JP6070246B2 - Kit containing test piece - Google Patents

Description

  The present invention relates to a kit including a test piece.

  Test pieces for detecting drug susceptibility in Mycobacterium tuberculosis (Patent Documents 1 and 2), test pieces for determining the drug resistance of Mycobacterium tuberculosis (Patent Documents 3 and 4), Mycobacterium tuberculosis and nontuberculous mycobacteria A test piece (Patent Document 5) and the like have been reported. These test pieces are means for quickly and easily determining drug sensitivity by detecting the presence or absence of hybridization with the target gene, but when the hybridization temperature in the test environment is not appropriate, It may lead to wrong judgment.

JP 2008-142075 A JP 2008-142076 A JP 2011-87465 A JP 2006-180746 A JP 2009-189283 A

  An object of the present invention is to provide a kit including a test piece that can prevent erroneous determination caused by an inappropriate hybridization temperature. There is.

  The present inventors use the first standard gene including the predetermined first part and the second standard gene including the second part to prevent erroneous determination caused by an inappropriate hybridization temperature. The present invention has been completed.

The kit for detecting a target gene of the present invention comprises a test piece, a first standard gene and a second standard gene, and can be hybridized with the target gene at a temperature of T _max ° C or lower. In addition, at least the first detection probe and the second detection probe are immobilized, and the presence or absence of mutation of the target gene can be determined at a temperature of T _min ° C to T _max ° C. The gene is an oligonucleotide having a sequence containing a first portion, wherein the first portion of the first standard gene hybridizes with the first detection probe at a temperature lower than T _max ° C, and T _At a temperature of _max ° C. or higher, the first part of the first standard gene is The second standard gene is an oligonucleotide that does not hybridize and has a sequence containing the second part, and the second part of the second standard gene is the first part at a temperature lower than T _min ° C. 2 Hybridizes with the detection probe, and the second portion of the second standard gene does not hybridize with the second detection probe at a temperature of T _min ° C or higher.

  In one embodiment, the first standard gene and the second standard gene are one in which the first part of the first standard gene and the second part of the second standard gene are continuous. It constitutes an oligonucleotide (hereinafter referred to as “linked standard gene”).

  In one embodiment, the first portion of the first standard gene and the second portion of the second standard gene are each 10 to 30 nucleotides in length.

  In one embodiment, the first portion of the first standard gene is a complementary sequence of the oligonucleotide of the first detection probe, and the second portion of the upper second standard gene is the first portion. 2 It is a complementary sequence of the oligonucleotide of the probe for detection, but is a sequence in which one mismatch base is introduced only at a substantially central position.

The present invention also provides a method for determining a hybridization temperature, wherein at least a first detection probe and a second detection probe that can hybridize with a target gene at a temperature of T _max ° C or less are immobilized. And a step of adding at least a first standard gene and a second standard gene to a test piece that enables discrimination of the presence or absence of mutation of the target gene at a temperature of T _min ° C to T _max ° C, and the above A step of confirming a hybridization state of the first detection probe and the first standard gene and the second detection probe and the second standard gene from a test piece, wherein the first detection probe Standard gene, an oligonucleotide consisting of a sequence comprising a first portion, than the T _max ° C. At low temperatures, the said first part of the first standard gene is hybridized with the first probe for detecting at T _max ° C. or higher temperatures, the first of said first portion of the standard gene the first The second standard gene is an oligonucleotide that does not hybridize with the detection probe and the second standard gene comprises a sequence containing the second portion, and the second standard gene has a temperature lower than T _min ° C. The portion hybridizes with the second detection probe, and the second portion of the second standard gene does not hybridize with the second detection probe at a temperature _{equal to} or higher than T _min ° C. First detection probe, first standard gene, second detection probe, and second stander The step of confirming the state of hybridization with the first gene comprises: (i) the first standard gene hybridized to the first detection probe and the second standard to the second detection probe. When the gene is hybridized, it is determined that the hybridization temperature in the test environment is too low, and (ii) the first standard gene hybridizes to the first detection probe, but the second detection probe If the second standard gene does not hybridize, it is determined that the hybridization temperature in the test environment is appropriate, (iii) the first standard gene does not hybridize to the first detection probe, If the second standard gene does not hybridize to the second detection probe, the test is performed. It is determined that the hybridizing temperature in the environment is too high.

  In one embodiment, in the determination method, the first standard gene and the second standard gene are the first part of the first standard gene and the second part of the second standard gene. Constitutes a continuous oligonucleotide (linked standard gene).

  According to the present invention, a kit including a target gene detection test piece for preventing an erroneous determination caused by an inappropriate hybridization temperature, and conditions for hybridization temperature using the kit are determined in advance. A method can be provided. The kit of the present invention enables simpler and more accurate determination in detection of pathogenic bacteria including Mycobacterium tuberculosis and confirmation of sensitivity and resistance of these bacteria to drugs. In particular, it can be easily used by medical personnel who are technically immature in harsh environments such as Southeast Asia.

