JP5838550B2 - Chlamydia trachomatis RNA detection method and detection reagent - Google Patents

Description

  The present invention relates to a method for detecting Chlamydia trachomatis RNA rapidly and with high sensitivity, and a detection reagent using the method. The present invention is useful in the fields of clinical testing, public health, food testing, and food poisoning testing.

  Chlamydia trachomatis is a Gram-negative, obligate intracellular parasitic eubacteria belonging to the Chlamydiaceae family and a causative microorganism of genital chlamydial infection. Genital chlamydial infections are primarily sexually transmitted, and men develop urethritis and epididymis, while women develop cervicitis and pelvic inflammatory disease but do not have special symptoms ( Many asymptomatic infections).

  In Japan, genital chlamydial infection is designated as a fixed-point grasp 4 disease by the Ministry of Health, Labor and Welfare, and an outbreak trend survey is being conducted. According to it, genital chlamydia infection accounts for about 40% of fixed point grasping 4 diseases in men, and it is about 60% in women (Non-patent Document 1). Leaving genital chlamydial infections can lead to epididymis and pelvic inflammatory disease, which may cause infertility and premature labor, and may cause neonatal conjunctivitis and neonatal pneumonia due to birth canal infection during delivery. May develop. Therefore, if infection with this bacterium is found, it is necessary to treat it with certainty, and it is very important to conduct Chlamydia trachomatis tests on patients and pregnant women suspected of infection.

  Conventionally, Chlamydia trachomatis has been detected clinically by antigen testing or nucleic acid testing. The antigen test is a method for detecting an antigen specific to Chlamydiaceae or Chlamydia trachomatis, and the procedure is simple and results can be obtained relatively quickly. Nucleic acid testing is a method for detecting genes specific to Chlamydia trachomatis (DNA and RNA), PCR method for amplifying and detecting DNA, SDA method (star displacement amplification), and TMA method for amplifying and detecting RNA. (Translation medium amplification) (Patent Document 1). Each nucleic acid test is characterized in that both sensitivity and specificity are higher than those of a conventional antigen test, and a non-invasive urine sample can be used.

  For the treatment of chlamydia, specimens (urine, urethral scrapings, cervical scrapings, pharyngeal scrapings, etc.) are collected from subjects, and detection results are obtained very quickly, and appropriate drugs are prescribed based on these results. This is very important. However, in the existing nucleic acid test, it takes about 4 to 5 hours to obtain a test result.

  In recent years, a mutant strain of Chlamydia trachomatis (CT Swedish strain) lacking a part of the cryptoplasmid DNA was discovered in Northern Europe (Non-patent Document 2). In which the DNA-deficient site is used as an amplification region, and this mutant strain cannot be detected (Non-patent Document 3).

Japanese Patent No. 3241717 JP 2000-14400 JP 2001-37500 A Japanese Patent No. 2650159 Japanese Patent No. 3152927 Japanese Patent Laid-Open No. 8-211050 JP 2001-13147 A

Onodera Shoichi (2009), Japan Lin, Vol. 67, no. 1, 5-15 Ripa. T. T. et al. et al. (2006), Eurosurveilance Vol. 11, (45) Pathogen detection information, Vol. 29, no. 9 (No. 343) September 2008, 9-10 Ishiguro T. et al. (2003) Anal. Biochem. 314-77-86 Ishiguro T. et al. (1996) Nucleic Acids Res. , 24, 4992-4997

  Accordingly, an object of the present invention is to provide a method for obtaining a detection result in a short time without impairing the features of high sensitivity and high specificity of nucleic acid testing. In addition to the above, an object of the present invention is to provide a method for detecting a portion of the Chlamydia trachomatis gene that is unlikely to undergo the mutation as described above.

