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  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/16—Primer sets for multiplex assays
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Definitions

• the present invention relates to the microbiological field of nucleotide based microorganism detection and identification.
• Microarrays are genomic tools originally developed to monitor gene expression, also applied for the detection of specific mutations in DNA sequences and lately employed in the parallel detection and identification of microorganisms in environmental or clinical samples.
• MDMs microbial diagnostic microarrays
• MDM sensitivity can be defined in different ways.
• MDMs for microbial community analysis need to be capable of detecting microbes from a broad phylogenetic range. In turn, their application can tolerate a relatively low sensitivity in terms of relative abundance. On the other hand, high specificity and sensitivity are primary requirements for MDMs applied for pathogen detection.
• MDMs used in clinical, veterinary, food and public health microbiology, typically rely on species or genus specific PCR amplifications to increase sensitivity and specificity of detection (Sergeev et al., 2004; Lee & Chao, 2005; Maynard et al . , 2005) . As a drawback, they are then limited to a narrow range of pathogens. To cover a range of different pathogens, this approach requires multiple PCR or multiplex PCR (PCR with multiple primer pairs at once) reactions to be performed. Epidemiological and public health surveys as well as the screening of veterin- ary, food or water samples for pathogens requires a different approach, combining broad coverage with high specificity and increased sensitivity.
• the CN 1396270 describes a DNA r ⁇ icroarray for detecting the frequently encountered pathogenic bacteria in water, such as Escherichia 's bacteria, salmonella, staphylococcus, etc., which uses the l ⁇ s rRNA gene, two primers designed in the preservation region of 16s rRNA gene, and a probe for the variable region of l ⁇ s rRNA gene.
• the DE 199 45 916 describes several oligonucleotide sequences for bacterial 23S/5S rRNA probes, which can be used in assays to distinguish bacteria of different categories.
• the WO 2004/046379 A describes the identification of microorganisms through variable regions of topoisomerase genes.
• a goal of the present invention is to provide oligonucleotide probes capable of the sensitive and specific detection of a broad range of microorganisms from samples harbouring complex microbial communities.
• the present invention provides a set of oligonucleotides specific for the gyrB gene of a microorganism, characterized in that at least one oligonucleotide comprises a sequence of SEQ ID NOs 1 to 71, preferably of SEQ ID NOs 1 to 69, or a fragment of a sequence of SEQ ID NOs 1 to 71, preferably of SEQ ID NOs 1 to 69, of at least 14 nucleotides (preferably up to 30 nucleotides) , with optionally 1 or 2 point mutations, and complementary reverse sequences thereto.
• the full nucleotides of the given sequences are preferred.
• the sequences may also comprise further nt sequences or not.
• the mutations allow the detection of other closely related microorgansims leading to mismatches in hybridization experiments with sample genetic material, or amplified material of one or more microorganism (s) .
• the suitability of such derivatives can be steered by the skilled man in the art when using the inventive set by varying standard assay conditions modifying stringency.
• a nucleotide exchange but also a deletion or an insertion is possible.
• the mutations are at the 3' end of the oligonucleotide sequences.
• the present invention also provides a set of oligonucleotides specific for the gyrB gene of at least two microorganisms of different genera, which can be used for the method described herein.
• the oligonucleotides comprise sequences selected from at least two, more preferably at least 10, most preferably at least 30 different sequences of SEQ ID NOs 1 to 71.
• the sequences of this set are specific for the gyrB gene of different species or genera of selected microorganisms and can be used for their detection or identification.
• each oligonucleotide can detect only one microorganism or closely related microorganisms (e.g. via lower stringency).
• the set comprises oligonucleotides, wherein each oligonucleotides is specific for the gyrB gene of one microorganism and the set comprises oligonucleotides specific for at least 2, 3, 4, 5, ⁇ , 7, 8, 9, 10, 12, 16, 24 or 32 microorganisms of different genera, thus allowing a broad scope of detectable microorganisms .
• the set comprises oligonucleotides, wherein different oligonucleotides comprise sequences selected from at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 24, 30 or 32 different sequences of SEQ ID NOs 1 to 71.
• a set of oligonucleotides comprises of oligonucleotides, which are complementary reversed to the oligonucleotides of a sequence of SEQ ID NOs 1 to 71.
• Such oligonucleotides, which sequence is complementary reversed to a template oligonucleotides can thus hybridize these template oligonucleotides.
