WO2013009084A9 - Composition for simultaneously detecting mycobacterium tuberculosis and nontuberculous mycobacteria by means of real-time multiplex polymerase chain reaction comprising nested hybridization pna probe system having parallel binding structure, and method for detection using same - Google Patents

Abstract

The present invention relates to a composition for simultaneously detecting Mycobacterium tuberculosis and nontuberculous mycobacteria based on a nested hybridization peptide nucleic acid (PNA) probe system having a parallel binding structure, and to a method for simultaneously detecting Mycobacterium tuberculosis and nontuberculous mycobacteria using the composition and real-time polymerase chain reaction. Also, the present invention relates to a primer, a probe composition, and a method for analyzing to simultaneously detect Mycobacterium tuberculosis and nontuberculous mycobacteria based on the nested hybridization PNA probe system having the parallel binding structure. When carrying out the real-time multiplexed polymerase chain reaction by using the composition comprising the nested hybridized PNA probe system having the parallel binding structure, according to the present invention, Mycobacterium tuberculosis and nontuberculous mycobacteria can be simultaneously and accurately detected in a single tube with high sensitivity, thereby providing the advantage of making a more rapid and accurate clinical diagnosis.

Description

Simultaneous Detection of Mycobacterium Tuberculosis and Non-tuberculosis Antibiotic by a Real-time Multiplex Polymerase Chain Reaction Comprising a Double Hybridized PNA Probe System of Parallel Binding Structure and a Detection Method Using the Same

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. In another aspect, 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 μm in thickness and 1-4 μm in length, and is the most infectious disease in human history. In general, 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. About one billion people, or one-third, are known to develop 8 million new tuberculosis (TB) patients each year.

It was discovered in 1885 that 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-γ 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.

Recently, the method of analyzing mycolic acid using high performance liquid chromatography (HPLC), nucleic acid probe method, polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-restriction fragment length polymorphism) analysis, PRA) and the like have been used to identify non-tuberculosis.

Recently, a method for screening for tuberculosis using molecular diagnosis has attracted attention. 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. In particular, 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.

Various detection methods are used for molecular diagnosis. 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.

Molecular diagnostics using real-time PCR : 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].

Detection of target nucleotides using DNA-based probes : There are two main types of DNA-based probes that are widely used. 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. 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 (MB, Molecular Beacon) 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].

However, 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. There are disadvantages.

Detection of target nucleotides using 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].

Recently, a new method using a dual linear probe structure has been reported. This method uses short secondary probes to improve the discrimination ability of low single nucleotide sequences, which is a disadvantage of long sequence DNA probes. The two probes are designed to form anti-parallel binding to each other. James M. Coull, et al., US 6607889.

Recently, various molecular diagnostic techniques have been developed to detect tuberculosis bacteria, but there is still a need for a technology development capable of detecting tuberculosis bacteria in a short analysis time with high sensitivity and specificity.

In the present invention, overcoming the above disadvantages of molecular diagnostic technology, PNA-based Mycobacterium tuberculosis (MTB) and non -tuberculous mycobacteria (NTM) for simultaneous detection of target nucleotides in a sample can be detected simultaneously. Provided is a composition and a method for simultaneous detection of MTB and NTM using the same.

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.

By using the detection method according to the present invention, MTB and NTM can be detected simultaneously with high sensitivity and specificity, which is very useful for the diagnosis of tuberculosis.

In another aspect, 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.

Other objects and advantages of the present invention will become apparent from the following description, the means for solving the problems and the detailed description of the invention.

In the present invention, in order to overcome the limitations of the existing DNA-based real-time PCR probes have thermal and biological stability, using a PNA having a higher recognition ability and binding ability than the target nucleotide than DNA, high sensitivity and specificity A composition capable of simultaneously detecting MTB and NTM, an MTB and NTM diagnostic kit comprising such a composition, and a method of detecting MTB and NTM through real-time PCR using the composition or kit have been developed.

