Patent application title: NUCLEIC ACID BEACONS FOR FLUORESCENT IN-SITU HYBRIDISATION AND CHIP TECHNOLOGY
Inventors:
Ian Thrippleton (Duesseldorf, DE)
Assignees:
MIACOM DIAGNOSTICS GMBH
IPC8 Class: AC12Q168FI
USPC Class:
506 9
Class name: Combinatorial chemistry technology: method, library, apparatus method of screening a library by measuring the ability to specifically bind a target molecule (e.g., antibody-antigen binding, receptor-ligand binding, etc.)
Publication date: 2012-10-11
Patent application number: 20120258876
Abstract:
The present invention relates to beacons for fluorescent in-situ
hybridisation and chip technology.Claims:
1. A nucleic acid capable of forming a hybrid with a target nucleic acid
sequence and capable of forming a stem-loop structure if no hybrid is
formed with the target sequence, said nucleic acid comprising (a) a
nucleic acid portion comprising (a1) a sequence complementary to the
target nucleic acid sequence, (a2) a pair of two complementary sequences
capable of forming a stem, (b) an effector and an inhibitor, wherein the
inhibitor inhibits the effector when the nucleic acid forms a stem-loop
structure, and wherein the effector is active when the nucleic acid is
not forming a stem-loop structure.
2. The nucleic acid of claim 1, wherein the nucleic acid is suitable for in-situ hybridisation, in particular FISH.
3. The nucleic acid of claim 1, wherein the hybridisation takes place within a cell.
4. The nucleic acid of claim 1, wherein the nucleic acid when covalently linked to a solid phase is suitable for hybridisation with the target nucleic acid sequence, wherein the target nucleic acid sequence is preferably provided in a cell-free sample.
5. The nucleic acid of claim 4, wherein the target nucleic acid sequence of (at) is a nucleic acid sequence of a microorganism.
6. The nucleic acid of claim 1, wherein the target nucleic acid sequence of (al) is a DNA sequence or a RNA sequence, in particular a rRNA sequence.
7. The nucleic acid of claim 1, wherein essentially the two complementary sequences of (a2) form the stem.
8. The nucleic acid of claim 1, wherein essentially the sequence complementary to the target nucleic acid sequence of (a1) forms the loop.
9. The nucleic acid of claim 1 wherein the sequence complementary to the target nucleic acid sequence of (a1) and at least one of two complementary sequences of (a2) overlap, preferably by 1, 2, 3, 4 or 5 nucleotides or/and nucleotide analogues.
10. The nucleic acid of claim 1, wherein the Tm of the hybrid of the sequences of (a2) is essentially equal or lower than the Tm, of the hybrid of the sequence of (a1) with the target sequence, e.g. at the maximum about 5.degree. C., about 4.degree. C., about 3.degree. C., about 2.degree. C., or about 1 ° C. lower.
11. The nucleic acid of claim 1, wherein the Tm, of the hybrid of the sequences of (a2) is essentially equal or lower than the Tm of the hybrid of the sequence of (a1) with the target sequence under essentially Mg2+ free conditions.
12. The nucleic acid of claim 1, wherein the ΔG of the hybrid of the sequences of (a2) is smaller than 0, preferably in the absence of the target sequence.
13. The nucleic acid of claim 1, wherein the ΔG of the hybrid of the sequences of (a2) is higher than the ΔG of the hybrid of the sequence of (a1) with a target sequence.
14. The nucleic acid of claim 1, wherein the ΔG of the hybrid of the sequence of (a2) is lower than the ΔG a hybrid of the nucleic acid with a mismatch sequence or/and a sequence different from the target sequence.
15. The nucleic acid of claim 1, wherein the ΔG of the hybrid of the sequence of (a1) with its target sequence is in the range of about -17 to about -25 kcal/mol under hybridisation conditions.
16. The nucleic acid of claim 1, wherein the stem formation takes place in the presence of Mg2+, in particular in the presence of about 1 to about 20 mM Mg2+, more particular in the presence of about 5 to about 10 mM Mg2+, most particular in the presence of about 8 to about 10 mM Mg2+.
17. The nucleic acid of claim 1, wherein the inhibitor is covalently bound to one end of the nucleic acid portion and the effector is bound to the other end of the nucleic acid portion.
18. The nucleic acid of claim 1, wherein the effector or/and the inhibitor is coupled to the nucleic acid portion via a linker, which linker preferably comprises building blocks selected from nucleotides, nucleotide analogues, amino acids, and amino acid analogues.
19. The nucleic acid of claim 1, wherein the inhibitor and the effector do not form part of the stem.
20. The nucleic acid of claim 1, wherein the effector is a luminescent label, in particular is fluorescent label, and the inhibitor is a quencher.
21. The nucleic acid of claim 20, wherein the effector and the inhibitor are suitable for fluorescence resonance transfer technology.
22. The nucleic acid of claim 1, wherein the effector is an enzyme and the inhibitor is an inhibitor of the enzyme.
23. The nucleic acid of claim 22, wherein the enzyme exerts an electrochemical signal.
24. The nucleic acid of claim 22, wherein the enzyme is a reporter enzyme, preferably selected from the group consisting of tyrosinase, peroxidase, sulfite oxidase, alkaline phosphatase, glucose oxydase, guanine oxidase.
25. The nucleic acid of claim 22, wherein the enzyme is derived from thermo- or/and hyperthermophylic organisms.
26. The nucleic acid of claim 22, wherein the enzyme is a recombinant enzyme.
27. The nucleic acid of claim 22 wherein the enzyme is glucose oxidase and the inhibitor is an adenine nucleotide.
28. The nucleic acid of claim 1, wherein the nucleic acid portion (a) consists of ribonucleotides, ribonucleotide analogues, deoxyribonucleotides or/and deoxyribonucleotide analogues, which nucleotide analogues are different from PNA building blocks.
29. The nucleic acid of claim 1, wherein at least one of the two complementary sequences of (a2) comprises at least one non-matching nucleotide.
30. The nucleic acid of claim 1, wherein the nucleic acid portion (a) is selected from the beacon sequences of Table 1.
31. A combination comprising at least two nucleic acids as claimed in claim 1.
32. The combination of claim 31, wherein the ΔG values of the hybrid of the sequences of (a2) or/and the hybrid of the sequence of (a1) with a target sequence of the individual nucleic acids differ at the maximum by about 4 kcal/mol.
33. The combination of claim 31, wherein the Tm values of the hybrid of the sequences of (a2) or/and the hybrid of the sequence of (a1) with a target sequence of the individual nucleic acids differ at the maximum by about 3.degree. C.
34. The combination of claim 31, wherein the individual nucleic acids function uniformly under hybridisation conditions required to hybridise under in-situ hybridisation conditions.
35. The combination of claim 31, wherein the individual nucleic acids when covalently linked to an inorganic solid phase function uniformly under hybridisation conditions required to hybridise DNA or RNA1 wherein DNA or/and RNA are preferably provided in a cell-free sample.
36. The combination of claim 31, wherein the individual nucleic acids when covalently linked to protein function uniformly under hybridisation conditions required to hybridise DNA or RNA, wherein DNA or/and RNA are preferably provided in a cell-free sample.
37. The combination of claim 36, wherein the individual nucleic acids function uniformly under hybridisation conditions wherein the protein is an enzyme linked to one end and an enzyme inhibitors linked to the other end of the nucleic acid portion.
38. The combination of claim 36, wherein the individual nucleic acids function uniformly under hybridisation conditions required to hybridise DNA or RNA, wherein the enzyme is an enzyme derived from thermo- or hyperthermophylic organisms.
39. The combination of claim 37, where the enzyme exerts an electrochemical signal.
40. A hybridisation method comprising (a) contacting at least one nucleic acid of claim 1 with a biological sample, (b) hybridising the nucleic acid or the combination of nucleic acid of (a) with the sample under conditions where the stem of the nucleic is open, and (c) inducing conditions which allow for stem formation in those nucleic acid molecules of (a) not forming a hybrid with the sample.
41. The method of claim 40, which is an in situ hybridisation, in particular FISH.
42. The method of claim 40, wherein hybridisation takes place within a cell.
43. The method of claim 40, wherein the sample comprises a microorganism to be detected.
44. The method of claim 40 wherein the target nucleic acid sequence of the nucleic acid is a nucleic acid sequence of a microorganism.
45. The method of claim 40 wherein the target nucleic acid sequence is a DNA sequence or a RNA sequence, in particular an rRNA sequence.
46. The method of claim 40 wherein the at least one nucleic acid is covalently linked to a solid phase and wherein the target nucleic acid is preferably provided in a cell-free sample.
47. The method of claim 40, wherein step (b) comprises hybridising with a buffer which is essentially free of Mg2+.
48. The method of claim 40 wherein step (c) comprises washing with a Mg2+ containing buffer.
49. The method of claim 40 wherein step (c) comprises washing at pH>8 or/and at room temperature.
50. A kit comprising a nucleic acid of claim 1.
51. A chip comprising a nucleic acid of claim 1.
52. The chip of claim 51 wherein the effector is an enzyme and the inhibitor is an inhibitor of the enzyme.
53. The chip of claim 51 wherein the enzyme is glucose oxidase and the inhibitor is an adenine nucleotide.
54. The chip of claim 50, which is an electrochemical chip.
55. Use of at least one nucleic acid of claim 1 to identify the presence or absence of one or a plurality of organisms, in particular microorganisms, within a biological sample such as a plurality of live or dead matter of human, animal or/and food origin.
56. Use of claim 55 comprising ISH or/and FISH.
57. Use of claim 55, which is a diagnostic use.
58. Use of any of claim 55, wherein at least two nucleic acids are functioning simultaneously under identical conditions.
59. A hybridisation method comprising (a) contacting a combination of nucleic acids of claim 31 with a biological sample, (b) hybridising the nucleic acid or the combination of nucleic acid of (a) with the sample under conditions where the stem of the nucleic is open, and (c) inducing conditions which allow for stem formation in those nucleic acid molecules of (a) not forming a hybrid with the sample.
Description:
[0001] The present invention relates to beacons for fluorescent in-situ
hybridisation and chip technology.
BACKGROUND/PRIOR ART
[0002] Since the wide-spread success of the polymerase chain reaction (PCR) technology, microbiology laboratories are waiting for the application of molecular biology to routine microbiology. This has been held back by an inherent and fundamental problem of molecular biology. Because of its precision, you need to know which tools (probes) to choose. The prerequisite is, that a request has to be specified with respect to organisms to be detected. In clinical samples, however, you do not know which of the over 2000 clinically relevant pathogens is the causative agent of an infection. A rational approach solves the problem [0003] Focus must be made on 95% of problem causing organisms [0004] If it is known where the sample was taken, and clinical data is present, the number of organisms can be reduced to between 2 and 16. [0005] The number of organisms to cover the 95-percentile in most clinical samples is in the order of 100
[0006] This rationale makes it economically feasible to run a DNA-probe based assay on a routine basis.
