KR20130008283A - Composition for detecting mycobacterium tuberculosis and non-tuberculous mycobacteria comprising parallel binding structured pna probe system and detection method using thereof - Google Patents
Composition for detecting mycobacterium tuberculosis and non-tuberculous mycobacteria comprising parallel binding structured pna probe system and detection method using thereof Download PDFInfo
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Abstract
Description
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 having a parallel binding structure.
Real-time multiplex polymerase chain reaction using a composition comprising a double-hybrid PNA probe system of parallel binding structure according to the present invention, it is possible to accurately detect both tuberculosis bacteria and non-tuberculosis antibacterial bacteria in one tube with excellent sensitivity 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 reported to develop 8 million new tuberculosis (TB) patients each year.
MTB and M. bovis (Mycobacterium It was discovered in 1885 that mycobacterium other than bovis was detected in the human body, but it was regarded as non-pathogenic bacterium only because of contamination or colonization, and its name was also atypical. mycobacteria ), anonymous mycobacteria ), nontuberculous mycobacteria (NTM) and mycobacterium other than tuberculosis (MOTT). 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 combines various conditions, even if no TB bacteria have been identified in the lesion. 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 difficult, time-consuming and trained personnel to accurately identify species.
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, the method of screening for tuberculosis using molecular diagnosis has attracted attention. Molecular diagnosis is an effective method to diagnose the infection of a disease by analyzing a human gene (DNA or RNA), or to identify a mutation or a nucleotide sequence of a gene, to predict and ascertain whether or not a disease has occurred. Especially, it is considered to be one of the most advanced technologies of existing disease diagnosis methods.
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 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 the primer-based detection method, which has drawbacks in that it is vulnerable to design difficulties 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. It 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 ability to distinguish single base sequence mutations. Therefore, in order to improve the ability to discriminate single base sequence mutations, MGB tachycardia which shortens the sequence length by introducing MGB (minor groove binder) TaqMan was also developed [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, et 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 (loop) and hairpin structure of the base sequence complementary to the target nucleotide. While this method has the advantage of distinguishing excellent single nucleotide sequence variants, it has the disadvantage of difficulty in designing and synthesizing 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 repulsive force in binding to complementary DNA oligomers with negative charges, which enables faster and stronger binding with target nucleotide sequences than DNA probes and shows high stability due to no 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 a short secondary probe to improve the ability to discriminate low single nucleotide sequence mutations, which is a disadvantage of DNA probes with long sequences, and the two probes are designed to have anti-parallel binding with 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 disadvantages of the above molecular diagnosis technology for the simultaneous detection of Mycobacterium tuberculosis (MTB) and non -tuberculous mycobacteria (NTM) based on PNA that can accurately detect the target nucleotides in the sample 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 more apparent from the following description of the invention, with reference to the accompanying drawings.
In the present invention, in order to overcome the limitations of the existing DNA-based real-time PCR probes have thermal and biological stability, high sensitivity and specificity by utilizing PNA having a better recognition ability and binding ability to the target nucleotide than DNA 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. The probes used at this time are divided into two types, and the pros and cons of each probe are compared in Table 2.
Structured probes in Table 2 are known to have excellent detection specificity for single nucleotide sequence variation. However, if the staple is not designed to have a stable hairpin structure due to the binding force of the stem, quenching will be incomplete and nonspecific fluorescence will be generated . Therefore, design and synthesis are difficult because the probe should be manufactured in consideration of the binding energy difference between the binding energy of the stem and the target nucleotide. On the other hand, linear probes have various advantages including convenience of fabrication, but they are disadvantageous in that the ability to detect single nucleotide sequence variation is deteriorated due to the absence of the 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.
PNA can be hybridized with PNA having a complementary base sequence in two forms of anti-parallel binding and parallel binding (Fig. 1) 2] [Stefano Sforza, Eur. J. Org. Chem., 197-204, 1999]. Due to this difference in binding energy, dual linear PNA probes having parallel binding sequences in the absence of target nucleotides in the sample do not fluoresce through complementary binding to each other. In addition, fluorescence is generated by dissociation between the existing PNA-PNA probes.
Therefore, in the present invention, the first PNA probe is synthesized in accordance with the target Tm, and the binding strength between the two PNA probes is completely complementarily combined with all the sequences of the PNA-DNA using the parallel binding, a second PNA probe was designed and synthesized such that some of the sequences of the perfect-match and PNA-DNA were in the middle of different incomplete mismatches. By using these two PNA linear probes, it is possible to improve the detection ability of single nucleotide sequence variation by providing the function of the stem and to develop a detection system which is not limited to a sequence but is easy to design and synthesize (see FIG. 3) .
