WO2001023609A2

WO2001023609A2 - Hybridization probes which detect bacteria of the genus thermobifida

Info

Publication number: WO2001023609A2
Application number: PCT/EP2000/009461
Authority: WO
Inventors: Olga Genilloud; Rafael P. Mellado; Victor Parro; Vicente Rodriguez
Original assignee: Merck Sharp & Dohme De Espana, S.A.E.
Priority date: 1999-09-27
Filing date: 2000-09-25
Publication date: 2001-04-05
Also published as: WO2001023609A3

Abstract

A method is provided herein which allows for the specific detection of bateria from the genus Thermobifida. This method makes use of nucleic acid probes that are complementary to a conserved region of the 16S rRNA genes present within the bacteria. The probes herein provided permit rapid detection of bacterial strains of Thermobifida in an accurate and reproducible fashion.

Description

TITLE OF THE INVENTION

HYBRIDIZATION PROBES WHICH DETECT BACTERIA OF THE GENUS

THERMOBIFIDA

CROSS-REFERENCE TO RELATED APPLICATIONS Not Applicable

STATEMENT REGARDING FEDERALLY-SPONSORED R&D Not Applicable

REFERENCE TO MICROFICHE APPENDIX Not Applicable

FIELD OF THE INVENTION The present invention relates to nucleic acid probes which specifically detect strains of the genus Thermobifida (formerly classified as Thermomonospora). Said probes are derived from the region coding for the mature 16S ribosomal RNA (rRNA) of said strains. The invention also relates to assays using these probes.

BACKGROUND OF THE INVENTION

Although much progress has been made in the last decade with regards to the identification of microorganisms, the procedures in use are often still laborious, non-sensitive and not specific. Traditional methods only permit the identification of those microorganisms that can be cultivated in the laboratory. This makes an accurate description of the composition of natural microbial communities very difficult. It is generally accepted that less than 20% of the microorganisms in nature have been discovered and, further, that the development of new culture-independent methods for studying the composition of microbial communities is well needed (Ward, D.M. et al, 1990 Nature, 45:63-64). Many of the pitfalls intrinsic to traditional methods have been overcome through the use of nucleic acid probes. These nucleic acid probes, made of genomic DNA, plasmids, riboprobes or synthetic oligonucleotides, are designed to target genomic DNA or certain RNA species present in biological samples. The use of synthetic oligonucleotides as probes is largely preferred because oligonucleotides can be rapidly synthesized in large amounts using chemical methods, have a long shelf-life, and are easy to purify and label.

The sequence of 16S rRNA, a common but distinctive cellular element, has been largely useful for this purpose, and has revealed the presence of numerous uncultured microorganisms in natural communities (Ward, D.M., et al., supra; Giovanni, S.J. et al, 1990 Nature 345:60-63; Barry, T. et al, 1991 In PCT Methods and Applications, Cold Spring Harbour, pp 51-56; Mehling, A et al, 1995 Microbiology 141:2139-2147; and Rheims, H. et al, 1996 Microbiology 142:2863- 2870). 16S and 23S rRNA genes are very often used for probe development given that sequences can readily be obtained using described methods and it is known that variable regions exist within these highly conserved genes which can be used for species-specific detection. Species-specific probes have been described for a large number of microorganisms. Universal probes for the detection of bacteria are also known (Giovannoni, S.J. et al, 1988 J. Bacteriol 170:720-726, and Barry, T et al, 1991 supra).

Modem methods, based on the 16S rRNA sequence have permitted the establishment of new evolutionary trees. From these analyses, the class Actinobacteria (Stackebrandt et al, 1997 Int. J. Syst. Bacteriol. 47:479-491) (comprising aerobic, gram-positive bacteria which form branching, usually non- fragmenting hyphae, and asexual spores borne on aerial mycelia) is shown to exhibit extraordinary complexity, encompassing many different genera clustered in at least 35 families.