[0007] Grouping of micro-organisms and the very rapid testing for presence/absence of specific or a range of micro-organisms is also of relevance in other fields of microbiological testing: Blood banks, Pharmaceutical industry, Cosmetic industry and the Food industry. Frequently the same organisms are of relevance throughout the disciplines and reaction conditions therefor need to be standardised for all probes.
[0008] The detection of ribosomal RNA via Fluorescent in-situ Hybridisation (FISH) or utilising chip technology represents an efficient way of utilising sensitivity and specificity of DNA-probes without having to use an enzymatic amplification step. FISH relies on two approaches to the in-situ detection of targets generating a signal strong enough to be detected with standard measuring devices such as an epifluorescence microscope: [0009] 1. Identical molecules are present within a cell in sufficient numbers to bind one specific oligonucletide or nucleotide analogue probe with one fluorophor each. [0010] 2. Large probes carrying a plurality of fluorophores e.g. labelled cosmids.
[0011] FISH technology for the identification of micro-organisms in their respective environments is well known in the art. Application of FISH for the detection of pathogens is of especial interest to the clinical microbiology and infectiology, where FISH excels in speed and cost efficiency.
[0012] Detecting rRNA with chip technology also relieves from the necessity to amplify the target. Total rRNA is extracted from a sample and placed on a chip. Specific probes are concentrated on a small surface area and attract respective rRNA-molecules to give specific presence/absence signals. In order to make such chips economically viable they need to be used repeatedly with as little manipulations as possible. Furthermore the standardisation of probe characteristics is paramount for the generation of reproducible results.
[0013] In order to gain acceptance in a routine environment probes must be designed in such a way that all probes for one disease state can be run simultaneously under identical conditions in or on one vessel (chips, micro-fluidic devices or micro titre plates). In the design of the probes and to make the probes economically viable, it must-be taken into account that one probe may be of relevance to different disease states. Therefor, not only one set of probes but all probes must work under identical hybridisation conditions.
[0014] Sequence and length of the working probes must be tested accordingly.
[0015] The selection and definition of a working probe cannot be performed by simple sequence comparison and determination of a theoretical Tm-value. Depending on the algorithm applied a wide set of values are obtained giving no guidance to the choice of probe sequence suitable for standardised hybridisation conditions.
[0016] The choice of algorithm and factors influencing the quality of a probe is discussed widely in the art (1-7). Further guidance may be sought comparing sequences and actual position in the three dimensional structure of the ribosome. In an attempt to rationalise the design of probes Behrens et al (8) investigated the correlation between hybridisation sites and actual accessibility with the help of the 3 Å three dimensional model of the ribosome. Their findings demonstrated that the SDS used in in-situ procedures has a predominant denaturing effect, not captured by algorithms predicting secondary structures.
[0017] A further problem in both FISH and chip technology is that the procedure calls for a stringent wash step to remove unbound probes, requiring additional handling steps, reagents and time. The success of a hybridisation may depend largely on the skill and precision applied to the washing step. However, routine applications call for minimal steps and hands-on time, most importantly they must be independent from individual skills.
[0018] One solution to the reduction of steps would be the application of fluorescence resonance energy transfer ("FRET") in an oligo-nucleotide or nucleotide analogue hairpin formation (molecular beacon). Several approaches to the development of beacons are known in the art and generalised descriptions to their construction are freely available (13). Beacons are widely used in real time PCR, where they anneal in solution to an increasing number of templates generated by amplifying enzymes (15). Only few attempts have been made to generate beacons for the detection/identification of bacteria on membranes (14). One successful beacon was constructed to detect E. coli in whole cells with a peptide nucleic acid (PNA) probe. The corresponding DNA-probe failed to give adequate performance (16). The production of further PNA-beacons is limited due to the poor solubility of PNA based oligonucleotides as laid down in design recommendations (17).
[0019] Patent CA 2176266/EP 0745690 gives guidance to the construction of universal stems for real time PCR (9). Surprisingly, these recommendations do not render working beacons when combined with probes designed to identify micro-organisms in-situ. Real time PCR is performed in solution while both ISH (in-situ hybridisation) and chips require fixed targets. Their thermodynamic details were not compatible with in-situ hybridisation and FRET requirements. Thus, universally working stems could not be predicted for applications with fixed targets. It was therefore necessary to empirically search for specific beacons fitting individual oligo-nucleotide or nucleotide analogues in order to accomplish a plurality of beacons working under identical ISH specifications.
[0020] In the selection of ISH-beacons care has to be taken that the stem does not hinder the delicate balance of hybridising towards RNA entwined in large protein/RNA complexes such as ribosomes. The accessibility of binding sites is widely discussed in the art and is summarised in (1).
[0021] Further limitations in the design of a beacon probe are given by the size of pores generated in the cell wall during the ISH procedure. Adding the same stem to different probes results in distinctly individual beacons. A plurality of probes already form hairpin loops and the addition of a stem does not result in a "beacon" formation. In addition, simply adding bases to form complementary pairs may increase the Tm to such an extent that the hairpin is thermodynamically preferred rather than the hybrid formation. Special stems have to be devised that pull the sequence into beacon formation while maintaining the Tm at or below that of the hybrid. The teachings with respect to the design of beacons (13) show that the increase of the stem length by one base pair increases the Tm by 5° C. and that the Tm of the stem should be 10° C. higher than the Tm of the hybridising sequence.
[0022] FISH with single microorganisms, such as bacteria, based upon specific rRNA sequences, may be difficult due to sterical hindrance of the rRNA in the ribosome. In other words, a beacon forming a hairpin may poorly anneal to the embedded rRNA target sequence.
[0023] It is therefore the subject of the present invention to provide molecular beacons which overcome the above described disadvantages at least partially. The solution provided in the present invention and preferred embodiments thereof are described in the claims.
[0024] Subject of the present invention is a nucleic acid capable of forming a hybrid with a target nucleic acid sequence and capable of forming a stem-loop structure if no hybrid is formed with the target sequence, said nucleic acid comprising [0025] (a) a nucleic acid portion comprising [0026] (a1) a sequence complementary to the target nucleic acid sequence, [0027] (a2) a pair of two complementary sequences capable of forming a stem, [0028] (b) an effector and an inhibitor, wherein the inhibitor inhibits the effector when the nucleic acid forms a stem-loop structure, and wherein the effector is active when the nucleic acid is not forming a stem-loop structure.
[0029] The nucleic acid of the present invention capable of forming a hybrid with a target nucleic acid sequence and capable of forming a stem-loop structure if no hybrid is formed with the target sequence is also referred herein as "beacon", "molecular beacon", "hairpin", or "hairpin loop", wherein the "open" form (no stem is formed) as well as the "closed" form (the beacon forms a stem) is included. The open form includes a beacon not forming a hybrid with a target sequence and a beacon forming a hybrid with the target sequence.
[0030] In particular, the two complementary sequences (a2) are flanking the sequence (a1), i.e. the first sequence (a2) is attached at the 3' end of the sequence (a1) and the second sequence (a2) is attached at the 5' end of the sequence (a1).
[0031] The hybrid of the sequence (a1) with the target sequence is also referred herein as "hybrid with the cognate sequence" or as "cognate hybrid".
[0032] In the present invention, the effector may be attached at one of the two complementary sequences capable of forming a stem, whereas the inhibitor may be attached at the other of the two complementary sequences, so that the inhibitor essentially inhibits the effector activity when a stem is formed, and that the effector is active when the hairpin is open. Preferably, the effector is attached at the 5' end or the 3' end of the beacon, respectively, or at a position which is 1, 2, 3, 4, or 5 nucleotides distant to the 5' end or the 3' end, respectively. The inhibitor is preferably attached at the other end not covered by the effector, i.e. at the 3' end or the 5' end, respectively, or at a position which is 1, 2, 3, 4, or 5 nucleotides distant to the 3' end or the 5' end, respectively.
[0033] The design of the hairpin loops disclosed herein therefore differs fundamentally from beacons well known in the art.
[0034] Hybridisation of the beacon of the present invention with target sequence may take place under conditions where the loop is open. A beacon which is not forming a stem when hybridizing is capable of annealing to a target rRNA sequence, for instance, and can therefor achieve successful hybridisation.
[0035] This goal is for instance achieved by a Tm of the beacon (i.e. the Tm of the stem) which is essentially equal to or lower than the Tm of the cognate hybrid (i.e. the hybrid of the beacon with the target sequence). Thus, hybridisation with the target sequence takes place when the stem is open, for instance if hybridisation takes place under essentially Mg2+ free conditions.
[0036] "Essentially equal Tm" of the cognate hybrid and the stem of the beacon refers to melting temperatures differing in less than 5° C., preferably less than 3° C., more preferably less than 2° C., more preferably less than 1° C., more preferably less than 0.5° C., even more preferably less than 0.2° C., most preferably less than 0.1° C.
[0037] In order to achieve an inhibition of the effector by the inhibitor, both of which form part of the beacon, in those beacon molecules not hybridising with the target sequence, stem formation must be induced after the hybridisation reaction. This may for instance be achieved by a beacon having a ΔG<0, so the hairpin will form spontaneously. Further, stem formation may be introduced by washing with a Mg2+ containing buffer as described herein.
[0038] In particular, the hairpin loops are constructed in such a way that under standardised hybridisation conditions (e.g. under essentially Mg2+ free conditions) the beacon stem is open so that possible sterical limitations do not hinder the hybridisation process. For instance, sterical limitations may be present when the target sequence is a rRNA sequence. If the effector is a fluorophor, the fluorophor will not be quenched by the close proximity of ribosomal proteins.
[0039] Suitable conditions for induction of stem formation after hybridisation include an. Mg2+ containing buffer, for instance containing about 1 to about 20 mM Mg2+, more particular about 5 to about 15 mM Mg2+, even more particular about 8 to about 12 mM Mg2+, most particular about 10 mM Mg2+. The buffer may have a pH>8.
[0040] Furthermore, the beacons function in their entirety and cannot be dissected into stem and loop as nearest neighbour and stacking effect have a profound influence in their thermodynamic properties. Preferred beacons of the present invention are summarised in Table 1. They clearly show that the preferred stem sequence is independent from the ΔG, Tm, GC content or length of the sequence chosen to identify a species.