• Complementary reversed oligonucleotides are also specific for the anti-sense strand of a genetic (or amplified) DNA strand.
• These sets may also comprise further oligonucleotides, especially microorgansim specific oligonucleotides, e.g. directly selected from SEQ ID NOs 1 to 71, thus forming mixtures in the specificity for the gyrB strand in sense and anti-sense direction.
• oligonucleotides of the set are labeled.
• the present invention provides a solid support, wherein oligonucleotides of the set or oligonucleotides which are complementary reversed to the oligonucleotides of the set (and can thus hybridise the oligonucleotides of the set) are immobilized on the solid support.
• the present invention provides a solid support comprising at least two probes specific for the gyrB gene of at least two microorganisms of different genera immobilized on the solid support.
• the probes on. the support comprise sequences that are selected from at least two, more preferably at least 10, most preferably at least 30 different sequences that are complementary reversed to SEQ ID NOs 1 to 71 (and therefore can hybridise these sequences) .
• said oligonucleotide molecules are immobilized in different spots on the surface.
• spots specifically defined regions comprising the immobilized oligonucleotide molecules are produced.
• one spot is specific for certain microorganism it is possible to exactly define the microorganism present in the sample analysed.
• a reference oligonucleotide molecule in the spots which is not only a positive control but can also be used as a reference for quantification.
• the solid support forms a microarray.
• DNA mi- croarrays are a powerful tool for the parallel, high-throughput detection and quantification of many genes. Originally developed for whole genome gene expression analyses, DNA microarrays have a very strong application potential in many areas of microbiology. Upon availability of corresponding probe sets, they enable the detection of up to several thousand microbial strains, species or groups of species (depending on the design of the probe) in a single assay. In clinical, veterinary and plant microbiology, food and water quality control, this means that a single test can be developed to detect all pathogenic/beneficial/contaminating bacteria which might be present in the investigated sample. The potential for environmental microbiology is even stronger. By applying nested sets of oligonucleotide probes which target genes reflecting the phylogeny of the carrying organism, it becomes possible to roughly assess the whole proka- ryotic diversity of an environment.
• a microarray comprises a large number of immobilized oligonucleotide molecules provided in high density on the solid support.
• a microarray is a highly efficient tool in order to detect hundreds to millions of target molecules in one single detection step.
• Such microarrays are often provided as slides or plates in particular microtiter plates.
• a microar- ray is both defined either as a miniaturized arrangement of binding sites (i.e. a material, the support) or as a support comprising miniaturized binding sites (i.e. the array) . Both definitions can be applied for the embodiment of the present invention.
• the preferred embodiment of the present invention is a miniaturized arrangement of the oligonucleotides of the present invention in a microarray.
• the oligonucleotide molecules are preferably immobilized onto the microarray with the help of a printing device which ensures immobilization in high density on the solid support.
• This microarray is particularly useful when analysing a large number of samples .
• the solid support comprises an aldehyde surface.
• An aldehyde surface can be used for the immobilisation of oligonucleotide probes, especially with a NH 2 -group at one end, preferably included by the linker.
• the linker is preferably a Ci to Cio, preferably a C 2 to C 6 linker bound to a short, e.g. 4, 5, 6 nt long, T-stretch.
• the present invention provides a method for identifying a microorganism, comprising i) providing DNA of the gyrB gene of the microorganism, ii) hybridizing the gyrB DNA with the set of oligonucleotides of the present or the solid support, wherein the oligonucleotides form immobilized probes, under stringent conditions, iii) optionally labelling hybridized oligonucleotides, iv) visualising hybridized oligonucleotides.
• a multitude of microorgansims can be detected and identified via the gyrB gene.
• the specificity of MDMs is predominantly defined by the degree of conservation of the marker gene and the length of the oligonucleotide probe (Loy & Bo- drossy, 2006) .
• the gyrB gene, encoding the subunit B of the bacterial gyrase meets all the requirements for a phylogenetic- ally useful protein-coding gene: it can be found in most bacterial species and it does not appear to be frequently horizontally transmitted.
• a method according to the present invention is capable of the sensitive and specific detection of a broad range of microorganisms from samples harbouring complex microbial communities.
