JPH09234084A - Oligonucleotide probe and determination of presence of procaryote using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain an oligonucleotide probe useful for simply, specifically and rapidly determining the presence of a procaryote, especially a mycoplasma with a high sensitivity.

SOLUTION: This oligonucleotide probe is shorter than a gene capable of coding a mycoplasma ribosomal RNA (rRNA) and comprises at least 9 bases and is further selected from 3'GCTTAGTCGATACAGC5', 5' CGAAUCAGCUAUGUCG3' and nucleic acid sequences complementary thereto. A sample is brought into contact with the oligonucleotide probe capable of specifically hybridizing with a nucleotide base sequence, present in a procaryote but absent in a eucaryote and hybridized with a nucleic acid present in the sample. Thereby, the presence of the procaryote containing the nucleotide base sequence is detected with the nucleic acid hybridized with the oligonucleotide probe.

COPYRIGHT: (C)1997,JPO

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION This invention relates generally to the field of biology, and more particularly to biomedical, biochemical and molecular biology.

[0002]

BACKGROUND OF THE INVENTION Mycoplasma is a group of pathogenic microorganisms characterized by a small shape and no cell walls, the Mollicutes web. These microorganisms are among the smallest known microorganisms that can survive independently and are important pathogens in humans, plants and animals. For example, atypical pneumonia and non-gonococcal urethritis are common mycoplasma infections in humans.
Mycoplasma also causes rheumatoid arthritis, spontaneous abortion, infertility and other genital tract illnesses,
And is also involved in certain autoimmune disease states. Further, mycoplasmas are common contaminants in cell culture.
In biological studies, mycoplasma contamination of tissue culture is a serious problem and requires constant monitoring and monitoring.

[0003] These organisms are extremely difficult to culture.
It is not surprising that there is currently no specific method for the cost of diagnosing an infection with mycoplasma. The most commonly used methods for detecting mycoplasma in clinical samples are serological and culture. Serological methods are easy to mistakenly judge positive,
Cultural methods are expensive, time consuming and tedious. Many of the biochemical techniques currently used to detect microbial contamination in cell culture do not specifically detect mycoplasma but rather prokaryote or simple eukaryotes such as yeast and fungi. Some even show presence and detect viruses. Such tests are advantageous when only interested in knowing the presence of microorganisms, but when looking for substances of suspicious etiology for animal or human disease, classify this substance as much as possible. Is not always sufficient.

Further, the above method is limited by special problems. For example, there are apparently "non-cultivable" mycoplasmas that are not detected by conventional culture methods. In addition, in the case of immunofluorescence tests, it is conceivable that one or more antibodies may be required for the identification of a particular organism, since nine different Mycoplasma species are common tissue culture contaminants. Also, DNA staining is not always specific to mycoplasma.

[0005]

Therefore, a simple, sensitive, specific, cost-effective and fast mycoplasma detection system has been urgently desired in the fields of diagnostic medicine and biological research. .

The use of nucleotide sequence homology and nucleic acid hybridization kinetics has become a widely used technique for detecting cells and various organisms in cell culture. However, prior to the present invention, reliable and specific DNA probes for mycoplasma detection were not available.

[0007]

