JP5700385B2 - Method and test piece for determining drug resistance of Mycobacterium tuberculosis - Google Patents

Description

  The present invention relates to a method and a test piece for determining the drug resistance level of Mycobacterium tuberculosis.

  Currently, it is said that thousands of people die in Japan each year, and millions of people die globally on a global scale. In the treatment of tuberculosis, medical therapy centered on chemotherapy is basically used, and surgical therapy is considered when it is impossible to achieve the purpose of the therapy.

  As drugs for treating tuberculosis used in medical therapy, isoniazid (INH), rifampicin (RFP), streptomycin (SM), ethambutol (EB), new quinolone (NQ), pyrazinamide (PZA) and the like are known. These drugs kill M. tuberculosis by inhibiting the growth of M. tuberculosis by the action of a drug-specific gene (drug resistance gene) unique to M. tuberculosis. For example, INH is taken into Mycobacterium tuberculosis, and then metabolized by INH oxidase (catalase peroxidase) unique to Mycobacterium tuberculosis encoded by the katG gene to produce a metabolite that inhibits the growth of Mycobacterium tuberculosis. However, when a drug is administered to a patient, there is a problem that a mutation causes a drug-resistant bacterium that has acquired resistance to the administered drug.

  Drug resistant bacteria acquire drug resistance by mutating drug resistance genes. For example, since the katG gene is mutated in INH resistant bacteria, even if INH is incorporated, metabolites that inhibit the growth of Mycobacterium tuberculosis cannot be generated sufficiently. For this reason, methods for detecting drug-resistant bacteria by detecting mutations in drug resistance genes have been developed. For example, an oligonucleotide synthesized based on the base sequence of the drug resistance gene on the Mycobacterium tuberculosis genome in a wild-type Mycobacterium tuberculosis that is sensitive to a tuberculosis treatment drug and a mutant Mycobacterium tuberculosis that is resistant to any drug is immobilized. A Mycobacterium tuberculosis diagnostic kit including the prepared substrate is disclosed (Patent Document 1). The currently developed DNA microarray kit "Oligo Array" (Nisshinbo Co., Ltd.) can detect mutations in drug resistance genes specific to five drugs, INH, RFP, SM, kanamycin (KM), or EB It is a kit.

  By the way, it has been clarified that drug-resistant bacteria include bacteria having high resistance to drugs and bacteria having moderate to low resistance. Since the method for treating tuberculosis caused by drug-resistant bacteria differs depending on whether the resistance to the drug is high or low, it is preferable to determine the degree of resistance of the tubercle bacillus to the drug.

  Conventionally, CLSI (Clinical and Laboratory Standards Institute) / NCCLS (National Committee for Clinical Laboratory Standards) is a method for determining the drug resistance of Mycobacterium tuberculosis (Non-patent Document 1). In Japan, as a kit according to this method, “drug-sensitive (acid-fast bacterium) kit“ Nichibi ”well pack medium S for acid-fast bacteria test” (manufactured by Nippon BCG Supply Co., Ltd.), “drug-sensitive (acid-fast bacterium) ) Kit Kyokuto Mycobacterium tuberculosis susceptibility bit spectrum-SR "(manufactured by Kyokuto Pharmaceutical Co., Ltd.) is commercially available. However, it takes a long time of about 40 days to determine the drug resistance of M. tuberculosis using these kits. Therefore, a quicker and simpler determination method is desired.

JP 2001-103981 A

GL Woods et al., `` Susceptibility Testing of Mycobacteria, Nocardiae, and Other Aerobic Actinomycetes; Approved Standard '', NCCLS document M24-A (ISBN 1-56238-500-3), NCCLS, 2003, Vol. 23, No. 18, p.3-19

  An object of the present invention is to provide a new means for quickly and easily determining the drug resistance level of Mycobacterium tuberculosis.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that the drug resistance level of Mycobacterium tuberculosis can be associated with the mutation of the drug resistance gene. Therefore, the present inventors simply identified the drug resistance gene mutation in Mycobacterium tuberculosis based on the database created for the degree of drug resistance exhibited by Mycobacterium tuberculosis having a known drug resistance gene mutation. It was found that the degree could be determined quickly and simply, and the present invention was completed.

  The present invention provides a test piece for determining the drug resistance level of Mycobacterium tuberculosis, wherein at least one probe is fixed to the test piece, and the at least one probe is an arbitrary region of the drug resistance gene. The region has a mutation position corresponding to the drug resistance of Mycobacterium tuberculosis, and the base at the mutation position is a wild type.

  In one embodiment, the test piece has an additional probe immobilized thereon, the additional probe comprising an oligonucleotide that can hybridize to any region of the drug resistance gene, the region comprising the drug resistance of Mycobacterium tuberculosis. The position of the mutation is associated with the degree, and the base at the position of the mutation is a mutant type.

  The present invention also provides a test piece for determining the degree of drug resistance of Mycobacterium tuberculosis, wherein the test piece has at least one probe immobilized thereon, and the at least one probe is an arbitrary drug resistance gene. It consists of an oligonucleotide that can hybridize with a region, and the region has a mutation position associated with the drug resistance of Mycobacterium tuberculosis, and the base at the mutation position is a mutant type.