It is a schematic diagram of the said test piece for demonstrating an example of the test piece which comprises the kit of this invention. It is a figure which represents typically an example of the said test piece at the time of selecting two detection probes from the test piece to which the detection probe which can hybridize with a target gene which comprises this invention was fix | immobilized. BRIEF DESCRIPTION OF THE DRAWINGS It is a figure for expressing typically an example of the production procedure of a connection standard gene which comprises this invention, Comprising: (a1) is the complement of the oligonucleotide of the 1st detection probe of the two selected detection probes It is a figure for showing the state which each prepared the 2nd part which consists of the 1st part which consists of a sequence, and the 2nd part which consists of the sequence | arrangement in which the mismatch base was introduce | transduced in the approximate center position of the complementary sequence of the oligonucleotide of a 2nd detection probe, b1) is a diagram for explaining a state in which the prepared linked standard gene is prepared. It is a figure for expressing typically an example of the preparation procedure of a connection standard gene which constitutes the present invention, Comprising: The complementary sequence (1st part) of the oligonucleotide of the 1st detection probe, and the 2nd detection probe For explaining a state in which one oligonucleotide consisting of a sequence having a sequence (second part) in which a mismatch base is introduced at a position substantially in the middle of a complementary sequence of an oligonucleotide is prepared to produce a linked standard gene FIG. It is a schematic diagram for explaining an example of a color development state of a test piece when determining a temperature suitable for hybridization using the kit of the present invention, wherein (a) shows a first detection probe and a first detection probe; (2) As a result of adding the linking standard gene to the detection probe, any position of the two detection probes selected in the test piece is colored and the hybridization temperature in the test environment for hybridization is too low. (B) shows the position of the first detection probe among the two detection probes selected in the test piece as a result of adding the linked standard gene to the first detection probe and the second detection probe. FIG. 2 shows that only the color develops and the hybridization temperature in the test environment is in a suitable state, and (c) is the first detection probe. As a result of adding the linking standard gene to the second detection probe, the position of the two detection probes selected in the test piece does not develop color, and the hybridization temperature in the test environment is too high. It is. A linked standard gene consisting of a sequence complementary to the probe 2 of Example 1 (first portion) and a sequence in which one mismatch base is introduced only at the approximately central position of the sequence complementary to the probe 4 (second portion). 1 is a schematic diagram showing 1. FIG. A linked standard gene comprising a sequence complementary to the probe 2 of Example 1 (first portion) and a sequence (second portion) into which one mismatch base has been introduced only at approximately the center of the sequence complementary to the probe 19 FIG.

(1. Kit)
The present invention provides a kit for detecting a target gene, and the kit includes a test piece, a first standard gene, and a second standard gene.

  The target gene is not particularly limited, and examples thereof include a gene for identifying a microorganism and a gene relating to drug sensitivity or resistance of the microorganism.

  The gene for identifying the microorganism is not particularly limited, and examples thereof include 16S ribosomal RNA.

  Examples of genes relating to drug sensitivity or resistance of microorganisms include wild-type or mutant genes. The drug is not particularly limited and includes, for example, pyrazinamide (PZA), isoniazid (INH), rifampicin (RFP), streptomycin (SM), and ethambutol (EB) in the case of Mycobacterium tuberculosis.

(1.1. Test piece)
FIG. 1 is a schematic diagram of the test piece for explaining an example of the test piece constituting the kit of the present invention.

  As shown in FIG. 1, a plurality of types of detection probes 12 are immobilized at different positions on a test piece 10 constituting the kit of the present invention. Preferably, each probe is fixed at regular intervals. Furthermore, a control or marker for confirming color development for detection may be immobilized.

(1.1.1. Detection probe)
The detection probe is a probe for detecting a target gene, and consists of an oligonucleotide that can hybridize with the target gene at a temperature of T _max ° C (the upper limit of the hybridization temperature) or lower. Preferably, the target gene consists of an oligonucleotide that does not hybridize with a gene that differs by one base at an arbitrary position (that is, a mismatch). For example, it consists of an oligonucleotide having a base sequence complementary to the base sequence of the target gene. The oligonucleotide length is not particularly limited, but is preferably 10 to 30 nucleotides, more preferably 12 to 26 nucleotides.

  The detection probe can be appropriately designed by those skilled in the art based on the base sequence of the target gene to be detected. The base sequences of at least two detection probes can be selected to target different regions within the target gene. The regions targeted by these detection probes may partially overlap.

  The oligonucleotide of the detection probe is not particularly limited. For example, the complementary base sequence can be appropriately designed by using a standard program and primer analysis software such as Primer Express (Perkin Elmer). They can be synthesized as appropriate using standard methods such as automated oligonucleotide synthesizers.

  The detection probe is preferably modified at either the 5 'or 3' end in order to be immobilized on the test strip.

  The test piece which comprises the kit of this invention is produced as follows, for example.

(1.1.2. Immobilization of detection probe)
The test piece can be prepared by physically or chemically immobilizing the at least two detection probes on the surface of the carrier. The carrier is not particularly limited, and examples thereof include organic materials such as vinyl polymer, nylon, polyester, polyethylene, polypropylene, polystyrene, polyethylene terephthalate, and nitrocellulose; inorganic materials such as glass and silica; metallic materials such as gold and silver. Is mentioned. An organic material is preferable in terms of easy molding, and polyesters such as polyethylene terephthalate are more preferable. These carriers are preferably white in order to make the color development easily visible in detection by a color development method.

