JPH0630795A - Nucleic acid probe for detecting mycoplasma pneumoniae - Google Patents

Abstract

PURPOSE: To obtain a nucleic acid sequence that can detect the causative factor of human primary atypical pneumonia.

CONSTITUTION: This probe is a nucleic acid as a composition that has about 10-250 nucleotides and can hybridize with the rRNA or rDNA of pathogenic Mycoplasma microorganisms associated with human primary atypical pneumonia with priority to the rRNA or rDNA of non-Mycoplasma bacteria and of human. In addition, this nucleic acid can hybridize with 16S rRNA or 16S rDNA area of Mycoplasma pneumoniae selected from the group consisting of the 1,110-1,160 area and the 1,220-1,270 area.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION This invention is Mycoplasma pneumoniae is the causative agent of human primary atypical pneumonia (Myco
The present invention relates to detection of plasma pneumoniae ). In particular, the present invention relates to Mycoplasma pneumoniae in clinical and other samples.
nucleic acid compositions and methods for use in the specific detection of Pneumoniae ). The nucleic acid composition features nucleic acid sequences of 10 to 250 nucleotides that can hybridize preferentially to Mycoplasma pneumoniae rRNA and rDNA.

[0002]

BACKGROUND OF THE INVENTION Mycoplasma are bacteria that do not have microscopic cell walls and were originally isolated from animal sources including humans. There are several related genera characterized as Mycoplasma (Mycoplasma) and bacteria that do not have a micro cell walls of over 70: they Spiroplasma sp (Spiro
plasma ), the genus Accoleplasma ( Acolepla)
sma ), Ureaplasma ( Ureaplasm
a), Aneropurazuma genus (Anaeroplasma)
And the genus Asteroleplasma
sma ). A handful of species within these genera have been found associated with humans--some thought to be "normal indigenous flora" and others suspected to be pathogenic. , And only one species (whenever it is isolated) is known to be an important factor in human pathological conditions. Mycoplasma pneumonia ( My
coplasma pneumoniae ) is an important factor in primary atypical pneumonia and some non-respiratory complications. Its presence always provides clinically relevant information.

Other pathogenic Mycoplasma organisms include Mycoplasma fermentans ( Mycoplasma
fermentans), Mycoplasma hominis (Mycoplasma hominis), Ureaplasma Urearichikumu (Ureaplasma ur
earyticum ) and Mycoplasma genitalium ( Mycoplasma genitalium )
Is included. Nucleic acid compositions and methods for the detection of these organisms are described as "Nucleic acid probes for the detection of genital mycoplasmas" and "Nucleic acid probes for the detection of Mycoplasma fermentans or AIDS-related viral-like infectious agents". Two simultaneously filed U entitled
SSN07 / 673,661 and USSN07 / 67
It is the subject of 3,687. At least one inventor is common to both of these applications and this application.

Mycoplasma pneumoniae ( Myco
Mycoplasma, such as plasma pneumoniae ), is a mess of culture conditions and requires a complex culture medium containing peptone, enzyme extracts, expensive animal serum and sterols. It grows relatively slowly and reaches low cell densities compared to most bacteria. Moreover, atmospheric conditions for cell growth require the addition of carbon dioxide. For these reasons, in many clinical laboratory Mycoplasma pneumoniae (M. Pneumo
niae ) could not be carried out, and as a result the true diagnosis of the presence of this important pathogenic bacterium remains undiagnosed. Since mycoplasma does not have a bacterial wall, antibiotics that target the bacterial cell wall, such as penicillin, do not have antimycoplasmal activity. Therefore, it is important for clinicians to diagnose atypical pneumonia and prescribe the appropriate antibiotic.

[0005] Several researchers have found serological (Lind,
et al. J. Clinical Microbi
ol. , 20, 1984) and within the DNA sequence containing the rRNA operon (Yogev and Razin, I.
nt. Jnl. System. Bacteriol. ，
36, 1986) Mycoplasma pneumoniae ( M
ycoplasma pneumoniae) and Mycoplasma genitalium (Mycoplasma ge
nitalium ).
Weisburg, et al. Discusses various other evolutionarily related species of Mycoplasma pneumoniae ( M. pneumoniae ) (Jnl. Of B.
actiol. , 171, 1989).

Mycoplasma genitalium ( M. ge
nitalium) is Mycoplasma pneumoniae (M. in co-culture with pneumoniae) or Mycoplasma pneumoniae, (M. pneumo
niae ) appears to play some role in respiratory infections even in the absence (Tully, Clinica)
l Micro. Newsletter, Volume 11, 19
89). Mycoplasma genitalium ( Mycopl
asa genitalium ) may account for some proportion of clinical atypical pneumonia.

