WO2013009084A9 - Composition pour la détection simultanée de mycobacterium tuberculosis et de mycobactéries non-tuberculeuses au moyen d'une réaction en chaîne de la polymérase multiplexe en temps réel comprenant un système de sonde pna d'hybridation nichée ayant une structure de liaison parallèle, et procédé de détection à l'aide de celle-ci - Google Patents

Composition pour la détection simultanée de mycobacterium tuberculosis et de mycobactéries non-tuberculeuses au moyen d'une réaction en chaîne de la polymérase multiplexe en temps réel comprenant un système de sonde pna d'hybridation nichée ayant une structure de liaison parallèle, et procédé de détection à l'aide de celle-ci Download PDF

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WO2013009084A9
WO2013009084A9 PCT/KR2012/005483 KR2012005483W WO2013009084A9 WO 2013009084 A9 WO2013009084 A9 WO 2013009084A9 KR 2012005483 W KR2012005483 W KR 2012005483W WO 2013009084 A9 WO2013009084 A9 WO 2013009084A9
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pna
probe
tuberculosis
composition
mycobacterium tuberculosis
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WO2013009084A3 (fr
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김성기
박창식
조군호
박희경
김지현
최지아
최성록
김수남
김용태
김세련
장현정
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주식회사 파나진
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/35—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycobacteriaceae (F)

Definitions

  • the present invention is a composition for simultaneous detection of Mycobacterium tuberculosis and non-tuberculosis acid bacterium based on a double hybridized Peptide Nucleic Acid (PNA) probe system of parallel binding structure, and a method for simultaneous detection of Mycobacterium tuberculosis and non-tuberculosis acid bacterium using the composition and real-time polymerase chain reaction. It is about.
  • the present invention provides a primer, a probe composition and an analysis method for the simultaneous detection of Mycobacterium tuberculosis and non-tuberculosis antibacterial bacterium based on a double-hybrid PNA probe system of parallel binding structure.
  • Real-time multiplex polymerase chain reaction using a composition comprising a double-hybrid hybridized PNA probe system of parallel binding structure according to the present invention it is possible to accurately detect tuberculosis bacteria and non-tuberculosis antibacterial bacteria in one tube with excellent sensitivity at the same time. Therefore, there is an advantage that more rapid and accurate clinical diagnosis is possible.
  • Tuberculosis is caused by Mycobacterium tuberculosis, a bacterium of 0.2-0.5 ⁇ m in thickness and 1-4 ⁇ m in length, and is the most infectious disease in human history.
  • tuberculosis is a wasting chronic disease caused by Mycobacterium tuberculosis (MTB), which is detected in the tuberculosis of a tuberculosis patient, according to the WHO report.
  • MTB Mycobacterium tuberculosis
  • mycobacterium other than MTB and Mycobacterium bovis was detected in the human body, but it was considered non-pathogenic bacterium just because of contamination or colonization.
  • Atypical mycobacteria anonymous mycobacteria , nontuberculous mycobacteria (NTM), mycobacterium other than tuberculosis (MOTT), etc. have been mixed. It is known in the 1950s that such non-tuberculosis mycobacterium can cause disease in humans, but since the 1980s, the mycobacterium avium complex causes systemic disease in many AIDS patients. From the known, increasing interest in non-tuberculosis mycobacterium disease has led to advances in diagnosis and treatment.
  • Tuberculosis can be diagnosed by a chest X-ray or by a doctor's clinical judgment that incorporates a variety of other conditions.
  • Laboratory diagnostic methods for tuberculosis include smear, culture, immunological and molecular diagnostic tests.
  • the smear test is simple and economical, and has the advantage of detecting infectious tuberculosis patients, but has the disadvantage of having to repeat two or three times the test.
  • the culture test is a method of isolating and identifying tuberculosis bacteria after the antibacterial culture test, but the only method for confirming tuberculosis has a disadvantage in that it takes a long time to culture.
  • Immunological diagnostic methods include tuberculin test and extracorporeal Interferon- ⁇ test to detect the presence of Mycobacterium tuberculosis antibodies by injecting Mycobacterium tuberculosis antigen into the skin layer.
  • Non-TB bacterium was classified by colony color, shape and growth rate and identified by biochemical methods such as niacin production, nitrate reduction and Tween-80 hydrolysis. This biochemical method requires accurate, time-consuming, and trained personnel.
