CN101171260A - PNA probe for detecting mycoplasma - Google Patents

Abstract

This invention is related to PNA probes, probe sets, mixtures, methods and kits pertaining to the determination of Mycoplasma and related Mollicutes.

Description

Be used to detect the PNA probe of gentle film body

The cross reference of related application:

The application requires to enjoy the U.S. Provisional Patent Application No.60/678 that submitted on May 6th, 2005,331 with the relevant rights and interests of the U.S. Patent application of submitting on May 4th, 2006 (application number is also specified), described application is this complete being incorporated herein by reference.

The sub-section titles of Shi Yonging is for clear layer herein, rather than in order to limit subject content.

1. technical field

The present invention relates to detection, analysis and/or quantitatively field, for example detection of mycoplasma (Mycoplasma) and relevant gentle film body (Mollicutes) of microorganism.

2. Introduce

The Mollicutes member belongs to minimum and the simplest prokaryotic organism.This class biology comprises that mycoplasma (Mycoplasma) belongs to, acholeplasma (Acholeplasma) belongs to and urea substance (Ureaplasma) belongs to, and they may be the xenobiotic pollutantss that exists in pathogenic agent and/or cell culture or some product and the method.Because size is little and lack the rigidity cell walls, Mollicutes member (after this abbreviating " gentle film body " as) can pass the filter that is used to remove bacterial pollutant easily.Because size is little, its biosynthesis ability also is limited, and they need rely on the essential nutriment and the cofactor of external source.Therefore the gentle film body of many kinds is evolved and is intracellular parasite.

The mycoplasma of all kinds all is considered to have the risk that becomes potential pollution of cell culture thing.As pollutent, Mycoplasma has shown can apply multiple negative impact to cultured cells.For example, Mycoplasma can with culturing cell competition must nutriment or generation cause the toxin of necrocytosis, all these can both influence the quality and the output of cell culture.Therefore biological-pharmacy is paid special attention to these biologies, because the production of medicine, vaccine and other " biological product " depends on the successive cell cultures.The member of Acholeplasma, especially Rider Lloyd's acholeplasma (Acholeplasma laidlawii) are also paid close attention to as the pollutent of cell cultures, product or the method for external source.

The culture method that is used for gentle film body detection now is subject to these required time of fastidious organisms growth.The result is, utilizing these methods to detect gentle film body may need 28 days or longer, and this time period is inconsistent with medication preparation and distribution fast now.Fast, the method for the gentle film body of simple and relatively inexpensive detection, will make the routine test of cell culture, starting material, device, equipment and analogue and active mass control become possibility.

3. Accompanying drawing

The technician will understand accompanying drawing as described below just for illustration purpose.Accompanying drawing is not the scope that is used to limit the present invention's instruction in any case.

Figure 1A and 1B are the legends that can be used for some nucleic acid bases (nucleobases) of the nucleic acid primer of the different embodiments of the present invention and/or PNA probe.

Fig. 2 is that a kind of self-indication PNA probe is not hybridized the legend of (a) and hybridization (b) state at it.Do not hybridize the basic up-quenching of state (a) fluorescence at it and go out, obviously increase and can detect fluorescence at hybridization state (b).

Fig. 3 A utilizes at least two kinds of nucleic acid primers described herein and at least a PNA probe gentle film body to be detected the ABI 7700 data output of real-time sensitivity and specificity analyses.

Fig. 3 B is that the Ct drawing of data among Fig. 3 A is analyzed.

Fig. 4 utilizes at least two kinds of nucleic acid primers described herein and at least a PNA probe that ABI 7700 data of gentle real-time sensitivity of film body and specific detection analysis are exported.

Fig. 5 utilizes at least two kinds of nucleic acid primers described herein and at least a PNA probe gentle film body to be detected the ABI 7700 data output of sensitive special analysis in real time.

Fig. 6 utilizes at least two kinds of nucleic acid primers described herein and at least a PNA probe gentle film body to be detected the ABI 7700 data output of sensitive special analysis in real time.

Fig. 7 utilizes nucleic acid primer described herein and at least a PNA probe gentle film body to be carried out the ABI 9800 data output of quick end point analysis.

Fig. 8 is the legend of dyestuff 1 chemical structure.

All documents that the application quotes and similar data, include but not limited to, patent, patent application, paper, books, disquisition and internet webpage, no matter which kind of form the document and similar data are, no matter for which kind of purpose, all complete herein clear being incorporated herein by reference.

4. Definition

In order to explain this specification sheets, use following definition, as long as suitable, the term that is used as odd number also comprises plural number, vice versa.(comprise) under the inconsistent situation of word usage in any definition that is elucidated later herein below and any other document, should be as the criterion with the definition of elucidated hereinafter herein with reference to any document of quoting.

A. " nucleic acid base " used herein is meant the heterocyclic group that those natural existence and non-natural exist, its be utilize the nucleic acid technology or utilize the peptide nucleic acid(PNA) technology to produce the technician of polynucleotide base chain thus known, but wherein said polynucleotide base chain sequence-specific ground bind nucleic acid.The non-limitative example of suitable nucleic acid base comprises: VITAMIN B4, cytosine(Cyt), guanine, thymus pyrimidine, uridylic, 5-proyl-uridylic, 2-sulfo--5-proyl-uridylic, 5-methylcytosine, false iso-cytosine, the 2-thiouracil, 2-sulphur thymus pyrimidine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), xanthoglobulin, N9-(7-denitrification-guanine), N9-(7-denitrification-8-azepine-guanine) and N8-(7-denitrification-8-azepine-VITAMIN B4).Other non-limitative example of suitable nucleic acid base comprises those nucleic acid bases shown in Figure 1A and the 1B (also can referring to US 6,357,163).

B. " nucleic acid base sequence " used herein is meant any fragment of polynucleotide base chain, perhaps two or more segmental aggregates.The non-limitative example of suitable polynucleotide base chain comprises oligodeoxynucleotide (for example DNA), oligoribonucleotide (for example RNA), peptide nucleic acid(PNA) (PNA), PNA mosaic, nucleic acid analog and/or nucleic acid mimics.

C. phrase used herein " subunit that contains nucleic acid base " is meant the subunit (subunit) of the polynucleotide base chain that comprises nucleic acid base.Concerning oligonucleotide, the subunit that contains nucleic acid base is a Nucleotide.Concerning peptide and albumen, subunit is an amino acid.For oligonucleotide and peptide, it will be understood to those of skill in the art that and the relevant subunit form of other kind polynucleotide base chain.

D. " target sequence " used herein is meant the nucleic acid base sequence of polynucleotide base chain to be detected.Target sequence can be gentle film body rRNA subsequence.Target sequence can be gentle film body rDNA subsequence.Target sequence can also be the cDNA subsequence of gentle film body rRNA.Target sequence can be the subsequence of gentle film body genomic dna.Target sequence can be the subsequence of gentle film body mRNA.Target sequence is produced the polynucleotide base chain subsequence of (comprising amplicon polynucleotide base chain) by nucleic acid amplification reaction.The non-limitative example of nucleic acid amplification reaction comprises: polymerase chain reaction (PCR), ligase chain reaction (LCR) (LCR), strand displacement amplification (SDA), the amplification of transcriptive intermediate (TMA), rolling circle amplification (RCA), cycling probe technology (CPT), the isothermal duplication (LAMP) of ring mediation, linear target sequence isothermal poly amplification (LIMA), and the amplification of Q-β replicative enzyme.

E. " polynucleotide base chain " used herein is meant complete poly strand, and it comprises the subunit that contains nucleic acid base.

F. " nucleic acid " used herein is meant polymer or the polynucleotide base chain that contains nucleic acid base sequence, and it has the skeleton of Nucleotide or the formation of its analogue.Preferred nucleic acid is DNA, RNA, and L-DNA, lock nucleic acid (locked nucleic acid, LNA).For avoiding any query, PNA is a nucleic acid mimics, rather than nucleic acid or nucleic acid analog.PNA is not a nucleic acid, because it is not made of Nucleotide.

G. " peptide nucleic acid(PNA) " used herein or " PNA " are meant any polynucleotide base chain or the polynucleotide base chain fragment that comprises two or more PNA subunits, and it includes but not limited to, in U.S. Patent number 5,539,082,5,527,675,5,623,049,5,714,331,5,718,262,5,736,336,5,773,571,5,766,855,5,786,461,5,837,459,5,891,625,5,972,610,5,986,053,6, the any polynucleotide base chain or the polynucleotide base chain fragment that are called as peptide nucleic acid(PNA) in 107,470 and 6,357,163.

Term " peptide nucleic acid(PNA) " or " PNA " also are applicable to any polynucleotide base chain or polynucleotide base chain fragment, and it comprises two or more subunits of this nucleic acid mimics that following publication is described: people such as Lagriffoul, Bioorganic ﹠amp; Medicinal Chemistry Letters, 4:1081-1082 (1994); People such as Petersen, Bioorganic ﹠amp; Medicinal Chemistry Letters, 6:793-796 (1996); People such as Diderichsen, Tett.Lett.37:475-478 (1996); People such as Fujii, Bioorg.Med.Chem.Lett.7:637-627 (1997); People such as Jordan, Bioorg.Med.Chem.Lett.7:687-690 (1997); People such as Krotz, Tett.Lett.36:6941-6944 (1995); People such as Lagriffoul, Bioorg.Med.Chem.Lett.4:1081-1082 (1994); Diederichsen, U., Bioorganic ﹠amp; Medicinal ChemistryLetters, 7:1743-1746 (1997); People such as Lowe, J.Chem.Soc.Perkin Trans.1, (1997) 1:539-546; People such as Lowe, J.Chem.Soc.Perkin Trans.11:547-554 (1997); People such as Lowe, J.Chem.Soc.Perkin Trans.11:555-560 (1997); People such as Howarth, J.Org.Chem.62:5441-5450 (1997); Altmann, people such as K-H, Bioorganic ﹠amp; Medicinal ChemistryLetters, 7:1119-1122 (1997); Diederichsen, U., Bioorganic ﹠amp; Med.Chem.Lett., 8:165-168 (1998); People such as Diederichsen, Angew.Chem.Int.Ed., 37:302-305 (1998); People such as Cantin, Tett.Lett., 38:4211-4214 (1997); People such as Ciapetti, Tetrahedron, 53:1167-1176 (1997); People such as Lagriffoule, Chem.Eur.J., 3:912-919 (1997); People such as Kumar, Organic Letters 3 (9): 1269-1272 (2001); With people such as Shah in WO96/04000 disclosed Peptide-Based Nucleic Acid Mimics (the peptidyl nucleic acid mimics, PENAM).