  In one embodiment, the test piece has an additional probe immobilized thereon, the additional probe comprising an oligonucleotide that can hybridize to any region of the drug resistance gene, the region comprising the drug resistance of Mycobacterium tuberculosis. The position of the mutation is associated with the degree, and the base at the position of the mutation is wild type.

  In one embodiment, the drug resistance gene is at least one selected from the group consisting of isoniazid resistance gene, rifampicin resistance gene, streptomycin resistance gene, ethambutol resistance gene, new quinolone resistance gene and pyrazinamide resistance gene.

  In one embodiment, the drug resistance gene is at least one selected from the group consisting of katG, inhA, rpoB, rpsL, rrs, gyrA, gyrB, embA, embB, embC and pncA.

  In one embodiment, the drug resistance gene is katG and the mutation of the drug resistance gene is H97R, Q127E, N133T, M176T, P232S, S315R, S315T, S383P, D419H, M420T, R489S, D542H, R632C, Δ191 -192, at least one selected from the group consisting of 124 frame shifts and 160 frame shifts.

  In one embodiment, the drug resistance gene is inhA, and the mutation of the drug resistance gene is at least one selected from the group consisting of t-8c and c-15t.

  In one embodiment, the drug resistance gene is rpoB, and the mutation of the drug resistance gene is at least one selected from the group consisting of D516V, S521L, H526D, and H526Y.

  In one embodiment, the drug resistance gene is gyrA, and the mutation of the drug resistance gene is at least one selected from the group consisting of G88C, A90V, S91P and D94G / A / H / N.

  In one embodiment, the drug resistance gene is gyrB and the mutation of the drug resistance gene is D495N.

  In one embodiment, the drug resistance gene is embB and the mutation of the drug resistance gene is selected from the group consisting of M306L / V / I, D328G / Y, G406C / A / D / S and Q497K / R. At least one kind.

  In one embodiment, the drug resistance gene is pncA and the mutation of the drug resistance gene is M1I, A3E, L4S, L4W, I5N, V7D, V7F, D8E, D8G, V9A, V9G, Q10P, Q10R, Q10stop , D12A, C14R, C14Y, G23V, A25E, R26G, A28D, Y34D, Y34stop, L35P, V45G, A46E, T47A, D49A, H51P, H51Q, H57D, H57P, F58L, T61P, D63H, Y64D, S67P, W67G, 68 , W68S, W68stop, H71P, T76I, T76P, G78D, L85P, L85R, K96E, K96Q, G97D, Y99D, Y99S, Y103H, Y103S, Y103stop, W119stop, R12 P, V128G, T135P, D136N, D136Y, H137P, C138R, C138S, V139A, V139L, V139M, Q141P, Q141stop, A146T, R148S, V155G, L156Q, L159R, L172P, V180F, Frame 180 It is at least one selected from the group consisting of shift, 88 frame shift, 123 frame shift, 141 frame shift, 162 frame shift, 164 frame shift and 177 frame shift.

  The present invention also provides a kit for determining the degree of drug resistance of Mycobacterium tuberculosis, and the kit includes the test piece.

  In one embodiment, the kit includes a reagent for detecting a hybridized probe and a primer pair for amplification of a drug resistance gene.

  The present invention further provides a method for determining the drug resistance level of Mycobacterium tuberculosis, the method comprising a step of amplifying the drug resistance gene of Mycobacterium tuberculosis, contacting the amplified drug resistance gene with the test piece, and A step of hybridizing with a probe fixed to a test piece, a step of identifying a probe hybridized with the drug resistance gene, and a step of determining the drug resistance level of the tuberculosis bacteria from the position of the identified probe.

  According to the present invention, the degree of drug resistance of Mycobacterium tuberculosis can be determined quickly and easily. For this reason, it becomes possible to appropriately treat tuberculosis caused by drug-resistant bacteria.

It is a graph which shows the time-dependent change of INH oxidase activity in various tuberculosis bacteria.

  In the present invention, the tuberculosis bacterium refers to a bacterium belonging to the genus Mycobacterium, a kind of mycobacteria. Examples of Mycobacterium tuberculosis include Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium bovis (Mycobacterium bovis), Mycobacterium africanum (African type). Mycobacterium tuberculosis).

  In the present invention, the drug refers to a drug for treating tuberculosis. Examples of the drug include isoniazid (INH), rifampicin (RFP), streptomycin (SM), ethambutol (EB), new quinolone (NQ), and pyrazinamide (PZA).

  In the present invention, the drug resistance gene of Mycobacterium tuberculosis refers to a gene related to the action of a drug for treating tuberculosis. The drug resistance genes of Mycobacterium tuberculosis include isoniazid (INH) resistance gene, rifampicin (RFP) resistance gene, streptomycin (SM) resistance gene, ethambutol (EB) related to the action of INH, RFP, SM, EB, NQ, and PZA, respectively. ) Resistance gene, new quinolone (NQ) resistance gene, pyrazinamide (PZA) resistance gene.

  Examples of the INH resistance gene include katG (encodes catalase peroxidase) and inhA (encodes enoyl CoA reductase). Examples of the RFP resistance gene include rpoB (encoding RNA polymerase B subunit). Examples of the SM resistance gene include rpsL (encoding ribosomal protein S12) and rrs (encoding 16S rRNA). Examples of the EB resistance gene include embA, embB, and embC (all of which encode arabinosylindolylacetylinositol synthase). Examples of the NQ resistance gene include gyrA (encoding DNA gyrase A) and gyrB (encoding DNA gyrase B). An example of the PZA resistance gene is pncA (encodes pyrazinamidase).