  • HPLC high performance liquid chromatography
  • nucleic acid probe method nucleic acid probe method
  • PCR-restriction fragment length polymorphism analysis polymerase chain reaction-restriction fragment length polymorphism analysis
  • PRA polymerase chain reaction-restriction fragment length polymorphism analysis
  • Molecular diagnostics is an effective method for analyzing human genes (DNA or RNA) to diagnose disease infection or to identify sequencing or mutations of genes to predict and identify disease outbreaks.
  • DNA or RNA human genes
  • it is considered as the best technology among the existing disease diagnosis methods, and it is one of the technologies currently attracting attention in the medical field.
  • Typical examples include methods using real-time PCR, methods using DNA-based probes, methods using PNA-based probes, and the like. The features of each method are briefly described below.
  • Real-time PCR analysis involves the PCR amplification product generation process through polymerase chain reaction (PCR) and the intensity of the fluorescence signal in real time by combining with primers or probes labeled with fluorescent material. By showing, more accurate quantitative analysis is possible.
  • the target nucleotide detection method used for real-time PCR can be largely divided into two types. The first is primer-based detection, which has the disadvantage of difficulty in design and quantitative analysis. The second method is a probe-based detection method, which is convenient in design and can be applied to both quantitative and qualitative analysis. The advantages and disadvantages of the two detection methods are shown in Table 1 [Meti Buh Ga, et al., Anal. Bioanal. Chem. 396, 2023, 2010].
  • TaqMan probes are linear probes that combine a reporter molecule and a quencher molecule at the ends of a DNA sequence capable of complementarily binding to a target nucleotide, and enzymatic cleavage of the probe sequence bound to the target nucleotide. This is a method of detecting a signal of a fluorescent (reporter) material that deviates from [Holland, PM, et al., Proc. Nat'l Acad. Sci. USA, 88, 7276-7280, 1991; Livak, KJ, et al., PCR Methods Appl., 4, 357-362.
  • This method has the disadvantage of lowering the discrimination ability of single nucleotide sequence mutations, and thus, MGB tags have been shortened by introducing a minor groove binder (MGB) with a matte material at the 3 'end for the purpose of improving the discrimination ability of single nucleotide sequences.
  • MGB minor groove binder
  • TaqMan has also been developed by Igor VK, et al, Nucl. Acids Res. 25, 3718-3723, 1997; Igor V. K., et al, Nucl. Acids Res. 28 (2): 655-661, 2000; I. A. Afonina, ea al, BioThechniques, 32, 940-949, 2002; I. A. Afonina, ea al, Nucleic Acids Research, 25, 2657-2660, 1997].
  • Molecular Beacons is a new type of probe consisting of a stem (stem) structure to form a loop and hairpin (hairpin) structure of the base sequence complementary to the target nucleotide. While this method has the advantage of distinguishing single nucleotide sequence variation, it is difficult to design and synthesize probes [US 20080064033 A; S. Tyagi, et al., Nat. Biotechnol., 16, 49, 1998; Stryer, L., Ann. Rev. Biochem., 47, 819-846, 1987; S. Tyagi, et al., Nat. Biotechnol., 14, 303-308, 1996; Bonnet, G., Proc. Natl Acad. Sci. USA, 96, 61716176, 1999].
  • DNA probe-based detection methods make DNA less stable by damage by enzymes such as nucleases and proteases [Demidov et al., Biochem. Phamacol. 48, 1310-1313, 1994], as well as weak DNA-DNA binding ability due to the charge repulsion between negative charges of the DNA backbone and low single nucleotide sequence discrimination ability due to the use of long sequences to overcome it.
  • enzymes such as nucleases and proteases
  • PNA-based probes In order to compensate for the shortcomings of using DNA probes, methods using PNA, an analog of DNA, have been studied. Since PNA has no charge in its backbone, it has less repulsion in binding to complementary DNA oligomers with negative charges, which enables faster and stronger binding to target nucleotide sequences than DNA probes and shows high stability without damage by enzymes. Egholm et al., Nature 365, 556-568, 1993 ,; Nielsen et al., Bioconjugate Chem, 5, 3-7, 1994; Demidov, et al., Biochem. Pharmacol. 48, 1310-1313, 1994].