Means for solving the above-mentioned problems are as follows. (1) 3'GCT, which is shorter than the gene encoding mycoplasma rRNA and consists of at least 9 bases
TAGTCGATACAGC 5 ', 5'CGAAUCA
It is an oligonucleotide probe selected from GCUAUGUCG3 'and nucleic acid sequences complementary thereto. (2) A method for determining the presence of a prokaryote containing a nucleotide base sequence present in a prokaryote but not in a eukaryote, wherein a sample is specifically hybridized to the nucleotide base sequence. The presence of a prokaryote containing the nucleotide base sequence is detected by contacting the obtained oligonucleotide probe, hybridizing the oligonucleotide probe with a nucleic acid present in the sample, and detecting the presence of a prokaryote containing the nucleotide base sequence by the nucleic acid hybridized with the oligonucleotide probe. And the oligonucleotide probe is the oligonucleotide probe according to (1). (3) The prokaryote is a species of the class Moricathes (species of
the Class Mollicutes), wherein the nucleotide base sequence is present only in prokaryotic nucleic acids derived from species of the class Moricates. (4) The method according to (3), wherein the species of the class Moricates is a species of the genus Mycoplasma, and the nucleotide base sequence is present only in prokaryotic nucleic acids derived from the species of the genus Mycoplasma. . (5) The mycoplasma species is mycoplasma.
Capricolane (Mycoplasma capricolun), wherein the nucleotide base sequence is present only in prokaryotic nucleic acids derived from Mycoplasma capricolane. (6) the mycoplasma species is mycoplasma;
Species PG50 (Mycoplasma species PG50)
Wherein the nucleotide base sequence is mycoplasma
The method according to (4), which is present only in a prokaryotic nucleic acid derived from PG50. (7) The method according to (4), wherein the Mycoplasma species is Mycoplasma pneumoniae, and the nucleotide base sequence is present only in prokaryotic nucleic acids derived from Mycoplasma pneumoniae. (8) The method according to (3), wherein the species of the class Moricates is a species of ureaplasma, and the nucleotide base sequence is present only in prokaryotic nucleic acids derived from the species of ureaplasma. (9) The method according to (3), wherein the species of the class Molacecates is a species of Spiroplasma, and the nucleotide base sequence is present only in prokaryotic nucleic acids derived from the species of Spiroplasma. (10) The method according to (3), wherein the species of the class Moricates is a species of Acholeplasma, and the nucleotide base sequence is present only in a prokaryotic nucleic acid derived from the species of Acholeplasma.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention is a radioisotope label,
Labeled by various techniques such as biotin labeling, PEI-peroxidase conjugation or fluorescent antibody labeling ELISA method (la.
beled or tagged) specific mycoplasma ribosomal RNA gene fragment is used for specifically and highly sensitively detecting mycoplasma in a clinical sample, cell or cell culture by DNA or RNA hybridization. .

In one aspect, the present invention provides a nucleotide sequence complementary to the mycoplasma 16S RNA gene, wherein the nucleotide sequence is a code for mycoplasma ribosomal RNA (rRNA), AACACGTATAC, CGA
ATCAGCTATGTCG, GAGGTT-AAC,
ATCCGGATTTATT, TCTCAGTTCGGG
ATTGA, AGGTGGGTGCATGGGTTG, TC
CTGGCTCAGGAT, ATACATAGGT, A
ACTATGTGC, AATTTTTCCACAATG and TCTCGGGTCT comprising a nucleotide base sequence selected from the group consisting of:

In this nucleotide base sequence, T
There thymine, G is guanine, A is adenine, C is cytosine, - representing the missing nucleotides when compared to a nucleotide base sequence of control in E. coli (E .coli). These fragments are significantly different from the 16S RNA gene of E. coli and constitute the basic part of a mycoplasma-specific probe consisting of a labeled nucleotide sequence complementary to that described above.

In another aspect, the present invention relates to a prokaryotic rR.
Code for NA, ACGGGTGAGT,
TAATACCGCAT, TAGGGGAGGCAGC
AGT, GTGGGGGAGCAA, AGGATTAG
ATACCCT, CCGTAACGAT, GAATT
GACGGGG, CCCGCACAAG, GGTGGA
GCATGT, TGTTGGGTTAAGTCCCGC
AACGA, GGGATGACGT, ACGTGCTA
CAATG, CTAGTAATCG, TGTACACA
CCGCCCGTCA, AAGTCGTAACAAGGG
TA and TGGATCACCCTCTT-16S RNA containing a nucleotide base sequence selected from the group
Provided is an identified DNA chlorine sequence of the gene.

These fragments represent a region in the 16S RNA gene common to E. coli and all mycoplasmas tested. A universal probe for all prokaryotes consists of a labeled nucleotide base sequence complementary to these fragments.

In general, the present invention is a method for determining the presence of a prokaryote containing a nucleic acid containing a specific nucleotide base sequence that is present in a prokaryotic nucleic acid but is not present in a eukaryotic nucleic acid. The method comprises providing a medium that may be derived from the prokaryote and that may contain a nucleic acid or nucleic acid fragment containing the particular nucleotide base sequence to an oligonucleotide comprising a nucleotide base sequence complementary to the particular nucleotide base sequence. Contacting with a nucleotide, thereby causing the oligonucleotide to hybridize to the nucleic acid or nucleic acid fragment from the prokaryote containing the particular nucleotide base sequence that may be present in the medium, and hybridizing to the oligonucleotide. Method performed by detecting the presence of a nucleic acid or nucleic acid fragment For free.

In another aspect, the present invention involves a specific biological probe generally used for the detection of mycoplasma or prokaryotes, which relies on the method described above and the identification and production of such a probe. .