Kohne et al. (Biophy
social Journal 8: 1104-111
8, 1968) discusses one method of producing a probe for an rRNA sequence, Mycoplasma pneumoniae ,
Or Mycoplasma genitalium ( M. genit
Mycoplasma pneumoniae with alium) (M. pneumoni a e ) it does not provide the teaching necessary to make a probe to detect.

Pace and Campbell (Jou
rnal of Bacteriology 107:
543-547, 1971) discusses the homology of ribosomal ribonucleic acids from various bacterial species and hybridization methods for the quantification of such homology levels. Similarly, Sogin, Sogin and W
oese (Journal of Molecular
Evolution 1: 173-184,1972)
Discusses theoretical and practical aspects for the evaluation of phylogenetic relationships using primary structural characterization of different ribosomal RNA molecules. Fox, Pechman and Woese (International Jou
rnal of Systematic Bacter
iology 27: 44-57, 1977) is a 16S ribosomal RN as a clue for prokaryotic systematics.
Discussing A's comparison catalog.

Hogan et al. (International Patent Application, Publication No. WO88 / 03957) is Mycoplasma pneumoniae, which is estimated five (M. Pneumon
IAE) has been described for specific probe, Mycoplasma genitalium by strict standards (M. ge
nitalium) it has not been tested in a manner to enable the evaluation of cross-reactivity. The mixture of the four probes is related to Mycoplasma gallisepticum ( M. galli
10 times greater than septicum) strongly Mycoplasma
Reacted with genitalium ( M. genitalium ). None of those probes target the 23S rRNA molecule. The disclosed probe seems incapable of distinguishing between Mycoplasma genitalium ( M. genitalium ) and known homologous sequences.

Zivin and Monahan (European Patent Application Publication No. 0305145A2) describe several probes designed based on the determination of the 307 bases at the 5'end of Mycoplasma pneumoniae ( M. pneumoniae ). They Mycoplasma genitalium with some of the probe (M. Genita
It is claimed anecdotally patents to exclude lium).

Gobel, et al. , Discusses probes for mycoplasma, including Mycoplasma pneumoniae ( M. pneumoniae ) (Gobel, et al., Israel Jn.
l. of Med. Sci. 23, 1987), remains unclear as to the structure of these probes. In addition, European patent application number EP02
50662 A1, in Gobel, et al. Is Mycoplasma pneumoni ( M. pneumoni
ae ) suggesting probes: However, such probes lack sensitivity or specificity for clinical applications and solution hybridization.
It is also not suitable for formatting.

Rogers, et al. Discusses the sequence of mycoplasma 5S rRNA (Ro
gers, et al. , Proc. Natl. Aca
d. Sci. USA, 82, 1985). Roger
s, et al. Does not suggest sequences that may be useful in facilitating detection of these organisms.
There is no discussion of S or 23S rRNA sequences. Woese, et al. Discusses a catalog of 16S rRNA oligonucleotides for selected Mycoplasma species.
Pneumonia ( Mycoplasma pneumon
iae ) is not discussed.

Bernet, et al. Is Mycoplasma pneumeu ( Mycoplasma pneu
moniee ) polymerase chain reaction detection, but not related to rRNA or rDNA sequences (Bernet, et al., Jn.
l. Clin. Micro. , 27, 1989). H
yman, et al. Momoplasma pneumonia
e ) and Mycoplasma genitalium ( Mycop
discusses the polymerase chain reaction detection of lasma genitalium) but, rRNA or r
We have not discussed the utility of DNA sequences (Hyma
n, et al. , Jnl. Clin. Micro. ，
25, 1987).

Gobel, et al. Discusses mycoplasma rRNA probes in a published paper (Gobel, et al., Jnl.G).
enl. Micro. , 133, 1987). However, Gobel, et al. Are other Mycoplasma species, especially Mycoplasma genitalium ( Mycopl
It does not recognize the significance of distinguishing the probe for Asma genitalium ).

Ribosomes are of great importance in all organisms because they serve as the only known means of translating genetic information into cellular proteins (the main structural and catalytic elements of life). A clear indication of this importance is that all cells have ribosomes.

Bacterial ribosomes are 5S, 16S and 2
Called 3S rRNA (at least in E. coli)
It contains three distinct RNA molecules. In eukaryotes, there are four distinct rRNA species commonly referred to as 5S, 18S, 28S and 5.8S. These names were historically R as determined by their sedimentation rate.
It is related to the size of the NA molecule. However, in reality, the size of ribosomal RNA molecules varies substantially between organisms. Nevertheless, 5S, 16S
And 23S rRNA are commonly used as common names for homologous RNA molecules in any bacterium, including mycoplasmas, and this tradition is inherited herein.