  The drug resistance gene of Mycobacterium tuberculosis encodes enzymes and drug-binding proteins, and the action of these proteins and drugs causes the growth of Mycobacterium tuberculosis to be inhibited and killed. When a mutation in a drug resistance gene results in an amino acid substitution, insertion and / or deletion in the amino acid sequence of the encoded protein, a decrease in enzyme activity of these proteins, a structural change in the drug binding site, or the like may occur. Due to such changes, when the action of a protein encoding a drug resistance gene and the drug is inhibited, a drug resistant bacterium that escapes death appears.

  For example, the katG gene encodes INH oxidase (catalase peroxidase), and cell wall synthesis is inhibited and killed by INH metabolites oxidized by INH oxidase. When oxidative activity is lost, INH resistant bacteria are filed.

  For example, rpoB gene encodes RNA polymerase B subunit, and when RFP binds to RNA polymerase B subunit, the initiation reaction of RNA synthesis is inhibited and killed. However, mutation of rpoB gene causes RNA polymerase to die. If the binding activity of the B subunit to RFP is lost, RFP-resistant bacteria are filed.

  For example, the gyrA gene encodes DNA gyrase A. When NQ binds to DNA gyrase A, M. tuberculosis is inhibited by DNA replication and killed. If the binding activity is lost, NQ resistant bacteria are filed.

  In the present invention, as a drug resistance gene mutation of Mycobacterium tuberculosis, for example, when the drug resistance gene is katG (accession number: NC_000962 REGION: 2153889..2156111), for example, H97R, Q127E, N133T, M176T, P232S, S315R, S315T, S383P, D419H, M420T, R489S, D542H, R632C, Δ191-192, 124 frame shift, and 160 frame shift.

  For example, when the drug resistance gene is inhA (accession number: NC — 000962 REGION: 1674202..1675011), examples include t-8c and c-15t.

  For example, when the drug resistance gene is rpoB (accession number: NC_000962 REGION: 759807..763325), for example, D516V, S521L, H526D, and H526Y can be mentioned.

  For example, when the drug resistance gene is gyrA (accession number: NC — 000962 REGION: 7302..9818), examples include G88C, A90V, S91P, and D94G / A / H / N.

  For example, when the drug resistance gene is gyrB (accession number: NC — 000962 REGION: 5123..7267), for example, D495N can be mentioned.

  For example, when the drug resistance gene is embB (accession number: NC — 000962 REGION: 4246514..4249810), for example, M306L / V / I, D328G / Y, G406C / A / D / S, and Q497K / R can be mentioned.

  For example, when the drug resistance gene is pncA (accession number: NC_000962 REGION: 2288681..2289241), for example, M1I, A3E, L4S, L4W, I5N, V7D, V7F, D8E, D8G, V9A, V9G, Q10P, Q10R , Q10stop, D12A, C14R, C14Y, G23V, A25E, R26G, A28D, Y34D, Y34stop, L35P, V45G, A46E, T47A, D49A, H51P, H51Q, H57D, H57P, F58L, T61P, G63H, P64W , W68R, W68S, W68stop, H71P, T76I, T76P, G78D, L85P, L85R, K96E, K96Q, G97D, Y99D, Y99S, Y103H, Y103S, Y103stop, W119stop p, R121P, V128G, T135P, D136N, D136Y, H137P, C138R, C138S, V139A, V139L, V139M, Q141P, Q141stop, A146T, R148S, V155G, L156Q, L159R, L172P, Vshift Examples include 80 frame shift, 88 frame shift, 123 frame shift, 141 frame shift, 162 frame shift, 164 frame shift, and 177 frame shift.

  Here, for example, “H97R” means that the 97th amino acid of the protein encoded by the drug resistance gene was mutated from histidine (H) to arginine (R). “H”, “R” and the like are single letter codes for amino acids. For example, “Q10stop” means that the 10th amino acid of the protein encoded by the drug resistance gene is mutated from glutamine (Q) to a stop codon. For example, "t-8c" means that the base at position -8 was mutated from thymine (t) to cytosine (c) with reference to the coding region of the drug resistance gene (with a as the first position of the start codon atg). Means. “T”, “c”, etc. are base notations. For example, “Δ191-192” means that the 191st to 192nd amino acids of the protein encoded by the drug resistance gene have been deleted, and “124 frame shift” means the 124th of the coding region of the drug resistance gene. It means a mutation in which the insertion or deletion of a base occurs in the second codon and the encoded amino acid is changed thereafter.

  The mutation of the drug resistance gene of Mycobacterium tuberculosis is not limited to the known mutation as described above, and may be an unknown mutation. A method for identifying an unknown mutation is not particularly limited. For example, a drug-resistant gene is isolated from a drug-resistant bacterium by PCR, etc., and the base sequence of the gene is decoded. Then, the base sequence of the decoded gene is compared with the base sequence of the wild-type drug-resistant gene. A method of identifying is mentioned.

  In the present invention, the drug resistance level of Mycobacterium tuberculosis is associated with the mutation of the drug resistance gene. For this purpose, the degree of drug resistance of Mycobacterium tuberculosis having a mutation in the drug resistance gene is determined.