[0041] In the present invention, the thermodynamic specifications for the individual construction of beacons suitable for standardised conditions are set: The Gibbs energy (ΔG) for the formation of the beacon has to be designed in such a way that [0042] The beacon will form spontaneously (ΔG<0) in the absence of a cognate target sequence under hybridisation conditions. [0043] The ΔG of the cognate hybrid is significantly lower (i.e. is more negative) than the ΔG of the beacon. [0044] The respective ΔG of the beacon is lower than a mismatch or non-cognate sequence. [0045] The Tm for the formation of the beacon has to be designed in such a way that the Tm of the beacon is lower than or essentially at the Tm of the hybrid.
[0046] It is preferred that the ΔG of the cognate hybrid is in the range of about -17 to about -25 kcal/mol, preferably about -18 to about -24 kcal/mol, more preferably about -19 to about -23 kcal/mol, most preferably about -20 to about -22 kcal/mol under hybridisation conditions.
[0047] It is also preferred that the ΔG of the cognate hybrids under hybridisation conditions do not vary more than 5 kcal/mol, preferably no more than 3 kcal/mol, more preferably 2 kcal/mol and most preferably 1 kcal/mol.
[0048] Occasionally cognate sequences may form spontaneous hairpin loops, where one arm only needs to be supplemented -to achieve the beacon formation. If the target sequence is a rRNA sequence, this, however renders the effector, e.g. the fluorophor, in very close proximity to potentially quenching proteins of the ribosome. In a preferred configuration the stem is extended. In order to conform with said thermodynamic specifications as described herein even with an extended stem a method was devised to keep both the Tm and ΔG within the specifications. According to the present invention, this can be achieved by the introduction of at least one non-matched nucleotide or nucleotide analogue. In the present invention, introduction of at least one non-matched nucleotide may be enhanced by the introduction of an additional nucleotide or nucleotide analogue, so that the two complementary sequences have a different length, and the stem becomes "bended" (see for example position 36 in SEQ ID NO:1), or/and may be achieved by a replacement of a matching nucleotide or nucleotide analogue by a non-matching nucleotide or nucleotide analogue (see for example position 5 in SEQ ID NO: 7). Thus, in the present invention, the "complementary sequences capable of forming a stem" may also include at least one non-matched nucleotide, preferably 1, 2, 3, 4 or 5 non-matched nucleotides.
[0049] As can be seen from Table 2 none of the sequences disclosed here could be devised as PNA-beacons due to the said limitations in the construction of PNA-oligonucleotides. The major limitation being in the oligonucleotide length required to have both sufficient specificity and a stem length sufficient to ensure the re-folding of the loop when not hybridised. It is therefore necessary to devise DNA-beacons that are able to hybridise with sufficient affinity and speed to enable the in-situ identification of micro-organisms.
[0050] The beacon of the present invention is not a PNA beacon. The backbone of the beacon is preferably a nucleic acid backbone. The beacon may comprise a nucleic acid analogue such as a deoxyribonucleotide analogue or a ribonucleotide analogue in the nucleic acid portion or/and in the linker if a linker is present. This analogue is preferably a nucleotide analogue modified at the sugar moiety, the base or/and the phosphate groups. The nucleotide analogue is preferably not a PNA building block.
[0051] Following the said 95-percentile in clinical samples, pathogens can be grouped into disease related groups. Probes towards these organisms must work simultaneously under the said conditions, especially if all probes are to be utilised on one chip. The chip application calls for a stringent standardisation of both the cognate and stem characteristics. If a combination of more than one probe is employed, i. e. at least two probes, all probes have to be designed to work on the same slide/chip simultaneously.
[0052] Another subject of the present invention is a combination comprising at least 2, preferably at least 10, at least 20, at least 30, at least 40, or at least 50 beacons. The combination may comprise but is not limited to all of the beacons of Table 1, preferably at the maximum 100, at the maximum 80, at the maximum 70, at the maximum 60, at the maximum 50, at the maximum 40, at the maximum 30 or at the maximum 20 beacons.
[0053] In a combination of the present invention, the beacons may have the same or different target sequences. It is preferred that the target sequences of individual beacons are different.
[0054] In a combination of beacons of the present invention, the ΔG difference of the individual beacons of the hybrid of the sequences of (a2) or/and the hybrid of the sequence of (a1) with a target sequence may be at the maximum about 4 kcal/mol, preferably at the maximum about 3 kcal/mol, more preferably at the maximum about 2 kcal/mol, and most preferably at the maximum about 1 kcal/mol with respect to the cognate sequence.
[0055] In a combination, the Tm values of individual beacons with respect to its respective cognate sequence may differ at the maximum by about 3° C., preferably at the maximum about 2° C., more preferably at the maximum about 1° C.
[0056] It is preferred that in the combination of the present invention the individual nucleic acids function uniformly. "Functioning uniformly" means that successful hybridisation can be achieved with different nucleic acids probes of the present invention under the same hybridisation conditions, for instance under standardised hybridisation conditions. In other words, uniformly functioning nucleic acids of the present invention do not require individual optimisation of the hybridisation conditions.
[0057] Depending on the disease state certain pathogens most frequently are the causative agents and can thus be compiled into diagnostic groups. Addition or omission of certain pathogens may be required depending on regional epidemiology in order to reach the 95-percentile. The preferred listing of Table 1 covers the requirements of Europe and most of North America.
[0058] Yet another aspect of the present invention is a kit or chip which may contain at least two beacons of Table 1 required to detect the listed organisms optionally together with the required hybridisation reagents, Preferably, the chip or kit contains at least 10, at least 20, at least 30, at least 40, or at least 50 beacons. The kit or chip may contain at the maximum all of the beacons of Table 1, preferably at the maximum 100, at the maximum 80, at the maximum 70, at the maximum 60, at the maximum 50, at the maximum 40, at the maximum 30 or at the maximum 20 beacons.
[0059] List of groupings and resulting kits for the detection, enumeration and identification of the listed organisms is compiled in Table 1.
[0060] The beacons can be applied to assays designed to be performed in tubes, microtitre plates, filtered microtitre wells, slides and chips. The detection can be made with fluorescence, time resolved fluorescence, with a plurality of fluorophores and utilising electrochemical enzymes.
[0061] In the preferred embodiment for FISH the assay is performed on glass slides designed to hold and separate several samples.
[0062] Another subject of the present invention is a hybridisation method comprising [0063] (a) contacting at least one nucleic acid of any of the present invention or a combination of nucleic acids of the present invention with a biological sample, [0064] (b) hybridising the nucleic acid or the combination of nucleic acid of (a) with the sample under conditions where the stem of the nucleic is open, e.g. hybridising with a buffer which is essentially free of Mg2+, and [0065] (c) inducing conditions which allow for stem formation in those nucleic acid molecules of (a) not forming a hybrid with the sample, e.g. washing with a Magnesium containing buffer, for instance at pH>8 or/and at room temperature.
[0066] The sample may be any sample of biological origin, such as a clinical or food sample, suspected of comprising a nucleic acid to be detected by the beacon. The sample may be a sample comprising microorganisms, such as bacteria, yeasts and molds, in particular Gram positive or/and Gram negative bacteria.
[0067] Also employed in the hybridisation method of the present invention can be a kit or chip as described herein.
[0068] "Essentially free of Mg2+" refers to a Mg2+ concentration of less than 1 mM, preferably less than 0.1 mM, more preferably less than 0.05 mM, most preferably less than 0.01 mM.
[0069] The buffer in step (c) may contain about 1 to about 20 mM Mg2+, more particular about 5 to about 15 mM Mg2+, even more particular about 8 to about 12 mM Mg2+, most particular about 10 mM Mg2+.
[0070] Any suitable hybridisation protocol comprising application of an essentially Mg2+ free solution and a Mg2+ containing solution as indicated above may be applied. For instance, the following protocol may be used: Aliquots of clinical samples are applied to defined fields on the slides. Preferably a defined quantity of 10 μl is applied and dried. [0071] 1. The samples are the heat fixed to the slides. [0072] 2. Gram positive organisms are subjected to a Lysozyme/Lysostaphin digestion following well published specifications. In a preferred embodiment the digestion is run for 7 minutes at 46° C. in a humidified chamber. [0073] 3. Pores are then formed for instance by immersing the slide 100% methanol or ethanol for several minutes. In a preferred embodiment the methanol or ethanol is ice cold and the immersion time is 7 minutes for Gram negative organisms and 3 minutes for Gram positive organisms. [0074] 4. The slide is then dried on a slide warmer, for instance at 55° C. [0075] 5. The beacons are dissolved in a hybridisation buffer (which may be essentially free of Mg2+) and then applied to each field of the slide while on the slide warmer. [0076] 6. The slide is placed in a hybridisation chamber, humidified with hybridisation buffer. In a preferred embodiment the slide is covered with a hydrophobic cover slip and placed on a covered slide warmer at 46° C. for 12 minutes. [0077] 7. The slide is then washed with a Magnesium containing buffer, for instance at pH>8 or/and at room temperature. The buffer main contain about 1 to about 20 mM Mg2+, more particular about 5 to about 15 mM Mg2+, even more particular about 8 to about 12 mM Mg2+, most particular 10 mM Mg2+ [0078] 8. The slide is then dried and may be mounted with mounting fluid and can be read under an epifluorescence microscope at a total magnification of for instance 400×, 600×, or 1000×.
[0079] Should other vessels be used for the hybridisation, the detection may be via flow-cytometry or automated fluorescence reader well known in the art.
[0080] Yet another embodiment of the present invention relates to Chip applications of the beacons of the present invention. For Chip applications the beacons need to be covalently attached to a carrier surface. To facilitate this, the 3'-terminal base of the designed beacons may be either biotinylated or linked via a hetero-bifunctional reagent to an enzyme using methods well known in the art of protein and nucleic acid chemistry. Biotinylated beacons may then be added to Streptavidin coated chips as can be obtained freely from commercial sources (19). In this application the respective biotinylated hairpin loops can be attached to plurality of distinct fields of one chip, for instance at least 10, at least 50, at least 100, at least 200, or at least 500 fields, or at the maximum 500, at the maximum 400 or at the maximum 300 fields. Total RNA can be extracted from samples using commercially available kits (20) and can be applied to the chip under hybridising conditions. After hybridisation the chip can be briefly washed with a magnesium containing buffer, for instance at pH>8. Fluorescence on a field marks the presence of specific target sequence, for instance a specific RNA indicating the presence of a respective organism in the sample.
[0081] In order to open hybridisation assays to large scale routine applications it is necessary to analyse a plurality of samples sequentially on one reusable chip. The design of the chip must allow large scale production, efficient quality control and long shelf live.
