Time-minimized determination of ribosome and tRNA levels in bacterial cells using flow field–flow fractionation

Arfvidsson, Cecilia; Wahlund, Karl-Gustav

doi:10.1016/s0003-2697(02)00541-9

Cited by 41 publications

(18 citation statements)

References 43 publications

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“…We have focused on rRNAs as the target for precise and sensitive quantification of commensal subdominant bacterial populations, since rRNA is a universal constituent of bacterial ribosomes and high copy numbers (10 3 to 10 4 molecules per actively growing cell) are present as housekeeping genes (1,17). Targeting these molecules has the potential to increase the detection sensitivity compared to the sensitivity of assays based on detection of a single copy or even multiple copies of genomic sequences.…”

mentioning

confidence: 99%

Sensitive Quantitative Detection of Commensal Bacteria by rRNA-Targeted Reverse Transcription-PCR

Matsuda

Tsuji

Asahara

et al. 2007

Appl Environ Microbiol

262

232

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A sensitive rRNA-targeted reverse transcription-quantitative PCR (RT-qPCR) method was developed for exact and sensitive enumeration of subdominant bacterial populations. Using group-or species-specific primers for 16S or 23S rRNA, analytical curves were constructed for Escherichia coli, Enterococcus faecalis, Staphylococcus aureus, Clostridium perfringens, and Pseudomonas aeruginosa, and the threshold cycle value was found to be linear up to an RNA amount of 10 ؊3 cell per RT-PCR. The number of bacteria in culture was determined by RT-qPCR, and the results correlated well with the CFU count over the range from 10 0 to 10 5 CFU. The bacterial counts obtained by RT-qPCR were the same as the CFU counts irrespective of the growth phase in vitro, except for C. perfringens during starvation periods; the viable cell counts obtained by using a combination of 4,6-diamidino-2-phenylindole (DAPI) staining and SYTO9-propidium iodide double staining were in good agreement with the RT-qPCR counts rather than with the CFU counts. The RT-qPCR method could detect endogenous Enterobacteriaceae and P. aeruginosa in feces of hospitalized patients (n ‫؍‬ 38) at a level of 10 3 cells per g of feces, and for enumeration of S. aureus or P. aeruginosa spiked into human peripheral blood, the lower detection limit for RT-qPCR quantification of the bacteria was 2 cells per ml of blood, suggesting that this method was equivalent to the conventional culture method. As only 5 h was needed for RT-qPCR quantification, we suggest that rRNA-targeted RT-qPCR assays provide a sensitive and convenient system for quantification of commensal bacteria and for examining their possible invasion of a host.For almost a century, culture techniques have been recognized as the "gold standards" for determining viable bacterial counts. As the human fecal flora has been reported to consist of approximately 400 bacterial species (12, 35) and these species are present at a concentration of 10 11 viable microorganisms per g of contents (42), conventional culture techniques for enumeration of different populations involve the use of selective microbiological media, followed by isolation of pure cultures and the use of confirmatory biochemical tests. Recently, a number of molecular methods based on immunological and genotypic techniques have been developed (41,48). In analyses of the gut microflora, a number of molecular methods have been used in place of cultivation-based techniques. Techniques such as the clone library method (42, 46), denaturing gradient gel electrophoresis (13), and terminal restriction fragment length polymorphism (31, 36) allow analysis of predominant bacteria that are difficult to culture. The fluorescent in situ hybridization method (18,43) and the quantitative PCR (qPCR) method with rRNA-targeted oligonucleotide probes or primers have also been used as culture-independent methods. Among these, PCR methods targeting mainly well-conserved 16S rRNA genes have prevailed for rapid quantification of bacteria and are recognized as having two advant...

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mentioning

confidence: 99%

Sensitive Quantitative Detection of Commensal Bacteria by rRNA-Targeted Reverse Transcription-PCR

Matsuda

Tsuji

Asahara

et al. 2007

Appl Environ Microbiol

262

232

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“…Extensive studies have been done on ribosomes using AsFlFFF [77][78][79][80][81][82]. Figure 6 shows an impressive fractionation of the 70S ribosome from the 30S and 50S subunits and tRNA and proteins.…”

Section: Ribosomesmentioning

confidence: 99%

“…This degree of resolution made it possible to monitor ribosomal composition and tRNA levels and correlate them to cell growth and protein production levels [77,81,82] (6 min ribosome preparation, 8 min separation, 2 min wash) made this a viable technique for at-line optimization of cultivation conditions in bioreactors. Additional experiments turned up a previously unreported 100S particle that was believed to be a dimer of the 70S ribosomal particle [78].…”

Section: Ribosomesmentioning

confidence: 99%

Field‐flow fractionation of proteins, polysaccharides, synthetic polymers, and supramolecular assemblies

Williams¹,

Lee

2006

J of Separation Science

125

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This review summarizes developments and applications of flow and thermal field-flow fractionation (FFF) in the areas of macromolecules and supramolecular assemblies. In the past 10 years, the use of these FFF techniques has extended beyond determining diffusion coefficients, hydrodynamic diameters, and molecular weights of standards. Complex samples as diverse as polysaccharides, prion particles, and block copolymers have been characterized and processes such as aggregation, stability, and infectivity have been monitored. The open channel design used in FFF makes it a gentle separation technique for high- and ultrahigh-molecular weight macromolecules, aggregates, and self-assembled complexes. Coupling FFF with other techniques such as multiangle light scattering and MS provides additional invaluable information about conformation, branching, and identity.

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“…All three mentioned FFF methods were used also for the separation and analysis of various bacteria [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16]. The first paper, published already in 1985 by Fox et al [1], concerned * Corresponding author.…”

Section: Introductionmentioning

confidence: 99%

“…the application of SFFF which, in the meantime, became the most frequently used FFF technique when taking into account the number of published papers [1][2][3][4][5][6][7][8][9][10][11]. However, it seems that the use of FFFF, firstly applied in 1996 by Ross et al [3], is more intensively studied in the last 10 years [1,3,7,10,[12][13][14][15][16]. The EFFF was only exceptionally used by Saenton et al [7] who compared it with SFFF and FFFF.…”

Section: Introductionmentioning

confidence: 99%

Micro-thermal field-flow fractionation of bacteria

Janča

Kašpárková

Halabalová

et al. 2007

Journal of Chromatography B

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Time-minimized determination of ribosome and tRNA levels in bacterial cells using flow field–flow fractionation

Cited by 41 publications

References 43 publications

Sensitive Quantitative Detection of Commensal Bacteria by rRNA-Targeted Reverse Transcription-PCR

Sensitive Quantitative Detection of Commensal Bacteria by rRNA-Targeted Reverse Transcription-PCR

Field‐flow fractionation of proteins, polysaccharides, synthetic polymers, and supramolecular assemblies

Micro-thermal field-flow fractionation of bacteria

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