JP2008142075A - Test piece for detecting pyrazinamide sensitivity in tubercle bacillus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a means for preventing a wrong detection related to pyrazinamide (PZA) sensitivity in PZA-sensitive tubercle bacillus having silent mutation. <P>SOLUTION: The present invention provides a test piece for detecting PZA sensitivity in tubercle bacillus. Various probes hybridizing with pncA gene encoding pyrazinamidase are immobilized in the test piece. At least one probe among the probes comprises an oligonucleotide hybridizing with a base sequence domain having the silent mutation of the pncA gene of the tubercle bacillus. The other probe is an oligonucleotide hybridizing with an arbitrary domain comprising the wild type base sequence of the pncA gene of the tubercle bacillus. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a test strip for detecting pyrazinamide sensitivity in 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. Treatment of tuberculosis is basically a medical therapy centered on chemotherapy, and surgical therapy is considered when it is impossible to achieve the purpose of treatment with medical therapy.

  Many types of drugs such as isoniazid (INH), rifampicin (RFP), streptomycin (SM), ethambutol (EB) are known as drugs for treating tuberculosis used in medical therapy. However, when a drug is administered to a patient, a problem arises that a mutation causes a resistant bacterium having resistance to the administered drug. For pyrazinamide (hereinafter also referred to as “PZA”) whose use has been started since 1996, the appearance of resistant bacteria is feared.

  In order to obtain information on the drug resistance of Mycobacterium tuberculosis possessed by tuberculosis patients, drug susceptibility testing of Mycobacterium tuberculosis has been conducted. The drug sensitivity test is generally performed by examining the presence or absence of resistance to a predetermined drug based on the presence or absence of bacterial growth in a drug sensitivity test medium containing the predetermined drug. For example, examples of the drug sensitivity test medium include Bactec MGIT ™ 960 (Nippon Becton Dickinson Co., Ltd.) and tuberculosis sensitive PZA liquid medium (Kyokuto Pharmaceutical Co., Ltd.). However, there is a problem that M. tuberculosis has a slow growth, and it takes several weeks to check the presence or absence of M. tuberculosis growth and determine the resistance.

  It has been reported that drug resistant bacteria acquire drug resistance by mutating specific genes. Therefore, a method for detecting drug-resistant bacteria by detecting a mutation in a specific gene has been developed. For example, an oligo synthesized based on the base sequence of a tuberculosis therapeutic drug resistance-related gene on the Mycobacterium tuberculosis genome in a wild-type tuberculosis bacterium sensitive to tuberculosis treatment drug and a mutant tuberculosis bacterium resistant to any drug. A tuberculosis diagnosis kit including a substrate on which nucleotides are immobilized has been disclosed (Patent Document 1). The currently developed DNA microarray kit “Oligo Array” (Nisshinbo Co., Ltd.) is a kit that can detect gene mutations involved in resistance to 5 drugs of INH, RFP, SM, kanamycin (KM), and EB. .

  It has been reported that resistant bacteria to pyrazinamide (PZA) become resistant by mutation of the pncA gene encoding pyrazinamidase (Patent Document 2 and Non-Patent Documents 1 to 5). However, it is known that many gene mutations related to PZA drug resistance exist throughout the entire region of the pncA gene, and it is said that PZA resistance is acquired by any one of these mutations. That is, in order to appropriately detect PZA resistant bacteria, it must be possible to detect any one mutation without leaking. There may be more unknown pncA gene mutations related to PZA resistance, and it is necessary to find these mutations immediately in order to detect PZA resistant bacteria.

Therefore, the present inventors have developed detection probe sets that can cover the entire region of the pncA gene in order to reliably detect mutations that exist over the entire region of the pncA gene (Patent Documents 3 and 4). This probe set for detection is designed so that each probe can hybridize with a wild-type pncA gene. Specifically, in this probe set for detection, since the color develops when the pncA gene binds to the probe, the color development of all the probes in the set indicates that there is no mutation, and thus is determined to be PZA sensitive. On the other hand, when there is a mutation in the pncA gene, the pncA gene does not bind to the probe corresponding to the mutation site, so that no color is developed. Therefore, if there is a probe that does not develop color, it can be seen that there is a mutation, that is, a PZA resistant bacterium.
JP 2001-103981 A JP 2000-512493 A JP 2006-180746 A Japanese Patent Laid-Open No. 2006-180753 J. Korean Med Sci., 16, 537-543 (2001) Epidemiol. Infect., 128, p.337-342 (2002) J. Clin. Microbiol., 40, 2, 501-507 (2002) Microbiol. Drug, 3, 7, p.223-228 (2001) Scorpio et al., Nat. Med., 2, 6, p. 662-667 (1996)

  Gene mutations include silent mutations in which a base is substituted but the encoded amino acid is not changed. When detecting mutations in a gene using a probe, if the mutation that affects the enzyme activity and the silent mutation exist at different positions, the probe is designed to detect only the mutation that affects the enzyme activity. There is no need to consider silent mutations. However, when mutations that affect enzyme activity and silent mutations are located very close to each other, the probe detection range overlaps with the position of these mutations, and a probe that can reliably distinguish each mutation. It is difficult to design. Therefore, a probe intended to detect a mutation that affects enzyme activity is detected if any one of the mutations is present. Thus, when only a silent mutation exists, there is a problem that an erroneous detection result that there is a mutation affecting the enzyme activity is obtained even though the enzyme activity is not affected.

  The present inventors have found that there is also a silent mutation in the pncA gene, and this silent mutation is located very close to the position of the mutation associated with PZA resistance. Furthermore, even when a new silent mutation is found in the pncA gene, it is highly likely that the mutation is very close to the position of the mutation related to PZA resistance. Therefore, in the probe set for detection described above, a tuberculosis bacterium that has only a silent mutation and is sensitive to PZA is erroneously determined to be a PZA resistant bacterium, and as a result, a tuberculosis patient having such a silent bacterium is treated. On the other hand, there arises a problem that treatment by PZA administration, which should be effective, is not given. Therefore, a means for preventing erroneous detection of PZA sensitive bacteria is desired.

  The present inventors have found that PZA sensitivity can be more reliably determined by using both a probe corresponding to the silent mutation and a probe corresponding to the wild type.