  In the present invention, the drug resistance level of Mycobacterium tuberculosis refers to the extent to which Mycobacterium tuberculosis does not die in the presence of the drug. The method for classifying the drug resistance is not particularly limited, but a simple method is preferable. As a simple method, for example, there is a method of classifying the drug resistance degree according to the concentration of the drug for which Mycobacterium tuberculosis is resistant. For example, there is a method of distinguishing tuberculosis bacteria that do not die in the presence of a high concentration of drug from tuberculosis bacteria that die in the presence of a high concentration of drug but not in the presence of a low concentration of drug. For example, a method for distinguishing between Mycobacterium tuberculosis that does not die in the presence of 1.0 μg / mL drug and Mycobacterium tuberculosis that does not die in the presence of 1.0 μg / mL drug but does not die in the presence of 0.2 μg / mL drug. Is mentioned. The number of sections (drug concentration) is not particularly limited, but is preferably 2 to 3.

  In the present invention, the method for determining the drug resistance level of Mycobacterium tuberculosis is not particularly limited, but a standard method is preferable. The standard method includes, for example, a method defined by CLSI (Clinical and Laboratory Standards Institute) / NCCLS (National Committee for Clinical Laboratory Standards). For example, in Japan, as a kit according to this method, “drug-sensitive (acid-fast bacilli) kit“ Nichibi ”well pack medium S for acid-fast bacilli test” (manufactured by Nippon BCG Supply Co., Ltd.) Acid bacteria) kit Kyokuto Mycobacterium tuberculosis susceptibility bit spectrum-SR "(manufactured by Kyokuto Pharmaceutical Co., Ltd.) is commercially available. In these kits, M. tuberculosis is cultured in the presence or absence of various drug concentrations, and after culturing for a certain period, for example, the viability of M. tuberculosis can be confirmed visually.

(probe)
The probe of the present invention comprises an oligonucleotide that can hybridize with an arbitrary region of a drug resistance gene, and this region has a mutation position associated with the drug resistance level of Mycobacterium tuberculosis. The base is wild type (hereinafter sometimes referred to as “wild type probe”). Wild-type probes cannot hybridize to these regions if the region consisting of a base sequence that can be mutated has a mutation. The length of the wild-type probe is generally 10 to 30 nucleotides, preferably 12 to 26 nucleotides, although it depends on the hybridization conditions.

  The probe of the present invention also comprises an oligonucleotide that can hybridize with any region of a drug resistance gene, and this region has a mutation position associated with the drug resistance level of Mycobacterium tuberculosis, and the position of the mutation. This base is a mutant type (hereinafter sometimes referred to as “mutant probe”). A mutant probe cannot hybridize to a region consisting of a base sequence that can be mutated if it does not have a mutation. The length of the mutant probe is generally 10 to 30 nucleotides, preferably 12 to 26 nucleotides, although it depends on the hybridization conditions. The position of the mutation in the mutant probe is preferably near the center of the oligonucleotide constituting the mutant probe in order to identify the mutation reliably.

  In the present invention, a wild type probe and a mutant type probe may be used in combination.

  Each probe above can be designed using standard programs and primer analysis software such as Primer Express (Perkin Elmer) and synthesized using standard methods such as automated oligonucleotide synthesizers. Can do.

  In addition, each probe may be modified at either the 5 'or 3' end for immobilization to a test strip, as described in detail below.

(Test pieces)
In the test piece of the present invention, at least one probe is fixed. At least one probe is a wild type probe, and preferably a mutant probe is further immobilized. Alternatively, at least one probe is a mutant probe, and preferably a wild type probe is further immobilized.

  The test piece of the present invention can be prepared by fixing the probe to the surface of the carrier at regular intervals. When a plurality of probes are fixed, the surface of the carrier is divided and fixed for each probe. Examples of the carrier include organic materials such as vinyl polymer, nylon, polyester, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, and nitrocellulose; inorganic materials such as glass and silica; and metal materials such as gold and silver. There is no particular limitation. An organic material is preferable in terms of easy molding processability, and polyesters such as polyethylene terephthalate are more preferable. When using the color development method in the following method for determining the drug resistance, the carrier is preferably white in order to make the color development easily visible.

  Examples of the method for immobilizing the probe on the surface of the carrier include a physical method and a chemical method. The physical method is not particularly limited. For example, a method of coating the carrier surface with a polycationic polymer such as polylysine, and when the carrier surface is a metal material such as gold, a thiol or disulfide compound having an amino group such as 2-aminoethanethiol A method for treating the surface of the carrier is mentioned. Since these methods use electrostatic interaction with a probe that is a polyanion, the immobilization efficiency can be increased. Furthermore, immobilization efficiency can be increased by adding an irrelevant base sequence (such as a polythymine chain) to the end of the probe and increasing the molecular weight of the probe itself to be immobilized. For example, the probe can be fixed relatively easily by discharging a solution containing a polythymine-added oligonucleotide probe onto a nitrocellulose membrane using a dispenser and then irradiating the nitrocellulose membrane with ultraviolet rays. Specifically, the solution containing each probe is placed in a dispenser equipped with a 24 gauge needle, and applied at a rate of 2.5 to 8.5 mm / sec while being discharged at a rate of 0.5 to 1.0 μL / min. When applied on the nitrocellulose film at a certain interval at a speed, a stripe having a width of about 1 to 2 mm is formed in which the probes are arranged at a certain interval. The probe is fixed on the surface of the nitrocellulose film by irradiating the nitrocellulose film on which the stripe of the probe is formed with ultraviolet rays. Furthermore, if necessary, if a thin cut is made so as to cross these stripes, a large number of test pieces in which the probes are sequentially fixed can be obtained at a time.