  The method for physically immobilizing the probe on the surface of the carrier is not particularly limited, and various known methods can be mentioned. 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 applied at a rate of 2.5 mm / sec to 8.5 mm / sec while discharging an amount of 0.5 μL / min to 1.0 μL / min. When applied on the nitrocellulose membrane at a constant rate, each probe is applied as a stripe having a width of about 1 mm to 2 mm arranged at a constant interval. The probe is immobilized 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 support is a metal material such as gold, the surface of the support is treated with a thiol having an amino group, such as 2-aminoethanethiol, or a disulfide compound, thereby causing electrostatic interaction with a probe that is a polyanion. It is also known that the efficiency of immobilization is improved.

  The method for chemically immobilizing a probe by introducing a functional group is not particularly limited, and various known methods can be mentioned. For example, when the carrier is an inorganic material such as glass or silicon, there is a method in which the end of the probe is modified with a functional group capable of silane coupling reaction such as trimethoxysilane or triethoxysilane, and a solution containing the modified probe A test piece is obtained by immersing the carrier in the substrate for 24 to 48 hours, removing it, and washing it. Alternatively, an inorganic carrier such as glass or silicon is treated with a silane coupling agent having an amino group such as aminoethoxysilane to aminate the surface of the carrier, and then the amino coupling is performed with a probe having a carboxylic acid introduced at the terminal. There is also a method of immobilizing the probe by reacting. Further, when 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 this modified probe for 24 hours to It can be fixed by immersing for 48 hours, taking out, and 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 immobilizing the synthesized probe.

  In this way, a test piece constituting the kit of the present invention is produced.

  In the present invention, as shown in FIG. 2, the first detection probe 102 and the second detection probe 104 are selected from the test piece 100 on which the at least two detection probes are immobilized.

  The positional relationship between the first detection probe 102 and the second detection probe 104 selected by the test piece 100 is not particularly limited. That is, the first detection probe 102 and the second detection probe 104 may be adjacent probes, or may be one or more other probes interposed therebetween.

(1.2. Standard gene)
There are two standard genes, a first standard gene and a second standard gene, which are genes for confirming whether the target gene and the detection probe on the test piece are hybridized at an appropriate temperature. The first standard gene and the second standard gene may be oligonucleotides (linked standard genes) having a sequence in which the first part of the first standard gene and the second part of the second standard gene are continuous. Good.

The nucleotide sequences of the oligonucleotides constituting the first standard gene and the second standard gene are not particularly limited, and the first detection probe and the second detection probe for detecting the target gene are changed from T _min ° C to T _{min. When} hybridizing with the target gene at a temperature of _max ° C, the first portion of the first standard gene hybridizes with the first detection probe at a temperature lower than T _min ° C, and the second standard gene Two portions hybridize with the second detection probe, and the first standard gene hybridizes with the first detection probe at a temperature of T _min ° C to T _max ° C. The second standard gene the second part is not the second detector probe hybridizes, T _max ° C. or more of In time, the first portion of the first standard gene without first detector probe hybridizes, and a second portion of the second standard gene does not hybridize with a second detector probe. In other words, the first standard gene hybridizes to the first detection probe at a temperature lower than T _max ° C, and the first standard gene hybridizes with the first detection probe at a temperature lower than T _max ° C. The second standard gene does not hybridize with the first detection probe, and the second standard gene does not hybridize with the first detection probe at a temperature lower than T _min ° C. And the second part of the second standard gene does not hybridize with the second detection probe at a temperature of T _min ° C or higher. The temperature range from T _min ° C to T _max ° C can be used to determine the presence or absence of the target gene mutation in the measurement of the presence or absence of the target gene mutation using the test piece to which the first probe and the second probe are fixed. As long as it is, it is not specifically limited, For example, 2-4 degreeC, Preferably it is 2 degreeC.

  The oligonucleotide lengths of the first part of the first standard gene and the second part of the second standard gene are not particularly limited. For example, the oligonucleotide length is 10 to 40 nucleotides, preferably 10 to 30 nucleotides. More preferably, the length is 15 to 25 nucleotides.

  The first part of the first standard gene is preferably a complementary sequence of the oligonucleotide of the first detection probe. The second part of the second standard gene is preferably a complementary sequence of the oligonucleotide of the second detection probe, but has a mismatch base at a base located almost at the center of the base sequence of the second part. The position of the mismatch base may depend on the length of the sequence of the second part including the mismatch base, but may be a position “substantially central” with respect to the entire length of the second part. When the length of the oligonucleotide of the second part containing a mismatch base is 18 nucleotides, a mismatch base can be introduced, for example, at positions 7 to 10, preferably 8 to 9.

  Here, the mismatch base means a base that is not complementary, that is, a base that does not form a base pair. For example, a base other than T which is a complementary base to the base A to be paired, for example, C, G or A. Preferably, it is the same base as the base to be paired. For example, the same base A is preferable with respect to the base A to be paired.

