Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay

Leskelä, Tarja T.; Tilsala-Timisjärvi, Anu; Kusnetsov, Jaana; Neubauer, Peter; Breitenstein, Antje

doi:10.1016/j.mimet.2005.02.008

Cited by 29 publications

(21 citation statements)

References 29 publications

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“…Interestingly, PCR in clinical applications may have a higher PPV (than for environmental samples) despite the lower sensitivity with nonrespiratory specimens (379). Two potential remedies for the low PPV with samples from environmental sources include reverse transcription-PCR, amplifying labile RNA targets present in metabolically active bacteria, and the use of a cell-impermeant chemical, such as ethidium monoazide (EMA) or propidium monoazide (PMA), to inhibit PCR amplification from nonviable cells or extracellular nucleic acids (385)(386)(387)(388)(389)(390). Notably, neither alternative protocol alone will discriminate VBNC legionellae; however, this cell population still poses a potential human health risk (391)(392)(393)(394).…”

Section: Nucleic Acid-based Molecular Diagnosticsmentioning

confidence: 99%

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

2015

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SUMMARY Legionnaires' disease (LD) is an often severe and potentially fatal form of bacterial pneumonia caused by an extensive list of Legionella species. These ubiquitous freshwater and soil inhabitants cause human respiratory disease when amplified in man-made water or cooling systems and their aerosols expose a susceptible population. Treatment of sporadic cases and rapid control of LD outbreaks benefit from swift diagnosis in concert with discriminatory bacterial typing for immediate epidemiological responses. Traditional culture and serology were instrumental in describing disease incidence early in its history; currently, diagnosis of LD relies almost solely on the urinary antigen test, which captures only the dominant species and serogroup, Legionella pneumophila serogroup 1 (Lp1). This has created a diagnostic “blind spot” for LD caused by non-Lp1 strains. This review focuses on historic, current, and emerging technologies that hold promise for increasing LD diagnostic efficiency and detection rates as part of a coherent testing regimen. The importance of cooperation between epidemiologists and laboratorians for a rapid outbreak response is also illustrated in field investigations conducted by the CDC with state and local authorities. Finally, challenges facing health care professionals, building managers, and the public health community in combating LD are highlighted, and potential solutions are discussed.

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Section: Nucleic Acid-based Molecular Diagnosticsmentioning

confidence: 99%

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

2015

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“…However, this SUC2 mRNA is highly abundant in S. cerevisiae. The hybridization assay was not very sensitive and had only a low degree of robustness (8,11,14). Major challenges to address before the usefulness of SBSH could be extended to low-abundance transcripts were high background levels of fluorescence in the fluorescence substrate reaction and the aggregation of the beads.…”

Section: Resultsmentioning

confidence: 99%

“…In addition to the preparation time needed, total-mRNA isolation procedures require the inactivation of ribonucleases and particular attention to rapid sample acquisition. Prokaryotic mRNAs have short half-lives, averaging 6.8 min in Escherichia coli (23), prohibiting extensive centrifugation steps for preparation, which may cause partial mRNA degradation and altered gene expression results.RNA-targeting bead-based sandwich hybridization is becoming more of an established method for the analysis of bacterial rRNAs (8,11,14) or for the analysis of dynamic changes in mRNA levels (13,17,19,22). Although sandwich hybridization can be applied for the quantitative detection of rRNAs in crude cell extracts (8, 11), so far the method has been inefficient in detecting mRNA transcripts in crude cell…”

mentioning

confidence: 99%

“…RNA-targeting bead-based sandwich hybridization is becoming more of an established method for the analysis of bacterial rRNAs (8,11,14) or for the analysis of dynamic changes in mRNA levels (13,17,19,22). Although sandwich hybridization can be applied for the quantitative detection of rRNAs in crude cell extracts (8,11), so far the method has been inefficient in detecting mRNA transcripts in crude cell lysates with good sensitivity and a high degree of robustness (19).…”

mentioning

confidence: 99%

“…Although sandwich hybridization can be applied for the quantitative detection of rRNAs in crude cell extracts (8,11), so far the method has been inefficient in detecting mRNA transcripts in crude cell lysates with good sensitivity and a high degree of robustness (19).…”