In some embodiments, term " peptide nucleic acid(PNA) " or " PNA " also are applicable to polynucleotide base chain or polynucleotide base chain fragment, and it comprises the two or more covalently bound subunit shown in the following chemical formula:

Wherein, each J is identical or different, is selected from: H, R ¹, OR ¹, SR ¹, NHR ¹, NR ¹ ₂, F, Cl, Br and I.Each K is identical or different, is selected from: O, S, NH and NR ¹Each R ¹Being identical or different, is the alkyl with 1 to 5 carbon atom, optional heteroatoms or replacement or the unsubstituted aryl of comprising.Each A is selected from: singly-bound, chemical formula-(CJ ₂) _S-group and chemical formula-(CJ ₂) _SC (O)-group, wherein, J is defined in the above, each s is 1 to 5 integer.Each t is 1 or 2, and each u is 1 or 2.Each L is identical or different, independently be selected from: VITAMIN B4, cytosine(Cyt), guanine, thymus pyrimidine, uridylic, 5-proyl uridylic, 2-sulfo--5-proyl uridylic, 5-methylcytosine, false iso-cytosine, 2-thiouracil and 2-sulphur thymus pyrimidine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), xanthoglobulin, N9-(7-denitrification-guanine), N9-(7-denitrification-8-azepine-guanine), N8-(7-denitrification-8-azepine-VITAMIN B4), the nucleic acid base (for example Figure 1A and 1B) that the nucleic acid base analogue that other exists natively and other non-natural exist.

In some embodiments, the PNA subunit can be the nucleic acid base that natural existence or non-natural exist, and it is connected to N-[2-(aminoethyl) by the methylene radical ketonic linkage] on the N-α-glycyl nitrogen of glycine skeleton; Current this is the peptide nucleic acid(PNA) subunit form of normal use.

H. term used herein " marker ", " report section " or " but test section " are general, be meant the part that can adhere to polynucleotide base chain or antibody, thereby or can be used for the part that reporting system can detect polynucleotide base chain or antibody by device or method.For example, mark can be following arbitrary portion: (i) provide detectable signal; (ii) interact to modify the detectable signal that first or second marker provides with second marker; Capturing function (for example hydrophobic avidity, antibody/antigen, ion complexation) perhaps (iii) is provided.

I. " sequence-specific " used herein is meant the base pairing hybridization by hydrogen bond action.The non-limitative example of standard base pairing comprises VITAMIN B4 and thymus pyrimidine or uridylic base pairing, guanine and cytosine(Cyt) base pairing.Other non-limitative example of base pairing basic model includes, but are not limited to: VITAMIN B4 and following any one base pairing: 5-proyl-uridylic, 2-sulfo--5-proyl-uridylic, 2-thiouracil or 2-sulphur thymus pyrimidine; Guanine and following any one base pairing: 5-methylcytosine or false iso-cytosine; Cytosine(Cyt) and following any one base pairing: xanthoglobulin, N9-(7-denitrification-guanine) or N9-(7-denitrification-8-azepine-guanine); Thymus pyrimidine or uridylic and following any one base pairing: 2-aminopurine, N9-(2-amino-6-chloropurine) or N9-(2,6-diaminopurine); And N8-(7-denitrification-8-azepine-VITAMIN B4), as a kind of universal base, with other nucleic acid base pairing arbitrarily, for example following any one: VITAMIN B4, cytosine(Cyt), guanine, thymus pyrimidine, uridylic, 5-proyl-uridylic, 2-sulfo--5-proyl uridylic, 5-methylcytosine, false iso-cytosine, 2-thiouracil and 2-sulphur thymus pyrimidine, 2-aminopurine, N9-(2-amino-6-chloropurine), N9-(2, the 6-diaminopurine), xanthoglobulin, N9-(7-denitrification-guanine) or N9-(7-denitrification-8-azepine-guanine) (referring to: people such as Seela, Nucl.Acids, Res.:28 (17): 3224-3232 (2000)).

J. term used herein " mosaic " or " chimeric oligomer " are meant the polynucleotide base chain that comprises two or more connection subunits, and described subunit is selected from dissimilar subunits.For example, the PNA/DNA mosaic can comprise with at least one 2 '-at least one PNA subunit that the thymus nucleic acid subunit is connected (the exemplary method of PNA/DNA mosaic preparation and form) referring to WO96/40709.The exemplary composition subunit of mosaic can be selected from the PNA subunit, naturally occurring amino acid subunit, DNA subunit, RNA subunit and nucleic acid analog or stand-in subunit.

K. term used herein " connection polymer " is meant and comprises the segmental polynucleotide base of two or more polymers chain that described polymer fragment connects by joint.Can be connected to form the polymeric polymer fragment of connection can be selected from: oligodeoxynucleotide, oligoribonucleotide, peptide, polymeric amide, peptide nucleic acid(PNA) (PNA) and mosaic.

1. term used herein " (one or more) soften film body " is meant one or more kinds of Mycoplasma, Acholeplasma or Ureaplasma.

M. " solid support " or " solid carrier " used herein are meant any solid phase material, can synthesize on it, adhesion, connection or fixing polynucleotide base chain.Solid support comprises term for example " resin ", " solid phase ", " surface " and " upholder ".Solid support can be by organic polymer, such as by polystyrene, and polyethylene, polypropylene, fluorinated ethylene propylene, polyoxyethylene, and polyacrylamide, and their multipolymer and grafts are formed.Solid support also can be an inorganics, for example glass, silica, controlled pore glass (CPG), perhaps anti-phase silica.The profile of solid support can be pearl, spheroid, particle, particulate, gel or surface.The surface can be flat, flat or uneven basically.Solid support can be porous or atresia, can have to expand or unexpansive characteristic.That solid support can be made is poroid, spill or other ware shape, container-like, profile or place.Multiple solid support can be placed on the different positions of array, it is addressable that described array is sent automatically to reagent, perhaps by comprising that laser radiation and copolymerization Jiao or deflection optically focused (deffective light gathering) method for scanning detects.

N. term used herein " upholder combination " is meant and is fixed on the solid support, perhaps is fixed in solid support.Should understand and to fix by any method, for example comprise: by covalent attachment, fix, adhere to, by contact or by depositing from the teeth outwards by part/ligand interaction by static.

5. Describe

I. introduction:

Should be appreciated that the following discussion that in this " introduction " chapters and sections, proposes, be applicable to described herein more of the present invention, perhaps whole different embodiments." perhaps " use represent " and/or ", except as otherwise noted.Similarly, " comprising " and " comprising " is general, and it does not think restrictive.Should also be clear that at different embodiments and describe the place of using terms " to comprise ", those skilled in the art should understand at some in particular cases, this embodiment can select to use " basically by ... form " or " by ... form " be described.

PNA is synthetic:

PNA chemistry assembling synthetic method is known (referring to for example: U.S. Patent number 5,539,082,5,527,675,5,623,049,5,714,331,5,718,262,5,736,336,5,773,571,5,766,855,5,786,461,5,837,459,5,891,625,5,972,610,5,986,053 and 6,107,470.The reference commonly used of PNA synthetic method sees also: people such as Nielsen, Peptide Nucleic Acids; Protocolsand Applications (" peptide nucleic acid(PNA); Principle and application "), Horizon Scientific Press, Britain Norfolk (1999).

Now be useful on chemicals and the device of peptide nucleic acid(PNA) upholder in conjunction with the robotics assembling.Mark and unlabelled PNA oligomer can be customized PNA oligomer from commercial company equally.It is synthetic that the chemistry assembling of PNA is similar to solid-phase peptide, and wherein oligomer has reactive alkylamino end in each assembling circulation, and described end can combine to be added to the synthesis unit on the polymer of growth with the next one.Because use the standard peptide chemical process, natural and non-natural amino acid can be introduced the PNA oligomer usually.Because PNA is a polymeric amide, so it has C-end (C-terminal) and N-end (N-terminal).In order to design a kind of hybridization probe that is suitable for antiparallel in conjunction with target sequence (preferred orientation), with PNA probe in detecting nucleic acid base (probing nucleobase) sequence N-end, be equal to 5 of DNA of equal value or RNA oligonucleotide '-C-terminal.But the direction of hybridization is unrestricted, because known PNA oligomer is also with parallel direction bind nucleic acid and other PNA oligomer.

The PNA mark:

The non-limiting method of mark PNA oligomer is described in U.S. Patent number 6,110, and in 676,6,355,421,6,361,942 and 6,485,901, perhaps it is that the synthetic field of PNA is known.Other limiting examples of mark PNA oligomer also sees people such as Nielsen, Peptide Nucleic Acids; Protocolsand Applications, (" peptide nucleic acid(PNA); Principle and application "), Horizon Scientific Press, Britain Norfolk (1999).PNA oligomer and oligonucleotide also can be with the protein of describing in the U.S. Patent number 6,197,513 (for example enzyme) and peptide-labeled.Therefore, can prepare or buy the PNA oligomer of various marks from commercial company.

Synthetic and the modification of nucleic acid

Nucleic acid oligomers (oligonucleotide and oligoribonucleotide) is synthetic to have become conventional.The nucleic acid synthetic is described in detail and is seen also Gait, M.J., Oligonucleotide Synthesis:a Practical Approach. (" oligonucleotide is synthetic: the practice progress ") IRL Press, England Oxford.Those of ordinary skills know that mark and unlabelled oligonucleotide (DNA, RNA and its synthetic analogues) can obtain easily.They can utilize commercial device and reagent to synthesize, and perhaps they can buy the commercial company from customization preparation oligonucleotide.

Synthetic and the modification of mosaic:

The PNA mosaic is the combination of nucleic acid and peptide nucleic acid(PNA) subunit.The PNA mosaic is synthetic, the visible U.S. Patent number 6,063,569 of suitable reference of mark and modification.In addition, above-mentioned PNA is synthetic can be used for modifying the chimeric PNA part of PNA usually with marking method.In addition, nucleic acid known method synthetic and mark can be used for modifying the chimeric nucleic acid moiety of PNA usually.Therefore, chimeric synthetic, mark of PNA and modification can utilize method known to those skilled in the art and top description or method as a reference.

Marker:

Can be used for that the mark embodiment of the present invention is used polynucleotide base chain (for example PNA probe) but or the detection moiety of antibody (mark) limiting examples comprise, dextran binding substances (dextran conjugate), the side chain nucleic acid detection system, chromophore, fluorophor, spin label, radio isotope, enzyme, haptens, acridinium ester or chemiluminescence compound.Other suitable labelled reagent and preferred method of attachment are that the synthetic field of PNA, peptide or nucleic acid those of ordinary skill is known.

Haptenic limiting examples comprises 5 (6)-Fluoresceincarboxylic acids, 2, and 4-dinitrophenyl, digoxin and vitamin H.

The limiting examples of fluorescence dye (fluorophor) comprises 5 (6)-Fluoresceincarboxylic acids (Flu); 6-((7-amino-4-methylcoumarin-3-ethanoyl) amino) caproic acid (Cou); 5 (with 6)-carboxyls-X-rhodamine (Rox); cyanine 2 (Cy2) dyestuff; cyanine 3 (Cy3) dyestuff, cyanine 3.5 (Cy3.5) dyestuff, cyanine 5 (Cy5) dyestuff; cyanine 5.5 (Cy5.5) dyestuff; cyanine 7 (Cy7) dyestuff, cyanine 9 (Cy9) dyestuff ( cyanine dyes 2,3; 3.5; 5 and 5.5 with the form of NHS ester available from Amersham, the highland, Arlington, IL) or Alexa series dye (Molecular Probes; the Eugene, OR).