[0082] In order to meet these specifications, in another embodiment of the present invention, a beacon of the present invention is covalently attached to an enzyme exerting a signal by catalysing a specific reaction. In particular, the enzyme may exert an electrochemical signal. Suitable enzymes comprise, but are not limited to tyrosinase, peroxidase, sulfite oxidase, alkaline phosphatase, glucose oxydase, guanine oxidase. In a preferred embodiment the enzyme is recombinantly derived from a genomic sequence of a thermo- or hyperthermophylic organism to render it stable under hybridisation conditions and elevated temperatures (21). The enzyme may be attached to the beacon at one end of the beacon molecule. At the other end of the molecule, an inhibitor may be attached which is capable of inhibiting the enzyme activity. When no cognate sequence to said hairpin loops is present the inhibitor inhibits the enzyme and no signal is generated. In the presence of a cognate sequence the loop will remain unfolded with the inhibitor well removed from the enzyme and the enzyme will produce an electrochemical signal which can be detected by devices well described in the art. A linker may be employed for the attachment of the enzyme or/and the inhibitor, in particular for the attachment of the inhibitor.
[0083] In a further preferred embodiment glucose oxidase is attached to one end of the said hairpin loops and a glucose oxidase inhibitor, such as an adenine nucleotide or adenine nucleotide analogue is attached to the other end of the hairpin loop. Adenine nucleotides are known inhibitors of glucose oxidase (22, 23). A linker may be employed for the attachment of the glucose oxidase or/and the glucose oxidase inhibitor, in particular for the attachment of the glucose oxidase inhibitor. When no cognate sequence to said hairpin loops is present the inhibitor, in particular the adenine nucleotide inhibits the enzyme and no signal is generated. In the presence of a cognate sequence the loop will remain unfolded with the inhibitor well removed from the enzyme and the enzyme will produce an electrochemical signal which can be detected by devices well described in the art.
[0084] To perform such an assay a large plurality of sequences with identical characteristics (Table I) have been developed, which may be applied to defined positions on the detecting device (chip) respectively. Total RNA is extracted from a sample utilising extraction procedure and kits readily available on the market (20) and placed on the chip under hybridisation conditions. After the hybridisation the chip is washed with substrate buffer at 46° C. and the signal is read. At the end of the cycle all hybridised RNA is washed off with hybridisation buffer at elevated temperature. Preferably the wash temperature is chosen 10° C. above the respective Tm. In a preferred embodiment the chip is washed at 60° C. with hybridisation buffer. The temperature may then dropped to 46° C. to equilibrate for the next analytical cycle.
LEGENDS
[0085] Table 1 describes beacon sequences of the present invention. Abbreviations: R&G: a red or/and a green fluorescent dye may be attached to the beacon, such as Cy3 or FITC or a derivative thereof.
[0086] Table 2 describes that PNA beacons are not suitable in the present invention. Calculations were performed with the sequences of Table 1 assuming the beacon to be a PNA beacon. In contrast to DNA beacons, all of the following five criteria have to be fulfilled: GC content <60%, <3 bases selfcomplementary, 4 purines in a row, length of maximal 18, inverse sequence palindromes or repeats or hairpins. "Yes" ("No") in Table 2 indicates that the criterion is fulfilled (not fulfilled). The column "Final" indicates if a PNA beacon is suitable in the present invention ("Yes") or not ("No"). "No" in final indicates that one of the five criteria is not met. "Yes" would indicate that all criteria are met. All sequences of Table 2 are judged to be "No". Thus, no one of the sequences of Table 1 would be suitable in a PNA beacon.
REFERENCES
[0087] 1. Sebastian Behrens, Caroline Ruhland, Joao Inacio, Harald Huber, A. Fonseca, I. Spencer-Martins, Bernhard M. Fuchs, and Rudolf Amann, In Situ Accessibility of Small-Subunit rRNA of Members of the Domains Bacteria, Archaea, and Eucarya to Cy3-Labeled Oligonucletide or nucleotide analogue Probes, Applied and Environmental Microbiology, March 2003, p. 1748-1758, Vol. 69, No. 3 [0088] 2. Wallace, R. B.; Shaffer, J.; Murphy, R. F.; Bonner, J.; Hirose, T.; Itakura, K. Nucleic Acids Res. 6, 3543 (1979). [0089] 3. Howley, P. M; Israel, M. F.; Iaw, M-F.; Martin, M. A. J. Biol. Chem. 254, 4876. [0090] The equations for RNA are:
[0090] Tm=79.8+18.5 log M+58.4(XG+XC)+11.8(XG+XC)2-820/L-0.35F [0091] And for DNA-RNA hybrids:
[0091] Tm=79.8+18.5 log M+58.4(XG+XC)+11.8(XG+XC)2-820/L-0.50F [0092] 4. Breslauer, K. J.; Frank, R.; Bl{hacek over (s)}cker, H.; Marky, L.A. Proc. Natl. Acad. Sci. USA 83, 3746-3750(1986). For RNA see: Freier, S. M.; Kierzek, R.; Jaeger, J. A.; Sugimoto, N.; Caruthers, M. H.; Neilson, T.; Turner, D. H. Proc. Natl. Acad. Sci. 83, 9373-9377 (1986). [0093] 5. Rychlik, W.; Spencer, W. J.; Rhoads, R. E. (1990) Nucl. Acids Res. 18 (21), 6409-6412. [0094] 6. Owczarzy R., You Y., Moreira B. G., Manthey J. A., Huang L., Behlke M. A., Walder J. A. (2004) Effects of Sodium Ions on DNA Duplex Oligomers: Improved Predictions of Melting Temperatures, Biochemistry, 43:3537-3554. [0095] 7. Sebastian Behrens,1 Bernhard M. Fuchs,1 Florian Mueller,2 and Rudolf Amann1 Appl Environ Microbiol. 2003 August; 69(8): 4935-4941. [0096] 8. HYBRIDIZATION PROBES FOR NUCLEIC ACID DETECTION-UNIVERSAL STEMS document view Patent number: CA2176266 Publication date: 1996 Nov. 13,/EP0745690 (A2) [0097] 9. Tyagi, S., D. P. Bratu, and F. R. Kramer. 1998. Multicolor molecular beacons for allele discrimination. Nat. Biotechnol. 16:49-53. [0098] 10. Tyagi, S., and F. R. Kramer. 1996. Molecular beacons: probes that fluoresce upon hybridization. Nat. Biotechnol. 14:303-308. [0099] 11. Schofield, P., A. N. Pell, and D. O. Krause. 1997. Molecular beacons: trial of a fluorescence-based solution hybridization technique for ecological studies with ruminal bacteria. Appl. Environ. Microbiol. 63:1143-1147. [0100] 12. www.molecular-beacons.org [0101] 13. Molecular Beacons: Trial of a Fluorescence-Based Solution by Hybridization Technique for Ecological Studies with Ruminal Bacteria PETER SCHOFIELD, ALICE N. PELL,* AND DENIS O. KRAUSE† APPLIED AND ENVIRONMENTAL MICROBIOLOGY, March 1997, p. 1143-1147 [0102] 14. Steven Park, May Wong, Salvatore A. E. Marras, Emily W. Cross, Timothy E. Kiehn, Vishnu Chaturvedi, Sanjay Tyagi, and David S. Perlin; Journal of Clinical Microbiology, August 2000, p. 2829-2836, Vol. 38, No. 8; Rapid Identification of Candida dubliniensis Using a Species-Specific Molecular Beacon [0103] 15. Chuanwu Xi, Michal Balberg, Stephen A. Boppart, and Lutgarde Raskin APPLIED AND ENVIRONMENTAL MICROBIOLOGY, September 2003, p. 5673-5678 Vol. 69, No. 9 Use of DNA and Peptide Nucleic Acid Molecular Beacons for Detection and Quantification of rRNA in Solution and in Whole Cells [0104] 16. Guidelines for Sequence Design of PNA Oligomers www.appliedbiosystems.com/support/seqguide.cfm [0105] 17. Tijssen, P. Hybridization with nucleic acid probes. part I. Theory and nucleic acid preparation, p. 268. Elsevier Science Publishers B.V., Amsterdam. [0106] 18. Nanogen, www.nanogen.com [0107] 19. Qiagene, www.Qiagene.com [0108] 20. Microbiology and Molecular Biology Reviews, March 2001, p. 1-43, Vol. 65, No. 1 Hyperthermophilic Enzymes: Sources, Uses, and Molecular Mechanisms for Thermostability, Claire Vieille and Gregory J. Zeikus [0109] 21. ELECTROCHEMICAL SENSORS FOR ENVIRONMENTAL MONITORING: A REVIEW OF RECENT TECHNOLOGY by JOSEPH WANG Department of Chemistry and Biochemistry, New Mexico State University Las Cruces, N.Mex. 88003 [0110] 22. Brenda, www.brenda.uni-koeln.de
Sequence CWU
1
358140DNAArtificial SequenceBeacon, target Acinetobacter 1tgccggatta
ccatcctctc ccatactcta aatcccggca
40221DNAArtificial SequenceProbe, target Acinetobacter 2accatcctct
cccatactct a
21321DNAUnknownTarget of SEQ ID NO 1 and 2, organism Acinetobacter
3tagagtatgg gagaggatgg t
21430DNAArtificial SequenceBeacon, target Acinetobacter baumannii
4gcgcgtccgg tagcaagcta ccttccgcgc
30520DNAArtificial SequenceProbe, target Acinetobacter baumannii
5tccggtagca agctaccttc
20620DNAAcinetobacter baumannii 6gaaggtagct tgctaccgga
20741DNAArtificial SequenceBeacon, target
Aspergillus flavus 7ccgccggcgt acagagttcg tggtgtctcc tcgcccagcg g
41820DNAArtificial SequenceProbe, target Aspergillus
flavus 8acagagttcg tggtgtctcc
20920DNAAspergillus flavus 9ggagacacca cgaactctgt
201023DNAArtificial sequenceBeacon, target
Aspergillus fumigatus 10cgtcgcctac agagcaggtg acg
231118DNAArtificial sequenceProbe, target Aspergillus
fumigatus 11gcctacagag caggtgac
181218DNAAspergillus fumigatus 12gtcacctgct ctgtaggc
181332DNAArtificial sequenceBeacon,
target Aspergillus niger 13ctctgaactg attgcattca atcaactcag ag
321425DNAArtificial sequenceProbe, target
Aspergillus niger 14actgattgca ttcaatcaac tcaga
251525DNAAspergillus niger 15tctgagttga ttgaatgcaa tcagt
251636DNAArtificial
sequenceBeacon, target Aspergillus terreus 16tccgtctgat tgcaaagaat
cacactcaga gacgga 361724DNAArtificial
sequenceProbe, target Aspergillus terreus 17tgattgcaaa gaatcacact caga
241824DNAAspergillus terreus
18tctgagtgtg attctttgca atca
241941DNAArtificial sequenceBeacon, targets Bacteroides/Prevotella
19gccgccggca tccaatgtgg gggaccttct agcccagcgg c