  As a result of intensive studies to achieve the above object, the present inventors have found a portion in the Chlamydia trachomatis gene that is unlikely to be mutated and highly sensitively and highly specifically identify the portion in a short time. A detectable method has been found and the present invention has been completed. That is, the present invention that achieves the above object is a method for amplifying and detecting a specific base sequence of Chlamydia trachomatis 23S rRNA in a sample, wherein the specific base sequence is amplified from the base sequence set forth in SEQ ID NO: 10. A first primer consisting of a sequence capable of hybridizing under stringent conditions to a base sequence of 15 bases or more selected continuously, and 15 bases of a base sequence selected from the base sequence set forth in SEQ ID NO: 19 This is a method for amplifying and detecting a specific base sequence of Chlamydia trachomatis 23S rRNA, characterized in that it is made using a second primer comprising a sequence that can hybridize to the base sequence under stringent conditions. The present invention is particularly preferably a method for amplifying and detecting a specific base sequence of Chlamydia trachomatis 23S rRNA, comprising the following steps.

(1) A second primer having a sequence complementary to a part of a specific base sequence in Chlamydia trachomatis 23S rRNA is hybridized to the RNA, and the RNA is used as a template by an enzyme having RNA-dependent DNA polymerase activity. Extending the second primer, synthesizing cDNA complementary to the specific base sequence to generate an RNA-DNA duplex with the RNA,
(2) a step of generating single-stranded DNA by degrading the RNA-DNA double-stranded RNA with an enzyme having ribonuclease H (RNase H) activity;
(3) A first primer having a sequence homologous to a part of the specific base sequence is hybridized to the single-stranded DNA obtained in (2), and the DNA is obtained by an enzyme having DNA-dependent DNA polymerase activity. The template is used as a template to extend the first primer (wherein either the second primer or the first primer has an RNA polymerase promoter sequence added to its 5 ′ end), a specific nucleotide sequence or a specific sequence Generating a double-stranded DNA containing a promoter sequence capable of transcribing RNA having a sequence complementary to the base sequence;
(4) generating an RNA transcript using the double-stranded DNA as a template by an enzyme having RNA polymerase activity;
(5) using the RNA transcript as a template for cDNA synthesis in the reaction of (1) above, a step of generating an RNA transcript in a chain; and
(6) A step of measuring the amount of RNA transcript produced.

  The present invention also provides a reagent for amplifying and detecting a specific base sequence of Chlamydia trachomatis 23S rRNA in a sample, comprising 15 bases or more continuously selected from the base sequence described in SEQ ID NO: 6 or 7 A first primer selected from sequences that can hybridize to a base sequence under stringent conditions, and a stringent condition to a base sequence of 15 or more bases selected continuously from the base sequence set forth in SEQ ID NO: 17 or 18. And the second primer selected from sequences capable of hybridizing in (1). Hereinafter, the present invention will be described in detail.

  In the present invention, food, drinking water, environmental water such as lake and river water, feces, vomit, and nasal cavity, pharynx, urethra, cervical and other swabs and gargles, blood, urine, and other A sample derived from a living body such as a secretory fluid, a body fluid, or a tissue washing solution can be used as a sample. In the present invention, a sample obtained by previously extracting a nucleic acid from the above-described biological sample or the like and suspending the nucleic acid in an appropriate solution can also be used. In the present invention, it is preferable to previously remove interfering substances that can inhibit an enzyme reaction or the like described later.

  Chlamydia trachomatis that is a detection target of the present invention belongs to the family Chlamydiaceae, but Chlamydiaceae is a causative bacterium of Chlamydia pneumonia in addition to Chlamydia trachomatis which is a causative agent of genital Chlamydia infection. A plurality of types having infectivity to humans are known, such as Chlamydophila pneumoniae and Chlamydophila sittashi which is a causative bacterium of parrot disease. In order to detect Chlamydia trachomatis to be detected from these Chlamydiaceae fungi with high sensitivity and high specificity, the present invention relates to ribosomal RNA (rRNA) having a high content per cell of Chlamydia trachomatis. It detects a portion specific to Chlamydia trachomatis. In addition, since such a portion is relatively highly preserved even when the strain itself changes as described above, the present invention can still be detected even if a mutation occurs in Chlamydia trachomatis.