The limiting examples of enzyme comprises polysaccharase (Taq polysaccharase for example, Klenow archaeal dna polymerase, T7DNA polysaccharase, Sequenase, archaeal dna polymerase 1 and phi29 polysaccharase), alkaline phosphatase (AP), horseradish peroxidase (HRP), soybean peroxidase (SBP), rnase and proteolytic enzyme.

Energy shifts

In certain embodiments, polynucleotide base chain can shift with energy and be combined into row labels.Available energy shifts when hybridizing in order to detect, and should have an energy to shift combination, and it comprises at least one energy transfer donator and at least one energy transfer receptor part.Usually, energy shifts combination and comprises single donor part and single receptor part, but this is not a kind of restriction.Energy shifts combination and can comprise above a donor part and/or above an acceptor portion.Donor and acceptor portion so act on so that one or more acceptor portions are accepted the energy that one or more donors partly shift, perhaps the signal of the one or more donors parts of cancellation.Therefore, in some embodiments, donor part and acceptor portion all are fluorophors.Though the fluorophor of before having listed (having suitable spectral quality) also can be used as the energy transfer receptor, acceptor portion also can be that non-fluorescent quenching part is such as 4-((4-(dimethylamino) phenyl) azo-group) M-nitro benzoic acid (dabcyl).The marker that energy shifts combination can be connected polynucleotide base chain end or be connected the inner site of polynucleotide base chain.For example, each in two marks of energy transfer combination can be connected the distal-most end of polynucleotide base chain.

Energy between donor and the acceptor shifts and can finish by any energy transfer process, such as the collision between combining closely partly by energy transfer combination or by for example fluorescence resonance energy transmission of non-radiative method (FRET).For FRET takes place, donor and the energy between acceptor portion that energy shifts combination shift, need these parts spatially close, and the emmission spectrum of donor and the absorption spectrum of acceptor are overlapping basically (referring to people Analytical Biochemistry such as Yaron, 95:228-235 (1979), especially 234 page of first hurdle, the 232nd page of first hurdle to the).Perhaps, (radiationless) energy of collision mediation shifts and can occur between the donor and acceptor portion of combining closely very much, no matter and whether the emmission spectrum of donor part is overlapping basically (referring to people such as Yaron with the absorption spectrum of acceptor portion, AnalyticalBiochemistry, 95:228-235 (1979), especially 232 page of first hurdle, the 229th page of first hurdle to the).This process is called as intramolecularly collision because generally believe cancellation result from donor and acceptor portion direct the contact (referring to: people such as Yaron).Energy shifts also and can take place by the process that mechanism of action is still waiting to describe.Should be appreciated that any reference that the application's energy shifts, relate to the phenomenon that comprises that all these mechanism are different.Should also be clear that energy shifts can be simultaneously be undertaken by the energy transfer process that surpasses, and the variation of detectable signal can be measure two or more energy transfer processes simultaneously and/or the successive related activity.Therefore, the energy metastasis is not the restriction to embodiment of the present invention.

Detected energy shifts in self-indication polynucleotide base chain:

When shifting composite marking with energy, we are called self-indication with polynucleotide base chain.The limiting examples of self-indication nucleic acid oligomers is described in U.S. Patent number 6,103, and 476,6,150,097 and 6,365,729.The limiting examples of self-indication PNA oligomer is described in U.S. Patent number 6,355, and 421,6,485,901,6,528,267 and 6,649,349.In some cases, the self-indication oligomer can be used as the primer (for example U.S. Patent number 5,866,366,6,090,552,6,117,635 and 6,365,729) of nucleic acid amplification reaction.

Shift at least one member's of combination at least a physical properties by detected energy, the hybridization that can monitor between self-indication oligomer and target sequence forms, wherein compare with the oligomer that is present in non-hybridization state, can to detect ground different for described physical properties when hybridization complex formed.We claim that this phenomenon is the self-indication character of polynucleotide base chain.The variation of this detectable signal is derived from the variation of energy transfer efficiency between donor and acceptor portion, and this is that hybridization by oligomer and target sequence causes.

For example, detection method may relate to the fluorescence that detected energy shifts combination donor or acceptor fluorescence group.In some embodiments, energy shifts combination can comprise at least one donor fluorophor and at least one acceptor (fluorescence or non-fluorescence) quenching group, so that the fluoroscopic examination of donor fluorophor can be used for detecting, identify or the quantitatively hybridization of oligomer and target sequence.For example, when oligomer and target sequence hybridization, the fluorescence measurability increase of donor fluorophor (referring to: Fig. 2).

In some embodiments, energy shifts combination can comprise at least one donor fluorophor and at least one acceptor fluorescence group, so that any one or boths' fluoroscopic examination can be used for detecting, identify and/or the quantitatively hybridization of self-indication oligomer and target sequence at least one donor part or the acceptor portion.

Self-indication PNA oligomer can be used for the in situ hybridization test.Self-indication PNA oligomer also can be used in real time or at the end point analysis nucleic acid amplification reaction.

Detectable and independent detectable part/multiple analysis:

Carry out the multiple crossing test according to embodiment of the present invention.In multiple test, can detect the multiple situation that merits attention simultaneously.During test is finished or afterwards, multiple analysis relies on the ability of graded samples composition or related data.In different embodiments of the present invention, but one or more different independence test section can be used for two or more different probes used in the mark test.But distinguish and/or quantitatively the ability of each independent test section provide and carried out the method that multiple crossing detects, because the related data of each uniqueness (independently) label probe and particular target sequence hybridization, with the existence of various biologies to be measured in the sample (for example gentle film body) whether and/or quantity exist related.Therefore to the existence of two or more different biological (for example gentle film bodies) in the same sample whether and/or quantity, multiple detection of the present invention is used in the same test,, detect simultaneously.For example, multiple detection can utilize two or more PNA probes, and wherein each independently can detect the fluorophor mark with independent fluorophor or a group of can detecting.

Spacer/shank:

Usually, spacer is used for the side effect of big labelled reagent to probe hybridization character minimized.Joint causes snappiness and randomness usually in polynucleotide base chain, perhaps be used to connect two or more nucleic acid base sequences of polynucleotide base chain.Preferred interval thing/the shank that is used for polynucleotide base chain described herein can comprise one or more aminoalkyl group carboxylic-acids (for example hexosamine), amino acid whose side chain (for example side chain of Methionin or ornithine), natural amino acid (for example glycine), amino oxygen alkyl acid (for example 8-amino-3, the 6-dioxy is sad), alkyl acid (for example Succinic Acid), alkyl oxygen diacid (for example diglycollic acid) or the alkyl diamino (for example, 1,8-diamino-3,6-two oxa-octanes).Spacer/shank also can be arbitrarily or is had a mind to make up to improve water-soluble (for example referring to: people such as Gildea, Tett.Lett.39:7255-7258 (1998) and U.S. Patent number 6,326,479 and 6,770,442) of polynucleotide base chain.

For example, spacer/shank can comprise one or more chemical formula-Y-(O that have _m-(CW ₂) _n) _oThe connection compound of-Z-.Y group can be selected from: singly-bound ,-(CW ₂) _p-,-C (O) (CW ₂) _p-,-C (S) (CW ₂) _P-and-S (O ₂) (CW ₂) _PThe Z group can have chemical formula NH, NR ², S or O.Each W can independently be H, R ²,-OR ², F, Cl, Br or I; Wherein, each R ²Can independently be selected from :-CX ₃,-CX ₂CX ₃,-CX ₂CX ₂CX ₃,-CX ₂CX (CX ₃) ₂With-C (CX ₃) ₃Each X can independently be H, F, Cl, Br or I.Each m can independently be 0 or 1.Each n, o and p can independently be the integers between 0 to 10.

Hybridization conditions/stringency:

The those of ordinary skill in nucleic acid hybridization field knows that the factor that is generally used for influencing or controls the hybridization stringency comprises methane amide concentration (perhaps other chemical modification reagent), salt concn (that is, ionic strength), hybridization temperature, detergent concentration, whether the existence of pH and chaotropic agent.Usually, measure the effect of certain single stringency factor of variation then by fixing several above-mentioned stringency factors, this known technology is found the best stringency of probe/target combination.Thereby therefore can regulate the stringency of these identical stringency factors control PNA and nucleic acid hybridization, unless the hybridization of PNA is obvious and ionic strength is irrelevant.Test definite best stringency that detects by examining and determine various stringency factors up to obtaining the required degree of discrimination.

Suitable hybridization conditions:

Usually, cause the background of contaminant nucleic acid relevant more, control stringency that must be careful more with target sequence.The sealing probe also can only carry out the restriction that optimization is obtained of stringency factor to break through as the means that improve ability to see things in their true light.Therefore suitable hybridization conditions comprises the condition that reaches the required degree of discrimination, so that should test can produce accurately (in the detection of desired tolerance) and repeatably result.Only according to normal experiment method and provided herein disclosing, those skilled in the art can determine the suitable hybridization conditions of utilizing method, test kit and composition to test described herein easily.Suitable hybridization conditions can be used for the hybridization of probe and target sequence, and for example hybridization of primer and primer sites in the real-time PCR reactions.Suitable in situ hybridization condition comprises the condition that is suitable for carrying out in-situ hybridization method.Therefore, suitable hybridization or suitable in situ hybridization condition are known in field of biology, utilize content disclosed herein comprising or do not comprising under other normal experiment situation, can realize reaching the specified conditions of required discriminating level.

The sealing probe:

Sealing probe (blocking probe) is nucleic acid or non-nucleic acid probe (for example PNA probe), and it can be used for suppressing the polymeric detection nucleic acid base sequence of probe and combines with non-target sequence.The PNA oligomer can be sealing probe (referring to people such as Coull, U.S. Patent number 6,110,676).Usually, sealing probe and detection nucleic acid base sequence height correlation, preferably they are compared with the detection nucleic acid base sequence and comprise a point mutation.Thereby generally believe the sealing probe operation by and non-target sequence hybridization form than detect between nucleic acid base sequence and the non-target sequence hybridization thermodynamics more stabilized complex work.Formation more stable and preferred mixture has stoped the formation that detects the not preferred mixture of less stable between nucleic acid base sequence and the non-target sequence.Therefore, the sealing probe can be used for the different embodiments of the present invention, thereby suppresses combining of PNA probe and the non-target sequence that may exist interference test also to carry out.The sealing probe can be used for the discriminating of simple point mutation.

Detect nucleic acid base sequence:

The detection nucleic acid base sequence of polynucleotide base chain is the distinguished sequence identification division of construct.For example, the detection nucleic acid base sequence of PNA probe is design and the nucleic acid base sequence of target sequence sequence specific hybridization, wherein the existence of target sequence whether and/or quantity can be used for biological in the test sample (for example gentle film body) existence whether and/or quantity.Therefore, with due regard to arrive of the requirement of selected test method, select the detection nucleic acid base sequence length of probe usually, make PNA probe and target sequence under suitable hybridization conditions or suitable in situ hybridization condition, form stable compound the PNA probe.