412018DNAArtificial sequenceProbe, targets Bacteroides/Prevotella
20ccaatgtggg ggaccttc
182118DNAUnknownTarget of SEQ ID NO 19 and 20, organisms
Bacteroides/Prevotella 21gaaggtcccc cacattgg
182239DNAArtificial sequenceBeacon, target Borrelia
burgdorferi 22tgccggattc atgcttaaga cgcactgcca atcccggca
392320DNAArtificial sequenceProbe, target Borrelia burgdorferi
23catgcttaag acgcactgcc
202420DNABorrelia burgdorferi 24ggcagtgcgt cttaagcatg
202540DNAArtificial sequenceBeacon, target
Bordetella pertussis 25tgccggattc agcactctgc aaagacgaaa aatcccggca
402621DNAArtificial sequenceProbe, target Bordetella
pertussis 26cagcactctg caaagacgaa a
212721DNABordetella pertussis 27tttcgtcttt gcagagtgct g
212839DNAArtificial sequenceBeacon,
target Burkholderia cepacia complex (Option I) 28accgctcttt
ctttccggac aaaagtgctt tgagcggct
392924DNAArtificial sequenceProbe, target Bukholderia cepacia complex
(Option I) 29tttctttccg gacaaaagtg cttt
243024DNAUnknownTarget of SEQ ID NO 28 and 29, organism
Burkholderia cepacia complex (Option I) 30aaagcacttt tgtccggaaa gaaa
243136DNAArtificial sequenceBeacon,
target Burkholderia cepacia complex (Option II = two beacons)
31cgccttcaga accaaggatt tctttccggg aaggcg
363222DNAArtificial sequenceProbe, target Burkholderia cepacia complex
(Option II = two beacons) 32agaaccaagg atttctttcc gg
223322DNAUnknownTarget of SEQ ID NO 31 and 32,
organism Burkholderia cepacia complex (Option II = two beacons)
33ccggaaagaa atccttggtt ct
223433DNAArtificial sequenceBeacon, target Burkholderia cepacia complex
(Option II = two beacons) 34acgcaagagc caaggttttc tttccgcttg cgt
333521DNAArtificial sequenceProbe, target
Burkholderia cepacia complex (Option II = two beacons) 35agagccaagg
ttttctttcc g
213621DNAUnknownTarget of SEQ ID NO 34 and 35, organism Burkholderia
cepacia complex (Option II = two beacons) 36cggaaagaaa accttggctc t
213728DNAArtificial
sequenceBeacon, target Burkholderia cepacia 37acgctcgtca tcccccggcc
atgagcgt 283816DNAArtificial
sequenceProbe, target Burkholderia cepacia 38gtcatccccc ggccat
163916DNABurkholderia cepacia
39atggccgggg gatgac
164029DNAArtificial sequenceBeacon, targets Burkholderia
pyrrocinia/stabilis/ambifaria 40cgctccgtca tcccccggct ataggagcg
294118DNAArtificial sequenceProbe, targets
Burkholderia pyrrocinia/stabilis/ambifaria 41cgtcatcccc cggctata
184218DNAUnknownTarget of
SEQ ID NO 40 and 41, organisms Burkholderia pyrrocinia / stabilis /
ambifaria 42tatagccggg ggatgacg
184329DNAArtificial sequenceBeacon, target Burkholderia
dolosa/anthina 43ccgctcgtca tcccccggct gtagagcgg
294417DNAArtificial sequenceProbe, target Burkholderia
dolosa / anthina 44gtcatccccc ggctgta
174517DNAUnknownTarget of SEQ ID NO 43 and 44, organism
Burkholderia dolosa / anthina 45tacagccggg ggatgac
174641DNAArtificial sequenceBeacon, target
Burkholderia multivorans 46gccgccggcg tcgtcatccc ccgatcgtat cgcccagcgg c
414718DNAArtificial sequenceProbe, target
Burkholderia multivorans 47cgtcatcccc cgatcgta
184818DNABurkholderia multivorans 48tacgatcggg
ggatgacg
184941DNAArtificial sequenceBeacon, target Burkholderia
cenocepacia/vietnamiensis 49gccgccggcg tcgtcatccc ccgactgtat cgcccagcgg c
415018DNAArtificial sequenceProbe, target
Burkholderia cenocepacia/vietnamiensis 50cgtcatcccc cgactgta
185118DNAUnknownTarget of SEQ
ID NO 49 and 50, organisms Burkholderia cenocepacia / vietnamiensis
51tacagtcggg ggatgacg
185218DNACampylobacter thermophiles 52gccctaagcg tccttcca
185331DNAArtificial sequenceBeacon,
target Campylobacter lari 53acgctcgaag tgtaagcaac taaatgagcg t
315419DNAArtificial sequenceProbe, target
Camplyobacter lari 54gaagtgtaag caactaaat
195519DNACampylobacter lari 55atttagttgc ttacacttc
195630DNAArtificial
sequenceBeacon, target Campylobacter jejuni 56acgctcagct aaccacttat
accggagcgt 305718DNAArtificial
sequenceProbe, target Campylobacter jejuni 57agctaaccac ttataccg
185818DNACampylobacter jejuni
58cggtataagt ggttagct
185932DNAArtificial sequenceBeacon, target Campylobacter upsaliensis
59ccgctccgtg tgtcgcccta ggcgtagagc gg
326020DNAArtificial sequenceProbe, target Campylobacter upsaliensis
60cgtgtgtcgc cctaggcgta
206120DNACampylobacter upsaliensis 61tacgcctagg gcgacacacg
206230DNAArtificial sequenceBeacon,
target Campylobacter coli 62ccgctctcga tggcatcagg ggttgagcgg
306318DNAArtificial sequenceProbe, target
Campylobacter coli 63tcgatggcat caggggtt
186418DNACampylobacter coli 64aacccctgat gccatcga
186518DNAArtificial
sequenceProbe, target Campylobacter coli (competitor) 65tcgacggcat
caggggtt
186618DNACampylobacter coli 66aacccctgat gccgtcga
186739DNAArtificial sequenceBeacon, targets
Chlamydia 67acgccggcgt tagctgatat cacatagatc gcccagcgt
396818DNAArtificial sequenceProbe, targets Chlamydia 68tagctgatat
cacataga
186918DNAUnknownTarget of SEQ ID NO 67 and 68, organisms Chlamydia
69tctatgtgat atcagcta
187041DNAArtificial sequenceBeacon, targets Chlamydiaceae 70tccgccggcg
tctttccgcc tacacgccct cgcccagcgg a
417118DNAArtificial sequenceProbe, targets Chlamydiaceae 71ctttccgcct
acacgccc
187218DNAUnknownTarget of SEQ ID NO 70 and 71, organisms
Chlamydiaceae 72gggcgtgtag gcggaaag
187341DNAArtificial sequenceBeacon, targets Chlamydiales
73tccgccggcg tcctccgtat taccgcagct cgcccagcgg a
417418DNAArtificial sequenceProbe, targets Chlamydiales 74cctccgtatt
accgcagc
187518DNAUnknownTarget of SEQ ID NO 73 and 74, organisms
Chlamydiales 75gctgcggtaa tacggagg
187639DNAArtificial sequenceBeacon, targets Chlamydophila
76acgccggcgt ctaactttcc tttccgcctc gcccagcgt
397718DNAArtificial sequenceProbe, targets Chlamydophila 77ctaactttcc
tttccgcc
187818DNAUnknownTarget of SEQ ID NO 76 and 77, organisms
Chlamydophila 78ggcggaaagg aaagttag
187941DNAArtificial sequenceBeacon, target Chlamydia
pneumoniae 79tccaccggcg tctcttcctc aaccgaaagt cgcccagtgg a
418018DNAArtificial sequenceProbe, target Chlamydia pneumoniae
80ctcttcctca accgaaag
188118DNAChlamydia pneumoniae 81ctttcggttg aggaagag
188241DNAArtificial sequenceBeacon, targets
"Chlamydia psittaci" group 82tcagccggcg taaggcaaaa ccaactccct cgcccagctg
a 418318DNAArtificial sequenceProbe, targets
"Chlamydia psittaci" group 83aaggcaaaac caactccc
188418DNAUnknownTarget of SEQ ID NO 82 and 83,
organisms "Chlamydia psittaci" group 84gggagttggt tttgcctt
188539DNAArtificial sequenceBeacon,
targets Subgroup of the Parachlamydiaceae 85ccgccggcgt tccgttttct
ccgcctactc gcccagcgg 398618DNAArtificial
sequenceProbe, targets Subgroup of the Parachlamydiaceae
86tccgttttct ccgcctac
188718DNAUnknownTarget of SEQ ID NO 85 and 86, organisms Subgroup of
the Parachlamydiaceae 87gtaggcggag aaaacgga
188841DNAArtificial sequenceBeacon, targets Chlamydia
ssp. 88tccgccggcg tgctcccctt gctttcgcgt cgcccagcgg a
418918DNAArtificial sequenceProbe, targets Chlamydia ssp. 89gctccccttg
ctttcgcg
189018DNAUnknownTarget of SEQ ID NO 88 and 89, organisms Chlamydia
ssp. 90cgcgaaagca aggggagc
189130DNAArtificial sequenceBeacon, target Chlamydia trachomatis
91acgctctcgg atgcccaaat atcggagcgt
309218DNAArtificial sequenceProbe, target Chlamydia trachomatis
92tcggatgccc aaatatcg
189318DNAChlamydia trachomatis 93cgatatttgg gcatccga
189429DNAArtificial sequenceBeacon, target
Candida albicans 94ggaatggcta cccagaagga aaccattcc
299520DNAArtificial sequenceProbe, target Candida albicans
95aatggctacc cagaaggaaa
209620DNACandida albicans 96tttccttctg ggtagccatt
209736DNAArtificial sequenceBeacon, target
Candida krusei 97ccgctctgta ttagctctag atttccacgg gagcgg
369824DNAArtificial sequenceProbe, target Candida krusei
98tgtattagct ctagatttcc acgg
249924DNACandida krusei 99ccgtggaaat ctagagctaa taca
2410032DNAArtificial sequenceBeacon, target Candida
dubliniensis 100gtttgccccg aaagagtaac ttgcaggcaa ac
3210120DNAArtificial sequenceProbe, target Candida
dubliniensis 101ccccgaaaga gtaacttgca
2010220DNACandida dubliniensis 102tgcaagttac tctttcgggg
2010340DNAArtificial
sequenceBeacon, target Candida glabrata 103gccgccggcg tggccaccca
ggcccaaatc gcccagcggc 4010417DNAArtificial
sequenceProbe, target Candida glabrata 104ggccacccag gcccaaa
1710517DNACandida glabrata
105tttgggcctg ggtggcc
1710643DNAArtificial sequenceBeacon, target Candida parapsilosis
106gccgccggcg tgccaaaaag gctagccaga atcgcccagc ggc
4310720DNAArtificial sequenceProbe, target Candida parapsilosis
107gccaaaaagg ctagccagaa
2010820DNACandida parapsilosis 108ttctggctag cctttttggc
2010924DNAArtificial sequenceBeacon,
targets Candida spp. 109acgcgcttgg ctggccggtc gcgt
2411016DNAArtificial sequenceProbe, targets Candida
spp. 110gaccggccag ccaagc
1611116DNAUnknownTarget of SEQ ID NO 109 and 110, organisms
Candida spp. 111gcttggctgg ccggtc
1611244DNAArtificial sequenceBeacon, target Candida
tropicales 112accgccggcg ttacgcatca gaaagatgga cctcgcccag cggt
4411321DNAArtificial sequenceProbe, target Candida tropicales
113tacgcatcag aaagatggac c
2111421DNACandida tropicalis 114ggtccatctt tctgatgcgt a
2111540DNAArtificial sequenceBeacon, target
Citrobacter freundii 115tgccggattc tacttgttag gtgactgcgt aatcccggca
4011621DNAArtificial sequenceProbe, target
Citrobacter freundii 116ctacttgtta ggtgactgcg t
2111721DNACitrobacter freundii 117acgcagtcac
ctaacaagta g
2111833DNAArtificial sequenceBeacon, target Clostridium botulinum
118tcttgtagtg ccgtttcatg cgaaactaca aga
3311922DNAArtificial sequenceProbe, target Clostridium botulinum
119gccgtttcat gcgaaactac aa
2212022DNAClostridium botulinum 120ttgtagtttc gcatgaaacg gc
2212146DNAArtificial sequenceBeacon,
target Clostridium difficile 121gccgccggcg tcgaagtaaa tcgctcaact
tgcatcgccc agcggc 4612223DNAArtificial sequenceProbe,
target Clostridium difficile 122cgaagtaaat cgctcaactt gca
2312323DNAClostridium difficile 123tgcaagttga
gcgatttact tcg
2312429DNAArtificial sequenceBeacon, targets Clostridium spp.