  The “part” of the rRNA to be amplified and detected for the detection of Chlamydia trachomatis according to the present invention is found in the 23S rRNA of Chlamydia trachomatis, and is from the 5 ′ end of the homologous region with the first primer described later. This is a portion up to the 3 ′ end of the complementary region of the second primer described later (hereinafter, this portion is referred to as “specific base sequence”). By carrying out the present invention, when Chlamydia trachomatis is present in a sample, a nucleic acid having a specific base sequence is amplified. Therefore, it can be determined whether or not Chlamydia trachomatis was present in the sample by detecting whether or not a nucleic acid having a specific base sequence is present in the amplification step or after the amplification step. The initial amount (the initial copy number of Chlamydia trachomatis 23S rRNA) can also be known.

The first primer in the present invention is a sequence that can hybridize under stringent conditions to a base sequence of 15 bases or more selected continuously from the base sequence set forth in SEQ ID NO: 10. Among such first primers, those comprising a sequence that can hybridize under stringent conditions to the sequence shown in SEQ ID NO: 6 or 7 are preferred, and particularly preferably complementary to the sequence shown in SEQ ID NO: 6 or 7 It consists of an array.
The term “stringent conditions” as used in the present invention refers to conditions in which DNA or DNA and RNA hardly form a double strand as compared with a normal state. For example, 50% at 42 ° C. (v / v ) Conditions in which formamide, 0.1% bovine serum albumin, 0.1% ficoll, 0.1% polyvinylpyrrolidone, 50 mM sodium phosphate buffer (pH 6.5), 150 mM sodium chloride and 75 mM sodium citrate coexist Is mentioned. Therefore, from a base sequence capable of hybridizing under stringent conditions to a base sequence of 15 bases or more selected continuously from the base sequence set forth in SEQ ID NO: 10 or the base sequence set forth in SEQ ID NO: 6 or 7. As the first primer, in addition to a base sequence that is completely complementary to the base sequence, substitution, deletion, addition, and modification of bases within a range that allows hybridization with the base sequence under stringent conditions It may be composed of a base sequence in which etc. occur.

  The length (number of bases) of the first primer is not particularly limited, but in order to obtain sufficient specificity for Chlamydia trachomatis 23S rRNA, it is 8 to 50 bases, preferably 10 to 50 bases, Preferably it is 15 to 50 bases.

  The second primer in the present invention comprises a sequence capable of hybridizing under stringent conditions to a base sequence of 15 bases or more selected continuously from the base sequence set forth in SEQ ID NO: 19. Among such second primers, those consisting of a sequence capable of hybridizing under stringent conditions to the sequence shown in SEQ ID NO: 17 or 18 are preferred, and particularly preferably complementary to the sequence shown in SEQ ID NO: 17 or 18. It consists of an array. The second primer also has a base sequence of 15 or more bases selected continuously from the base sequence described in SEQ ID NO: 19, or a base that is completely complementary to the base sequence described in SEQ ID NO: 17 or 18 In addition to the sequence, it may be composed of a base sequence in which base substitution, deletion, addition, modification, or the like has occurred within a range that allows hybridization with the base sequence under stringent conditions. The length (number of bases) of the second primer is not particularly limited, but is 8 to 50 bases, preferably 10 to 50 bases, in particular, in order to obtain sufficient specificity for 23S rRNA of Chlamydia trachomatis. Preferably it is 15 to 50 bases.

  The method of the present invention using the first primer and the second primer described above uses these primers in a method of amplifying a specific base sequence as DNA, such as a PCR (Polymerase Chain Reaction) method or a LAMP method. A method for detecting trachomatis is included. The principle and detailed implementation method such as the PCR method are already widely known. In the present invention, the first primer and the second primer are used as a primer pair, and RNA is reverse transcribed in the first step. There are no special conditions other than performing the operation (so-called RT-PCR method or RT-LAMP method). In the PCR method, when a specific base sequence, that is, Chlamydia trachomatis 23S rRNA is present in a sample, DNA containing the specific base sequence and DNA containing a complementary sequence of the specific base sequence are amplified. Therefore, the presence or the like of Chlamydia trachomatis can be detected by detecting a specific base sequence or a complementary sequence of the specific base sequence in the amplification step or after the end of the amplification step.

