JP2003159067A - Oligonucleotide probe and method for determining existence of prokaryote using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a probe and a method for simply, highly sensitively, specifically and rapidly determining existence of a prokaryote, especially a mycoplasma. <P>SOLUTION: Disclosed is a method for determining the existence of the prokaryote in a medium comprising a process for contacting a medium containing a target nucleic acid sequence with a synthesized and isolated oligonucleotide under the hybridization condition selected in advance so that a non-target nucleic acid sequence is not hybridized and the target nucleic acid sequence derived from the prokaryote is hybridized in the medium, and a process for detecting the existence of the target nucleic acid sequence under the hybridization condition selected in advance. <P>COPYRIGHT: (C)2003,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. In addition, mycoplasma are a common contaminant in cell culture.
In biological studies, mycoplasma contamination of tissue culture is a serious problem and requires constant monitoring.

These organisms have extremely troublesome culture conditions,
It is not surprising that there is currently no specific way to pay the cost of diagnosing a mycoplasma infection. The most commonly used methods for detecting mycoplasma in clinical samples are serological and culture. Serological method is easy to mistakenly judge as 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.

Moreover, 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 various organisms in cells and cell cultures. 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) A method for determining the presence of a prokaryotic organism in a medium, which does not hybridize to a non-target nucleic acid sequence in the medium but hybridizes to a target nucleic acid sequence derived from the prokaryotic organism. Contacting the medium containing the target nucleic acid sequence with the synthesized and separated oligonucleotides under preselected hybridization conditions, and, under the preselected hybridization conditions, And a step of detecting the presence. (2) The method according to (1) above, wherein the preselected hybridization conditions are stringent. (3) The method according to (1) above, wherein the target nucleic acid sequence is an rRNA sequence. (4) The method according to (1) above, wherein the target nucleic acid sequence is a DNA sequence. (5) A method for determining the presence of a prokaryotic organism containing a particular nucleotide sequence, wherein the hybridization is not carried out to a non-target nucleic acid sequence but to a target nucleic acid sequence derived from said prokaryote. Contacting the target nucleic acid sequence with an oligonucleotide under selected hybridization conditions, and detecting the presence of a nucleic acid sequence with which the oligonucleotide hybridizes under the preselected hybridization conditions, It is a method including. (6) The method according to (5), wherein the preselected hybridization condition is stringent. (7) A composition comprising a synthesized and isolated oligonucleotide that is substantially complementary to a preselected prokaryotic target nucleic acid sequence but not substantially complementary to a non-target nucleic acid sequence. (8) The oligonucleotide sequence has at least 9
The composition according to (7) above, which is one nucleotide. (9) The composition according to (7), wherein the target nucleic acid sequence is an rRNA sequence. (10) The composition according to (7), wherein the target nucleic acid sequence is a DNA sequence. (11) Synthesized and isolated oligonucleotides that hybridize to preselected prokaryotic nucleic acid sequences under preselected hybridization conditions but not to unselected nucleic acid sequences. It is a composition containing. (12) The composition according to (11), wherein the oligonucleotide sequence is at least 9 nucleotides. (13) The preselected hybridization condition is stringent (1)
It is the composition described in 1). (14) A target nucleic acid sequence consisting of at least 9 nucleotides, which hybridizes under stringent hybridization conditions to a preselected prokaryotic target nucleic acid sequence but not to a non-target nucleic acid sequence. A composition comprising synthetic and isolated oligonucleotides substantially complementary to. (15) The composition according to (14), wherein the target nucleic acid sequence is an rRNA sequence. (16) The composition according to (14), wherein the target nucleic acid sequence is a DNA sequence.

[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 base sequence complementary to the 16S RNA gene of mycoplasma, wherein the nucleotide base sequence is the ribosomal RN of mycoplasma.
AACACG, a code for A (rRNA)
TATC, CGAATCAGCTATGTCG, GAG
GTT-AAC, ATCCGGATTTATT, TCT
CAGTTCGGATTGA, AGGTGGTGCAT
GGTTG, TCCTGGCTCAGGAT, ATAC
ATAGGT, AACTATGTGC, AATTTTT
It contains a nucleotide base sequence selected from the group consisting of CACAATG and TCTCGGGTCT.