• Preferred stringent conditions are 60 0 C annealing temperature for labelling, and 55 0 C for hybridization with the probes of the solid support, with ⁇ xSSC, lxDenhardt's reagent (Sigma, St. Louis, MO, USA), 0,1% SDS, especially in cases where the reactive oligonucleotides are designed for a melting temperature of 60 0 C (e.g. +/- 2 0 C, however a significant proportion of oligos with lower Tm can still work) , for both the hybridization of the reactive oligonucleotides (RC oligos) to the sample (e.g. the PCR product) and/or the probes on the support.
• ⁇ xSSC lxDenhardt's reagent
• SDS lxDenhardt's reagent
• oligonucleotides with different overall melting temperatures can also be designed, e.g. 55°C by selecting gyrB stretches with different GC content, thus lowering the preferred stringent temperature to the melting temperature.
• the stringency is increased by increasing the designed melting temperature by 5 0 C above the average melting temperature of the oligonucleotides .
• a method for identifying a microorganism comprising a) providing the inventive set of oligonucleotides specific for the gyrB gene of the microorgansim, b) providing a support with immobilized probes, the probes being capable of hybridizing to the oligonucleotides under stringent conditions, c) providing a sample of microorganism DNA or RNA, which is preferably amplified by PCR, most preferred PCR specific for the gyrB gene, d) hybridizing the oligonucleotides with the sample under stringent conditions, e) labelling hybridized oligonucleotides, f) hybridizing the oligonucleotides against the probes on the support under stringent conditions, g) visualising the labelled oligonucleotides bound to the probes .
• the probes comprise the reverse complement sequences of the oligonucleotides (to allow the hybridization to the oligonucleotides), and preferably a linker for binding to the support.
• the support preferably solid support, and the immobilized probes form a microarray.
• the probes are oligonucleotide probes, in particular DNA or RNA with lengths between 10 to 30 nts.
• the labelling is performed by addition of at least one labelled ddNTP.
• the addition of ddNTPs prevents further labelling and signal alteration.
• the label is a fluorescence label or a radioactive label, e.g. 32 P.
• the labeled nucleotides are ddCTP.
• competitive oligonucleotides are present, which differ from the oligonucleotides of the set by at least one mismatch, preferably 1 to 6 mismatches, most preferred 1 to 3 mismatches.
• These competitive oligonucleotides can bind to the sample with less specificity than a respective RC oligo and help to reduce or prevent false positive signals.
• the competitive oligonucleotides cannot bind a label, preferably have a 3' phosphate modification incompatible with a nucleotide addition reaction (e.g. sequence specific end labeling) - thus the competitive oligonucleotides cannot bind a label.
• a nucleotide addition reaction e.g. sequence specific end labeling
• the competitive oligonucleotides comprise at least one, preferably at least 3, most preferred at least 6 different sequences selected from SEQ ID NOs 74 to 84, given in table 3 below.
• the microorganism is a pathogen, in particular selected from E. coli, Shigella spp, Salmonella spp, A. hydro- phila, V. cholerae, M. avium, M. tuberculosis, H. pylori, P. mirabilis, Y. enterocolitica and C. jejuni.
• pathogen in particular selected from E. coli, Shigella spp, Salmonella spp, A. hydro- phila, V. cholerae, M. avium, M. tuberculosis, H. pylori, P. mirabilis, Y. enterocolitica and C. jejuni.
• steps (e.g. steps i) to iv) or a) to g) of the methods above) of a first analysis are repeated in at least a second analysis with a set of oligonucleotides ex- eluding oligonucleotides for microorganisms used in a previous analysis. Especially with environmental samples this improves the detection and identification of less abundant microbes within a complex microbial community.
• At least 4, 9, 10, 12, 16, 18, 20, 24 or 26, preferably at least 30, 36 or 40, most preferred at least 50, 60 or 70 different probes are immobilized on the support.
• the oligonucleotides comprise sequence (s) selected from at least one, more preferably at least 4, 9, 10, 12, 16, 18, 20, 24 or 26, preferably at least 30, 36 or 40, most preferred at least 50, 60 or 70 different sequences of SEQ ID NOs 1 to 71, given in table 1 below.
• the probes on the support comprise sequence (s) that is/are selected from at least 4, 9, 10, 12, 16, 18, 20, 24 or 26, preferably at least 30, 36 or 40, most preferred at least 50, 60 or 70 different sequences that are complementary reversed to SEQ ID NOs 1 to 71, given in table 1 below.
• Fig. 1 Probe set validation.
• Fig. 2 Sensitivity of detection.