  The detection may be performed according to a conventionally known method. Examples of conventionally known methods include electrophoresis, liquid chromatography, and hybridization using a nucleic acid probe labeled with a detectable label. Specifically, the method using a nucleic acid probe includes a solid phase on which a nucleic acid complementary to a nucleic acid sequence (or its complementary sequence) is immobilized, a labeling substance that generates a detectable signal, and a specific base sequence (or its complementary sequence). ) And complementary nucleic acid conjugates, each is hybridized to form a complex containing a specific base sequence (or its complementary sequence), and the labeling substance not involved in complex formation is removed. A method of detecting a labeling substance that has not been removed after (so-called B / F separation) can be exemplified. In particular, the use of a labeled probe designed to change its optical properties by hybridizing with a specific base sequence (or its complementary sequence) to be detected eliminates the aforementioned B / F separation operation. Etc., from the viewpoints of easy and quick operation. In addition, since such a labeled probe does not require B / F separation, detection can be performed in a sealed container, and it is possible to prevent nucleic acid amplification products from scattering into the environment when the container is opened and closed. It is.

Examples of the labeled probe include a fluorescent labeled probe using FRET (fluorescence resonance energy transfer), an intercalator labeled probe labeled with an intercalating fluorescent dye, and the like. When a nucleic acid probe labeled with an intercalating fluorescent dye forms a double strand complementary to a specific base sequence (or its complementary sequence) to be detected, the intercalating fluorescent dye portion becomes the complementary double strand. Particularly preferred is a method using a nucleic acid probe designed to intercalate into a portion and change its fluorescence characteristics (for example, fluorescence intensity).
Although it does not specifically limit as an intercalating fluorescent dye, The oxazole yellow, thiazole orange, ethidium bromide, hemicyanine, or these derivatives which are used widely can be used.

  For example, in the case of oxazole yellow, it is known that fluorescence at 510 nm (excitation wavelength: 490 nm) is significantly increased by intercalating with double-stranded DNA. In order to bind such a dye to an oligonucleotide, for example, it may be bonded to an intercalating fluorescent dye via a suitable linker at the phosphodiester part or the base part at the 3 'end of the oligonucleotide. When a nucleic acid probe labeled with an intercalating fluorescent dye is allowed to coexist, for example, in steps (1) to (4) in the preferred method of the present invention described later, the hydroxyl group at the 3 ′ end of the oligonucleotide It is preferable to modify the hydroxyl group at the 3 ′ end for the purpose of preventing elongation from the above.

  Another method of the present invention using the first primer and the second primer is a method for detecting Chlamydia trachomatis using these primers in a method of amplifying a specific base sequence as RNA. Examples of the method for amplifying a specific base sequence (RNA) as RNA include TRC method (Patent Documents 2 and 3, Non-Patent Document 4), NASBA method (Patent Documents 4 and 5), and TMA method. These methods can amplify and detect Chlamydia trachomatis extremely quickly, and can be carried out by simply putting a certain amount of sample into a container filled with a reagent under a constant temperature condition. In addition to the above, even if various DNAs are mixed in the sample, they are not particularly affected by the present invention.