The present invention provides a test strip for detecting pyrazinamide sensitivity in Mycobacterium tuberculosis,
At least two probes are fixed to the test piece,
At least one of the probes is composed of an oligonucleotide capable of hybridizing with a region of a base sequence having a silent mutation of the Mycobacterium tuberculosis pyrazinamidase gene, and the silent mutation in the region of the base sequence having the silent mutation The base sequence other than is wild type,
The other probes of the probes consist of oligonucleotides that can hybridize with any region consisting of the wild-type base sequence of the Mycobacterium tuberculosis pyrazinamidase gene, and the other probes are respectively located at different positions on the test strip. The region of the base sequence that is fixed and has the silent mutation is approximately the same region as one of the arbitrary regions of the wild-type base sequence.

  In a preferred embodiment, a plurality of oligonucleotides capable of hybridizing with any region of the wild-type base sequence hybridize with different regions of the base sequence of the pyrazinamidase gene, and the pyramidase gene of the pyrazinamidase gene is hybridized by the different regions. Nearly the entire coding sequence or its complementary sequence is covered.

  In a further embodiment, the oligonucleotide capable of hybridizing with the region of the base sequence having the silent mutation and the oligonucleotide capable of hybridizing with the region of the wild-type base sequence in the same region as the region are located at the same position on the test strip. Fixed to.

  In one embodiment, the base sequence consisting of the wild type of the pyrazinamidase gene is the base sequence set forth in SEQ ID NO: 1 in the sequence listing or a complementary sequence thereof.

  In one embodiment, the silent mutation is a mutation from base C to T at position 275 of the base sequence set forth in SEQ ID NO: 1 in the sequence listing or its complementary sequence.

  In a specific embodiment, the oligonucleotide capable of hybridizing with the region of the base sequence having the silent mutation consists of the base sequence set forth in SEQ ID NO: 22 in the sequence listing or a complementary sequence thereof.

  In a more specific embodiment, at least one of the oligonucleotides capable of hybridizing with any region of the wild-type base sequence consists of the base sequence set forth in SEQ ID NO: 21 of the sequence listing or a complementary sequence thereof.

  In a more specific embodiment, the plurality of other oligonucleotides that can hybridize to any region of the base sequence consisting of the wild type are SEQ ID NOs: 3 to 18, 21, and 23 to 52 in the sequence listing, respectively. The base sequence or a complementary sequence thereof.

  In another embodiment, the silent mutation is a mutation from base C to T at position 260 of the base sequence set forth in SEQ ID NO: 1 in the sequence listing or a complementary sequence thereof.

  In a specific embodiment, the oligonucleotide capable of hybridizing with the region of the base sequence having a silent mutation consists of the base sequence set forth in SEQ ID NO: 20 in the sequence listing or a complementary sequence thereof.

  In a more specific embodiment, at least one of the oligonucleotides capable of hybridizing with any region of the wild-type base sequence consists of the base sequence set forth in SEQ ID NO: 19 in the sequence listing or a complementary sequence thereof.

  In a more specific embodiment, the other plurality of oligonucleotides that can hybridize to any region of the base sequence consisting of the wild type is SEQ ID NOs: 3 to 17, 19, 21, and 23 in the sequence listing, respectively. To 52 base sequences or a complementary sequence thereof.

  The present invention also provides a kit for detecting pyrazinamide sensitivity in Mycobacterium tuberculosis, comprising any one of the above test pieces.

  In one embodiment, the kit further includes a coloring reagent and a pyrazinamidase gene amplification primer pair.

The present invention further provides a method for detecting pyrazinamide sensitivity in Mycobacterium tuberculosis, the method comprising:
Extracting a Mycobacterium tuberculosis pyrazinamidase gene in a sample;
Contacting the extracted pyrazinamidase gene with any of the test strips described above to bind to the probe immobilized on the test strip, and coloring the pyrazinamidase gene bound to the probe.

  According to the present invention, when a silent mutation is newly found, a probe corresponding to the mutation may be designed and placed on a test piece together with a probe corresponding to the wild type. Since the test piece for detecting PZA sensitivity in Mycobacterium tuberculosis of the present invention has such a probe, when this test piece is used, a PZA sensitive bacterium having only a silent mutation is erroneously detected as being PZA resistant. Therefore, PZA sensitivity can be detected more reliably. As a result, an appropriate treatment can be provided for tuberculosis patients who possess pyrazinamide-sensitive tuberculosis bacteria.

(PncA gene encoding pyrazinamidase)
As mentioned above, PZA resistance in Mycobacterium tuberculosis can be caused by mutations in the pncA gene encoding pyrazinamidase. The base sequence of the pncA gene possessed by wild-type tuberculosis is the base sequence described in SEQ ID NO: 1 in the sequence listing or a complementary sequence thereof. The base ATG at positions 81 to 83 shown in SEQ ID NO: 1 corresponds to the start codon for protein synthesis, and positions 559 to 561 correspond to the stop codon. That is, positions 81 to 561 are a pyrazinamidase coding sequence, and the amino acid sequence of pyrazinamidase is as shown in SEQ ID NO: 2 in the sequence listing. The bases at positions -80 to -1 correspond to the upstream part of protein synthesis.

(Mutation of the pncA gene)
The position of the mutation of the pncA gene that can be a PZA resistant bacterium in the present invention is within the coding sequence of the base sequence described in SEQ ID NO: 1 of the Sequence Listing (from position 1 to position 561), and further from position -20 to position 561. Of the pncA gene that may be present in There are many known mutations in the pncA gene that induce PZA resistance, and these are disclosed in, for example, Patent Documents 3 and 4 and Non-Patent Documents 1-5. Any one of these mutations can be a PZA-resistant Mycobacterium tuberculosis.

  The pncA gene was newly found to have a silent mutation in addition to the above mutation. The silent mutation refers to a mutation that does not affect the enzyme activity because the wild-type base is mutated (substituted) as described above, but the encoded amino acid does not change. Specifically, a mutation from base C to T at position 275 and a mutation from base C to T at position 260 of the base sequence set forth in SEQ ID NO: 1 in the sequence listing or its complementary sequence (see FIG. 1). The position of the white arrow). These silent mutations are located very close to the mutations related to the above-mentioned PZA resistance (the positions of the thin and thick arrows in FIG. 1).