The PNA probe according to the present invention has a form in which a reporter substance and a light extinction substance are bound to one or both ends of a PNA oligomer having a certain base sequence.
The PNA probe according to the present invention is preferably a structure in which a site for controlling property of property and / or a reporter substance and a extinction substance are bonded to both ends as shown in the following formula (1), but the present invention is not limited thereto. It is obvious to a person skilled in the art that a 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 refers to a part that forms parallel binding or anti-parallel binding to a target nucleotide. A and A 'may be the same or different substances as a reporter molecule or a quencher molecule, but either or both may be present. X and X 'may be the same or different from each other as a physical property regulating part, none of them may be included, or one or more may be included. N ' and C ' denote N -terminus and C -terminus, respectively.
Particularly, P, which is a PNA base moiety, may have a structure as shown in the following formula (2), but it is not limited thereto. It is obvious to a person skilled in the art that a PNA base having any structure can be used as long as the object of the present invention can be achieved .
In the above formula (2), B is selected from natural nucleobases or non-natural nucleobases including adenine, thymine, guanine, cytosine and uracil as nucleotide bases, and R or S is hydrogen (H) May not be present, but may be transformed into an isomeric substituent. Also, R or S may be in the form of a modified reporter molecule or an isomeric substituent labeled with a quencher molecule [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.
Therefore, 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, so that whether MTB and NTM infection is accurate and rapid, That can be used to diagnose tuberculosis.
BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram showing a parallel and anti-parallel binding structure between PNA and PNA; FIG.
2 is a graph showing the relative binding force intensities of PNA-PNA and PNA-DNA.
Figure 3 shows a method of 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 is a diagram showing the stability of a probe.
(a) PCR result immediately after mixing the first probe and the second probe.
(b) PCR result after incubation for 6 months at room temperature with mixed first probe and second probe.
5 is a diagram illustrating fluorescence intensity enhancement through dual fluorescence labeling.
(a) PCR was performed by mixing a second probe and a first probe that were not fluorescently labeled.
(b) PCR was performed by mixing the fluorescent probe-labeled second probe with the first probe.
FIG. 6 is a graph showing the result of performing PCR and confirming calibration curve for confirming the detection sensitivity of warfarin single nucleotide sequence mutation detection and confirming the applicability of the quantitative method, with reference to Example 8 using the method of the present invention.
FIG. 7 is a graph showing the results of PCR and the calibration curve for confirming the sensitivity of ITS gene detection and the applicability of the quantitation method, in relation to Example 9 using the method of the present invention. FIG.
8 is a view showing a PCR result using a DNA probe.
(a) PCR using wild type and single nucleotide sequence variants of warfarin metabolism-related gene CYP2C9 430 gene (○ indicates wild type gene detection line and × indicates detection line of single nucleotide sequence variant) using a DNA probe.
(b) PCR using wild-type and single-nucleotide sequence variants of the VKORC1 3730 gene for warfarin metabolism-related genes using a DNA probe (O indicates a wild type gene detection line and X indicates a detection sequence of a single nucleotide sequence variant).
(c) PCR using wild type and single nucleotide sequence variants of the CYP2C9 430 gene of warfarin metabolism related genes 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 using wild type and single nucleotide sequence variants of VKORC1 3730 gene (○ indicates wild type gene detection line and straight line indicates detection sequence of single nucleotide sequence variant) using the PNA probe.
Fig. 9 is a graph showing the fluorescence intensity of Mycobacterium < RTI ID = 0.0 > tuberculosis, MTB) and non-tuberculosis mycobacteria (Non - tuberculous A diagram showing PCR results of specifically and simultaneously detecting mycobacteria and NTM (○ represents a detection line of Mycobacterium tuberculosis and Δ represents a detection line of non-TB bacterium).
FIG. 10 shows the change of the melting temperature with respect to the single base sequence mutant by the second probe (the right peak represents the melting temperature of the wild type, and the left peak represents the melting temperature of the single base sequence mutant).
Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are intended to further illustrate the present invention, and it is to be understood 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.
The 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: When a pair of PNAs are hybridized, the N-term is complementary in the same direction. In the case of DNA, the 5'-end (5'-end) Refers to a form having a complementary bond in the same direction. When the PNA and DNA are hybridized, 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 a spacer, or for labeling a reporter material or a quenching material, such as between a PNA and a 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; IS Blagbrough, Biochemical
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
Single nucleotide sequence mutation ( SNP ): One DNA sequence of a particular gene means different, and includes both germline mutations and somatic mutations (somatic mutations).
