JP4176134B2 - Method for detecting pathogenic microorganisms - Google Patents

Description

  The present invention relates to oligonucleotide probes and primers useful for the detection of pathogenic microorganisms, a method for detecting pathogenic microorganisms using these, and a kit for the method.

  Known methods for detecting a pathogenic microorganism include a method for immunologically detecting a protein derived from the microorganism, a method for detecting a specific region of a gene derived from the microorganism by a nucleic acid amplification reaction, and the like. Examples of the method for detecting a specific region of the gene include a detection method using a nucleic acid amplification reaction. The nucleic acid amplification reaction is described in US Pat. Nos. 4,683,195 (Patent Document 1), 4,683,202 (Patent Document 2) and 4,800,159 (Patent Document 3). Polymerase chain reaction method (PCR method), Trends in Biotechnology, Volume 10, pages 146-152 (1992) (Non-patent Document 1) and reverse transcription combined with reverse transcriptase reaction Copy PCR method (RT-PCR method), ligase chain reaction (LCR) method described in European Patent Application No. 320,308 (Patent Document 4) published on June 14, 1989, Transcription-based amplification system (TAS) described in PCR Protocols, Academic Press. Inc., 1990, pages 245-252 (Non-Patent Document 2) ) Method.

  In addition, nucleic acid amplification methods that can be performed in an isothermal state have been developed. For example, the strand displacement amplification (SDA) method, the self-sustained sequence replication (3SR) method described in Japanese Patent Publication No. 7-114718 (Patent Document 5), Japanese Patent No. 2650159 ( Nucleic acid sequence amplification (NASBA) method described in Patent Document 6), TMA (transcription-mediated amplification) method, Qβ replicase method described in Japanese Patent No. 2710159 (Patent Document 7), US Pat. No. 5,824,517 (Patent Document 8), International Publication No. 99/09211 (Patent Document 9), International Publication No. 95/25180 (Patent Document 10), or International Publication No. 99 / Examples include various improved SDA methods described in pamphlet No. 49081 (Patent Document 11). US Pat. No. 5,916,777 describes a method for enzymatic synthesis of oligonucleotides in an isothermal state. Furthermore, a LAMP (Loop-mediated Isothermal Amplification) method described in International Publication No. 00/28082 pamphlet (Patent Document 13) and an ICAN (Isothermal and Chimeric primer) described in International Publication No. 00/56877 Pamphlet (Patent Document 14). -initiated Amplification of Nucleic acids).

  As described above, there are various nucleic acid amplification methods that can be used in the detection method of pathogenic microorganisms, but target nucleic acid regions suitable for each nucleic acid amplification reaction so as to satisfy the detection sensitivity required by the actual site for testing pathogenic microorganisms, It has been difficult to select a primer suitable for each nucleic acid amplification method and a probe suitable for the target nucleic acid detection method following each nucleic acid amplification reaction. Furthermore, there has been a demand for a method for detecting pathogenic microorganisms that can obtain reproducible results at a low running cost. Hereinafter, examples of the pathogenic microorganisms include Mycobacterium tuberculosis, Phosphorus, Chlamydia, and hepatitis C virus (HCV).

(A) Mycobacterium tuberculosis tuberculosis is a disease that occupies the top cause of human death in the past, and there are still a large number of patients even now that various treatments have been developed.

  Tuberculosis has been conventionally diagnosed by culture and smear methods. However, since tuberculosis bacteria are slow to grow, the results are 1 to 8 weeks until test results are obtained, usually about 1 month, which is not rapid and accurate. Moreover, it was not a sufficient diagnostic method such as 70% or less. Against this background, in recent years, direct, rapid, and accurate detection methods and reagents from sputum and the like have been developed that target the gene of Mycobacterium tuberculosis. The Mycobacterium tuberculosis group includes Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium africanum, Mycobacterium microti, and Mycobacterium canetti.

  For example, a method of extracting DNA from a specimen such as sputum and rapidly detecting the presence of M. tuberculosis by PCR [Lancet, No. 8671, page 1069 (1989) (Non-patent Document 3)] has been reported. Has been. This method is a method in which a gene encoding the 65 kDa antigen of Mycobacterium tuberculosis is used as a target, amplified by PCR, and detected by electrophoresis. A kit for amplifying 16S ribosomal RNA and detecting the amplified product with a chemiluminescent probe is also commercially available [Gen-Probe]. Furthermore, PCR kits targeting DNA encoding 16S ribosomal RNA are also commercially available (Roche).

  On the other hand, the sequence of a gene called IS6110 specific for Mycobacterium tuberculosis has been published [Nucleic Acids Research, Vol. 18, 188 (1990) (Non-patent Document 4)] It is described that it will be useful as a probe for the detection of Mycobacterium tuberculosis. In fact, a method for detecting the IS6110 gene by PCR has been reported [J. Clin. Microbiol., 28, 2668 (1990) (Non-patent Document 5). ]. Since then, many papers on PCR methods targeting the IS6110 gene have been reported and their usefulness has been described. In addition, Japanese Patent No. 2814422 (Patent Document 15) relating to detection of IS6110 gene by SDA method, which is a nucleic acid amplification method different from PCR method, and Japanese Patent Publication No. 10-500023 relating to detection of IS6110 gene by LCR method (Patent Document) 16).

  However, the above method for detecting Mycobacterium tuberculosis is still not a satisfactory method in clinical practice, and further improvement is desired. For example, nucleic acid amplification detection methods targeting ribosomal RNA genes have been widely reported, but only a part common to the genus Mycobacterium is amplified, and tuberculosis bacteria are identified with a species-specific probe. In this method, it is only the probe sequence that determines the specificity for Mycobacterium tuberculosis, and there are many reports that there are anxieties including detection sensitivity and there are cases that cannot be detected even with culture positive samples. ing. On the other hand, in order to increase specificity and detection sensitivity, a PCR method targeting a Mycobacterium tuberculosis-specific gene called IS6110 has been reported as described above. However, in a comparative study of tests targeting IS6110 at seven institutions, it is clear that the false positive rate by this method is 3 to 77% and the sensitivity is 2 to 90%, and the test results vary greatly from one facility to another. [Journal of Clinical Microbiology, Vol. 32, p. 277 (1994) (Non-patent Document 6)].

  On the other hand, the method for extracting nucleic acid from a specimen is complicated, and the extraction method is not sufficiently considered in terms of infection of the inspector.

  In addition, the hybridization method is used in the detection method of the amplification product. Conventionally, the amplified double-stranded nucleic acid is first denatured with strong alkali in the liquid layer, and then the denatured single-stranded nucleic acid is peeled off. A method of hybridizing with a probe under neutral conditions in the presence of a target nucleic acid was used (Roche amplicore kit instructions). In this case, there has been a problem that the hybridization of the probe is competitively hindered by the complementary amplified nucleic acid peeled off from the double strand and the sensitivity and specificity are not increased. That is, the amplified double-stranded DNA was once converted into a single strand by alkali denaturation, neutralized, and then hybridized with the probe under neutral conditions. In some cases, heat denaturation may be performed while disabling alkali denaturation, but this makes it impossible to accurately test a large number of specimens. For this reason, there are many steps, and one of the single-stranded DNAs competes with the probe and hybridizes, thereby reducing the detection sensitivity of the target nucleic acid.

  In addition, conventional methods and commercially available kits require about 6 hours or more from sample collection to report of results, and because of the infectious disease of tuberculosis, measures such as diagnosis and patient isolation are required as soon as possible. However, the problem of public health that the transmission of tuberculosis infection cannot be kept to a minimum cannot be solved, since the sample collection and the result report and the corresponding treatment to the patient cannot be handled on the same day, and the transmission of tuberculosis infection cannot be minimized. It was.

  From the information published as described above, it has been clarified that there is no reliable and useful medicine for M. tuberculosis.

(B) Phosphorus Phosphorus (ie Neisseria gonorrhoeae, Neisseria gonorrhoeae) is a mania pathogen that is one of the most commonly reported bacterial infections in the United States. Miyada and Born, 1991, Mol. Cell. Probes 5: 327-335 (Non-patent Document 7); US Pat. No. 5,256,536 (Patent Document 17); and US Pat. No. 5,525,717 (patent) Reference 18) describes N.I. N. cerevisiae using a DNA probe derived from a genomic fragment containing an open reading frame (ORF1) having significant homology to the sequence of the gonorhua cytosine DNA methyltransferase gene (M.Ngo PII). The detection of gonorjoule is described. However, there has been no method that can specifically detect phosphorus bacteria with sufficient sensitivity.

  In addition, it has been reported that patients infected with phosphorus bacteria are also infected with Chlamydia trachomatis.

(C) Chlamydia “Chlamydia” refers to a microorganism belonging to the genus Chlamydia. Three species of the genus Chlamydia, C. trachomatis, C. psittaci, and C. pneumoniae are clinically important because they can infect human hosts and cause disease. Chlamydia trachomatis, in particular, is reported to be the most common sexually transmitted disease in developed societies that causes genital infections in both sexes. Therefore, there is a need for methods and reagents that can specifically and timely detect Chlamydia trachomatis.

(D) HCV
Conventionally, detection of HCV in a sample such as serum has been performed by a reverse transcription PCR (RT-PCR) method. This method consists of (1) extraction of HCV RNA from serum, (2) synthesis of cDNA using the extracted RNA as a template, (3) amplification by PCR method for raising and lowering the temperature, and (4) immobilization probe. Hybridization, (5) washing and removal of unreacted reagent, (6) reaction with labeling reagent, (7) washing and removal of unreacted reagent, (8) addition of coloring reagent, (9) addition of coloring stop agent, ( 10) A total of 10 steps of measuring absorbance are performed. In addition, J.H. Virol. Methods Vol. 64, pp 147-159 (1997) (Non-patent Document 8), HCV detection by a homogeneous detection method using a real-time detection PCR method has also been studied.

  As described above, HCV can be measured with high sensitivity, and a large amount of specimens can be measured quickly and inexpensively. Blood preparation, blood transfusion, blood donation quality control, treatment progress determination, case monitoring, or treatment cost reduction It is very important from the point of view. However, in the PCR method, it is essential to strictly control the temperature, and the measuring machine for that purpose is complicated and large. On the other hand, the processing capacity is limited to 96 samples in 5 hours at present. Therefore, it was impossible to measure a large number of samples of 1000 samples or more in 2 hours required in blood centers and clinical laboratory centers.

  As described above, the conventional methods have various problems, and a technique for simply and quickly measuring HCV nucleic acids in a large number of specimens within a certain period of time has been demanded.

US Pat. No. 4,683,195 U.S. Pat.No. 4,683,202 US Pat. No. 4,800,159 European Patent Application No. 320,308 Japanese Patent Publication 7-114718 Japanese Patent No. 2650159 Japanese Patent No. 2710159 US Patent No. 5,824,517 International Publication No. 99/09211 Pamphlet WO95 / 25180 pamphlet WO99 / 49081 pamphlet US Patent No. 5,916,777 International Publication No. 00/28082 Pamphlet International Publication No. 00/56877 Pamphlet Japanese Patent No. 2814422 Special table hei 10-500023 US Pat. No. 5,256,536 US Pat. No. 5,525,717 Trends in Biotechnology, Vol. 10, pp. 146-152 (1992) PCR Protocols (Academic Press. Inc., 1990), pages 245-252 Lancet, No. 8671, page 1069 (1989) Nucleic Acids Research, Vol. 18, 188 (1990) Journal of Clinical Microbiology, 28, 2668 (1990) Journal of Clinical Microbiology, 32, 277 (1994) Miyada and Born, 1991, Mol. Cell. Probes 5: 327-335 J. et al. Virol. Methods Vol. 64, pp 147-159 (1997)

  The main object of the present invention is to provide means for measuring pathogenic microorganisms for a large number of specimens within a predetermined time with high sensitivity, accuracy, simply, in a short time, and at a low cost.

  As a result of intensive studies, the present inventors have found a method for detecting pathogenic microorganisms using a nucleic acid amplification method superior to conventional nucleic acid amplification methods. In addition, the present inventors have found a chimeric oligonucleotide primer for target nucleic acid amplification and a probe for detecting a target nucleic acid for the method. Furthermore, a kit for the method was constructed and the present invention was completed.

  That is, the first invention of the present invention is a probe having the sequence number of Mycobacterium tuberculosis, Mycobacterium bovisBCG, Mycobacterium africanum, Mycobacterium microti and / or Mycobacterium canti which can detect a sequence number of Mycobacterium canti The present invention relates to a probe containing a part thereof or a probe characterized by being a base sequence described in SEQ ID NO: 11 in the Sequence Listing.

  A second invention of the present invention is a probe capable of detecting Neisseria gonorrhoeae of phosphorous fungus, comprising a base sequence described in SEQ ID NO: 27 of the sequence listing or a part thereof, The probe has the base sequence described in SEQ ID NO: 21.

  3rd invention of this invention is a probe which can detect Chlamydia trachomatis of Chlamydia, Comprising: The probe which contains the base sequence of sequence number 22 of a sequence table, or its part, The sequence of a sequence table It is related with the probe characterized by being the base sequence of No. 20.

  A fourth invention of the present invention relates to a probe capable of detecting HCV and having the base sequences set forth in SEQ ID NOs: 34 and 35 in the sequence listing.

  The fifth invention of the present invention relates to a probe capable of hybridizing to a target nucleic acid of a pathogenic microorganism in an alkaline region. In the fifth invention of the present invention, a probe capable of hybridizing to a target nucleic acid of a pathogenic microorganism in an alkaline region of pH 8 to 14 is suitable, and the target nucleic acid of the pathogenic microorganism is a target derived from Mycobacterium tuberculosis, phosphorus, chlamydia, or HCV. Probes that are any of the nucleic acids are preferred. Further, the target nucleic acid of the pathogenic microorganism is selected from the base sequence of the IS6110 gene of Mycobacterium tuberculosis, the cppB gene of Phosphorus, the pLGV440 of Chlamydia, and the 5 ′ untranslated region of HCV, and a probe capable of hybridizing to the gene is preferred. Can be used. The probe is a probe comprising a base sequence represented by SEQ ID NO: 39 in the sequence listing present in the IS6110 gene of the Mycobacterium tuberculosis group, or a base sequence containing a part thereof, preferably represented by SEQ ID NO: 11 of the sequence listing. A probe comprising a base sequence is exemplified. Further, the probe comprises a base sequence shown in SEQ ID NO: 27 of the sequence listing present in the cppB gene of the bacterium or a part thereof, preferably a base sequence shown in SEQ ID NO: 21 of the sequence listing. A probe is exemplified. Further, there is a probe comprising a base sequence shown in SEQ ID NO: 22 in the Chlamydia pLGV440 or a base sequence containing a part thereof, preferably a probe consisting of the base sequence shown in SEQ ID NO: 20 in the Sequence Listing. Illustrated. Further, a probe comprising a base sequence shown in SEQ ID NO: 34, 35 in the sequence listing existing in the 5 ′ untranslated region of HCV or a base sequence containing a part thereof, preferably shown in SEQ ID NO: 34, 35 in the sequence listing. A probe having a base sequence is exemplified.

  In the probes of the first to fifth inventions of the present invention, a label may be added. The probe is an oligonucleotide having a base sequence consisting of 8 to 53 bases in the base sequence represented by SEQ ID NO: 11, 20, 21, 34, 35 in the sequence listing, and hybridized with the target nucleic acid. In some cases, a probe for detecting a pathogenic microorganism may be used, in which the fluorescence intensity is not suppressed, and when it does not hybridize with the target nucleic acid, the probe is fluorescently labeled so that the fluorescence intensity is suppressed. Further, among the base sequences shown in SEQ ID NOs: 11, 20, 21, 34, and 35 in the sequence listing, a probe for detecting pathogenic microorganisms comprising 8 or more consecutive bases may be used, and the 5 ′ end thereof is rhodamine-based fluorescent. A probe labeled with a dye or an oxazine-based fluorescent dye, wherein the probe comprises a base sequence represented by SEQ ID NO: 34 in the sequence listing and comprises 8 or more consecutive bases. Good. The labeled fluorescent dye is a probe having a reporter fluorescent dye and a quencher dye, and is composed of 8 or more consecutive bases among the base sequences shown in SEQ ID NOs: 11, 20, 21, 34, and 35 in the sequence listing. It may be a probe for detecting pathogenic microorganisms. The reporter dye may be a fluorescein dye, and the quencher dye may be a DABCYL dye. Furthermore, a probe to which a label selected from a fluorescent substance, a dye, an enzyme, biotin, a gold colloid, and a radioisotope is added can be preferably used.