Currently the art is silent as to a simple yet robust method to discriminate between close groups of Actinomycetes that occur in natural isolates, particularly for those bacteria that develop aerial mycelia prior to release of their spores. It would, thus, be desirable to have nucleic acid probes that could be used in fast, accurate assays to detect specific strains of Actinomycetes. Of particular interest in this respect are strains belonging to the genus Thermobifida (of the family Nocardiopsaceae) which comprises two species of the former Thermomonospora group, recently revised (Zhensui et al, 1998 Int. J. Syst. Bacteriol. 48:411-422). SUMMARY OF THE INVENTION

This invention relates to nucleic acid probes which hybridize under hybridization conditions to nucleic acids encoding a portion of the 16S rRNA of bacteria belonging to the genus Thermobifida. Another aspect of this invention is a method for detecting the presence of bacteria related to the genus Thermobifida, comprising:

(a) contacting the sample with a nucleic acid probe; wherein said probe specifically hybridizes to nucleic acid encoding 16S rRNA from bacteria of the genus Thermobifida; (b) imposing hybridization conditions; and

(c) determining if hybridization has occurred.

Yet another aspect of this invention is a kit for the detection of bacteria from the genus Thermobifida which comprises specific probes from 10 to 250 nucleotides in length which are complementary to or homologous with at least 90% of a nucleic acid sequence comprising Thermobifida nucleic acids corresponding to base pairs 430 to 445 of Thermobifida fusca DNA encoding the mature 16S rRNA molecule (Zhensui et al, 1998 Int. J. Syst. Bacteriol. 48:411-422).

The kit may additionally comprise reagents, compositions, instructions, disposable hardware and suitable packaging.

BRIEF DESCRIPTION OF THE DRAWINGS

Not Applicable

DETAILED DESCRIPTION OF THE INVENTION A method is provided herein which allows for the specific detection of bacteria from the genus Thermobifida. The method makes use of nucleic acid probes that are complementary to a conserved region of the 16S rRNA genes present within the bacteria. The probes herein provided permit rapid detection of bacterial strains of Thermobifida in an accurate and reproducible fashion. As used throughout this application and claims, the term "probe" will refer to synthetic or biologically produced nucleic acids, between 10 and 250 base pairs in length which contain specific nucleotide sequences that allow specific and preferential hybridization under predetermined conditions to target nucleic acid sequences, and optionally contain a moiety for detection or for enhancing assay performance. A minimum of ten nucleotides is generally necessary in order to statistically obtain specificity and to form stable hybridization products, and a maximum of 250 nucleotides generally represents an upper limit for length in which reaction parameters can be easily adjusted to determine mismatched sequences and preferential hybridization. Probes may optionally contain certain constituents that contribute to their proper or optimal functioning under certain assay conditions. For example, probes may be modified to improve their resistance to nuclease degradation (e.g., by end capping), to carry detection ligands (e.g., fluorescein, 32p_; biotin, etc.) or to facilitate their capture onto a solid support (e.g., poly-deoxyadenosine "tails").

"Preferential hybridization" or "hybridizing preferentially" means that hybridization with the intended target nucleic acid results in a hybridization reaction product which is more stable than any hybridization reaction product resulting from hybridization with a non-target nucleic acid under identical conditions. It is well within the skill of the ordinary artisan to compare stability of hybridization reaction products and evaluate which one is more stable, i.e., determine which one has bound "preferentially."

The terms "homology" and "homologous to" are meant to refer to the degree of similarity between two or more nucleic acid sequences and is not meant to imply any taxonomic relatedness between organisms. The degree of similarity is expressed as a percentage, i.e., 90% homology between two sequences will mean that 90% of the bases of the first sequence are identically matched to the bases of the second sequence.

"Specific" means that a nucleotide sequence will hybridize to a predetermined target sequence and will not substantially hybridize to a non-target sequence. "Specifically discriminate" means that a probe will substantially hybridize to a predetermined target sequence and will not substantially hybridize to a non-target sequence.