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confidence: 99%

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Sandwich Hybridization Assay for Sensitive Detection of Dynamic Changes in mRNA Transcript Levels in Crude Escherichia coli Cell Extracts in Response to Copper Ions

Thieme

Neubauer

Nies

et al. 2008

Appl Environ Microbiol

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Transcript quantification techniques usually rely on purified mRNAs. We report here a solution-based sandwich hybridization assay for the quantification of mRNAs from Escherichia coli without the need of prior RNA isolation. This assay makes use of four DNA oligonucleotide probes adjacently hybridizing to target RNA in clarified cell extracts. Two helper probes facilitate the hybridization of a detection and a capture probe. The latter is biotin labeled, allowing binding to streptavidin-coated paramagnetic beads and the separation of the RNA-DNA hybrid from cellular constituents. Added antidigoxigenin Fab fragments conjugated to alkaline phosphatase bind to the digoxigenin-labeled detection probe, completing the sandwich of the paramagnetic bead, mRNA, probes, and alkaline phosphatase. The target transcript can be quantified by assessing phosphatase activity on a substrate that is converted into a fluorescent product. The amount of target mRNA is calculated from the fluorescence output and from a calibration curve for a known concentration of in vitro-synthesized target mRNA. This technique was used in time course experiments to investigate the expression of three genes responsible for the copper resistance of E. coli. The induction of gene expression by copper cations was rapid, but under aerobic conditions, the levels of expression returned to low, prestress levels within minutes. In anaerobiosis, high-level expression continued for at least 1 h. When cultures were shifted from anaerobiosis to aerobiosis, expression levels were diminished within minutes to prestress levels. The improved technique presented here is relatively simple, has very high degrees of sensitivity and robustness, is less laborious than other RNA quantification methods, and is not negatively affected by genomic DNA. These characteristics make it a powerful complementary application to genetic reporter fusions and to reverse transcription-PCR.Several methods for the quantification of transcripts in different organisms are available. These techniques differ largely in the amounts of time and expense required for data acquisition. In general, these methods can be grouped into those that target single transcripts and those that target whole transcriptomes. Northern hybridization has the disadvantage of a rather low level of sensitivity, limited quantification potential, and a large time commitment but is comparatively inexpensive. On the other hand, global and quantitative assays like transcriptome microarray analysis call for major investments in equipment and disposables, aside from limitations in view of quantitative data evaluation/expression analysis. For the quantification of single transcripts, reverse transcription (RT)-PCR is currently the method of choice. RT-PCR can be performed as a quantitative or semiquantitative assay depending on whether absolute transcript numbers are required or whether relative expression levels are investigated.A major drawback of RT-PCR is the necessity of purified high-quality RNA that is virtually free o...

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The changing face of microbial quality control practices in the brewing industry: Introducing mass spectrometry proteomic fingerprinting for microbial identification

et al. 2017

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Microbiological quality control testing in breweries is essential for maintaining high product quality and should be performed routinely. The effects of microbial contamination, derived from organisms including bacteria, wild yeast and mould, can range from comparatively minor changes in beer flavour and fermentation performance to gross flavour and aroma defects, turbidity problems and reduced yeast performance. Further implications of batch contamination include significant financial loss and decreased consumer confidence. This review discusses current microbiology testing techniques and their limitations, then focuses on new developments and technologies for routine microbiological control testing. The adoption of new technologies such as proteomic fingerprinting by mass spectrometry would allow breweries to conduct more extensive microbiological analyses that are currently cost‐ and time‐prohibitive, particularly for small breweries. Copyright © 2017 The Institute of Brewing & Distilling

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Sensitive genus-specific detection of Legionella by a 16S rRNA based sandwich hybridization assay

Cited by 29 publications

References 29 publications

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

Current and Emerging Legionella Diagnostics for Laboratory and Outbreak Investigations

Sandwich Hybridization Assay for Sensitive Detection of Dynamic Changes in mRNA Transcript Levels in Crude Escherichia coli Cell Extracts in Response to Copper Ions

The changing face of microbial quality control practices in the brewing industry: Introducing mass spectrometry proteomic fingerprinting for microbial identification

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