As used herein, a probe is designed or selected to include a specific nucleic acid sequence that allows it to preferentially hybridize to a target nucleic acid sequence under hybridization conditions. , Nucleic acids produced synthetically or by organisms (DNA or RN
A) is shown. The term "predominantly" is used in a relative sense; one hybridization reaction product is more stable than another under the same conditions.

In addition to its hybridization characteristics, the probe may also contain certain constituents involved in the proper or optimal functionalization under the particular assay conditions. For example, to improve resistance to nuclease degradation (eg, by endcapping), to carry detection ligands (eg, fluorescein, biotin and avidin), to facilitate direct or indirect detection ( ³² P, And fluorescent and chemiluminescent substances), or probes may be modified to facilitate capture onto a solid support (eg homopolymer “tail”). Such modifications are elaborated on the basis of their basic probe function, which is the ability to usefully distinguish between target and non-target organisms during hybridization.

Hybridization is traditionally 2
Two partially or completely complementary strands of nucleic acids are joined together in an antiparallel fashion (one oriented 5'to 3'and the other 3'to 5 '), and the nucleobases are It is understood to be the process of forming double-stranded nucleic acids by specific and stable hydrogen bonding according to the unequivocal rules related to pairing with each other. The high specificity of the probe relies on the low statistical probability of a randomly generated unique sequence dictated by the product by a multiplicative increase in individual probabilities. About 10 to 25
Normal hybridization conditions for a 0 nucleotide nucleic acid would be at a temperature of about 60 ° C. in the presence of 1.08 M sodium chloride, 60 mM sodium phosphate and 6 mM ethylenediaminetetraacetic acid (pH 7.4).

Hybridization conditions are readily modified to suit nucleic acids of different sequence. Factors that will influence the hybridization conditions for a particular nucleic acid composition are the base composition of the probe / target duplex and the level of mispairing and binding structure between the two nucleic acids.

The reaction parameters usually adjusted are the concentration and type of ionic species present in the hybridization solution,
The concentration of denaturant present and the temperature of hybridization. Generally, the more stringent the hybridization conditions (or the less likely it is that hybridization will occur), the longer the probe will be required to form stable hybrids.

[0022]

SUMMARY OF THE INVENTION The invention features nucleic acid compositions and sets of compositions and methods for the specific detection or identification of pathogenic mycoplasma bacteria associated with human primary atypical pneumonia. One embodiment of the present invention provides, as a composition, a non-mycoplasma bacterium and a human rRN.
RRNA or r of a pathogenic mycoplasma bacterium associated with human primary atypical pneumonia than to A or rDNA
It features nucleic acids with about 10 to 250 nucleotides that can preferentially hybridize to DNA.
The nucleic acid composition is Mycoplasma pneumoniae ( Myco
plasma pneumoniae ) and Mycoplasma genitalium ( Mycoplasma gen)
Italium ).

The embodiments of the present invention is Mycoplasma pneumoniae (Mycoplasm a pneumonia
e ) 16S rRNA or 16S rDNA and Mycoplasma pneumoniae ( Mycoplasma p
neumoniae ) 23SrRNA or 23Sr
It features a nucleic acid capable of hybridizing to DNA.
One embodiment of the present invention is a Mycoplasma pneumoniae .
And Mycoplasma genitalium ( Mycopla
16SrRN of both sma genitalium )
A or 16S rDNA or 23S rRNA or 2
It features a nucleic acid that can hybridize to 3S rDNA. The nucleic acid composition is Mycoplasma pneumoniae ( M
ycoplasma pneumoniae ) 16Sr
1110 to 1160 or 1220 to 12 of RNA
70th place, Mycoplasma pneumoniae ( Mycopl
asma pneumoniae ) 23S rRNA 1
50 to 200, 260 to 340, 1590 to 16
30, 2080 to 2190 or 2600 to 266
0th place, Mycoplasma pneumoniae ( Mycopla
sma pneumoniae) and Mycoplasma genitalium (Mycoplasmagenital
ium ) 16S rRNA 50 to 100, 140 to 230, 440 to 500, 620 to 670, 810
To 860, 980 to 1030 or 1210 to 1
270th place, and Mycoplasma pneumoniae ( My
coplasma pneumoniae ) and Mycoplasma genitalium ( Mycoplasma g)
enitalium ) 330-37 of 23S rRNA
RRNA or r selected from the group of regions consisting of position 0
It is complementary or homologous to the DNA region. All such designations are nucleotide positions, counted from the 5'end of the RNA molecule, as is well known to those skilled in the art.

