ArticlePloS one2009
Optimizing Taq polymerase concentration for improved signal-to-noise in the broad range detection of low abundance bacteria.
Article in PloS one, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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Who cites it
22 citing papers in PubMed, 50 citations in OpenAlex.
- Development of Single Molecule Techniques for Sensing and Manipulation of CRISPR and Polymerase Enzymes.Small (Weinheim an der Bergstrasse, Germany) · 2023Review
- Contamination Issue in Viral Metagenomics: Problems, Solutions, and Clinical Perspectives.Frontiers in microbiology · 2021Review
- Equivolumetric Protocol Generates Library Sizes Proportional to Total Microbial Load in 16S Amplicon Sequencing.Frontiers in microbiology · 2021Article
- Precision public health to inhibit the contagion of disease and move toward a future in which microbes spread health.BMC infectious diseases · 2019Review
- Novel properties of recombinant Sso7d-Taq DNA polymerase purified using aqueous two-phase extraction: Utilities of the enzyme in viral diagnosis.Biotechnology reports (Amsterdam, Netherlands) · 2018Article
- The fallopian tube microbiome: implications for reproductive health.Oncotarget · 2018Article
- Changes in the lung bacteriome in relation to antipseudomonal therapy in children with cystic fibrosis.Folia microbiologica · 2018Article
- Massively parallel digital high resolution melt for rapid and absolutely quantitative sequence profiling.Scientific reports · 2017Article
- Construction, Expression, and Characterization of Recombinant Pfu DNA Polymerase in Escherichia coli.The protein journal · 2016Article
- Performance evaluation of the Verigene® (Nanosphere) and FilmArray® (BioFire®) molecular assays for identification of causative organisms in bacterial bloodstream infections.European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology · 2015Article
- The Characterization of Novel Tissue Microbiota Using an Optimized 16S Metagenomic Sequencing Pipeline.PloS one · 2015Article
- How to deal with PCR contamination in molecular microbial ecology.Microbial ecology · 2014Article
- Article
- Analysis of Carotenoid Production by Halorubrum sp. TBZ126; an Extremely Halophilic Archeon from Urmia Lake.Advanced pharmaceutical bulletin · 2014Article
- Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples.Nucleic acids research · 2013Article
- Rapid 16S rRNA next-generation sequencing of polymicrobial clinical samples for diagnosis of complex bacterial infections.PloS one · 2013Article
- Prosthetic joint infection diagnosis using broad-range PCR of biofilms dislodged from knee and hip arthroplasty surfaces using sonication.Journal of clinical microbiology · 2012Article
- Pyrosequencing reveals the complex polymicrobial nature of invasive pyogenic infections: microbial constituents of empyema, liver abscess, and intracerebral abscess.European journal of clinical microbiology & infectious diseases : official publication of the European Society of Clinical Microbiology · 2012Article
- Comparison of DNA extraction methods for microbial community profiling with an application to pediatric bronchoalveolar lavage samples.PloS one · 2012Article
- An efficient strategy for broad-range detection of low abundance bacteria without DNA decontamination of PCR reagents.PloS one · 2011Article
Corrections and comments
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Authors and funding
3 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundPCR in principle can detect a single target molecule in a reaction mixture. Contaminating bacterial DNA in reagents creates a practical limit on the use of PCR to detect dilute bacterial DNA in environmental or public health samples. The most pernicious source of contamination is microbial DNA in DNA polymerase preparations. Importantly, all commercial Taq polymerase preparations inevitably contain contaminating microbial DNA. Removal of DNA from an enzyme preparation is problematical. METHODOLOGY/PRINCIPAL
findingsThis report demonstrates that the background of contaminating DNA detected by quantitative PCR with broad host range primers can be decreased greater than 10-fold through the simple expedient of Taq enzyme dilution, without altering detection of target microbes in samples. The general method is: For any thermostable polymerase used for high-sensitivity detection, do a dilution series of the polymerase crossed with a dilution series of DNA or bacteria that work well with the test primers. For further work use the concentration of polymerase that gave the least signal in its negative control (H(2)O) while also not changing the threshold cycle for dilutions of spiked DNA or bacteria compared to higher concentrations of Taq polymerase. CONCLUSIONS/SIGNIFICANCE: It is clear from the studies shown in this report that a straightforward procedure of optimizing the Taq polymerase concentration achieved "treatment-free" attenuation of interference by contaminating bacterial DNA in Taq polymerase preparations. This procedure should facilitate detection and quantification with broad host range primers of a small number of bona fide bacteria (as few as one) in a sample.
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