[0015]

The Examples detail first the specific steps involved in the production and use of the biological probes of the present invention, and then describe how the specific nucleotide base sequences useful in the present invention are determined. The present invention will now be described. - Synthesis of deoxyoligonucleotides - 16 bp oligodeoxynucleotide which is complementary to one of the 16S mycoplasma RNA gene base sequence listed above, 3'GCTTAGTCGATACAG
C5 'is a reference (MHCaruth
ers et al. , Gonetic Engineering , Vol.4, p.1-17, Plenum
Synthesized by the phosphoric acid triester solid phase method described in Publishing Co. (1982)). Other deoxyoligonucleotides described above or other desired oligonucleotides are synthesized as well. For example, instead of a DNA probe, it may be desirable to synthesize an RNA probe such as a recombinant SP6 vector transcript containing a 5'CGAAUCAGCUAUGUCG3 'nucleotide sequence.

DNA-R for ribosomal RNA molecules
NA or RNA-RNA hybridization is performed using an oligonucleotide probe as a genomic DNA.
DNA-DNA or RNA-D used for base sequence
The detection sensitivity is several hundred times higher than NA hybridization. This is because the use of such oligonucleotide probes detects multiple copies of ribosomal RNA per mycoplasma or eubacterial cell. - testing of deoxyoligonucleotides - Additional deoxy oligonucleotides literature (CCRichardson to be inserted herein by reference, Procedure in Nucl
eic Acid Research (Cantoni, GLand Davies, DR, ed
s.), Vol.2, pp.815-828, Harper and Row, New York (197
1)) method was ^{3 2} P-labeled 5 'ends using the. The resulting labeled deoxyoligonucleotide was then used as an oligonucleotide probe specific for mycoplasma.

The specificity for mycoplasmas is described in the literature (M. Cunningham , Anal.
m. 128: 415-421 (1983)). For this purpose, a 1600 bp DNA fragment of Mycoplasma pneumoniae cloned in pUC8 was used. The 1600 bp cross section contains the 16 base deoxyoligonucleotide described above. A typical prokaryotic eubacter
The genomic digest of Escherichia coli is the enzyme Hind I.
Produced by digestion with II. Digested E. coli D
NA and a 1600 bp pneumonia mycoplasma DNA fragment are described in the literature (JJLeary et al., Incorporated herein by reference).
L. , Proc . Natl . Acad . Sci . USA 80: 4045-4049 (1983)) and transferred onto a nitrocellulose filter. D
Nitrocellulose filters containing NA fragments are heated for 2 hours under reduced pressure at 80 ° C., it was hybridized to ^{3 2} P-labeled deoxyoligonucleotides. The generated slot blot was developed as shown in the figure to yield 0.00034 ng, 0.0034 ng, 0.0
There were high intensity blots for 34 ng, 0.34 ng, 3.4 ng (calculated for 16 bp) Mycoplasma pneumoniae DNA fragment, 0.00015 μg, 0.00
15 μg, 0.015 μg, 0.15 μg, 1.5 μg
It was revealed that there was no blot for the E. coli DNA fragment of E. coli, indicating the specificity of the deoxyoligonucleotide probe for mycoplasma.

3'GCTTAGTCGATACAGC
The deoxyoligonucleotide having the 5'base sequence is effective as an oligonucleotide probe specific to mycoplasma which hybridizes with the DNA of mycoplasma and does not hybridize with the DNA of other prokaryotes.

On the other hand, for example, TGCCCA which is complementary to one of the gene base sequences which are the codes of prokaryotes
Deoxyoligonucleotides having a CTCA base sequence are effective as prokaryotic-specific oligonucleotide probes that hybridize to prokaryotes and do not hybridize to eukaryotes.

The following method was found to be effective for detecting mycoplasma in infected cells. Cells were used in a 1-2T75 tissue culture flask and trypsin EDTA (0.05% trypsin in PBS, 0.04
% EDTA) at 37 ° C. for 2 minutes. The trypsinized cells were resuspended in 1-2 ml of growth medium and subjected to Minifold II slot blot hybridization (Schleich).
er and Schuell, inc., Keene, NH).
SC (1 × SSC is 15 mM Na citrate, 150 m
A volume of 50-100 μl (1 × 10 ⁵ -1 × 10 ⁶ cells) was spotted on a nitrocellulose membrane moistened with M NaCl, pH 7.4). DNA samples applied to slot blots were alkaline (0.5 M NaOH,
(1.5 M NaCl) for 5-10 minutes at room temperature, 0.5 M Tris, pH 7.2 and 3.0 M Na
Neutralized with Cl for 5-10 minutes at room temperature. The filters were then washed with 2 × SSC at room temperature for 5 minutes and heated at 80 ° C. for 2 hours in a vacuum oven. The filter was 0.5 mM EDTA, 5 mM Tris, pH 7.0.
5, 5 × Denhardt and 100 μg / ml
Were prehybridized at 65 ° C. for 2 hours using a prehybridization buffer consisting of the heat denatured denatured sperm DNA. 10 mM Tris, pH 7.5, 1
mM EDTA, 0.75 M NaCl, 1 × Denhards, 0.5% SDS, 10% dextran sulfate and 10%
0 Pg / hybridization buffer consisting ml of heat denatured the sparse sperm DNA, ^{3 2} P-labeled 1 to 2 × 10 ⁶ cpm of oligodeoxynucleotide specific activity 10 ⁸ c
Using the test means of the present invention at a concentration measured as ≥ pm / μg, hybridization was carried out at 65 ° C for 16
Made time.

