ArticleJournal of clinical microbiology2006
Real-time quantitative broad-range PCR assay for detection of the 16S rRNA gene followed by sequencing for species identification.
Article in Journal of clinical microbiology, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 35 papers.
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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
35 citing papers in PubMed, 95 citations in OpenAlex.
- One Health Perspective of Mycoplasmas: Proposals for Standardizing In Vitro PK/PD Models of Human Mycoplasma Species by Referencing Veterinary Experiences.Microorganisms · 2026Review
- Enhanced Detection of Bacterial Ocular Pathogens: A Comparative Study of Broad-Range Real-Time PCR and Conventional Culture Methods.Diagnostics (Basel, Switzerland) · 2025Article
- Molecular Discovery of Filarial Nematode DNA in an Endangered Wild Pinniped (Galapagos Sea Lion,Ecology and evolution · 2024Article
- Overexpressing CsSABP2 enhances tolerance to Huanglongbing and citrus canker inFrontiers in plant science · 2024Article
- Estimation of Red-complex Bacteria in Diode Laser Treated Chronic Periodontitis Patients: A Clinical and Microbiological Study.Journal of pharmacy & bioallied sciences · 2020Article
- Serum separator tube method for matrix-assisted laser desorption/ionization time-of-flight analysis.Access microbiology · 2019Article
- Assessment and comparison of bacterial load levels determined by quantitative amplifications in blood culture-positive and negative neonatal sepsis.Revista do Instituto de Medicina Tropical de Sao Paulo · 2018Article
- Beyond Blood Culture and Gram Stain Analysis: A Review of Molecular Techniques for the Early Detection of Bacteremia in Surgical Patients.Surgical infections · 2016Review
- 16S rRNA gene sequencing is a non-culture method of defining the specific bacterial etiology of ventilator-associated pneumonia.International journal of clinical and experimental medicine · 2015Article
- Massive parallel sequencing provides new perspectives on bacterial brain abscesses.Journal of clinical microbiology · 2014Article
- Bacteremia caused by Comamonas kerstersii in a patient with diverticulosis.Journal of clinical microbiology · 2014Article
- Real-time PCR TaqMan assay for rapid screening of bloodstream infection.Annals of clinical microbiology and antimicrobials · 2014Article
- A dual molecular beacon approach for fast detection of Mycobacterium tuberculosis.Molecular biology reports · 2013Article
- Development of a broad-range 23S rDNA real-time PCR assay for the detection and quantification of pathogenic bacteria in human whole blood and plasma specimens.BioMed research international · 2013Article
- Broad-range real-time PCR assay for detection of bacterial DNA in ocular samples from infectious endophthalmitis.Japanese journal of ophthalmology · 2012Article
- Development of PCR-based method for detection of Enterobacteriaceae in septicemia.Journal of research in medical sciences : the official journal of Isfahan University of Medical Sciences · 2012Article
- Dual priming oligonucleotides for broad-range amplification of the bacterial 16S rRNA gene directly from human clinical specimens.Journal of clinical microbiology · 2012Article
- Molecular methods for pathogen and microbial community detection and characterization: current and potential application in diagnostic microbiology.Infection, genetics and evolution : journal of molecular epidemiology and evolutionary genetics in infectious diseases · 2012Review
- Evaluation of DNA extraction methods and their clinical application for direct detection of causative bacteria in continuous ambulatory peritoneal dialysis culture fluids from patients with peritonitis by using broad-range PCR.Annals of laboratory medicine · 2012Article
- Design and Construction of a Single-Tube, LATE-PCR, Multiplex Endpoint Assay with Lights-On/Lights-Off Probes for the Detection of Pathogens Associated with Sepsis.Journal of pathogens · 2012Article
Corrections and comments
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Authors and funding
7 authors at 5 institutions in 2 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Here we determined the analytical sensitivities of broad-range real-time PCR-based assays employing one of three different genomic DNA extraction protocols in combination with one of three different primer pairs targeting the 16S rRNA gene to detect a panel of 22 bacterial species. DNA extraction protocol III, using lysozyme, lysostaphin, and proteinase K, followed by PCR with the primer pair Bak11W/Bak2, giving amplicons of 796 bp in length, showed the best overall sensitivity, detecting DNA of 82% of the strains investigated at concentrations of < or =10(2) CFU in water per reaction. DNA extraction protocols I and II, using less enzyme treatment, combined with other primer pairs giving shorter amplicons of 466 bp and 342 or 346 bp, respectively, were slightly more sensitive for the detection of gram-negative but less sensitive for the detection of gram-positive bacteria. The obstacle of detecting background DNA in blood samples spiked with bacteria was circumvented by introducing a broad-range hybridization probe, and this preserved the minimal detection limits observed in samples devoid of blood. Finally, sequencing of the amplicons generated using the primer pair Bak11W/Bak2 allowed species identification of the detected bacterial DNA. Thus, broad-spectrum PCR targeting the 16S rRNA gene in the quantitative real-time format can achieve an analytical sensitivity of 1 to 10 CFU per reaction in water, avoid detection of background DNA with the introduction of a broad-range probe, and generate amplicons that allow species identification of the detected bacterial DNA by sequencing. These prerequisites are important for its application to blood-containing patient samples.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.