  The chemical method is not particularly limited. For example, when the carrier material is an inorganic material such as glass or silicon, the end of the probe is modified with a functional group capable of silane coupling reaction such as trimethoxysilane or triethoxysilane, and the carrier is modified with a modified probe. A method of immersing in a solution containing selenium for 24 to 48 hours, taking out, and then washing. Alternatively, there is a method in which the carrier is treated with a silane coupling agent having an amino group such as aminoethoxysilane so that the surface of the carrier is aminated and then subjected to an amino coupling reaction with a probe having a carboxylic acid introduced at the terminal. . When the material of the carrier is a metal material such as gold or silver, the end of the probe is modified with a functional group capable of binding to a metal such as a thiol group or a disulfide group, and the carrier is added to a solution containing the modified probe in 24 to A method of immersing for 48 hours, taking it out, and washing it.

  As a method for immobilizing the probe on the surface of the carrier, in addition to the method for immobilizing the synthesized probe, a method for synthesizing the probe on the surface of the carrier using a lithography technique or the like can be mentioned.

(Method to determine the drug resistance of Mycobacterium tuberculosis)
The method for determining the drug resistance level of M. tuberculosis according to the present invention includes a step of amplifying a drug resistance gene of M. tuberculosis, a probe fixed on the test piece by contacting the amplified drug resistance gene with the test piece. And the step of identifying a probe hybridized with the drug resistance gene, and the step of determining the drug resistance level of the tubercle bacilli from the position of the identified probe.

1. Specimen In the present invention, a specimen refers to a substance containing Mycobacterium tuberculosis that is a target for determining the degree of drug resistance. Samples were obtained from, for example, body fluids such as sputum, pharyngeal swab, gastric fluid, bronchoalveolar lavage fluid, endotracheal aspirate, throat wipes and / or tissue biopsy, and cultures thereof. Mycobacterium tuberculosis itself can be mentioned.

2. Sample pretreatment (DNA extraction)
As a pretreatment of the specimen, DNA is extracted from the specimen. The method for extracting DNA from the specimen is not particularly limited. For example, a phenol extraction method, a guanidine thiocinate extraction method, and a vanadyl ribonucleoside complex extraction method may be mentioned.

3. Amplification of drug resistance gene The method for amplifying the drug resistance gene is not particularly limited. For example, PCR method, LAMP method, and ICAN method are mentioned. In each method, the conditions for amplifying the drug resistance gene are appropriately set. For example, when a drug resistance gene is amplified by the PCR method, a primer pair may be designed so that a region consisting of a base sequence having a mutation of the target drug resistance gene can be amplified. Preferably, a primer pair may be designed so that a drug resistance gene region capable of covering all mutations can be amplified, and more preferably, a drug resistance gene coding region can be amplified. For example, when the INH resistance gene katG is amplified by the PCR method, examples of the primer pair include an oligonucleotide having a base sequence upstream and downstream of the coding region of the katG gene or a base sequence complementary thereto. For example, the oligonucleotide which consists of a base sequence shown to the sequence number 7 and 8 as described in the following examples is mentioned.

4). Determination of the drug resistance level of Mycobacterium tuberculosis The method for bringing the amplified drug resistance gene into contact with the test piece and hybridizing with the probe fixed to the test piece is not particularly limited. For example, a method of immersing a test piece in a solution containing an amplified drug resistance gene for a predetermined time can be mentioned. Conditions such as the temperature during the immersion are appropriately set in consideration of suppression of non-specific hybridization.

  The method for identifying the probe hybridized with the drug resistance gene is not particularly limited. For example, when a drug resistance gene is amplified by a PCR method, a primer pair modified with a labeling substance such as a radioisotope, a fluorescent substance, or a chemiluminescent substance is used to detect these labeling substances added to the drug resistance gene. A method is mentioned. Alternatively, a method may be mentioned in which a probe is modified with a labeling substance such as a radioisotope, a fluorescent substance, or a chemiluminescent substance, and a probe to which a drug resistance gene is hybridized is detected using these labeling substances. The method for detecting the labeling substance is not particularly limited. For example, a nitro blue tetrazolium (NBT) / 5-bromo-4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) coloring method can be mentioned.

  Specifically, when a drug resistance gene is amplified by PCR, a primer pair in which the 3 'or 5' end is modified with biotin is used. Next, the test piece is immersed in a solution containing a gene having biotin at the amplified end. In this step, the drug resistance gene can hybridize with the probe immobilized on the test strip. The specimen is then washed and immersed in a solution containing alkaline phosphatase labeled streptavidin. In this step, alkaline phosphatase-labeled streptavidin binds to the drug resistance gene hybridized with the probe fixed to the test piece. Next, NBT / BCIP is added to the washed test piece, and color development due to the reaction of alkaline phosphatase is detected. This color development is visible. In order to clarify the presence / absence of color development, a difference in color development between the wild type probe and the mutant type probe may be confirmed.