  The silent mutation was found as follows. First, sputum is collected from a tuberculosis patient, germs are removed and cultured. Examples of the method for removing germs include the N-acetyl-L-cysteine (NALC) / sodium hydroxide method. Specifically, NALC-NaOH (4% NaOH (ml): 2.9% aqueous sodium citrate solution (mL): NALC (g) = 100: 100: 1) was added to the jar twice as much For 5 to 20 seconds. Thereafter, the mixture was allowed to stand at room temperature for 15 minutes with occasional mixing, and phosphate buffered saline (PBS, pH 6.8) was further added and mixed. After centrifugation, the supernatant is discarded, the sediment is suspended in PBS, and the resulting tuberculosis is cultured in a tuberculosis isolation medium of Middlebrook 7H9 liquid medium (Becton Dickinson). A PZA sensitivity test was performed on a Middlebrook 7H12 liquid medium (Becton Dickinson) containing PZA at a concentration of 100 mg / L. Specifically, PZA was added to Mycobacterium tuberculosis and cultured for several days, and then ammonium sulfate was added to measure the presence or absence of pyrazinamidase activity. From the above tests, genomic DNA was extracted from Mycobacterium tuberculosis determined to be PZA sensitive by the phenol extraction method, and the nucleotide sequence was analyzed with a sequencer.

As a method for detecting the above mutation on the pncA gene, Nollau et al., Clin. Chem., 43, 1114-1120 (1997), “Laboratory protocol for mutation detection” (Landegren, U. et al., Oxford University). Press (1996)), and “PCR” 2nd edition (Newton et al., BIOS Scientific Publishers Limited (1997)) and the like can be applied. Specifically, for example, PCR fragment sequencing method, single-stranded conformation polymorphism method (SSCP method: Orita, M. et al., Proc. Natl. Acad. Sci. USA., 86 (8), 2766- 2770 (1989)), heteroduplex denaturing gradient gel electrophoresis (DGGE method: Sheffield, VC et al., Proc. Natl. Acad. Sci. USA., 86 (1), 232-236 (1989)) Invader method (Griffin, TJ et al., Trend Biotech, 18, 77 (2000)), SniPer ™ method (Amersham pharmacia biotech), Tackman PCR method (Livak, KJ, Genel. Anal., 14, 143 (1999); Morris, T. et al., J. Clin. Microbiol., 34, 2933 (1996)), MALDI-TOF / MS method (Griffin, TJ et al., Proc. Natl. Acad. Sci. USA., 96 (11), 6301-6 (1999)), restriction enzyme length polymorphism analysis method (RFLP: Murray, JC et al., Proc. Natl. Acad. Sci. USA., 80 (19), 5951-5955 (1983)), DNA chip hybridization method ( Kokoris, K. et al., J. Med. Genet., 36, 730 (1999)), Masscode ^TM method (Q iagen Genomics) and the like, but can be selected from, but not limited to, known methods. Any means can be applied as long as it is a method for detecting a gene mutation. For example, the PCR-sequence-specific oligonucleotide probes (SSOP) method can also be used. The PCR-SSOP method is a method in which a probe that is completely complementary to one allele sequence of about 10 to about 30 bases including a mutation site is prepared, DNA containing the mutation site is amplified by the PCR method, and then hybridized. This is a method for discriminating gene polymorphism based on the presence or absence of hybridization.

(probe)
As a probe used for a test piece for detecting pyrazinamide sensitivity in Mycobacterium tuberculosis of the present invention, an oligonucleotide probe capable of binding to a region having a silent mutation of the pncA gene (hereinafter sometimes referred to as “silent mutation probe”) As well as oligonucleotide probes that can bind to any region of the wild-type pncA gene (hereinafter sometimes referred to as “wild-type probes”). The length of each probe is generally 10 to 30 nucleotides, preferably 12 to 26 nucleotides, although it depends on the temperature at which it is bound (hybridized).

  In the present invention, the silent mutation probe can bind (hybridize) to a region having only the silent mutation, and the region having only the silent mutation has a wild-type base sequence other than the silent mutation. If this region is wild-type, as well as if it contains other mutations, silent mutation probes cannot bind to this region. Examples of the silent mutation include a mutation from base C to T at position 275 of the base sequence shown in SEQ ID NO: 1 in the sequence listing or its complementary sequence; and the base sequence shown in SEQ ID NO: 1 in the sequence listing or its complementary sequence Of the base at position 260 of C to T. Moreover, in order to recognize silent mutation reliably, it is preferable to design so that the position of the silent mutation exists in the middle of the oligonucleotide constituting the silent mutation probe.

  In the present invention, the wild-type probe can bind to any region of the wild-type pncA gene. In order to detect PZA sensitivity, it is important to detect at least all the mutations associated with PZA resistant bacteria. Therefore, in the present invention, preferably, the wild-type probe covers, for example, −20 so as to cover almost the entire coding sequence of the nucleotide sequence set forth in SEQ ID NO: 1 of the sequence listing or a nucleotide sequence complementary thereto. It is designed to be able to bind (hybridize) to the bases from position 560 to position 560, that is, total 580 bases.

  For example, if a probe is designed in order from the upstream side or the downstream side of the pncA gene and arranged so as to overlap with the adjacent probe by about 4 to 7 bases, even if there is a mutation at the boundary position of the arranged region, Are preferred because they can bind. Therefore, for example, the total number of probes for detecting a total of 580 bases from the -20th position to the 560th position is assumed to be the same length of all the probes, and each probe overlaps the adjacent probe by 4 bases. Thus, it is at least 22 or more, preferably 30 or more, more preferably 35 or more, and further preferably 40 or more.

  In the present invention, a wild-type probe designed to be able to bind to substantially the same region as the binding region of each silent mutation probe described above (hereinafter referred to as “silent-compatible wild-type probe”). )is required. For example, a silent-type mutant probe that can bind to a region containing a mutation at position 275 or 260 of the nucleotide sequence of SEQ ID NO: 1 in the sequence listing or its complementary sequence has a wild-type probe that can bind to almost the same region. Designed. The substantially the same region may be the same region or a region having a range of 1 to 2 bases long / short. Therefore, in these regions, at least one probe can bind both in the presence of a silent mutation and in the case of a wild type (no mutation), and when a mutation other than the silent mutation exists. Neither probe binds.

