JP2001516590A - Hybridization probe for distinguishing between Streptomycetes and Maduromycetes - Google Patents

Abstract

(57) Abstract: The present invention provides for discrimination between bacteria from Maduromycetes and Streptomycetes taxa using specific nucleic acid probes complementary to conserved regions of the Maduromycetes 16S rRNA gene. To provide a way. The probe allows for the rapid detection of 16S rRNA-encoding DNA in a sample and distinguishes Streptomycetes from Maduromycetes within the order Actinomycetales. The method is accurate and reproducible.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION [0001] The present invention relates to streptomycetes (streptomycetes) and Maduromycetes (
maduromycetes), and a nucleic acid probe derived from a region encoding mature 16S ribosomal RNA (rRNA) of Maduromycetes. The present invention also relates to assays using these probes.

BACKGROUND OF THE INVENTION [0002] Although the identification of microorganisms has progressed significantly during the past decade, the methods used therefor are still laborious, insensitive and not specific. Many of these disadvantages can be overcome by using nucleic acid probes. These nucleic acid probes, consisting of genomic DNA, plasmids, riboprobes or synthetic oligonucleotides, can target genomic DNA or certain RNA species present in a biological sample.
It is generally preferred to use synthetic oligonucleotides as probes. This is because oligonucleotides can be rapidly synthesized in large quantities by chemical methods, have a long shelf life, and are easily purified and labeled.

[0003] Traditional methods for describing the composition of natural microbial communities are problematic. Because they are expensive and labor intensive, they only allow the identification of microorganisms that can be cultured in a research facility. It is generally believed that less than 20% of natural microorganisms are currently discovered, and new culture-independent methods for studying microbial community composition are needed (Ward, DM et al., 1990
Nature, 345: 63-64). A sequence of 16S rRNA, a common but distinctive cellular element, has already been used for this purpose, indicating the presence of a large number of uncultured microorganisms in natural communities (Ward, DM et al., Supra; Giovannoni, SJ et al., 1990 Nat
ure, 345: 60-63; Barry T. et al., 1991, PCR Methods and Applications, Cold Sp.
ring Harbor. pp 51-56; Mehling, A. et al., Microbiology, 141: 2139-2147; and Rheims, H. et al., 1996, Microbiology, 142: 2863-2870).

[0004] For a number of microorganisms, species-specific probes have already been described. The 16S and 23S rRNA genes are very frequently used for probe development. This is because sequences can easily be obtained using the described methods, and it is known that variable regions that can be used for species-specific detection are present in these highly conserved genes. Universal probes for the detection of bacteria are known (eg, Giovannoni, SJ et al., 1988, J. Bacteriol. 170: 720-726 and Barry, T. et al.,
1991, supra).

[0005] Actinomycetes is an aerobic Gram-positive bacterium that forms branched, usually non-disruptive hyphae and asexual spores (produced on aerial hyphae). The 16S rRNA sequence data has allowed the construction of a very complex evolutionary tree of this order, containing about 37 genera in at least seven groups, including Streptomycetes and Maduromycetes (Go
odfellow, M., 1989, “Suprageneric classification of Actinomycetes”, Be
rgey's Manual of Systematic Bacteriology Vol. 4, Williams and Wilkins
pp. 2333-2339).

A simple but powerful method for distinguishing between actinomycetes closely related groups found in natural isolates (particularly bacteria that develop aerial hyphae prior to release of spores) No mention in the art at present. It would be desirable to have a nucleic acid probe that can be used in a rapid and accurate assay that can distinguish between Maduromycetes and Streptomycetes.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a nucleic acid encoding a portion of the 16S rRNA of a Maduromycetes bacterium, which hybridizes under hybridization conditions to a nucleic acid encoding a portion of a Streptomycetes bacterium.
The present invention relates to a nucleic acid probe that does not hybridize to a nucleic acid encoding a portion of 16S rRNA under the same hybridization conditions.

[0008] Another embodiment of the invention is a method for detecting the presence of a Maduromycetes bacterium in a sample, the method comprising: a) using a nucleic acid probe (the probe comprises a Maduromycetes 16S rRNA); That hybridizes to the encoding nucleic acid, but does not hybridize to the nucleic acid encoding Streptomycetes 16S rRNA), b) subjected to hybridization conditions, and c) determines whether hybridization has occurred The method comprises:

[0009] Yet another embodiment of the present invention is a method of distinguishing Maduromycetes from Streptomycetes in a bacterial sample, comprising: a) lysing the bacteria to release the bacterial DNA; C) bringing a probe containing the sequence of the CNB-ESP probe into contact with the extracted DNA under hybridization conditions; and d) determining whether hybridization of the probe to the extracted DNA has occurred. A method comprising:

[0010] Another embodiment of the present invention provides a nucleic acid sequence comprising a Maduromycetes nucleic acid corresponding to base pairs 68-90 of Streptomyces ambofaciens DNA encoding a mature 16S rRNA molecule. A kit for the detection of bacteria from the Maduromycetes group, comprising a 10-250 nucleotide long probe that is at least 90% complementary or homologous.