Mycoplasma pneumoniae ( Myco
plasma pneumoniae ) and Mycoplasma genitalium ( Mycoplasma gen)
italium ) to both rRNA or rDNA. These nucleic acid compositions of the present invention include
One of the two probes is useful in a two-probe system that can distinguish between the two organisms.
Therefore, one embodiment of the present invention includes an article of manufacture. The article of manufacture contains a nucleic acid set consisting of at least two nucleic acids. Each nucleic acid has 10 to 250 nucleotides, and each nucleic acid has a different base sequence composition. At least one nucleic acid in the set is 2295,228.
9, 2296, 2298, 2299, 2300, 229
4,2167,2196,2297,2192,221
9, 2162, 2202, 2201, 1166, 222
Any 10 selected from the group of sequences defined by probes consisting of 4,2195, 2225 and 2230.
Is complementary or homologous to at least 90% of the sequence containing contiguous nucleotides of.

Suitably, at least two nucleic acid compositions are at least 90% complementary or homologous to a sequence comprising 10 contiguous nucleotides selected from the group defined by the probes, probe 2196 and Probe 2167; probe 2162 and probe 2202; probe 2224 and probe 2166; probe 2162 and probe 2230; probe 2167 and probe 2219; probe 2192 and probe 2202; probe 2296 and probe 2297; and probe 2299 and probe 2300 To be selected.

A further embodiment of the present invention comprises the
One or more mycoplasma (essentially Mycoplasma pn
It features a method of detecting the presence of Eumoniae) and consisting of Mycoplasma genitalium (Mycoplasmagenitalium)]. The method involves contacting the sample with at least one nucleic acid having about 10 to 250 nucleotides that can preferentially hybridize to mycoplasma rRNA and rDNA. The method includes forced hybridization conditions to the sample, which conditions allow the preferential binding of nucleic acids to mycoplasma rRNA and rDNA, forming nucleic acid complexes. The nucleic acid complex is detected as an indicator of mycoplasma.

Preferably, the nucleic acid in the contacting step is the probe 22.
95, 2289, 2296, 2298, 2299, 23
00, 2294, 2167, 2196, 2297, 21
92, 2219, 2162, 2202, 2201, 21
At least 90% is complementary or homologous to a sequence containing any 10 contiguous nucleotides selected from the group of sequences defined by 66, 2224, 2195, 2225 and 2230.

One embodiment of the present invention features a contacting step that utilizes two nucleic acid compositions. Each nucleic acid has a base sequence of 10 to 250 and can preferentially hybridize to mycoplasma rRNA and rDNA, and each has a different base composition. Nucleic acid is at least 90 with a sequence containing any 10 contiguous nucleotides selected from the group of sequences defined by the probe.
% Are complementary or homologous: probe 2196 + probe 2167, probe 216
2+ probe 2202 probe 2224+ probe 2166, probe 216
2+ probe 2230 probe 2167+ probe 2219, probe 219
2+ probe 2202 probe 2296+ probe 2297, probe 229
9+ probe 2300 selected from the group of sets.

Another embodiment of the invention features a kit for detecting pathogenic mycoplasma bacteria associated with human primary atypical pneumonia. The kit has a nucleic acid composition with a base sequence of 10 to 250 that can preferentially hybridize to non-mycoplasma and rRNA or rDNA of pathogenic Mycoplasma bacteria over human rRNA and rDNA.

Typically, the kit will include reagents, compositions, instructional materials, disposable hardware and packaging suitable for allowing convenient assembly into the market.

The nucleic acid compositions, kits and methods of the present invention provide the basis for the development of effective nucleic acid hybridization assays for the specific detection of atypical pneumonia or its causative agents. The types of samples that will resist it include sputum, throat swabs, blood, urine, spinal fluid, skin, biopsy samples, saliva, synovial fluid, bronchial lavage fluid, bronchial lavage fluid, or human patients or Liquid samples from affected livestock are included.

Mycoplasma pneumoniae ( M. pneumonia ) does not necessarily cause cross-reaction between species.
e ) and Mycoplasma genitalium ( M. gen)
With regard to the detection of a panel of italium ) isolates, the discovery that the probe was able to generate with the surprisingly comprehensive and exclusive properties of the present invention was unexpected and not expected.

A further understanding of the principles and aspects of the present invention may be gained by reference to the tables and figures set forth below: Tables 1 and 2 describe the structure of the target region and The physical structure is shown and Table 3 shows the hybridization behavior of the probe to a panel of representative mycoplasma species in clinical and environmental settings. Also, Figure 1 is a schematic representation of a dual probe capture / detection assay.