"Hybridization" is a process by which, under predetermined reaction conditions, two partially or completely complementary strands of nucleic acid are allowed to come together in an antiparallel fashion to form a double-stranded nucleic acid with specific and stable hydrogen bonds, following explicit rules pertaining to which nucleic acid bases may pair with one another.

"Substantial hybridization" means that the amount of hybridization observed will be such that one observing the results would consider the result positive in a clinical setting. Data which is considered "background noise" is not substantial hybridization.

"Stringent hybridization conditions" means approximately 35°C to 65°C in a salt solution of approximately 0.9 molar NaCl. Stringency may also be governed by such reaction parameters as the concentration and type of ionic species present in the hybridization solution, the types and concentrations of denaturing agents present, and the temperature of hybridization. Generally as hybridization conditions become more stringent, longer probes are preferred if stable hybrids are to be formed. As a rule, the stringency of the conditions under which hybridization is to take place will dictate certain characteristics of the preferred probes to be employed. Such relationships are well understood and can be readily manipulated by those skilled in the art.

In designing a probe for identification purposes, it is preferred that the probe be as specific as necessary (i.e., it should not cross-react with undesired nucleic acids) and highly sensitive (i.e., most, if not all, strains of the organism to be detected should react with the probe). Hence, the preferred target sequences should have the following characteristics:

(a) the sequence should be present in the genome of each strain of the microorganism concerned; and (b) the evolutionary diversity of the sequence should be such that, on the one hand, there is sufficient sequence-diversity to allow differentiation of the species concerned from other closely related species and, on the other hand, sufficient sequence-conservation to allow detection of the strain of concern with the probe used. Comparison and alignment of 16S rDNA sequences present in databases for the many actinomycetes and bacteria of the genus Thermobifida confirmed the existence of the so-called variable regions that appear in all 16S rDNA of actinomycetes, and allowed for the identification of a particular region within the Thermobifida strains which seemed suitable to derive probes that would be specific for bacteria of the genus Thermobifida. Sequence comparison and analysis were carried out using programs from the UWGCG package (Version 9.0, December 1996) and the Edit- View 1.0 DNA sequencer viewer (Applied Biosystems). 16S rDNA sequences were obtained from the NCBI Genbank database. Probes were checked against all 16S rRNA available via Ribosomal Database Project World Wide Web server (Bonnie, L. et al, 1999 A new version of the RDP (Ribosomal Database Project), Nucleic Acids Res. 27:171-173). As a result of the sequence comparison, two primers were designed. The first is designated BLT-N: 5' CCCAGACTCCTACGGGAGGCAGCAG 3' (SEQ ID NO:l) corresponding to base pairs 300-324 from Thermomonospora fusca DNA encoding mature 16S rRNA (Zhensui, Z et al, 1998 Int. J. Syst. Bacteriol. 48:411- 422). The second is designated BIT-R: 5' CCGCCTACGAGCTCTTTACGCCCA 3' (SEQ ID NO: 2) corresponding to base pairs 526-549 from Thermomonospora fusca DNA encoding mature 16S rRNA (Zhensui et al, supra.). Both of these can be used as primers in polymerase chain reaction technology to amplify the DNA region in order to obtain a preferred probe of this invention. Designated large quantities of the probe can be generated using known PCR techniques such as those in U.S. patents 4,683,202 and 4,683,195.