  • PNA-based Mycobacterium tuberculosis (MTB) and non -tuberculous mycobacteria (NTM) for simultaneous detection of target nucleotides in a sample can be detected simultaneously.
  • MTB Mycobacterium tuberculosis
  • NTM non -tuberculous mycobacteria
  • the detection method according to the present invention is characterized by performing a real-time PCR method using two real-time nucleic acid amplification PNA probes capable of specifically binding to MTB and NTM and having dual hybridization in a parallel binding structure. It is done.
  • MTB and NTM can be detected simultaneously with high sensitivity and specificity, which is very useful for the diagnosis of tuberculosis.
  • the present invention provides a kit for diagnosing tuberculosis, comprising two PNA probe systems for amplifying nucleic acids in real time, capable of specifically binding to the MTB and NTM, and capable of double hybridization.
  • probe-based detection generally has many advantages over primer-based detection in detecting target nucleotides. Probes used at this time are largely divided into two types, and the pros and cons of each probe are compared in Table 2.
  • the structured probe is known to have excellent detection specificity against single nucleotide sequence mutations, but unless designed to have a stable hairpin structure due to the binding force of the stem, quenching may be incomplete and generate nonspecific fluorescence. Can be. Therefore, design and synthesis are difficult because the probe must be manufactured in consideration of the binding energy difference between the binding energy of the stem and the target nucleotide.
  • the linear probe has various advantages including convenience of fabrication, but has a disadvantage in that the detection ability against a single nucleotide sequence variation is poor due to the absence of a stem.
  • the inventors of the present invention have attempted to fabricate a PNA probe system having both the advantages of a linear probe, which is easy to design and synthesize, and a molecular beacon, which exhibits high detection of single nucleotide sequences.
  • PNAs can hybridize with PNAs having complementary sequences in two forms, anti-parallel binding and parallel binding [FIG. 1], and the binding energy between them is shown in [FIG. 2] [Stefano Sforza, Eur. J. Org. Chem., 197-204, 1999]. Due to this difference in binding energy, the dual linear PNA probes with parallel binding sequences in the absence of the target nucleotides in the sample do not fluoresce through complementary binding to each other. In addition, fluorescence is generated by dissociation of existing PNA-PNA probes.
  • the first PNA probe is synthesized according to the target Tm, and the binding strength between the two PNA probes is completely complementary to each other in the sequence of the PNA-DNA by using the parallel binding, which is relatively weak in binding strength and easy to control.
  • the second PNA probe was designed and synthesized such that some of the sequences of perfect-match and PNA-DNA were intermediate between different incomplete complementary mismatches.
  • the PNA probe according to the present invention has a form in which a reporter material and a quencher material are bound to one or both ends of a PNA oligomer having a predetermined sequence.
  • PNA probe according to the invention is preferably in the form of a combination of the physical properties control site and / or reporter material and the matting material at both ends, such as the structure of formula (1), but is not limited to this, to achieve the object of the present invention It will be apparent to those skilled in the art that any PNA probe structure having any structure can be used.
  • P is a PNA base moiety having a sequence complementary to the target nucleotide
  • N in the subscript is the number of PNA bases, preferably an integer of 7 to 25, more preferably an integer of 8 to 18. It is the part which forms parallel binding or anti-parallel binding to a target nucleotide.
  • a and A ′ may be the same or different materials as reporter molecules or quencher molecules, or only one of them may be present.
  • X and X ' may be the same or different materials as the physical property control site, none may be included, and one or more may be included.
  • N ' and C' mean N -terminal and C -terminal, respectively.
  • the PNA base portion P may have a structure as shown in Chemical Formula 2, but is not limited thereto. It will be apparent to those skilled in the art that PNA base having any structure can be used as long as the object of the present invention can be achieved. .
  • B is selected from a natural nucleic acid base or a non-natural nucleic acid base including adenine, cymine, guanine, cytosine, and uracil as a nucleic acid base, and in the simplest case, R or S is hydrogen (H). It may not be present but may be modified with isomeric substituents. R or S may also be in a modified form of a reporter molecule or a quencher molecule with labeled isomeric substituents [Ethan A. et al., Organic Lett. 7 (16), 3465-3467, 2005].
  • PNA probes are dual linear structures that form parallel bonds, are easy to design and synthesize, and have high sensitivity and specificity by rapid complementary binding with target nucleotides without non-specific signals.