After hybridization, the filters were 2 × SET, 0.2% SDS (1 × SET was 30 m
(Tris M, ph 8.0, 150 mM NaCl) for 2-4 hours at 65 ° C. and 1-2 hours at room temperature with 4 mM Tris base. The filter is then dried and one or two Dupont Cronex
nex) Exposed on X-ray film using intensifying screen. -Determination of Specific Nucleotide Base Sequence-While the specific nucleotide base sequence was prepared and used as described above, in order to understand the broad scope of the present invention, a specific nucleotide sequence for mycoplasma is described below. Oligonucleotide probe, oligonucleotide probe specific to common prokaryotes, mycoplasma, ureaplasma (ureaplasma) acholeplasma (acholeplasm)
a) and an individual species-specific probe of spiroplasma, or an effective species-specific probe of Eubacterium,
A test of the technique used to determine a particular nucleotide base sequence is described.

Such a determination is made in the following steps. 1. 1. Cloning the complete genome of ribosomal RNA of a specific species of Mycoplasma into a bacteriophage or plasmid vector. 2. Testing the resulting ribosomal RNA gene fragment with a non-mycoplasmal prokaryotic ribosomal RNA operon. 3. characterizing fragments that hybridize to this non-mycoplasmal prokaryotic ribosomal RNA operon. References (Gobel, U. and St.
anbridge, EJ , Science , Vol.226, pp.1211-1213 (1984)
4. Identifying mycoplasma-specific fragments by differential hybridization as described in 5). 5. subcloning a fragment specific for Mycoplasma into a plasmid that is undergoing nucleotide sequence 6. The step of arranging the generated subcloned Mycoplasma-specific fragment with the base sequence. 7. Repeating steps 1-6 for other species of mycoplasma and non-mycoplasma prokaryotes; A step of comparing the nucleotide sequences obtained in steps 6 and 7.
As a result, a nucleotide sequence that is common to all species of Mycoplasma and different from the corresponding nucleotide sequence of a non-Mycoplasma prokaryote is effective as an oligonucleotide probe specific for Mycoplasma, and all species of Mycoplasma and non-Mycoplasma prokaryotes A nucleotide sequence common to living organisms is effective as an oligonucleotide probe specific to general prokaryotes, and a nucleotide sequence specific to any species of Mycoplasma, Acoreplasma, Ureaplasma or Spiroplasma. , And any given Eubacterium species-specific base sequence is effective as an oligonucleotide probe specific for each individual species of Mycoplasma, Acoreplasma, Ureaplasma or Spiroplasma.

The cloning of the ribosomal RNA genome of Mycoplasma pneumoniae was performed using the total DNA of Mycoplasma pneumoniae.
Of the Hind III fragment and the H of the Hind III fragment
This was achieved by ligation into the vector pUC8 digested with indIII. Fragments of the resulting ribosomal RNA gene can be found in the literature (Gobel et al., Scienc
e, 226: 1211-1213 (1984)).
Tests were performed using the E. coli ribosomal RNA operon in 5 plasmids and a cloned fragment containing the ribosomal base sequence was identified.

Hybridizing under stringent hybridization conditions to a species of Mycoplasma,
A 1600 bp fragment was chosen on the basis of no hybridization to E. coli or mammalian DNA. This 1600 bp section was removed from the pUC8 vector by Hind III digestion and inserted here as a reference (Sanger et al., Proc. Natl. Acad . Sci . US) .
A. , 74: 5463-5467 (1977)), using the Sanger dideoxy method described in M13Mp8D for sequencing.
Was linked to the NA bacterial virus.