  The sixth invention of the present invention relates to a method for detecting a pathogenic microorganism comprising the step of performing hybridization with a target nucleic acid of a pathogenic microorganism using the probe of the first to fifth inventions of the present invention. In the sixth aspect of the present invention, hybridization with a target nucleic acid of a pathogenic microorganism can be performed in an alkaline region. In addition, examples of the pathogenic microorganism include Mycobacterium tuberculosis group, phosphorous fungus, chlamydia, and HCV. When the pathogenic microorganism is a Mycobacterium tuberculosis group, the target nucleic acid is preferably the IS6110 gene or a fragment thereof, and after amplification of the Mycobacterium tuberculosis-derived IS6110 gene and / or fragment thereof, the amplified product and the first or fifth of the present invention are used. It is preferable to perform hybridization with the probe of the invention. Further, the IS6110 gene derived from Mycobacterium tuberculosis and / or a fragment thereof is amplified using a primer having a base sequence shown in each of SEQ ID NOS: 36 and 37 in the sequence listing, or a sequence partially overlapping with the sequence. Is preferred. Further, when the pathogenic microorganism is phosphorus, the target nucleic acid is preferably the cppB gene or a fragment thereof, and after amplification of the cppB gene and / or fragment thereof derived from the bacterium, the amplified product and the second or fifth of the present invention are used. It is preferable to perform hybridization with the probe of the invention. In addition, the cppB gene and / or a fragment thereof derived from a bacterium may be amplified using a primer having a base sequence shown in each of SEQ ID NOs: 28 and 29 in the sequence listing or a sequence partially overlapping with the sequence. preferable. Further, when the pathogenic microorganism is chlamydia, the target nucleic acid is preferably pLGV440 or a fragment thereof, and after amplification of the chlamydia-derived pLGV440 and / or its fragment, the amplified product and the probe of the third or fifth invention of the present invention It is preferable to perform hybridization with Further, it is preferable that pLGV440 derived from Chlamydia and / or a fragment thereof is amplified using a primer having a base sequence shown in each of SEQ ID NOS: 23 to 26 in the sequence listing or a sequence partially overlapping with the sequence. Further, when the pathogenic microorganism is HCV, the target nucleic acid is preferably a 5 ′ untranslated region of HCV or a fragment thereof, and after amplifying the 5 ′ untranslated region and / or fragment thereof derived from HCV, the amplified product and the present invention It is preferable to perform hybridization with the probe of the fourth or fifth invention. In addition, the HCV-derived 5 ′ untranslated region and / or a fragment thereof is amplified using a primer having a base sequence shown in each of SEQ ID NOS: 30 to 33 in the sequence listing or a sequence partially overlapping with the sequence. It is preferable.

  The seventh invention of the present invention is a pathogenic microorganism comprising a step of detecting a nucleic acid derived from Mycobacterium tuberculosis, Phosphorus, Chlamydia, HCV using the method for detecting a target nucleic acid of a pathogenic microorganism of the sixth invention of the present invention. It relates to the detection method.

According to an eighth aspect of the present invention, there is provided a method for detecting a pathogenic microorganism, comprising detecting an amplified nucleic acid obtained by a nucleic acid amplification method including the following steps.
(A) A step of preparing a reaction mixture by mixing a template nucleic acid, deoxyribonucleotide triphosphate, DNA polymerase having strand displacement activity, at least one primer, and RNase H; where the primer serves as a template It is substantially complementary to the base sequence of the nucleic acid and contains at least one selected from deoxyribonucleotides or nucleotide analogs and ribonucleotides, and the ribonucleotides are arranged at the 3 ′ end or 3 ′ end side of the primer A chimeric oligonucleotide primer; and
(B) incubating the reaction mixture for a time sufficient to produce a reaction product;
About. In the eighth aspect of the present invention, it is preferable to use a reaction mixture containing a chimeric oligonucleotide primer having a sequence substantially homologous to the base sequence of the nucleic acid serving as a template. In addition, a chimeric oligonucleotide primer represented by the following general formula can be preferably used as the chimeric oligonucleotide primer.
General formula: 5'-dNa-Nb-dNc-3 '
(A: an integer of 11 or more, b: an integer of 1 or more, c: 0 or an integer of 1 or more, dN: deoxyribonucleotide and / or nucleotide analog, N: unmodified ribonucleotide and / or modified ribonucleotide, dNa A part of dN may be substituted with N)

  Further, c of the chimeric oligonucleotide primer may be 0, the nucleotide analog may be deoxyriboinosine nucleotide or deoxyribouracil nucleotide, and the modified ribonucleotide may be (α-S) ribonucleotide. Moreover, it is preferable to use the chimera oligonucleotide primer which consists of a base sequence each represented by sequence number 13-16 of a sequence table, 23-26, 28-31. Furthermore, you may include the process of detecting amplified nucleic acid using the probe of the 1st-5th invention of this invention.

A ninth invention of the present invention relates to a chimeric oligonucleotide primer for detecting pathogenic microorganisms represented by the following general formula.
General formula: 5'-dNa-Nb-dNc-3 '
(A: an integer of 11 or more, b: an integer of 1 or more, c: 0 or an integer of 1 or more, dN: deoxyribonucleotide and / or nucleotide analog, N: unmodified ribonucleotide and / or modified ribonucleotide, dNa A part of dN may be substituted with N)

  In the ninth invention of the present invention, those in which c is 0 can be used preferably, the nucleotide analog is deoxyriboinosine nucleotide or deoxyribouracil nucleotide, and the modified ribonucleotide is (α-S) ribonucleotide Nucleotide primers are preferred. Although not particularly limited, for example, chimeric oligonucleotide primers for detecting pathogenic microorganisms represented by SEQ ID NOS: 13 to 16, 23 to 26, and 28 to 31 in the sequence listing are preferable.

  The tenth aspect of the present invention amplifies the Mycobacterium tuberculosis-derived IS6110 gene and / or a fragment thereof having the nucleotide sequences shown in SEQ ID NOs: 36 and 37 in the sequence listing, or sequences partially overlapping with the sequences. Primer for amplification, primer for amplifying the cppB gene derived from Phosphorus and / or a fragment thereof having the base sequence shown in SEQ ID NO: 27 of the sequence listing, or a sequence partially overlapping with the sequence, sequence of the sequence listing A primer for amplifying the chlamydia-derived pLGV440 and / or a fragment thereof having the base sequence shown in No. 22 or a sequence partially overlapping with the base sequence, and the base sequences shown in SEQ ID Nos. 41 to 43 in the sequence listing, respectively Or a 5 ′ untranslated region derived from HCV and / or a fragment thereof having a sequence partially overlapping with the sequence Regarding a primer for width.

  In the tenth aspect of the present invention, the primer may be a chimeric oligonucleotide primer in which a part of the base sequence is substituted with a ribonucleotide. In the ninth and tenth aspects of the present invention, a label may be added. Examples of the label include fluorescent substances, dyes, enzymes, biotin, and gold colloids.

  The eleventh aspect of the present invention relates to a target nucleic acid detection composition comprising the probe of the first to fifth aspects of the present invention.

  A twelfth aspect of the present invention relates to a target nucleic acid detection composition comprising the primer according to the ninth to tenth aspects of the present invention.

  The eleventh and twelfth inventions of the present invention can be used for detection of pathogenic microorganisms.

  A thirteenth aspect of the present invention is a composition for use in the sixth to eighth pathogenic microorganism detection methods of the present invention, wherein at least one reagent for performing amplification or hybridization of a target nucleic acid is provided. And a composition for detecting pathogenic microorganisms.

  In the thirteenth aspect of the present invention, a reagent selected from DNA polymerase having strand displacement activity, RNase H, and deoxyribonucleotide triphosphate may be contained. The DNA polymerase may be a 5 ′ → 3 ′ exonuclease-deficient Bca DNA polymerase derived from Bacillus caldotenax, and the RNase H is derived from a genus Pyrococcus and / or from the genus Arcaeoglobus It may be a type II RNase H derived from bacteria.

  A fourteenth invention of the present invention relates to a kit for detecting a pathogenic microorganism, comprising the probe of the first to fifth inventions of the present invention.

  In the fourteenth aspect of the present invention, the ninth to tenth primers of the present invention may be contained. The kit can be used for detection of Mycobacterium tuberculosis, Phosphorus, Chlamydia, and HCV. The kit is used for the sixth to eighth pathogenic microorganism detection methods of the present invention, and includes at least one reagent for performing amplification or hybridization of a target nucleic acid. Also good. Moreover, you may contain the reagent selected from DNA polymerase which has strand displacement activity, RNaseH, and deoxyribonucleotide triphosphate. The DNA polymerase is preferably a 5 '→ 3' exonuclease-deficient Bca DNA polymerase derived from Bacillus cardotenax, and the RNase H may be a type II RNase H derived from a genus Pyrococcus and / or an bacterium belonging to the genus Arcaeoglobus. Furthermore, a carrier for capturing the amplification product may be contained, and the carrier is preferably a carrier selected from microtiter plates, beads, magnetic beads, membranes, and glass.

  A fifteenth aspect of the present invention relates to a method for detecting the Mycobacterium tuberculosis group, comprising a step of treating a Mycobacterium tuberculosis-containing specimen with muramidase and extracting a nucleic acid in the method for detecting the Mycobacterium tuberculosis group.

  Provided is a means for measuring pathogenic microorganisms for a large number of specimens within a certain period of time with high sensitivity, accuracy, simply, in a short time, and at a low cost.

  As used herein, deoxyribonucleotide (also referred to herein as dN) refers to a nucleotide whose sugar moiety is composed of D-2-deoxyribose. For example, the base moiety includes adenine, cytosine, guanine, The thing which has thymine is mentioned. Furthermore, deoxyribonucleotide analogs such as deoxyribonucleotides and deoxyinosine nucleotides having modified bases such as 7-deazaguanosine are also included in the above deoxyribonucleotides.

  In the present specification, ribonucleotide (also referred to as N in the present specification) refers to a nucleotide whose sugar moiety is composed of D-ribose, and whose base moiety has adenine, cytosine, guanine, uracil. Can be mentioned. Furthermore, the ribonucleotide includes a modified ribonucleotide. For example, a modified ribonucleotide in which the oxygen atom of the phosphate group at the α-position is replaced with a sulfur atom [(α-S) ribonucleotide, (α-S) N is also described. And other derivatives.

  As used herein, a chimeric oligonucleotide primer refers to a primer containing deoxyribonucleotides and ribonucleotides. The primer may contain nucleotide analogs and / or modified nucleotides.

  In the chimeric oligonucleotide primer used in the present invention, a ribonucleotide is arranged on the 3 ′ end or 3 ′ end side of the primer, and in the method of the present invention, the nucleic acid strand can be extended, cleaved with an endonuclease, and subjected to a strand displacement reaction. Anything that can be performed is encompassed by the chimeric oligonucleotide primer of the present invention.

  In this specification, the 3 'terminal side refers to a part of a nucleic acid, for example, a primer from the center to the 3' terminal. Similarly, the 5 ′ end side refers to a portion of the nucleic acid from the center to the 5 ′ end.

  In this specification, endonuclease refers to a substance that acts on double-stranded DNA produced by extending DNA from the above-mentioned chimeric oligonucleotide primer annealed to a template nucleic acid to specifically cleave the ribonucleotide portion of the primer. If it is.

  In this specification, DNA polymerase refers to an enzyme that synthesizes a new DNA strand using a DNA strand as a template, and includes naturally occurring DNA polymerase and a mutant enzyme having the above activity. Examples of the enzyme include a DNA polymerase having strand displacement activity, a DNA polymerase not having 5 '→ 3' exonuclease activity, and a DNA polymerase having both reverse transcriptase activity and endonuclease activity.

  In this specification, the term “strand displacement activity” means that when DNA replication is carried out according to a nucleic acid sequence as a template, it proceeds while replacing the DNA strand and releases the complementary strand annealed to the template strand, ie, strand displacement (strand substitution). activity that can be displaced). In the present specification, a DNA chain released from a nucleic acid sequence serving as a template by strand replacement is referred to as a “substituted chain”.

  In this specification, the alkali region refers to a region where the pH exceeds 7.

  In the present specification, pathogenic microorganisms refer to pathogenic bacteria and viruses.

  In this specification, the target nucleic acid refers to an arbitrary region of a gene derived from a pathogenic microorganism to be amplified or detected, and includes both DNA and RNA.

Hereinafter, the present invention will be described in detail.
(1) Probe of the present invention The probe of the present invention is characterized in that it can detect pathogenic microorganisms, such as Mycobacterium tuberculosis, Phosphorus, Chlamydia, or HCV. As a preferred embodiment of the probe of the present invention, one that can stably hybridize to the target nucleic acid in the alkaline region is exemplified. That is, the probe capable of hybridizing in the alkaline region of the present invention is not particularly limited, but for example, in an alkaline region exceeding pH 7.0, preferably in the range of pH 8 to 14, particularly preferably in the range of pH 8 to pH 10. And a probe capable of hybridizing with a target nucleic acid having a sequence complementary to the base sequence. Although it does not specifically limit, For example, what contains the base sequence of sequence number 11, 20, 21, 34, 35 of a sequence table is mentioned. The probe of the present invention can be used for conventional hybridization in a neutral region.

  When using the probe of the present invention, a double-stranded nucleic acid can be denatured into a single strand by alkali treatment and then used in a hybridization step without neutralization. This simplifies the process and improves the sensitivity by more than 10 times compared to the conventional method. Moreover, the hybridization specificity can be improved by hybridization in the alkaline region.

  There is no particular limitation on the method for detecting the probe hybridized with the nucleic acid derived from the specimen, and any known detection method can be applied to the present invention.

  By using the probe of the present invention labeled with an appropriate method, the target nucleic acid present in the sample can be detected efficiently and / or with high sensitivity.

The labeling method of the probe is not limited, and for example, a radioisotope (such as ³² P), a dye, a fluorescent substance, a luminescent substance, various ligands (such as biotin and digoxigenin), an enzyme, and the like can be used. The presence of the labeled probe can be confirmed by a detection method corresponding to the label. In the case of a ligand that cannot be directly detected, it may be combined with a ligand-binding substance labeled with a detectable label. For example, a target nucleic acid can be detected with high sensitivity by combining a ligand-labeled probe with an enzyme-labeled anti-ligand antibody and amplifying the signal.

  Furthermore, the probe may have a labeling substance for detection. The labeling substance is not particularly limited, and for example, any of ligands or receptor substances, radioisotopes, fluorescence, luminescence, and color development can be preferably used.

  In the method of the present invention, a method of hybridizing a probe having a labeling substance and a nucleic acid obtained by amplifying a target region and detecting a free probe after washing, or a so-called homogeneous detection method in which the washing step is omitted. Either can be suitably used. Examples of the homogeneous detection method using fluorescence include Proc. Natl. Acad. Sci. USA, vol. 85, p 8790-8794 (1988), Fluoresence resonance energy transfer (FRET) method, Nature Biotechnology, vol. 14, p 303-308 (1996), Molecular Beacons method, Anal. Chem. , Vol. 72, p3717-3724 (2000), and the like can be suitably used.