  • composition or kit comprising the PNA probe specific for MTB and NTM according to the present invention has high sensitivity and specificity and can simultaneously detect MTB and NTM. That can be used to diagnose tuberculosis.
  • 1 is a diagram showing a parallel binding and anti-paralle binding structure between PNA-PNA.
  • FIG 3 shows a method for detecting a target nucleotide using a dual hybridized PNA probe system.
  • a second PNA probe is used as a detection probe in the method for detecting a target nucleotide using a double hybridized PNA probe system having a parallel binding structure.
  • Example 6 is a view showing a calibration curve and a result of performing PCR for confirming the detection sensitivity of warfarin single nucleotide sequence detection and the applicability of the quantitative method in relation to Example 8 using the method of the present invention.
  • Example 7 is a view showing a calibration curve and a result of performing PCR for confirming the ITS gene detection sensitivity and the applicability of the quantitative assay in relation to Example 9 using the method of the present invention.
  • FIG. 9 is a diagram showing PCR results of detecting simultaneously Mycobacterium tuberculosis (MTB) and Non-tuberculous mycobacteria (NTM) using PNA probes labeled with different fluorescence.
  • delta) means the detection line of non-tuberculous acid bacterium).
  • FIG. 10 is a diagram showing the change in melting temperature for a single nucleotide variant by a second probe (right peak represents the melting temperature of the wild type, the left peak represents the melting temperature of the single sequence variant).
  • the present invention relates to a method for detecting the presence or the amount of target nucleotides present in a target sample or detecting sequence mutations using a PNA-based real-time PCR probe.
  • Hybridization A state in which complementary base pairs form a double helix structure through hydrogen bonding.
  • N-terminal forms a complementary relationship in the same direction when a pair of PNAs are hybridized.
  • 5'-end is It means the form of complementary binding in the same direction, and when the PNA and DNA are hybridized, it means the form in which the N-terminal of the PNA and the 5'-terminal of the DNA form a complementary relationship in the same direction.
  • N-terminal forms complementary binding in opposite directions when a pair of PNAs are hybridized.
  • DNA the 5'-terminal complementary relationship is opposite to each other.
  • the N-terminus of the PNA and the 3'-end of the DNA form a complementary bond in the same direction.
  • Complementary bond refers to a bond in which the base (A, T, G, C) forms a double strand structure through hydrogen bonding, and in the present invention, 5 'of a single strand forming a double strand.
  • the parallel bonds in which the bases in the complementary relationship are hydrogen-bonded in the state in which the 5'-ends face the same direction it also means.
  • Double hybridization means that two PNA probes bind to sense and anti-sense DNA, respectively, to form two double helix structures.
  • Reporter molecule A material that absorbs and emits light of a specific wavelength and emits light, and refers to a material capable of labeling a probe and confirming whether hybridization between the target nucleic acid and the probe has been performed.
  • Quencher molecule A material that absorbs light generated by a reporter material and reduces fluorescence intensity.
  • Physical property control site means a material for controlling the solubility of a probe, such as a linker or spacer, or for labeling a reporter material or a quenching material, such as a material between PNA and fluorescent or quenching material
  • Linkers to facilitate linking, spacers to control distance, materials for improving solubility and binding to target nucleotides known in the art, and the like. Linkers are described in Akira Kishimoto, Chem. Commun., 742 743, 2003; Peter E. Nielsen, Chem Bio Chem, 6668, 2005; Vladimir Guelev, JACS, 2864-2865, 2002; Ethan A. Englund and Daniel H.
  • Appella, Organic Lett., 3465-3467, 2005 and the like can be used, but are not limited to such spacers, OlafKchler, ChemBioChem, 6977, 2005; Liisa D., J. Med. Chem., 2326-2340, 2007 and the like can be used, but not limited to, materials used for controlling solubility and binding strength include Irina V. Smolina, Vadim V. Demidov, Nucleic Acids Research, e146, 2005; I.S. Blagbrough, Biochemical Society Transactions part 2, 397-406, 2003; Nathalie Berthet, J. Med.
  • the materials described in Chem., 3346-3352, 1997, and the like are possible, but are not limited thereto. Any material may be used when the technical characteristics of the present invention are satisfied. It will be obvious to them.