[0011] The kit may further include reagents, compositions, instructions, disposable hardware and suitable packaging.

The term “probe” as used throughout the present application and claims is 10-25.
Means a synthetic or biologically obtained nucleic acid of 0 base pairs in length, which contains a specific nucleotide sequence that allows specific and preferential hybridization to a target nucleic acid sequence under defined conditions. , For detection and for enhancing the performance of the assay, if desired. Generally, a minimum of 10 nucleotides is required to achieve satisfactory specificity and to form a stable hybridization product, and generally a maximum of 250 nucleotides is required for mismatching and preferential hybridization. This corresponds to an upper limit on the length at which the reaction parameters can be easily adapted to determine. Probes may optionally contain certain constructs that contribute to proper or optimal function under certain assay conditions. For example, to improve the resistance of the probe to nuclease degradation (eg, by end capping), or to retain a detection ligand (eg, fluorescein, ³² P, biotin, etc.), or to allow the probe to bind to a solid support. Probes can be modified to facilitate capture (eg, polydeoxyadenosine “tail”).

“Preferential hybridization” or “preferentially hybridizes” means that hybridization to an intended target nucleic acid is greater than any hybridization reaction product resulting from hybridization to a non-target nucleic acid under the same conditions. Also means providing a stable hybridization reaction product. Comparing the stability of hybridization reaction products and assessing which is the most stable (ie, determining which are "preferentially" bound) is within the skill of the artisan. Fully included.

The terms “homology” and “homology” refer to the degree of similarity between two or more nucleic acid sequences and do not imply any systematic relationship between the organisms. The degree of similarity is expressed as a percentage. That is, a 90% homology between the two sequences means that 90% of the bases in the first sequence match identically to the bases in the second sequence.

“Specific” means that the nucleotide sequence hybridizes to a given target sequence but does not substantially hybridize to a non-target sequence.

“Specifically discriminates” means that the probe substantially hybridizes to a given target sequence but does not substantially hybridize to a non-target sequence.

“Hybridization” refers to the joining of two partially or completely complementary nucleic acid strands in antiparallel under defined reaction conditions, according to obvious rules about which nucleobases can pair with each other. Is a process of forming a double-stranded nucleic acid by specific and stable hydrogen bonding.

“Substantial hybridization” means that an amount of hybridization is observed such that an observer of the result determines the result as positive in a clinical setting. Data considered "background noise" is not substantial hybridization.

“Stringent hybridization conditions” include about 0.9 molar NaC
means about 35 ° C to 65 ° C in 1 salt solution. Stringency can also be affected by reaction parameters such as the concentration and type of ionic species present in the hybridization solution and the hybridization temperature. In general, longer probes are preferred as hybridization conditions become more stringent if stable hybrids are to be formed. Typically, the stringency of the conditions under which the hybridization is performed will determine certain characteristics of the preferred probe used. Such a relationship is well understood by those skilled in the art and can be readily modified.

When designing a probe for identification purposes, the probe is optionally specific (ie, it does not cross-react with undesired nucleic acids).
Preferably, it is highly sensitive (ie most, if not all, of the organisms to be detected react with the probe). Therefore, a preferred target sequence should have the following properties: a) the sequence should be present in the genome of each strain of the microorganism of interest; and b) the evolutionary diversity of the sequence With regard to evolutionary diversity, on the one hand, there is sufficient sequence diversity to be able to distinguish the species from other closely related species, while on the other hand, using the probes used, There is sufficient sequence conservation to be able to detect the strain of interest.

[0021] The Maduromycetes and Streptomycetes present in the database
Comparison and alignment of 16S rDNA sequences is performed on all 16S of Actinomycetes.
In addition to proving the existence of so-called variable regions that appear in rDNA, it is possible to identify specific regions in Actinomadura 16S rDNA that are considered to have low homology to sequences equivalent to Streptomyces (Streptomyces) I made it. Sequence comparison and analysis were performed using the UWGCG package (Version 9.0, December 1996) and programs from the Edit-View 1.0 DNA Sequencer Viewer (Applied Biosystems). The 16S rDNA sequence was obtained from the NCBI Genbank database.