  As one aspect of the present invention, the 5 ′ end of the probe is labeled with a rhodamine-based or oxazine-based dye, the 3 ′ end is labeled with an oligonucleotide or a reporter dye that is a base sequence that causes quenching, and the probe Examples of the molecular beacon method or the smart probe method in which the 5 ′ end side and the 3 ′ end side are self-annealed.

  When the above-described probe is used and the nucleic acid amplified by the amplification method of the present invention is present, the probe hybridizes to the amplified nucleic acid, the self-annealing structure of the probe is eliminated, and as a result, the fluorescence intensity is suppressed. Without a fluorescence signal. On the other hand, when no amplified nucleic acid is present, the probe retains a self-annealing structure and the fluorescence intensity is suppressed, so that no fluorescence signal is detected.

  That is, in the case of the smart probe method, the fluorescent dye is preferably a rhodamine dye or an oxazine dye, and the dye is bound to the 5 ′ end of the probe and cooperates with the base sequence at the 3 ′ end to bind the fluorescent light to the target nucleic acid. The fluorescent intensity may be suppressed when the probe is not bound, and the fluorescent intensity may not be suppressed when the fluorescent probe is bound to the target nucleic acid.

  On the other hand, in the case of the molecular beacon method, the fluorescence intensity is suppressed by fluorescence resonance energy transfer when the fluorescent probe is not bound to the target nucleic acid, and the fluorescence intensity is not suppressed when bound. That's fine.

  As these fluorescent dyes, FAM (6-carboxy-fluorescein), TAMRA (6-carboxytetramethylrhodamine), JA242 (oxazine) and DABCYL (4-dimethylaminoazobenzene-4′-sulfonyl chloride) are preferable. . These fluorescent dyes are known and are commercially available.

  A method for fluorescently labeling oligonucleotides is described in, for example, Nuc. Acids Res. , Vol. 12, p3387-3403 (1984), J. MoI. Am. Chem. Soc. , Vol. 112, p1253-1254 (1990) or Anal. Chem. , Vol. 70, p4771-4779 (1998).

  The chain length of the probe of the present invention is not particularly limited, but is, for example, 12 mer or more, preferably 20 mer or more, particularly preferably 40 mer or more.

  The tuberculosis group detection probe of the present invention can detect Mycobacterium tuberculosis, Mycobacterium bovisBCG, Mycobacterium africanum, Mycobacterium microti and / or Mycobacterium cantetti. The probe is not particularly limited as long as the target nucleic acid is selected according to the nucleic acid to be detected or the organism. For example, in the detection of Mycobacterium tuberculosis, the IS6110 gene is used as the target nucleic acid. A probe can be used, and can be designed by selecting an appropriate region from the base sequence of the Mycobacterium tuberculosis-derived IS6110 gene shown in SEQ ID NO: 12 in the Sequence Listing. Although not particularly limited, for example, it is selected from the region containing the nucleotide sequence set forth in SEQ ID NO: 39 in the sequence listing, preferably from the region containing SEQ ID NO: 40 in the sequence listing, more preferably from the region containing SEQ ID NO: 38 in the sequence listing. can do. In particular, it can be selected from a region containing the base sequence described in SEQ ID NO: 11 in the sequence listing. The above probe is particularly useful for detection of the Mycobacterium tuberculosis group since it hybridizes stably and with high specificity to the IS6110 gene derived from Mycobacterium tuberculosis group or a fragment thereof.

  The probe for detecting a bacterium of the present invention can detect Neisseria gonorrhoeae. The probe is not particularly limited as long as the target nucleic acid is selected according to the nucleic acid to be detected or the organism to be detected. For example, in the detection of gonococci, a cryptoplasmid protein B (cppB) gene is used. A probe serving as a target nucleic acid can be used, and can be designed by selecting an appropriate region from the base sequence of the bacterium-derived cppB gene shown in SEQ ID NO: 27 in the sequence listing. Although not particularly limited, for example, it can be selected from a region containing the base sequence described in SEQ ID NO: 27 in the sequence listing, preferably from a region containing SEQ ID NO: 21 in the sequence listing. Since the above probe hybridizes stably and with high specificity to the cppB gene derived from Phosphorus or a fragment thereof, it is particularly useful for the detection of the gene, and further for the detection of Phosphorus.

  The chlamydia detection probe of the present invention can detect Chlamydia trachomatis. The probe is not particularly limited as long as the target nucleic acid is selected according to the nucleic acid to be detected or the organism, and for example, in the detection of chlamydia, cryptic plasmid pLGV440. Can be used, and can be designed by selecting an appropriate region from the base sequence of Chlamydia-derived pLGV440 shown in SEQ ID NO: 22 in the Sequence Listing. Although not particularly limited, for example, it is selected from the region containing the nucleotide sequence set forth in SEQ ID NO: 22 in the sequence listing, preferably from the region containing SEQ ID NO: 44 in the sequence listing, more preferably from the region containing SEQ ID NO: 20 in the sequence listing. can do. The above probe is particularly useful for detection of the gene and further detection of chlamydia since it hybridizes stably and highly specifically with pLGV440 derived from chlamydia or a fragment thereof.

  Further, the HCV detection probe of the present invention is not particularly limited as long as it is a probe that can hybridize to the base sequence in the region sandwiched between the forward primer and the reverse primer used for target nucleic acid amplification. Further, the length of the probe is not particularly limited, but is preferably selected in the range of 8 bases to 53 bases, particularly preferably in the range of 8 bases to 36 bases. For example, a probe having a base sequence of 8 bases or more continuous in the above range may be selected from the base sequence shown in SEQ ID NO: 43 in the sequence listing, and although not particularly limited, it is shown in SEQ ID NO: 34 or 35 in the sequence listing. And a probe having 8 or more consecutive bases in the base sequence.

(2) Detection method of the present invention The method for detecting a target nucleic acid of the present invention comprises the step of neutralizing the target nucleic acid from a double strand to a single strand by using the probe described in (1) above. Can be omitted. That is, in the method for detecting a target nucleic acid of the present invention, it is possible to hybridize with a nucleic acid having a sequence complementary to the base sequence under alkaline conditions where the pH exceeds 7, particularly preferably under alkaline conditions in the range of pH 8-14. It is. The hybridization conditions are not particularly limited, and examples of the “strict conditions” include those known to those skilled in the art. Such conditions are described in 1989 by Cold Spring Harbor Laboratory, T.W. Edited by T. Maniatis et al., Molecular Cloning: A Laboratory Manual Second Edition (Molecular Cloning: A Laboratory Manual 2nd ed.) And those described in the Examples of the present application can be used. .

  Exemplary hybridization solutions include the following: 0.5% SDS, 5 × Denhardt's; 0.1% bovine serum albumin (BSA), 0.1% polyvinyl pyrrolidone, 0.1 % Ficoll 400] and 6 × SSC containing 100 μg / ml salmon sperm DNA (1 × SSC is 0.15 M NaCl, 0.015 M sodium citrate). In the present invention, the pH of the hybridization solution may be adjusted in the range of 8 to 14.

  The hybridization temperature is not particularly limited, but is performed, for example, at a temperature lower by 5 ° C. or more than the Tm value of the probe.

Here, the Tm value of the probe is, for example, the following formula:
Tm = 81.5−16.6 (log ₁₀ [Na +]) + 0.41 (% G + C) − (600 / N)
(Where N is the chain length of the probe and% G + C is the content of guanine and cytosine residues in the probe)
Is required.

In addition, when the chain length of the probe is shorter than 18 bases, the Tm value is represented by the following formula:
Tm = (% A + T) × 2 + (% G + C) × 4]
(Where (% A + T) is the content of adenine and thymine residues in the probe, and (% G + C) is the content of guanine and cytosine residues in the probe)
Can be estimated as

  By confirming hybridization between the probe and the target nucleic acid under the above hybridization conditions, a probe suitable for detection of the target nucleic acid can be selected.

  The hybridization conditions in the detection method of the present invention are not particularly limited, and known hybridization conditions, preferably strict hybridization conditions are used. Moreover, you may adjust the pH of a hybridization solution to the range of 8-14.

  As one aspect of the method for detecting a target nucleic acid of the present invention, for example, in a method for detecting Mycobacterium tuberculosis group, a probe suitable for detecting IS6110 gene derived from Mycobacterium tuberculosis group can be preferably used, but is not particularly limited. For example, the base sequence shown in SEQ ID NO: 39 or a part thereof, preferably the base sequence shown in SEQ ID NO: 40 or a part thereof, more preferably the base sequence shown in SEQ ID NO: 38 or a sequence thereof Some, particularly preferably, a probe containing SEQ ID NO: 11 in the sequence listing can be suitably used in the present invention. In addition, for example, in a method for detecting a bacterium, a probe suitable for detection of a cppB gene derived from a bacterium can be suitably used. Although not particularly limited, for example, the nucleotide sequence described in SEQ ID NO: 27 in the sequence listing or A probe containing a part thereof, preferably the base sequence described in SEQ ID NO: 21 in the sequence listing or a part thereof can be suitably used in the present invention. Further, in the detection method for chlamydia, a probe suitable for detection of pLGV440 derived from chlamydia can be suitably used, and is not particularly limited. For example, the base sequence described in SEQ ID NO: 22 in the sequence listing or a part thereof, Preferably, a probe containing the base sequence described in SEQ ID NO: 44 or a part thereof in the sequence listing, and more preferably a probe containing the base sequence described in SEQ ID NO: 20 in the sequence listing or a portion thereof can be suitably used in the present invention. Furthermore, in the method for detecting HCV, although not particularly limited, a 5 ′ untranslated region is suitable. For example, the base sequence described in SEQ ID NO: 43 in the sequence listing or a part thereof, preferably the sequence in the sequence listing A probe containing the base sequence described in No. 34 or a part thereof, or the base sequence shown in SEQ ID No. 35 or a part thereof can be preferably used in the present invention.

  The pathogenic microorganism detection method of the present invention can specifically detect individual pathogenic microorganisms by using the probe described in (1) above. That is, since the above probe hybridizes stably and with high specificity to a gene derived from each pathogenic microorganism or a fragment thereof, it is particularly useful for detecting the gene and further detecting each pathogenic microorganism.

  Furthermore, in the detection method of the present invention, by using the probe described in (1) above, between the probe and the sample containing the nucleic acid under alkaline conditions exceeding pH 7, preferably pH 8 to 14, particularly preferably Hybridization can be performed under conditions of pH 8-10. That is, a method for detecting a gene derived from a pathogenic microorganism or a fragment thereof without the neutralization step following the target nucleic acid denaturation step is provided by this method, and the method detects M. tuberculosis contained in a specimen. can do.

  The method of the present invention can be used for detection and quantification of a specific gene present in a sample. That is, a specific gene can be detected and quantified from any sample that may contain a nucleic acid such as DNA or RNA. Examples of the sample include, but are not limited to, whole blood, serum, buffy coat, urine, feces, cerebrospinal fluid, semen, saliva, tissue (eg, cancer tissue, lymph node, etc.), cell culture ( Biological samples such as mammalian cell cultures and bacterial cultures), samples that may be contaminated or infected with microorganisms such as viruses or bacteria (food, biological preparations, etc.), or Specific genes can be detected and quantified from samples that may contain organisms such as soil and wastewater. Further, for example, it can be used for the detection and quantification of the above-mentioned pathogenic microorganisms depending on the presence or absence of the target nucleic acid. In particular, the method for detecting pathogenic microorganisms is useful in the hygiene and environmental fields. As the nucleic acid used as a template for the detection method, either RNA or DNA can be preferably used.

  The preparation of the nucleic acid-containing preparation from these materials is not particularly limited, and can be performed, for example, by dissolution treatment with a surfactant, ultrasonic treatment, shaking and stirring using glass beads, use of a French press, or the like. In some instances, it may be advantageous to further manipulate and purify the nucleic acid (eg, when an endogenous nuclease is present). In these examples, nucleic acid purification is performed by known methods such as phenol extraction, chromatography, ion exchange, gel electrophoresis, or density gradient centrifugation.

  When targeting a nucleic acid having an RNA-derived sequence, the method of the present invention may be carried out using cDNA synthesized by reverse transcription using the RNA as a template as a template.

  The primer used for the reverse transcription reaction is not particularly limited as long as it anneals to the template RNA under the reaction conditions used. The primer may be a primer having a base sequence complementary to a specific template RNA (specific primer), an oligo dT (deoxythymine) primer, or a primer having a random sequence (random primer). The length of the primer for reverse transcription is preferably 6 nucleotides or more, more preferably 9 nucleotides or more from the viewpoint of specific annealing, and preferably 100 nucleotides or less from the viewpoint of oligonucleotide synthesis. More preferably, it is 30 nucleotides or less. Furthermore, as a primer for reverse transcription, a chimeric oligonucleotide primer that can be used as a primer for a strand displacement reaction when performing the nucleic acid amplification method of the present invention using cDNA after reverse transcription as a template can be used. Such a primer is not particularly limited as long as it has the properties described in (3) below and can be used for reverse transcription reaction from RNA. For example, SEQ ID NOs: 32 and 33 in the Sequence Listing are used. Primers having the base sequences described can be preferably used.

  The enzyme used in the reverse transcription reaction is not particularly limited as long as it has cDNA synthesis activity using RNA as a template. For example, avian myeloblastosis virus-derived reverse transcriptase (AMV RTase), Moloney murine Examples include reverse transcriptases of various origins such as leukemia virus-derived reverse transcriptase (MMLV RTase) and rous-associated virus 2 reverse transcriptase (RAV-2 RTase). In addition, a DNA polymerase having reverse transcription activity can also be used. For the purpose of the present invention, an enzyme having reverse transcription activity at high temperature is suitable. For example, a DNA polymerase derived from a Thermus bacterium (Tth DNA polymerase or the like), a DNA polymerase derived from a thermophilic Bacillus bacterium or the like can be used. . Although not particularly limited, for example, a DNA polymerase derived from a thermophilic Bacillus bacterium is preferable. St-derived DNA polymerase (Bst DNA polymerase) and Bca DNA polymerase are preferred. For example, Bca DNA polymerase does not require manganese ions for reverse transcription reaction, and can synthesize cDNA while suppressing secondary structure formation of template RNA under high temperature conditions. As for the enzyme having the above-mentioned reverse transcriptase activity, either a natural body or a mutant can be used as long as it has the activity.

  Any of double-stranded DNA such as genomic DNA and PCR fragments isolated by the above method and single-stranded DNA such as cDNA prepared by reverse transcription from total RNA or mRNA can be used in the present invention. It can be suitably used as a template DNA in the nucleic acid amplification method. In the case of the above double-stranded DNA, one that has been subjected to a step (denature) for denaturation into single-stranded DNA can be suitably used.

  As described above, a region on the target nucleic acid that hybridizes with the probe of the present invention can be amplified by an appropriate nucleic acid amplification method and used for hybridization. The nucleic acid amplification method to be used is not particularly limited as long as it can amplify a region on the target nucleic acid. For example, polymerase chain reaction (PCR; polymerase chain reaction, US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), ligase chain reaction (LCR), strand displacement amplification (SDA), strand displacement amplification, No. 7-11718), self-sustained sequence replication (3SR), nucleic acid sequence amplification method (NASBA method; nucleic acid sequence based amplification, Patent No. 2650159), TMA method (transcription-mediated amplification), Qβ replicase method (Patent No. 2710159), ICAN method (Isothermal and Chimeric primer-initiated Amplification of Nucleic acids, International Publication No. 00/56877 pamphlet) and the like can be used.

  The ICAN method is a method of continuously amplifying DNA having a specific base sequence on a template nucleic acid under isothermal conditions using a chimeric nucleotide primer, an endonuclease, and a DNA polymerase having strand displacement activity.

  Here, “continuously” means that the reaction proceeds without changing the reaction temperature and the composition of the reaction solution. In the present specification, “isothermal” means a substantially constant temperature condition in which the enzyme and the nucleic acid chain function in each of the above steps.