This particularly preferred embodiment of the method of the present invention is characterized by including the following steps.
(1) A second primer having a sequence complementary to a part of a specific base sequence in Chlamydia trachomatis 23S rRNA is hybridized to the RNA, and the RNA is used as a template by an enzyme having RNA-dependent DNA polymerase activity. Extending the second primer, synthesizing cDNA complementary to the specific base sequence to generate an RNA-DNA duplex with the RNA,
(2) a step of generating single-stranded DNA by degrading the RNA-DNA double-stranded RNA with an enzyme having ribonuclease H (RNase H) activity;
(3) A first primer having a sequence homologous to a part of the specific base sequence is hybridized to the single-stranded DNA obtained in (2), and the DNA is obtained by an enzyme having DNA-dependent DNA polymerase activity. The template is used as a template to extend the first primer (wherein either the second primer or the first primer has an RNA polymerase promoter sequence added to its 5 ′ end), a specific nucleotide sequence or a specific sequence Generating a double-stranded DNA containing a promoter sequence capable of transcribing RNA having a sequence complementary to the base sequence;
(4) generating an RNA transcript using the double-stranded DNA as a template by an enzyme having RNA polymerase activity;
(5) using the RNA transcript as a template for cDNA synthesis in the reaction of (1) above, a step of generating an RNA transcript in a chain; and
(6) A step of measuring the amount of RNA transcript produced.

In the present invention, an enzyme having RNA-dependent DNA polymerase activity used in step (1), an enzyme having ribonuclease H (RNaseH) activity used in step (2), and a DNA-dependent DNA used in step (3) Any enzyme having polymerase activity is a known enzyme, and various enzymes can be selected. In the present invention, separate enzymes can be used as the enzymes, but it is preferable to use enzymes having two or more kinds of activities from the viewpoint of reducing the number of reagents. Particularly preferably, an enzyme having the above three activities is used, and a retrovirus derived from retrovirus can be exemplified as such an enzyme. More specifically, for example, AMV (Avian Myeloblastosis Virus) reverse transcriptase, MMLV (Molone Murine Leukemia Virus) reverse transcriptase, RAV (Rous Associated Virus) reverse transcriptase, HIV (HumanVir reverse transcriptase), Specific examples of these derivatives (these enzymes whose activities and the like have been improved by introducing mutations in the amino acid sequence) can be specifically exemplified.
The enzyme having RNA polymerase activity used in the step (4) is not particularly limited as long as it corresponds to the promoter sequence added to the 5 ′ end of either the first primer or the second primer. For example, the use of bacterial phage-derived T7 RNA polymerase and the corresponding promoter sequence, T3 RNA polymerase and the corresponding promoter sequence, or SP6 RNA polymerase and the corresponding promoter sequence can be exemplified. In the present invention, a transcription initiation region known to affect transcription efficiency may be added to the promoter sequence.

  In the above method, the double-stranded DNA produced in step (3) has a specific base sequence in the sense strand or antisense strand downstream of the promoter sequence of RNA polymerase. Specifically, when a second primer having an RNA polymerase promoter sequence added to its 5 ′ end is used, the double-stranded DNA produced in the step (3) is an anti-stream downstream of the RNA polymerase promoter sequence. When a first primer having a specific base sequence in the sense strand and having an RNA polymerase promoter sequence added to its 5 ′ end is used, the double-stranded DNA produced in step (3) It has a specific base sequence in the sense strand downstream of the promoter sequence. As a result, in the former case, RNA having a sequence complementary to the specific base sequence is generated in step (4), and in the latter case, RNA having a sequence homologous to the specific base sequence is generated in step (4). The RNA generated in step (4) becomes a template for cDNA synthesis in step (1), and steps (1) to (5) are generated in a chain, and RNA transcripts are generated in a chain. become.

  In order to advance such a chain reaction, the method of the present invention is carried out under conditions necessary for each enzyme to exert its activity. As such conditions, for example, it is important to use an appropriate buffer, magnesium salt, potassium salt, nucleoside-triphosphate, or ribonucleoside-triphosphate in the reaction. In addition, for example, dimethyl sulfoxide (DMSO), dithiothreitol (DTT), bovine serum albumin (BSA), sugar and the like are preferably used in order to adjust the efficiency of the amplification reaction. As other conditions, for example, when using AMV reverse transcriptase or T7 RNA polymerase, it is preferable to set the reaction temperature in the range of 35 ° C. to 65 ° C., particularly in the range of 40 ° C. to 50 ° C. preferable. In a preferred embodiment of the present invention, RNA chain amplification proceeds at a constant temperature, and the reaction temperature can be set to an appropriate temperature so that reverse transcriptase and RNA polymerase exert their activities.