As a result of the sequence comparison, two primers were designed. The first primer was called NVR1 (5 'CAC GGA GAG TTT GAT CCT GGC 3') (SEQ ID NO: 1), which
Streptomyces ambofaciens encoding S rRNA (Streptomyces a
mbofaciens) base pairs 2-12 from DNA (Pernodet, JL. et al., 1989, Gene, 79
: 33-46). The second primer is SOR (5'GTA TTA GAC CCA GTT TCC CGG GC 3 ')
(SEQ ID NO: 2), which is base pairs 144-17 from Streptomyces ambofaciens DNA encoding mature 16S rRNA.
This is Maduromycetes DNA corresponding to No. 6 (Pernodet, 1989, supra). Both of these can be used as primers in the polymerase chain reaction technique for amplifying a DNA region to obtain the preferred probes of the present invention. Large quantities of the probe can be obtained using known PCR techniques (eg, those described in US Pat. Nos. 4,683,202 and 4,683,195).

A preferred probe, CNB-ESP, was derived from the hypervariable region of the 16S rRNA molecule. It contains the sequence 5 'GAA AGG CCC TTC GGG GGT ACT CG 3' (SEQ ID NO: 3), which contains 68-bp base pairs from Streptomyces ambofaciens DNA encoding mature 16S rRNA. It is Maduroma Isetes DNA corresponding to 90. In the present invention, a probe similar to CNB-ESP can be produced by increasing or decreasing the length of CNB-ESP. In the case of longer probes, the additional nucleotides (3 'or 5') are replaced by the corresponding Streptomyces ambofaciens encoding the mature 16S rRNA.
It is preferably a nucleotide of DNA.

One embodiment of the present invention is a nucleic acid designated CNB-ESP, which is specific for chromosomal DNA encoding the mature part of a bacterial 16S rRNA molecule from the Maduromycetes group. It results in binding and does not substantially hybridize to equivalent chromosomal regions from bacteria belonging to the closely related Streptomycetes taxon.

Preferred probes of the present invention generally have at least about 23 nucleotides to about 16 nucleotides.
Contains 6 nucleotides (precursor region + mature region + maximum number of regulatory region nucleotides of the gene encoding the 16S rRNA). More preferably, the probe comprises 23
It contains about 23 to about 166 nucleotides resulting from PCR amplification of a DNA fragment containing a DNA sequence that hybridizes to a nucleotide-long CNB-ESP probe.

A particularly preferred probe is shown in FIG.

[0027] The present invention also provides a chromosomal DN encoding a mature 16S rRNA from Maduromycetes.
Oligonucleotides that are sufficiently complementary to hybridize to the sequence in the A region but not sufficiently complementary to hybridize to equivalent regions from distantly related Gram-positive bacteria such as the bacterium Bacillus subtilis For probes for use in hybridization assays using

A particularly preferred assay in the present invention is a Southern blot. One probe that can be used for Southern blot assays is approximately 23-166 bp in length obtained by PCR amplification of a DNA fragment obtained using two primers, NVR1 and SOR. Southern blot or dot blot assays can be performed using well-known methods. Generally, it involves immobilizing the target nucleic acid or nucleic acid population on a filter such as nitrocellulose, nylon or other commercially available derivatized membrane. The immobilized nucleic acid is then tested for hybridization to the probe of interest under defined stringency conditions.
Under stringent conditions, probes with nucleotide sequences having higher homology to the target will show higher levels of hybridization than probes with sequences having lower homology. Hybridization can be detected in a number of ways. For example, the probe can be isotopically labeled by adding a ³² P-phosphorus moiety to the 5 ′ end of the oligonucleotide by a conventional polynucleotide kinase reaction. After hybridization has occurred, unhybridized probe is removed by washing. The filter is exposed to x-ray film and the intensity of the hybridization signal is evaluated.

The probes of the present invention can be chemically synthesized using commercially available methods and equipment. For example, short oligonucleotides 15-30 nucleotides in length can be obtained using the solid phase phosphoramidite method. In the present invention, Applied Bi
It is preferred to chemically synthesize short DNA oligonucleotides using any of the osystems DNA Synthesizers and reagents supplied by the supplier. The chemically synthesized oligonucleotide was obtained from Boehringer Mannheim.

The Maduromycetes strains that can be detected by the probes of the present invention can be obtained from the ATCC collection and are listed in the table below.

[0031]

[Table 1]

In the present invention, the following Streptomyces strains can be used: Streptomyces ambofaciens, S. antibioticus, S. cinnamonensis. cinnam
onensis), S. coelicolor (S. coelicolor) A3 (2), S. fragier (S.
fradiae), S. lividans TK21, S. Nataliensis (S.
nataliensis), S. peucetius, S. violasens
S violascens) and Streptomyces sp.
.