  Although there is no particular limitation on the preparation of one oligonucleotide consisting of a sequence in which the first part of the first standard gene and the second part of the second standard gene are continuous, for example, by directly synthesizing the linked standard gene Alternatively, the first standard gene and the second standard gene may be synthesized and then combined. The method for synthesizing the standard gene is not particularly limited. For example, the method for synthesizing the oligonucleotide of the detection probe may be used. The method for linking the first part of the first standard gene and the second part of the second standard gene is not particularly limited, and for example, DNA ligase may be used.

  The method for preparing an oligonucleotide into which a mismatch base has been introduced is not particularly limited. For example, an automated oligonucleotide synthesizer is designed by appropriately designing a base sequence designed in advance so as to introduce a mismatch base at the approximately central position of the second part. The method of synthesize | combining suitably using standard methods, such as these is mentioned.

  An example of a method for producing a linked standard gene constituting the kit of the present invention will be described.

  FIG. 3 is a diagram schematically showing an example of a procedure for preparing a linked standard gene constituting the present invention.

  As shown in FIG. 3 (a1), the ligation standard gene is composed of a first portion composed of the complementary sequence of the oligonucleotide of the selected first detection probe and a complementary sequence of the oligonucleotide of the second detection probe. A second portion 204 having a sequence in which a mismatch base 208 is introduced at the central position is prepared separately. Next, as shown in FIG. 3 (b 1), the first part 202 of the first standard gene and the second part 204 of the second standard gene are connected to each other via a connection point 206. The means for linking the first part 202 of the first standard gene and the second part 204 of the second standard gene is not particularly limited. For example, DNA ligase may be used.

  Another example of a method for producing a linked standard gene constituting the kit of the present invention will be described.

  FIG. 4 is a diagram schematically showing another example of a procedure for producing a linked standard gene constituting the present invention.

  As shown in FIG. 4, the linked standard gene has a sequence (first portion) 302 complementary to the base sequence of the first detection probe and a sequence complementary to the base sequence of the oligonucleotide of the second detection probe. It is produced by preparing an oligonucleotide having a sequence in which a sequence (second portion) 304 having a mismatch base 308 introduced at a substantially central position is continuous.

  In order to detect hybridization with the probe on the test piece, the first standard gene and the second standard gene preferably have either a 5 ′ or 3 ′ end as a radioisotope, a fluorescent substance, and a chemiluminescent substance. It is modified with biotin. In addition, when the first standard gene and the second standard gene are linked standard genes, either the 5 ′ or 3 ′ end of the linked standard gene is a radioisotope, a fluorescent substance, a chemiluminescent substance, It will be modified with biotin.

(1.3. Other reagents)
The reagent for detecting hybridization with the probe on the test piece is not particularly limited. For example, when alkaline phosphatase-labeled streptavidin or avidin is used, nitro blue tetrazolium (NBT) / 5-bromo-4- Examples include chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) coloring reagent.

  In addition to the test piece, the first standard gene and the second standard gene or the linked standard gene, the kit of the present invention further includes, for example, a reagent for extracting gene DNA from a specimen, a reagent for gene amplification, a gene It includes a denaturing reagent, a hybridization reagent, and a reagent for detecting hybridization between the probe and the gene.

  The method for extracting the gene DNA is not particularly limited, and examples thereof include a phenol extraction method, a guanidine thiocinate extraction method, and a vanadyl ribonucleoside complex extraction method. The reagent for extracting gene DNA may be a reagent generally used in these methods.

  The gene amplification reagent includes a gene amplification primer pair, and the gene amplification primer is preferably a radioisotope, a fluorescent substance, a chemiluminescent substance, or biotin in order to detect hybridization between a gene to be amplified and a probe. Etc. The gene amplification method is not particularly limited, and examples thereof include a PCR method, a LAMP method, and an ICAN method.

  The reagent for gene denaturation is not particularly limited as long as the gene can be denatured into single-stranded DNA.

  The hybridization reagent contains a salt and a surfactant so that oligonucleotides having complementary base sequences hybridize with each other, and the concentration thereof is appropriately set.

  The reagent for detecting hybridization with the probe on the test piece is not particularly limited. For example, when the gene amplification primer is modified with alkaline phosphatase, nitro blue tetrazolium (NBT) / 5-bromo- Examples include 4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) coloring reagent.

(2. Method for detecting target gene)
(2.1. Sample)
The specimen is not particularly limited, and examples thereof include body fluids such as sputum, pharyngeal swab, gastric fluid, bronchoalveolar lavage fluid, intratracheal aspirate, throat wipe and / or tissue biopsy, and culture from these And cultures obtained by combining these, and combinations thereof.

(2.2. Extraction of gene DNA)
The method for extracting gene DNA from the specimen is not particularly limited, and examples thereof include a phenol extraction method, a guanidine thiocinate extraction method, and a vanadyl ribonucleoside complex extraction method. This step may be omitted.

(2.3. Amplification of target gene)
The gene amplification method is not particularly limited, and examples thereof include a PCR method, a LAMP method, and an ICAN method. The gene amplification primer is preferably modified with a radioisotope, a fluorescent substance, a chemiluminescent substance, biotin or the like in order to detect hybridization between the probe and the gene. The target gene may be amplified directly from the sample without extracting the gene DNA from the sample. In order to increase the detection sensitivity, for example, when a target gene is directly amplified from a specimen, the target gene may be amplified by the Nested PCR method (Japanese Patent Publication No. 6-81600) before the PCR method. As a primer in the Nested PCR method, an oligonucleotide having a base sequence located upstream or downstream of the target gene is usually used than the primer used in the PCR method.