Probe complex:

In some embodiments, two probes can share with target sequence hybridizes, the nucleic acid base sequence of each probe comprises half or only about half of detection nucleic acid base sequence, and the complete target sequence hybridization of biology to be measured is required in described detection nucleic acid base sequence and the test.As limiting examples, the detection nucleic acid base of two probes can be according to people such as H.Orum (U.S. Patent number 6,027,893, be entitled as: " Method of identifying a nucleic acid using triple helix formation of adjacentlyannealed probes " " adopting adjacent annealing probe triple helical to form the method for differentiating nucleic acid " is hereby incorporated by) the method design described.Utilize this method, with the probe of target sequence hybridization can mark, also mark not.But, detection be the probe complex that adjacent probe annealing forms.The similar constituent that includes only the PNA probe is described in U.S. Patent number 6,287,772.

Connect polymer:

In some embodiments, connect polymer and can be used as probe.As preceding definition, the connection polymer is meant and comprises the segmental polynucleotide base of two or more polymers chain that described polymer fragment connects by joint.Therefore, in some embodiments, the PNA probe can be designed as and comprises two nucleic acid base sequences, each of two nucleic acid base sequences includes half or only about half of detection nucleic acid base sequence, and described detection nucleic acid base sequence is that the complete target sequence hybridization of biology to be measured is required in the test.When using above two polymer fragments, two or more connection polymer fragments can make up, so that the gathering nucleic acid base sequence of different fragments comprises the required detection nucleic acid base sequence of biological target sequence hybridization to be measured in the test.

Primer:

In different embodiments, polynucleotide base chain can be used as the primer in the nucleic acid amplification reaction.Primer can be an oligonucleotide.Primer can be the PNA/DNA mosaic.In some embodiments, primer can be labeled.In some embodiments, primer can not be labeled.Primer can be used to for example pcr amplification reaction of nucleic acid amplification reaction.

Bu Duicheng ﹠amp; Asynchronous PCR:

In some embodiments, can use in test asymmetric (asymmetric) or asynchronous (asynchronous) PCR.People such as Gyllensten (U.S. Patent number 5,066,584) have described asymmetric PCR, its can with U.S. Patent number 6,485, the self-indication probe coupling in 901 and 6,649,349.People such as Chen (WO01/94638) have described asynchronous PCR.In both cases, a polynucleotide base chain of the producible double-stranded template of amplified reaction is more than another chain, and wherein the polynucleotide base chain that excessively produces comprises target sequence.

Inner positive control:

In certain embodiments, the augmentation detection test can comprise inner positive control (IPC).IPC can be set up jointly by the independent sets of primer, probe and target DNA.Can comprise that IPC increases in test with confirmation.Increase in reaction by confirmation, under the situation that obtains negative findings, can get rid of the amplification failure.Can be in isolating pipe or hole or with the probe of one or more detection mycoplasmas and relevant gentle film body and primer together, carry out the IPC amplification.Target DNA also can be cloned into plasmid or as oligonucleotide.Inner positive control is a commercialized supply.

II. different embodiments of the present invention:

A. The PNA probe:

In some embodiments, the present invention leads and relates to one or more PNA probes.Can select described one or more PNA probes to be used for the mycoplasma and relevant gentle film body (for example acholeplasma and/or urea substance) of test sample.Calibrating can comprise mycoplasma, acholeplasma and/or the urea substance kind in detection, evaluation and/or the quantitative sample.The PNA probe can be used for analyzing in the biology of concern whether have associated nucleic acid.Therefore, the present invention can be used for bioanalysis (for example original position analysis) and from the biological extraction paid close attention to or the analysis (for example pcr analysis) of source nucleic acid.Therefore, the source of target sequence is not construed as limiting.

Note:

1) nucleic acid base sequence for nucleic acid be 5 '-3 ', be that N holds the C end for PNA.

2) carry out database search and be used to detect the Mollicutes mycoplasma and related specy has the similarity that PCR draws primer sequence now with disclosed so that determine the nucleic acid primer sequence.Utilize " comparing two sequences " function that the state-run center of biotechnology information (National Center for Biotechnology Information) provides ( Http:// www.ncbi.nlm.nih.gov/blast/bl2seg/bl2.html), with SEQ ID.Nos.1-9 with surpass 50 disclosed mycoplasma target primer sets (Dussurget and Roulland-Dussoix, 1994; People such as Kong, 2001; People such as Tang, 2000; Uphoff and Drexier, 1999; Van Kuppeveld waits the people, and 1994) compare.SEQ.ID.No.2,3,5 and 6-9 not overlapping with the primer of any screening, and SEQ.ID.No.1 and 4 and known primer demonstrate portion homologous.

Usually, the present invention can be used for the detection of gentle film body.The PNA probe can comprise a kind of detection nucleic acid base sequence that helps the gentle special detection of film body of one or more kind.Be suitable for detecting the detection nucleic acid base sequence of the different PNA probes of one or more gentle film body target sequences, shown in the SEQ ID No.6-9 of table 1.Therefore, in some embodiments, the PNA probe can comprise SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, the complementary sequence of SEQ.ID.NO:6, the complementary sequence of SEQ.ID.NO:7, the complementary sequence of SEQ.ID.NO:8 or the complementary sequence of SEQ.ID.NO:9.With primer coupling shown in the SEQ ID No.1-5, can carry out one or more pcr amplification reaction with the listed biology of detection table 2.Table 2 has provided and has surpassed 100 kinds of biologies that belong to Mollicutes.

In some embodiments, the PNA probe can have the subunit that contains nucleic acid base of long 12-20.In some embodiments, the PNA probe can have the subunit that contains nucleic acid base of long 13-18.In some embodiments, the PNA probe can have the subunit that contains nucleic acid base of long 15-17.In some embodiments, the detection nucleic acid base sequence of PNA probe can select to have the listed nucleic acid base sequence of definite table 1.Therefore, in some embodiments, the detection nucleic acid base sequence of PNA probe is SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, the complementary sequence of SEQ.ID.NO:6, the complementary sequence of SEQ.ID.NO:7, the complementary sequence of SEQ.ID.NO:8 or the complementary sequence of SEQ.ID.NO:9.The complementary sequence of table 1SEQ ID No.6-9 listed detection nucleic acid base sequence can be used for preparing suitable PNA probe because what may prepare or amplify be the copy of target sequence, wherein should copy with the complementary sequence of associative list 1 listed nucleic acid base sequence.

The PNA probe can have the detection nucleic acid base sequence with target complement sequence.In addition, can using basically, complementary detects nucleic acid base sequence, because it is verified when utilizing when having the relevant probe of one or more point mutation (base mispairing) between probe and target sequence, can obtain bigger recognition sequence ability (referring to people such as Guo, Nature Biotechnology 15:331-335 (1997)).

The present invention expection, the variant of the listed detection nucleic acid base sequence of table 1 can provide the suitable PNA probe of the listed biology of special detection table 2.Common variant comprises, deletion, insertion and frameshit.Detection nucleic acid base sequence variant in the described herein parameter is considered to the part of the specific embodiment of the invention.

The PNA probe can include only detect nucleic acid base sequence (as herein before as described in) or can comprise other parts.But the limiting examples of other parts comprises other subunit of test section (marker), joint, spacer, natural or non-natural amino acid, peptide, enzyme and/or PNA, DNA or RNA.Other parts can be in test that function or non-functional is arranged.But usually, in the test design of using the PNA probe, select other parts that function is arranged.For example, but the PNA probe can be with one or more test sections mark, but perhaps with two or more test section marks independently.Independently but the test section can be independently can detect fluorophor.No matter be detectable or independent detectable, the PNA probe can be a self-indication.The limiting examples of marker was before existing to be described.

The PNA probe can be used in situ hybridization (ISH) and fluorescence in situ hybridization (FISH) test (for example referring to U.S. Patent number 6,280,946,6,649,349,6,656,687 and 6,664,045).The PNA probe can be used for nucleic acid amplification reaction (for example referring to U.S. Patent number 6,355,421,6,361,942,6,441,152,6,485,901 and 6,649,349).

Sometimes remove in test excess probe, thereby but make the detection moiety of specific hybridization probe in background signal, can detect, the probe that described background signal is not hybridized by still existing is produced.But, but because some self-indication probe only produces a little or do not produce detection background, so they do not need to remove (washing off) excess probe fully from sample in use.

Unlabelled non-nucleic acid probe:

But probe of the present invention can not need to carry out mark with exercisable test section.When utilizing the PNA probe, can detect by the detection nucleic acid base sequence and the target sequence of probe and hybridize formed PNA probe/target sequence mixture.For example, hybridize formed PNA/ nucleic acid complexes by PNA detection nucleic acid base sequence and target sequence, can be under the antibodies condition, with detecting with the antibody of mixture specific action.Suitable PNA/ nucleic acid complexes antibody and their preparation and use for example are described in U.S. Patent number 5,612, in 458.

Antibody/PNA/ nucleic acid complexes that interaction by α-PNA/ nucleic acid antibody and PNA/ nucleic acid complexes forms can detect by several methods.For example, but α-PNA/ nucleic acid antibody can be used the test section mark.Described but suitable test section is before existing.Therefore, but whether and/or quantity the existence of test section, can with the existence of antibody/PNA/ nucleic acid complexes and one or more gentle film bodies to be measured whether and/or quantity be associated.In addition, but can utilize the second antibody of test section mark, detect antibody/PNA/ nucleic acid complexes.Usually second antibody is under the antibodies condition, with α-PNA/ nucleic acid antibody specific combination.Therefore, but whether and/or quantity the existence of test section, can with the existence of antibody/antibody/PNA/ nucleic acid complexes and one or more gentle film bodies to be measured whether and/or quantity be associated.Term antibody used herein comprises the antibody fragment of other antibody of specific combination or other antibody fragment.

From the teeth outwards fixing of probe:

In some embodiments, can in solution, use the PNA probe.In some embodiments, the PNA probe can be fixed on the upholder.Therefore, one or more PNA probes can randomly be fixed to the surface and go up the target sequence of paying close attention to the target biology to detect.

Can utilize known UV-cross-linking method with the PNA probe stationary to the surface.Can be from the teeth outwards to be suitable for deprotection but not from synthesizing the mode that upholder cuts; synthetic PNA probe (referring to: Weiler; J. wait the people; Hybridization based DNA screening on peptide nucleic acid (PNA) oligomer arrays. " DNA based on hybridization on peptide nucleic acid(PNA) (PNA) oligomer array screens "; Nucl.Acids Res.; 25,14:2792-2799 (in July, 1997)).In another embodiment, by the appropriate functional group of probe and the reaction of lip-deep functional group, the PNA probe can be by covalently bound to the surface (referring to U.S. Patent number 6,475,721).