The methods used for the growth and manipulation of bacteria and general DNA manipulations in connection with the present invention are as described in the literature (Hopwood et al., 1985, Genetic Manipulation of Str.).
eptomyces. A Laboratory Mannual. John Innes Foundation Norwich; and Sam
brook et al., 1989, Molecular Cloning: A Laboratory Manual. Cold Spring Harbo
r Laboratory Press. Cold Spring Harbor, New York). For DNA extraction, bacteria were grown in LB or YEME medium at the temperature recommended by the ATCC. Description of corn (Dellaporta et al., 1985, “Maize DNA Miniprep”. Molecula
r Biology of Plants- a Laboratory Course Mannual.Cold Spring Harbor, N.
Y. Cold Spring Harbor Laboratory, NY USA. Pp. 36-37) applied to Streptomyces (Mehling et al. 1995, Microbiology, 141: 2139-).
2147), Chromosomal DNA was purified from cultures growing in late exponential phase.

The following non-limiting examples further illustrate the present invention.

Example 1Streptomycetes and Maduromycetes by Southern analysis of genomic DNA Identification  To demonstrate the utility of the CNB-ESP probe to distinguish between Streptomycetes and Maduromycetes, bacterial strains were obtained and grown to mid-log phase. Geno
DNA was prepared as follows. About 0.5-1.0 g of mycelium was resuspended in 2 ml of lysis buffer (0.1 M NaCl, 50 mM EDTA, pH 8.0) containing glass beads (3 mm diameter), and the suspension was vortexed for 2 minutes. Then 2 ml lysis buffer + 10-15 mg
Lysozyme and 50mg ml^-1RNase (without DNase) was added. The suspension was incubated at 37 ° C for 30-80 minutes. 400 ml of 10% SDS (w / v) was added
Subsequently, the solution was incubated at 37 ° C. for 15 minutes. The glass beads are removed and the DN
Apply A four times with one volume of phenol / chloroform / isoamyl alcohol (25: 24: 1).
And extracted once more with one volume of chloroform. The extracted DNA was precipitated with ethanol, dried and resuspended in 500 ml of distilled water.

20 mg of chromosomal DNA extracted from each strain is incubated with 50 units of the endonuclease BamHI or PstI by incubating for 16 hours at 37 ° C. in the respective buffer as recommended by the supplier (Boehringer Mannheim). After restriction, the sample was fractionated by electrophoresis in a 0.8% agarose gel. The fractionated DNA fragment was
Transfer to a Hybond N + membrane (Amersham, plc.) By time capillary transfer. Subsequently, the DNA immobilized on the solid support was washed with a hybridization buffer containing 5 × SSC, 5 × Denhardt's solution and 0.5% SDS, and 10 pmol of the radiolabeled CNB-ESP probe was added in the same buffer. Hybridization was performed at 45 ° C for 16 hours. The solid support was then washed at this hybridization temperature with 1 x SSC (0.15 M NaCl + 0.015 M sodium citrate, pH 7.2) and 0.5% SDS.
Was washed three times. The solid support was then exposed to an X-ray film at -70 ° C and imaged.

The probe hybridized to all of the Maduromycetes strains listed in the table above, but did not hybridize to the Streptomyces strain. As expected, the probe also gave a negative result for low G + C content genomic DNA from the distant relative Bacillus subtilis, which was carried as a negative control. The results obtained indicate that CNB-ESP can discriminate between Maduromycetes and Streptomycetes in hybridization to genomic DNA.

Example 2Streptomycetes and Maduromycetes by Southern analysis of PCR amplified DNA Identification  Chromosomal DNA from all strains used in Example 1 was extracted as described.
And primer NVR1 (5'-CAC GGA GAG TTT GAT CCT GGC 3 '(SEQ ID NO: 1))
And SOR (5'-GTA TTA GAC CCA GTT TCC CGG GC 3 '(SEQ ID NO: 2))
The extracted DNA was used for PCR amplification. 16.6mM (NH_Four)_TwoSO_Four, 67 mM Tris-HCl (pH8.8), 0.1% Tween-20 and 1 mM MgCl_TwoApproximately 0.5-1.0 mg of genomic DNA template with 280 ng of each primer in a final reaction volume of 100 ml of incubation buffer containing
Used for Incubation at 95 ° C (5 minutes) followed by 1 unit of EcoTaq polymer
30 cycles of incubation at 95 ° C (1 minute), 55 ° C (1 minute) and 72 ° C (1 minute) in the presence of Rase (Ecogen), and a final extension cycle at 72 ° C for 10 minutes.
The amplification was performed in an automatic thermocycler.