  In the above preferred method of the present invention, when an RNA polymerase promoter sequence is added to the 5 ′ end of the first primer and RNA having a sequence homologous to the specific base sequence is generated in step (4), Prior to 3), the Chlamydia trachomatis 23S rRNA is homologous to the first primer at the 5 ′ end site of the homologous region with the first primer, ie, within a specific base sequence in the Chlamydia trachomatis 23S rRNA. This is a partial sequence including the 5 ′ end of the region. As a cleavage method, for example, an oligonucleotide having a sequence complementary to a region adjacent to the 5 ′ direction of Chlamydia trachomatis 23S rRNA from a partial sequence to be cleaved and an enzyme having ribonuclease H (RNase H) activity are preferably used. . More specifically, the oligonucleotide has a base sequence of about 15 bases complementary to the region adjacent to the 5 ′ direction of Chlamydia trachomatis 23S rRNA from the partial sequence to be cleaved, and at the 3 ′ end thereof. For example, those in which a certain hydroxyl group is appropriately modified such as amination so as not to cause an elongation reaction starting from the oligonucleotide can be exemplified. The oligonucleotide is hybridized to generate an RNA-DNA double strand, and an enzyme having ribonuclease H (RNase H) activity is allowed to act to digest the RNA portion of the RNA-DNA hybrid, whereby Chlamydia trachomatis 23S rRNA is Can be cut at a part. Thus, by cleaving Chlamydia trachomatis 23S rRNA at the 5 ′ end site of the specific base sequence, at the same time as the extension of the 3 ′ end of the first primer using DNA as a template in step (3), This is because a DNA strand complementary to the promoter sequence of the first primer hybridized to DNA can be formed by extension of the 3 ′ end, and as a result, a functional double-stranded DNA promoter structure can be formed.

  Among the preferable methods of the present invention described above, the following steps (1) to (6) are included, and prior to step (3), it overlaps with the 5 ′ terminal site of the homologous region with the first primer. From a sequence that can hybridize under stringent conditions to a base sequence of 15 bases or more selected in succession from the base sequence set forth in SEQ ID NO: 5 that is complementary to the region adjacent in the 5 ′ direction from the site. The method comprising the step of cleaving Chlamydia trachomatis 23S rRNA at the 5 ′ end site of the specific base sequence using an oligonucleotide having the above and an enzyme having ribonuclease H (RNase H) activity comprises: Chlamydia trachomatis 23S rRNA This method is a preferred method for amplifying and detecting a specific base sequence of swiftly with high sensitivity and specificity.

(1) A second primer having a sequence complementary to a part of a specific base sequence in Chlamydia trachomatis 23S rRNA is hybridized to the RNA, and the RNA is used as a template by an enzyme having RNA-dependent DNA polymerase activity. Extending the second primer, synthesizing cDNA complementary to the specific base sequence to generate an RNA-DNA duplex with the RNA,
(2) a step of generating single-stranded DNA by degrading the RNA-DNA double-stranded RNA with an enzyme having ribonuclease H (RNase H) activity;
(3) A first primer selected from sequences that can hybridize to the nucleotide sequence of SEQ ID NO: 6 or 7 under stringent conditions is hybridized to the single-stranded DNA obtained in (2) above, and DNA The first primer is extended using the DNA as a template with an enzyme having dependent DNA polymerase activity (here, a promoter sequence of RNA polymerase is added to the 5 ′ end of the first primer), and a specific base sequence Or a step of generating double-stranded DNA containing a promoter sequence capable of transcribing RNA having a sequence complementary to a specific base sequence,
(4) generating an RNA transcript using the double-stranded DNA as a template by an enzyme having RNA polymerase activity;
(5) using the RNA transcript as a template for cDNA synthesis in the reaction of (1) above, a step of generating an RNA transcript in a chain; and
(6) A step of measuring the amount of RNA transcript produced.

