ArticleJournal of clinical microbiology2000
Contamination and sensitivity issues with a real-time universal 16S rRNA PCR.
Article in Journal of clinical microbiology, 2000. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 161 papers.
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Who cites it
161 citing papers in PubMed, 452 citations in OpenAlex.
- Quantitative real-time PCR assays to identify and quantify fecal Bifidobacterium species in infants receiving a prebiotic infant formula.Applied and environmental microbiology · 2005Trial
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- A broad-range polymerase chain reaction on a formalin-fixed, paraffin-embedded tissue is a powerful diagnostic tool, but requires cautious interpretation: a case report.Frontiers in medicine · 2026Article
- Unveiling pathogens and contaminants: refining metagenomics for clinical diagnostics.Frontiers in microbiology · 2026Article
- Peptide-Based Plasmon-Enhanced Spectroscopic Immunoassay to Detect Immunity Against Cytomegalovirus.Biosensors · 2025Article
- The eukaryotic horizontal gene transfer dataset a compendium.bioRxiv : the preprint server for biology · 2025Article
- CleanSeqU algorithm for decontamination of catheterized urine 16S rRNA sequencing data.Scientific reports · 2025Article
- De-biasing microbiome sequencing data: bacterial morphology-based correction of extraction bias and correlates of chimera formation.Microbiome · 2025Article
- Exploring the Pathogen Profiles of Ancient Feces.bioRxiv : the preprint server for biology · 2025Article
- Targeted pathogen profiling of ancient feces reveals common enteric infections in the Rio Zape Valley, 725-920 CE.PloS one · 2025Article
- Validating digital polymerase chain reaction for 16S rRNA gene amplification from low biomass environmental samples.ISME communications · 2025Article
- Detecting life by behavior, the overlooked sensitivity of behavioral assays.Scientific reports · 2024Article
- Occurrence of recreational water quality monitoring general fecal indicator bacteria and fecal source identification genetic markers in gray seal scat.The Science of the total environment · 2024Article
- Unveiling challenging corneal infections: a comprehensive etiological diagnosis through metagenomic next-generation sequencing (mNGS) of corneal tissue samples.International ophthalmology · 2024Article
- Host evolution shapes gut microbiome composition inEcology and evolution · 2024Article
- 16S rRNA Next-Generation Sequencing May Not Be Useful for Examining Suspected Cases of Spontaneous Bacterial Peritonitis.Medicina (Kaunas, Lithuania) · 2024Article
- Novel rapid method for identifying and quantifying pathogenic bacteria within four hours of blood collection.Scientific reports · 2024Article
- Sample Collection Methods in Upper Gastrointestinal Research.Journal of Korean medical science · 2023Review
- Development of a Simple Direct and Hot-Start PCR UsingInternational journal of molecular sciences · 2023Article
- Blood Culture Negative Endocarditis: A Review of Laboratory Diagnostic Approaches.International journal of general medicine · 2023Review
101 more citing papers are in PubMed but not listed here.
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
6 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
A set of universal oligonucleotide primers specific for the conserved regions of the eubacterial 16S rRNA gene was designed for use with the real-time PCR Applied Biosystems 7700 (TaqMan) system. During the development of this PCR, problems were noted with the use of this gene as an amplification target. Contamination of reagents with bacterial DNA was a major problem exacerbated by the highly sensitive nature of the real-time PCR chemistry. This was compounded by the use of a small amplicon of approximately 100 bases, as is necessary with TaqMan chemistry. In an attempt to overcome this problem, several methodologies were applied. Certain treatments were more effective than others in eliminating the contaminating DNA; however, to achieve this there was a decrease in sensitivity. With UV irradiation there was a 4-log reduction in PCR sensitivity, with 8-methoxypsoralen activity facilitated by UV there was between a 5- and a 7-log reduction, and with DNase alone and in combination with restriction digestion there was a 1.66-log reduction. Restriction endonuclease treatment singly and together did not reduce the level of contaminating DNA. Without the development of ultrapure Taq DNA polymerase, ultrapure reagents, and plasticware guaranteed to be free of DNA, the implementation of a PCR for detection of eubacterial 16S rRNA with the TaqMan system will continue to be problematical.
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Registered trials
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