The obtained amplified DNA fragment having a length of about 166 bp was fractionated by electrophoresis in a 1.5% agarose gel. The fractionated DNA fragment was transferred to Hybond N + membrane (Amersham, plc.) By capillary transfer for 16 hours. Subsequently, the DNA immobilized on the solid support is washed with a hybridization buffer containing 5 × SSC, 5 × Denhardt's solution and 0.5% SDS, and 10 pmol of radioactively labeled in the same buffer.
The CNB-ESP probe was hybridized at 45 ° C. for 16 hours. The solid support was then washed three times at the hybridization temperature with 1 x SSC (0.15 M NaCl + 0.015 M sodium citrate, pH 7.2) and 0.5% SDS. The solid support is then X-treated at -70 ° C.
Exposure to linear film and subsequent development.

The probes were derived from all of the Maduromycetes strains listed in the table above.
It hybridized to all of the 166 bp PCR amplified fragments, but did not hybridize to the Streptomyces strain. The obtained results were obtained from CNB-ES
P shows that P can discriminate between Maduromycetes and Streptomycetes in hybridization to genomic DNA.

[Sequence list]

[Brief description of the drawings]

FIG. 1 is a particularly preferred probe of the present invention.

──────────────────────────────────────────────────続 き Continuation of front page (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), CA, JP, US (72) Inventor Mézillado, Lahua El Pé Spain, E-28049 Madrid, Camps de Canto Branco, Centro Nacional de Biotechnolohia (Se Esse) (I-Se) (No address) (72) Inventor Paro, Victor Spain, E-28049 Madrid, Camps de Canto Blanco, Centro Nacional de Biotechnolohia (Se Ese (Lee Se) (No address) (72) Inventor Rodriguez, Vicente Spain, A-28049 Madrid Do, Camps de Canto Branco, Centro Nacional de Biotechnolohia (Se Ese I Se) (No address) F-term (reference) 4B063 QA01 QA18 QQ06 QQ42 QQ43 QR32 QR56 QR62 QS25 QS34 QX07

Claims (12)

[Claims] 1. A nucleic acid encoding a 16S rRNA portion of a Maduromycetes bacterium hybridizes under hybridization conditions to a nucleic acid encoding a portion thereof, and a nucleic acid encoding a 16S rRNA portion of a Streptomycetes bacterium under the same hybridization conditions. Non-hybridizing nucleic acid probe. 2. The probe according to claim 1, which is DNA. 3. The probe according to claim 2, which has 10 to 250 base pairs. 4. A nucleic acid sequence comprising at least 90% complementary or homologous to a nucleic acid sequence comprising a Maduromycetes nucleic acid corresponding to 68-90 base pairs of Streptomycetes ambofaciens DNA encoding mature 16S rRNA. Item 4. The probe according to Item 3. 5. The probe according to claim 3, comprising 5 ′ GAA AGG CCC TTC GGG GGT ACT CG 3 ′ (SEQ ID NO: 3). 6. The probe according to claim 4, according to FIG. 7. A method for detecting the presence of a Maduromycetes bacterium in a sample, the method comprising: a) binding the sample to a nucleic acid probe (the probe hybridizes to a nucleic acid encoding Maduromycetes 16S rRNA). But does not hybridize to the nucleic acid encoding Streptomycetes 16S rRNA), b) subjecting to hybridization conditions, and c) determining whether hybridization has occurred. 8. The method of claim 7, further comprising the step of lysing the bacteria in said sample prior to step a). 9. The method according to claim 8, wherein said probe is a radioactively labeled probe. 10. The method according to claim 9, wherein the hybridization conditions are stringent hybridization conditions. 11. A method for distinguishing between Maduromycetes bacteria and Streptomyces bacteria in a sample, comprising: a) immobilizing a nucleic acid derived from a putative Maduromycetes bacteria and / or Streptomyces bacteria; The probe is labeled with the immobilized nucleic acid. The probe hybridizes to a nucleic acid encoding Maduromycetes 16S rRNA, but does not hybridize to a nucleic acid encoding Streptomycetes 16S rRNA. C) subjecting to hybridization conditions; and d) detecting hybridization of the probe to Maduromycetes nucleic acid. 12. The probe was obtained by PCR amplification using: a) a nucleic acid comprising SEQ ID NO: 3; b) a primer comprising SEQ ID NO: 1 and SEQ ID NO: 2.
12. The method according to claim 11, comprising a nucleic acid comprising a 166 bp amplification product, and c) a nucleic acid selected from the group consisting of the nucleic acids shown in FIG.