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 probe specific for mycoplasma composed of a labeled nucleotide base sequence complementary to the one described above.

In another aspect, the present invention provides a prokaryotic rR
A code for NA, ACGGGTGAGT,
TAATACCGCAT, TACGGGAGGCAGC
AGT, GTGGGGGAGCAA, AGGATTTAG
ATACCCT, CCGTAAACGAT, 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
The identified DNA chlorine sequences of the gene are provided.

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 present in a prokaryote-derived nucleic acid and not in a eukaryote-derived nucleic acid. The method may include a medium which is derived from this prokaryote and which may contain a nucleic acid or nucleic acid fragment containing this specific nucleotide base sequence, and an oligo containing a nucleotide base sequence complementary to this specific nucleotide base sequence. Contacting with a nucleotide whereby the oligonucleotide is hybridized with a nucleic acid or nucleic acid fragment from the prokaryote that contains this particular nucleotide base sequence that may be present in the medium, and is hybridized with the oligonucleotide. A method performed by detecting the presence of a nucleic acid or nucleic acid fragment For free.

In another aspect, the present invention relates generally to specific biological probes used in the detection of mycoplasma or prokaryotes, which rely on the above methods and the identification and production of such probes. .

[0014]

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 by using an oligonucleotide probe as a DNA for a genome.
DNA-DNA or RNA-D used for nucleotide sequences
The sensitivity of detection is increased several hundred times over that of 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.

Specificity for mycoplasma is described in the literature (M. Cunningham , Anal. Bioche) , which is hereby incorporated by reference.
m. 128: 415-421 (1983)) by means of slot blot hybridization. For this purpose a 1600 bp DNA fragment of Mycoplasma pneumoniae cloned in pUC8 was used. The 1600 bp section contains the 16 base deoxyoligonucleotide described above. A typical prokaryotic eubacter
E. coli genomic digest is the enzyme Hind I.
Produced by digestion with II. Escherichia coli D digested
The NA and 1600 bp Mycoplasma pneumoniae DNA fragments are incorporated by reference (JJLeary et a
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 resulting slot blots were developed as shown in the figure to 0.00034ng, 0.0034ng, 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 for prokaryotes
The deoxyoligonucleotide having the base sequence of CTCA is effective as a prokaryotic-specific oligonucleotide probe that hybridizes with a prokaryote but does not hybridize with a eukaryote.

The following method has been found to be effective for detecting mycoplasma in infected cells. Cells were used in 1-2T75 tissue culture flasks with trypsin EDTA (0.05% trypsin in PBS, 0.04
% EDTA) and trypsinized for 2 minutes at 37 ° C. Trypsinized cells were resuspended in 1-2 ml of growth medium and a Minifold II Slot Blot Hybridizer (Schleich).
er and Schuell, inc., Keene, NH) 10 × S
SC (1 x SSC is 15 mM Na citrate, 150 m
An amount of 50-100 μl (1 × 10 ⁵ -1 × 10 ⁶ cells) was spotted onto a nitrocellulose membrane moistened with M NaCl, pH 7.4). The DNA sample applied to the slot blot was alkaline (0.5M NaOH,
1.5M NaCI) at room temperature for 5-10 minutes, 0.5M Tris, pH 7.2 and 3.0M Na.
Neutralized with Cl for 5-10 minutes at room temperature. The filters were then washed at room temperature in 2 × SSC for 5 minutes and heated in a vacuum oven at 80 ° C. for 2 hours. The filter is 0.5 mM EDTA, 5 mM Tris, pH 7.
5, 5 x Denhardt and 100 μg / ml
Was prehybridized for 2 hours at 65 ° C. using a prehybridization buffer consisting of heat-denatured denatured sperm DNA. 10 mM Tris, pH 7.5, 1
mM EDTA, 0.75M NaCl, 1 × Denher's, 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 or more, the hybridization was 1 ° C at 65 ° C.
It was held for 6 hours.