• Fig. 3. Application of competitive oligonucleotides. Microarray images showing hybridisation results of the En- terobacter cloacae labelled without (A) and with (B) competitive oligonucleotides. Each array contains the same probes in triplicates. Images were scanned at 100% laser power, 750 V PMT and are displayed in rainbow colour mode.
• 1 Internal control Msi_294. 2: Eco_1402 and 3 - Eco_1404 false positive signals that could be successfully silenced with the addition of the competitive oligonucleotide C0_2.
• 3 Yer_1740 false positive signal that could be successfully silenced with the addition of the competitive oligonucleotides C0_7
• Fig. 4 Sequence specific end-labelling of oligonucleotides (SSELO) principle.
• Detection and identification methods for pathogens need in many cases detect less abundant microbes within a complex microbial community. In addition, a high specificity is required, providing robust, reliable identification at least at the species level.
• a microbial diagnostic microarray approach, combining a unique labelling method, gyrB as the marker gene, and the application of competitive oligoprobes was developed, enabling the sensitive and specific detection of a broad range of pathogenic bacteria from samples harbouring complex microbial communities. The approach was first tested with a set of 35 oligoprobes targeting E. coli, Shigella spp, Salmonella spp, A. hydrophila, V. cholerae, M. avium, M. tuberculosis, H.
• the gyrB sequence database was established by downloading sequences from the NCBI database (http://www.ncbi.nlm.nih.gov) and by the sequencing of the strains used for microarray validation. Alignment and neighbour joining phylogenetic tree of the gyrB sequences were constructed using the ARB software package (Ludwig et al., 2004), which was subsequently also used for the probe design. The most important point considered during probe design was the placement of the diagnostic mismatch (es) as close to the 3' end as possible. This feature is very important for the sequence-specific end labelling of the oligonucleotides.
• the probes are designed in a way, that:
• Mismatches of non-targeted, but related sequences are preferably located towards the 3' end of the probe
• target Salmonella
• non-target organism has a mismatch at the 3' position (highlighted in italic) .
• Some probes have mismatches further away from the 3' end. More like those highlighted in bold - even though the 3' mismatches are already enough to guarantee specificity.
• RC oligo Sal_1451 name fullname mis N_mis wmis pos ecoli rev ' AGGCACCCCOGGCCG ⁇ C'
• Table 1 Oligonucleotide probe set. Sequences listed are those of the RC oligo probes that were used in the labelling reaction. All sequences are listed without the 3' terminal C residue. Indicated Tm and G+C % values were calculated using CalcOligo 2.03. SEQ ID Nr are given for PCR reaction oligonucleotides ("RC oligos") of the set. The sequences of the capture oligonucleotides, immobilized on the support, are complementary and reverse (to allow hybridization with the RC oligos) and include an oligo T tail linker for immobilization.
• a - numbers at the end of the probe names refer to their relative positions on the E. coli gyrB gene b - M. avium, M. intracellular, M. maloense c - M. tuberculosis, M. bovis, M. gastri
• Oligonucleotides for immobilisation were custom synthesised (VBC Genomics, Vienna, Austria) with a 5' aminolink followed by five thymidine residues preceding the probe sequence.
• a 384 well flat bottom plate (Ritter GmbH, Schwabmunchen, Germany) was prepared with 30 ⁇ l of 50 ⁇ M oligonucleotide solution in Arraylt spotting buffer (TeleChem Inc., Sunnyvale, CA, USA).
• Microarrays were spotted with an OmniGrid spotter (1 TeleChem SMP3 pin) at • 55% relative humidity and 21°C onto aldehyde silylated slides (CEL Associates Inc., Pearland, TX, USA) . Arrays were spotted in triplicate to allow a statistical correction of the errors. Slide processing was carried out as described before (Stralis- Pavese et al., 2004). Processed slides were stored desiccated at room temperature in dark.
• Genomic DNA samples from pure cultures were purified using the DNeasy extraction kit (QIAGEN, Hilden, Germany) , following manufacturer's instructions. Archived veterinary samples used for the testing of the method' s performance were kindly provided by Clyde Hutchinson (Institute of Zoology, London, UK) . These DNA samples were prepared from animal specimens known to be contaminated with one or more bacterial pathogens using the DNeasy extraction kit (QIAGEN, Hilden, Germany) . Genomic DNA from soil samples was extracted using UltraClean Soil DNA Kit (MO BIO Laboratories, Carlsbad, CA, USA) following the manufacturer's instructions .