A preferred probe CNB-THER.2 was derived from a variable region of the 16S rRNA molecule. It comprises the sequence 5' TTTCTGGGGGTTGACG 3' (SEQ ID NO:3), which is DNA corresponding to base pairs 430-445 from Thermomonospora fusca DNA encoding mature 16S rRNA. In accordance with this invention, probes similar to CNB-THER.2 may be made by increasing or decreasing the length of CNB-THER.2. For a longer probe, it is preferred that additional nucleotides (either 3' or 5') be those of the corresponding DNA of Thermomonospora fusca encoding mature 16S rRNA. One embodiment of this invention is a nucleic acid, designated CNB-

THER.2, which provides specific binding to chromosomal DNA encoding the mature part of the 16S rRNA molecule of bacteria from the genus Thermobifida and does not substantially hybridize to the equivalent chromosomal region from bacteria belonging either to closely related actinomycetes taxa or to other bacteria. The preferred probes of this invention generally contain from at least about 10 nucleotides to about 250 nucleotides (the maximum number of nucleotides of the mature region of the genes coding for the 16S rRNA). More preferably, the probe will contain from about 10 nucleotides to about 250 nucleotides resulting from the PCR amplification of a DNA fragment comprising a DNA sequence hybridizing with the 16 nucleotide-long CNB-THER.2 probe.

The invention also relates to probes for use in hybridization assays, which use an oligonucleotide sufficiently complementary to hybridize to a sequence of chromosomal DNA region encoding the mature 16S rRNA from bacteria of the genus Thermobifida, yet not complementary enough to hybridize to the equivalent region from far-related Gram positive bacteria (e.g., Bacillus subtilis). A particularly preferred assay in accordance with this invention is a Southern blot. One probe which can be used for a Southern blot assay is about 250 bp long, obtained by PCR amplification of the DNA fragment obtained by use of the two primers BLT-N and BIT-R. The Southern blot, or dot blot assay can be conducted using well known procedures. Generally, it involves the steps of immobilizing a target nucleic acid or population of nucleic acids on a filter such as nitrocellulose, nylon or other derivatized membranes which are readily commercially available. The immobilized nucleic acids are then tested for hybridization under predetermined stringency conditions with the probe of interest. Under stringent conditions probes with nucleotide sequences with greater complementary to the target will exhibit a higher level of hybridization than probes whose sequences have less homology. Hybridization can be detected in a number of ways. For example, the probe can be isotopically labeled with the addition of a 32p_p osphorous moiety to the 5'-end of the oligonucleotide by the conventional polynucleotide kinase reaction. After hybridization has occurred, non-hybridized probe is removed by washing. The filters are exposed to x-ray film and the intensity of the hybridization signals is evaluated.

Another preferred assay in accordance with this invention is PCR. The probe CNB-THER.2 may be used as a primer in a Polymerase Chain Reaction in combination with the primer RVR.3 (SEQ ID NO:4, 5' AAGCTTTTAAGCAACATGCTCCGCCG 3 ') so as to specifically amplify a DNA fragment only when genomic DNA of the genus Thermobifida is used. The temperature of annealing for specific detection is 50°C.

The probes of this invention may be chemically synthesized using commercially available methods and equipment. For example, the solid phase phosphoramidite methods can be used to produce short oligonucleotides between 15 and 30 nucleotides long. Preferably, short DNA oligonucleotides are synthesized chemically using any of the Applied Biosystems DNA Synthesizers with reagents supplied by the same company. Chemically synthesized oligonucleotides can be obtained from Boehringer Mannheim and Perkin-Elmer. Strains which are detectable by the probes of this invention may be obtained from the ATCC collection and are the following:

Thermomonospora fusca (Thermobifida fusca comb, nov.)

ATCC 27730 Thermomonospora mesouviformis (Thermobifida mesouviformis comb. nov.) ATCC 27644 Other actinomycetes used in this invention also available from the ATCC collection are included in the following table:

ATCC ATCC

Strain Name Strain Name Number Number

Actinomadura citrea ATCC 27887 Actinomadura viridis ATCC 27103

Actinomadura coerulea ATCC 33576 Microbispora aerata ATCC 15448

Actinomadura cremea ATCC 33577 Microbispora ATCC 15740 amethystogenes

Actinomadura spadix ATCC 27298 Microbispora diastatica ATCC 33325

Nonomuria spiralis ATCC 35114 Microbispora echinospora ATCC 27300

Nonomuria fastidiosa ATCC 33516 Microbispora parva ATCC 33326

Nonomuria ferruginea ATCC 35575 Microbispora rosea ATCC 12950

Nonomuria helvata ATCC 27295 Microtetraspora flexuosa ATCC 35864

Actiniomadura livida ATCC 33578 Microtetraspora fusca ATCC 23058

Actinomadura madurae ATCC 19425 Microtetraspora glauca ATCC 23057

Actinomadura malachitica ATCC 27888 Microtetraspora ATCC 27301 niveoalba

Nonomuria pusilla ATCC 27296 Planobispora rosea ATCC 23866

Nonomuria roseola ATCC 33579 Planomonospora ATCC 23863 parontospora

Nonomuria rubra ATCC 27031 Strepto sporangium ATCC 12428 roseum

Nonomuria salmonea ATCC 33580 Streptosporangium ATCC 33329 vulgare

The following stremptomycetes strains may be used in this invention: Streptomyces ambofaciens, S. antibioticus, S. cinnamonensis, S. coelicolor A3 (2), S. fradiae, S. lividans TK21, S. nataliensis, S. peucetius, S. violascens and Streptomyces sp.

Procedures used for the growth and manipulation of the bacteria related to this invention and general DNA manipulation are as described (Hopwood et al, 1985 Genetic Manipulation of Streptomyces. A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York). For DNA extraction, bacteria are grown in LB or YEME media at the ATCC recommended temperatures. Chromosomal DNA is purified from cultures growing in late exponential phase as described for maize (Dellaporta et al, 1985 "Maize DNA Miniprep," Molecular Biology of Plants - a Laboratory Course Manual. Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory, N.Y. USA. Pp. 36-37), as adapted for Streptomyces (Mehling et al, 1995 Microbiology 141:2139-2147).

The following non-limiting examples are presented to better illustrate the invention.

EXAMPLE 1 Identification of Thermobifida Strains by Southern Analysis of PCR Amplified DNA

Strains of bacteria were obtained and grown until mid-logarithmic phase to validate the usefulness of the CNB-THER.2 probe to identify bacteria of the genus Thermobifida. Genomic DNA was prepared as follows: Approximately 0.5-1.0 g mycelia were resuspended in 2 ml lysis buffer (NaCl 0.1M, EDTA 50 mM, pH 8.0) containing glass beads (3 mm diameter) and the suspension was vortexed for 2 minutes before adding 2 ml of lysis buffer plus 10-15 mg lysozyme and 50 μg ml-1 Rnase Dnase-free. The suspension was incubated for 30-80 minutes at 37°C. After the addition of 500 μl 10% SDS (w/v), the solution was incubated at 37°C for 15 min. The glass beads were removed and the DNA extracted four times with 1 volume of phenol/chloroform/isoamyl alcohol (25:24: 1) and once more with 1 volume of chloroform. The extracted DNA was ethanol precipitated, dried and resuspended in 500 μl distilled water. Chromosomal DNA from all the strains listed above was used for PCR amplification using primers BLT.N: 5' CCCAGACTCCTACGGGAGGCAGCAG 3' (SEQ ID NO:l) and BIT.R: 5' CCGCCTACGAGCTCTTTACGCCCA 3' (SEQ ID NO:2). Approximately 0.5-1.0 μg genomic DNA template was used with 280 ng of each primer in a final reaction volume of 100 μl of an incubation buffer containing 16.6 mM (NH_i)2SO4, 67 mM Tris-HCL (pH8.8), 0.1% Tween-20 and ImM MgCl2-

Amplifications were performed in automated thermocyclers by incubation at 95 °C (5 min) followed by 30 cycles of incubation at 95°C (1 min), 55°C (1 min) and 72°C (1 min) in the presence of one unit of Eco Taq polymerase (Ecogen).

