DNA Pyrosequencing-Based Bacterial Pathogen Identification in a Pediatric Hospital Setting

Luna, Ruth Ann; Fasciano, Lea R.; Jones, Shaunte C.; Boyanton, Bobby L.; Ton, Trang T.; Versalovic, James

doi:10.1128/jcm.00630-07

Cited by 65 publications

(45 citation statements)

References 33 publications

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“…A robust method, required for bacterial identification, has been perused by several investigators [4,14,26,[36][37][38][39][40]; studies on universal and multiplex primers]. With the current number of eubacterial species surpassing 7166 species [41], the UM described here [42] should fulfil this requirement.…”

Section: Introductionmentioning

confidence: 99%

A Universal Method for the Identification of Bacteria Based on General PCR Primers

Barghouthi

2011

Indian J Microbiol

106

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The Universal Method (UM) described here will allow the detection of any bacterial rDNA leading to the identification of that bacterium. The method should allow prompt and accurate identification of bacteria. The principle of the method is simple; when a pure PCR product of the 16S gene is obtained, sequenced, and aligned against bacterial DNA data base, then the bacterium can be identified. Confirmation of identity may follow. In this work, several general 16S primers were designed, mixed and applied successfully against 101 different bacterial isolates. One mixture, the Golden mixture7 (G7) detected all tested isolates (67/67). Other golden mixtures; G11, G10, G12, and G5 were useful as well. The overall sensitivity of the UM was 100% since all 101 isolates were detected yielding intended PCR amplicons. A selected PCR band from each of 40 isolates was sequenced and the bacterium identified to species or genus level using BLAST. The results of the UM were consistent with bacterial identities as validated with other identification methods; cultural, API 20E, API 20NE, or genera and species specific PCR primers. Bacteria identified in the study, covered 34 species distributed among 24 genera. The UM should allow the identification of species, genus, novel species or genera, variations within species, and detection of bacterial DNA in otherwise sterile samples such as blood, cerebrospinal fluid, manufactured products, medical supplies, cosmetics, and other samples. Applicability of the method to identifying members of bacterial communities is discussed. The approach itself can be applied to other taxa such as protists and nematodes.

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Section: Introductionmentioning

confidence: 99%

A Universal Method for the Identification of Bacteria Based on General PCR Primers

Barghouthi

2011

Indian J Microbiol

106

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“…DNA arrays have two main uses in microbiology: Detection of known pathogens, and assessment of diversity 113) . In general, a PCR amplification step using specific primer sets is used to generated labeled DNA 9,11,27,58,79,95,96,100,105,136,156,173,182) . This requirement of predesigned oligomers for amplification and hybridization opens the possibility that many unknown organisms remain undetected (especially viruses which have no universal homologous gene).…”

Section: Introductionmentioning

confidence: 99%

Global Sequencing: A Review of Current Molecular Data and New Methods Available to Assess Microbial Diversity

Christen

2008

Microb. Environ.

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Roughly estimating the diversity of life has for a long time been seen as a challenge. Sequences obtained from the environment by direct amplification are now seen as the sole mean to provide information for at least 99% of the prokaryotes in natural communities. Recent massive parallel sequencing technologies now allow to sequence nearly every sequence resulting from such amplification. Are we ready to deal with such a deluge of data? Using three recent publications (16S rRNA genes amplification in soil and seawater followed by massive parallel sequencing) as case examples, this review analyses the state of the field.

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“…As we found in this study, pyrosequencing of a single region within the 16S rRNA gene using the small Pyromark Q24 instrument can still not deliver the identification to the species level which can be achieved by conventional sequencing. Combining pyrosequence data from more than one fragment of the 16S rRNA gene has been more successful at differentiation [7,8], but a combination of sequences from hypervariable regions 2, 3 and 6 appears to be optimal [15]. This requires multiple reactions and more sequence analysis, which is less convenient for a routine laboratory.…”

Section: Discussionmentioning

confidence: 99%

“…Pyrosequencing requires significantly less hands-on time, with fewer steps and less complexity compared to the conventional (Sanger) sequencing process, however, pyrosequencing read lengths are limited compared to those of over 500 nucleotides achieved by the conventional method. Despite this, pyrosequencing has recently been applied in 16S rDNA PCR studies; for example, differentiation of bacteria into either Gram-positive or Gram-negative groups was possible using only 3 nucleotides of pyrosequence data [6] and bacteria were identified to at least the genus level using 20 -30 nucleotides from more than one locus in the 16S and/or 23S rRNA genes [7][8][9]. However, as sequence read lengths have improved over time to around 50 -80 nucleotides, pyrosequencing may now be a more feasible option for identification to the species level [10].…”

Section: Introductionmentioning

confidence: 99%

Evaluation of Real-Time 16S rDNA PCR and Pyrosequencing for Routine Identification of Bacteria in Joint Fluid and Tissue Specimens

Gadsby¹,

Önen²,

Phillips³

et al. 2011

OJMM

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Abstract16S rDNA PCR and sequencing are powerful tools for bacterial detection and identification, although their routine use is not currently widespread in the field of clinical microbiology. The availability of pyrosequencing now makes 16S rDNA assays more accessible to routine diagnostic laboratories, but this approach has had limited evaluation in general diagnostic practice. In this study we evaluated a real-time 16S rDNA PCR and pyrosequencing assay for use in a routine microbiology laboratory, by retrospectively testing joint fluid and joint tissue specimens received for conventional culture. We found that use of the real-time 16S rDNA assay was clinically valuable in this specimen type because it enabled us to identify a small number of culture-negative infections. Although faster and less labour-intensive, we found that the utility of pyrosequencing for pathogen identification is still hampered by shorter read lengths compared to conventional (Sanger) sequencing. Combining results from both molecular and conventional culture methods, bacteria were only detected in 11.8% specimens in this study. However, the detection rate was increased to 18.6% if specimens were only included from patients with a documented clinical suspicion of infection. In conclusion, while pyrosequencing had significant advantages in speed and ease-of-use over conventional sequencing, multiple reactions will be required to deliver comparable species-level identification, thus negating many of the benefits of using the technique. We found that 16S rDNA PCR and sequencing should be rationally targeted on the basis of good clinical information in the routine diagnostic setting, and not used as a general screening test for the exclusion of bacterial infection in joint specimens.

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DNA Pyrosequencing-Based Bacterial Pathogen Identification in a Pediatric Hospital Setting

Cited by 65 publications

References 33 publications

A Universal Method for the Identification of Bacteria Based on General PCR Primers

A Universal Method for the Identification of Bacteria Based on General PCR Primers

Global Sequencing: A Review of Current Molecular Data and New Methods Available to Assess Microbial Diversity

Evaluation of Real-Time 16S rDNA PCR and Pyrosequencing for Routine Identification of Bacteria in Joint Fluid and Tissue Specimens

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