After hybridization, the filter was 2 × SET, 0.2% SDS (1 × SET was 30 m).
M Tris, ph 8.0, 150 mM NaCl) at 65 ° C. for 2-4 hours and 4 mM Tris base at room temperature for 1-2 hours. The filter is then dried and one or two Dupont Cronex
nex) Exposed on X-ray membrane 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,
Describes the testing of techniques used to determine specific nucleotide base sequences.

Such a determination is made in the next step.

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 base sequence different from the corresponding base sequence of a prokaryote that is common to all species of Mycoplasma and is not mycoplasma is effective as an oligonucleotide probe specific to Mycoplasma, and all species of Mycoplasma and prokaryotes that are not Mycoplasma. 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.

Cloning of the ribosomal RNA genome of Mycoplasma pneumoniae was performed using the total DNA of Mycoplasma pneumoniae.
HindIII digestion of HindIII and H of this HindIII fragment
It was achieved by ligation to a vector pUC8 digested with ind III. The resulting ribosomal RNA gene fragment is incorporated by reference (Gobel et al., Scienc) .
e, 226: 1211-1213 (1984)), pKK353
Tested with the E. coli ribosomal RNA operon in 5 plasmids to identify the cloned fragment containing the ribosomal base sequence.

Hybridize to the species of Mycoplasma under stringent hybridization conditions,
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.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Wolf Gobel             Federal Republic of Germany Day-7800 Fribe             Luc Hermann Helder Strasse             11 F-term (reference) 4B024 AA13 CA01 CA11 HA14                 4B063 QA01 QA18 QA19 QQ06 QQ15                       QQ42 QR32 QR35 QR55 QS34                       QX01

Claims (14)

[Claims] 1. A method for determining the presence of a prokaryotic organism in a medium, which hybridizes to a non-target nucleic acid sequence in said medium but to a target nucleic acid sequence derived from said prokaryotic organism. Contacting the medium containing the target nucleic acid sequence with the synthesized and separated oligonucleotide under such preselected hybridization conditions, and under the preselected hybridization conditions,
Detecting the presence of the target nucleic acid sequence. 2. The method of claim 1, wherein the preselected hybridization conditions are stringent. 3. The method of claim 1, wherein the target nucleic acid sequence is a rRNA sequence. 4. The method according to claim 1, wherein the target nucleic acid sequence is a DNA sequence. 5. A method for determining the presence of a prokaryote comprising a particular nucleotide sequence, which hybridizes to a non-target nucleic acid sequence but to a target nucleic acid sequence derived from said prokaryote. Contacting the target nucleic acid sequence with an oligonucleotide under different preselected hybridization conditions, and under the preselected hybridization conditions,
Detecting the presence of a nucleic acid sequence with which the oligonucleotide hybridizes. 6. The method of claim 5, wherein the preselected hybridization conditions are stringent. 7. A composition comprising a synthesized and isolated oligonucleotide that is substantially complementary to a preselected prokaryotic target nucleic acid sequence, but not substantially complementary to a non-target nucleic acid sequence. 8. The composition of claim 7, wherein the oligonucleotide sequence is at least 9 nucleotides. 9. The composition of claim 7, wherein the target nucleic acid sequence is a rRNA sequence. 10. The composition according to claim 7, wherein the target nucleic acid sequence is a DNA sequence. 11. A synthesized and isolated oligo that hybridizes under preselected hybridization conditions to a preselected prokaryotic nucleic acid sequence but not to a nonselected nucleic acid sequence. A composition comprising a nucleotide. 12. The composition of claim 11, wherein the oligonucleotide sequence is at least 9 nucleotides. 13. The composition of claim 11, wherein the preselected hybridization conditions are stringent. 14. A target nucleic acid sequence of at least 9 nucleotides, which under stringent hybridization conditions hybridizes to a preselected prokaryotic target nucleic acid sequence but not to a non-target nucleic acid sequence. A composition comprising synthetic and isolated oligonucleotides that are substantially complementary to a nucleic acid sequence.