Adaption of a fragment analysis technique to an automated high-throughput multicapillary electrophoresis device for the precise qualitative and quantitative characterization of microbial communities

Trotha, R.; Reichl, Udo; Thiès, Frank; Sperling, Danuta; König, Wolfgang; König, Brigitte

doi:10.1002/1522-2683(200204)23:7/8<1070::aid-elps1070>3.0.co;2-h

Cited by 32 publications

(33 citation statements)

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“…Despite these techniques, terminal restriction fragment length polymorphism (T-RFLP) analysis of the glyceraldehyde-3-phosphate dehydrogenase gene (gap) represents a high-throughput reproducible method that allows the identification of distinct Staphylococcus species (22). The aim of the present study was to evaluate the performances of VITEK 2 in combination with the newly developed VITEK 2 ID-GP identification card (bioMérieux, Marcy l'Etoile, France) and the BD Phoenix system (Becton Dickinson Diagnostic Systems) for identification of Staphylococcus species in comparison with gap-based T-RFLP analysis as a reference method for accuracy.…”

Section: Other Significant Opportunistic Pathogens Include Staphylocomentioning

confidence: 99%

“…Escherichia coli C2 was used to obtain additional fragments for the internal size standard. The PCR conditions used for amplification of the 730-bp and 981-bp fragments of the genomic E. coli C2 DNA, fragment cloning, sequencing of selected plasmids, and generation of fluorescently labeled fragments from the plasmids were those previously described by Trotha et al (22), with some modifications. The primers used for amplification of the 730-bp fragment were 776F (5Ј-GCAAACAGGATTAGATA-3Ј) and 1492R (5Ј-TACCTTGTTACGACTT-3Ј), and those used for the 981-bp fragment were 515F (5Ј-GCCAGCAGCCGGGGTAA-3Ј) and 1492R (5Ј-TAC CTTGTTACGACTT-3Ј).…”

Section: Other Significant Opportunistic Pathogens Include Staphylocomentioning

confidence: 99%

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Comparative Study Using Various Methods for Identification of Staphylococcus Species in Clinical Specimens

et al. 2006

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Coagulase-negative staphylococci (CNS) play a predominant role in nosocomial infections. Rapid, reliable identification of these organisms is essential for accurate diagnosis and prompt effective treatment of these infections. Quite recently, the VITEK 2 g-positive (gram-positive [GP]) identification card (bioMérieux) has been redesigned for greater accuracy in the identification of gram-positive cocci. We compared the BD Phoenix (Becton Dickinson) and VITEK 2 (bioMérieux) automated microbiology systems, using their respective update version cards, and the API ID32 STAPH test. The glyceraldehyde-3-phosphate dehydrogenase (gap) gene-based T-RFLP (terminal restriction fragment length polymorphism) method was used for verifying the results. In total, 86 clinical isolates of CNS and 27 reference strains were analyzed. The results show that for identification of CNS, the automated identification methods using the newest VITEK 2 and BD Phoenix identification cards are comparable. However, API ID32 STAPH revealed more correct results compared to both automated microbiology systems. Despite the increased performance of the phenotypic automated identification systems compared to the former versions, molecular methods, e.g., the gap-based T-RFLP method, still show superior accuracy in identifying Staphylococcus species other than Staphylococcus aureus. So far, 40 species of the genus Staphylococcus have been identified (8).Staphylococcus aureus is the best known and has been frequently implicated in the etiology of a series of infections and intoxications in humans, whereas coagulase-negative staphylococci (CNS), representing the majority of the species, have been considered to be saprophytic or rarely pathogenic. Currently, several species of CNS are recognized as potential pathogens, mainly causing nosocomial infections, often involved in infections related to implanted medical devices such as intravenous catheters, prosthetic heart valves, and orthopedic implants. The species that most frequently cause diseases in humans are Staphylococcus epidermidis, Staphylococcus haemolyticus, and Staphylococcus saprophyticus. (6,12,17,20). Other significant opportunistic pathogens include Staphylococcus hominis, Staphylococcus warneri, Staphylococcus capitis, Staphylococcus simulans, Staphylococcus cohnii, Staphylococcus xylosus, Staphylococcus saccharolyticus, and Staphylococcus lugdunensisIn this regard, comprehensive and accurate identification of the distinct Staphylococcus species is of great importance. A variety of methods have been proposed for identification schemes based on commercial tests and in relation to the publication of Kloos and Schleifer (15). Various automated identification and susceptibility test systems are currently on the market, among them VITEK 2 (bioMérieux, Marcy l'Etoile, France) and the BD Phoenix system (Becton Dickinson Diagnostic Systems, Sparks, MD.). Quite recently, the VITEK 2 gram-positive (GP) identification card (bioMérieux, Marcy l'Etoile, France) has been redesigned for increased accuracy...