(2.4. Determination of hybridization temperature for detecting target gene)
Prior to the detection of the target gene, the first standard gene and the second standard are used to determine the appropriate temperature (T _min ° C to T _max ° C) for the target gene to hybridize with the detection probe on the test strip. The gene or linked standard gene is hybridized with the test piece.

  The first standard gene and the second standard gene or the linked standard gene are provided so that both the first detection probe and the second detection probe on the test piece are approached. Two standard genes or linked standard genes are given to the first detection probe and the second detection probe on one test piece.

  Briefly, the hybridization treatment is mixing the gene and the test piece under a certain condition, and preferably mixing in the presence of the hybridization reagent. As a result of the hybridization treatment, for example, when the first standard gene and the second standard gene or the linked standard gene whose 5 ′ or 3 ′ end is modified with biotin are hybridized with the probe on the test piece, By reacting with alkaline phosphatase-labeled streptavidin and then with NBT / BCIP, alkaline phosphatase binds to the probe on the test piece, and color develops with NBT / BCIP.

  By observing whether or not each test piece is colored, it is determined whether the temperature is suitable for hybridization in each test environment as follows.

  That is, as shown in FIG. 5 (a), when both the first detection probe 102 and the second detection probe 104 are colored on the test piece 100, the test environment for hybridization is shown. The hybridization temperature is too low. Therefore, those skilled in the art can recognize that the temperature environment is not suitable for hybridization of the target gene under such a test environment.

  Further, for example, when one of the first detection probe 102 and the second detection probe 104 is colored on the test piece 100 (when only the position of the first detection probe 102 is colored in FIG. 5B). The position of the second detection probe 104 indicated by a broken line indicates that no color is developed), and the test environment is in a state suitable for hybridization. For this reason, those skilled in the art can recognize that it is in the temperature environment suitable for hybridization of a target gene in such a test environment.

  Further, as shown in FIG. 5 (c), when neither the first detection probe 102 nor the second detection probe 104 is colored as indicated by the broken line in the test piece 100, The hybridization temperature in the test environment for hybridization is too high. For this reason, those skilled in the art can recognize that the temperature environment is not suitable for hybridization of the target gene in such a test environment.

(2.5. Detection of target gene)
The amplified target gene is denatured into single-stranded DNA, and hybridized with the test piece at the proper hybridization temperature determined above. For example, if the gene amplification primer of the target gene is modified with biotin, the amplified biotin-labeled target gene will react with alkaline phosphatase-labeled streptavidin when hybridized with the detection probe immobilized on the test strip. Then, by reacting with NBT / BCIP, alkaline phosphatase binds to the probe on the test piece, and color develops with NBT / BCIP. When the color develops, the target gene is hybridized with the detection probe fixed to the test piece, indicating that the target gene has been detected.

  Based on the results of the detection, for example, the presence or absence of pathogenic bacteria including Mycobacterium tuberculosis can be confirmed, and the sensitivity and resistance of these bacteria to drugs can be confirmed. Furthermore, based on the confirmed result, for example, a treatment policy can be determined.

  EXAMPLES Hereinafter, although an Example demonstrates this invention concretely, this invention is not limited to these Examples.

(Example 1: Preparation of test piece)
(Probe selection)
The 11th, 3rd, 8th, 11th and 14th positions of the sequence complementary to the base sequence of the pyrazinamide (PZA) resistance gene (pncA gene) represented by SEQ ID NO: 1 in the Sequence Listing 19th, 20th, 23rd, 24th, 26th, 28th, 29th, 35th, 40th, 41st, 42nd, 68th, 69th, 74th, 75th, 77th, 78th, 78th, 83rd, 100th, 102nd, 104th, 134th, 137th, 139th, 143rd 146, 151-230, 152, 153, 157, 158, 161, 162, 169, 170, 172, 181 182nd, 185th, 187th, 190th, 199th, 199th, 202nd, 203rd, 212th, 216th, 226th, 227th, 231st, 233rd, 233rd, 254th, 261st, 286th, 287th, 288th, 290th, 295th, 296th 307, 308, 309, 307-311, 341, 351, 356, 362, 363, 369, 381-382, 383, 382-390, 386, 386-389, 392, 393, 403, 406, 410, 412, 415, 416 421, 422, 436, 442, 449, 452, 452, 466, 467, 476, 481, 486-496 494th, 515th, 525th, 533th, As a probe capable of detecting any one of the nucleotide mutations of # 538, prepared probe 1-47 (respectively, corresponding to SEQ ID NO: 2-48) shown below, respectively.