Structural Probe (structured probe ): Probe forming secondary structure.
Linear probe (linear probe ): Oligonucleotide labeled 5 'end with fluorescent material and 3' end with quencher, meaning 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.
Fully complementary binding (perfect match ): When two strands of DNA or PNA hybridize, the complementary base pairs match perfectly.
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 matting material ( Black Hole Quencher , BHQ TM ): A matting material sold by Biosearch Technologies Inc. (USA), classified into BHQ1, BHQ2, BHQ3 according to structure and wavelength difference.
Blackberry Quencher (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 prepared so as to specifically bind to the IS6110 gene of Mycobacterium tuberculosis and the ITS gene of non-tuberculous acidophilic bacteria, respectively. It was designed and constructed to be able to bind perfectly to the wild-type gene and the single-nucleotide sequence mutant gene of warfarin metabolism-related genes CYP2C9 430 and VKORC1 3730, respectively.
For example, the PNA probe of the present invention can be composed of the nucleotide sequence of any one of SEQ ID NOS: 1 to 14 shown in Table 3 below. All of the PNA probe sequences within a range that can be easily modified by those skilled in the art from the above base sequence will be considered to be within the scope of the present invention. Is within the scope of the present invention as long as it can detect the target nucleotide using the PNA real-time PCR according to the present invention as a PNA probe system capable of parallel bonding.
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), (+) 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. For the identification of the CYP2C9 430 gene, SEQ ID NOs: 15 and 16 primer sets and VKORC1 3730 genes were designed for SEQ ID NOS: 17 and 18 primers. 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 primer was synthesized by Biona (Korea).
In Table 4, Y means a mixed base of C and T.
[ Example 3] Warfarin Metabolism-related gene CYP2C9 430 and VKORC1 3730 clone acquisition
The amplified products were purified using a Labopass TM PCR purification kit (Cosmos TECH, Korea) in combination of SEQ ID NOS: 23 and 24, 25 and 26, respectively, for cloning of CYP2C9 430 and VKORC1 3730, (Promega, USA) and transformed into E. coli JM109 cells to obtain a large amount of DNA. In order to obtain a single nucleotide sequence variant, a clone having a mutated gene was obtained using a normal clone prepared by the above method and using a site-specific mutagenization kit (Stratagene, USA), and mutation was confirmed by base sequence analysis Respectively. Clones with confirmed genotypes were used as reference materials in the amplification of the gene of the present invention.
[ Example 4] IS6110 And Non-tuberculosis Antibiotic acid gene ITS Securing clones
To obtain a clone for the target nucleic acid, Mycobacterium tuberculosis {ATCC 25177, USA} and Mycobacterium asiaticum {KCTC 9503, Korea Life Resource Center, Korea}. DNA was extracted from the cultured strains using an InstaGene Matrix (Biorad, USA), and the combination of SEQ ID NOS: 27 and 28, 29 and 30, and Mycobacterium of tuberculosis, MTB) IS6110 gene and non-tuberculosis mycobacteria (Non -tuberculous mycobacteria, NTM) was amplified ITS gene. The amplified product was purified using a Labopass TM PCR purification kit (Kosomjin Tech, Korea) and then ligated into a pGEM-T isotype vector (Promega, USA) and transformed into E. coli JM109, Respectively.
[ Example 5] Real time PCR reaction
A real-time detection method using a PNA probe using clones obtained by the methods of Examples 3 and 4 was established. 10 μl of a mixed solution of 2 μl of template DNA (10 5 copies / μl), the first probe (5 pmoles / μl) and the second probe (10 pmoles / μl), 10 μl of a primer set 5 μl of a 10 × Taq polymerase buffer solution (Solgent, Korea), 10 μl of a 10 mM mixed solution of dNTPs (solution of 10 μM dNTPs, Pleasant, South Korea) 1 ㎕, Taq polymerase (5 U / ㎕, brush Gentry, Korea) 0.4 ㎕, after a mixture of distilled water and 27.6 ㎕ real-time gene amplifier (Real-time PCR machine, CFX96 TM Real-time PCR System, bio Rad, USA) at 95 ° C for 3 minutes, followed by a reaction at 95 ° C for 10 seconds and at 60 ° C for 30 seconds and at 72 ° C for 15 seconds to hybridize the first and second probes together with the primers. Fluorescence intensity was measured after hybridization at 60 ° C.