DETAILED DESCRIPTION OF THE INVENTION AND BEST MODE PROBE DEVELOPMENT STRATEGY The first step taken in the development of the nucleic acid compositions of the invention is that of 16S rRNA that can serve as a target site for a specific probe with the desired sensitivity. Identification of the area.
As mentioned previously, to obtain this desired sensitivity, the Mycoplasma pneum
umoniae ), a unique probe target, and Mycoplasma pneumoni ( M. pneumoni)
ae ) and Mycoplasma genitalium ( M. ge)
The probe target that is common to both N. This is (1) Mycoplasma pneumoniae ( M. pneum
oniae ) and Mycoplasma genitalium ( M. g .
are different Tsu between Enitalium), and (2) is substantially the same sequences in the two species, finding a site are different in the next closest species sequence on the evolution Inevitably accompanied.

For this analysis, the exact sequence of the Mycoplasma 16S and 23S rRNA sequences was investigated. Mycoplasma pneumonia ( M. pneumonia
e ) and Mycoplasma genitalium ( M. gen)
italium ) both essentially complete 16S rRNA
And 23 rRNA sequences were determined as part of this study. Such nucleotide sequences were determined by standard experimental protocols familiar to those skilled in probe design. The 16S and 23S rDNA were cloned into a plasmid vector from the product produced by enzymatic amplification. Mycoplasma pneumonia ( My
coplasma pneumoniae) and Mycoplasma genitalium (M. genitaliu
m ) sequences were compared side by side with homologous sequences of other mycoplasma and non-mycoplasma ribosomal RNA sequences. Mycoplasma pneumonia ( M. pneumo
niae ) and Mycoplasma genitalium ( M. ge)
clinical and evolutionary relationship close of nitalium) In addition to specially research, the following 16SrRNA sequences were evaluated can be distinguished:.. Mycoplasma agalactiae (M agalactiae), Mycoplasma arginine (M arginini), Mycoplasma Ars utility disk (M. arthritidis), mycoplasma ball Vie genitalium (M. bovigen
italium), Mycoplasma Karihorunikumu (M. calfornicum), Mycoplasma capricolum (M.capricolum), Mycoplasma Erikunie (M. ellychniae), Mycoplasma Meraryukumu (M. melaleucu
m ), Mycoplasma fermentans ( M. fer
mentans ), Mycoplasma gallisepticum ( M. gallisepticum ), Mycoplasma hominis ( M. hominis ), Mycoplasma
M. hyopneumouia
e ), Mycoplasma hyorinis ( M. hyorhi
nis ), Mycoplasma iowae ( M. iowa
e ), Mycoplasma lipofilm ( M. lipop
hilum ), Mycoplasma mobile ( M. mobi
le ), Mycoplasma murris ( M. muris ),
Mycoplasma mycoides ( M. mycoide
s), Mycoplasma neuro utility Kum (M. ne
urolyticum ), Mycoplasma olere ( M. orale ), Mycoplasma pilum ( M. p .
irum ), Mycoplasma pneumoniae ( M. pn
eumoniae), Mycoplasma pulmonis (M. pulmonis), Mycoplasma script referencing tumefaciens (M. putrefaciens), Mycoplasma Saribariumu (M. salivariu
m), Mycoplasma Suaruvi (M. sualv
i ) 10 Spiroplasms
a ) Sequence of species, one Asteroleplasma ( Aster)
aleplasma array and Ureaplasma urealytict ( Ureaplasmaurealyt)
icum ) 16S rRNA sequence. (GenBank accession number is Weisburg et al., Jnl.o.
f Bacterology, Volume 171 1989,
Shown).

20 Designed, Synthesized and Tested
The probes are listed in Tables 1 and 2.

[0037]

[Table 1]

[Table 2] Nine probes specifically differentiate Mycoplasma pneumoniae ( M. pneumoniae ) and Mycoplasma genitalium ( M. genitalium ), and 11 probes react specifically with these two species. Probes that hybridize to both these species have at least two advantages: (1) As explained previously, Mycoplasma genitalium ( M. genitalium )
Pneumoniae (M. Pneumoniae) by would cause significant morbidity indistinguishable from pathological conditions caused, and (2) only only one probe is required for the desired specificity, dual probe assays Probe can be used.

Description of Probes The probe selection strategy is useful for hybridization to the causative agent of primary atypical pneumonia in a sample.
0 probes were obtained.