Usually, the chimeric oligonucleotide primer is composed of deoxyribonucleotide and ribonucleotide, and ribonucleotide is arranged at the 3 ′ end or 3 ′ end side. For example, an oligonucleotide having a structure represented by the following general formula can be used as a primer for the ICAN method.
General formula: 5'-dNa-Nb-dNc-3 '
(A: an integer of 11 or more, b: an integer of 1 or more, c: 0 or an integer of 1 or more, dN: deoxyribonucleotide and / or nucleotide analog, N: unmodified ribonucleotide and / or modified ribonucleotide, dNa A part of dN may be substituted with N)

  For example, deoxyriboinosine nucleotides can be used as the nucleotide analogs, and (α-S) ribonucleotides can be used as the modified ribonucleotides. The nucleotide analog can be contained as long as the function as a primer is not substantially lost.

  The primer has a sequence homologous to the 5′-side base sequence based on the 5′-side or 3′-side base sequence of the region desired to be amplified on the template, and is complementary to the 3′-side base sequence. Two kinds of typical ones are created and used for amplification.

The endonuclease used in the present invention acts on a double-stranded DNA generated by extending the DNA from the above-described chimeric oligonucleotide primer annealed to a template nucleic acid, so that a strand displacement reaction occurs. Any material capable of cleaving the chain may be used. That is, it is an enzyme that can generate a nick in the chimeric oligonucleotide primer portion of the above double-stranded DNA. Although not particularly limited, for example, ribonuclease can be used in the present invention, and in particular, endoribonuclease H (RNase H) acting on the RNA portion of a double-stranded nucleic acid formed from DNA and RNA can be preferably used. . Moreover, as long as it has the said effect | action as this ribonuclease, all of normal temperature to heat-resistant ribonuclease can be used for this invention suitably. For example, as shown in the Examples below, RNase H derived from E. coli can be used in the method of the present invention in a reaction at about 50 ° C. to about 70 ° C. In the method of the present invention, a heat-resistant ribonuclease can also be preferably used. The thermostable ribonuclease is not particularly limited. For example, in addition to commercially available Hybridase ^™ Thermostable RNase H (manufactured by Epicenter Technologies), thermophilic Bacillus bacteria, Thermus bacteria, Pyrococcus bacteria, Thermotoga bacteria, Alkae RNase H and the like derived from bacteria belonging to the genus Oglobus and Methanococcus can also be preferably used. Furthermore, as the ribonuclease, any of natural products and mutants can be preferably used. In addition, the enzyme unit of RNaseH described in this specification is a numerical value displayed based on the enzyme unit measurement method shown in the reference examples in the examples.

  The RNase H is not particularly limited as long as it can be used in the method of the present invention. For example, any of various viruses, phages, prokaryotes, and eukaryotes may be used. Further, it may be either cellular RNase H or viral RNase H. For example, E. coli RNaseHI is exemplified as the cellular RNaseH, and HIV-1-derived RNaseH is exemplified as the viral RNaseH. In the method of the present invention, RNase H can be any of type I, type II, and type III. Although not particularly limited, for example, RNaseHI derived from Escherichia coli, Pyrococcus bacteria or RNaseHII derived from Alkaeoglobus bacteria can be suitably used.

  In addition, the efficiency of the cleavage reaction of the endonuclease used in the method of the present invention, for example, RNase H, depends on the base sequence near the 3 ′ end of the primer, and it is considered that the amplification efficiency of the desired DNA is affected. It is natural to design an optimal primer for the RNase H to be used.

  In the ICAN method, a DNA polymerase having an activity of performing strand displacement of DNA is used. Those having substantially no 5 '→ 3' exonuclease activity are particularly preferably used.

  The DNA polymerase used in the ICAN method is not particularly limited as long as it has the above-described strand displacement activity. For example, Bacillus caldotenax (hereinafter referred to as B. ca) or Bacillus stearothermophilus. A mutant lacking the 5 ′ → 3 ′ exonuclease activity of a thermophilic Bacillus bacterium-derived DNA polymerase such as Bacillus stearothermophilus (hereinafter referred to as “B.st”), or a large fragment of Escherichia coli-derived DNA polymerase I (Klenow fragment) Etc. In addition, as the DNA polymerase that can be used in the present invention, any of room temperature to heat resistant can be suitably used. The enzyme may be either purified and obtained from its original origin or a recombinant protein produced by genetic engineering. In addition, the enzyme may be modified by substitution, deletion, addition, insertion or the like by genetic engineering or other methods. Examples of such an enzyme include 5 ′ → 3 ′ exonuclease. Examples include BcaBEST DNA polymerase (Takara Shuzo Co., Ltd.) which is a Bca DNA polymerase deficient in activity. The enzyme exhibits activity at 50 ° C. to 70 ° C. and can be suitably used in the method.

Some DNA polymerases are known to have endonuclease activity, for example, RNase H activity, under specific conditions. Such a DNA polymerase can be used in the method of the present invention. That is, there is an embodiment in which the DNA polymerase is used under conditions such that RNase H activity is expressed, for example, in the presence of Mn ²⁺ . In this embodiment, the method of the present invention can be carried out without adding the RNase H. That is, the above-mentioned Bca DNA polymerase can exhibit RNase H activity in a buffer containing Mn ²⁺ , and the above embodiment is not limited to Bca DNA polymerase, and is known to have RNase H activity. Known DNA polymerases such as Tth DNA polymerase from Thermus thermophilus can also be used in the present invention.

  In the ICAN method, a chimeric oligonucleotide primer, a sample containing a nucleic acid serving as a template, endonuclease, DNA polymerase, deoxyribonucleotide triphosphate (dNTP) and the like are mixed in a buffer solution having a composition that allows the enzyme to exhibit activity, The reaction is carried out by incubating the reaction solution at an appropriate temperature for a time sufficient to produce a reaction product.

  Prior to the reaction, the chimeric oligonucleotide primer and the template nucleic acid are mixed, held at a temperature at which the double-stranded nucleic acid is denatured, for example, at 90 ° C. or higher, and then below the reaction temperature used in the method of the present invention. Although annealing may be performed by cooling, this is not an essential operation for the amplification reaction.

  In the detection method of the present invention, when RNA is used as a template, the reverse transcription reaction and the nucleic acid amplification reaction may be performed in one step. Although not particularly limited, for example, a combination of reverse transcriptase and strand displacement DNA polymerase can be suitably used, for example, AMV RTase, MMLV RTase, or a combination of RAV-2 RTase and Bca DNA polymerase. Furthermore, one kind of DNA polymerase having reverse transcriptase activity and strand displacement activity may be used.

  The target nucleic acid detection method of the present invention can be carried out by directly amplifying a target nucleic acid from a sample containing the nucleic acid. In this case, the length of the target nucleic acid to be amplified is not particularly limited, but from the viewpoint of detecting the target nucleic acid with high sensitivity, for example, a region of 200 bp or less, more preferably 150 bp or less is effective. By setting the chimeric oligonucleotide primer of the present invention so as to have the amplified chain length, the target nucleic acid in the sample can be detected with high sensitivity.

  Furthermore, in the detection method of the present invention, a reaction buffer containing bicine, tricine, hepes, phosphate or tris buffer components, and an annealing solution containing spermidine or propylenediamine are used to further increase the amount of nucleic acid samples. The target nucleic acid can be detected with high sensitivity. In this case, the endonuclease and DNA polymerase to be used are not particularly limited. For example, a combination of RNase H derived from Escherichia coli, Pyrococcus bacteria or Alcaeoglobus bacteria and BcaBEST DNA polymerase is preferable. In particular, it is expected that the number of units that can be suitably used differs depending on the type of the endonuclease and the DNA polymerase. In this case, using the improvement in detection sensitivity or the increase in the amount of amplified product as an index, What is necessary is just to adjust a composition and the addition amount of an enzyme.

  In addition, in the nucleic acid amplification method using a double-stranded template DNA and two chimeric oligonucleotide primers, depending on the reaction conditions, etc., each primer-extension chain intermediate during the extension reaction is used. Template exchanges may occur between the bodies, producing double-stranded nucleic acids in which the synthesized primer extension strands are annealed. This double-stranded nucleic acid has a chimeric oligonucleotide primer at both ends, and then a complementary strand extension reaction can be started again from both ends by strand displacement. As a result of this reaction, an amplification product having a primer sequence at one end is generated. Furthermore, when the template exchange occurs during this reaction, the same double-stranded nucleic acid as described above is generated again.

  In the present invention, a nucleic acid amplification method including a step of performing a template exchange reaction using a DNA polymerase having the above strand displacement activity can be used.

  As a DNA polymerase having the ability to perform the above-described template exchange reaction during the strand displacement reaction, for example, a Bca DNA polymerase mutant enzyme lacking 5 '→ 3' exonuclease activity is particularly preferably used. The enzyme is commercially available as BcaBEST DNA polymerase (Takara Shuzo Co., Ltd.), and Escherichia coli HB101 / pUI205 (FERM BP-3720, Tsukuba, Ibaraki, Japan, 1-1-1 Higashi, Tsukuba, Japan, which contains the gene for the enzyme. Prepared by the method described in Japanese Patent No. 2978001 from Chuo No. 6 (zip code 305-8666), National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center on May 10, 1991 (original deposit date). You can also

  In the detection method of the present invention, dUTP can be incorporated as a substrate during amplification of the target nucleic acid. Therefore, when dUTP is used as a substrate, the amplified product can be degraded using uracil N-glycosidase (UNG), and false positives due to carry-over contamination of the amplified product can be prevented. .

  In order to detect the target nucleic acid amplified by the nucleic acid amplification method in the detection method of the present invention, a known nucleic acid detection method, for example, a method of detecting a reaction product of a specific size by electrophoresis, or by hybridization with a probe Detection or the like can be used. Furthermore, a detection method combining magnetic beads or the like can also be suitably used. For the detection by electrophoresis, a fluorescent substance such as ethidium bromide is usually used, but hybridization with a probe may be combined. In addition to labeling with a radioisotope, a probe with a non-radioactive label such as biotin or a fluorescent substance can be used. In addition, by using a labeled nucleotide in the above step (b), it is possible to facilitate the detection by incorporating the labeled nucleotide into the amplification product and to enhance the detection signal using the label. Detection using fluorescence polarization, fluorescence energy transfer, or the like is also possible. Furthermore, by constructing an appropriate detection system, it is possible to automatically detect the target nucleic acid or to quantify the target nucleic acid. Moreover, the naked eye detection method by a hybrid chromatography method can also be used conveniently.

  The detection method of the present invention is any of a ribonucleotide (RNA) probe labeled with two or more kinds of fluorescent substances arranged at such a distance as to be quenched, or a chimeric oligonucleotide probe composed of ribonucleotide and deoxyribonucleotide. Can be used for The probe does not emit fluorescence, but RNase H degrades the probe when annealed to amplified DNA derived from a target nucleic acid complementary thereto. As a result, the distance between the fluorescent substances on the probe increases and fluorescence is emitted, and the presence of the target nucleic acid can be known. When the nucleic acid amplification method of the present invention is carried out using RNase H, the target nucleic acid can be detected simply by adding the probe to the reaction solution. As a fluorescent substance used for labeling the probe, for example, a combination of 6-FAM (6-carboxyfluorescein) and TAMRA (N, N, N ′, N′-tetramethyl-6-carboxyrhodamine) can be preferably used. .

  When the isothermal amplification method is used in the detection method of the present invention, an apparatus such as a thermal cycler is not required. Further, in the amplification method of the present invention, one or two primers can be used and less than the conventional method. Since a reagent such as dNTP used in PCR or the like can be used, the running cost can be reduced as compared with the conventional method. Therefore, it can be suitably used in fields such as genetic testing where routine work is performed. Furthermore, the method of the present invention can be used as a gene detection method that is simple, rapid, and highly sensitive because many amplification products can be obtained in a shorter time than the PCR method.

  In the case of using the detection method of the present invention, in order to analyze a large amount of specimen, a reaction system may be miniaturized and further combined with means for increasing the degree of integration. As one of the means, using the most advanced ultra-fine processing technology, the basic process of the detection method of the present invention, such as extraction of DNA from cells, nucleic acid amplification reaction, detection of target DNA, etc. You may combine what was integrated on the microchip of cm square-fingertip size. Furthermore, if necessary, a gel or capillary electrophoresis, or a hybridization process with a detection probe may be combined. The system is also called a microchip, a micro CE (capillary electrophoresis) chip, or a nanochip.

  As the nucleic acid amplification reaction in such a system, any nucleic acid amplification reaction can be used as long as the target DNA fragment can be amplified. Although not particularly limited, for example, a method capable of amplifying a nucleic acid under isothermal conditions such as the ICAN method can be suitably used. Combining the methods makes it possible to simplify the system and is very suitable for use in an integrated system as described above. Furthermore, it is possible to construct a system with a higher degree of integration using the technology of the present invention.

  In the detection method of the present invention, although not particularly limited, for example, a chimeric oligonucleotide primer for detecting Mycobacterium tuberculosis group having base sequences represented by SEQ ID NOs: 13 to 16 in the sequence listing, SEQ ID NOs: 28 to 29 in the sequence listing, respectively. Chimeric oligonucleotide primers for detection of phosphorous bacteria each having a base sequence represented by SEQ ID NO: 23, Chimeric oligonucleotide primers for detection of chlamydia having base sequences represented by SEQ ID NOs: 23 to 26 of the sequence listing, SEQ ID NO: 30 to Chimeric oligonucleotide primers for detecting HCV each having a nucleic acid sequence represented by 31 can be preferably used. A target nucleic acid in a sample can be amplified using the chimeric oligonucleotide primer, and the target amplification product can be confirmed by electrophoresis or real-time detection.

  Furthermore, in the detection method of the present invention, a tuberculosis group detection probe selected from regions containing the nucleotide sequences represented by SEQ ID NOs: 11, 12, and 38 to 40 in the sequence listing, and SEQ ID NO: 27 in the sequence listing A probe for detecting a bacterium selected from a region containing a base sequence represented, a probe for detecting chlamydia selected from a region containing a base sequence represented by SEQ ID NOs: 22 and 44 of the sequence listing, The target nucleic acid can be detected using an HCV detection probe selected from the region containing the base sequences represented by SEQ ID NOs: 43 to 45, respectively.

  When the isothermal nucleic acid amplification method is used in the method of the present invention, a necessary reaction apparatus may be a constant temperature layer, and a strict apparatus such as a thermal cycler is not necessary. As a device for measuring the label signal, a commercially available spectrophotometer, a fluorescence detector, a plate reader, or the like can be used. Therefore, it is possible to measure a large amount of samples quickly in a place where a normal inspection operation is performed. Furthermore, reagents necessary for the method of the present invention are also commercially available.

  When HCV is detected using the method of the present invention, HCV RNA extracted from a sample by a conventional method, a primer for reverse transcription reaction, reverse transcriptase (reverse transcription when using a DNA polymerase having reverse transcription activity) No enzyme is required), a dNTP mixed solution containing dATP, dGTP, dCTP, and dTTP is prepared. Using the cDNA generated from HCV-RNA as a template at a constant temperature, the target nucleic acid is prepared with the above-mentioned chimeric oligonucleotide primer and ICAN DNA polymerase. Amplification is performed, and this is hybridized with the above-described fluorescently labeled probe in a homogeneous system, and the fluorescence intensity is measured. Specific conditions for the reaction are detailed in the examples below.

  In the reaction, cDNA is first synthesized using HCV-RNA as a template, and then DNA is amplified by the ICAN method using this cDNA as a template. Since the amplified DNA includes a region complementary to the fluorescent probe, the fluorescent probe hybridizes to the amplified DNA in a single-stranded state. When the fluorescent probe is hybridized, the suppression of the fluorescence intensity is released and the fluorescence intensity increases. On the other hand, when no HCV RNA is present in the sample, hybridization does not occur, and the fluorescence intensity is suppressed and the fluorescence intensity is weak. Therefore, by measuring the fluorescence intensity, HCV RNA in the sample can be detected simply, rapidly and with high sensitivity.