124cgctcacacc cgtccgccgc taatgagcg
2912518DNAArtificial sequenceProbe, targets Clostridium spp.
125cacccgtccg ccgctaat
1812618DNAUnknownTarget of SEQ ID NO 124 and 125, organisms
Clostridium spp. 126attagcggcg gacgggtg
1812747DNAArtificial sequenceBeacon, target Clostridium
perfringens 127gccgccggcg tgattgctcc tttggttgaa tgatgtcgcc cagcggc
4712824DNAArtificial sequenceProbe, target Clostridium
perfringens 128gattgctcct ttggttgaat gatg
2412924DNAClostridium perfringens 129catcattcaa ccaaaggagc
aatc 2413031DNAArtificial
sequenceBeacon, target Clostridium perfringens 130acgctcggtt gaatgatgat
gccatgagcg t 3113123DNAArtificial
sequenceProbe, target Clostridium perfringens 131ggttgaatga tgatgccatc
ttt 2313223DNAClostridium
perfringens 132aaagatggca tcatcattca acc
2313327DNAArtificial sequenceBeacon, target Clostridium tetani
133gcggacctgt gttactcacc cgtccgc
2713420DNAArtificial sequenceProbe, target Clostridium tetani
134ctgtgttact cacccgtccg
2013520DNAClostridium tetani 135cggacgggtg agtaacacag
2013640DNAArtificial sequenceBeacon, targets
Enterobacteriaceae 136tgccggattt tcgtgtttgc acagtgctgt aatcccggca
4013721DNAArtificial sequenceProbe, targets
Enterobacteriaceae 137ttcgtgtttg cacagtgctg t
2113821DNAUnknownTarget of SEQ ID NO 136 and 137,
organisms Enterobacteriaceae 138acagcactgt gcaaacacga a
2113937DNAArtificial sequenceBeacon,
targets Enterobacteriaceae 139tgccggattt ctcgcgaggt cgcttctaat cccggca
3714018DNAArtificial sequenceProbe, targets
Enterobacteriaceae 140tctcgcgagg tcgcttct
1814118DNAUnknownTarget of SEQ ID NO 139 and 140,
organisms Enterobacteriaceae 141agaagcgacc tcgcgaga
1814239DNAArtificial sequenceBeacon,
targets Enterobacteriaceae 142tgccggattc ccccwctttg gtcttgcgaa atcccggca
3914320DNAArtificial sequenceProbe, targets
Enterobacteriaceae 143cccccwcttt ggtcttgcga
2014420DNAUnknownTarget of SEQ ID NO 142 and 143,
organisms Enterobacteriaceae 144tcgcaagacc aaagwggggg
2014537DNAArtificial sequenceBeacon,
targets Enterococci 145tgccggatta tccatcagcg acacccgaat cccggca
3714618DNAArtificial sequenceProbe, targets
Enterococci 146atccatcagc gacacccg
1814718DNAUnknownTarget of SEQ ID NO 145 and 146, organisms
Enterococci 147cgggtgtcgc tgatggat
1814837DNAArtificial sequenceBeacon, target Enterococcus
faecalis 148tgccggattc cctctgatgg gtaggttaat cccggca
3714918DNAArtificial sequenceProbe, target Enterococcus faecalis
149ccctctgatg ggtaggtt
1815018DNAEnterococcus faecalis 150aacctaccca tcagaggg
1815147DNAArtificial sequenceBeacon,
target Enterococcus faecium 151gccgccggcg tttcaaatca aaaccatgcg
gtttctcgcc cagcggc 4715224DNAArtificial sequenceProbe,
target Enterococcus faecium 152ttcaaatcaa aaccatgcgg tttc
2415324DNAEnterococcus faecium 153gaaaccgcat
ggttttgatt tgaa
2415440DNAArtificial sequenceBeacon, target Escherichia coli
154tgccggattg gaagaagctt gcttctttgc aatcccggca
4015521DNAArtificial sequenceProbe, target Escherichia coli 155ggaagaagct
tgcttctttg c
2115621DNAEscherichia coli 156gcaaagaagc aagcttcttc c
2115728DNAArtificial sequenceBeacon, targets
EU-bacteria 157cgctcgctgc ctcccgtagg agtgagcg
2815818DNAArtificial sequenceProbe, targets EU-bacteria
158gctgcctccc gtaggagt
1815918DNAUnknownTarget of SEQ ID NO 157 and 158, organisms
EU-bacteria 159actcctacgg gaggcagc
1816030DNAArtificial sequenceBeacon, target Gardnerella
vaginalis 160acgctccacc atgaagcaac ccgtgagcgt
3016118DNAArtificial sequenceProbe, target Gardnerella vaginalis
161caccatgaag caacccgt
1816218DNAGardnerella vaginalis 162acgggttgct tcatggtg
1816335DNAArtificial sequenceBeacon,
target Haemophilus influenzae 163acccgctatt ccgataatac gcggtattag cgggt
3516423DNAArtificial sequenceProbe, target
Haemophilus influenzae 164ttccgataat acgcggtatt agc
2316523DNAHaemophilus influenzae 165gctaataccg
cgtattatcg gaa
2316639DNAArtificial sequenceBeacon, target Haemophilus influenzae
166cggtgctcta atacgcggta ttagcgacag agagcaccg
3916722DNAArtificial sequenceProbe, target Haemophilus influenzae
167taatacgcgg tattagcgac ag
2216822DNAHaemophilus influenzae 168ctgtcgctaa taccgcgtat at
2216942DNAArtificial sequenceBeacon,
target Klebsiella pneumoniae 169acgccggcgt aggttattaa cctcatcgcc
ttcgcccagc gt 4217021DNAArtificial sequenceProbe,
target Klebsiella pneumoniae 170aggttattaa cctcatcgcc t
2117121DNAKlebsiella pneumoniae 171aggcgatgag
gttaataacc t
2117235DNAArtificial sequenceBeacon, target Klebsiella oxytoca
172ggaagggata taggttatta acctcactcc cttcc
3517324DNAArtificial sequenceProbe, target Klebsiella oxytoca
173aggttattaa cctcactccc ttcc
2417424DNAKlebsiella oxytoca 174ggaagggagt gaggttaata acct
2417532DNAArtificial sequenceBeacon, target
Lactobacillus brevis 175cgctcattca acggaagctc gttcgatgag cg
3217622DNAArtificial sequenceProbe, target
Lactobacillus brevis 176tcattcaacg gaagctcgtt cg
2217722DNALactobacillus brevis 177cgaacgagct
tccgttgaat ga
2217837DNAArtificial sequenceBeacon, target Legionella pneumophila
178tgccggatta tctgaccgtc ccaggttaat cccggca
3717918DNAArtificial sequenceProbe, target Legionella pneumophila
179atctgaccgt cccaggtt
1818018DNALegionella pneumophila 180aacctgggac ggtcagat
1818130DNAArtificial sequenceBeacon,
target Listeria monocytogenes 181acgctcataa gatgtggcgc atgcgagcgt
3018218DNAArtificial sequenceProbe, target
Listeria monocytogenes 182ataagatgtg gcgcatgc
1818318DNAListeria monocytogenes 183gcatgcgcca
catcttat
1818418DNAArtificial sequenceProbe, target Mycoplasma hominis
184attgctaacc tcgctcga
1818518DNAMycoplasma hominis 185tcgagcgagg ttagcaat
1818630DNAArtificial sequenceBeacon, targets
Proteus mirabili / vulgaris 186ggcgtcacac cggatacgta gtgctacgcc
3018718DNAArtificial sequenceProbe, targets
Proteus mirabili / vulgaris 187ggcgtcacac cggatacg
1818819DNAUnknownTarget of SEQ ID NO 186 and
187, organisms Proteus mirabili/vulgaris 188ccgtatccgg tgtgacgcc
1918926DNAArtificial
sequenceBeacon, target Pneumocystis-1 189actcggcttc atgccaacag tcgagt
2619018DNAArtificial sequenceProbe,
target Pneumocystis-1 190ggcttcatgc caacagtc
1819118DNAUnknownTarget of SEQ ID NO 189 and 190,
organism Pneumocystis-1 191gactgttggc atgaagcc
1819228DNAArtificial sequenceBeacon, target
Pneumocystis-2 192gacaccataa gatgccgagc gaggtgtc
2819318DNAArtificial sequenceProbe, target Pneumocystis-2
193cataagatgc cgagcgag
1819418DNAUnknownTarget of SEQ ID NO 192 and 193, organism
Pneumocystis-2 194ctcgctcggc atcttatg
1819543DNAArtificial sequenceBeacon, target Pseudomonas
aeruginosa 195accgccggcg taagacgact cgtcatcacc ttcgcccagc ggt
4319620DNAArtificial sequenceProbe, target Pseudomonas
aeruginosa 196aagacgactc gtcatcacct
2019720DNAPseudomonas aeruginosa 197aggtgatgac gagtcgtctt
2019844DNAArtificial
sequenceBeacon, target Pseudomonas spp. 198gccgccggcg tggcagattc
ctaggcatta cttcgcccag cggc 4419921DNAArtificial
sequenceProbe, target Pseudomonas spp. 199ggcagattcc taggcattac t
2120021DNAUnknownTarget of SEQ ID
NO 198 and 199, organism Pseudomonas spp. 200agtaatgcct aggaatctgc c
2120131DNAArtificial
sequenceBeacon, targets Salmonellae 201agctctgcgc ttttgtgtac ggggctgagc t
3120221DNAArtificial sequenceProbe,
targets Salmonellae 202tgcgcttttg tgtacggggc t
2120321DNAUnknownTarget of SEQ ID NO 201, 202 and 204,
organisms Salmonellae 203agccccgtac acaaaagcgc a
2120419DNAArtificial sequenceProbe, targets
Salmonellae (331 competitor) 204gtgcatttgt gtacggggc
1920529DNAArtificial sequenceBeacon, targets