Probe 1: 5'-cgcatacgtccaccatacgttcgg-3 '(SEQ ID NO: 2)
Probe 2: 5'-atgatcaacgcccgcatac-3 '(SEQ ID NO: 3)
Probe 3: 5'-cacgtcgacgatgatcaacg-3 '(SEQ ID NO: 4)
Probe 4: 5′-gtcgttctgcacgtcgac-3 ′ (SEQ ID NO: 5)
Probe 5: 5'-ccaccctcgcagaagtcgtt-3 '(SEQ ID NO: 6)
Probe 6: 5′-cgagccaccctcgcagaag-3 ′ (SEQ ID NO: 7)
Probe 7: 5'-gttaccgccagcgagccacc-3 '(SEQ ID NO: 8)
Probe 8: 5'-agcgcggcgccaccggttaccg-3 '(SEQ ID NO: 9)
Probe 9: 5'-gtagtcgctgatggcgcgggccagcgcg-3 '(SEQ ID NO: 10)
Probe 10: 5′-gccaggtagtcgctgatggcgc-3 ′ (SEQ ID NO: 11)
Probe 11: 5'-tggtagtccgccgcttcggccaggta-3 '(SEQ ID NO: 12)
Probe 12: 5′-gtatgcgggcgttgatcatgtcgacgtgccgaacgac-3 ′ (SEQ ID NO: 13)
Probe 13: 5′-gtatgcgggcgttgatcatcaccgcattgggtcagcg-3 ′ (SEQ ID NO: 14)
Probe 14: 5′-cgcatacgtccaccatacgttcgg-3 ′ (SEQ ID NO: 15)
Probe 15: 5′-atgatcaacgcccgcatac-3 ′ (SEQ ID NO: 16)
Probe 16: 5′-cacgtcgacgatgatcaacg-3 ′ (SEQ ID NO: 17)
Probe 17: 5′-gtcgttctgcacgtcgac-3 ′ (SEQ ID NO: 18)
Probe 18: 5′-ccaccctcgcagaagtcgtt-3 ′ (SEQ ID NO: 19)
Probe 19: 5′-cgagccaccctcgcagaag-3 ′ (SEQ ID NO: 20)
Probe 20: 5′-gttaccgccagcgagccacc-3 ′ (SEQ ID NO: 21)
Probe 21: 5′-agcgcggcgccaccggttaccg-3 ′ (SEQ ID NO: 22)
Probe 22: 5′-gtagtcgctgatggcgcgggccagcgcg-3 ′ (SEQ ID NO: 23)
Probe 23: 5′-gccaggtagtcgctgatggcgc-3 ′ (SEQ ID NO: 24)
Probe 24: 5'-tggtagtccgccgcttcggccaggta-3 '(SEQ ID NO: 25)
Probe 25: 5′-cgcatacgtccaccatacgttcgg-3 ′ (SEQ ID NO: 26)
Probe 26: 5′-atgatcaacgcccgcatac-3 ′ (SEQ ID NO: 27)
Probe 27: 5′-cacgtcgacgatgatcaacg-3 ′ (SEQ ID NO: 28)
Probe 28: 5′-gtcgttctgcacgtcgac-3 ′ (SEQ ID NO: 29)
Probe 29: 5′-ccaccctcgcagaagtcgtt-3 ′ (SEQ ID NO: 30)
Probe 30: 5′-cgagccaccctcgcagaag-3 ′ (SEQ ID NO: 31)
Probe 31: 5′-gttaccgccagcgagccacc-3 ′ (SEQ ID NO: 32)
Probe 32: 5'-agcgcggcgccaccggttaccg-3 '(SEQ ID NO: 33)
Probe 33: 5′-gtagtcgctgatggcgcgggccagcgcg-3 ′ (SEQ ID NO: 34)
Probe 34: 5′-gccaggtagtcgctgatggcgc-3 ′ (SEQ ID NO: 35)
Probe 35: 5'-tggtagtccgccgcttcggccaggta-3 '(SEQ ID NO: 36)
Probe 36: 5′-gtatgcgggcgttgatcatgtcgacgtgccgaacgac-3 ′ (SEQ ID NO: 37)
Probe 37: 5′-gtatgcgggcgttgatcatcaccgcattgggtcagcg-3 ′ (SEQ ID NO: 38)
Probe 38: 5′-cgcatacgtccaccatacgttcgg-3 ′ (SEQ ID NO: 39)
Probe 39: 5′-atgatcaacgcccgcatac-3 ′ (SEQ ID NO: 40)
Probe 40: 5′-cacgtcgacgatgatcaacg-3 ′ (SEQ ID NO: 41)
Probe 41: 5′-gtcgttctgcacgtcgac-3 ′ (SEQ ID NO: 42)
Probe 42: 5′-ccaccctcgcagaagtcgtt-3 ′ (SEQ ID NO: 43)
Probe 43: 5′-cgagccaccctcgcagaag-3 ′ (SEQ ID NO: 44)
Probe 44: 5′-gttaccgccagcgagccacc-3 ′ (SEQ ID NO: 45)
Probe 45: 5′-agcgcggcgccaccggttaccg-3 ′ (SEQ ID NO: 46)
Probe 46: 5′-gtagtcgctgatggcgcgggccagcgcg-3 ′ (SEQ ID NO: 47)
Probe 47: 5′-gccaggtagtcgctgatggcgc-3 ′ (SEQ ID NO: 48)

(Immobilization of probe)
Polythymine is added to each 5 ′ end of oligonucleotide probes 1 to 47 shown in SEQ ID NOs: 2 to 48 using a terminal transferase. Specifically, 0.4 μL of terminal transferase (30 unit / μL, manufactured by Promega), 2 μL of thymidine triphosphate (10 pmol / μL), 2 μL of oligonucleotide probe (50 mM), 2 μL of reaction buffer attached to the product Then, 3.6 μL of purified water was added to prepare a reaction solution, reacted at 37 ° C. for 4 hours, and 90 μL of 10 × SSC buffer was added to prepare 1 pmol / μL of each oligonucleotide probe to which polythymine was added. To do.

