Identification of mixed bacterial DNA contamination in broad-range PCR amplification of 16S rDNA V1 and V3 variable regions by pyrosequencing of cloned amplicons

Grahn, Niclas; Olofsson, Margaretha; Ellnebo-Svedlund, Katarina; Monstein, Hans‐Jürg; Jonasson, Jon

doi:10.1016/s0378-1097(02)01190-4

Cited by 134 publications

(84 citation statements)

References 18 publications

(29 reference statements)

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“…Using a sensitive and rapid method combining broad-range PCR amplification of bacterial 16S rDNA fragments and pyrosequencing for detection, identification and typing, Grahn et al (11) have found contaminating bacterial DNA in reagents used for PCR reactions, further identified as water-borne bacteria. Among the PCR-positive and culture-negative group (Table 3) we have identified Xanthomonas campestris on three samples and Burkolderia sp.…”

Section: Comparison Of Pcr and Culture Resultsmentioning

confidence: 99%

Application of broad-range bacterial PCR amplification and direct sequencing on the diagnosis of neonatal sepsis

Ruppenthal¹,

Pereira²,

Cantarelli³

et al. 2005

Braz. J. Microbiol.

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A broad-range bacterial PCR target to conserved regions of the 23S rDNA was applied to 306 blood culture samples from 295 infants (up to one year of age) admitted to a neonatal intensive care unit. Classic blood culture results were compared to DNA sequencing analysis of the PCR amplification products. Culture results were in agreement to DNA sequencing in 90.5% (277) of 306 samples tested, including 263 PCR and culture negative samples and 29 culture and PCR positive samples. The sensitivity of the PCR method combined with sequencing was 88%, and the specificity was 96.3%, with positive and negative predictive values of 74.3 e 98.5%, respectively. The PCR-based approach directly applied to blood culture samples, correlated well with blood culture results from neonates with presumptive diagnosis of bacterial sepsis. The PCR/sequencing approach is suggested to be a valuable complementary data for diagnosis of neonatal sepsis. This methodology is relatively easy and reliable giving accurate results that can be applied to samples colleted during antimicrobial treatment or by a hospital clinical procedure, especially when routine cultures are negative. It can also be useful for the identification of rare bacterial species and for those isolates not readily identified by microbiological tests.

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Section: Comparison Of Pcr and Culture Resultsmentioning

confidence: 99%

Application of broad-range bacterial PCR amplification and direct sequencing on the diagnosis of neonatal sepsis

Ruppenthal¹,

Pereira²,

Cantarelli³

et al. 2005

Braz. J. Microbiol.

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show abstract

“…MPS is also now used to access metagenomes or viruses 7,24,178) or to identify bacteria after PCR (Polymerase Chain Reaction) or MDA (Multiple Displacement Amplification) amplifications 49,50,74,76,85,116,120,129,168) . Parallel sequencing would also have the capability to distinguish a low abundance true pathogen, when increased sensitivity of detection is needed and that low contamination exists in molecular biology reagents, including ultra-pure water 50) .…”

Section: Introductionmentioning

confidence: 99%

“…Parallel sequencing would also have the capability to distinguish a low abundance true pathogen, when increased sensitivity of detection is needed and that low contamination exists in molecular biology reagents, including ultra-pure water 50) .…”

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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“…Several assays using pyrosequencing have been reported. [29][30][31][32] We have developed a unique assay that combines realtime PCR and pyrosequencing for the detection and gram morphotype categorization of most clinically important bacteria. 13 One attractive feature of this approach is the rapid time to detection; in addition the detection of a single nucleotide polymorphism (i.e., nucleotide position three of the pyrogram) is all that is required to differentiate most gram positives from gram negatives.…”

Section: Discussionmentioning

confidence: 99%

The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections

Kobayashi

Bauer

Tuohy

et al. 2006

Journal Orthopaedic Research

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ABSTRACT:We have developed a combined real-time PCR and pyrosequencing assay that successfully differentiated the vast majority of gram-positive and gram-negative bacteria when bacterial isolates were tested. The purpose of this study was to evaluate this assay on clinical specimens obtained from orthopedic surgeries, and to prospectively compare the results of ''molecular Gram stain'' with culture and conventional direct Gram stain. Forty-five surgical specimens were obtained from patients who underwent orthopedic surgery procedures. The DNA was extracted and a set of broad-range PCR primers that targeted a part of the 16S rDNA gene was used for pan-bacterial PCR. The amplicons were submitted for pyrosequencing and the resulting molecular Gram stain characteristics were recorded. Culture and direct Gram staining were performed using standard methods for all cases. Surgical specimens were reviewed histologically for all cases that had a discrepancy between culture and molecular results. There was an 86.7% (39/45) agreement between the traditional and molecular methods. In 12/14 (85.7%) culture-proven cases of bacterial infection, molecular Gram stain characteristics were in agreement with the culture results, while the conventional Gram stain result was in agreement only for five cases (35.7%). In the 31 culture negative cases, 27 cases were also PCR negative, whereas 4 were PCR positive. Three of these were characterized as gram negative and one as gram positive by this molecular method. Molecular determination of the Gram stain characteristics of bacteria that cause orthopedic infections may be achieved, in most instances, by this method. Further studies are necessary to understand the clinical importance of PCR-positive/culture-negative results. ß

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Identification of mixed bacterial DNA contamination in broad-range PCR amplification of 16S rDNA V1 and V3 variable regions by pyrosequencing of cloned amplicons

Cited by 134 publications

References 18 publications

Application of broad-range bacterial PCR amplification and direct sequencing on the diagnosis of neonatal sepsis

Application of broad-range bacterial PCR amplification and direct sequencing on the diagnosis of neonatal sepsis

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

The comparison of pyrosequencing molecular Gram stain, culture, and conventional Gram stain for diagnosing orthopaedic infections

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