• Table 2 List of bacterial species used for microarray validation. Strains were environmental or clinical isolates from the culture collection of the University of Sassari, Department of Biological Sciences, except for Pseudomonas putida, which was obtained from DSMZ (German Collection of Microorganisms and Cell Cultures) . gyrB ac ⁇
• EHEC slt-I E. coli enterohaemorragic
• EIEC E. coli enteroinvasive
• EPEC E. coli enteropathogenic
• DNA amplification gyrB gene was amplified using universal primers UPl ( 5 ? -GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-S ' ) and UP2r ( 3 ' -TACTGNCTRCGNCTRCANCTRCCGAGCGTGTAGGCATGGGACGA-S ' ) .
• UPlG 5'-GAAGTCATCATGACCGTTCTG- CAYGCNGGNGGNAARTTYGG-3'
• UP2Ar 3 -TACTGNCTRCGNCTRCANCTRCCGAGCGTGTAGGCATGGGACGA-S '
• PCR reactions were performed in 100 ⁇ l aliquots, consisting of Ix PCR buffer, 2 mM MgCl 2 , 4U Taq DNA polymerase (Invitro- gen, Carlsbad, CA, USA), 50 ⁇ M for each of the four dNTPs, 150 nM for each primer, with 50 - 100 ng DNA as template.
• amplification was performed with all four primers in one "multiplex" reaction, using the FailSafe PCR Pre- Mix E (Epicentre, Madison, WI, USA) .
• Amplification conditions were 95°C for 5 min, followed by 35 cycles of: 1 min at 95°C, 1 min at 58 0 C, 2 min at 72°C, followed by a final elongation step of 10 min at 72 0 C
• PCR products were subsequently purified using a commercial PCR purification kit (QIAGEN, Hilden, Germany) according to manufacturer's instructions and eluted in 30 ⁇ l dH 2 O. DNA concentration was measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and adjusted to a final concentration of 50 ng/ ⁇ l.
• PCR products were cloned using the TOPO TA Cloning Kit for Sequencing (Invitrogen, Carlsbad, CA, USA) following manufacturer's instructions.
• 25 ⁇ l of the chemically competent cells (provided with the cloning kit) were transformed with 6 ⁇ l of the ligation reaction.
• 100 ⁇ l of the transformation reaction were plated onto LB plates containing 100 ⁇ g/ml ampicillin and incubated overnight at 37 0 C.
• Template for the colony PCR was prepared by resuspending single colonies in 20 ⁇ l dH 2 O and denaturing the cell suspension for 10 min at 95°C in a thermocycler with subsequent cooling down to 4 0 C.
• PCR reactions 1 ⁇ l of this suspension was used as a template for 50 ⁇ l PCR reactions.
• M13 PCR reactions were performed as described above, but using M13 forward (5' GTAAAACGACGGCCAG 3') and M13 reverse primer (3' GTCCTTTGTCGATACTG 5') and 50 0 C annealing temperature.
• PCR products were subsequently purified using PCR purification kit (QIAGEN, Hilden, Germany) according to manufacturer's instructions and eluted in 30 ⁇ l dH 2 O. DNA concentration was measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) .
• Sequencing was performed using the BigDye Terminator vl.l Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) . Sequencing reaction was done in 10 ⁇ l reaction aliquots consisting of 4 ⁇ l purified PCR product, 2 ⁇ l sequencing mix and 400 nM primer (M13 forward or M13 reverse) . Sequencing conditions were 95 0 C for 2 min, followed by 25 cycles of: 30 sec at 95°C, 15 sec at 5O 0 C, 4 min at 60 0 C with no final elongation step.
• Sequencing reactions were purified using Sepha- dex G-50 (Amersham Biosciences, Piscataway, NJ, USA) columns and run on the automated ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA) . Sequences were analysed using Sequencher v 4.5 (Gene Codes Corporation, Ann Arbor, MI, USA) - -
• a pmoA PCR product from Methylosinus trichosporium 0B3b was included in each labelling and hybridisation experiment and subsequently applied for normalisation of the results. Genomic DNA extraction and pmoA PCR amplification were performed as described before (Bodrossy et al, 2003) . PCR product was purified using PCR purification kit (QIAGEN, Hilden, Germany) according to manufacturer' s instructions . DNA concentration was measured using a NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and adjusted to a final concentration of 50 ng/ ⁇ l.