  • Isomer Substituents Compounds that have the same molecular formula and method of linking members but have different spatial arrangements between atoms are called isomers, and are usually present in the case of carbon compounds in which all four atomic groups linked to carbon have different asymmetric carbons. That is, two different kinds of isomers are formed according to the three-dimensional arrangement of the substituents, and the isomeric substituents in the present invention mean substituents that form only one isomer in one direction.
  • R or S is based on hydrogen (H), but a natural or unnatural amino acid residue (Anca Dragulescu-Andrasi, JACS, 10258-10267, 2006; Filbert Totsingan, Chirality, 245253, 2009; Stefano Sforza, Eur. J. Org.Chem., 1056-1063, 2003), and alkyl groups, amines, alcohols, carboxylic acids (Shabih Shakeel, Sajjad Karim, J. Chem. Technol. Biotechnol., 892899, 2006), etc. It may be used as a substituent, but is not limited thereto.
  • SNP Single nucleotide sequence variation
  • Structured probe refers to a probe that forms a secondary structure.
  • Linear probe oligonucleotide labeled 5 'end with a fluorescent material, 3' end with a matting material, means a probe that does not form a secondary structure because there is no stem.
  • Double linear probe A type of probe in which two linear oligonucleotides in which a reporter material and a quencher material are respectively bonded to each other form a complementary bond.
  • Incomplete Complementary Mismatch When two strands of DNA or PNA hybridize, one or more base pairs in complementary relationship do not match.
  • Black Hole Quencher (BHQ TM ): A matte material sold by Biosearch Technologies Inc. (USA), classified into BHQ1, BHQ2 and BHQ3 according to the structure and wavelength difference.
  • Blackberry Quencher A matting material sold by Berry & Associates, USA, having the following structure:
  • the PNA probe of the present invention was designed to specifically bind to the IS6110 gene of Mycobacterium tuberculosis and the ITS gene of Non-TB tuberculosis. In addition, it was designed and manufactured to perfectly bind to warfarin metabolism related genes CYP2C9 430 and VKORC1 3730 wild type gene and single nucleotide sequence gene.
  • the PNA probe of the present invention may be composed of any one of SEQ ID NOs: 1 to 14 shown in Table 3 below. It will be appreciated that all of the PNA probe sequences within the range that can be easily modified by those skilled in the art from the above nucleotide sequences are within the scope of the present invention. As long as the PNA probe system capable of parallel binding can detect a target nucleotide using PNA real-time PCR according to the present invention, it is included within the scope of the present invention.
  • O is a linker
  • bold letters and underlined letters are ⁇ -lysine ( ⁇ -lysine) or ⁇ -glutamic acid-PNA monomer (monomer)
  • K is lysine (lysine)
  • (+) is aeg [ N- ( ⁇ - alanine)]
  • (+) means aeg [ N- (succinicacid)].
  • PNA probes were synthesized by solid phase synthesis from a PNA monomer protected with benzothiazolesulfonyl (Bts) and functionalized resin according to the method described in Korean Patent No. 464,261 [Lee et al. , Org. Lett., 2007, 9, 3291-3293].
  • PNA can also be synthesized using known 9-fluorenylmetholoxycarbonyl (Fmoc: 9-flourenylmethloxycarbonyl) or t-Boc (t-butoxycarbonyl) synthesis methods [Kim L. et al., J. Org. . Chem. 59, 5767-5773, 1994; Stephen A. et al., Tetrahedron, 51, 6179-6194, 1995]. Reporter materials and quenching materials were labeled on the PNA probe according to methods well known in the art.
  • SEQ ID NO: 19 20 primer set for identification of IS6110 gene of Mycobacterium tuberculosis (MTB) and Internal transcribed spacer (ITS) gene for ITS ( Non-tuberculous mycobacteria (NTM) gene) Primer sets were designed. The designed primers were used by Synthetic Co., Ltd. (Korea).
  • Y means a mixed base of C and T.
  • amplification products were purified using a combination of SEQ ID NOs: 23, 24, and 25 and 26, respectively, using Labopass TM PCR purification kit (Cosmogenetech, Korea), and then pGEM-T.
  • a large amount of DNA was obtained by binding to Easy Vector (Promega, USA) and transforming E. coli JM109 cells.
  • using a normal clone prepared by the above method using a site-specific mutagenesis kit (stratazine, USA) to obtain a clone with a mutant gene and confirm the mutation by sequencing It was.