  From the position of the probe to which the identified drug resistance gene has hybridized, it can be seen which probe has hybridized. Since each probe is associated with the drug resistance level of M. tuberculosis, the drug resistance level of M. tuberculosis in the sample can be determined.

(Determination kit for the drug resistance of Mycobacterium tuberculosis)
The kit of this invention contains said test piece. Preferably, any reagent suitable for determining the drug resistance of Mycobacterium tuberculosis can be included. Examples of the reagents include a DNA extraction reagent, a gene amplification reagent, and a hybridized probe detection reagent. The gene amplification reagent may also include a primer pair for amplification of a drug resistance gene. Examples of reagents for detecting hybridized probes include biotin, streptavidin-modified alkaline phosphatase, NBT, and BCIP in the case of the NBT / BCIP color development method.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited thereto.

(Example 1: Determination of INH resistance of Mycobacterium tuberculosis)
Wild strains of Mycobacterium tuberculosis shown in Table 1 and mutant strains having mutations in the INH resistance gene katG were obtained from the National International Medical Center.

  Using the “Drug-sensitive (acid-fast bacilli) kit Far East tuberculosis susceptibility bit spectrum-SR” manufactured by Kyokuto Pharmaceutical Co., Ltd., which is widely used as the CLSI / NCCLS method, according to the method attached to the kit, Tolerance was determined.

1. Pre-culture Far-east 1% Ogawa medium (1 g of potassium dihydrogen phosphate, 1 g of sodium glutamate, 100 mL of distilled water, 200 mL of whole egg solution, 6 mL of glycerin, 2 mL of malachite green 6 mL, pH 6.5) And suspended in 4 mL of a pre-culture liquid medium (“Myco Broth” manufactured by Kyokuto Pharmaceutical Co., Ltd.) contained in a 12 mL sterilized test tube. The test tube was allowed to stand at 37 ° C., and the bacteria in the test tube were grown. The bacteria were cultured until the absorbance at 530 nm (OD (530 nm)) reached 0.1 (corresponding to a bacterial cell concentration of 1 mg / mL) or more.

2. Preparation of the bacterial solution The bacterial solution in the test tube after the pre-culture was stirred, and the test tube was allowed to stand for about 15 minutes until the bacterial mass subsided. Next, the upper layer of the bacterial solution in the test tube was diluted with sterilized distilled water to prepare a bacterial solution having an OD (530 nm) of 0.1. 0.5 mL of this bacterial solution was added to 4.5 mL of sterilized distilled water contained in a 50 mL sterilized test tube and mixed to obtain a 10-fold diluted bacterial solution. Further, 0.1 mL of 10-fold diluted bacterial solution was added to 10 mL of sterile distilled water contained in a 50 mL volume sterile test tube and mixed to obtain a 1000-fold diluted bacterial solution.

3. The medium sealing tape on the microtiter plate of the main culture kit was peeled off, and the lid was removed. Then, using the dedicated dropper attached to the kit, 1 drop (about 0. 1) of the 10-fold diluted solution to the well of “C +” and INH at a concentration of 0.2 μg / mL or 1.0 μg / mL. 02 times), add 1 drop (about 0.02 mL) of the 1000-fold diluted solution to the “C1 / 100” well, and 1 drop (about 0.02 mL) of sterile distilled water to the “C-” well. 02 mL) was added in portions. Next, after adding 1 to 2 mL of sterilized distilled water to the filter paper placed around the well on the microtiter plate, cover the microtiter plate and seal the gap between the lid edge and the microtiter plate with tape. did. The microtiter plate was allowed to stand at 37 ° C. to initiate main culture.

4). Determination The microtiter plate was observed on the 7th, 10th, and 14th days after the start of the main culture, and the determination was made when the liquid in the well was colored red in 3/4 or more of the “C +” well. went. In the determination, the coloration of “C−”, “C +”, and “C1 / 100” is “0”, “4+”, and “1+”, respectively, and INH is a concentration of 0.2 μg / mL or 1.0 μg / mL. The coloration of the wells contained in was scored from “0” to “4+” compared to the coloration of the wells of “C−”, “C +” and “C1 / 100”. The case where the score was “0” or “1+” was regarded as sensitive, and the case where the score was “2+” to “4+” was regarded as resistant. And, a well containing INH at a concentration of 1.0 μg / mL is resistant to 1.0 μg / mL INH resistance, and a well containing INH at a concentration of 1.0 μg / mL is sensitive but is resistant to INH. Wells containing at a concentration of 0.2 μg / mL had an INH resistance of 0.2 μg / mL indicating resistance. The determination results are shown in Table 2. The determined strain could be classified into an INH resistance of 1.0 μg / mL and an INH resistance of 0.2 μg / mL.

(Reference example: activity measurement of INH oxidase encoded by INH resistance gene)
It was examined how the activity of INH oxidase encoded by the INH resistance gene katG is affected by the mutation of katG. Wild-type katG and mutant-type katG were isolated from the wild and mutant strains of Mycobacterium tuberculosis shown in Table 1 above using the primer pairs shown below, respectively, and the expression vectors pTrcHis2-TOPO (Invitrogen) were used. Inserted into the multiple cloning site via TA ligation.