  In the method of the present invention, RNA of HCV is measured as fluorescence intensity without a washing step in a homogeneous system. The present invention is advantageous in that it can cope with multi-item simultaneous measurement by changing the fluorescent dye and changing the measurement wavelength. In other words, not only HCV but also HBV, HIV, and HTLV are important test items at the blood center, and specific ICAN primers and probes are prepared, and each probe is labeled with a fluorescent dye having a different measurement wavelength. The above items can be measured simultaneously. In addition, measuring fluorescence intensity at the reaction end point enables high-sensitivity measurement of a large amount of specimens such as blood centers as a qualitative measurement, while kinetic fluorescence measurement of the reaction as a quantitative measurement for treatment monitoring Quantitative measurement is also possible. Therefore, according to the method of the present invention, there is no need for complicated and special equipment for raising and lowering the temperature as in the conventional RT-PCR method, and the inconvenience that a large amount of specimens cannot be measured in the facility for a limited time is improved. The cleaning process is unnecessary and is very convenient.

(3) Primer of the present invention The primer used in the detection method of the present invention is exemplified by a chimeric oligonucleotide primer. The primer is composed of deoxyribonucleotides, nucleotide analogs, unmodified or modified ribonucleotides, and ribonucleotides arranged on the 3 ′ end or 3 ′ end side thereof. For example, an oligonucleotide having a structure represented by the following general formula can be used as a primer for the ICAN method.
General formula: 5'-dNa-Nb-dNc-3 '
(A: an integer of 11 or more, b: an integer of 1 or more, c: 0 or an integer of 1 or more, dN: deoxyribonucleotide and / or nucleotide analog, N: unmodified ribonucleotide and / or modified ribonucleotide, dNa A part of dN may be substituted with N)

  As the above-mentioned nucleotide analogs, for example, deoxyriboinosine nucleotides, deoxyribouracil nucleotides or modified deoxyribonucleotides can be used, and as modified ribonucleotides, for example, (α-S) ribonucleotides can be used. .

  The primer has a sequence homologous to the 5′-side base sequence based on the 5′-side or 3′-side base sequence of the region desired to be amplified on the template, and is complementary to the 3′-side base sequence. Two kinds of typical ones are created and used for amplification.

  In the present invention, for example, a nucleic acid amplification reaction according to the above-described method is performed on a sample for detecting the Mycobacterium tuberculosis group, and then hybridization between the amplified product and the probe of the present invention is performed to obtain the Mycobacterium tuberculosis group. Can be detected. Primers for nucleic acid amplification can be designed and produced by referring to the base sequence of the Mycobacterium tuberculosis group IS6110 gene shown in SEQ ID NO: 12 in the sequence listing, and suitable for use in various nucleic acid amplification methods. . Although not particularly limited, for example, a region containing the base sequence shown in SEQ ID NO: 39 or a part thereof in the IS6110 gene, preferably a region containing the base sequence shown in SEQ ID NO: 40 or a part thereof More preferably, one capable of amplifying a region containing the base sequence shown in SEQ ID NO: 38 of the sequence listing or a part thereof is suitable for the present invention. Furthermore, what can amplify the area | region containing the base sequence of sequence number 11 of a sequence table is preferable.

  Furthermore, the primer of the present invention is particularly preferable by analyzing a polymorphism of the previously reported base sequence of Mycobacterium tuberculosis group IS6110 from the GenBank gene database and examining closely related sequences homologous to Mycobacterium tuberculosis group IS6110. Can be used for highly sensitive and quantitative nucleic acid amplification when nucleic acid amplification primers having the nucleotide sequences shown in SEQ ID NOs: 36 and 37 in the sequence listing are used. Therefore, the primer is useful for amplifying Mycobacterium tuberculosis group IS6110 gene or a fragment thereof by various nucleic acid amplification methods.

  In the present invention, as in the detection of the Mycobacterium tuberculosis group, the detection can be suitably performed by using a probe for detecting bacterium, chlamydia, and HCV, and a chimeric oligonucleotide primer.

  The primer is not limited to the above-described base sequence, and a primer having a sequence around the base sequence, that is, a part of the sequence overlapping with the base sequence can be suitably used in the present invention. That is, a primer can be prepared by selecting an appropriate base sequence overlapping with the sequence according to the characteristics of the nucleic acid amplification method to be used.

  These chimeric oligonucleotide primers can be synthesized by the phosphoamidite method using, for example, a DNA synthesizer type 394 of Applied Biosystems Inc. (ABI, Applied Biosystems Inc.) so as to have an arbitrary nucleic acid sequence. As another method, there are a phosphoric acid triester method, an H-phosphonate method, a thiophosphonate method, and the like, but they may be synthesized by any method.

  As a primer for performing nucleic acid amplification by the ICAN method, the deoxyribonucleotide at the 3 'terminal portion of the primer may be substituted with a ribonucleotide so that it can be used in the method. As an example of such a primer, chimera oligonucleotide primer which shows a base sequence in sequence number 13-16, 23-26, 28-31 of a sequence table, respectively is mentioned.

  Further, these primers may be labeled with an appropriate substance, such as a fluorescent substance, a dye, a ligand (biotin, digoxigenin, etc.), or a gold colloid, for example, by using a primer labeled with a ligand. Thus, there is provided a measurement method having high sensitivity and simple quantitativeness, such as capturing the amplified target nucleic acid on a carrier. In this case, examples of the solid phase include microtiter plates, beads, magnetic beads, membranes, and glass.

  Extracted DNA samples from sputum specimens contain not only human DNA but also DNA derived from airway resident bacteria and viruses. Therefore, in order to examine the specificity of the primer developed in the present invention, an attempt was made to amplify the Mycobacterium tuberculosis group gene using a sputum from a volunteer with no history of infection with M. tuberculosis as a specimen. The lack of a positive result in such a test can be evidence of the specificity of the primer. The IS6110 gene fragment was amplified by the ICAN method using the above-mentioned chimeric oligonucleotide primer on DNA extracted from sputum collected from 38 volunteers. As a result, no positive results were observed, demonstrating the high specificity of the above-mentioned primers for Mycobacterium tuberculosis, indicating that non-IS6110 target DNA was not made false positive.

(4) Composition of this invention This invention provides the composition used for the detection method of this invention of the above-mentioned (2). Examples of the composition include those containing the probe described in (1) above and the primer described in (3) above. Or the form of the composition for a detection containing the said probe and the composition for amplification containing the said primer may be sufficient. Further, it may be in the form of a composition containing magnesium acetate and a composition containing a primer. Furthermore, a buffer component, dNTP, etc. may be included. Further, it may contain a modified deoxyribonucleotide or deoxynucleotide triphosphate analog. Furthermore, an internal standard (IC; internal control) for confirming the non-amplification status (false negative) due to decomposition / deactivation of the reagent in the composition of the present invention may be contained. The internal standard can be amplified with the primers of the present invention.

  Another embodiment includes a composition suitable for the detection method of the present invention and containing the above-mentioned various components. The composition can be obtained by adding an appropriate template and a chimeric oligonucleotide primer. An amplification reaction can be performed. Furthermore, when the amplification target is clear in advance, a composition containing a chimeric oligonucleotide primer suitable for amplification of the amplification target is preferable.

  As a particularly preferred embodiment, a composition suitable for the nucleic acid amplification method of the present invention and containing the above-mentioned various components can be mentioned, and the composition performs an amplification reaction only by adding an appropriate template. can do. Furthermore, when a detection probe is included, a target nucleic acid derived from a pathogenic microorganism can be detected in real time.

  Although not particularly limited, for example, by using the method of the present invention, a composition or a kit for the method in detecting HCV, the conventional amplicore HCV kit has a measurement range of 2 orders, compared to 3 orders. The measurement range can be expanded up to. Also, in the conventional amplicore HCV kit, the required time can be significantly reduced to about 45 minutes in the present invention, compared to the required time of about 5 hours. Therefore, it is possible to increase the number of samples that can be processed per day than before.

(5) Kit of the present invention The kit of the present invention is a kit for detecting a target nucleic acid derived from a pathogenic microorganism, and is not particularly limited, but a probe capable of hybridizing with a target nucleic acid under alkaline conditions exceeding pH 7, Although not particularly limited, for example, the probe contains the probe described in (1) above.

  One embodiment of the kit of the present invention includes a kit for detecting Mycobacterium tuberculosis group. The kit contains a probe capable of specifically detecting Mycobacterium tuberculosis, Mycobacterium bovis BCG, Mycobacterium africanum, Mycobacterium microtti and / or Mycobacterium canti. The kit can also be used for detection of Mycobacterium tuberculosis under alkaline conditions exceeding pH 7.

  One embodiment of the kit of the present invention includes a kit for detecting phosphorus bacteria. The kit is characterized by containing a probe capable of specifically detecting Neisseria gonorrhoeae. The kit can also be used for detection of phosphorous bacteria under alkaline conditions exceeding pH 7.

  One embodiment of the kit of the present invention includes a kit for detecting chlamydia. The kit is characterized by containing a probe capable of specifically detecting Chlamydia trachomatis. The kit can also be used for the detection of chlamydia under alkaline conditions above pH 7.

  One embodiment of the kit of the present invention includes a kit for detecting HCV. The kit is characterized by containing a probe capable of specifically detecting HCV. The kit can also be used for detection of HCV under alkaline conditions exceeding pH7.

  Furthermore, in the said kit, you may contain a primer as another embodiment. Furthermore, it may be a kit containing a reagent for nucleic acid amplification method (a DNA polymerase or the like) for amplifying a target gene, a reagent used for probe detection reaction, a reagent used for nucleic acid extraction from a specimen, etc. . The kit is suitable for carrying out the method for detecting the Mycobacterium tuberculosis group and the like of the present invention simply and quickly. This kit can give a test result with high reproducibility and reliability, particularly when examining the presence of M. tuberculosis group or the like for a large number of samples. Furthermore, an internal standard (IC; internal control) for false negative confirmation may be included, and a probe for detecting the internal standard. The internal standard can be amplified with the primers of the present invention.

  In a further embodiment, the kit is characterized in that it contains instructions for use of the probes, primers, DNA polymerase and endonuclease of the invention in packaged form. In addition, commercially available DNA polymerases and / or endonucleases may be selected and used according to the instructions. Furthermore, a reagent for reverse transcription reaction using RNA as a template may be included. The DNA polymerase can be selected from the DNA polymerases described above. The endonuclease can be selected from either Pfu-derived RNase HII or Afu-derived RNase HII.

  The above “instructions” are printed materials that describe how to use the kit, for example, how to prepare a reagent solution for strand displacement reaction, recommended reaction conditions, etc. Including labels attached to, packages written in kits, etc. Furthermore, information disclosed and provided through an electronic medium such as the Internet is also included.

  The kit of the present invention may contain a reaction buffer containing bicine, tricine, hepes, phosphate, or a tris buffer component, and an annealing solution. Moreover, DNA polymerase and RNaseH may be contained. Further, it may contain a modified deoxyribonucleotide or deoxynucleotide triphosphate analog.

  In the kit of the present invention, a kit capable of detecting a target nucleic acid derived from a pathogenic microorganism in real time by appropriately selecting a detection probe is provided.

(6) Nucleic acid extraction method of the present invention The present invention provides a nucleic acid extraction method for detecting Mycobacterium tuberculosis or the like with high sensitivity. In other words, nucleic acid extraction from clinical specimens is a delicate and difficult process, such as liquid specimens such as urine, pleural effusion, cerebrospinal fluid, blood, thick viscous specimens such as pus and sputum, and cells and tissues. The scope is wide. At present, there is no standard method for extracting a gene such as Mycobacterium tuberculosis from these specimens, and therefore, various nucleic acid extraction methods are used in reports on genetic tests such as Mycobacterium tuberculosis reported to date. In these conventional methods, mixing is performed in a reagent containing various surfactants, alkalis, organic solvents, or chaotropic reagents, or sonication is performed in the presence of glass beads.

  The present inventors have made various mutanolysin from the above-mentioned various conventional methods and muramidase (muramidase, actinomycete globisporus), which has been used for nucleic acid extraction from the genus Listeria, Lactobacillus, and Streptococcus. We compared the method of using Sigma for M. tuberculosis, and found that the method of digesting M. tuberculosis was the best. Moreover, it discovered that the further excellent result was obtained by boiling the microbial cell digested with this enzyme for 5 minutes.

  A sample containing M. tuberculosis was treated with muramidase for 30 minutes, then treated at 96 ° C. for 5 minutes, and then the gene was amplified by the ICAN method to try to detect M. tuberculosis. As a result, surprisingly, it is possible to detect Mycobacterium tuberculosis DNA with a sensitivity 10 times higher than that when using a reagent such as a conventional surfactant, which has a detection sensitivity corresponding to 5 genome copies of Mycobacterium tuberculosis. I understood that there was.

  The series of detection operations described above can eliminate the inhibitory effect on the nucleic acid amplification reaction of components mixed in the nucleic acid extract in the conventional method using a surfactant or chaotropic reagent. Furthermore, by adding heat treatment, a bactericidal effect against M. tuberculosis and the like is also born, and there is an advantage that an effect of preventing infection such as M. tuberculosis in a laboratory that has been troubled by inspectors has been produced. Therefore, the invention of this protocol can rapidly extract DNA such as Mycobacterium tuberculosis with high sensitivity.

  For example, when using cocoon as a sample, first, the cocoon is treated by the NALC (N-acetyl-L-cysteine) -NaOH method, which is a known cocoon treatment method, and then the above-described nucleic acid extraction method of the present invention is performed. It is preferable to do. That is, the detection method of the present invention may include a step of treating a sample containing M. tuberculosis or the like with muramidase and extracting a nucleic acid.

  All the protocols used in the detection method of the present invention, that is, the probe, primer, nucleic acid extraction method, and nucleic acid amplification method, enable extremely fast, reliable, and highly sensitive detection of Mycobacterium tuberculosis. To do. Using this method, sample collection and result reporting can be completed within 3 hours, and the test time is reduced by half compared to conventional kits that require 6 hours (for example, Roche kits). It was done. In other words, the method of the present invention can provide test results on the same day, enables the isolation of infected patients as soon as possible, and brings about a great social contribution in that the transmission of tuberculosis can be prevented early on public health.

  The present invention will be described in more detail with reference to the following examples. However, the present invention is not limited to the scope of the examples. Further, based on the present invention, improvements to prevent contamination of amplification products due to carry-over, and improvements for simultaneous amplification and detection of genes such as internal standard substances and other tuberculosis bacteria are easily inferred by those skilled in the art. It is an improvement.

  EXAMPLES The present invention will be specifically described below based on examples, but the present invention is not limited to the following examples.

Reference Example 1 Cloning of Pyrococcus furiosus RNaseHII gene (1) Preparation of Pyrococcus furiosus genomic DNA Tryptone (Difco Laboratories) 1%, Yeast extract (Difco Laboratories) 0.5%, Soluble starch (Nacalai Tesque) 1%), Jamarin S. Solid (Jamarine Laboratory) 3.5%, Jamarin S. Liquid (Jamarine Laboratory) 0.5%, MgSO ₄ 0.003%, NaCl 0.001%, FeSO ₄ · 7H ₂ O 0.0001%, CoSO ₄ 0.0001%, CaCl ₂ · 7H ₂ O 0.0001%, ZnSO ₄ 0.0001%, CuSO ₄ · 5H ₂ O 0.1 ppm, KAl (SO _{4 ) 2 0.1ppm, H 3 BO 4} 0.1ppm, Na 2 MoO 4 · 2H 2 O 0.1p m, put Medium 2 liters of the composition of NiCl ₂ · 6H ₂ O 0.25ppm to Mejuumubotoru 2 liter, 120 ° C., was sterilized for 20 minutes, bubbled with nitrogen gas to remove dissolved oxygen, to Pyrococcus furiosus (Pyrococcus furiosus, purchased from Deutsche Zamlunk von Microorganismen: DSM3638) was inoculated and incubated at 95 ° C. for 16 hours, and then the cells were obtained by centrifugation.