  • the genotype confirmed clone was used as a standard in gene amplification of the present invention.
  • Mycobacterium tuberculosis ⁇ ATCC 25177, USA ⁇ and Mycobacterium asiaticum ⁇ KCTC 9503, Korea Life Resource Center, Korea ⁇ were distributed to obtain clones for the target nucleic acid.
  • DNA was extracted from InstaGene Matrix (Biorad, USA) from the strains that had been distributed, and the Mycobacterium tuberculosis (MTB) IS6110 gene and non-TB bacterium were combined with the combination of SEQ ID NOs: 27, 28, and 29, 30, respectively.
  • ITS gene of non-tuberculous mycobacteria (NTM) was amplified.
  • the amplification product was purified using Labopass TM PCR purification kit (Cosmogenetech, Korea), then bound to pGEM-T easy vector (Promega, USA) and transformed into E. coli JM109 to bulk DNA. Secured.
  • Example 5 using a mixed solution of the first probe of SEQ ID NO: 4 and the second probe of SEQ ID NO: 6 to measure the change in fluorescence intensity when the reporter material is introduced into the second probe as well as the first probe Real time detection PCR was performed. The results are shown in FIG. When the reporter material was introduced into the second probe, the fluorescence intensity increased by about 50% compared to the case where the reporter material was introduced only into the first probe.
  • the detection C T value increases as the concentration of the reference material decreases. It was confirmed that it can also be applied to quantification. The results are shown in FIG.
  • Tests were performed to confirm the sensitivity and quantitative applicability to target nucleotide detection using a first PNA probe (SEQ ID NO: 12) and a second PNA probe (SEQ ID NO: 13).
  • the detection limits were determined by diluting the non-tuberculosis mycobacterium clones 10 times from 10 9 copies / ⁇ l to 10 1 copies / ⁇ l, respectively, and detected up to 10 1 copies / ⁇ l.
  • the detection C T value increases as the concentration of the standard decreases. Confirmed. The results are shown in FIG.
  • DNA probes are used to detect single nucleotide sequence mutations of the warfarin metabolism related genes CYP2C9 430 and VKORC1 3730 of SEQ ID NOs: 30 and 31.
  • the corresponding PNA probes used SEQ ID NOs: 8, 9.
  • the results of comparing the detection of single nucleotide sequence mutations using the respective probes are shown in FIG. 8. While both DNA probes for detecting a target gene did not detect a single nucleotide sequence, the PNA probe could reliably detect a single nucleotide sequence of two different target genes.
  • Example 11 Mycobacterium tuberculosis using different reporter materials ( Mycobacterium tuberculosis , MTB) and non-TB bacteria ( Non-tuberculous mycobacteria , NTM) simultaneous detection method
  • PNA probes labeled with different reporter materials were used to detect whether two different types of target nucleic acids could be detected simultaneously.
  • MTB Mycobacterium tuberculosis
  • NTM non-tuberculous mycobacteria
  • the first and second probes (SEQ ID NO: 3) and the second gene, warplin metabolism-related gene CYP2C9 430, were used to confirm that the specificity of the detection of a single nucleotide sequence could be increased when the first and second probes were mixed.
  • the melting curve analysis to measure the fluorescence while increasing by 0.5 °C from 25 °C to 95 °C. The results are shown in FIG. There was no change in melting temperature for the wild type with and without the second probe. However, when the second probe was mixed together, it was confirmed that the melting temperature of the single nucleotide sequence variation decreased by about 4 to 6 ° C. That is, when the second probe is used together due to the melting temperature drop for the single nucleotide sequence variation, it was confirmed that the detection specificity for the single nucleotide sequence variation was improved.
  • SEQ ID Nos: 1 to 14 are base sequences of PNA probes according to the present invention.
  • SEQ ID NO: 15 to 30 is the base sequence of the primer according to the present invention.
  • SEQ ID Nos: 31 and 32 are base sequences of DNA probes according to the invention.