PNA probe chemistry is connected to the method on surface, is usually directed to treat the nucleophilic group (for example amine or mercaptan) and the reaction of the electrophilic group on the upholder to be finished of stationary probe.Perhaps, nucleophilic reagent may reside on the upholder, and electrophilic reagent (for example activatory carboxylic acid) is present on the probe.Because natural PNA has N-terminal, so PNA does not need to modify and just can be fixed on the surface (referring to U.S. Patent number 6,475,721).

Be suitable for the PNA probe stationary is arrived lip-deep condition, similar with those conditions that are suitable for the polymer mark usually.Fixation reaction is identical with labeled reactant in fact, just replaces marker with the surface for the treatment of to be connected with polymer.

All kinds of is commercialized supplies with amino, hydroxy-acid group, isocyanic ester, lsothiocyanates and the various surfaces of maleimide group deutero-.The limiting examples of suitable surface comprises film, chip (for example silicone resin chip), glass, controlled pore glass, granules of polystyrene (pearl), silicon and gold nano grain.

The array of PNA probe:

Array is the surface that is fixed with two or more probes, and each probe all is fixed on the specific position.For example U.S. Patent number 6,475,721 and Weiler, J. wait people (Hybridization based DNAscreening on peptide nucleic acid (PNA) oligomer arrays. " DNA based on hybridization on peptide nucleic acid(PNA) (PNA) oligomer array screens ", Nucl.Acids Res., 25,14:2792-2799 (in July, 1997)) the PNA probe array is described.Detection nucleic acid base sequence that can careful selection stationary probe is to detect the sample that may comprise from one or more target biotinylated nucleic acids.Because the position of each stationary probe and form known, so array can be used for detecting simultaneously, identify and/or quantitatively can be present in two or more target biologies (for example one or more the gentle film bodies) nucleic acid in the sample.In addition, in each test back by the nucleic acid of the hybridization arbitrarily of the dissociating PNA probe array of can regenerating, thereby the method for utilization with a large amount of samples of an array replicate analysis is provided.Therefore, PNA probe array or PNA probe groups can be used for paying close attention to the screening that repeats of target biological sample.Array can comprise at least one PNA probe or more than a PNA probe.Comprise the PNA probe that detects nucleic acid base sequence shown in the table 1, applicable to detecting one or more listed gentle film bodies of table 2.

B. The PNA probe groups:

In some embodiments, the invention still further relates to and be suitable for detecting, identify and/or the quantitative probe groups of one or more gentle film bodies in the sample.General feature and the preferred feature that is suitable for detecting one or more gentle film body PNA probes described in this paper front.Be suitable for the exemplary detection nucleic acid base sequence that in this probe, uses, list in table 1 SEQ ID No.6-9.One or more this PNA probes can form probe groups with one or more other probe combinations.

Therefore, in some embodiments, at least one PNA probe of described group can comprise SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, the complementary sequence of SEQ.ID.NO:6, the complementary sequence of SEQ.ID.NO:7, the complementary sequence of SEQ.ID.NO:8 or the complementary sequence of SEQ.ID.NO:9 are as detecting nucleic acid base sequence.In some embodiments, probe groups can comprise and comprises SEQ.ID.NO:6 as the PNA probe that detects nucleic acid base sequence, comprise SEQ.ID.NO:7 as the PNA probe that detects nucleic acid base sequence, comprise SEQ.ID.NO:8 as the PNA probe that detects nucleic acid base sequence and/or comprise SEQ.ID.NO:9 as the PNA probe that detects nucleic acid base sequence.In some embodiments, the PNA probe groups can comprise the PNA probe shown in the SEQ.ID.NO:6, the PNA probe shown in the SEQ.ID.NO:7, the PNA probe shown in PNA probe shown in the SEQ.ID.NO:8 and/or the SEQ.ID.NO:9.

It is the very strong embodiment of the present invention that the PNA probe is divided into the group that is characterised in that special one or more gentle film bodies of detection.Probe groups can comprise at least one PNA probe, but need not to include only the PNA probe.For example, probe groups can comprise the mixture of PNA probe and nucleic acid probe and/or nucleic acid primer, and prerequisite is described group and comprises at least one PNA probe described herein.Be used to detect one or more gentle film bodies the PNA probe can be used for other biological probe combinations.Probe groups can with primer and/or other agent combination, described primer and/or other reagent are selected to be tested, for example in situ hybridization test or PCR test.In some embodiments, the sealing probe that can form by PNA or nucleic acid of some probes of described group.

Table 1 is listed the two or more detection nucleic acid base sequences that are suitable for detecting one or more gentle film bodies.When having selectable detection nucleic acid base sequence, can use the probe groups that contains two or more PNA probes, can increase the detectable signal in the test thus.Two or more PNA probes also can be combined into one group, can increase the number that can detect kind in the single test thus.

In some embodiments, probe groups can comprise two or morely independently can detect the PNA probe, but wherein each independently detection probes be suitable for gentle film bodies different in the test sample, perhaps gentle film body group.This test can be multiple test, if each of the wherein two or more gentle film body genus or the film body kind that softens is present in the sample, then can be detected independently.In some multiple tests, but suitable independence detection probes can be used for detecting each in to be identified, detection and/or the quantitative various different sorts.

C. Mixture:

In some embodiments, the invention still further relates to and be suitable for detecting, identify and/or the quantitative mixture of one or more gentle film bodies in the sample.Mixture can comprise one or more PNA probes described herein.General and the preferred feature of the PNA probe that is suitable for detecting gentle film body has been described in this paper front.Be suitable for the exemplary detection nucleic acid base sequence of this probe, list in the SEQ ID No.6-9 of table 1.

With the mixture of the gentle film body of the synthetic special detection of probe groups, be the very strong embodiment of the present invention.This mixture can comprise at least one PNA probe, but need not to include only the PNA probe.This mixture can be with the PNA probe, nucleic acid probe, and/or nucleic acid primer makes up.This mixture can will be used to detect the PNA probe of one or more gentle film bodies, and is used to detect other biological probe and makes up.The mixture that can select PNA probe and primer and/or other agent combination is to test, and for example in situ hybridization test or PCR test.In some embodiments, the probe of mixture can comprise the sealing probe of being made up of PNA or nucleic acid.

In some embodiments, the component that can select to be combined to form mixture is tested.For example, can select the component of mixture to carry out nucleic acid amplification reaction, described nucleic acid amplification reaction is the part of the test of one or more gentle film bodies in detection, evaluation and/or the quantitative sample.This mixture can comprise the nucleic acid primer of at least two long 15-30 Nucleotide, wherein each primer can both be hybridized the primer sites of gentle film body nucleic acid, with at least one 12-20 PNA probe that contains the subunit of nucleic acid base, wherein said PNA probe can be hybridized the target sequence of gentle film body nucleic acid.

For example, at least two nucleic acid primers can comprise separately, perhaps are made up of following sequence: SEQ ID.NO:1, SEQ ID.NO:2, SEQ ID.NO:3, SEQ ID.NO:4 or SEQ ID.NO:5.Similar, the detection nucleic acid base sequence of one or more PNA probes can comprise respectively, perhaps form: SEQ.ID.NO:6 by following sequence, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, the complementary sequence of SEQ.ID.NO:6, the complementary sequence of SEQ.ID.NO:7, the complementary sequence of SEQ.ID.NO:8 or the complementary sequence of SEQ.ID.NO:9.A kind of such mixture can comprise: a) primer shown in SEQ ID NO:1; Primer shown in SEQ ID NO:2; Primer shown in SEQ ID NO:3; Primer shown in SEQ ID NO:4; And/or the primer shown in SEQ ID NO:5; And, b) the PNA probe of forming as the detection nucleic acid base sequence by SEQ.ID.NO:6; By SEQ.ID.NO:7 as detecting the PNA probe that nucleic acid base sequence is formed; By SEQ.ID.NO:8 as detecting the PNA probe that nucleic acid base sequence is formed; And/or by SEQ.ID.NO:9 as detecting the PNA probe that nucleic acid base sequence is formed.

In some embodiments, mixture can also comprise and chooses other reagent that carries out pcr amplification reaction.In some embodiments, mixture can also comprise inner positive control.This reagent and contrast are commercialized supplies, or known to a person of ordinary skill in the art.

In some embodiments, the component that can choose mixture is carried out the in situ hybridization test.The original position analysis that utilizes the PNA probe is known (referring to U.S. Patent number 6,280,946,6,649,349,6,656,687 and 6,664,045).Therefore, adopt not transnormal experiment and information disclosed herein, can select to be suitable for detecting the component of mixture of one or more gentle film bodies.

In some embodiments, can select the component of mixture to carry out multiple test, can detect more than a kind of biology thus.In some cases, can design multiple test, detect two or more gentle film bodies.In some cases, can design multiple test, detect the mycoplasma that surpasses two kinds.

According to the test needs, select the quantity of every kind of component in the mixture.Therefore, the specific design of test will determine the amount of probe and primer.Because it all is known utilizing PNA probe and primer major part in the different sorts test, so not transnormal experiment and disclosed content combination herein just can determine to prepare the amount that is suitable for carrying out the required component of this test mixture.

D. Method:

In some embodiments, the invention still further relates to the method that is suitable for one or more gentle film bodies in the test sample.General and the preferred feature of the PNA probe that is suitable for detecting gentle film body has been described in this paper front.Exemplary detection nucleic acid base sequence is listed in the SEQ ID No.6-9 of table 1.

In some embodiments, described method comprises: a) sample and one or more are contained 12-20 PNA probe that contains the subunit of nucleic acid base and contact, wherein each in one or more PNA probes comprises, the detection nucleic acid base sequence that comprises following sequence or form: SEQ.ID.NO:6 by following sequence, SEQ.ID.NO:7, SEQ.ID.NO:8 or SEQ.ID.NO:9; And b) under suitable hybridization conditions or suitable in situ hybridization condition, the hybridization of at least one target sequence in the detection nucleic acid base sequence that detects the PNA probe and the sample, and with the existence of one or more gentle film bodies in detected result and the sample whether and/or quantity foundation related.By the direct or indirect measurement of probe/target sequence mixture, can set up this association.

In some embodiments, described method is to carry out in situ hybridization.Can be used for the biology of in-situ method analysis to analyze by the different methods preparation.Cell can be fixed on the slide glass, observes by film, photographic camera, slide scanner or microscope.Perhaps, cell can be fixed in flow cytometer then and analyze.Slide scanner and flow cytometer are particularly conducive to fast quantification and pay close attention to the biological number of the target that exists in the sample.

Can also analyze the nucleic acid in the biology.Usually these are analyzed, the nucleic acid of collection of biological, after the biomass cells cracking, collect nucleic acid usually.

After nucleic acid is collected, can carry out nucleic acid amplification reaction.Therefore, in some embodiments, described method also comprises: c) with at least two nucleic acid primers of sample and a long 15-30 Nucleotide with choose other reagent that carries out pcr amplification reaction, contact; And d) thus carrying out pcr amplification reaction produces one or more amplicons that comprise target sequence.In some embodiments, nucleic acid primer can comprise separately, perhaps is made up of following sequence: SEQ ID.NO:1, SEQ ID.NO:2, SEQ ID.NO:3, SEQ ID.NO:4 or SEQ ID.NO:5.In some embodiments, the PCR test is terminal point PCR test or PCR in real time test.In some embodiments, one or more PNA probes are self-indication PNA probe (for example embodiment 1).