  Each of the above probes can be obtained by using a standard program and primer analysis software such as Primer Express (Perkin Elmer), and can be synthesized using a standard method such as an automated oligonucleotide synthesizer. .

  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 for detecting pyrazinamide sensitivity in Mycobacterium tuberculosis according to the present invention, at least two probes are fixed. At least one of the immobilized probes is the silent mutant probe, and the other probes are wild-type probes immobilized at different positions on the test strip, including silent-corresponding wild-type probes. .

  As described above, preferably, the wild-type probe fixed to the test strip is designed to cover almost the entire coding sequence of the pncA gene or its complementary sequence, and more preferably, the probe comprises: They are arranged at regular intervals from the upstream side or the downstream side in the order of the base sequences to be bound. Furthermore, a control or marker for confirming color development may be fixed.

  More preferably, the silent mutant probe and the silent-corresponding wild type probe are fixed at the same position on the test strip. The silent mutation probe and the silent wild-type probe may be fixed separately by overlapping each other at the same position (in any order), or these probes may be mixed and fixed. At this position, the pncA gene does not bind to the region to which these probes bind, and if there is a mutation other than the silent mutation, the pncA gene binds when there is no mutation or only the silent mutation exists. Therefore, it does not bind in the case of PZA resistance and binds in the case of PZA sensitivity.

  The test piece of the present invention can be prepared by physically or chemically fixing the probe on the surface of the carrier. 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; metal materials such as gold and silver, and the like. Not. An organic material is preferable in terms of easy molding processability, and polyesters such as polyethylene terephthalate are more preferable. These carriers are preferably white in order to make the color development easily visible in the detection by the color development method.

  The probes are arranged on the carrier at regular intervals for each type. As a method for physically immobilizing the probe on the surface of these carriers, various known methods are used. For example, there is a method of coating the carrier surface with a polycationic polymer such as polylysine. According to this method, the efficiency of immobilization can be increased by electrostatic interaction with a probe that is a polyanion. There is also a method of increasing the efficiency of immobilization by adding an irrelevant base sequence (such as a polythymine chain) to the end of the probe and increasing the molecular weight of the immobilized probe itself. For example, various probes can be immobilized relatively easily by discharging a solution containing a polythymine-added oligonucleotide probe from a dispenser onto a nitrocellulose membrane and irradiating with ultraviolet rays. Specifically, each probe is placed in a dispenser equipped with a 24-gauge needle, and an amount of 0.5 to 1.0 μL / min is discharged at a coating speed of 2.5 to 8.5 mm / sec. When applied on the nitrocellulose membrane at regular intervals, each probe is applied as a stripe having a width of approximately 1 to 2 mm arranged at regular intervals. The probe is fixed on the carrier by irradiating the carrier coated with the stripe of the probe 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 arranged in sequence can be obtained at a time.

  When the carrier surface is a metal material such as gold, the surface of the carrier is treated with a thiol having an amino group such as 2-aminoethanethiol or a disulfide compound, thereby causing electrostatic interaction with the probe which is a polyanion. It is also known that the efficiency of immobilization is improved.

  As a method for chemically immobilizing a probe by introducing a functional group, various known methods are used. For example, when the carrier material is an inorganic material such as glass or silicon, there is a method of modifying the end of the probe with a functional group capable of silane coupling reaction such as trimethoxysilane or triethoxysilane. A test piece is obtained by immersing the carrier in the solution to be contained for 24 to 48 hours, taking it out, and washing it. Alternatively, by treating an inorganic material carrier such as glass or silicon with a silane coupling agent having an amino group such as aminoethoxysilane, the surface of the carrier is aminated, and then a probe having an carboxylic acid introduced at the terminal and an aminocup There is also a method of immobilizing the probe by ring reaction. Further, when the carrier material 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. It can be fixed by immersing for ~ 48 hours, taking out and then washing.

  There is also known a method of directly synthesizing a probe having a desired sequence on the surface of a carrier using a lithography technique instead of fixing the synthesized probe.

(PZA sensitivity detection method using a test piece)
1. Specimen In the present invention, the “specimen” may generally be a specimen necessary for the tubercle bacillus test. For example, body fluids such as sputum, pharyngeal swab, gastric juice, bronchoalveolar lavage fluid, intratracheal aspirate, throat wipes and / or tissue biopsy, and tuberculosis bacteria obtained from these cultures themselves Can be mentioned.

2. Sample pretreatment (DNA extraction)
Extraction of DNA from the specimen can be performed by a known method. Examples thereof include a phenol extraction method, a guanidine thiocinate extraction method, and a vanadyl ribonucleoside complex extraction method. A sample that has been pretreated is referred to as a “sample” in this specification.

3. Amplification of nucleic acid When nucleic acid is amplified by PCR, for example, the following steps are performed: (1) By heat-treating double-stranded genomic DNA under reaction conditions of about 92 to 95 ° C. for about 30 seconds to 1 minute. (2) PCR reaction by binding at least two kinds of amplification primers to each of the single-stranded DNAs under reaction conditions of about 50 to 65 ° C. for about 20 seconds to 1 minute. An annealing step for producing a double-stranded portion serving as a starting point; (3) a strand extension step in which a DNA polymerase is reacted at about 70 to 75 ° C. under reaction conditions for about 20 seconds to 5 minutes; and (1) to ( The process of repeating the process of 3) 20-40 times by a normal method. As a primer pair used to amplify the pncA gene by PCR, a sequence upstream from the sequence site containing all the mutation sites and a sequence from the polymorphic site so that the pncA gene containing all the mutation sites can be amplified. An oligonucleotide having the same or complementary base sequence as the downstream sequence can be mentioned. Examples of preferred primer pairs include oligonucleotides of SEQ ID NOs: 53 and 54 in the sequence listing or oligonucleotides having complementary sequences thereof.