Table 1 shows Mycoplasma pneumoniae ( M. pneumoniae ) and Mycoplasma pneumoniae .
Genitalium ( Mycoplasma genetic)
ium ) shows the exact sequence of the probe region for both, as well as the probe sequence designed from the aligned targets. Table 2 summarizes the particularly suitable nucleic acid compositions along with their length and G + C content.

The nucleic acid composition of the present invention should not be construed as limited to the specific nucleotides identified by the named probe. Optimal probe length is related to the stringency of the chosen hybridization conditions. The sequences identified by the named probes can be altered and modified somewhat. In fact, one of skill in the art will readily adjust the probe length and change the nucleotides to suit the specific assay and detection required. For example, the length of these particular oligonucleotides was optimized for use in dot blot assays and sandwich formats. When considering a set containing more than one probe, it is desirable that all probes behave in a consistent manner. Therefore, the exact length of a particular probe will, to some extent, reflect its particular intended use.

The nucleic acid composition of the present invention comprises a sequence which can be used as an oligonucleotide probe or incorporated into a larger polynucleotide of ribonucleic acid or deoxyribonucleic acid. Sequences complementary to the probes described here can be used as probes for the rRNA gene. Suitable probes or their complements can be used in sequencing or other applications as chain extension initiators for the polymerase chain reaction.

[0042]

【Example】

Example 1. Dot-Blot Analysis of Probe Hybridization Behavior For dot-blot analysis, nucleic acids or populations of nucleic acids are readily available as commercial products, such as nitrocellulose, nylon or other derivatized membranes, and specially designed for this purpose. Includes immobilization on the filter. Either DNA or RNA is readily immobilized on such filters, and hybridization under various conditions (ie, stringency) can then be demonstrated or tested for hybridization with the nucleic acid composition or probe of interest. Nucleic acids with sequences that have greater complementarity to the target will form hybridization reaction products, which are more stable than probes that contain lesser complementarity.
In fact, this added stability can be exploited to allow nucleic acids with sequences complementary to the target to bind preferentially to the target without substantial hybridization to non-target nucleic acids. .

The probe of the present invention is a dot-blot.
Tested in format. 100 nanograms of RNA purified by phenol extraction and centrifugation with a cesium trifluoroacetate gradient were denatured and spotted on nylon membranes. ³² P to the 5'end of the oligonucleotide
The probe is labeled with an isotope by linking the moieties. 1.08 M sodium chloride, 60 mM sodium phosphate and 6 mM ethylenediaminetetraacetic acid, p
Hybridization of the probe occurred at a temperature of 60 ° C. in the presence of H7.4. Unhybridized probe was removed by washing with a salt concentration of one third of the hybridization conditions. The filters were exposed to X-ray film and the intensity of the hybridization signal was evaluated 3 hours after exposure.

The experimental specificity of the preferred probe described in Example 1 is summarized in Table 2 above and Table 3 below.

[0045]

[Table 3] Mycoplasma pneumoniae ( Mycoplasma
Typical strains and Mycoplasma genitalium of 10 of pneumoniae) (M. genitaliu
m ) three strains are National Institute
e of Allergy and Infectio
us Disease's Mycoplasma S
This shows selection from section. Additional bacterial species and some fungal species were added to the panel to show the breadth of known bacterial taxa. Mycoplasma gallisep
ticum) is Mycoplasma pneumoniae (M. p
neumoniae ) / Mycoplasma genitalium ( M. genitalium ) cluster is clinically important as a representative closely related species.

All species on the panel are represented as 100 ng of purified, denatured RNA. The probe was synthesized (by conventional methods and chemistry well known to those skilled in the art), labeled with ³² P, hybridized to that of the panel under standard conditions (at the indicated temperature) and evaluated by autoradiography. did. "++++" represents the strongest hybridization signal after 3 hours exposure, the slightly weaker signal is represented by "++++", becoming "++" then "+" as it becomes weaker. "+
The "-" is virtually absent and the "-" does not indicate probe hybridization to the target. The specificity is empirical and some Mycoplasma pneumoniae ( M. pneumoniae ) + Mycoplasma genitalium ( M The S. genitalium probe showed a slight deviation from the expected specific behavior.