E. Test kit:

In some embodiments, the present invention relates to be suitable for carrying out the test kit of the correlation test of one or more gentle film bodies in the test sample.This paper front has been described and has been suitable for detecting, identify and/or the quantitative general and preferred feature of one or more gentle film body PNA probes.

Test kit of the present invention can comprise one or more PNA probes and other reagent or composition, described other reagent or composition be select to test or the short form test process required.Test kit can, for example comprise and carry out required damping fluid and/or other reagent of PNA-ISH or PNA-FISH test.In other embodiments, can select damping fluid and/or other reagent to carry out nucleic acid amplification reaction, for example PCR reaction.Those skilled in the art will know that the reagent which kind of type is arranged in the test kit usually, be used to carry out PNA-ISH, PNA-FISH test or PCR test.

F. Utilize the example use of embodiment of the present invention:

PNA probe described herein, probe groups, mixture, method and test kit be verified to can be used for detecting one or more gentle film bodies.Detection be meant establish one or more gentle film bodies in the sample existence whether and/or quantity.In addition, as long as available immediate mode is carried out test described herein (in some embodiments 35 minutes; Fig. 6 for example).Described test is responsive, stable and effective in the single test of detection and/or count table 2 listed biologies.

No matter be incorporated into upholder or in solution, PNA probe described herein, probe groups, mixture, method and/or test kit can be used for the cell culture of Mammals, insect or plant origin, the medicament of making, vaccine or other cell culture deutero-biological product, perhaps be used to produce the raw material of personal care product, milk-product or bottled water, perhaps in the analysis of environmental sample one or more gentle film bodies fast, sensitivity and stable detection.The analysis that these especially can be used to make or store raw material, equipment, product or the step of food, water, medicament, personal care product, milk-product perhaps is used for the analysis of environmental sample.

No matter be incorporated into upholder still is that PNA probe described herein, probe groups, mixture, method and/or test kit can be used for the detection of one or more gentle film bodies in clinical or animal doctor's sample and the environment in solution.The limiting examples of clinical sample comprises: phlegm, throat's swab, liquid of gastric lavage, segmental bronchus washings, slicer, aspirate, begma, body fluid (ridge for example, pleura, pericardium, synovia, blood, purulence, amnion, and urine), marrow and tissue slice.Suitable PNA probe, probe groups, mixture, method and test kit especially also can be used for treating the analysis of clinical sample, device, equipment or the product of people or animal.

6. Embodiment

Can further understand all respects of this instruction according to the following example, this is not the scope that is used to limit this instruction in any case.

Utilize conventional synthetic and purification process to prepare whole PNA oligomers.All nucleic acid primer is available from commercial source.Is [this correct for Jens/Byron-? ]

The detection of embodiment 1 mycoplasma and relevant genus

Although designed the relevant genus that the test of many polymerase chain reactions is used to detect Mycoplasma and gentle film body (Dussurget waits the people, 1994; People such as Haier, 1999; People such as Jensen, 1999; People such as Kidder, 1993; People such as Tang, 2000; People such as van Kuppeveld, 1992; People such as van Kuppeveld, 1994), should the fact but these primer sets can not show to the complete specificity of its pre-determined target target, reflect to identify to be used for selective amplification and to belong to the difficulty that the primer of various biological group takes place in this huge and system people such as (, 2001) Kong.Described hereinly be used to detect mycoplasma and the dna primer of relevant genus and being used in combination of PNA probe, higher than the specificity of existing test, because it comprises two-layer specificity.The first layer specificity is the combination (SEQ ID No 1-5) of 5 kinds of dna primers described herein, and described primer is selected to comprise the sequence polymorphism of this extensive target biological group.Under the test conditions that present embodiment is described, expect that these primers can produce amplicon, for example acholeplasma and Ureaplasma to the relevant genus with gentle film body of a large amount of Mycoplasmas.

Note: 1) glu=L-glutamic acid; Lys=Methionin; 2) for the nucleic acid primer nucleic acid base sequence be 5 ' to 3 ' direction, be that N-terminal is to C-terminal for the PNA probe; NH ₂Show that PNA ends up with amide group; 3) between some primers and some probes, there is partly overlapping nucleic acid base sequence.This overlapping with the boldface type explanation.

Although design these primers and be the non-target bacterium DNA cloning of bacillus (Bacillus), lactobacillus (Lactobacillus), staphylococcus (Staphylococcus), suis (Streptococcus) and clostridium (Clostridium) for example that is used to avoid the most relevant genus, but still might take place and the unexpected cross reaction of other non-target bacterium (people such as Kong, 2001; People such as Jensen, 1999).Second layer specificity is the use (sequence 6-9) of 4 kinds of PNA probes described herein.The sequence-specific hybridization of these PNA probes is used to report that 4 kinds of experimental test targets exist to mycoplasma and other gentle film body expection amplicon.Introduce in test this two-layer specificity, unexpected primer and non-target biological gene group DNA or artifactitious cross reaction and side reaction for example primer dimer form, and their formed amplicons will can not obscured the explanation test-results.

The explanation of table 3:

Primer sequence:

The general reactive forward primer of SEQ ID No.1=.

SEQ ID No.2=is at mycoplasma arginini (Mycoplasma arginini), mycoplasma salivarium (Mycoplasma salivarium), Mycoplasma orale (Mycoplasma orale), mycoplasma arthritidis (Mycoplasma arthriditis), the reverse primer of mycoplasma hominis (Mycoplasma hominis) and related specy.

SEQ ID No.3=is at the reverse primer of mycoplasma fermentans (Mycoplasma fermentans) and related specy.

SEQ ID No.4=is at the reverse primer of Rider Lloyd's's acholeplasma (Acholeplasma laidlawii) and related specy.

SEQ ID No.5=is at pears shape mycoplasma (Mycoplasma pirum), mycoplasma pneumoniae (Mycoplasma pneumoniae), the reverse primer of genital tract mycoplasma (M.genitalium) and related specy.

Probe sequence:

SEQ ID No.6=is used for detecting the self-indication PNA probe that utilizes SEQ ID No.1 (forward primer) and SEQ ID No.2 (reverse primer) to make up the amplicon that is produced.

SEQ ID No.7=is used for detecting the self-indication PNA probe that utilizes SEQ ID No.1 (forward primer) and SEQ ID No.3 (reverse primer) to make up the amplicon that is produced.

SEQ ID No.8=is used for detecting the self-indication PNA probe that utilizes SEQ ID No.1 (forward primer) and SEQ ID No.4 (reverse primer) to make up the amplicon that is produced.

SEQ ID No.9=is used for detecting the self-indication PNA probe utilize the amplicon that SEQ ID No.1 (forward primer) and SEQ ID No.5 (reverse primer) produced.

Detect explanation:

This detection comprises 4 PNA probes, 1 forward PCR primer and 4 inverse PCR primers.Select this probe groups to identify nearly all known mycoplasma kind, comprise it is reported 8 " problem " kinds that cause 95% clone to pollute.This probe groups can also be identified Rider Lloyd's's acholeplasma, and as the pollution of cell culture thing, it is also paid close attention to very much.The target biology that other can utilize this detection to identify is listed in the table 2.This detection is at Spiroplasma (Spiroplasma), worm Ureaplasma (Entomoplasma), and middle Ureaplasma (Mesoplasma) and non-gentle film body belong to lactobacillus (Lactobacillus).

The PCR condition:

Real-time PCR reactions uses following condition: add 40 μ LPCR mixtures and 10 μ L mycoplasmas or acholeplasma genomic dna (American Type Culture Collection in the PCR pipe, American type culture collection, Manassas VA) or its suitable mixture.The PCR mixture comprises: and 1 * buffer A (Applied Biosystems, the Foster city, Foster City, CA), 3.0mM MgCl ₂, every kind of 0.25mM of 4 kinds of dNTP (deoxynucleoside triphosphate), 500nM PNA probe, 100nM forward primer, 1000nM reverse primer, 2 AmpliTaq of unit ^TMThe Gold polysaccharase (AppliedBiosystems, the Foster city, CA).

In 7000 sequence detection systems, utilize following circulation step to carry out the PCR reaction:

Preheating: 95 ℃ 10 minutes.

50 circulations:

95 ℃ of o 15 seconds.

60 ℃ of o 1 minute.

Terminal point PCR reaction conditions is as follows fast: add 20 μ LPCR mixtures and 5 μ l mycoplasma argininis (Mycoplasma arginini) and subtilis (Bacillus subtilis) genomic dna mixture (U.S. representative microbial preservation center in the PCR pipe, the Manassas, VA).Add various DNA ultimate density be 140fg.The ultimate density of PCR mixture is: and 1 * KOD Hot Start dna polymerase buffer liquid (Novagen, Madison, WI), 2.0mM MgCl ₂Every kind of 0.25mM of 4 kinds of dNTP (deoxynucleoside triphosphate), 500nM PNA probe (SEQ.ID NO.6), 100nM forward primer (SEQ.IDNO.1), 1000nM reverse primer (SEQ.ID NO.2), (Novagen, Madison WI) with in 1 * (50nM) ROX are marked dyestuff to 1 unit K OD Hot Start archaeal dna polymerase.In ABI 9800 fast PCR systems, carry out the PCR reaction by following circulation step:

50 circulations:

95 ℃ of o 2 seconds.

60 ℃ of o 5 seconds.

After PCR finishes, the fast PCR system board is transferred to ABI Prism 7700 sequence detection systems, collection spectrum and data are quantitative.Utilize ultraviolet diaphane also can observe fluorescence easily.

Inner positive control:

The following component of having described the inside positive control that uses in the PCR detection:

PNA probe: Vic-Glu-CTC-GTT-GAT-CTT-CCG-Lys-Lys (Dabcyl)

Forward primer: SEQ ID NO.10 CAT-CCG-CAC-ACT-ATC-TCA-TCG-T

Reverse primer: SEQ ID NO.11 CCA-CAC-TAT-CAA-TGC-CAG-AAC-GG

The partial sequence of plasmid-SEQ ID NO.12

TTT-TTT-TTT-TTT-TTT-TTT- TTC-ATC-CGC-ACA-CTA-TCT-CAT-CGT-TA

T-CGT-TCC-ATC-AGC-TCG-TTG-ATC-TT C-CGT-TCT-GGC-

ATT-GAT-AGT-GTG-GCG-GTT-GGA-TCC-CTA-TAG-TGA-GTC-GTA-TTA

The primer sites among the IPC represented in black matrix, and underscore is represented PNA probe binding site.The PNA probe of Vic mark is available from Applied Biosystems.