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Section: Other Significant Opportunistic Pathogens Include Staphylocomentioning

confidence: 99%

Section: Other Significant Opportunistic Pathogens Include Staphylocomentioning

confidence: 99%

Comparative Study Using Various Methods for Identification of Staphylococcus Species in Clinical Specimens

et al. 2006

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“…TRFLP is not a perfect method, and as with any PCRbased method there are potential limitations of primer binding bias (Leuders and Friedrich 2003), operon copy number heterogeneity (Crosby and Criddle 2003), and polymerase error (Osborn et al 2000), although many of these issues can be ameliorated by procedural modification (Polz andCavanaugh 1998, Osborn et al 2000). When conducted with appropriate methodological and statistical treatment, TRFLP has been shown to yield reliable, accurate, pseudo-quantitative representations of complex microbial community structure (Blackwood et al 2003, Leuders and Friedrich 2003, Liu et al 1997, Osborn et al 2000, Schutte et al 2008, Trotha et al 2002.…”

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confidence: 99%

Profiling the Yeast Communities of Wine Fermentations Using Terminal Restriction Fragment Length Polymorphism Analysis

Bokulich¹,

Hwang²,

Liu³

et al. 2011

Am. J. Enol. Vitic.

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Abstract:Terminal restriction fragment length polymorphism (TRFLP), used most often to describe bacterial communities, presents a high-throughput, low-cost solution for analyzing mixed yeast communities in wine and other fermentations. In this study, a TRFLP approach was developed for the identification and discrimination of yeasts and used to construct a TRFLP database comprising 121 strains of yeast (representing 24 genera and 72 species) associated with wine and food fermentations. This database exhibits sensitive discrimination among species and robust intraspecific conservation of TRFLP profiles, enabling reliable characterization of mixed yeast communities. The yeast ecology of sweet, botrytized wine fermentations from two separate vintages was analyzed using this database, demonstrating the utility of this method for fast-paced, qualitative detection and identification of differences in yeast community structures over time in a complex, diverse fermentation system.

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“…This discussion comes all the way from the time when only gel electrophoresis was used in T-RFLP analysis. More recently, capillary and multicapillary electrophoresis has been the choice for higher sample throughput and highly precise determination of fragment lengths (11,14). Even then, there's still no consensus.…”

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confidence: 99%

“…Even then, there's still no consensus. While the use of peak area was recommended (14), some prefer to use peak heights (2,7).…”

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confidence: 99%

Quantitative analysis of Terminal Restriction Fragment Length Polymorphism (T-RFLP) microbial community profiles: peak height data showed to be more reproducible than peak area

Caffaro-Filho

Fantinatti-Garboggini

Durrant

2007

Braz. J. Microbiol.

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Adaption of a fragment analysis technique to an automated high-throughput multicapillary electrophoresis device for the precise qualitative and quantitative characterization of microbial communities

Cited by 32 publications

References 0 publications

Comparative Study Using Various Methods for Identification of Staphylococcus Species in Clinical Specimens

Comparative Study Using Various Methods for Identification of Staphylococcus Species in Clinical Specimens

Profiling the Yeast Communities of Wine Fermentations Using Terminal Restriction Fragment Length Polymorphism Analysis

Quantitative analysis of Terminal Restriction Fragment Length Polymorphism (T-RFLP) microbial community profiles: peak height data showed to be more reproducible than peak area

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