Mycoplasma pneumoniae, Mycoplasma capricolum ( M. capricolum ) and Mycoplasma sp. PG
Comparison of the nucleotide sequences of 50 ( Mycoplasma species PG50 ) and E. coli showed that a certain nucleotide sequence is common to all these species of mycoplasma and different to E. coli. These base sequences are synthesized, labeled and used as an oligonucleotide probe specific for mycoplasma. For example, 3'GCTTAGTCGATACAGC5 'constituted an oligonucleotide probe specific for mycoplasma. Other base sequences were common to these species of Mycoplasma as well as E. coli. These latter base sequences are synthesized, labeled and used as oligonucleotide probes specific for all prokaryotes. In addition, other base sequences are unique to one Mycoplasma species and are synthesized, labeled and used as oligonucleotide probes specific for Mycoplasma species.

The present invention thus provides a specific, sensitive and rapid method for detecting mycoplasma in contaminated cell cultures or other biological environments. The present invention can also provide a ribosomal DNA probe derived from a region retained in all prokaryotes.
Such a probe would be very effective in the rapid and sensitive diagnosis of human or animal bacteremia or sepsis.

The present invention can also provide a ribosomal DNA probe specific for each of Mycoplasma, Acoreplasma, Ureaplasma, Spiroplasma and Eubacterium. These probes will be particularly effective against those organisms with little or no known genetic makeup.

Although the present invention has been described in detail with reference to certain specific deoxyoligonucleotides and mycoplasma species, it will be apparent to those skilled in the art that many variations are possible. The scope of the invention includes such variations, and
The scope of the invention is limited only by the claims.

[Brief description of drawings]

FIG. 1 is a diagram showing slot blot development.

Continuation of the front page (51) Int.Cl. ⁶ Identification number Office reference number FI Technical display part // (C12N 15/09 ZNA C12R 1:35) (C12Q 1/68 C12R 1:35) (72) Inventor Eric Jay Stanbridge California, USA 92625 Corona del Mar Seward 501 (72) Inventor Wolf Gobel Germany Day-7800 Freiberg Hermann Helderstraße 11

Claims (10)

[Claims] 1. A 3 ', which is shorter than the gene encoding mycoplasma rRNA and consists of at least 9 bases.
GCTTAGTCGATACAGC 5 ', 5'CGAA
An oligonucleotide probe selected from UCAGCUAUGUCG3 'and nucleic acid sequences complementary thereto. 2. A method for determining the presence of a prokaryote that comprises a nucleotide sequence that is present in a prokaryote but not in a eukaryote, wherein the sample is high-specific for the nucleotide sequence. By contacting with a hybridizable oligonucleotide probe, the oligonucleotide probe is hybridized with a nucleic acid present in the sample, the presence of a prokaryote containing the nucleotide base sequence by the nucleic acid hybridized with the oligonucleotide probe. Detecting, wherein the oligonucleotide probe is the oligonucleotide probe of claim 1. 3. The prokaryote is a species of the class Molycates.
ies of the Class Mollicutes), wherein the nucleotide base sequence is present only in prokaryotic nucleic acids derived from species of the class Molycates. 4. The method according to claim 3, wherein the species of the class Moricates is a species of the genus Mycoplasma, and the nucleotide sequence is present only in a prokaryotic nucleic acid derived from the species of Mycoplasma. Method. 5. The method according to claim 4, wherein the species of the genus Mycoplasma is Mycoplasma capricolun, and the nucleotide base sequence is present only in a prokaryotic nucleic acid derived from Mycoplasma capricolans. 6. The Mycoplasma species PG5 is a Mycoplasma species PG5.
0), wherein the nucleotide base sequence is present only in the prokaryotic nucleic acid derived from Mycoplasma species PG50. 7. The method according to claim 4, wherein the species of the genus Mycoplasma is Mycoplasma pneumoniae, and the nucleotide sequence is present only in a prokaryotic nucleic acid derived from Mycoplasma pneumoniae. 8. The method according to claim 3, wherein the species of the class Moricates is a species of Ureaplasma and the nucleotide base sequence is present only in a prokaryotic nucleic acid derived from the species of Ureaplasma. 9. The method according to claim 3, wherein the species of the class Molycates is a species of Spiroplasma, and the nucleotide sequence is present only in a prokaryotic nucleic acid derived from a species of Spiroplasma. 10. The method according to claim 3, wherein the species of the class Moricates is a species of Acholeplasma, and the nucleotide base sequence is present only in a prokaryotic nucleic acid derived from the species of Accoleplasma.