In a preferred embodiment of the present invention, the oligonucleotide for cleavage is selected from sequences that can hybridize under stringent conditions to the base sequence set forth in SEQ ID NO: 5 that is homologous to a portion of Chlamydia trachomatis 23S rRNA. Nucleotides can be used.
In a more preferred embodiment of the present invention, the cleavage oligonucleotide consists of an oligonucleotide selected from sequences that can hybridize to the sequence described in SEQ ID NO: 1 or 2 under stringent conditions.

In the present invention, SEQ ID NOs: 5, 10, 19, and 22 are homologous or complementary strands of partial sequences of Chlamydia trachomatis D / UW-3 / CX (GenBank No. NC — 000117).
The target RNA in the present invention indicates a sequence other than the homologous or complementary region of the primer among the specific base sequence on the RNA transcript, and can be complementarily bound to a nucleic acid probe labeled with a fluorescent dye. Have the sequence Therefore, the nucleic acid probe labeled with the fluorescent dye has a sequence complementary to a part of the specific base sequence in the present invention.

  In the present invention, the amount of RNA transcript is measured in the presence of a fluorescent dye-labeled oligonucleotide probe designed to change the fluorescence property when a double strand complementary to the target RNA is formed. By using a method characterized in that it is done by doing, the simplicity, rapidity, and specificity are greatly improved.

  In a more preferred embodiment, the fluorescent dye-labeled oligonucleotide probe is an intercalator fluorescent dye-labeled oligonucleotide probe in which an intercalator fluorescent dye is bound via a linker.

  As one embodiment of the present invention, a nucleic acid probe labeled with an intercalating fluorescent dye is stringent to the nucleotide sequence of SEQ ID NO: 22 complementary to a part of Chlamydia trachomatis 23S rRNA or a complementary sequence thereof. An oligonucleotide having a sequence that can hybridize under conditions can be used.

  In a preferred embodiment of the present invention, a nucleic acid probe labeled with an intercalating fluorescent dye is a sequence described in SEQ ID NOs: 20 and 21 complementary to a part of Chlamydia trachomatis 23S rRNA, or a string in the complementary sequence thereof. It consists of an oligonucleotide selected from sequences that can hybridize under gentle conditions.

  In one embodiment of the present invention, the cleavage oligonucleotide comprises at least one oligonucleotide selected from oligonucleotides that are sequences that can hybridize to the sequence shown in SEQ ID NO: 5 under stringent conditions. The primer has a promoter sequence at its 5 ′ end, and is composed of an oligonucleotide that is a sequence that can hybridize to the sequence shown in SEQ ID NO: 10 under stringent conditions. The second primer is shown in SEQ ID NO: 19 It consists of an oligonucleotide that is a sequence that can hybridize to the sequence described under stringent conditions, and the intercalator fluorescent dye-labeled nucleic acid probe is stringent to the nucleotide sequence set forth in SEQ ID NO: 22 or a complementary sequence thereof. Is a sequence that can hybridize under various conditions. It is preferable that a Gore-nucleotide.

  More preferably, the cleavage oligonucleotide consists of an oligonucleotide selected from sequences that can hybridize under stringent conditions to the sequences shown in SEQ ID NOs: 1 and 2, and the first primer is SEQ ID NO: 6 Or an oligonucleotide selected from sequences that can hybridize to the sequence described in 7 under stringent conditions, and the second primer can hybridize to the sequences described in SEQ ID NOs: 17 and 18 under stringent conditions. It consists of an oligonucleotide selected from the sequence.