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Abstract

La présente invention concerne une composition pour la détection simultanée de Mycobatérium tuberculosis et de mycobactéries non-tuberculeuses sur la base d'un système de sonde acide nucléique peptidique (PNA) d'hybridation nichée ayant une structure de liaison parallèle, et un procédé de détection simultanée de Mycobacterium tuberculosis et de mycobactéries non-tuberculeuses à l'aide de la composition et d'une réaction en chaîne de la polymérase en temps réel. La présente invention concerne également une amorce, une composition de sonde et un procédé d'analyse pour détecter simultanément Mycobarterium tuberculosis et des mycobactéries non tuberculeuses sur la base du système de sonde PNA d'hybridation nichée ayant une structure de liaison parallèle. Lors de la mise en œuvre de la réaction en chaîne de la polymérase multiplexe en temps réel par l'utilisation de la composition comprenant le système de sonde PNA hybridé niché ayant la structure de liaison parallèle selon la présente invention, Mycobacterium tuberculosis et des mycobactéries non-tuberculeuses peuvent être détectés simultanément et de façon précise dans un tube unique avec une sensibilité élevée, permettant ainsi l'avantage de réaliser un diagnostic clinique plus rapide et plus précis.
PCT/KR2012/005483 2011-07-12 2012-07-11 Composition pour la détection simultanée de mycobacterium tuberculosis et de mycobactéries non-tuberculeuses au moyen d'une réaction en chaîne de la polymérase multiplexe en temps réel comprenant un système de sonde pna d'hybridation nichée ayant une structure de liaison parallèle, et procédé de détection à l'aide de celle-ci WO2013009084A2 (fr)

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KR10-2011-0068939 2011-07-12
KR1020110068939A KR20130008283A (ko) 2011-07-12 2011-07-12 평행결합 구조의 이중 혼성화 ｐｎａ 프로브 시스템을 포함하는 실시간 다중 중합효소 연쇄반응에 의한 결핵균 및 비결핵 항산균의 동시 검출용 조성물 및 이를 이용한 검출 방법

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KR102008454B1 (ko) * 2012-08-28 2019-08-08 주식회사 파나진 평행결합 구조의 이중 혼성화 ｐｎａ 프로브 시스템을 포함하는 ｃ형 간염바이러스(ｈｃｖ) 유전자형 감별용 조성물 및 이를 이용한 감별방법
KR101403507B1 (ko) * 2013-03-21 2014-06-09 주식회사 현일바이오 결핵균 및 비결핵 마이코박테리아의 선택적 검출 방법, 그리고 이를 이용한 키트
CN106536483B (zh) * 2014-05-09 2020-02-07 生物检索技术股份有限公司 Cosmic猝灭剂
US10087490B2 (en) * 2014-12-18 2018-10-02 Roche Molecular Systems, Inc. Compositions and methods for detection of drug resistant Mycobacterium tuberculosis
CN105671211A (zh) * 2016-04-07 2016-06-15 广东省农业科学院动物卫生研究所 小反刍兽疫病毒、施马伦贝格病毒和库布病毒分子鉴别诊断方法及其应用
KR101985378B1 (ko) 2017-05-02 2019-06-10 주식회사 레이 치아 촬영용 엑스레이 촬영기
KR101899371B1 (ko) 2017-07-25 2018-10-29 (주)엔바이오텍 핵산 복합체 페어, 핵산 복합체 페어를 포함하는 pcr용 키트, 및 핵산 복합체 페어를 이용한 타겟 검출 방법
KR20200045212A (ko) * 2018-10-22 2020-05-04 (주)바이오니아 옥타민 또는 옥타민 유도체가 결합된 프로브 및 이의 용도
KR102097721B1 (ko) * 2019-01-24 2020-04-06 주식회사 시선바이오머티리얼스 태그서열 snp를 이용한 단일 검출 프로브 기반 다중 표적 검출방법
CN113025729B (zh) * 2020-12-24 2022-11-04 复旦大学 结核分枝杆菌对氨基水杨酸耐药相关的基因突变位点及其应用
CN114592036B (zh) * 2022-04-02 2023-03-31 予果生物科技（北京）有限公司 放线菌核酸提取检测试剂、试剂盒及其方法和应用
KR20240028084A (ko) 2022-08-24 2024-03-05 주식회사 멀티렉스 표지자가 결합된 태그 프로브 및 이의 용도

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999022018A2 (fr) * 1997-10-27 1999-05-06 Boston Probes, Inc. Procedes, trousses et compositions ayant trait a des balises moleculaires de pna (acide nucleique peptidique)

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