The result:

Fig. 3 A has shown that the mycoplasma PCR in real time based on PNA of wide dynamic range detects.In this experiment, pears shape mycoplasma (Mycoplasma pirum) genomic dna of various amounts is used as reaction template.Being used to produce the amount of pears shape mycoplasma (M.pirum) genomic dna of each curve, is nanogram, pik or method gram (femptogram).Corresponding target copy number calculates according to the amount of adding DNA and the genome size of pears shape mycoplasma (M.pirum), provides in bracket.This figure has proved that described detection is good to the DNA target level performance that surpasses 6-logarithm (6-log) scope.Fig. 3 B is the Ct curve of SEQ.ID.NO.9.This curve can illustrate that the relation-described Ct value of adding between reaction system target DNA concentration known and the gained Ct value is the point that amplification curve and detection threshold intersect.This linear relationship can be used for calculating the target DNA concentration that is present at first in the unknown sample.In addition, the slope of this line also can be used for calculating the efficient of PCR reaction.

Fig. 4 shows that the PNA probe of SEQ.ID.NO:6 correspondence is to its expection target group (mycoplasma arginini (Mycoplasma arginini), mycoplasma arthritidis (Mycoplasma arthriditis), mycoplasma hominis (Mycoplasma hominis), Mycoplasma orale (Mycoplasma orale), mycoplasma salivarium (Mycoplasma salivarium) and related specy) specificity.Non-target mycoplasma kind (mycoplasma fermentans (Mycoplasma fermentans), Rider Lloyd's's acholeplasma (Acholeplasma laidlawii), pears shape mycoplasma (Mycoplasma pirum), mycoplasma pneumoniae (Mycoplasma pneumoniae)) do not produce signal, although there is the primer that can produce amplicon in the reaction from these kinds type genomic dna.

Fig. 5 shows the specificity of the PNA probe of SEQ.ID.NO:8 correspondence to its expection target group (Rider Lloyd's's acholeplasma (Acholeplasma laidlawii) and related specy).Non-target mycoplasma kind (mycoplasma arginini (Mycoplasma arginini), mycoplasma fermentans (Mycoplasma fermentans), pears shape mycoplasma (Mycoplasma pirum), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and subtilis (Bacillus subtilis)) do not produce signal, although there is the primer that can produce amplicon in the reaction from these kinds type genomic dna.

The mycoplasma PCR in real time that shows Fig. 6 detects to be differentiated the selectivity of mycoplasma kind.To comprise the multiple detection of the PNA probe of the dna primer of corresponding SEQ.ID No.1-5 and corresponding SEQ.ID.No.6-9, be combined in the single test tube that contains target and non-target bacterial genomes DNA.Utilize this method to be easy to detect the DNA of mycoplasma and 4 target groups of representativeness of relevant gentle film body.Do not detect the DNA of the non-target bacterial species of representative (subtilis (Bacillus subtilis), Pseudomonas aeruginosa (Pseudomonas aeruginosa), lactobacillus delbruckii (Lactobacillus delbrueckii)).

Fig. 7 shows dna primer described herein and PNA probe and the coupling of fast PCR system, is used for fast and the gentle film body of selectivity end point determination, and gentle film body in this example is mycoplasma arginini (Mycoplasma argnini).Target biology (mycoplasma arginini (Mycoplasma argnini), Δ Δ Rn～1.72) and non-target organism (subtilis (Bacillus subtilis), Δ Rn～0.59) greatest differences between the final fluorescent value in 530nm place, the specificity of having given prominence to reaction.The secondary fluorescence maximum at～605nm place belongs to interior mark, do not have diagnostic value.

Discuss:

Asymmetric PCR allows chain of the preferential amplification of another chain relatively.This realizes that by adding more the primer of volume described primer instructs needs the synthetic of excessive chain.The primer of excessive existence is limited resources of vast scale (Nucleotide, magnesium, or the like) more in the consumption reaction pipe, and this will cause the deflection of product in forming, especially in the reaction logarithmic phase.In the PCR of the PNA-probe that utilizes present embodiment to describe detects, expect that excessive product is the polynucleotide base chain (the amplification subchain that promptly contains PNA probe complementary nucleic acid base sequence) that contains target sequence.In the detection that present embodiment is described, forward primer (SEQ ID No.1) exists with the level of 0.1 μ M, and reverse primer (SEQ ID No.2-5) separately the concentration with 1 μ M exist.Used PNA concentration and probe concentration is 0.5 μ M.

Except can be used for detecting the mycoplasma contamination in biopharmaceutical material or the process, the existence that the unique combination of primer that present embodiment is described and probe also can be used for detecting relevant mycoplasma medically whether, because chip specificity (in silico specific) check shows, they can detect most of mycoplasma relevant with illness or urea substance (Ureaplasma) kind, for example relevant with other urogenical infection (people such as Yoshida, 2002) with people's nongonococcal urethritis (nongnococcal urethritis).These primers and probe also can be used for animal doctor's diagnostic use, because their expections can detect and the mycoplasma kind of important economic animal disease-related people such as (, 2002) Persson.

The target of this detection is to detect the kind that belongs to the relevant genus with gentle film body of mycoplasma of maximum quantity.Consider target group's size and system's generation diversity, before experiment and do not know whether the primer sequence of selecting has and detect required operation specificity.Therefore, it is that some is surprising, although biological mycoplasma arginini (M.argininiit) of target and Mycoplasma orale (M.orale) have a mispairing with reverse primer shown in the sequence No.2, under used terminal point PCR condition, still can detect these kinds easily.Similarly, mycoplasma salivarium (M.salivarium) has two mispairing with this sequence, also can detect easily in final the detection.

Although this instruction is described in conjunction with different embodiments, this does not also mean that this instruction is subject to these embodiments.On the contrary, this instruction comprises different replacements, change and coordinator, and this point it will be appreciated by those skilled in the art that.

7. Reference

1.Dussurget, O., and D.Roulland-Dussoix.1994.Rapid, sensitive PCR-baseddetection of Mycoplasmas in simulated samples of animal sera. (detection based on sensitive PCR of mycoplasma " in the animal serum analog sample to "), Appl.Environ.Microbiol.60:953-959.

2.Haier, J., M.Nasralla, A.R.Franco, with G.L.Nicolson.1999.Detection ofmycoplasmal infections in blood of patients with rheumatoid arthritis. (" detection of mycoplasma infection in the rheumatic arthritis blood samples of patients "), Rheumatology 38:504-509.

3.Hart, M.K., R.A.Del.Giudice, and G.W.Korch, Jr.2002.Absence ofMycoplasma contamination in the anthrax vaccine. (" disappearance of mycoplasma contamination in the anthrax vaccine "), Emerg.Infect.Dis.8:94-96.

4.Jensen, J., Bruun, B., with B.Gahrn-Hansen.1999.Unexpected cross-reactionwith Fusobacterium necrophorum in a PCR for detection of Mycoplasmas. (the unexpected cross reaction of fusobacterium necrophorum " among the detection of mycoplasma PCR with "), J.Clin.Microbiol.37:828-829.

5.Kidder, M., P.J.Chan, I.M.Seraj, W.C.Patton, with A.King.1998.Assessmentof archived paraffin-embedded cervical condyloma tissues formycoplasma-conserved DNA using sensitive PCR-ELISA. (" adopting responsive PCR-ELISA "), Gynecol.Oncol.71:254-257. to preserving the assessment of mycoplasma conserved dna in the paraffin embedding uterine neck condyloma tissue

6.Kong, F., James, G.,-Gordon, S.Zelynski, A. and G.L.Gilbert.2001.Species-specific PCR for identification of common contaminant mollicutes in cellculture. (" the specific specificity PCR that is used for the common gentle film body Identification of pollutants of cell culture ") Appl.Environ.Microbiol.67:3195-3200.

7.Persson, A., Jacobsson, K., Frykberg, L., Johansson, K.-E. and F.Poumarat.2002.Variable surface protein Vmm of Mycoplasma mycoides subsp.mycoidessmall colony type. (" the variable surface albumen of mycoplasma mycoides sheep subspecies bacterium gill fungus shape small colonies class ") J.Bacteriol.184:3712-3722.

8.Razin, S., D.Yogev and Y.Naot.1998.Molecular biology and pathogenicity ofMycoplasmas. (" mycoplasma molecular biology and pathogenicity bo ") Microbiol.MoI.Biol.Rev.62:1094-1156.

9.Rottem, S. and M.F.Barile.1993.Beware of mycoplasmas. (" guarding against mycoplasma ") Trends Biotechnol.11:143-151.

10.Tang, J., M.Hu, S.Lee and R.Roblin.2000.A polymerase chain reactionbased method for detecting Mycoplasmal Acholeplasma contaminants in cellculture. (" detecting ") J.Microbiol.Methods 39:121-126. based on mycoplasma acholeplasma pollutent in the cell culture of polymerase chain reaction

11.Uphoff, CC and H.G.Drexler.1999.Detection of Mycoplasma contaminationsin cell cultures by PCR analysis.Hum. (" by the detection of mycoplasma contamination in the pcr analysis pair cell culture ") Cell 12:229-236.

12.van Kuppeveld, F.J.M., J.T.M.van der Logt, A.F.Angulo, MJ.van Zoest, G.G.V.Quint, H.G.M.Niesters, J.M.D.Galama and W.J.G.Melchers.1992.Genus-and species-specific identification of mycoplasmas by 16S rRNAamplification. (" genus and the specific specificity of mycoplasma being differentiated ") Appl.Environ.Microbiol.58:2606-2615. by 16S rRNA amplification

13.van Kuppeveld, F.J.M., K.-E.Johansson, J.M.D.Galama, J.Kissing, G.B ó lske, J.T.M.va der Logt and W.J.G.Melchers.1994.Detection of Mycoplasmacontamination in cell cultures by a Mycoplasma group-specific PCR. (" by the detection of mycoplasma contamination thing in the mycoplasma group-specific PCR pair cell culture ") Appl.Environ.Microbiol.60:149-152.

14.Yoshida, T., Maeda, S.-l., Deguchi, T. and H.Ishiko.2002.Phylogeny-basedrapid identification of mycoplasmas and ureaplasmas from urethritis patients. (" differentiating ") J.Clin.Microbiol.40:105-110. based on phylogenetic mycoplasma and urea substance available from the urethritis patient

Sequence table

＜110〉Applera Corp. (APPLERA CORPORATION)

J.J. Yi Digenilesen (HYLDIG-NIELSEN, JENS j.)

S. league (unit of length) than (RIGBY, SUSAN)

B.F. cloth rem-Si Teqieer (BREHM-STECHER, BYRON F.)

D.S. Lee (LEE, DITTE S.)

M. smooth receiving (TANNER, MICHAEL)

＜120〉detect relevant PNA probe, mixture, method and test kit with mycoplasma and relevant gentle film body

<130>5387US

<150>US?60/678,331

<151>2005-05-06

＜150〉US is undetermined

<151>2006-05-04

<160>12

<170>PatentIn?version?3.3

<210>1

<211>25

<212>DNA

＜213〉mycoplasma kind (Mycoplasma sp.)