Salmonellae 205cgctccttca cctacgtgtc agcggagcg
2920619DNAArtificial sequenceProbe, targets Salmonellae
206cttcacctac gtgtcagcg
1920719DNAUnknownTarget of SEQ ID NO 205, 206, 210 and 211,
organisms Salmonellae 207cgctgacacg taggtgaag
1920820DNAArtificial sequenceProbe, targets
Salmonellae 208tcacctacat atcagcgtgc
2020920DNAUnknownTarget of SEQ ID NO 208, organisms
Salmonellae 209cgctgacacg taggtgaaga
2021038DNAArtificial sequenceBeacon, target Salmonella
210tgccggattc ttcacctacg tgtcagcgaa tcccggca
3821119DNAArtificial sequenceProbe, target Salmonella 211cttcacctac
gtgtcagcg
1921243DNAArtificial sequenceBeacon, target Serratia marcescens
212gccgccggcg tcgagactct agcttgccag ttcgcccagc ggc
4321320DNAArtificial sequenceProbe, target Serratia marcescens
213cgagactcta gcttgccagt
2021420DNASerratia marcescens 214actggcaagc tagagtctcg
2021543DNAArtificial sequenceBeacon, target
Staphylococcus aureus 215tgccggattt tctcgtccgt tcgctcgact tgcaatcccg gca
4321624DNAArtificial sequenceProbe, target
Staphylococcus aureus 216ttctcgtccg ttcgctcgac ttgc
2421724DNAStaphylococcus aureus 217gcaagtcgag
cgaacggacg agaa
2421828DNAArtificial sequenceBeacon, targets Staphylococci 218gcaactttcg
cacatcagcg tcagttgc
2821921DNAArtificial sequenceProbe, targets Staphylococci 219tttcgcacat
cagcgtcagt t
2122021DNAUnknownTarget of SEQ ID NO 218 and 219, organisms
Staphylococci 220aactgacgct gatgtgcgaa a
2122130DNAArtificial sequenceBeacon, target Stenotrophomonas
maltophilia 221ccctctacca cactctagtc gggtagaggg
3022221DNAArtificial sequenceProbe, target Stenotrophomonas
maltophilia 222ccctctacca cactctagtc g
2122321DNAStenotrophomonas maltophilia 223cgactagagt
gtggtagagg g
2122444DNAArtificial sequenceBeacon, target Streptococcus agalactiae
224gccgccggcg tactcctacc aacgttcttc tctcgcccag cggc
4422521DNAArtificial sequenceProbe, target Streptococcus agalactiae
225actcctacca acgttcttct c
2122621DNAStreptococcus agalactiae 226gagaagaacg ttggtaggag t
2122735DNAArtificial sequenceBeacon,
targets Streptococci 227ggccttcgcc gtccctttct ggttagttga aggcc
3522821DNAArtificial sequenceProbe, targets
Streptococci 228gccgtccctt tctggttagt t
2122921DNAUnknownTarget of SEQ ID NO 227 and 228, organisms
Streptococci 229aactaaccag aaagggacgg c
2123036DNAArtificial sequenceBeacon, target Streptococcus
pneumoniae 230gcgttaagca aatgtcatgc aacatctact taacgc
3623125DNAArtificial sequenceProbe, target Streptococcus
pneumoniae 231ttaagcaaat gtcatgcaac atcta
2523225DNAStreptococcus pneumoniae 232tagatgttgc atgacatttg
cttaa 2523338DNAArtificial
sequenceBeacon, target Streptococcus pyogenes 233tgccttcgag caattgcccc
ttttaaatta cgaaggca 3823424DNAArtificial
sequenceProbe, target Streptococcus pyogenes 234gagcaattgc cccttttaaa
ttac 2423524DNAStreptococcus
pyogenes 235gtaatttaaa aggggcaatt gctc
2423630DNAArtificial sequenceBeacon, target Ureaplasma
urealyticum 236acgctcgttc cccaactccc tactgagcgt
3023718DNAArtificial sequenceProbe, target Ureaplasma
urealyticum 237gttccccaac tccctact
1823818DNAUreaplasma urealyticum 238agtagggagt tggggaac
1823926DNAArtificial
sequenceBeacon, targets Urogenital-Peptostreptococci 239actccccctt
gtgtaaggca gggagt
2624018DNAArtificial sequenceProbe, targets Urogenital-Peptostreptococci
240ccccttgtgt aaggcagg
1824118DNAUnknownTarget of SEQ ID NO 239 and 240, organisms
Urogenital-Peptostreptococci 241cctgccttac acaagggg
1824230DNAArtificial sequenceBeacon, target
Yersinia enterocolitica 242acgctcccca ctttggtccg aagagagcgt
3024318DNAArtificial sequenceProbe. target
Yersinia enterocolitica 243cccactttgg tccgaaga
1824418DNAYersinia enterocolitica 244tcttcggacc
aaagtggg
1824536DNAArtificial sequenceBeacon, target Acinetobacter spp.
245tgccggtttt aggccagatg gctgccaatc ccggca
3624628DNAArtificial sequenceBeacon, target Aspergillus fumigatus
246ccggcccccg agaggtgata catgccgg
2824731DNAArtificial sequenceBeacon, target Aspergillus niger
247ccggcaatta caatgcggac tccgaagccg g
3124829DNAArtificial sequenceBeacon, target Mycobacterium avium Complex
248cccggtgttg atataaggca ggtgccggg
2924930DNAArtificial sequenceBeacon, target Mycobacterium chelonae
249cccggcatga agtgtgtggt cctatccggg
3025030DNAArtificial sequenceBeacon, target Mycobacterium fortuitum
250cccggtgaag cgcgtggtca tattcccggg
3025130DNAArtificial sequenceBeacon, target Mycobacterium gordonae
251cccggtgtgt cctgtggtcc tattcccggg
3025230DNAArtificial sequenceBeacon, target Mycobacterium intracellulare
252cccggacatg cgtctaaagg tcctaccggg
3025330DNAArtificial sequenceBeacon, targets Mycobacterium
kansasii/gastri 253cccggtagag ctgagacgta tcgatccggg
3025428DNAArtificial sequenceBeacon, target Mycobacterium
malmoense 254ccgcgccact gaaacgccct attcgcgg
2825528DNAArtificial sequenceBeacon, target Mycobacterium
smegmatis 255cccggcacgt cgagggctct gacccggg
2825624DNAArtificial sequenceBeacon, target Mycobacterium
tuberculosis complex 256ccaccggaga ggaaaaggag gtgg
2425727DNAArtificial sequenceBeacon, target
Mycobacterium xenopi 257ccgcgccgct accaaacgct ttcgcgg
2725830DNAArtificial sequenceBeacon, target Shigella
258ccgggtcacc ctgtatcgca cgcctcccgg
3025934DNAArtificial sequenceBeacon, target Citrobacter freundii
259cccggtcgct tcattacgct atgtatccac cggg
3426034DNAArtificial sequenceBeacon, target Streptococcus pyogenes
260cgctcgagca attgcccctt ttaaattacg agcg
3426136DNAArtificial sequenceBeacon, target Streptococcus pneumoniae
261ccgttaagca aatgtcatgc aacatctact taacgg
3626235DNAArtificial sequenceBeacon, targets Streptococci (F111)
262cgccttcgcc gtccctttct ggttagttga aggcg
3526331DNAArtificial sequenceBeacon, target Streptococcus agalactiae
263ccgctactcc taccaacgtt cttctcagcg g
3126432DNAArtificial sequenceBeacon, target Serratia marcescens
264ccgctccgag actctagctt gccagtgagc gg
3226531DNAArtificial sequenceBeacon, targets Pseudomonas spp.
265cgctcggcag attcctaggc attactgagc g
3126628DNAArtificial sequenceBeacon, targets Staphylococci 266ccaactttcg
cacatcagcg tcagttgg
2826730DNAArtificial sequenceBeacon, target Pseudomonas aeruginosa
267cccggaagac gactcgtcat cagctccggg
3026826DNAArtificial sequenceBeacon, target Legionella pneumophila
268ccggatctga ccgtcccagg ttccgg
2626935DNAArtificial sequenceBeacon, target Klebsiella oxytoca
269ccgaggtagg ttattaacct cactcccttc ctcgg
3527029DNAArtificial sequenceBeacon, target Staphylococcus aureus
270cccctcaagc ttctcgtccg ttcgagggg
2927130DNAArtificial sequenceBeacon, target Listeria monocytogenes
271cctagcatgc gccacatctt atcagctagg
3027229DNAArtificial sequenceBeacon, target Klebsiella pneumoniae
272caggcttagg ttattaacct catcgcctg
2927330DNAArtificial sequenceBeacon, target Haemophilus influenzae
273cccggccgca ctttcatctt ccgatccggg
3027430DNAArtificial sequenceBeacon, target Burkholderia spp.
274cccggccagt caccaatgca gttccccggg
3027527DNAArtificial sequenceBeacon, targets Burkholderia cepatia and
Burkholderia cenocepatia 275cctgcctatg tattcagcca tggcagg
2727629DNAArtificial sequenceBeacon, targets
Burkholderia malii and Burkholderia pseudomalei 276gggcctcgcc
tcactagacc tatgccggg
2927729DNAArtificial sequenceBeacon, target Burkholderia vietnamensis
277cccggtcgct tctctggacc tatgccggg
2927828DNAArtificial sequenceBeacon, target Burkholderia multivorans
278cccggcttca cccttccagc gcaccggg
2827930DNAArtificial sequenceBeacon, target Burkholderia gladioli
279ccagcggtac ggtcactgtt aaactgctgg
3028030DNAArtificial sequenceBeacon, targets Acinetobacter spp.