  In order to improve the sensitivity of the measurement, for example, when DNA is directly amplified from a specimen, the nucleic acid may be further amplified by the Nested PCR method (Japanese Patent Publication No. 6-81600) before the PCR reaction. As a primer in the Nested PCR method, a sequence outside the primer used in the PCR reaction can be selected. Examples thereof include oligonucleotides having SEQ ID NOs: 55 and 56 in the sequence listing or oligonucleotides having complementary sequences thereof.

  The above probe and primer sequences can be obtained 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.

4). Method for detecting PZA sensitivity of Mycobacterium tuberculosis In the present invention, PZA sensitivity of Mycobacterium tuberculosis can be detected by the PCR-SSOP method. More simply, it can be detected by comparing the number of immobilized probes with the number of spots detected in the PCR-SSOP method. For example, when using a test piece on which the above probe is immobilized, hybridization in the PCR-SSOP method is preferably performed at a temperature of about 60 to 65 ° C. in that nonspecific adsorption reaction is unlikely to occur. If the number of spots detected is the same as the number of immobilized probes, the M. tuberculosis is not mutated, i.e. PZA sensitive, and if the number of detection spots is less than the number of fixed probes, M. tuberculosis is PZA resistant. Can be determined.

  In order to easily count the number of spots detected in the PCR-SSOP method, it is preferable to amplify a gene using a primer pair in which a labeling substance such as a radioisotope, a fluorescent substance, or a chemiluminescent substance is modified. Among the detection methods using the above-mentioned labeling substances, nitroblue tetrazolium (NBT) / 5-bromo-4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) color development is relatively inexpensive and can be easily carried out. The method is preferred. Specifically, it is performed as follows. First, a gene having biotin at the end is amplified using a primer pair in which biotin is modified at the 3 'or 5' end of the primer. Subsequently, the amplified gene and the probe immobilized on the test piece are bound to the probe by hybridization, and further contacted with alkaline phosphatase-labeled streptavidin to bind to biotin present at the end of the gene bound to the probe. . Furthermore, by adding NBT / BCIP and reacting with alkaline phosphatase, color is developed at the position where alkaline phosphatase bound to the probe is present. This color development is visible.

(Detection kit including test piece)
The detection kit of the present invention includes the above-described test piece. Suitably any reagent suitable for detecting Mycobacterium tuberculosis sensitivity to pyrazinamide may be included. For example, a reagent for extracting the above DNA, a reagent for gene amplification, a reagent for detecting the binding of the gene to the probe, and the like can be mentioned. As a specific kit, a kit for carrying out the PCR-SSOP method is exemplified.

  The reagent for DNA extraction that can be included in the kit of the present invention is a reagent used in any of the above-described techniques.

  A primer pair for amplifying the pncA gene by PCR may also be included in the kit of the present invention. The primer pair has a base sequence that is the same as or complementary to a sequence upstream from the sequence site including all the mutation sites and a sequence downstream from the polymorphic site so that the pncA gene including all the mutation sites can be amplified. Is an oligonucleotide. Examples of preferred primer pairs include oligonucleotides of SEQ ID NOs: 53 and 54 in the sequence listing or oligonucleotides having complementary sequences thereof. Examples of the primer pair in the Nested PCR method for improving the sensitivity of the measurement include the oligonucleotides of SEQ ID NOs: 55 and 56 in the above sequence listing or oligonucleotides having complementary sequences thereof.

  Furthermore, the primer pair is preferably modified with a radioisotope, a fluorescent substance, a chemiluminescent substance, or the like in order to facilitate detection in the PCR-SSOP method.

  In addition to the PCR method, gene amplification includes known techniques such as the LAMP method and the ICAN method. In the present invention, any of these methods may be used, and the present invention also includes a kit containing any reagent used in these methods.

  Furthermore, the kit of the present invention may contain reagents for detection. For example, when the NBT / BCIP color development method is employed, streptavidin-modified alkaline phosphatase, NBT, and BCIP are exemplified.

(Example 1: Pyrazinamide sensitivity test)
1. Preparation of test solution To middle book liquid medium (Becton Dickinson), pyrazinamide with a final concentration of 100 mg / L and a fluorescent substrate, Tris-4,7-diphenyl-1,10-phenanthroline ruthenium chloride pentahydrate were added. A test medium was prepared by dissolution.

2. Pyrazinamide susceptibility test Tuberculosis bacteria purified and cultured from 255 sputum of tuberculosis patients were added to the test solution and cultured at 37 ° C. for 4 to 21 days. The growth was observed in the tube to which PZA was not added, and the case where the growth was not observed in the tube to which PZA was added was regarded as sensitivity. Growth was confirmed by the presence or absence of fluorescent coloration that increased with growth. As a result, it became clear that 227 specimens were sensitive to PZA.

(Example 2: Genetic analysis of pyrazinamide-sensitive tuberculosis bacteria)
1. Extraction of pncA gene Genomic DNA was extracted and purified from the 227 samples determined to be pyrazinamide-sensitive tuberculosis bacteria in Example 1 by the phenol extraction method.

2. Amplification of pncA gene Next, the pncA gene was amplified using the primers shown below. PCR reaction conditions were 94 ° C. and 30 seconds for the denaturation process, 55 ° C. and 20 seconds for the annealing process, and 72 ° C. and 20 seconds for the chain extension process, and amplification was performed in 30 cycles of this process.
Primer 1 ggcagtttgc ctaccgacgc (SEQ ID NO: 53)
Primer 2 ccaacagttc atcccggttc g (SEQ ID NO: 54)

3. Gene analysis of pyrazinamide-sensitive Mycobacterium tuberculosis The nucleotide sequences of 227 amplified pncA genes were examined with a sequencer. As a result, a new mutation: Ser65Ser [tcC → tcT] (open arrow on the probe 17 in FIG. 1) was found in 6 of 227 cases. This mutation was a silent mutation in which the encoded amino acid did not change despite the base substitution.

(Example 3: Preparation of test piece)
1. Design of wild-type probe Any of the mutation of the base described in Patent Documents 3 and 4 (position of the thin arrow in FIG. 1) and the newly found mutation Pro62Leu [cCg → cTg] (position of the thick arrow in FIG. 1) Probes 1 to 47 shown below (corresponding to SEQ ID NOs: 3 to 18, 21 and 23 to 52, respectively) were constructed as probes capable of detecting these. FIG. 1 shows the positional relationship between these probes.