<400>1

acaggattag?ataccctggt?agtcc 25

<210>2

<211>23

<212>DNA

＜213〉mycoplasma kind (Mycoplasma sp.)

<400>2

cctttgagtt?tcactcttgc?gag 23

<210>3

<211>23

<212>DNA

＜213〉mycoplasma kind (Mycoplasma sp.)

<400>3

cctttaagtt?ttattcttgc?gaa 23

<210>4

<211>26

<212>DNA

＜213〉mycoplasma kind (Mycoplasma sp.)

<400>4

gtcaattcct?ttgagtttca?tacttg 26

<210>5

<211>24

<212>DNA

＜213〉mycoplasma kind (Mycoplasma sp.)

<400>5

aattccgttt?gagtttcatt?cttg 24

<210>6

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<212>DNA

＜213〉mycoplasma kind (Mycoplasma sp.)

<400>6

ctgagtagta?tgctcg 16

<210>7

<211>16

<212>DNA

＜213〉mycoplasma kind (Mycoplasma sp.)

<400>7

ctgagtagta?cgttcg 16

<210>8

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＜213〉mycoplasma kind (Mycoplasma sp.)

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agtacgtacg?caagt 15

<210>9

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＜213〉mycoplasma kind (Mycoplasma sp.)

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gtagtacatt?cgcaaga 17

<210>10

<211>22

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＜213〉mycoplasma kind (Mycoplasma sp.)

<400>10

catccgcaca?ctatctcatc?gt 22

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＜213〉mycoplasma kind (Mycoplasma sp.)

<400>11

ccacactatc?aatgccagaa?cgg 23

<210>12

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＜213〉mycoplasma kind (Mycoplasma sp.)

<400>12

tttttttttt?tttttttttt?catccgcaca?ctatctcatc?gttatcgttc?catcagctcg 60

ttgatcttcc?gttctggcat?tgatagtgtg?gcggttggat?ccctatagtg?agtcgtatta 120

Claims (41)

1. PNA probe, it has 12-20 subunit that contains nucleic acid base, and described PNA probe comprises and is used for detecting, identifying and/or the quantitative detection nucleic acid base sequence of one or more gentle film bodies of sample.

2. PNA probe as claimed in claim 1 is characterized in that, described one or more gentle film bodies are mycoplasma, acholeplasma or urea substance.

3. PNA probe as claimed in claim 1 is characterized in that, described probe comprises the detection nucleic acid base sequence that is selected from following sequence:

SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, the complementary sequence of SEQ.ID.NO:6, the complementary sequence of SEQ.ID.NO:7, the complementary sequence of SEQ.ID.NO:8 and the complementary sequence of SEQ.ID.NO:9.

4. PNA probe as claimed in claim 1, it is characterized in that, the detection nucleic acid base sequence of described PNA probe is SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, the complementary sequence of SEQ.ID.NO:6, the complementary sequence of SEQ.ID.NO:7, the complementary sequence of SEQ.ID.NO:8 or the complementary sequence of SEQ.ID.NO:9.

5. PNA probe as claimed in claim 1 is characterized in that described probe is unlabelled.

6. PNA probe as claimed in claim 1 is characterized in that, but described probe is with at least a test section mark.

7. PNA probe as claimed in claim 6 is characterized in that, but described test section is selected from: dextran binding substances, side chain nucleic acid detection system, chromophore, fluorophor, spin label, radio isotope, enzyme, haptens, acridinium ester and chemiluminescence compound.

8. PNA probe as claimed in claim 1 is characterized in that, probe is and the upholder bonded.

9. PNA probe as claimed in claim 1 is characterized in that probe is a self-indication.

10. PNA probe groups, it is suitable for detecting, identifies and/or quantitative one or more gentle film bodies in the sample.

11. PNA probe groups as claimed in claim 10 is characterized in that, described one or more gentle film bodies are mycoplasma, acholeplasma or urea substance.

12. probe groups as claimed in claim 10 is characterized in that, every kind of PNA probe comprises 12-20 subunit that contains nucleic acid base.

13. probe groups as claimed in claim 10 is characterized in that, described group at least a PNA probe comprises the detection nucleic acid base sequence that is selected from following sequence:

SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, the complementary sequence of SEQ.ID.NO:6, the complementary sequence of SEQ.ID.NO:7, the complementary sequence of SEQ.ID.NO:8 and the complementary sequence of SEQ.ID.NO:9.

14. probe groups as claimed in claim 10 is characterized in that, described group comprises and comprises SEQ.ID.NO:6 as the PNA probe that detects nucleic acid base sequence; Comprise SEQ.ID.NO:7 as the PNA probe that detects nucleic acid base sequence; Comprise SEQ.ID.NO:8 as the PNA probe that detects nucleic acid base sequence; And/or comprise SEQ.ID.NO:9 as the PNA probe that detects nucleic acid base sequence.

15. probe groups as claimed in claim 10 is characterized in that, described group comprises by SEQ.ID.NO:6 as detecting the PNA probe that nucleic acid base sequence is formed; By SEQ.ID.NO:7 as detecting the PNA probe that nucleic acid base sequence is formed; By SEQ.ID.NO:8 as detecting the PNA probe that nucleic acid base sequence is formed; And/or by SEQ.ID.NO:9 as detecting the PNA probe that nucleic acid base sequence is formed.

16. probe groups as claimed in claim 10 is characterized in that, but whole probes of described group are with one or more test section marks.

17. probe groups as claimed in claim 10 is characterized in that, described group at least a probe is a self-indication.

18. probe groups as claimed in claim 10 is characterized in that, described group at least a probe is and the upholder bonded.

19. a mixture, it comprises:

A) nucleic acid primer of at least two kinds long 15-30 Nucleotide, wherein every kind can both with the hybridization of primer sequence in one or more gentle film body nucleic acid and

B) at least a 12-20 PNA probe that contains the subunit of nucleic acid base, it can be hybridized with the target sequence in one or more gentle film body nucleic acid.

20. mixture as claimed in claim 19 is characterized in that, described one or more gentle film bodies are mycoplasma, acholeplasma or urea substance.

21. mixture as claimed in claim 19, it also comprises:

C) choose other reagent that carries out pcr amplification reaction.

22. mixture as claimed in claim 21, it also comprises inner positive control.

23. mixture as claimed in claim 19, it also comprises:

C) choose other reagent that carries out the in situ hybridization detection.

24. mixture as claimed in claim 19 is characterized in that, described at least two kinds of nucleic acid primers independently are SEQ ID.NO:1, SEQ ID.NO:2, SEQ ID.NO:3, SEQ ID.NO:4 or SEQ ID.NO:5 separately.

25. mixture as claimed in claim 19, it is characterized in that, one or more described PNA probes comprise the detection nucleic acid base sequence that independently is selected from following sequence: SEQ.ID.NO:6 separately, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, the complementary sequence of SEQ.ID.NO:6, the complementary sequence of SEQ.ID.NO:7, the complementary sequence of SEQ.ID.NO:8 and the complementary sequence of SEQ.ID.NO:9.

26. mixture as claimed in claim 19 is characterized in that, the detection nucleic acid base sequence of every kind of probe independently is:

SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, the complementary sequence of SEQ.ID.NO:6, the complementary sequence of SEQ.ID.NO:7, the complementary sequence of SEQ.ID.NO:8 or the complementary sequence of SEQ.ID.NO:9.

27. mixture as claimed in claim 19 is characterized in that, one or more described PNA probes are self-indications.

28. mixture as claimed in claim 19, it comprises:

A) primer shown in SEQ ID NO:1; Primer shown in SEQ ID NO:2; Primer shown in SEQ IDNO:3; Primer shown in SEQ ID NO:4; And/or the primer shown in SEQ ID NO:5; And,

B) the PNA probe of forming as the detection nucleic acid base sequence by SEQ.ID.NO:6; By SEQ.ID.NO:7 as detecting the PNA probe that nucleic acid base sequence is formed; By SEQ.ID.NO:8 as detecting the PNA probe that nucleic acid base sequence is formed; And/or by SEQ.ID.NO:9 as detecting the PNA probe that nucleic acid base sequence is formed.

29. a method, it comprises:

A) sample and one or more being contained 12-20 PNA probe that contains the subunit of nucleic acid base contacts, each the self-contained detection nucleic acid base sequence that independently is selected from following sequence: SEQ.ID.NO:6 of one or more described PNA probes wherein, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, the complementary sequence of SEQ.ID.NO:6, the complementary sequence of SEQ.ID.NO:7, the complementary sequence of SEQ.ID.NO:8 and the complementary sequence of SEQ.ID.NO:9; With

B) under suitable hybridization conditions or suitable in situ hybridization condition, hybridization at least a target sequence in one or more described PNA probe in detecting nucleic acid base sequences and the sample, detect, with the existence of one or more gentle film bodies in result and the sample whether and/or quantity set up related.

30. method as claimed in claim 29 is characterized in that, described one or more gentle film bodies are mycoplasma, acholeplasma or urea substance.

31. method as claimed in claim 29, it also comprises:

C) with at least two kinds of nucleic acid primers of sample and a long 15-30 Nucleotide with choose other reagent that carries out pcr amplification reaction and contact; With

D) thus carrying out pcr amplification reaction produces the amplicon that one or more contain one or more target sequences.

32. method as claimed in claim 31 is characterized in that, described pcr analysis is terminal point pcr analysis or PCR in real time analysis.

33. method as claimed in claim 29 is characterized in that, one or more described PNA probes are self-indications.

34. method as claimed in claim 32, it also comprises:

E) sample is contacted with at least a inner positive control.

35. a test kit, it comprises:

A) nucleic acid primer of at least two kinds long 15-30 Nucleotide wherein can both be hybridized with the primer sequence in one or more gentle film body nucleic acid for every kind; With

B) at least a PNA probe, it has 12-20 subunit that contains nucleic acid base, and described PNA probe can be hybridized with the target sequence in one or more gentle film body nucleic acid.

36. test kit as claimed in claim 35 is characterized in that, described one or more gentle film bodies are mycoplasma, acholeplasma or urea substance.

37. test kit as claimed in claim 35, it also comprises:

C) choose other reagent that carries out pcr amplification reaction.

38. test kit as claimed in claim 35, it also comprises:

C) at least a inner positive control.

39. test kit as claimed in claim 38 is characterized in that, described at least two kinds of nucleic acid primers independently are separately:

SEQ ID.NO:1, SEQ ID.NO:2, SEQ ID.NO:3, SEQ ID.NO:4 or SEQID.NO:5.

40. test kit as claimed in claim 38 is characterized in that, one or more described PNA probes comprise the detection nucleic acid base sequence that independently is selected from following sequence separately:

SEQ.ID.NO:6, SEQ.ID.NO:7, SEQ.ID.NO:8, SEQ.ID.NO:9, the complementary sequence of SEQ.ID.NO:6, the complementary sequence of SEQ.ID.NO:7, the complementary sequence of SEQ.ID.NO:8 and the complementary sequence of SEQ.ID.NO:9.

41. test kit as claimed in claim 35 is characterized in that, one or more described PNA probes are self-indications.

Images