  Next, the obtained bacterial cells were suspended in 4 ml of 25% sucrose and 50 mM Tris-HCl (pH 8.0), 0.4 ml of 10 mg / ml lysozyme chloride (Nacalai Tesque) aqueous solution was added, The reaction was carried out at 1 ° C. for 1 hour. After completion of the reaction, 24 ml of 150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH 8.0), 0.2 ml of 20 mg / ml proteinase K (Takara Shuzo) and 2 ml of 10% aqueous sodium lauryl sulfate solution were added to the reaction solution. In addition, it was kept at 37 ° C. for 1 hour.

  After completion of the reaction, phenol-chloroform extraction and ethanol precipitation were performed to prepare about 1 mg of genomic DNA.

(2) Cloning of RNase HII gene The entire genome sequence of Pyrococcus horikoshii has been published [DNA Research, Vol. 5, pp. 55-76 (1998)], and encodes a homologue of RNase HII. It has been clarified that one gene (PH1650) exists (SEQ ID NO: 1, Japan Incorporated Administrative Agency National Institute of Technology and Evaluation, website: http://www.nite.go.jp/).

  Therefore, the PH1650 gene (SEQ ID NO: 1) and Pyrococcus furiosus genome sequence (University of Utah, Utah Genome Center homepage: http://www.genome.utah.edu/sequence.html) are partially homologous. I did a search. As a result, a highly homologous sequence was found.

  Based on the obtained sequence, primers 1650Nde (SEQ ID NO: 2) and 1650Bam (SEQ ID NO: 3) were synthesized.

  PCR was performed in a volume of 100 μl using 20 ng of 1650 Nde and 20 pmole of 1650 Bam as primers using 200 ng of Pyrococcus furiosus genomic DNA obtained in (1) above as a template. As a DNA polymerase in PCR, Takara Ex Tac (Takara Shuzo Co., Ltd.) was used according to the attached protocol, and PCR was performed for 30 cycles at 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 1 minute. The amplified DNA fragment of about 0.7 kb is digested with NdeI and BamHI (both manufactured by Takara Shuzo Co., Ltd.), and a plasmid pPFU220 in which the obtained DNA fragment is inserted between NdeI and BamHI of plasmid vector pET3a (manufactured by Novagen) is prepared. did.

(3) Determination of the base sequence of a DNA fragment containing the RNase HII gene
The base sequence of the inserted DNA fragment of pPFU220 obtained in (2) above was determined by the dideoxy method.

  When the result of the obtained base sequence was analyzed, an open reading frame (ORF, open reading frame) thought to encode RNaseHII was found. The base sequence of this open reading frame is shown in SEQ ID NO: 4 in the sequence listing. In addition, the amino acid sequence of RNase HII deduced from the base sequence is shown in SEQ ID NO: 5 in the sequence listing.

  E. coli JM109 transformed with plasmid pPFU220 is named and displayed as Escherichia coli JM109 / pPFU220, and is a patent biological deposit center of the National Institute of Advanced Industrial Science and Technology [1st, 1st East, 1-chome, Tsukuba, Ibaraki, Japan. 6 (postal code 305-8565)] was deposited under the accession number FERM P-18020 from September 5, 2000 (original deposit date) and deposited with the Patent Organism of the National Institute of Advanced Industrial Science and Technology under the Budapest Treaty. It has been transferred to the Center as FERM BP-7654 since July 9, 2001 (International Transfer Date).

(4) Preparation of purified RNase HII preparation The pPFU220 obtained in (2) above was transformed into E. coli HMS174 (DE3) (manufactured by Novagen), and the obtained E. coli HMS174 (DE3) containing pPFU220 was converted to 100 μg / ml. Inoculated into 2 liters of LB medium containing ampicillin and cultured with shaking at 37 ° C. for 16 hours. After completion of the culture, the cells collected by centrifugation were suspended in 66.0 ml of sonication buffer [50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM phenylmethanesulfonyl fluoride] and subjected to an ultrasonic crusher. . The crushed liquid was centrifuged at 12,000 rpm for 10 minutes, and the resulting supernatant was subjected to heat treatment at 60 ° C. for 15 minutes. Thereafter, centrifugation was again performed at 12000 rpm for 10 minutes, and the supernatant was collected to obtain 61.5 ml of a heat-treated supernatant.

  This heat-treated supernatant was applied to a RESOURSE Q column (Amersham Pharmacia Biotech) equilibrated with buffer A [50 mM Tris-HCl (pH 8.0), 1 mM EDTA], and an FPLC system (Amersham Pharmacia Biotech) was used. Chromatography. As a result, RNase HII passed through the RESOURCE Q column.

  Apply 60.0 ml of the RNase HII fraction passed through to a RESOURSE S column (Amersham Pharmacia Biotech) equilibrated with buffer A, and elute with a linear gradient of 0 to 500 mM NaCl using the FPLC system, at about 150 mM NaCl. The eluted RNase HII fraction was obtained.

  2.0 ml of this RNase HII fraction was concentrated by ultrafiltration using Centricon-10 (Amicon), and 250 μl of the concentrated solution was added with 50 mM Tris-HCl (pH 8.0) containing 100 mM NaCl and 0.1 mM EDTA. As a result of using an equilibrated Superdex 200 gel filtration column (Amersham Pharmacia Biotech) and elution with the same buffer, RNaseHII was eluted at a position corresponding to a molecular weight of 17 kilodaltons. This molecular weight corresponds to the case where RNaseHII is present as a monomer.

The thus eluted RNase HII was used as a Pfu RNase HII preparation.
RNase H activity was measured by the following method using the Pfu RNase HII preparation obtained above.

  10 mM Tris-HCl (pH 8.0), 1 mM dithiothreitol (manufactured by Nacalai Tesque), 0.003% bovine serum albumin (fraction V, manufactured by Sigma), 4% glycerol, 20 μg / ml poly (dT) (Amersham) Pharmacia Biotech) and 30 μg / ml poly (rA) (Amersham Pharmacia Biotech) were mixed and incubated at 37 ° C. for 10 minutes. This was used as a substrate solution for measuring RNase H activity.

1 μl of 1M MnCl ₂ was added to 100 μl of the substrate solution, and the mixture was kept at 40 ° C., and the Pfu RNase HII preparation was appropriately diluted to the reaction, and the reaction was started. After reacting at 40 ° C. for 30 minutes, 10 μl of 0.5 M EDTA was added to stop the reaction, and the absorbance at 260 nm was measured.

  As a result, in the reaction solution to which the above Pfu RNase HII preparation was added, the absorbance value at 260 nm was higher than that in which 10 μl of 0.5 M EDTA was added first and then added. Therefore, it was revealed that the sample has RNase H activity.

Reference Example 2 Preparation of Afu RNase HII Cloning of the RNase HII gene of Alcaeoglobus fulgidus (1) Preparation of Alkaeoglobus fulgidus genomic DNA Archaeobus fulgidus Purchase: DSM4139) Collect 8ml equivalent of cells, suspend in 100μl 25% sucrose, 50mM Tris-HCl (pH 8.0), 20μl 0.5M EDTA, 10μl 10mg / ml lysozyme chloride (Nacalai) Tesque) aqueous solution was added and reacted at 20 ° C. for 1 hour. After completion of the reaction, 800 μl of 150 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH 8.0), 10 μl of 20 mg / ml proteinase K (Takara Shuzo) and 50 μl of 10% aqueous sodium lauryl sulfate solution were added to the reaction solution. Incubated at 37 ° C. for 1 hour. After completion of the reaction, phenol-chloroform extraction, ethanol precipitation, and air drying were followed by dissolution in 50 μl of TE to obtain a genomic DNA solution.

(2) Cloning of RNase HII gene The entire genome sequence of Arcaeoglobus fulgidus has been published [Klenk, HP et al., Nature, 390, 364-370 (1997)], It has been revealed that there is one gene (AF0621) encoding a homologue of RNaseHII (SEQ ID NO: 6, http://www.tigr.org/tdb/CMR/btm/htmls/SplashPage.html).

  Therefore, primers AfuNde (SEQ ID NO: 7) and AfuBam (SEQ ID NO: 8) were synthesized based on the sequence of the AFO621 gene (SEQ ID NO: 6).

  PCR was performed in a volume of 100 μl using 30 ng of Arcaeoglobus fulgidus genomic DNA obtained in (1) above as a template, 20 pmol AfuNde and 20 pmol AfuBam as primers. As the DNA polymerase for PCR, Pyrobest DNA polymerase (Takara Shuzo) was used according to the attached protocol, and PCR was performed for 40 cycles with 94 ° C for 30 seconds, 55 ° C for 30 seconds, and 72 ° C for 1 minute. . The amplified DNA fragment of about 0.6 kb is digested with NdeI and BamHI (both manufactured by Takara Shuzo Co., Ltd.), and the obtained DNA fragment is between NdeI and BamHI of plasmid vector pTV119Nd (the NcoI site of pTV119N is converted to NdeI site). Plasmid pAFU204 integrated into was prepared.

(3) Determination of base sequence of DNA fragment containing RNase HII gene The base sequence of the inserted DNA fragment of pAFU204 obtained in (2) above was determined by the dideoxy method.

  When the result of the obtained base sequence was analyzed, an open reading frame thought to encode RNaseHII was found. The base sequence of this open reading frame is shown in SEQ ID NO: 9 in the sequence listing. Further, the amino acid sequence of RNase HII deduced from the base sequence is shown in SEQ ID NO: 10 of the sequence listing.

  Escherichia coli JM109 transformed with plasmid pAFU204 is named and displayed as Escherichia coli JM109 / pAFU204, and is an independent administrative agency, National Institute of Advanced Industrial Science and Technology (AIST). 6 (postal code 305-8565)], deposited on February 22, 2001 (original deposit date) as deposit number FERM P-18221, and under the Budapest Treaty, the National Institute of Advanced Industrial Science and Technology patent biological deposit It has been transferred to the center as FERM BP-7691 from August 2, 2001 (International Transfer Date).

(4) Preparation of purified RNase HII preparation E. coli JM109 was transformed with the pAFU204 obtained in (2) above, and the obtained E. coli JM109 containing pAFU204 was inoculated into 2 liters of LB medium containing 100 μg / ml ampicillin. And cultured with shaking at 37 ° C. for 16 hours. After completion of the culture, the cells collected by centrifugation were suspended in 37.1 ml of sonication buffer [50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 2 mM phenylmethanesulfonyl fluoride] and subjected to an ultrasonic crusher. . The crushed liquid was centrifuged at 12,000 rpm for 10 minutes, and the resulting supernatant was subjected to heat treatment at 70 ° C. for 15 minutes. Thereafter, centrifugation was again performed at 12000 rpm for 10 minutes, and the supernatant was collected to obtain 40.3 ml of a heat-treated supernatant.

  This heat-treated supernatant was applied to a RESOURSE Q column (Amersham Pharmacia Biotech) equilibrated with buffer A [50 mM Tris-HCl (pH 8.0), 1 mM EDTA], and an FPLC system (Amersham Pharmacia Biotech) was used. Chromatography. As a result, RNase HII passed through the RESOURCE Q column.

  The sample was applied to a RESOURSE S column (Amersham Pharmacia Biotech) equilibrated with buffer A, and chromatographed using an FPLC system (Amersham Pharmacia Biotech). As a result, RNase HII passed through the RESOURCE S column.

  Dialysis was carried out 3 times for 2 hours using 40.0 ml of the RNaseHII fraction passed through as buffer 2 [50 mM Tris-HCl (pH 7.0), 1 mM EDTA] 2 l containing 50 mM NaCl. The dialyzed enzyme solution (40.2 ml) was applied to a HiTrap-heparin column (manufactured by Amersham Pharmacia Biotech) equilibrated with buffer B containing 50 mM NaCl, and eluted with a 50 to 550 mM NaCl linear concentration gradient using the FPLC system. As a result, an RNase HII fraction eluted at about 240 mM NaCl was obtained.

  7.8 ml of this RNase HII fraction was concentrated by ultrafiltration using Centricon-10 (Amicon), and about 600 μl of the concentrated solution was divided into 4 portions and 50 mM Tris-HCl containing 100 mM NaCl and 0.1 mM EDTA. As a result of using a Superose 6 gel filtration column (Amersham Pharmacia Biotech) equilibrated with (pH 7.0) and elution with the same buffer, RNaseHII was eluted at a position corresponding to a molecular weight of 30.0 kilodaltons. . This molecular weight corresponds to the case where RNaseHII is present as a monomer. The thus eluted RNase HII was used as an Afu RNase HII preparation.

  Using the Afu RNase HII sample obtained above, the enzyme activity was measured by the method described in Reference Example 1- (4). As a result, RNase H activity was observed in the Afu RNase HII sample.

  The number of heat-resistant RNase H units used in the method of the present invention was calculated by the following method.

  1 mg of poly (rA) and poly (dT) (both manufactured by Amersham Pharmacia Biotech) are dissolved in 1 ml of 40 mM Tris-HCl (pH 7.7) each containing 1 mM EDTA to prepare a poly (rA) solution and a poly (dT) solution. did.

Next, in 40 mM Tris-HCl (pH 7.7) containing 4 mM MgCl ₂ , 1 mM DTT, 0.003% BSA, 4% glycerol, a poly (rA) solution with a final concentration of 20 μg / ml, a final concentration of 30 μg / ml A poly (dT) solution was added, reacted at 37 ° C. for 10 minutes, and then cooled to 4 ° C. to prepare a poly (rA) -poly (dT) solution. Add 1 μl of the enzyme solution diluted arbitrarily to 100 μl of this poly (rA) -poly (dT) solution, react at 40 ° C. for 10 minutes, stop the reaction by adding 10 μl of 0.5M EDTA, and then absorb the absorbance at 260 nm. It was measured. As a control, 10 μl of 0.5 M EDTA was added to the reaction solution, followed by reaction at 40 ° C. for 10 minutes, and the absorbance was measured. Thereafter, a value (absorbance difference) obtained by subtracting the absorbance of the control from the absorbance obtained by the reaction in the absence of EDTA was obtained. That is, the concentration of nucleotide released from the poly (rA) -poly (dT) hybrid by the enzyme reaction was determined from the difference in absorbance. One unit of RNase H was calculated according to the following equation, with the amount of enzyme increasing A ₂₆₀ corresponding to the release of 1 nmol of ribonucleotide in 10 minutes.
Unit = [absorbance difference × reaction volume (ml)] / 0.0152 × (110/100) × dilution rate

Reference example 3
The number of units of normal temperature RNase H used in the method of the present invention was measured by the following method.

(1) Preparation of reagent solution to be used Reaction solution for titer measurement: final concentration of 40 mM Tris-hydrochloric acid (pH 7.7, 37 ° C.), 4 mM magnesium chloride, 1 mM DTT, 0.003% BSA, 4% glycerol, Sterile water was used to prepare 24 μM poly (dT).
Poly [8- ³ H] adenylic acid solution: 370KBq poly [8- ³ H] adenylic acid solution of was dissolved in sterile water 200 [mu] l.
Polyadenylic acid solution: Polyadenylic acid was diluted with sterile ultrapure water to 3 mM.
Enzyme dilution: final concentrations of 25 mM Tris-HCl (pH 7.5, 37 ° C.), 5 mM 2-mercaptoethanol, 0.5 mM EDTA (pH 7.5, 37 ° C.), 30 mM sodium chloride, 50% glycerol, respectively Prepared with sterile water.
Preparation of heat-denatured calf thymus DNA: 200 mg of calf thymus DNA was suspended in 100 ml of TE buffer and swollen. The absorbance at 260 nm of the solution was measured and diluted with sterile ultrapure water to a concentration of 1 mg / ml. Next, the solution was heated at 100 ° C. for 10 minutes and then rapidly cooled in an ice bath.