The resulting about 250 bp long amplified DNA fragments were fractionated by electrophoresis in 1.5% agarose gels. The fractionated DNA fragments were transferred to Hybond N+ membranes (Amersham, pic.) by capillary transfer for 16 h. The DNA immobilized in the solid support was then washed with a hybridization buffer containing 5 x SSC, 5 x Denhardt's solution and 0.5% SDS and set to hybridize with 10 pmol of the radioactively labeled CNB-THER.2 probe in the same buffer for 16 h at 50°C. The solid supports were then washed three times with 1 x SSC (0.15 M NaCl plus 0.015 M sodium citrate, pH 7.2) and 0.5% SDS at the hybridization temperature. The solid supports were then set to exposure in X-ray films at -80°C prior to being developed.

The CNB-THER.2 probe hybridized with the Thermobifida strains named above but it did not hybridize with any of the other actinomycetes mentioned. As expected, the probe also gave a negative result with low G+C content genomic DNA from the far-related bacterium Bacillus subtilis which served as a negative control. The results, as well as the comparison of the 16S rDNA sequence of the probe CNB-THER.2 with the equivalent sequence of many other actinomycetes, indicate that the probe CNB-THER.2 specifically detects strains of the genus Thermobifida.

EXAMPLE 2 Detection of Bacteria from the Genus Thermobifida by PCR The probe CNB-THER.2 can be used as a forward primer for PCR in order to specifically amplify fragments from the target strains only. As a reverse primer the conserved oligonucleotide RVR.3 (SEQ ID NO:4; 5' AAGCTTTTAAGCAACATGCTCCGCCG 3') can be used in all cases.

Approximately 0.5-1.0 μg genomic DNA template of representative strains of each genera of bacteria listed above was used with 280 ng of each primer in a final reaction volume of 100 μl of an incubation buffer containing 16.6 mM (NH4)2SO4, 67 mM Tris-HCl (pH8.8), 0.1% Tween-20 and 1 mM MgCl2-

Amplifications were performed in automated thermocyclers by incubation at 95°C (5 min) followed by 30 cycles of incubation at 95°C (1 min), 50°C (1 min) and 72°C (1 min) in the presence of one unit of Eco Taq polymerase (Ecogen).

DNA fragments of the expected size were obtained only when the target organisms DNA were present in the reaction.

Claims

WHAT IS CLAIMED:

1. A nucleic acid probe which hybridizes under hybridization conditions to nucleic acid encoding a portion of 16S rRNA of bacteria belonging to the genus Thermobifida.

2. A probe according to claim 1 which is DNA.

3. A probe according to claim 2 which is between 10 and 250 base pairs.

4. A probe according to claim 3 which is complementary to or homologous with at least 90% of a nucleic acid sequence comprising a Thermobifida nucleic acid corresponding to base pairs 430-445 of Thermomonospora fusca DNA encoding mature 16S rRNA.

5. A probe according to claim 3 which comprises 5' TTTCTGGGGGTTGACG 3' (SEQ ID NO:3).

6. A method for detecting the presence of bacteria belonging to the genus Thermobifida, in a sample comprising:

(a) contacting the sample with a nucleic acid probe; wherein said probe specifically hybridizes to nucleic acid encoding 16S rRNA of bacteria of the genus Thermobifida; (b) imposing hybridization conditions; and

(c) determining if hybridization has occurred.

7. A method according to claim 6 further comprising the step of lysing bacteria in the sample prior to step (a).

8. A method according to claim 7 wherein the probe is a radioactively labeled probe.

9. A method according to claim 8 wherein hybridization conditions are stringent hybridization conditions.

10. A method according to claim 9 wherein said probe comprises nucleic acids selected from the group consisting of:

(a) nucleic acids comprising SEQ ID NO:3; and (b) nucleic acid comprising a 250 bp amplification product made by PCR amplification using primers SEQ ID NO:l and SEQ ID NO:2.