Magnetic capture–hybridization method for purification and probing of mRNA for neutral protease of Bacillus cereus

Bach, H.-J.; Hartmann, Anton; Trevors, J. T.; Munch, Jean Charles

doi:10.1016/s0167-7012(99)00066-4

Cited by 14 publications

(7 citation statements)

References 12 publications

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“…Bacteria only posses a poly(A)-tail on their mRNA in rare cases [18,19]. Although methods exist to isolate mRNA from bacteria [20,21], it is generally considered to be more challenging as compared to eukaryotic RNA, where Oligo(dT) primers are sufficient [22]. Therefore, we refrained from isolating the mRNA prior to reverse transcription.…”

Section: Introductionmentioning

confidence: 99%

Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni

2013

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Campylobacter jejuni remains one of the major gut pathogens of our time. Its zoonotic nature and wide-spread distribution in industrialized countries calls for a quick and reliable diagnostic tool. Antibody-based detection presents a suitable means to identify pathogenic bacteria. However, the knowledge about immunodominant targets is limited. Thus, an approach is presented, which allows for the rapid screening of numerous cDNA derived expression clones to identify novel antigens. The deeper understanding of immunodominant proteins assists in the design of diagnostic tools and furthers the insight into the bacterium’s pathogenicity as well as revealing potential candidates for vaccination. We have successfully screened 1536 clones of an expression library to identify 22 proteins that have not been described as immunodominant before. After subcloning the corresponding 22 genes and expression of full-length proteins, we investigated the immunodominant character by microarrays and ELISA. Subsequently, seven proteins were selected for epitope mapping. For cj0669 and cj0920c linear epitopes were identified. For cj0669, specificity assays revealed a specific linear epitope site. Consequently, an eleven amino acid residue sequence TLIKELKRLGI was analyzed via alanine scan, which revealed the glycine residue to be significant for binding of the antibody. The innovative approach presented herein of generating cDNAs of prokaryotes in combination with a microarray platform rendering time-consuming purification steps obsolete has helped to illuminate novel immunodominant proteins of C.jejuni. The findings of a specific linear epitope pave the way for a plethora of future research and the potential use in diagnostic applications such as serological screenings. Moreover, the current approach is easily adaptable to other highly relevant bacteria making it a formidable tool for the future discovery of antigens and potential biomarkers. Consequently, it is desirable to simplify the identification of structural epitopes, as this would extend the spectrum of novel epitopes to be detected.

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Section: Introductionmentioning

confidence: 99%

Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni

2013

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“…Biotin‐labelled oligonucleotide probe (Interactiva, Ulm, Germany) was added, and the mixture was incubated overnight at room temperature on a Dynal Sample Mixer 159.02 (Dynal Inc., Long Island, NY) at approximately 10 r.p.m. The following day, a volume of Dynabeads M‐280 Streptavidin (Deutsche Dynal GmbH, Hamburg, Germany) sufficient for 50 μl per hybridization reaction were transferred to an Eppendorf tube, washed three times with an equal volume of 0.5X SSC (20X SSC: 3 M NaCl, 0.3 M sodium citrate), resuspended in 0.1% blocking reagent solution in 0.5X SSC, aliquoted into reaction tubes, and incubated for 1 h on an end‐over‐end mixer (Bach et al , 1999). A magnet was used to collect the beads on the tube wall and the blocking mixture was removed by pipetting.…”

Section: Methodsmentioning

confidence: 99%

Isolation of small‐subunit rRNA for stable isotopic characterization

MacGregor

Brüchert

Fleischer

et al. 2002

Environmental Microbiology

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Small-subunit ribosomal RNA (SSU rRNA) has several characteristics making it a good candidate biomarker compound: it is found in bacteria, archaea and eukaryotes; it is quickly degraded extracellularly, hence SSU rRNA extracted from a sample probably derives from the currently active population; it includes both conserved and variable regions, allowing the design of capture probes at various levels of phylogenetic discrimination; and rRNA sequences from uncultured species can be classified by comparison with the large and growing public database. Here we present a method for isolation of specific classes of rRNAs from mixtures of total RNA, employing biotin-labelled oligonucleotide probes and streptavidin-coated paramagnetic beads. We also show that the stable carbon isotope composition of Escherichia coli total RNA and SSU rRNA reflects that of the growth substrate for cells grown on LB, M9 glucose and M9 acetate media. SSU rRNA is therefore a promising biomarker for following the flow of carbon, and potentially nitrogen, in natural microbial populations. Some possible applications are discussed.

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“…The blocking-reagent solution was removed using a magnetic-particle concentrator. The beads were resuspended in 0.5ϫ SSC, and 25 l was dispensed per reaction tube and incubated with probe-target hybrid at room temperature on a rotator for 2 h (3,26). The concentration of the beads was 10 mg/ml, and the binding capacity of the beads was 400 pmol/mg (according to the manufacturer), which was approximately four times more than the amount of probes added in the protocol.…”

Section: Methodsmentioning

confidence: 99%

Linking Microbial Community Function to Phylogeny of Sulfate-Reducing Deltaproteobacteria in Marine Sediments by Combining Stable Isotope Probing with Magnetic-Bead Capture Hybridization of 16S rRNA

Miyatake

MacGregor

Boschker

2009

Appl Environ Microbiol

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We further developed the stable isotope probing, magnetic-bead capture method to make it applicable for linking microbial community function to phylogeny at the class and family levels. The main improvements were a substantial decrease in the protocol blank and an approximately 10-fold increase in the detection limit by using a micro-elemental analyzer coupled to isotope ratio mass spectrometry to determine 13 C labeling of isolated 16S rRNA. We demonstrated the method by studying substrate utilization by Desulfobacteraceae, a dominant group of complete oxidizing sulfate-reducing Deltaproteobacteria in marine sediments. Stable-isotopelabeled [13 C]glucose, [ 13 C]propionate, or [ 13 C]acetate was fed into an anoxic intertidal sediment. We applied a nested set of three biotin-labeled oligonucleotide probes to capture Bacteria, Deltaproteobacteria, and finally Desulfobacteraceae rRNA by using hydrophobic streptavidin-coated paramagnetic beads. The target specificities of the probes were examined with pure cultures of target and nontarget species and by determining the phylogenetic composition of the captured sediment rRNA. The specificity of the final protocol was generally very good, as more than 90% of the captured 16S rRNA belonged to the target range of the probes. Our results indicated that Desulfobacteraceae were important consumers of propionate but not of glucose. However, the results for acetate utilization were less conclusive due to lower and more variable labeling levels in captured rRNA. The main advantage of the method in this study over other nucleic acid-based stable isotope probing methods is that 13 C labeling can be much lower, to the extent that ␦ 13 C ratios can be studied even at their natural abundances.

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Magnetic capture–hybridization method for purification and probing of mRNA for neutral protease of Bacillus cereus

Cited by 14 publications

References 12 publications

Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni

Rapid Identification of Novel Immunodominant Proteins and Characterization of a Specific Linear Epitope of Campylobacter jejuni

Isolation of small‐subunit rRNA for stable isotopic characterization

Linking Microbial Community Function to Phylogeny of Sulfate-Reducing Deltaproteobacteria in Marine Sediments by Combining Stable Isotope Probing with Magnetic-Bead Capture Hybridization of 16S rRNA

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