Primer 1: CCCGAGCAACACCCACCCATTACAGAAAC (SEQ ID NO: 1)
Primer 2: TCAGCGCACGTCGAACC (SEQ ID NO: 2)

  pTrcHis2-TOPO is a vector that can add a c-myc tag and a 6 × His tag to the C-terminus of the protein encoded by the DNA inserted into the multiple cloning site, but since the inserted katG has a stop codon, These tags are not added to the INH oxidase encoded by. Subsequently, the resulting plasmid was used to transform Escherichia coli ΔkatG strain (owned by the National International Medical Center Research Institute, Department of Infectious Disease Control) according to a conventional method. Transformed E. coli is cultured, and after 3 hours from the start of culture, IPTG (isopropyl-β-thiogalactopyranoside) is added to the medium to overexpress katG in E. coli, and after 5 hours from the start of the culture, E. coli is recovered. did. The recovered Escherichia coli was crushed by ultrasonic waves (using a UR-20P ultrasonic generator UR-20P manufactured by Tommy Seiko Co., Ltd., and repeated a cycle of ultrasonic treatment for 30 seconds and cooling for 60 seconds 5 times in power9), followed by centrifugation. (12,000 rpm, 5 minutes, 4 ° C .: Centrifuge5415R manufactured by Eppendorf), and the supernatant was used for the following measurement.

The total protein in the supernatant was quantified by BCA assay (BCA Protein Assay Kit: Thermo Scientific), and the INH oxidase activity in the supernatant was measured by Wei et al., Antimicrob. Agents Chemother., 2003, 47, p. 670- Measured with reference to 675. The INH oxidase activity can be measured spectroscopically by the absorbance at 560 nm (OD (560 nm)) of reduction of NBT (Nitroblue Tetrazolium) accompanying free radical generation due to oxidation of INH in the presence of H ₂ O ₂ .

  Specifically, NBT (manufactured by Sigma) having a final concentration of 0.2 mM in a supernatant corresponding to 200 μg of total protein in a 50 mM phosphate buffer (pH 7.0) so that the total amount becomes 1 mL, 5 mg glucose oxidase (manufactured by Sigma), glucose having a final concentration of 4 mM, and INH (manufactured by Sigma) having a final concentration of 9 mM were added and mixed at room temperature. The OD (560 nm) of this mixed solution was measured with a spectrophotometer for 200 seconds. Each supernatant was measured three times, and an average value ± standard deviation was calculated. The time course of OD (560 nm) is shown in FIG.

  According to FIG. 1, the activity of INH oxidase encoded by katG is divided into three categories according to the mutation of katG: “activity comparable to wild type”, “activity lower than wild type”, and “little activity”. These classifications are respectively “no resistance”, “0.2 μg / mL INH resistance” and “1.0 μg / mL” for the resistance of Mycobacterium tuberculosis to INH according to the mutation of katG shown in Table 2. It was found that it completely corresponds to the category of “INH resistance of mL”.

(Example 2: Preparation of test piece for determining the degree of INH resistance of Mycobacterium tuberculosis)
1. Probe synthesis The following detection of the S315T mutation associated with an INH resistance of 1.0 μg / mL and an H97R mutation associated with an INH resistance of 0.2 μg / mL, respectively, was performed. Mutant probes 1 and 2 corresponding to the obtained wild type probes 1 and 2 are prepared.

Wild type probe 1: GCGATCACCAGCGGCATCGA (SEQ ID NO: 3)
Wild-type probe 2: GACTACGGCCACTACGGGCC (SEQ ID NO: 4)
Mutant probe 1: GCGATCACCACCGGCATCGA (SEQ ID NO: 5)
Mutant probe 2: GACTACGGCCGCTACGGGCC (SEQ ID NO: 6)

2. Probe Immobilization Polythymine is added to the 5 ′ end of each probe using terminal transferase (Promega). Specifically, 0.4 μL of terminal transferase (30 units / μL), 2 μL of thymidine triphosphate (10 pmol / μL), 2 μL of oligonucleotide probe (50 mM), 2 μL of reaction buffer attached to the product, and purified water A reaction solution was prepared by mixing 3.6 μL, reacted at 37 ° C. for 4 hours, and then added with 90 μL of 10 × SSC buffer solution to the reaction solution, thereby containing a solution containing an oligonucleotide probe to which polythymine was added ( 1 pmol / μL).

  Next, the solution containing each probe to which polythymine has been added is placed in a dispenser equipped with a 24-gauge needle, and discharged at a rate of 0.7 mm / min. It is applied in a vertical direction at intervals of 2 mm on a nitrocellulose film (length 75 mm × width 150 mm: manufactured by Whatman) so as to form a stripe of Next, the probe is fixed to the nitrocellulose membrane by irradiating these nitrocellulose membranes with ultraviolet rays of 312 nm for 2 minutes. Next, the nitrocellulose membrane is cut so as to include all the stripes, thereby producing a test piece of 5 mm × 150 mm.

(Example 3: Determination of INH resistance of Mycobacterium tuberculosis using a test piece for determining INH resistance)
1. Amplification of katG gene In this example, wild strains, S315T mutant strains resistant to 1.0 μg / mL INH, and H97R mutant strains resistant to 0.2 μg / mL INH were used as specimens. The INH resistance is determined using the test piece prepared in Example 2.

  The sample INH resistance gene katG was mixed with primer 3 consisting of the base sequence shown in SEQ ID NO: 7, primer 4 consisting of the base sequence shown in SEQ ID NO: 8 and biotin bound to the 5 ′ end, and Taq DNA polymerase (Roche Amplification is performed by a PCR method using a diagnostics company.