• gyrB PCR products were treated with shrimp alkaline phosphatase (SAP) (Roche Diagnostics GmbH, Pen- zbeg, Germany) in order to dephosphorylate remaining nucleotides.
• SAP shrimp alkaline phosphatase
• oligonucleotides For the labelling a set of reverse complement oligonucleotides, lacking the 3' terminal cytosine residue, was custom synthesised (VBC Genomics, Vienna, Austria) . Lyophilised oligonucleotides were dissolved to a final concentration of 100 pmol/ ⁇ l and stored at -20 0 C. For the labelling reaction an oligonucleotides mix pRC mix ("PRC mix”) , containing each reverse complement oligonucleotide at a final concentration of 1 pmol/ ⁇ l, was pre- pared. Competitive oligonucleotides, when used, were added at the same concentration to the RC mix.
• the cyclic labelling was performed in 10 ⁇ l aliquots consisting of Ix Thermo Sequenase reaction buffer, 10 ng SAP treated control pmoA PCR product, 1 pmol of each reverse complement oligonucleotide, 10 pmol Tamra-ddCTP (PerkinElmer Life and Analytical Sciences, Boston, MA, USA) , 10 pmol of each ddATP, ddTTP, ddGTP (Roche Diagnostics GmbH, Penzbeg, Germany) , 3 U Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) or 3.2 ⁇ Thermo Sequenase (Amersham Biosciences, Piscataway, NJ, USA) and 100 ng of SAP treated gyrB PCR product.
• Reaction conditions were 25 cycles of 30 s at 95°C followed by 75 s at 60 0 C, carried out in a thermocycler. After cyclic labelling, samples were used directly for hybridisation, without further purification. For experiments investigating the possibility of using up to 1000 different probes, RC oligos were added at a final concentration of 0.1 pmol/ ⁇ l. Labelling experiments were also performed using all four nucleotides in form of Tamra-labelled ddNTPs (PerkinElmer Life and Analytical Sciences, Boston, MA, USA) at the final concentration of 10 pmol and omitting all silencing ddNTPs.
• Hybridisation was carried out as described before (Stralis- Pavese et al, 2004) . Labelled targets (10 ⁇ l) were mixed with 200 ⁇ l hybridisation buffer (pre-warmed to 65 0 C) . Final concentration of the hybridisation buffer was: ⁇ xSSC, ixDenhardt's reagent (Sigma, St. Louis, MO, USA), 0.1% SDS. Hybridisation was performed overnight at 55 0 C. After hybridisation, slides were washed in a 2xSSC, 0.1% SDS wash solution for 5 minutes, followed by two wash cycles for 5 minutes in 0.2xSSC, and a final wash for 5 minutes in 0. IxSSC, all at room temperature. Slides were dried with an oil-free air gun and scanned immediately.
• Microarrays were scanned at three lines to average and at 10 ⁇ m resolution using a GenePix 4000A laser scanner (Axon Instruments, Foster City, CA, USA) . PMT gain was adjusted to scan the spots below the saturation level. Scanned images were saved as multilayer tiff images and analyzed with the GenePix Pro 6.0 software (Axon Instruments, Foster City, CA, USA) . Microsoft Excel was used for statistical analysis and presentation of the results. Microarray hybridisation results were normalised to the signal obtained from the internal control oligonucleotide (Msi_294) and expressed as percentage, 100% equalling the signal of the control probe. Probes were considered to be positive during validation if their normalised signal was at least 10% (of the control signal, Msi_294) .
• Sequence specific end-labelling of oligonucleotides (SSELO) (Rudi et al., 1998; Rudi et al., 2003; Rudi et al., 2002b), originally developed for membrane-based macroarrays, offers a significant improvement in the detection limit of MDMs by focusing labelling onto the regions actually used in hybridisation to the microarray.
• the principle of the method is shown in Figure 4.
• Capture oligonucleotides are immobilised on the microarray.
• Reverse complements of the capture oligos (RC oligos) are end labelled in a linear amplification reaction, upon the availability of the corresponding target sequence. The entire mixture is then hybridised to the microarray to sort out the sequences which have been labelled.
• the specificity of the assay was shown to be determined primarily by the stringency of the annealing step during labelling, rather than that of the subsequent hybridisation.
• the optimum annealing temperature under the conditions applied was shown to be 6O 0 C, below and above which false positive and negative results started to appear in increasing numbers.