280ccgtagacca tcctctccca tactctacgg
3028123DNAArtificial sequenceBeacon, target Acinetobacter baumannii
281ccgctaggtc cggtagcaag cgg
2328228DNAArtificial sequenceBeacon, target Aspergillus flavus
282cggcctacat tccgggagcc tttggccg
2828329DNAArtificial sequenceBeacon, target Aspergillus terreus
283ccgatcagac accccgcccc atagatcgg
2928431DNAArtificial sequenceBeacon, targets Bacteroides / Prevotella
284ccgcggtgtc tcagttccaa tgtgggcgcg g
3128532DNAArtificial sequenceBeacon, targets Borrelia burgdorferi/
garinii/afzelii/valaisiana 285cccggggtaa cagataacaa gggttgcccg gg
3228629DNAArtificial sequenceBeacon, target
Bordetella pertussis 286ccgggctccc cacactttcg tgcacccgg
2928728DNAArtificial sequenceBeacon, target
Escherichia coli 287ccggcaaaga agcaagcttc ttccccgg
2828830DNAArtificial sequenceBeacon, target Gardnerella
vaginalis 288ccgctccacc atgaagcaac ccgtgagcgg
3028929DNAArtificial sequenceBeacon, targets Eu-bacteria
289ccgcgtgctg cctcccgtag gagtcgcgg
2929033DNAArtificial sequenceBeacons, target Enterococcus faecium
290cttcaaatca aaaccatgcg gtttcatttg aag
3329132DNAArtificial sequenceBeacon, target Stenotrophomonas maltophilia
291cccggaccct ctaccacact ctagtcgccg gg
3229225DNAArtificial sequenceBeacon, target Enterococcus faecalis
292caaccaccct ctgatgggta ggttg
2529329DNAArtificial sequenceBeacons, targets Enterococci 293cccggcatcc
atcagcgaca cccgccggg
2929428DNAArtificial sequenceBeacon, targets Enterobacteriaceae
294cccggtctcg cgaggtcgct tctccggg
2829531DNAArtificial sequenceBeacon, target Clostridium perfringens
295gcatcagatt gctcctttgg ttgaatgatg c
3129628DNAArtificial sequenceBeacon, targets Clostridium spp.
296cccggtaccg tcattatcgt cccccggg
2829725DNAArtificial sequenceBeacon, target Chlamydia trachomatis
297ccgctcggat gcccaaatat cgcgg
2529832DNAArtificial sequenceBeacon, target Candida dubliniensis
298cccggcccga aagagtaact tgcaaaaccg gg
3229927DNAArtificial sequenceBeacon, target Candida glabrata
299cccggaggca aggggcgcaa aaccggg
2730029DNAArtificial sequenceBeacon, target Candida krusei (Issatchenkia
orientalis) 300ccgtgacctg cagcaagaac cgatcacgg
2930129DNAArtificial sequenceBeacon, target Candida
lusitaniae (Clavispora lusitaniae) 301cactgccgac tcagaccacg
aaagcagtg 2930230DNAArtificial
sequenceBeacon, target Candida albicans 302ccgcgtttac acagacccgg
gtcatcgcgg 3030334DNAArtificial
sequenceBeacon, target Candida tropicalis 303cctcggacat tccaacgcaa
ttctcctacc gagg 3430434DNAArtificial
sequenceBeacon, target Candida parapsilosis 304cccggcacat ttctttgcac
ttatcctacc cggg 3430530DNAArtificial
sequenceBeacon, target Chlamydia pneumoniae 305ccgggctctt cctcaaccga
aaggtcccgg 3030628DNAArtificial
sequenceBeacon, targets Chlamydia psittaci group 306ccggaaggca aaaccaactc
ccatccgg 2830728DNAArtificial
sequenceBeacon, targets Campylobacter (pathogenic thermophiles)
307cccgggccct aagcgtcctt ccaccggg
2830828DNAArtificial sequenceBeacon, target Campylobacter coli
308cgctctcgat ggcatcaggg gttgagcg
2830934DNAArtificial sequenceBeacon, target Campylobacter lari
309cccggcccga agtgttagca actaaatcgc cggg
3431033DNAArtificial sequenceBeacon, target Campylobacter jejuni
310ccgggtaagc taaccacacc ttataccgcc cgg
3331134DNAArtificial sequenceBeacon, target Campylobacter upsaliensis
311cccgggccgt gtgtcgccct aggcgtagcc cggg
3431233DNAArtificial sequenceBeacon, target Pneumocystis carinii
312ccctgctatc cagtaactga aaccgatgca ggg
3331332DNAArtificial sequenceBeacon, target Mycoplasma pneumoniae
313cctccgtgat agctgtttcc aactaccgga gg
3231429DNAArtificial sequenceBeacon, target Cryptococcus neoformans
314cctggtatga ttcaccatag agggccagg
2931529DNAArtificial sequenceBeacon, target EHEC 315cccggtcacc ccataaaaga
ggctccggg 2931625DNAArtificial
sequenceBeacon, target Neisseria meningitidis 316caccgttatc ccccactact
cggtg 2531728DNAArtificial
sequenceBeacon, target Neisseria gonorrhoeae 317cccggacccc gccaaccagc
taaccggg 2831832DNAArtificial
sequenceBeacon, target Clostridium difficile 318cgggtcgaag taaatcgctc
aacttgcacc cg 3231928DNAArtificial
sequenceBeacon, target Clostridium botulinum 319cgttgccgtt tcatgcgaaa
ctacaacg 2832027DNAArtificial
sequenceBeacon, target Clostridium tetani 320ccggaactgt gttactcacc
cgtccgg 2732128DNAArtificial
sequenceBeacon, target Peptostreptococcus anaerobius 321ccggcctttg
atatatctac gatgccgg
2832228DNAArtificial sequenceBeacon, target Peptostreptococcus magnus
322ccgcctaatc cgaaatgaat tctggcgg
2832332DNAArtificial sequenceBeacon, target Peptostreptococcus magnus
323ccgccatgtg tttctacgat tttatgcggc gg
3232431DNAArtificial sequenceBeacon, target Peptostreptococcus micros
324cccggacttt catttcattt ccattcccgg g
3132531DNAArtificial sequenceBeacon, target Enterococcus faecalis
325ccatcggcac tcgggaggaa agaagcgatg g
3132631DNAArtificial sequenceBeacon, target Enterococcus faecium
326cgcccatgcg gttttgattg ttatacgggc g
3132732DNAArtificial sequenceBeacon, target Enterococcus casseliflavus
327ccgcgcaagg gacgaacatt ttactctcgc gg
3232830DNAArtificial sequenceBeacon, target Enterococcus gallinarum
328ccgcgcaagg gatgaacgtt ctactcgcgg
3032930DNAArtificial sequenceBeacon, target Candida albicans
329cccggtttcc ttctgggtag ccattccggg
3033032DNAArtificial sequenceBeacon, target Morganella morganii (Proteus
morganii) 330ccggcaagac tctagctgac cagtatcgcc gg
3233128DNAArtificial sequenceBeacon, target Proteus
mirabilis 331cgccgatagt gcaaggtccg aagcggcg
2833227DNAArtificial sequenceBeacon, target Proteus vulgaris
332ccgccgtaga cgtcatgcgg taggcgg
2733330DNAArtificial sequenceBeacon, target Treponema pallidum
333cccggtccgc cactctagag aaacgccggg
3033430DNAArtificial sequenceBeacons, target Trichonomas vaginalis
334cccgggaatg gcgtgcctct gatgaccggg
3033527DNAArtificial sequenceBeacon, targets Micrococci 335cccggacctc
acagtatcgc aaccggg
2733626DNAArtificial sequenceBeacon, target Lactobacillus brevis
336cggccgcggg atcatccaga aggccg
2633733DNAArtificial sequenceBeacon, target Yersinia enterocolitica
337cccggatctc tgctaaattc cgtggatgcc ggg
3333828DNAArtificial sequenceBeacon, target Bacillus cereus 338ccataccact
ctgctcccga aggtatgg
2833930DNAArtificial sequenceBeacon, target Vibrio cholerae 339ctgatgcata
tccggtagcg caagcatcag
3034032DNAArtificial sequenceBeacon, target Vibrio parahaemolyticus
340cccggtgcag ctattaacta cactaccccg gg
3234132DNAArtificial sequencebeacon, targets Cryptosporidium spp.
341ccgtacataa ggtgctgaag gagtaagtac gg
3234228DNAArtificial sequenceBeacon, target Coxiella burnetii
342ccgggaccct tgagaatttc ttccccgg
2834332DNAArtificial sequenceBeacon, targets Bartonella spp.
343ccggcacaaa tttctctgtg ttattccgcc gg
3234432DNAArtificial sequenceBeacon, target Enterobacter sakazakii
344cccggtctct gcaggattct ctggatgccg gg
3234530DNAArtificial sequenceBeacon, targets Ehrlichia spp. 345ccgcgctaat
ctaacgtagg ctcatcgcgg
3034632DNAArtificial sequenceBeacon, targets Rickettsia spp.
346ccgcgcactc actcggtatt gctggatcgc gg
3234727DNAArtificial sequenceBeacon, target Rickettsia spotted fever
complex 347ctagccccaa ttagtccgtt cggctag
2734829DNAArtificial sequenceBeacon, targets Rickettsia typhi
complex 348cgcccgtctg ccactaatta actagggcg
2934924DNAArtificial sequenceBeacon, targets Leishmania spp.
349cccggaaaag gcgttacggc cggg
2435027DNAArtificial sequenceBeacon, target Toxoplasma gondii
350ccggctccag gggaagaggc atgccgg
2735134DNAArtificial sequenceBeacon, targets Yeast spp. 351cccgggtatt
tacattgtac tcattccaac cggg
3435228DNAArtificial sequenceBeacon, target Francisella tularensis
352ccatgcgaca gcccgaaagc cagcatgg
2835328DNAArtificial sequenceBeacon, targets Lactobacillus spp. A
353cccggagttc cactgtcctc ttcccggg
2835428DNAArtificial sequenceBeacon, targets Lactobacillus spp. B
354cccggatcag tctctcaact cggccggg
2835528DNAArtificial sequenceBeacon, target Burkholderia cepacia complex
355cccggttggc aaccctctgt tccccggg
2835627DNAArtificial sequenceBeacon, target Staphylococcus aureus
356cctgcaagct tctcgtccgt tcgcagg
2735729DNAArtificial sequenceBeacon, target Staphylococcus aureus
357cccctcaagc ttctcgtccg ttcgagggg
2935829DNAArtificial sequenceBeacon, targets Eu-bacteria 358ccgcgtgctg
cctcccgtag gagtcgcgg 29
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