As is already known, 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.

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. On the other hand, 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.

Therefore, 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.

Therefore, in the present invention, 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. By using these two PNA linear probes to have a stem function, a detection system that is convenient for design and synthesis has been developed because it is not limited to a sequence while improving the detectability of a single nucleotide sequence (see FIG. 3). .

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.

In Formula 1, P is a PNA base moiety having a sequence complementary to the target nucleotide, and 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.

Particularly, 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. .

In Formula 2, 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.

Accordingly, the 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.

2 shows the relative binding strength of PNA-PNA and PNA-DNA.

3 shows a method for detecting a target nucleotide using a dual hybridized PNA probe system.

(a) When a first PNA probe is used as a detection probe in the detection method of the target nucleotide using the double hybridized PNA probe system of a parallel binding structure.

(b) 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.

4 shows the stability of the probe.

(a) PCR results immediately after mixing the first and second probes.

(b) PCR results after 6 months of storage at room temperature with the first and second probes mixed.

5 shows the increase in fluorescence intensity through double fluorescent labeling.

(a) PCR was performed by mixing the first probe with the second unfluorescent label.

(b) PCR was performed by mixing the first and second fluorescently labeled probes.

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.

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.

8 shows PCR results using DNA probes.

(a) PCR results using wild type and single sequence variants of the warfarin metabolism related gene CYP2C9 430 gene using a DNA probe (○ indicates a wild type gene detection line and × indicates a detection line of a single nucleotide sequence variant).

(b) PCR results using wild type and single nucleotide sequence variants of the warfarin metabolism related gene VKORC1 3730 gene using a DNA probe (○ indicates a wild type gene detection line, and × indicates a detection line of a single nucleotide sequence variant).

(c) PCR results using wild-type and single nucleotide variants of the warfarin metabolism-related gene CYP2C9 430 gene using a PNA probe (○ indicates a wild-type gene detection line, and a straight line indicates a detection line of a single nucleotide sequence variant).

(d) PCR results using wild type and single nucleotide sequence variants of the warfarin metabolism related gene VKORC1 3730 gene using a PNA probe (○ indicates a wild type gene detection line, and a straight line indicates a single nucleotide sequence detection line).

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. Detection line of tuberculosis bacterium, and (triangle | delta) means the detection line of non-tuberculous acid bacterium).

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).

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are intended to illustrate the present invention in more detail, it is clear that the scope of the present invention is not limited to the examples.

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.

Definitions of terms and abbreviations used in the present invention are as follows.

Hybridization: A state in which complementary base pairs form a double helix structure through hydrogen bonding.

Parallel binding: N-terminal (N-terminal) forms a complementary relationship in the same direction when a pair of PNAs are hybridized. In the case of DNA, 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.

Anti-parallel binding: N-terminal forms complementary binding in opposite directions when a pair of PNAs are hybridized. In the case of DNA, the 5'-terminal complementary relationship is opposite to each other. When the PNA and DNA are hybridized, 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. In addition to the antiparallel bonds in which the bases in the complementary relationship are hydrogen-bonded in the opposite directions, 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 (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. In Formula 2 of the present invention, 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.

Single nucleotide sequence variation (SNP): One DNA sequence of a particular gene means different, and includes both germline and somatic mutations.

Structured probe: refers to a probe that forms a secondary structure.

Linear probe (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.

Perfect match: When two strands of DNA or PNA hybridize, it means that the base pairs in complementary relationship are perfectly matched.

Incomplete Complementary Mismatch (mis-match): When two strands of DNA or PNA hybridize, one or more base pairs in complementary relationship do not match.

FAM: 6-Carboxyfluorescein

Dabcyl: 4,4-Dimethylamino-azobenzene-4'-carboxylic acid

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:

Example 1 Design and Fabrication of PNA Probes

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.

For example, 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.