Primer 3: CAACTCCTGGAAGGAATGCT (SEQ ID NO: 7)
Primer 4: GCAGGGCCGATCAACCCGAA (SEQ ID NO: 8)

  As PCR reaction conditions, the denaturation step is 94 ° C. for 30 seconds, the annealing step is 55 ° C. for 20 seconds, the chain extension step is 72 ° C. for 20 seconds, and these steps are performed for 30 cycles to amplify the katG gene. Biotin is bound to the end of the amplified gene.

2. Detection of hybridized probe To 10 μL of the solution containing the amplified gene, a solution (10 μL) of sodium hydroxide (5 M) and ethylenediaminetetraacetic acid (0.05 M) was added, stirred well, and allowed to stand for 5 minutes to amplify. The resulting gene is denatured into a single strand. To this solution, a solution of sodium dodecyl sulfate (0.01 w / v%), sodium chloride (1.8 w / v%), sodium citrate (1.0 w / v%) (1 mL), and made in Example 2 The test piece is added and the mixture is shaken at 62 ° C. for 30 minutes. Next, the test piece is taken out from the mixed solution, washed, alkaline phosphatase-labeled streptavidin is added to the test piece, and nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphatase p-toluidinyl salt are added. (BCIP) is added to develop the test piece.

  According to the present invention, the degree of drug resistance of Mycobacterium tuberculosis can be determined quickly and easily. For this reason, it becomes possible to appropriately treat tuberculosis caused by drug-resistant bacteria. Appropriate implementation of tuberculosis treatment not only reduces the burden on patients and doctors, but also contributes to reducing the medical costs of society as a whole.

Claims (6)

A kit for classifying and determining the drug resistance of Mycobacterium tuberculosis,
Including specimens,
The test piece has at least one probe fixed thereto;
The at least one probe comprises an oligonucleotide that hybridizes to any region of the drug resistance gene;
The region has a mutation location associated with the drug resistance of Mycobacterium tuberculosis;
The drug resistance gene is katG and the mutations are H97R, Q127E, N133T, M176T, P232S, S315R, S315T, S383P, D419H, M420T, R489S, D542H, R632C, Δ191-192, 124 frame shift and 160 frame shift Any one mutation selected from the group consisting of:
The M176T, S315R, S315T, D419H, M420T, D542H, R632C, Δ191-192, 124 frameshift and 160 frameshift are more than the concentration of isoniazid to which the H97R, Q127E, N133T, P232S, S383P and R489S are resistant. A mutation that is resistant to high concentrations of isoniazid,
The base at the position of the mutation is wild type;
kit. A further probe is fixed to the test piece ,
The further probe consists of an oligonucleotide that hybridizes to the region where the base at the mutation is mutated ,
The kit according to claim 1. A kit for classifying and determining the drug resistance of Mycobacterium tuberculosis,
Including specimens,
The test piece has at least one probe fixed thereto;
The at least one probe comprises an oligonucleotide that hybridizes to any region of the drug resistance gene;
The region has a mutation location associated with the drug resistance of Mycobacterium tuberculosis;
The drug resistance gene is katG and the mutations are H97R, Q127E, N133T, M176T, P232S, S315R, S315T, S383P, D419H, M420T, R489S, D542H, R632C, Δ191-192, 124 frame shift and 160 frame shift Any one mutation selected from the group consisting of:
The M176T, S315R, S315T, D419H, M420T, D542H, R632C, Δ191-192, 124 frameshift and 160 frameshift are more than the concentration of isoniazid to which the H97R, Q127E, N133T, P232S, S383P and R489S are resistant. A mutation that is resistant to high concentrations of isoniazid,
The base at the position of the mutation is a mutant type,
Kit . A further probe is fixed to the test piece ,
The further probe consists of an oligonucleotide that hybridizes to the region where the base at the mutation is wild type ;
The kit according to claim 3. The kit according to any one of claims 1 to 4, further comprising a detection reagent for a hybridized probe and a primer pair for amplification of a drug resistance gene. A method of determining by dividing the drug resistance of Mycobacterium tuberculosis,
Amplifying the drug resistance gene of Mycobacterium tuberculosis,
Amplified drug resistance gene is contacted with test pieces, the step of hybridized with a probe fixed to the test piece,
Identifying a probe hybridized with the drug resistance gene, and determining a drug resistance level of the tubercle bacilli from the position of the identified probe,
At least one of the probes immobilized on the specimen consists of an oligonucleotide that hybridizes to any region of the drug resistance gene;
The region has a mutation location associated with the drug resistance of Mycobacterium tuberculosis;
The drug resistance gene is katG and the mutations are H97R, Q127E, N133T, M176T, P232S, S315R, S315T, S383P, D419H, M420T, R489S, D542H, R632C, Δ191-192, 124 frame shift and 160 frame shift Any one mutation selected from the group consisting of:
The M176T, S315R, S315T, D419H, M420T, D542H, R632C, Δ191-192, 124 frameshift and 160 frameshift are more than the concentration of isoniazid to which the H97R, Q127E, N133T, P232S, S383P and R489S are resistant. A mutation that is resistant to high concentrations of isoniazid,
The base at the position of the mutation is wild type or mutant,
Method.

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