Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

Bushon, Rebecca N.; Kephart, Christopher M.; Koltun, G.F.; Francy, Donna S.; Schaefer, Frank W.; Lindquist, Heather

doi:10.1111/j.1472-765x.2009.02788.x

Cited by 15 publications

(10 citation statements)

References 21 publications

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“…Different batches of DNA extraction kit reagents can be a significant source of variation for longitudinal studies [23, 81]. It is wise to purchase all the extraction kits needed at the start of the study, or store samples and extract all at the same time, to minimize the effects of this variable.…”

Section: Planning a Microbiome Experimentsmentioning

confidence: 99%

Optimizing methods and dodging pitfalls in microbiome research

Kim¹,

Hofstaedter²,

Zhao³

et al. 2017

Microbiome

439

393

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Research on the human microbiome has yielded numerous insights into health and disease, but also has resulted in a wealth of experimental artifacts. Here, we present suggestions for optimizing experimental design and avoiding known pitfalls, organized in the typical order in which studies are carried out. We first review best practices in experimental design and introduce common confounders such as age, diet, antibiotic use, pet ownership, longitudinal instability, and microbial sharing during cohousing in animal studies. Typically, samples will need to be stored, so we provide data on best practices for several sample types. We then discuss design and analysis of positive and negative controls, which should always be run with experimental samples. We introduce a convenient set of non-biological DNA sequences that can be useful as positive controls for high-volume analysis. Careful analysis of negative and positive controls is particularly important in studies of samples with low microbial biomass, where contamination can comprise most or all of a sample. Lastly, we summarize approaches to enhancing experimental robustness by careful control of multiple comparisons and to comparing discovery and validation cohorts. We hope the experimental tactics summarized here will help researchers in this exciting field advance their studies efficiently while avoiding errors.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-017-0267-5) contains supplementary material, which is available to authorized users.

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Section: Planning a Microbiome Experimentsmentioning

confidence: 99%

Optimizing methods and dodging pitfalls in microbiome research

Kim¹,

Hofstaedter²,

Zhao³

et al. 2017

Microbiome

439

393

View full text Add to dashboard Cite

show abstract

“…Thus, AS had been adopted as good material for molecular methodological assessment in FISH and terminal restriction fragment length polymorphism (Wagner et al 1993; Liu et al 1997). Several evaluation studies on DNA extraction kits or methods for AS samples have been performed in recent years (Vanysacker et al 2010; Bushon et al 2010; Bonot et al 2010). However, these studies never tested the effectiveness of different kits by high throughput sequencing and, thus, were based on detailed taxonomic information, which is the more important index for community structure analysis.…”

Section: Introductionmentioning

confidence: 99%

Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing

Guo

Zhang

2012

Appl Microbiol Biotechnol

145

110

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“…DNA extraction was performed using a modified version (preventing the loss of sample material during the extraction process) of the procedure supplied with a MoBio PowerSoil DNA extraction kit (MoBio, Carlsbad, CA, USA) (21,22). Environmental water samples were centrifuged at 3,220 ϫ g for 30 min, the supernatant was carefully decanted, and the pellet was resuspended in sterile tap water and transferred into 2-ml tubes (21).…”

Section: Development Of the Qpcr Assay (I)mentioning

confidence: 99%

A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

Bliem

Schauer

Plicka³

et al. 2015

Appl Environ Microbiol

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Vibrio cholerae is a severe human pathogen and a frequent member of aquatic ecosystems. Quantification of V. cholerae in environmental water samples is therefore fundamental for ecological studies and health risk assessment. Beside time-consuming cultivation techniques, quantitative PCR (qPCR) has the potential to provide reliable quantitative data and offers the opportunity to quantify multiple targets simultaneously. A novel triplex qPCR strategy was developed in order to simultaneously quantify toxigenic and nontoxigenic V. cholerae in environmental water samples. To obtain quality-controlled PCR results, an internal amplification control was included. The qPCR assay was specific, highly sensitive, and quantitative across the tested 5-log dynamic range down to a method detection limit of 5 copies per reaction. Repeatability and reproducibility were high for all three tested target genes. For environmental application, global DNA recovery (GR) rates were assessed for drinking water, river water, and water from different lakes. GR rates ranged from 1.6% to 76.4% and were dependent on the environmental background. Uncorrected and GR-corrected V. cholerae abundances were determined in two lakes with extremely high turbidity. Uncorrected abundances ranged from 4.6 ؋ 10 2 to 2.3 ؋ 10 4 cell equivalents liter ؊1 , whereas GR-corrected abundances ranged from 4.7 ؋ 10 3 to 1.6 ؋ 10 6 cell equivalents liter ؊1 . GR-corrected qPCR results were in good agreement with an independent cellbased direct detection method but were up to 1.6 log higher than cultivation-based abundances. We recommend the newly developed triplex qPCR strategy as a powerful tool to simultaneously quantify toxigenic and nontoxigenic V. cholerae in various aquatic environments for ecological studies as well as for risk assessment programs. Vibrio cholerae is a waterborne bacterium found worldwide in brackish water, coastal areas, and estuarine environments (1). Although the species V. cholerae comprises more than 200 serotypes, only serotypes O1 and O139 are currently able to cause epidemic and pandemic cholera outbreaks, with more than 100,000 reported death cases per year (2). All other non-O1/non-O139 serotypes are usually associated with less-severe gastrointestinal, blood, wound, and ear infections (3). V. cholerae has been classified as a potential category B terrorism agent by the U.S. Centers for Disease Control and Prevention (4). Detection and quantification of V. cholerae, especially of serogroup O1/O139 strains in environmental samples, are still difficult tasks, and no international standard is available. Currently accepted cultivation-based methods take at least 48 h to obtain a final result (5) and severely underestimate the presence of O1/O139 serogroups, due to the fact that these strains predominantly enter a viable but nonculturable (VBNC) state once released from the human intestine into the environment (6, 7). Alternatively, fluorescence in situ hybridization (FISH) and the direct fluorescent-antibody assay (DFA) are direct cell-...

show abstract

Statistical assessment of DNA extraction reagent lot variability in real-time quantitative PCR

Cited by 15 publications

References 21 publications

Optimizing methods and dodging pitfalls in microbiome research

Optimizing methods and dodging pitfalls in microbiome research

Biases during DNA extraction of activated sludge samples revealed by high throughput sequencing

A Novel Triplex Quantitative PCR Strategy for Quantification of Toxigenic and Nontoxigenic Vibrio cholerae in Aquatic Environments

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