In Table 3, O is a linker, bold letters and underlined letters are γ-lysine (γ-lysine) or γ-glutamic acid-PNA monomer (monomer), K is lysine (lysine), and (+) is aeg [ N- (β - 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]. In addition to this method, 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.

Example 2 Synthesis and Use of Primers to Amplify Target Nucleic Acids of CYP2C9 430, VKORC1 3730 and Tuberculosis / N.

In the present invention, specific amplification by analyzing the site of each gene for amplification of target nucleic acids of warfarin metabolism-related genes CYP2C9 430, VKORC1 3730 and Mycobacterium tuberculosis (MTB) and non -tuberculous mycobacteria (NTM) genes A primer was prepared to achieve this. The primer sets SEQ ID NO: 15, 16 for the CYP2C9 430 gene identification and the primer set SEQ ID NO: 17, 18 for the VKORC1 3730 gene identification were designed. In addition, 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).

In Table 4, Y means a mixed base of C and T.

[Example 3] Warfarin metabolism related genes CYP2C9 430 and VKORC1 3730 clones secured

To obtain clones of CYP2C9 430 and VKORC1 3730, 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. In order to obtain a single nucleotide sequence variant, 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.

Example 4 Cloning of Mycobacterium Tuberculosis Gene IS6110 and Non-tuberculosis Antibiotic Gene ITS

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 ^™ 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 Real-Time PCR Reaction

Using the clones obtained by the method of Examples 3 and 4 above, a real-time detection method using a PNA probe was established. 2 μl of template DNA (10 ⁵ copies / μl), 10 μl of the mixed solution of the first probe (5 pmoles / μl) and the second probe (10 pmoles / μl), primer set (SEQ ID NOs: 15, 16 sets or 17, 18 Set or 19, 20 or 21, 22 sets, forward primer 50 pmoles / μl, reverse primer 6.25 pmoles / μl, 2 μl, 10 × Taq polymerase buffer solution (Solgent, Korea) 5 μl, 10 mM dNTP mixed solution (sol Gent, Korea) 1 μl, Taq polymerase (5 U / μl, Solgent, Korea), 0.4 μl, distilled water 27.6 μl, and then real-time PCR (Real-time PCR machine, CFX96 ^™ Real-time PCR System, Bio) RAD, USA) for 3 minutes at 95 ℃, the reaction was repeated for 60 seconds 30 seconds, 72 ℃ 15 seconds to hybridize the first probe and the second probe with 95 ℃ 10 seconds, and primers. Fluorescence intensity was measured after 60 ° C. hybridization.

Example 6 Stability of Parallel Bound PNA-based Real-Time PCR Probes

In order to test the storage stability of the first probe (SEQ ID NO: 2) and the second probe (SEQ ID NO: 5), real-time detection PCR is performed using the first probe and the second probe mixed solution according to the method of Example 5, respectively. After storage at room temperature for 6 months, real-time detection PCR was performed in the same manner and the effects were compared. The results are shown in FIG. As a result, it was confirmed that the fluorescence intensity did not decrease even when stored for 6 months at room temperature.

Example 7 Signal Strength Increase by Introducing Additional Reporter Material into the Second Probe

Method of 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.

Example 8 Confirmation of Sensitivity and Detection of a Single Base Sequence Mutation of the First Probe and Applicability of Quantitative Methods

A test was performed to confirm the sensitivity and the applicability of the quantitative method for detecting a single nucleotide sequence mutation using a first PNA probe (SEQ ID NO: 1) and a second PNA probe (SEQ ID NO: 5). Detection limits were determined by diluting CYP2C9 430 wild-type and single nucleotide variant clones 10-fold copies from 10 ⁹ copies / μl to 10 ¹ copies / μl, respectively, and detectable up to 10 ¹ copies / μl. In addition, as a result of analyzing the correlation between the C _T (cycle threshold) according to the number of copies of the single nucleotide sequence mutation gene, 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.