Probe 1 cgcatacgtc caccatacgt tcgg (SEQ ID NO: 3)
Probe 2 atgatcaacg cccgcatac (SEQ ID NO: 4)
Probe 3 cacgtcgacg atgatcaacg (SEQ ID NO: 5)
Probe 4 gtcgttctgc acgtcgac (SEQ ID NO: 6)
Probe 5 ccaccctcgc agaagtcgtt (SEQ ID NO: 7)
Probe 6 cgagccaccc tcgcagaag (SEQ ID NO: 8)
Probe 7 gttaccgcca gcgagccacc (SEQ ID NO: 9)
Probe 8 agcgcggcgc caccggttac cg (SEQ ID NO: 10)
Probe 9 gtagtcgctg atggcgcggg ccagcgcg (SEQ ID NO: 11)
Probe 10 gccaggtagt cgctgatggc gc (SEQ ID NO: 12)
Probe 11 tggtagtccg ccgcttcggc caggta (SEQ ID NO: 13)
Probe 12 ggttgccacg acgtgatggt ag (SEQ ID NO: 14)
Probe 13 gatgtggaag tccttggttg c (SEQ ID NO: 15)
Probe 14 cgggtcgatg tggaagtc (SEQ ID NO: 16)
Probe 15 tggtcacccg ggtcgatgt (SEQ ID NO: 17)
Probe 16 cggtgtgccg gagaagtggt ca (SEQ ID NO: 18)
Probe 17 cgacgaggaa tagtccggtgt (SEQ ID NO: 21)
Probe 18 tgcggtggcc acgacga (SEQ ID NO: 23)
Probe 19 cgctgacgca atgcggtg (SEQ ID NO: 24)
Probe 20 cgccgggagt accgctg (SEQ ID NO: 25)
Probe 21 aagtccgcgc cgggagta (SEQ ID NO: 26)
Probe 22 gtccagactg ggatggaagt cc (SEQ ID NO: 27)
Probe 23 cctcgattgc cgacgtgtcc ag (SEQ ID NO: 28)
Probe 24 ccttgtagaa caccgcctcga (SEQ ID NO: 29)
Probe 25 tccggtgtag gcacccttgt ag (SEQ ID NO: 30)
Probe 26 aagccgctgt acgctccggt (SEQ ID NO: 31)
Probe 27 ctcgtcgact ccttcgaagc cg (SEQ ID NO: 32)
Probe 28 tggcgtgccg ttctcgtcga (SEQ ID NO: 33)
Probe 29 gcagccaatt cagcagtggc gt (SEQ ID NO: 34)
Probe 30 cgccgcgttg ccgcagccaa (SEQ ID NO: 35)
Probe 31 atcgacctca tcgacgccgc (SEQ ID NO: 36)
Probe 32 tggcaatacc gaccacatcg ac (SEQ ID NO: 37)
Probe 33 cacacaatga tcggtggcaa ta (SEQ ID NO: 38)
Probe 34 ggccgtctgg cgcacacaat (SEQ ID NO: 39)
Probe 35 cgtaccgcgt cctcggccgt (SEQ ID NO: 40)
Probe 36 aagccattgc gtaccgcgtc (SEQ ID NO: 41)
Probe 37 ccagcaccct ggtggccaag ccat (SEQ ID NO: 42)
Probe 38 gtcaggtcca ccagcaccct gg (SEQ ID NO: 43)
Probe 39 cacccgctgt caggtccacc ag (SEQ ID NO: 44)
Probe 40 tcggccgaca cacccgct (SEQ ID NO: 45)
Probe 41 ggtggtatcg gccgacac (SEQ ID NO: 46)
Probe 42 cggcgacggt ggtatcgg (SEQ ID NO: 47)
Probe 43 ccagcgcggc gacggt (SEQ ID NO: 48)
Probe 44 cgcatctcct ccagcgcggc (SEQ ID NO: 49)
Probe 45 cggtgcgcat ctcctccag (SEQ ID NO: 50)
Probe 46 actcgacgct ggcggtgc (SEQ ID NO: 51)
Probe 47 gagctgcaaa ccaactcgac (SEQ ID NO: 52)

2. Design of Silent Mutation Probe Silent mutation found by the analysis in Example 2 is present in a region where the probe 17 can bind. Therefore, the following silent mutation probe (probe 17S: SEQ ID NO: 22) was constructed (see FIG. 1).
Probe 17S acgacgaaga atagtccggt gt (SEQ ID NO: 22)

3. Immobilization of probe Terminal transferase at the 5 ′ end of each of the above probes 1-47 (base sequences described in SEQ ID NOs: 3-18, 21, and 23-52) and probe 17S (base sequences described in SEQ ID NO: 22) (Promega) was used to add polythymine. 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 3.6 μL was mixed to prepare a reaction solution, reacted at 37 ° C. for 4 hours, and then 90 μL of 10 × SSC buffer solution was added to obtain 1 pmol / μL of a polythymine-added oligonucleotide probe.

  Next, each of the wild type probes (probes 1 to 47) to which polythymine was added was placed in a dispenser equipped with a 24 gauge needle, and 2 mm was applied at a coating speed of 2.5 mm / second while discharging an amount of 0.7 μL / min. It was applied in a vertical direction at intervals of 2 mm on a nitrocellulose film (length 75 mm × width 150 mm: Whatman) so as to form a stripe having a width of 1 mm. Further, a silent mutation probe (probe 17S) was also put in a dispenser and applied in the same manner in a manner overlapping the application position of the probe 17. Thereafter, these nitrocellulose membranes were irradiated with 312 nm ultraviolet rays for 2 minutes to immobilize the probes. Next, the nitrocellulose film was cut so as to include all the stripes, thereby preparing a test piece of 5 mm × 150 mm (referred to as mixed-type test piece A).

(Comparative Example 1: Production of conventional test piece)
Example 3 3. above except that the silent mutation probe (probe 17S) was not applied. In the same manner as described above, a test piece (referred to as a conventional test piece) in which only the wild type probe (probes 1 to 47) was fixed was prepared.