  Next, 0.5 μL of each oligonucleotide probe to which polythymine is added is applied on the polyester film at regular intervals, and fixed by irradiation with 312 nm ultraviolet rays for 2 minutes.

(Preparation of linked standard gene)
As temperature confirmation genes, linked standard genes 1 and 2 consisting of oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 49 and 50 below are prepared. Schematic diagrams representing ligation standard genes 1 and 2 are shown in FIGS. 6 and 7, respectively. 6 and 7, the bases shown in capital letters represent mismatch bases.

  The ligation standard gene 1 is prepared as follows. A sequence complementary to the base sequence of the second standard gene and probe 2 consisting of a sequence (second portion) in which a mismatched base is introduced at a position approximately in the middle of the sequence complementary to the base sequence of probe 4 (first portion) Are designed and prepared, and the first part of the first standard gene is linked to the 3 ′ end of the second part of the second standard gene. Next, biotin is bound to the 5 'end of the ligation standard gene 1.

  Ligation standard gene 1: 5′-cagcaagCcgtgcagctgtactagttgcgggcgtatg-3 ′ (SEQ ID NO: 49: “C” is mismatched base)

  The ligation standard gene 2 is prepared as follows. A second standard gene including a sequence (second part) in which a mismatch base is introduced at a substantially central position of a sequence complementary to the base sequence of the probe 19, and a sequence complementary to the base sequence of the probe 2 (first sequence) An oligonucleotide (linked standard gene) consisting of a continuous sequence with a first standard gene containing a portion) is designed and prepared, and then biotin is bound to the 5 ′ end of the standard gene.

  Linkage standard gene 2: 5′-gcgactgGgttacgccactactagttgcgggcgtatg-3 ′ (SEQ ID NO: 50: “G” is mismatched base)

(Example 2: Determination of hybridization temperature for detecting pyrazinamide resistance)
(2-1: Determination of hybridization temperature using linked standard gene 1)
In the hybridization reagent containing the ligation standard gene 1, sodium dodecyl sulfate (0.01 w / v%), sodium chloride (1.8 w / v%), sodium citrate (1.0 w / v%) 1 mL of the solution and one test specimen for detection prepared in Example 1 are added and reacted by shaking for 30 minutes. Add alkaline phosphatase-labeled streptavidin, and then add nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) to hybridize with each probe on the test strip. The alkaline phosphatase bound to the linked standard gene 1 containing biotin is developed. Since only the position of the first detection probe of the detection test piece is colored, it can be determined that the temperature of the test environment is suitable for hybridization.

(2-2: Determination of hybridization temperature using linked standard gene 2)
In the hybridization reagent containing the ligation standard gene 2, sodium dodecyl sulfate (0.01 w / v%), sodium chloride (1.8 w / v%), sodium citrate (1.0 w / v%) 1 mL of the solution and one test specimen for detection prepared in Example 1 are added and reacted by shaking for 30 minutes. Add alkaline phosphatase-labeled streptavidin, and then add nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) to hybridize with each probe on the test strip. The alkaline phosphatase bound to the linked standard gene 2 containing biotin is developed. Since only the position of the first detection probe of the detection test piece is colored, it can be determined that the temperature of the test environment is suitable for hybridization.

(Examples 3 to 14: Detection of pyrazinamide resistance)
(Extraction of pncA gene)
In this example, the detection of mutations in the pncA gene as a target gene is performed using tuberculosis bacteria as a specimen. Table 1 below shows the tubercle bacillus used as a sample in each example. Genomic DNA is extracted and purified from each specimen by the phenol extraction method, and used as a sample.

(Amplification of pncA gene)
A forward primer consisting of the base sequence shown in SEQ ID NO: 51 of the sequence listing, a reverse primer consisting of the base sequence shown in SEQ ID NO: 52 and having biotin bound to the 5 ′ end, and Taq DNA polymerase (Roche Diagnostics) The pncA gene contained in the sample is amplified by a PCR method using a sticky product). The base sequence of the primer is as shown below.

Forward primer: 5'-ggcgtcatggaccctatatc-3 '(SEQ ID NO: 51)
Reverse primer: 5'-caacagttcatcccggttc-3 '(SEQ ID NO: 52)
PCR reaction conditions include a denaturation process at 94 ° C. for 30 seconds, an annealing process at 55 ° C. for 20 seconds, and a chain extension process at 72 ° C. for 20 seconds. This cycle is repeated 30 times for amplification. As a result, biotin is bound to the DNA to be amplified.