Example 9 Confirmation of Sensitivity and Detection of Target Nucleotide in the First Probe and Applicability of Quantitation

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 / μl to 10 ¹ copies / μl, respectively, and detected up to 10 ¹ copies / μl. In addition, as a result of analyzing the correlation between the C _T according to the number of copies of the target nucleotide, the detection C _T value increases as the concentration of the standard decreases. Confirmed. The results are shown in FIG.

Example 10 Comparison of Detecting Single Base Sequence Mutations of PNA Probes and DNA Probes

Like PNA probes, DNA probes (tagman 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. Was used by Bioneer Co., Ltd. (Korea) for synthesis (see Table 5). 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.

In bold font in Table 5 indicates a single nucleotide sequence position.

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.

Mycobacterium tuberculosis (MTB) specific PNA probes according to SEQ ID NOs: 10 and 14 and non-tuberculous mycobacteria (NTM) specific PNA probes according to SEQ ID NOs: 11 and 13 Antibiotic bacteria were specifically detected at the same time, and the results are shown in FIG. 9.

Example 12 Confirmation of Detection Specificity for Single Base Sequence Variation

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. After performing a real-time PCR reaction according to the method of Example 5 using a probe (SEQ ID NO: 7), the melting curve analysis to measure the fluorescence while increasing by 0.5 ℃ from 25 ℃ to 95 ℃. 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.

Claims (11)

1. PNA probes that bind specifically to Mycobacterium tuberculosis (MTB) and PNA probes that bind in parallel to PNA probes that specifically bind to Mycobacterium tuberculosis ; And

   PNA probes that specifically bind to non-tuberculous mycobacteria (NTM) and PNA probes that bind in parallel to PNA probes that specifically bind to non-tuberculous mycobacteria ;

   Tuberculosis bacteria and non-tuberculosis anti-bacterial bacterium simultaneously detecting composition comprising a.
2. The method of claim 1,

   The Mycobacterium tuberculosis specific PNA probe and a PNA probe bound to the same have a sequence represented by SEQ ID NO: 10 and SEQ ID NO: 14,

   A non-tuberculosis antimicrobial-specific PNA probe and a PNA probe that binds parallel thereto have a composition selected from SEQ ID NO: 11 or 12 and SEQ ID NO: 13.
3. The method according to claim 1 or 2,

   Each of the PNA probes, characterized in that it further comprises a physical control site at one or more ends.
4. The method of claim 1,

   Wherein each PNA probe further comprises one or more materials selected from a reporter molecule and a quencher molecule.
5. The method of claim 4, wherein

   The reporter material or the quencher is directly bonded to the PNA terminal, or a composition characterized in that it is bound through a physical control site connected to the PNA terminal.
6. The method of claim 4, wherein

   The reporter material is fluorescein (fluorescein), fluorescein chlorotriazinyl, rhodamine green, rhodamine red, rhodamine red, tetramethylrhodamine, FITC, oregon green (Oregon green), Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, Cyanine-based dyes and thiadicarbocyanine dyes At least one fluorescent material selected from the group consisting of a composition.
7. The method of claim 4, wherein

   The matting material is dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa black FQ, Iowa Black RQ, IRDye QC- At least one selected from the group 1;
8. The method of claim 3, wherein

   The physical property control region bound to the PNA probe terminal is selected from the group consisting of hydrophilic residues, hydrophobic residues, ionic residues and hydrogen bond residues.
9. The method of claim 3, wherein

   The physical property control region bound to each of the PNA probe ends is an ionic moiety, characterized in that it comprises a positive charge, a negative charge or a zwitter ion.
10. A kit for simultaneously detecting Mycobacterium tuberculosis and non-tuberculosis anti-bacterial bacterium comprising the composition according to claim 1.
11. A method for simultaneously detecting tuberculosis and non-tuberculosis anti-bacterial bacteria using any one of the compositions selected from claims 1 and 2, or a kit including the composition.

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