(2) and held activity measurement method described above (1) poly [8- ³ H] Preparation titers measurement reaction solution 985μl in 10 minutes at 37 ° C. was added adenylate solution 7 [mu] l. Next, 8 μl of polyadenylic acid was added to a final concentration of 24 μM, and the mixture was further maintained at 37 ° C. for 5 minutes. The thus prepared poly [8- ³ H] rA- poly dT reaction 1000μl with. Next, after separating 200 μl of the reaction solution and keeping it at 30 ° C. for 5 minutes, 1 μl of enzyme solution diluted in an arbitrary dilution series was added, and 50 μl of these reaction solutions were sampled over time. Used for measurement. The time from enzyme addition to sampling was defined as Y minutes. Further, 50 μl of the total CPM reaction solution and 50 μl of the blank reaction solution were prepared by adding 1 μl of enzyme dilution instead of the enzyme solution. 100 μl of 100 mM sodium pyrophosphate, 50 μl of heat-denatured calf thymus DNA solution and 300 μl of 10% trichloroacetic acid (300 μl of ultrapure water in the case of total CPM measurement) are added to the sampling solution, held at 0 ° C. for 5 minutes, and then at 10,000 rpm Centrifuge for 10 minutes. After centrifugation, 250 μl of the obtained supernatant was put into a vial, 10 ml of Aquazolu 2 (manufactured by NEN Life Science Products) was added, and CPM was measured with a liquid scintillation counter.

(3) Unit calculation The number of units of each enzyme was calculated by the following formula.
Unit / ml = {(measured CPM-blank CPM) × 1.2 ^* × 20 × 1000 × dilution ratio} × 200 (μl) / (total CPM × Y minute × 50 (μl) × 9 ^** )
1.2 ^* Poly [8- ³ H] rA- nmol number per 50μl poly dT 9 ^** included in the total CPM: correction factor

Example 1
(1) DNA extraction from cocoon The cocoon was processed by the NALC method recommended by CDC (Centers for Disease Control and Prevention) in the United States. That is, put the sputum in a 50 ml tube with a screw cap, and add 0.5 g of N-acetyl-L-cysteine to an equal volume of NALC solution (mixture of 50 ml of 0.1 M sodium citrate and 50 ml of 4% sodium hydroxide). The sample was mixed well for 20 seconds with a test tube mixer to make the sample liquid. After standing at room temperature for 15 minutes, the total volume was adjusted to 50 ml with phosphate buffered saline (PBS), and then centrifuged at 3000 g or more for 20 minutes to collect the precipitate. 50 μl of lysis buffer (10 mM HEPES buffer (pH 7.8) containing 1 unit of mutanolysin (Sigma))] is added to the precipitate and mixed well. The mixture was reacted at 37 ° C. for 30 minutes, then boiled in a water bath for 5 minutes or heat-treated in a 96 ° C. heat block. When necessary, it was centrifuged with a micro high speed centrifuge, and the supernatant was collected. The supernatant prepared by the above operation was used for subsequent operations as a DNA extract from sputum. Furthermore, Gen Toru-kun yeast (Takara Shuzo) was also used for nucleic acid extraction according to the instruction manual.

(2) Preparation of Probe and Chimeric Oligonucleotide Primer A specific oligonucleotide probe for detecting IS6110 gene derived from Mycobacterium tuberculosis is GenBank Accession No. It was prepared based on the base sequence described in X52471. That is, an oligonucleotide probe MTIS-2BF consisting of the base sequence shown in SEQ ID NO: 11 in the sequence listing and having 5TC end attached with FITC (fluorescein isothiocyanate) was synthesized by a DNA synthesizer.

  A chimeric oligonucleotide primer for synthesizing a DNA fragment derived from the Mycobacterium tuberculosis group IS6110 gene by the ICAN method was prepared. That is, based on the base sequence of the Mycobacterium tuberculosis group IS6110 gene shown in SEQ ID NO: 12 in the sequence listing, forward primer MTIS-2F consisting of the base sequence shown in SEQ ID NO: 13 in the sequence listing, SEQ ID NO: 14 in the sequence listing The reverse primers MTIS2-RBio, which consisted of the base sequence shown in Fig. 5 and had biotin added at the 5 'end, were each synthesized by a DNA synthesizer. Similarly, forward primer K-F1033-2 consisting of the base sequence shown in SEQ ID NO: 15 of the sequence listing, reverse primer K-R1133 consisting of the base sequence shown in SEQ ID NO: 16 of the sequence listing and biotin added to the 5 ′ end. -2Bio was synthesized with a DNA synthesizer.

(3) Amplification by ICAN Method Reaction solutions for ICAN reaction shown in Table 1 were prepared and incubated at 60 ° C. for 30 minutes.

5 × Reaction Buffer Composition:
100 mM HEPES-potassium hydroxide (pH 7.8)
500 mM potassium acetate 5% dimethyl sulfoxide (DMSO)
0.05% bovine serum albumin (BSA)

  Similarly, the ICAN amplification reaction was also performed using a combination of K-F1033-3 / K-R1133-3 primers. After completion of the reaction, a part of the reaction solution was subjected to electrophoresis to confirm the target amplified fragment.

(4) Detection by solid-layer plate luminescence method Streptavidin (manufactured by Nacalai) is dissolved in PBS to a concentration of 2 μg / ml, and this solution is added to a white well detection 96-well microtiter plate at 150 μl / well. And left overnight at 4 ° C. to immobilize. After discarding the streptavidin solution, a 1% BSA-PBS solution was dispensed at 200 μl / well and allowed to stand overnight at 4 ° C. for blocking. The solution discarded was used as a streptavidin-coated plate in the following experiment.

  50 μl / well of a hybridization buffer [5 × SSC containing 1% TritonX-100, 1% BSA] was added to the above streptavidin-coated plate, and the MTIS-2F / MTIS2-RBio primer was added in Example 1- (3). The ICAN reaction solution containing the amplified fragments obtained by the combination was added by 10 μl / well, mixed well, reacted at room temperature for 15 minutes, and captured on the plate surface. Next, add 5 μl / well of DNA denaturation solution (0.1 N NaOH solution), mix well, perform 3 minutes of DNA denaturation, and denature the double-stranded DNA captured on the plate surface into single-stranded DNA. . Subsequently, the probe MTIS2BF was diluted in a hybridization buffer so as to be 5 pmol / ml, and 100 μl / well was added and mixed well, followed by reaction at room temperature for 40 minutes. At this time, the pH of each well was 13-14. After the reaction, the solution in the well is discarded, and the well is washed twice with 200 μl / well of washing buffer [25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% BSA, 0.05% Tween 20]. Thereafter, 100 μl / well of peroxidase-labeled anti-FITC rabbit antibody (Chemicon) dissolved in hybridization buffer at 2 μg / ml was added and reacted at room temperature for 20 minutes. After the reaction, the solution is discarded, and the wells are washed 4 times with 200 μl / well of washing buffer. Then, a luminescent substrate [SuperSignal ELISA Pico Substrate (Pierce)] is added in an amount of 100 μl / well, and immediately a luminescence plate reader (Lab Systems) Relative emission intensity was measured.

(5) Examination of Detection Sensitivity Detection of Mycobacterium tuberculosis group DNA was performed as described above with respect to moth-derived DNA samples containing 0, 5 and 10 copies of the Mycobacterium tuberculosis group genome. As a result, as shown in Table 2, strong luminescence of about 300 times in S / N ratio could be detected even with 5 copies.

  As described above, it was shown that the present invention can test for Mycobacterium tuberculosis quickly and with high sensitivity. In addition, it was confirmed that the method for detecting Mycobacterium tuberculosis of the present invention can be suitably used in any of the nucleic acid extraction method of the present invention and the method using the Gentoru-kun yeast reagent. Therefore, the usefulness of the nucleic acid extraction method of the present invention was confirmed from the viewpoint of simplicity of the operation method.

Example 2
Spiders were collected from 26 patients suspected of having M. tuberculosis infection, and M. tuberculosis DNA was detected according to the procedure described in Example 1. In addition, using the same specimen, detection using Amplicor Mycobacterium tuberculosis (Roche), which is a kit based on the PCR method targeting DNA encoding existing 16S rRNA, and culture method using Ogawa medium A tuberculosis test was performed. The results are shown in Table 3.

  As shown in Table 3, the method of the present invention was in good agreement with the results of the culture test, but in the PCR method, two cases that were negative in the culture method were detected as false positives, and one case that was positive in the culture method was Detected as false negative.

  That is, it was confirmed that the method of the present invention can obtain an accurate test result very quickly and easily compared with the conventional method.

Example 3
The specificity of the detection method of the present invention for Mycobacterium tuberculosis was examined. As genomic DNA, 37 strains shown in Table 4 were used.

For each genomic DNA, the copy number was calculated from the OD value, and diluted to 2 × 10 ⁷ copies. Detection was performed with the reaction solution composition shown in Table 5 below using the genomic DNA as the template.

  The reaction solution was held at 60 ° C. for 1 hour. Detection was performed by the method described in Example 1 (4). As a result, only the Mycobacterium tuberculosis strain and the Mycobacterium bovis BCG strain could be specifically detected. From this, it was confirmed that the method of the present invention is a highly specific detection method.

Example 4
(1) The detection of Mycobacterium tuberculosis group using magnetic beads was examined. As the primers, MTIS-2F primer and MTIS-2RBio primer were used. Next, the extracted genome derived from the positive specimen in Example 2 was diluted and prepared with sterilized water so as to be 30 times, 300 times, and 3000 times as a template. The reaction was performed as follows. That is, final concentration 32 mM Hepes-potassium hydroxide buffer (pH 7.8), 100 mM potassium acetate, 1% DMSO, 0.01% BSA, 4 mM magnesium acetate, 500 μM dNTPs, 50 pmol each of MTI-2F and MTIS-2R primers 8.75 U of Afu-derived RNase H, 8 U of BcaBEST DNA polymerase, and 1 μl of each template amount were added to a final volume of 50 μl with sterilized water. The reaction solution was set in a thermal cycler personal set at 60 ° C. and held for 60 minutes. The obtained amplified fragment was set in an automatic detection device, Lumipulse (Fujirebio Co., Ltd.), and detected with streptavidin-coated magnetic beads (Pierce Co., Ltd.). Streptavidin solid-layered magnetic beads with a biotin binding capacity of 100 pmol were reacted with the biotinylated amplified fragment in the cuvette first layer for 5 minutes, then 0.1N NaOH was added and hybridized with the FITC-labeled probe MTIBF for 5 minutes, after washing A POD-labeled anti-FITC antibody was added, washed after reaction for 5 minutes, and a luminescent substrate was added. As a result, it was shown that semi-quantification can be performed in a short time of 20 minutes using magnetic beads in an existing automated detection apparatus. The reagent used was the same as in Example 1. Detection was measured by photocounting the amount of luminescence. The results are shown in Table 6.

  From the results shown in Table 6, it was confirmed that detection was possible with the same sensitivity as that of the conventional plate emission method.

(2) The detection system in the case of combining an internal standard (IC) was examined. The internal standard was prepared as follows. That is, PCR amplification was performed using human genomic DNA as a template and the primers described in SEQ ID NOs: 17 and 18 in the sequence listing. The obtained amplified fragment was purified by a conventional method and inserted into pT7 Blue T-vector (manufactured by Novagen). The plasmid was used as an internal standard. The reaction was carried out under the same conditions as described in Example 3, and 1, 3, 10 pg of the above plasmid was added. The template of Mycobacterium tuberculosis genome was 0, 2 and 20 copies. For detection, an IC probe (ICF probe) having a base sequence described in SEQ ID NO: 19 in the Sequence Listing and having a FITC label at the 5 'end and a FITC-labeled probe MTISBF were used. As a result, the Mycobacterium tuberculosis genome could be detected at any IC plasmid concentration. In particular, it was confirmed that it was suitable when the concentration of the IC plasmid was 3 pg.

(3) As a method for detecting the amplified fragment, a hybrid chromatography method was examined. That is, streptavidin (manufactured by Nacalai Tesque) was immobilized on a nitrocellulose membrane and a water absorbing pad was connected to prepare a hybrid chromatographic strip. Using this, the amplified fragment obtained in Example 1 was detected by hybrid chromatography. Detection was performed by color development using 1-step TMB-Blotting (Pierce). That is, a reaction solution containing the amplified fragment was developed on a nitrocellulose membrane, and then a 0.1N NaOH solution, a FITC labeled probe, a washing solution, and a coloring solution were developed in that order. As a result, a blue band was detected in the amplified fragment positive for M. tuberculosis. In addition, by using this method, the results were found with the naked eye within 5 to 10 minutes after the method of the present invention was implemented, so that it was confirmed that the method was useful as a rapid genetic test method.

Example 5
Based on the results of Examples 1-4, a tuberculosis group kit was constructed. Each component is shown below.

(1) ICAN amplification reagent: 50 times 5 × reaction buffer 500 μl
100 mM magnesium acetate 100 μl
10 mM dNTP mix 125 μl
0.1 mM MTI-2F primer 25 μl
0.1 mM MTI-SRBio primer 25 μl
Afu RNase HII (17.5 U / μl) 25 μl
BcaBEST DNA polymerase (16 U / μl) 25 μl

(2) Reagent for detection (50 times)
Streptavidin immobilized 96-well plate 50 hybrid buffers (5 × SSC, 1% Triton X100,
1% BSA) 2.5ml
Denaturant (0.1N NaOH) 0.5ml
MT probe solution for detection (25 nM MTI-2BF probe) 5 ml
IC probe solution for detection (25 nM ICF probe) 5 ml
Labeled antibody solution (Block Ace (Dainippon Pharmaceutical Co., Ltd.): PBS = 1: 3,
500Unit POD-anti-FITC antibody) 5ml
10 × washing solution (250 mM Tris buffer solution (pH 7.5),
1.5M NaCl, 1% BSA, 0.5% Tween 20)
20ml
Luminescent reagent A (SuperSignal ELISA Pico Substrate A solution (Pierce)) 2.5ml
Luminescent reagent B (SuperSignal ELISA Pico Substrate B solution (Pierce)) 2.5ml
Internal standard solution (IC plasmid, TE buffer) 0.1ml
Positive control solution (IS6110-containing plasmid, TE buffer)
0.02ml
Negative control solution (TE buffer) 0.02ml

  When the Mycobacterium tuberculosis strain and the Mycobacterium bovis BCG strain were detected using the above reagents, it was confirmed that they could be detected rapidly, with high sensitivity, and with high specificity.

Example 6
(1) DNA extraction from a swab sample To a swab sample obtained by wiping the male urethra and a female cervix with a cotton swab, an extraction buffer containing a surfactant (Analyte STD sample treatment reagent (Roche)) is added to a temperature of 95 ° C to 100 ° C. For 10 minutes.

(2) Preparation of probe and chimeric oligonucleotide primer A specific oligonucleotide probe for detecting Chlamydia cryptic plasmid was prepared. That is, an oligonucleotide probe CT1234 consisting of the base sequence shown in SEQ ID NO: 20 in the sequence listing and having FITC (fluorescein isothiocyanate) attached to the 5 ′ end was synthesized by a DNA synthesizer. Moreover, a specific oligonucleotide probe for detecting the cppB gene of the bacterium was prepared. That is, an oligonucleotide probe CppB3 consisting of the base sequence shown in SEQ ID NO: 21 of the sequence listing and having FITC (fluorescein isothiocyanate) attached to the 5 ′ end was synthesized by a DNA synthesizer.

  A chimeric oligonucleotide primer for synthesizing a DNA fragment derived from Chlamydia cryptic plasmid by the ICAN method was prepared. That is, based on the base sequence of the Chlamydia cryptic plasmid shown in SEQ ID NO: 22 in the sequence listing, forward primers F1212-22, F1162-22 consisting of the base sequences shown in SEQ ID NO: 23 and 24 in the sequence listing Reverse primers R1272-22Bio and R1379-22Bio, which consisted of the nucleotide sequences shown in SEQ ID NOs: 25 and 26 in the sequence listing and were added with biotin at the 5 ′ end, were synthesized by a DNA synthesizer.