(Detection using probe)
Add 10 μL of a solution of sodium hydroxide (5 M) and ethylenediaminetetraacetic acid (0.05 M) to 10 μL of the solution containing the DNA to be amplified, stir well and let stand for 5 minutes to denature the DNA to be amplified into a single strand. . In this sample solution, 1 mL of a solution of sodium dodecyl sulfate (0.01 w / v%), sodium chloride (1.8 w / v%), sodium citrate (1.0 w / v%) and prepared in Example 1 One test specimen for detection is added and reacted by shaking for 30 minutes at the temperature determined in Example 2. Next, alkaline phosphatase-labeled streptavidin was added, and further nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphatase p-toluidinyl salt (BCIP) were added to each probe on the detection specimen. The color of alkaline phosphatase bound to the amplified DNA containing hybridized biotin is developed.

(Detection result)
The results of Examples 3 to 15 are shown in Table 2.

  When the kit of the present invention is used, a PZA drug-resistant bacterium produced by mutating at least one of the mutation positions of the pncA gene can be detected at an appropriate hybridization temperature. As a result, bacteria possessed by tuberculosis patients can be accurately monitored, and an appropriate treatment method can be provided for tuberculosis patients possessing PZA-resistant bacteria. Thus, unnecessary drug administration can be prevented, the burden on the patient can be reduced, and unnecessary medical expenses can be reduced.

  ADVANTAGE OF THE INVENTION According to this invention, the kit containing the test piece for target gene detection for preventing the misjudgment produced by the hybridization temperature being improper can be provided. The kit of the present invention enables simpler and more accurate determination in detection of pathogenic bacteria including Mycobacterium tuberculosis and confirmation of sensitivity and resistance of these bacteria to drugs. In particular, it can be easily used by medical personnel who are technically immature in harsh environments such as Southeast Asia.

10,100 Specimen 12 Detection probe 102 First detection probe 104 Second detection probe 200,300 Linked standard gene 202,302 First portion of first standard gene 204,304 Second of second standard gene Part 206 Connection point 208,308 Mismatch base

Claims (6)

A kit for detecting a target gene,
The kit includes a test strip, a first standard gene and a second standard gene;
At least a first detection probe and a second detection probe that can hybridize with the target gene at a temperature of T _max ° C or less are immobilized on the test piece, and the test piece has a temperature of T _min ° C to T _max ° C. It is possible to determine the presence or absence of mutations in the target gene,
The first standard gene is an oligonucleotide comprising a sequence containing a first portion,
The first portion of the first standard gene hybridizes with the first detection probe at a temperature lower than T _max ° C, and the first portion of the first standard gene at a temperature of T _max ° C or higher. The portion does not hybridize to the first detection probe;
The second standard gene is an oligonucleotide composed of a sequence containing a second portion, and the second portion of the second standard gene and the second detection probe are at a temperature lower than T _min ° C. Hybridizes, and the second portion of the second standard gene does not hybridize to the second detection probe at a temperature of T _min ° C or higher,
kit.   The first standard gene and the second standard gene constitute one oligonucleotide in which the first part of the first standard gene and the second part of the second standard gene are continuous; The kit according to claim 1.   The kit according to claim 1 or 2, wherein the nucleotide lengths of the first portion of the first standard gene and the second portion of the second standard gene are each 10 to 30 nucleotides in length. The first portion of the first standard gene is an oligonucleotide complementary sequence of the first detection probe;
The second portion of the second standard gene is a complementary sequence of the oligonucleotide of the second detection probe, but is a sequence in which one mismatch base is introduced only at a substantially central position. 4. The kit according to any one of 3. A method for determining a hybridization temperature comprising:
At least a first detection probe and a second detection probe that can hybridize with a target gene at a temperature of T _max ° C or less are immobilized, and whether there is a mutation in the target gene at a temperature of T _min ° C to T _max ° C. A step of providing at least a first standard gene and a second standard gene to a test piece that can be discriminated; and the first detection probe, the first standard gene, and the second detection probe from the test piece; And confirming the state of hybridization between the second standard gene and the second standard gene;
here,
The first standard gene is an oligonucleotide composed of a sequence containing a first portion, and the first portion of the first standard gene and the first detection probe are at a temperature lower than T _max ° C. The first portion of the first standard gene does not hybridize with the first detection probe at a temperature of T _max ° C or higher;
The second standard gene is an oligonucleotide composed of a sequence containing a second portion, and the second portion of the second standard gene and the second detection probe are at a temperature lower than T _min ° C. Hybridizes, and at a temperature of T _min ° C or higher, the second part of the second standard gene does not hybridize with the second detection probe;
Confirming the state of hybridization between the first detection probe and the first standard gene and the second detection probe and the second standard gene from the test piece,
(I) When the first standard gene hybridizes to the first detection probe and the second standard gene hybridizes to the second detection probe, it is determined that the hybridization temperature in the test environment is too low. And
(Ii) When the first standard gene hybridizes to the first detection probe, but the second standard gene does not hybridize to the second detection probe, the hybridization temperature in the test environment is appropriate. Judgment
(Iii) When the first standard gene does not hybridize to the first detection probe and the second standard gene does not hybridize to the second detection probe, the hybridization temperature in the test environment is too high. to decide,
Method.   The first standard gene and the second standard gene constitute one oligonucleotide in which the first part of the first standard gene and the second part of the second standard gene are continuous; The method of claim 5.

Images