16S rRNA targeted sandwich hybridization method for direct quantification of mycobacteria in soils

Nieminen, Tea; Pakarinen, Jaakko; Tsitko, Irina; Salkinoja‐Salonen, Mirja; Breitenstein, Antje; Ali-Vehmas, Terhi; Neubauer, Peter

doi:10.1016/j.mimet.2006.02.015

Cited by 19 publications

(13 citation statements)

References 37 publications

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“…High concentrations of detergent as in the original assay (8,11,14) were found not only to hamper sonication but also to lead to clumping of the magnetic beads. Lower detergent concentrations in buffers prevented bead aggregation and also facilitated the resuspension of the beads and diminished sticking of the beads to plastic surfaces during the assay.…”

Section: Resultsmentioning

confidence: 96%

“…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).…”

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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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Section: Resultsmentioning

confidence: 96%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

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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“…Due to these obstacles, the use of molecular methods for detection and identification of mycobacteria in the environment is favorable. Mycobacterial DNA has been isolated from soil in previous studies for the purpose of an ecological study or genus-specific identification, although no further species identification was done (30,33,34).…”

Section: Discussionmentioning

confidence: 99%

“ Mycobacterium avium subsp. hominissuis ” in Neck Lymph Nodes of Children and their Environment Examined by Culture and Triplex Quantitative Real-Time PCR

et al. 2011

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"Mycobacterium avium subsp. hominissuis" often causes cervical lymphadenitis in children; its prompt and accurate identification enables adequate therapy, tracing, and prevention. The aims of this study were to determine the causative agent of lymphadenitis using culture, PCR, and triplex quantitative real-time PCR (qPCR) methods with DNA directly isolated from tissue, as well as to identify possible sources of infection from the environment. We confirmed the diagnoses by detecting M. avium subsp. hominissuis using qPCR with DNA directly isolated from lymph node biopsy specimens of two patients. In order to trace the source of infection from the environment, a method of DNA isolation from soil and other environmental samples, such as dust, cobwebs, and compost, was developed. The triplex qPCR examination revealed the presence of M. avium subsp. hominissuis in a high proportion of the environmental samples (42.8% in the first patient's house and 47.6% in the second patient's house). Both patients were also exposed to M. avium subsp. avium, which was present due to the breeding of infected domestic hens. The high infectious dose of M. avium subsp. hominissuis or the increased susceptibility of humans to M. avium subsp. hominissuis compared to M. avium subsp. avium could be the reason why the children were infected with M. avium subsp. hominissuis.

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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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16S rRNA targeted sandwich hybridization method for direct quantification of mycobacteria in soils

Cited by 19 publications

References 37 publications

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

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

“ Mycobacterium avium subsp. hominissuis ” in Neck Lymph Nodes of Children and their Environment Examined by Culture and Triplex Quantitative Real-Time PCR

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

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