[ Example 6] parallel coupling PNA Based real-time PCR Of the probe stability
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] 2nd In the probe Through the introduction of additional reporter materials Signal strength increase
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] 1st Of the probe Single nucleotide sequence variation Confirm sensitivity of detection and applicability of quantification method
A test was conducted to confirm the applicability of the sensitivity and quantification method for detection of single nucleotide sequence variation using the first PNA probe (SEQ ID NO: 1) and the second PNA probe (SEQ ID NO: 5). CYP2C9 430 wild type and single nucleotide sequence variant clones were sequentially diluted 10 times from 10 9 copies / μl to 10 1 copies / μl, respectively, and the detection limit was measured. It was confirmed that detection was possible up to 10 1 copies / μl. In addition, analysis of the correlation between C T (cycle threshold) according to the number of copies of single nucleotide sequence mutation gene showed that the detected C T value increases as the concentration of the reference material decreases. It is confirmed that this method can be applied to quantitative analysis. The results are shown in Fig.
[ Example 9] First Of the probe Target Nucleotides Confirm sensitivity of detection and applicability of quantification method
A test was conducted to confirm the sensitivity and applicability of the target nucleotide detection using a first PNA probe (SEQ ID NO: 12) and a second PNA probe (SEQ ID NO: 13). The detection limit was determined by diluting the non-tuberculous mycobacterial clones sequentially from 10 9 copies / μl to 10 1 copies / μl by 10 times, and it was confirmed that detection was possible 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, so the present invention can be applied to the quantification of nucleic acid. Confirmed. The results are shown in Fig.
[ Example 10] PNA Probe And DNA Of the probe Single nucleotide sequence variation Detectability compare
In order to confirm whether single base sequence mutation can be detected using a DNA probe (Tackman probe) as in the case of the PNA probe, DNA probes for detecting single nucleotide sequence mutations of the warfarin metabolism-related genes CYP2C9 430 and VKORC1 3730 of SEQ ID NOS: 30 and 31, Was used for synthesis of Bioneer (Korea) (see Table 5). The corresponding PNA probes used SEQ ID NOS: 8 and 9. The results of comparing the detection ability of single nucleotide sequence variation using each probe are shown in FIG. While DNA probes for two target gene detection did not detect single nucleotide sequence mutations, PNA probes were able to detect single base sequence mutations 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
In order to confirm that the detection specificity of the single base sequence mutation can be enhanced when the first probe and the second probe are mixed, a first probe (SEQ ID NO: 3) using the warfarin metabolism related gene CYP2C9 430 as a target gene (SEQ ID NO: 3) Real-time PCR was carried out using the probe (SEQ ID NO: 7) according to the method of Example 5, followed by a melting curve analysis in which the fluorescence was measured at 25 ° C to 95 ° C by 0.5 ° C. The results are shown in Fig. There was no change in the melting temperature for the wild type with or without the second probe. However, when the second probes were mixed together, it was confirmed that the melting temperature for the single base sequence mutation decreased by about 4 to 6 ° C. That is, the detection specificity for single base sequence mutation was improved when the second probe was used together due to the lowering of the melting temperature for the single base sequence mutation.
<110> PANAGENE INC. <120> Parallel binding structured PNA probe system <160> 32 <170> Kopatentin 1.71 <210> 1 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> IMB-3 PNA probe <400> 1 acacggtcct caa 13 <210> 2 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> IMB-1 PNA probe <400> 2 acacggtcct caa 13 <210> 3 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> DFP-0207-26 PNA probe <400> 3 acacggtcct caak 14 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DFP-0207-22 PNA probe <400> 4 kkacacggtc ctcaakkk 18 <210> 5 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Q-B-13 PNA probe <400> 5 tgtgccagga gtt 13 <210> 6 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> IMB-0125-2 PNA probe <400> 6 kktgtgccag gagttkkk 18 <210> 7 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> DFP-0208-10 PNA probe <400> 7 tgtgccagga gttk 14 <210> 8 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Warfarin-W-4 PNA probe <400> 8 aacacggtcc tc 12 <210> 9 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> 3730-M1-2 PNA probe <400> 9 atgtgtgggt 10 <210> 10 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> TB-10608-2 PNA probe <400> 10 ttcgcctacg tg 12 <210> 11 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> ITS-20608-1 PNA probe <400> 11 gtgtggtgtt tga 13 <210> 12 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> ITS-20608-3 PNA probe <400> 12 gtggtgtttg a 11 <210> 13 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> ITS-20608-4 PNA probe <400> 13 cacaccacaa actkkk 16 <210> 14 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> TB-10616-1 PNA probe <400> 14 agcggatgca ck 12 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 15 gctgcggaat tttgggatgg 20 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 16 gatgtggggc ttctagatta cc 22 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 17 gatgtggggc ttctagatta cc 22 <210> 18 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 18 tgtaaaaaag agcgagcgtg tg 22 <210> 19 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 19 cgcttcggac caccagca 18 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 20 caggatcctg cgagcgtag 19 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 21 ccacctccyt tctaaggagc acc 23 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 22 gggatgctcg caaccactat yca 23 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 23 gaagcctgtg tggctgaata 20 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 24 ccattcccac catgttgact 20 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 25 gcaaggctaa gaggcactga 20 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 26 accacagtcc atggcagac 19 <210> 27 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 27 tcgtccagcg ccgcttcg 18 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 28 cgggtccaga tggcttgc 18 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer <400> 29 gattgggacg aagtcgtaac aag 23 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer <400> 30 agcctcccac gtccttcatc ggc 23 <210> 31 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> CYP2C9-430W DNA probe <400> 31 cctcttgaac acggtcctca atgct 25 <210> 32 <211> 25 <212> DNA <213> Artificial Sequence <220> ≪ 223 > VKORC1-3730M DNA probe <400> 32 cattgtcatg tgtgggtatg gcagg 25
Claims (11)
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 ;
The composition for the simultaneous detection of tuberculosis bacteria and non-tuberculosis acid bacilli.
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 sequence selected from SEQ ID NO: 11 or 12 and SEQ ID NO: 13.
Wherein each PNA probe further comprises a property-modifying moiety at one or more ends.
Wherein each of the PNA probes further comprises at least one substance selected from a reporter molecule and a quencher molecule.
Wherein the reporter substance or the minerals are bound to the PNA end directly or through a property-controlling site connected to the PNA end.
The reporter material may be selected from the group consisting of fluorescein, fluorescein chlorotriazinyl, rhodamine green, rhodamine red, tetramethylrhodamine, FITC, Oregon green, Alexa Fluor, FAM, JOE, ROX, HEX, Texas Red, TET, TRITC, TAMRA, Cyanine dyes and thiadicarbocyanine dyes ≪ / RTI > wherein the composition is at least one fluorescent material selected from the group consisting of < RTI ID = 0.0 >
The minerals may be selected from the group consisting of Dabcyl, TAMRA, Eclipse, DDQ, QSY, Blackberry Quencher, Black Hole Quencher, Qxl, Iowa black FQ, Iowa Black RQ, IRDye QC- Lt; RTI ID = 0.0 > 1 < / RTI >
Wherein the property-modifying moiety attached to the PNA probe end is selected from the group consisting of hydrophilic moieties, hydrophobic moieties, ionic moieties, and hydrogen bonding moieties.
Wherein the property-modifying moiety attached to the end of each PNA probe is an ionic moiety and comprises a positive charge, a negative charge or a zwitterion ion.
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| KR1020110068939A KR20130008283A (en) | 2011-07-12 | 2011-07-12 | Composition for detecting mycobacterium tuberculosis and non-tuberculous mycobacteria comprising parallel binding structured pna probe system and detection method using thereof |
| PCT/KR2012/005483 WO2013009084A2 (en) | 2011-07-12 | 2012-07-11 | 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 |
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Cited By (9)
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| KR20140028338A (en) * | 2012-08-28 | 2014-03-10 | 주식회사 파나진 | Composition for detecting hepatitis c virus(hcv) genotypes comprising parallel binding structured pna probe system and detection method using thereof |
| WO2014148817A1 (en) * | 2013-03-21 | 2014-09-25 | 주식회사 현일바이오 | Selective detection method for mycobacterium tuberculosis and nontuberculous mycobacteria and kit using same |
| KR20170003984A (en) * | 2014-05-09 | 2017-01-10 | 바이오서치 테크날로지즈, 인크. | Cosmic quenchers |
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| WO1999022018A2 (en) * | 1997-10-27 | 1999-05-06 | Boston Probes, Inc. | Methods, kits and compositions pertaining to pna molecular beacons |
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2011
- 2011-07-12 KR KR1020110068939A patent/KR20130008283A/en not_active Application Discontinuation
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Also Published As
| Publication number | Publication date |
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| WO2013009084A9 (en) | 2013-03-28 |
| WO2013009084A3 (en) | 2013-05-16 |
| WO2013009084A2 (en) | 2013-01-17 |
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