Example 2. Dual Probe Hybridization In practical practice, many applications of these probes will use a "sandwich" hybridization scheme in which a set of probes is used simultaneously. Turning now to FIG. 1, a sandwich assay featuring capture probe 12 and detection probe 13 is depicted. The capture probe 12 would ideally be a bifunctional nucleic acid made by adding a homopolymer 3'tail to a probe with high target specificity. The tail is
It will then hybridize to the complementary homopolymer 11 on the surface of the solid support 10 such as glass beads or filter disks. Hybridization of the capture probe 12 to its target 15 (in this case mycoplasma rRNA) will cause the target 15 to complex with the solid support 10. The detection probe 13 (conveniently with some specificity) is part of a detection scheme that depends on radioactivity, fluorescence, chemiluminescence, color and other detection means, and the detection portion 14 is a complete hybridization. It will indicate the presence of the complex. The detection probe is Kra
mer and Lizardi (Nature, 339)
Vol., 1989), and can be incorporated into mutants in Q-beta that can potentially be amplified as RNA sequences.

Example 3. Clinical diagnosis of Mycoplasma pneumoniae primary atypical pneumonia Clinical samples such as swab, sputum or tissue
Treated to release all the nucleic acids it contains. Samples containing destroyed mycoplasma are capture probes,
Incubate in chaotropic buffer such as guanidinium isothiocyanate in the presence of detection probe and magnetic particle beads (derivatized with oligo-thymidine as in Example 2).

If Mycoplasma pneumoniae ( M
ycoplasma pneumoniae) or Mycoplasma genitalium (M. genitaliu
m ) If a target molecule such as 16S or 23S rRNA (depending on which probe set is used) is present, a bead + capture probe + target + detector probe hybridization complex is formed as in FIG. It
The presence of a magnet near the bottom of the reaction tube will cause the magnetic particles + hybridization complex to adhere to the side of the tube, allowing removal of the sample matrix, unbound probe, etc. Repeating, rehydrating and denaturing the bead + probe + target complex could significantly reduce background (USSN922,
155, Collins, 1986). In this example, final detection can involve spotting beads on the membrane and assaying by autoradiography. Alternatively, the detection probe may be an amplifiable medium mutant probe.

The following capture and detection probes are an example of a suitable set for this particular assay: probe 2196 + probe 2167, probe 216.
2+ probe 2202 probe 2224+ probe 2166, probe 216
2+ probe 2230 probe 2167+ probe 2219, probe 219
2+ probe 2202 probe 2296+ probe 2297, probe 229
9+ probe 2300 Example 4. Samples are processed to obtain clinical diagnosis DNA of Mycoplasma pneumoniae ( M. pneumoniae ) primary atypical pneumonia from human samples using polymerase chain reaction amplification of Mycoplasma rDNA. One of the probes described herein is linked to the antiparallel complement of one of the probes described herein to encode Mycoplasma rRNA in the polymerase chain reaction Mycoplasma pneumoniae ( Mycoplasma)
Pneumoniae ) or Mycoplasma genitalium ( M. genitalium ) gene used for enzymatic amplification. The resulting material can be assayed with any of the probes herein in a "sandwich" hybridization assay. The polymerase chain reaction can itself be made highly specific by using the probes / primers described herein,
Alternatively, the reaction can be performed more generally with less specific probes, and the amplification products identified as Mycoplasma pneumoniae ( M. pneumoniae ) with the probes described here with great specificity. can do.

Example 5. In situ Hybridization as Cytological Stain The probe of the present invention can be used as a cytological staining reagent. For example, a sputum sample is smeared on a microscope slide. After proper fixation and lysis, probe hybridization is performed at that position (in situ).
In this example, Mycoplasma pneumoniae ( M
ycoplasma pneumoniae ) can be visualized in the specimen with a fluorescently labeled probe 2167, and the slides examined using a fluorescence microscope looking for small fluorescent bodies.

Example 6. Mycoplasma pneumoniae with culture ( Mycoplasma pneumonia)
e ) Confirmation Mycoplasma pneumoniae ( Mycoplasma
pneumoniae ) or Mycoplasma genitalium ( M. genitalium ) after standard culture steps (eg, on H-agar plates or SP-6 broth), using Mycoplasma
The presence of Pneumoniae ( M. pneumoniae ) is tested.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing a dual probe capture / detection assay.

[Explanation of symbols]

 10 Solid support 11 Complementary homopolymer 12 Capture probe 13 Detection probe 14 Detection moiety 15 Target

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI technical display area C12R 1:35)

Claims (12)