(Example 4: Detection of PZA sensitivity of pncA gene using mixed specimen A)
1. Amplification of the pncA gene In this example, M. tuberculosis itself was used as a sample to detect the PZA sensitivity of the pncA gene. The Mycobacterium tuberculosis of the specimen used was wild type, S65S silent mutant type, and P62L mutant type. In order to detect PZA resistant bacteria, it is necessary to amplify the pncA gene, which is a PZase-related gene that may be present in a specimen. Therefore, first, the pncA gene was amplified from the specimen by the nested PCR method. The following were used as Nested primers (see FIG. 1).
Nested primer 1 cagatatagg gccatgacgc c (SEQ ID NO: 55)
Nested primer 2 cttgcggcga gcgctc (SEQ ID NO: 56)

  Nested PCR reaction conditions were 90 ° C. for 60 seconds, annealing step at 55 ° C. for 60 seconds, chain extension step at 27 ° C. for 60 seconds, and 30 cycles of these steps, and the upstream part of the pncA gene. A gene in a slightly wider range from to the downstream part was amplified.

Next, using the gene amplified by the Nested PCR method as a template, the pncA gene was further amplified by the PCR method using the following primers (see FIG. 1) to obtain a DNA solution. Primer 1 consisting of the base sequence set forth in SEQ ID NO: 53 of the Sequence Listing, Primer 2 consisting of the base sequence set forth in SEQ ID NO: 54 and having biotin bonded to the 5 ′ end, and Taq DNA polymerase (Roche Diagnostics) The pncA gene was amplified by a PCR method using The base sequence of each primer is as shown below.
Primer 1 ggcagtttgc ctaccgacgc (SEQ ID NO: 53)
Primer 2 ccaacagttc atcccggttc g (SEQ ID NO: 54)

  The PCR reaction conditions were 94 ° C. for 30 seconds for the denaturation step, 55 ° C. for 20 seconds for the annealing step, 72 ° C. for 20 seconds for the chain extension step, and 30 cycles of these steps were performed to amplify the pncA gene. Biotin is bound to the terminal of the amplified DNA.

2. Detection using probe To 10 μL of amplified DNA solution, a solution (10 μL) of sodium hydroxide (5 M) and ethylenediaminetetraacetic acid (0.05 M) is added and stirred well, and left for 5 minutes. Denatured to a main chain. In this sample 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 the above example The mixed specimen A prepared in 3 was added, and the reaction was performed by shaking for 30 minutes at a reaction temperature of 62 ° C. Thereafter, alkaline phosphatase-labeled streptavidin was added and bound to biotin present at the end of the gene bound to each probe on the test piece. Furthermore, nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) are added to cause color development at the position where alkaline phosphatase bound to each probe on the test piece exists. It was. The results are shown in FIG.

  In the mixed specimen A, both wild type S. tuberculosis and S65S silent mutant M. tuberculosis were confirmed to be colored at all probe positions, determined to be PZA sensitive, and the P62L mutant M. tuberculosis was probed. No color development was observed at position 17 (position indicated by the black arrowhead in FIG. 2), and it was determined to be PZA resistant. Thus, by using the mixed type test piece A, it was possible to correctly determine that the S65S silent mutant tuberculosis bacterium is PZA sensitive.

(Comparative Example 2: Detection of PZA sensitivity of pncA gene using conventional test piece)
PZA sensitivity was detected for each Mycobacterium tuberculosis in the same manner as in Example 4 except that the conventional test piece prepared in Comparative Example 1 was used instead of the mixed test piece A. The results are summarized in FIG.

  In the conventional test piece, only the wild-type tuberculosis bacteria were confirmed to develop color at all probe positions and judged to be PZA sensitive. Both the S65S silent mutant M. tuberculosis and the P62L mutant M. tuberculosis did not show color development at the position of the probe 17 (position indicated by the black arrowhead in FIG. 2), and were determined to be PZA resistant. As described above, according to the conventional test piece, an erroneous determination was made for the S65S silent mutant tuberculosis bacterium that is sensitive to PZA.

(Example 5: Gene analysis and probe design of pyrazinamide-sensitive tuberculosis bacteria)
In the same manner as in Example 2, the base sequences of the 227 amplified pncA genes were further examined with a sequencer. As a result, a further new mutation: Gly60Gly [ggC → ggT] (the position of the white arrow on the probe 16 in FIG. 1) was found in 2 out of 227 cases. This mutation was also a silent mutation.

The new silent mutation found by the above analysis is present in a region where the probe 16 can bind. Therefore, the following silent mutation probe (probe 16S: SEQ ID NO: 20) was constructed (see FIG. 1). Furthermore, in consideration of the combined use with the probe 17S, a wild-type probe 16 ′ (SEQ ID NO: 19) shorter by 2 bases than the wild-type probe 16 was newly designed for the purpose of more reliably detecting in these regions. (See FIG. 1).
Probe 16S ggtgtaccgg agaagtggtca (SEQ ID NO: 20)
Probe 16 ′ gtgtgccgga gaagtggtca (SEQ ID NO: 19)

  A mixed test piece B was prepared in the same manner as in Example 3 except that the probe 16 'was used instead of the wild type probe 16, and the probe 16S was used instead of the silent mutation probe 17S.

(Example 6: Detection of PZA sensitivity of pncA gene using mixed specimen B)
In this example, M. tuberculosis itself was used as a specimen to detect PZA sensitivity of the pncA gene. The Mycobacterium tuberculosis of the specimen used was wild type, G60G silent mutant type, and F58V mutant type.

  In the same manner as in Example 5, after the pncA gene was amplified, the mixed type test piece B was used to detect PZA sensitivity. The results are shown in FIG.

  In the mixed specimen B, both wild type tuberculosis and G60G silent mutant tuberculosis were confirmed to be colored at all probe positions, determined to be PZA sensitive, and F58V mutant tuberculosis No color development was observed at position 16 (the position indicated by the black arrowhead in FIG. 3), and it was determined to be PZA resistant. Thus, by using the mixed test piece B, it was possible to correctly determine that the G60G silent mutant tuberculosis bacterium is PZA sensitive.