  A chimeric oligonucleotide primer for synthesizing a DNA fragment derived from the bacterium cppB gene by the ICAN method was prepared. That is, based on the base sequence of Chlamydia cryptic plasmid shown in SEQ ID NO: 27 in the sequence listing, forward primer pJDBF-1 consisting of the base sequence shown in SEQ ID NO: 28 in the sequence listing, SEQ ID NO: in the sequence listing Reverse primers pJDBR-3Bio consisting of the base sequence shown in No. 29 and having biotin added at the 5 ′ end were respectively synthesized by a DNA synthesizer.

(3) Amplification by ICAN Method Reaction solutions for ICAN reaction shown in Table 7 were prepared and incubated at 55 ° C. for 60 minutes.

5 × Reaction Buffer Composition:
100 mM HEPES-potassium hydroxide (pH 7.8)
500 mM potassium acetate 5% dimethyl sulfoxide (DMSO)
0.05% bovine serum albumin (BSA)

  After completion of the reaction, a part of the reaction solution was subjected to electrophoresis to confirm the target amplified fragment.

(4) Detection by solid-layer plate luminescence method Streptavidin (manufactured by Nacalai) is dissolved in PBS to a concentration of 2 μg / ml, and this solution is added to a white well detection 96-well microtiter plate at 150 μl / well. And left overnight at 4 ° C. to immobilize. After discarding the streptavidin solution, a 1% BSA-PBS solution was dispensed at 200 μl / well and allowed to stand overnight at 4 ° C. for blocking. The solution discarded was used as a streptavidin-coated plate in the following experiment.

  50 μl / well of hybridization buffer [5 × SSC containing 1% TritonX-100, 1% BSA] was added to the above streptavidin-coated plate, and F1212-22 / R1272-22Bio and pJDBF were used in Example 1- (3). -1 / pJDBR-3Bio Primer-containing ICAN reaction solution containing the amplified fragment was added to 2 wells per sample at 10 μl / well, mixed well, reacted at room temperature for 15 minutes, and captured on the plate surface. It was. Next, add 5 μl / well of DNA denaturation solution (0.1 N NaOH solution), mix well, perform 3 minutes of DNA denaturation, and denature the double-stranded DNA captured on the plate surface into single-stranded DNA. . Subsequently, the probe CT1234 or CppB3 was diluted in a hybridization buffer so as to have a concentration of 5 pmol / ml, and 100 μl / well was added and mixed well, followed by reaction at room temperature for 40 minutes. At this time, the pH of each well was 13-14. After the reaction, the solution in the well is discarded, and the well is washed twice with 200 μl / well of washing buffer [25 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% BSA, 0.05% Tween 20]. Thereafter, 100 μl / well of peroxidase-labeled anti-FITC rabbit antibody (Chemicon) dissolved in hybridization buffer at 2 μg / ml was added and reacted at room temperature for 20 minutes. After the reaction, the solution is discarded, and the wells are washed 4 times with 200 μl / well of washing buffer. Then, a luminescent substrate [SuperSignal ELISA Pico Substrate (Pierce)] is added in an amount of 100 μl / well, and immediately a luminescence plate reader (Lab Systems) Relative emission intensity was measured.

(5) Examination of detection sensitivity Chlamydia DNA was detected as described above for DNA samples containing 0, 10 and 100 copies of the chlamydia genome. As a result, as shown in Table 8, strong luminescence of about 30 times in S / N ratio could be detected even with 10 copies. In addition, for DNA samples containing 0, 10 and 100 copies of the bacterium genome, the bacterium DNA was detected as described above. As a result, as shown in Table 9, even at 10 copies, strong luminescence of about 300 times in S / N ratio could be detected.

  As described above, according to the present invention, it was shown that it is possible to quickly and highly sensitively detect chlamydia and phosphorus bacteria.

Example 7
According to the procedure described in Example 1, chlamydia and gonococcal DNA were simultaneously amplified and detected from 12 samples suspected of having mixed infection with Chlamydia and Phosphorus.

  As a result, 5 cases were positive only for Chlamydia, 1 case was positive only for Phosphorus, 3 cases were positive for both Chlamydia and Phosphorus, and 3 cases were both negative. This result was consistent with the result of the existing PCR method kit (Amplico STD (Roche)).

Example 8
Preparation of primers and probes According to the base sequence of the HCV genome, HCV-F and HCV-R1 chimeric oligonucleotide primers having the base sequences described in SEQ ID NOs: 30 and 31 of the sequence listing and SEQ ID NOs: 32 and 33 of the sequence listing An oligonucleotide primer for reverse transcription having a base sequence was synthesized. Furthermore, oligonucleotide probes having the base sequences shown in SEQ ID NOs: 34 and 35 in the sequence listing were synthesized. Furthermore, TAMRA (N, N, N ′, N′-tetramethyl-6-carboxy-rhodamine, manufactured by ABI) was prepared at the 5 ′ end of the oligonucleotide probe by an automatic DNA synthesizer, and used as a fluorescently labeled probe.

(2) Preparation of synthetic RNA Copy number of HCV-RNA positive sera obtained with informed consent was calculated with Amplicor HCV monitor kit (manufactured by Roche). A 10-fold dilution series from 10 ⁷ copies to 1 copy / μl was prepared and used as HCV-RNA.

(3) Reverse Transcription Reaction cDNA was synthesized from the HCV-RNA prepared in (2) above using the reverse transcription reaction primer prepared in (1) above and First-strand cDNA Synthesis Kit (Takara Shuzo).

(4) Preparation of ICAN reaction solution ICAN reaction was performed using the cDNA solution. Table 10 shows the composition of the reaction solution per tube.

(5) Homogeneous detection For each reaction tube, 47 μl of the above reaction solution was added, and 3 μl of the cDNA solution prepared in (4) was added thereto, and kept at 56 ° C. for 30 minutes. After completion of the reaction, the fluorescently labeled probe prepared in (1) above was added to this reaction solution so that the final concentration was 300 nM, treated at 98 ° C. for 2 minutes, then cooled to 25 ° C. by ice cooling and held. The reaction solution was measured for fluorescence intensity with a fluorescence detector, fluoroscan (manufactured by Ravao Systems). Furthermore, as a control, the same sample was also used for measurement with a commercially available PCR-based Amplicor HCV kit (Roche). The result is shown in FIG. FIG. 1 is a graph showing changes in fluorescence intensity when various HCV-RNA concentrations are measured and comparison of measurement ranges with the conventional method, where the left vertical axis is the OD450 value, and the right vertical axis is the fluorescence intensity (S / N ratio). The horizontal axis indicates the amount of HCV-RNA. Moreover, the black square of FIG. 1 shows the result by the method of this invention, and the black circle shows the result by an amplicore HCV kit.

As shown in FIG. 1, in the method of the present invention, it was confirmed that the fluorescence intensity (S / N ratio) increases as the number of HCV-RNA copies increases. The required time for the HCV detection method of the present invention was 45 minutes. On the other hand, in the amplicore HCV kit as a control, the required time was about 5 hours. Furthermore, as for the detection range of HCV-RNA, in the amplicore HCV kit indicated by absorbance as shown in FIG. 1, the measurement range is 2 orders of magnitude, although it increases as the copy number of HCV-RNA increases. It was confirmed that there was no quantitative property at ⁵ copies or more. On the other hand, it was confirmed that the method of the present invention has a wide detection range of 3 orders. Furthermore, the method of the present invention is excellent in terms of simplicity and speed, and it has been confirmed that the method is suitable for processing multiple samples.

Example 9
(1) Preparation of HCV-RNA from patient serum Detection of HCV from clinical specimens was examined. RNA was prepared from 100 μl each of 28 HCV patient sera from which informed consent was obtained, using an HCV-RNA extraction reagent (Roche) attached to the Amplicor HCV kit.

(2) Detection of HCV-RNA in patient serum HCV-RNA was amplified by the same method as described in Example 1 (3) and (4), and used for the detection method of the present invention. In this example, the average value of fluorescence intensity of 10 negative controls performed on a sample containing no control HCV-RNA + 3SD (three times the standard deviation value) was set as a cutoff value, and this cutoff value was exceeded. Samples showing fluorescence intensity were positive. On the other hand, the same specimen was measured with a commercially available Amplicor HCV kit, and positive / negative was determined according to the instruction manual of the kit. Table 11 shows a comparison of the results of the detection method of the present invention and the conventional amplicore HCV kit.

  As shown in Table 11, it was confirmed that the measurement results obtained by the method of the present invention correlated well with the results obtained by the conventional method. Further, it was confirmed that the method of the present invention can be measured quickly and easily with high sensitivity as compared with the conventional method.

  According to the present invention, a method and primer capable of measuring pathogenic microorganisms with high sensitivity, high specificity, rapid and simple, and capable of processing multiple samples in a limited time without using a special instrument. And probes and kits are provided.

When various HCV-RNA density | concentrations are measured by the method of this invention, it is the figure which compared the relationship of the change of HCV-RNA density | concentration and fluorescence intensity, and the difference of the measurement range with the conventional method.

SEQ ID NO: 2: PCR primer 1650Nde for cloning a gene encoding a polypeptide having a RNaseHII activity from Pyrococcus furiosus
SEQ ID NO: 3: PCR primer 1650Bam for cloning a gene encoding a polypeptide having a RNaseHII activity from Pyrococcus furiosus
SEQ ID NO: 7: PCR primer AfuNde for cloning a gene encoding a polypeptide having a RNaseHII activity from Archaeoglobus fulgidus
SEQ ID NO: 8: PCR primer AfuBam for cloning a gene encoding a polypeptide having a RNaseHII activity from Archaeoglobus fulgidus
SEQ ID NO: 11: Oligonucleotide probe to detect the DNA derived from Mycobacterium tuberculosis
SEQ ID NO: 13: Chymeric oligonucleotide primer to amplify the DNA fragment of IS6110 gene derived from Mycobacterium tuberculosis. “Nucleotides 16 to 18 are ribonucleotides-other nucleotides are deoxyribonucleotides”
SEQ ID NO: 14: Chymeric oligonucleotide primer to amplify the DNA fragment of IS6110 gene derived from Mycobacterium tuberculosis. “Nucleotides 19 to 21 are ribonucleotides-other nucleotides are deoxyribonucleotides”
SEQ ID NO: 15: Chimeric oligonucleotide primer to amplify the DNA fragment of IS6110 gene derived from Mycobacterium tuberculosis. “Nucleotides 19 to 21 are ribonucleotides-other nucleotides are deoxyribonucleotides”
SEQ ID NO: 16: Chimeric oligonucleotide primer to amplify the DNA fragment of IS6110 gene derived from Mycobacterium tuberculosis. “Nucleotides 20 to 22 are ribonucleotides-other nucleotides are deoxyribonucleotides”
SEQ ID NO: 17: Oligonucleotide primer C4-MT2F-S100 to amplify the DNA fragment of metaroprotease gene from human
SEQ ID NO: 18: Oligonucleotide primer C4-MT2R-A to amplify the DNA fragment of metaroprotease gene from human
SEQ ID NO: 19: Oligonucleotide probe to detect the internal control DNA
SEQ ID NO: 20: Oligonucleotide probe CT1234 to detect the Chramydia criptic plasmid
SEQ ID NO: 21: Oligonucleotide probe CppB3 to detect Neisseria gonorrhoeae cppB gene
SEQ ID NO: 23: Chimeric oligonucleotide primer to amplify the DNA fragment of Chramydia criptic plasmid. “Nucleotides 20 to 22 are ribonucleotides-other nucleotides are deoxyribonucleotides”
SEQ ID NO: 24: Chimeric oligonucleotide primer to amplify the DNA fragment of Chramydia criptic plasmid. “Nucleotides 20 to 22 are ribonucleotides-other nucleotides are deoxyribonucleotides”
SEQ ID NO: 25: Chimeric oligonucleotide primer to amplify the DNA fragment of Chramydia criptic plasmid. “Nucleotides 20 to 22 are ribonucleotides-other nucleotides are deoxyribonucleotides”
SEQ ID NO: 26: Chimeric oligonucleotide primer to amplify the DNA fragment of Chramydia criptic plasmid. “Nucleotides 20 to 22 are ribonucleotides-other nucleotides are deoxyribonucleotides”
SEQ ID NO: 28: Chimeric oligonucleotide primer to amplify the DNA fragment of Neisseria gonorrhoeae cppB gene. “Nucleotides 18 to 20 are ribonucleotides-other nucleotides are deoxyribonucleotides”
SEQ ID NO: 29: Chimeric oligonucleotide primer to amplify the DNA fragment of Neisseria gonorrhoeae cppB gene. “Nucleotides 15 to 17 are ribonucleotides-other nucleotides are deoxyribonucleotides”
SEQ ID No: 30: Designed chimeric oligonucleotide primer designated as HCV-F to amplify a portion of HCV. “Nucleotides 19 to 21 are ribonucleotides-other nucleotides are deoxyribonucleotides”
SEQ ID No: 31: Designed chimeric oligonucleotide primer designated as HCV-R3 to amplify a portion of HCV. “Nucleotides 16 to 18 are ribonucleotides-other nucleotides are deoxyribonucleotides”
SEQ ID No: 32: Designed oligonucleotide primer to synthsize cDNA of HCV
SEQ ID No: 33: Designed oligonucleotide primer to synthsize cDNA of HCV
SEQ ID No: 34: Designed oligonucleotide probe to detect a DNA fragment amplifying a portion of HCV
SEQ ID No: 35: Designed oligonucleotide probe to detect a DNA fragment amplifying a portion of HCV
SEQ ID No: 36: Oligonucleotide primer to amplify the DNA fragment of IS6110 gene derived from Mycobacterium tuberculosis
SEQ ID No: 37: Oligonucleotide primer to amplify the DNA fragment of IS6110 gene derived from Mycobacterium tuberculosis
SEQ ID No: 41: Primer area to amplify a portion of HCV
SEQ ID No: 42: Primer area to amplify a portion of HCV
SEQ ID No: 43: Probe area to detect a DNA fragment amplifying a portion of HCV

Claims (4)

It is derived from Chlamydia by a reaction solution containing chimeric oligonucleotide primers represented by SEQ ID NOs: 23, 24, 28 and 29 in the sequence listing, a sample containing nucleic acid, a DNA polymerase having strand displacement activity, RNase H and deoxyribonucleotide triphosphate, respectively. amplifying a fragment of pLGV440 and a fragment of the cppB gene from Phosphorus;
Hybridization in the alkaline region is carried out between the amplified product and the probes shown by the nucleotide sequences of SEQ ID NOs: 20 and 21 in the sequence listing, and the chlamydia-derived pLGV440 fragment and the phosphine-derived cppB gene fragment are detected. The step of:
For detecting Chlamydia trachomatis and Neisseria gonorrhoeae, including A composition for use in the method of detecting Chlamydia trachomatis and Neisseria gonorfoea according to claim 1,
A composition comprising chimeric oligonucleotide primers represented by SEQ ID NOs: 23, 24, 28 and 29 in the sequence listing, DNA polymerase having strand displacement activity, RNase H and deoxyribonucleotide triphosphate, respectively. A kit for use in the method for detecting Chlamydia trachomatis and Neisseria gonorfoea according to claim 1,
Probes represented by the nucleotide sequences of SEQ ID NOS: 20 and 21 in the sequence listing, chimeric oligonucleotide primers respectively represented by SEQ ID NOS: 23, 24, 28 and 29 of the sequence listing, DNA polymerase having strand displacement activity, RNase H, deoxyribo A kit comprising nucleotide triphosphate. The kit according to claim 3, further comprising a carrier for capturing an amplification product selected from microtiter plates, beads, magnetic beads, membranes, and glass.

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