[Claims] 1. Non-mycoplasma bacteria and human rR
A nucleic acid as a composition having about 10 to 250 nucleotides that can preferentially hybridize to rRNA or rDNA of a pathogenic mycoplasma bacterium associated with human primary atypical pneumonia over NA or rDNA. 2. The nucleic acid comprises 1110 to 1160,122.
Mycoplasma pn selected from the group of positions 0 to 1270
eumoniae ) 16S rRNA or 16SrD
The nucleic acid according to claim 1, which is capable of hybridizing to the NA region. 3. The nucleic acid comprises 150 to 200, 260 to 340, 1590 to 1630, 2080 to 219.
Mycoplasma pneumoniae selected from the group of positions consisting of 0,2600 to 2660.
capable of hybridizing to the 23SrRNA or 23SrDNA region of ma pneumoniae), according to claim 1 nucleic acid. 4. The nucleic acid comprises 50 to 100, 140 to 2
30, 440 to 500, 620 to 670, 810 to 860, 980 to 1030, 1210 to 1270
Mycoplasma pneumonia selected from the group consisting of
e ) and Mycoplasma genitalium ( Mycop
16Sr of both lasma genitalium)
The nucleic acid according to claim 1, which is capable of hybridizing to a region of RNA or 16S rDNA. 5. The nucleic acid comprises 330 to 370 16.
Mycoplasma pnee selected from the group of SrRNA and rDNA
2. The nucleic acid according to claim 1, which is capable of hybridizing to the region of 23S rRNA or 23S rDNA of both Mycoplasma genitalium and Mycoplasma genitalium . 6. The nucleic acid is 2295, 2289, 229.
6,2298,2299,2300,2294,216
7, 2196, 2297, 2192, 2219, 216
2,2202,2201,2166,2224,219
A complementary or homologous to at least 90% of a sequence containing any 10 contiguous nucleotides within the sequence selected from the group of sequences defined by the probe consisting of 5,2225 and 2230. The described nucleic acid. 7. A set of nucleic acids comprising at least two nucleic acids, each nucleic acid being Mycoplasma pneumoniae ( M
rRN of ycoplasma pneumoniae )
Can hybridize preferentially to A and rDNA 10
To 250, and each nucleic acid has a different nucleotide sequence composition, and the probes 2295, 22
89, 2296, 2298, 2299, 2300, 22
94, 2167, 2196, 2297, 2192, 22
19, 2162, 2202, 2201, 1166, 22
The set of nucleic acids as an article of manufacture which is complementary or homologous to at least 90% of a sequence comprising any 10 contiguous nucleotides selected from the group of sequences defined by 24, 2195, 2225 and 2230. . 8. At least two nucleic acid compositions are complementary or homologous to at least 90% of a sequence containing any 10 contiguous nucleotides selected from the group of sequences defined by the probe, and From probe 2196 and probe 2167; probe 2162 and probe 2202; probe 2224 and probe 2166; probe 2162 and probe 2230; probe 2167 and probe 2219; probe 2192 and probe 2202; probe 2296 and probe 2297; and probe 2299 and probe 2300 8. The article of manufacture of claim 7, selected from the group of the set: 9. A) contacting the sample with at least one nucleic acid having about 10 to 250 nucleotides capable of preferentially hybridizing to mycoplasma rRNA and rDNA; b) adding hybridization conditions to the sample. In a sample, the conditions comprising preferentially binding the nucleic acid to mycoplasma rRNA and rDNA to form a nucleic acid complex; and c) detecting the nucleic acid complex as an indicator of the presence of the mycoplasma. One or more Mycoplasma [essentially Mycoplasma pneumoniae ( Mycoplasma
Pneumoniae ) and Mycoplasma genitalium ( M. genitalium )]. 10. The probe 229 wherein the nucleic acid in the contacting step is
5,2289,2296,2298,2299,230
0,2294,2167,2196,2297,219
2,2219,2162,2202,2201,216
10. The method of claim 9, which is complementary or homologous to at least 90% of the sequence containing any 10 contiguous nucleotides selected from the group of sequences defined by 6,2224, 2195, 2225 and 2230. . 11. The contacting step comprises at least two nucleic acid compositions, each nucleic acid capable of predominantly hybridizing to rRNA and rDNA of Mycoplasma.
A sequence having 50 base sequences, each of different base composition, containing at least 90 contiguous nucleotides selected from the group of sequences defined by the probe.
% Complementary or homologous to% and probe 2196 + probe 2167, probe 216
2+ probe 2202 probe 2224+ probe 2166, probe 216
2+ probe 2230 probe 2167+ probe 2219, probe 219
2+ probe 2202 probe 2296+ probe 2297, probe 229
10. The method of claim 9 selected from the group of groups of groups consisting of 9+ probes 2300. 12. Non-mycoplasma and human rRN
A pathogenic mycoplasma bacterium associated with human primary atypical pneumonia comprising a nucleic acid composition having a 10 to 250 base sequence capable of preferentially hybridizing to rRNA or rDNA of pathogenic mycoplasma bacterium over A and rDNA Kit for detection.