(Comparative Example 3: Detection of PZA sensitivity of pncA gene using conventional test piece)
Other than using the conventional test piece prepared in Comparative Example 1 above in place of the mixed type test piece B, and using the G60G silent mutant and the F58V mutant instead of the S65S silent mutant and the P62L mutant, respectively. Were operated in the same manner as in Example 4 to detect PZA sensitivity for each Mycobacterium tuberculosis. The results are summarized in FIG.

  In the conventional test piece, only the wild-type tuberculosis bacteria were confirmed to develop color at all probe positions and judged to be PZA sensitive. Both the G60G silent mutant M. tuberculosis and the F58V mutant M. tuberculosis did not show color development at the position of the probe 16 (position indicated by the black arrowhead in FIG. 3), and were determined to be PZA resistant. Thus, according to the conventional test piece, it was erroneously determined for the P60-sensitive G60G silent mutant tuberculosis.

  When the test piece for detecting pyrazinamide sensitivity in M. tuberculosis of the present invention is used, PZA sensitivity can be more reliably detected without erroneously detecting PZA-sensitive bacteria having only silent mutations as being resistant to PZA. . In particular, patients with PZA-sensitive Mycobacterium tuberculosis with only silent mutations were previously misdiagnosed as being resistant to PZA, so treatment with pyrazinamide was not immediately selected. It can be readily diagnosed as being treatable with pyrazinamide. As a result, appropriate treatment can be provided to such patients. Therefore, unnecessary drug administration and testing can be prevented, the burden on the patient can be reduced, and unnecessary medical expenses can be reduced.

It is a figure which shows the relationship between the base sequence of the pncA gene of Mycobacterium tuberculosis, and the position of the designed probe. It is a photograph which shows the result of PZA sensitivity detection of each Mycobacterium tuberculosis of the wild type, S65S silent mutant type, and P62L mutant type using the test piece of the present invention (mixed type test piece A) and the conventional type test piece. It is a photograph which shows the result of PZA sensitivity detection of each wild-type, G60G silent mutant type, and F58V mutant type tuberculosis bacteria using the test piece of the present invention (mixed type test piece B) and the conventional type test piece.

Claims (15)

A test strip for detecting pyrazinamide sensitivity in Mycobacterium tuberculosis,
At least two probes are fixed,
At least one of the probes is composed of an oligonucleotide capable of hybridizing with a region of a base sequence having a silent mutation of the Mycobacterium tuberculosis pyrazinamidase gene, and the silent mutation in the region of the base sequence having the silent mutation The base sequence other than is wild type,
The other probes of the probes consist of oligonucleotides that can hybridize with any region consisting of the wild-type base sequence of the Mycobacterium tuberculosis pyrazinamidase gene, and the other probes are respectively located at different positions on the test strip. The region of the base sequence that is fixed and has the silent mutation is substantially the same region as one of the arbitrary regions of the wild-type base sequence;
Test pieces.   A plurality of oligonucleotides capable of hybridizing with an arbitrary region of the wild-type base sequence hybridize with different regions of the base sequence of the pyrazinamidase gene, and a coding sequence of the pyrazinamidase gene or its complementary sequence depending on the different region The test piece according to claim 1, wherein substantially the whole is covered.   The oligonucleotide capable of hybridizing with the region of the base sequence having the silent mutation and the oligonucleotide capable of hybridizing with the region of the wild-type base sequence in substantially the same region as the region are fixed at the same position of the test piece. The test piece according to claim 1 or 2.   The test piece according to any one of claims 1 to 3, wherein the base sequence comprising the wild type of the pyrazinamidase gene is the base sequence set forth in SEQ ID NO: 1 in the sequence listing or a complementary sequence thereof.   The test piece according to any one of claims 1 to 4, wherein the silent mutation is a mutation from base C to T at position 275 of the base sequence shown in SEQ ID NO: 1 of the sequence listing or a complementary sequence thereof.   The test piece according to claim 5, wherein the oligonucleotide capable of hybridizing with the region of the base sequence having a silent mutation comprises the base sequence set forth in SEQ ID NO: 22 of the sequence listing or a complementary sequence thereof.   The test according to claim 5 or 6, wherein at least one of the oligonucleotides capable of hybridizing with any region of the base sequence consisting of the wild type consists of the base sequence set forth in SEQ ID NO: 21 of the sequence listing or a complementary sequence thereof. Fragment.   A plurality of other oligonucleotides that can hybridize to any region of the base sequence consisting of the wild type are the base sequences of SEQ ID NOs: 3 to 18, 21, and 23 to 52 of the sequence listing, or their complementary sequences, respectively. The test piece according to claim 5 or 6, comprising:   The test piece according to any one of claims 1 to 7, wherein the silent mutation is a mutation from base C to T at position 260 of the nucleotide sequence set forth in SEQ ID NO: 1 in the sequence listing or a complementary sequence thereof.   The test piece according to claim 9, wherein the oligonucleotide that can hybridize with the region of the base sequence having a silent mutation consists of the base sequence shown in SEQ ID NO: 20 of the sequence listing or a complementary sequence thereof.   The test according to claim 9 or 10, wherein at least one of the oligonucleotides capable of hybridizing with any region of the base sequence consisting of the wild type consists of the base sequence set forth in SEQ ID NO: 19 of the sequence listing or a complementary sequence thereof. Fragment.   A plurality of other oligonucleotides that can hybridize with an arbitrary region of the base sequence consisting of the wild type are the base sequences of SEQ ID NOs: 3 to 17, 19, 21, and 23 to 52 of the sequence listing, or The test piece according to claim 9 or 10, comprising a complementary sequence.   A kit for detecting pyrazinamide sensitivity in Mycobacterium tuberculosis, comprising the test piece according to any one of claims 1 to 12.   Furthermore, the kit of Claim 13 containing the coloring reagent and the primer pair for pyrazinamidase gene amplification. A method for detecting pyrazinamide sensitivity in Mycobacterium tuberculosis,
Extracting a Mycobacterium tuberculosis pyrazinamidase gene in a sample;
The extracted pyrazinamidase gene is brought into contact with the test piece according to any one of claims 1 to 12 to bind to a probe fixed to the test piece, and the pyrazinamidase gene bound to the probe is colored. A method comprising the steps.

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