• using targets labelled under optimised conditions increasing the hybridisation temperature from the standard (55 0 C) to 60 0 C did not improve specificity further. In specific, - -
• One of the mechanisms conferring specificity to the labelling step is the application of labelled ddCTP in combination with the other three ddNTPs unlabelled (silencing ddNTPs) .
• This way the chance of an unexpected hybridisation of the RC oligo to a PCR product, yielding a labelled oligo is decreased by 75% (assuming a random chance of the next nucleotide in the 3' direction on the PCR product being G, serving as template for the addition of the labelled ddCTP) .
• this system seriously limits the options for probe design. This way one is allowed to select only probes where the nucleotide upstream of the RC oligo is a cytosine.
• a crucial factor determining the potential performance of the method is the maximal number of probes that could be employed in parallel.
• the standard protocol applies 1 ⁇ l of a mixture of RC oligonucleotides with 1 pmol/ ⁇ l final concentration for each oligo.
• Oligonucleotide stock solutions are usually 100 pmol/ ⁇ l of concentration, further increase of which is quickly leading to an unstable solution, resulting in precipitation, uneven mixing, etc.
• the maximal number of probes is limited in the range of 100-200 (our current array and hence the labelling procedure employs 35 oligonucleotide probes in one assay) .
• CO oligos competitive oligonucleotides
• Competitive oligonucleotides were designed as a variation of an RC oligo showing false positive signals with non-targeted strains.
• the probe sequence was altered in a way to design a new probe that was a perfect match towards the strain exhibiting false positive signals.
• These CO oligos should therefore have a higher specificity towards the sequence giving rise to the false positive signal than the corresponding RC oligos.
• CO oligos were synthesised with a 3' phosphate modification (VBC Genomics, Vienna, Austria) .
• CO oligos were included in the labelling reaction at the same concentration as the RC oligos.
• 7 competitive oligonucleotides developed (Table 3) , 6 silenced false positive signal they were targeted against.
• Figure 3 shows hybridisation profiles of E. cloacae obtained with and without CO oligos.
• One of the competitive oligos (C0_l) failed to completely silence the false positive signal (i.e., below detection threshold). Still, a significant decrease in the signal was achieved. It has to be noted, that this was the strongest false positive signal obtained.
• Table 3 Set of competitive oligonucleotides. Positions of mismatches with the original probe (RC oligo, see sequence in Table 1) are indicated by capital letters.
• probe design essential requirements during probe design are to tune the probe set in a way that their behaviour is compatible with the conditions of the cyclic labelling reaction and that probes have sufficient discriminating power.
• Three essential guidelines for the probe design were defined: i) position of the diagnostic mismatch (es) as close to the 3' end as possible, ii) similar melting temperature (60 ⁇ 2 0 C, if possible) and iii) similar probe length (17-28 nt) .
• Probes potentially forming hairpin structures or 3' self-dimers that could lend themselves to self-priming were avoided.
• Probe-target pairs with weighted mismatch (wMM) values of up to 0.5 were expected to yield positive hybridisation under the conditions applied.
• the sensitivity of MDMs is usually defined as the lowest relative abundance of the target group detectable (within the analysed community) . When reported, it is found to be around 5% (Loy et al., 2002; Bodrossy et al., 2003; Tiquia et al., 2004; Denef et al., 2003) .
• this limitation is due to the target consisting of labelled nucleic acid fragments (several hundred nucleotides long) , spanning the whole target gene amplified, increasing the potential for the accumulation of background signal arising from a low rate of non-specific hybridisation.
• each probe develops a low background signal of around 1-5% in respect of the maximal signal obtainable (Bodrossy & Sessitsch, 2004) .
• the SSELO approach minimises this background signal.
• the sensitivity of the analysis is also influenced by the inherent bias in the PCR amplification of the gyrB gene.
• the applicability of the microarray was also demonstrated by analysing two archived veterinary samples (courtesy of Clyde Hutchinson, Institute of Zoology, London, UK) .
• the two samples were pathological stool samples from a harbor porpoise and a greenfinch, both having shown pathological signs of Salmonella infection.
• the microarray indicated the presence of Salmonella in both samples, as well as a lower, but clear signal for Vibrio cholerae. Unfortunately the second finding could not be confirmed by cultivation based experiments as the storage of the samples did not allow for the long-term survival of bacteria.

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