity results in a better understanding of process LOD and contributing factors to suboptimal assay performance. The samples used demonstrated the use of different DNA characterization methods presented but did not encompass the full range of DNA extract characteristics.

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Section: Discussionmentioning

confidence: 95%

DNA extract characterization process for microbial detection methods development and validation

Olson

Morrow

2012

BMC Res Notes

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“…Biosensor platforms have evolved alongside biomarker-based diagnostic assays [10,11]. Portable biosensors are sought as a deployable means to monitor water quality, measure environmental pollution, assess warfighter health, detect pathogens in a point-of-care setting, and more [12][13][14][15][16][17][18]. Techniques such as interferometry, microwave sensing, surface plasmon resonance, fluorimetry, and Bloch surface wave sensing have been applied to the development of portable biosensing technologies [19][20][21][22][23][24][25][26][27].…”

Section: Introductionmentioning

confidence: 99%

Portable Waveguide-Based Optical Biosensor

et al. 2022

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Rapid, on-site diagnostics allow for timely intervention and response for warfighter support, environmental monitoring, and global health needs. Portable optical biosensors are being widely pursued as a means of achieving fieldable biosensing due to the potential speed and accuracy of optical detection. We recently developed the portable engineered analytic sensor with automated sampling (PEGASUS) with the goal of developing a fieldable, generalizable biosensing platform. Here, we detail the development of PEGASUS’s sensing hardware and use a test-bed system of identical sensing hardware and software to demonstrate detection of a fluorescent conjugate at 1 nM through biotin-streptavidin chemistry.

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“…The ratio of absorbance at 260 and 230 nm (A 260 /A 280 ) is used as a secondary measure of nucleic acid purity (Boehm et al, 2009;Lim et al, 2009;Ning et al, 2009;Wilfinger et al, 2006).…”

Section: Choice Of a Protocol For Direct Extraction Of Dna From Soilmentioning

confidence: 99%

Choice of DNA extraction protocols from Gram negative and positive bacteria and directly from the soil

Tiago¹,

Naiara²,

Shana³

et al. 2015

Afr. J. Microbiol. Res.

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DNA extraction is a fundamentally important step for the implementation of genotypic techniques in microbial identification, and the use of such techniques has become essential for the analysis of soil microbial diversity. Considering culture independent methodologies, it is still necessary to ensure that DNA is extracted in appropriate amounts and that extracted DNA is inhibitor-free. This study aimed at selecting a single protocol suitable for the extraction of total DNA from Gram positive and negative bacteria isolated from different sources, as well as a protocol for the direct extraction of DNA from soil. Four experimental protocols and a commercial kit were tested for the extraction of total DNA from isolated bacteria. Among the protocols, the detergent + salt + thermal incubation method (based on Harju et al., 2004) was considered the most promising because it produced satisfactory yields of DNA, with adequate quality for all isolates studied, especially Staphylococcus aureus, without the need to use enzymes and glass beads which can make the extraction process more expensive. Three experimental protocols and the commercial kit were tested for the direct extraction of DNA from soil. Regarding PCR amplification, the amount of total DNA extracted is less limiting than its quality. Thus, commercial kit PowerMax™ Soil DNA Isolation (MoBio) offered more promising results, because although this provided low yields of DNA, it was sufficient for polymerase chain reaction (PCR) amplification.

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Sample preparation and assay refinements for pathogen detection platforms

Cited by 3 publications

References 32 publications

DNA extract characterization process for microbial detection methods development and validation

DNA extract characterization process for microbial detection methods development and validation

Portable Waveguide-Based Optical Biosensor

Choice of DNA extraction protocols from Gram negative and positive bacteria and directly from the soil

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