  Furthermore, preferably, the cleavage oligonucleotide is composed of an oligonucleotide selected from the complementary strands of the sequences described in SEQ ID NOs: 1 and 2, and the first primer is a complementary strand of the sequences described in SEQ ID NO: 6 or 7 The second primer is composed of an oligonucleotide selected from a complementary strand of the sequences described in SEQ ID NOs: 17 and 18.

  In one embodiment of the present invention, the sample has at least a first primer having a T7 promoter sequence at the 5 ′ end (the T7 at the 5 ′ end of the oligonucleotide hybridizable to the sequence described in SEQ ID NO: 10 under stringent conditions). A sequence to which a promoter sequence (SEQ ID NO: 23) is added, a second primer (an oligonucleotide that can hybridize to the sequence described in SEQ ID NO: 19 under stringent conditions), a nucleic acid probe labeled with an intercalating fluorescent dye ( The sequence shown in SEQ ID NO: 22, or a sequence that can hybridize to the complementary sequence under stringent conditions), a cleavage oligonucleotide (an oligonucleotide that can hybridize to the sequence shown in SEQ ID NO: 5 under stringent conditions) ), AMV reverse transcriptase, T7 RNA poly An amplification reagent containing lyase, buffer, magnesium salt, potassium salt, nucleoside-triphosphate, ribonucleoside-triphosphate, dimethyl sulfoxide (DMSO) is added, and the reaction temperature is 35 to 65 ° C. (preferably 40 to 50 ° C. And a method of measuring the fluorescence intensity of the reaction solution over time at the same time.

  In the above embodiment, most preferably, the cleaving oligonucleotide consists of an oligonucleotide selected from sequences complementary to the base sequences described in SEQ ID NOs: 1 and 2, and the first primer is described in SEQ ID NO: 6 or 7 The second primer is composed of an oligonucleotide selected from sequences complementary to the base sequences described in SEQ ID NOs: 17 and 18.

  In the above embodiment, since the fluorescence intensity is measured over time, the measurement can be completed at any time when a significant increase in fluorescence is observed, and the nucleic acid amplification and measurement are usually completed within 30 minutes. Is possible.

It should be noted that all the samples contained in the measurement reagent can be sealed in a single container. That is, as long as an operation of dispensing a certain amount of sample into such a single container is performed, Chlamydia trachomatis 23S rRNA can be automatically amplified and detected thereafter.
This container only needs to be made of a transparent material at least partially so that the signal emitted from the fluorescent dye can be measured from the outside, and any container that can be sealed after dispensing a sample is contaminated. It is particularly preferable in terms of preventing nations.

  Since the RNA amplification / measurement method of the above embodiment can be carried out at a constant temperature in one step, it can be said that it is a simpler method suitable for automation than RT-PCR. According to the present invention, Chlamydia trachomatis 23S rRNA can be measured with high specificity, high sensitivity, rapid, simple, constant temperature, and one-step simultaneous measurement.

The present invention
(1) a step of amplifying a nucleic acid containing the specific base sequence using a specific base sequence in Chlamydia trachomatis 23S rRNA as a template and using a first primer, a second primer, a nucleic acid polymerizing enzyme such as DNA polymerase,
(2) measuring the nucleic acid amplification product,
A method for detecting Chlamydia trachomatis 23S rRNA comprising an oligonucleotide that is a sequence hybridizable under stringent conditions to the sequence of the first primer SEQ ID NO: 10; The primer is characterized by comprising at least one or more kinds of oligonucleotides selected from sequences that can hybridize under stringent conditions to the sequence shown in SEQ ID NO: 19.
According to the present invention, it is possible to amplify and detect a small amount of Chlamydia trachomatis 23S rRNA contained in a sample quickly and with high sensitivity regardless of the gene group / genotype.

  Furthermore, in the process of measuring the amount of RNA transcripts obtained using the first primer or the second primer having a promoter sequence, the signal characteristics are changed when a complementary double strand is formed with the target RNA. The detection of Chlamydia trachomatis 23S rRNA is simple, rapid, and highly sensitive by measuring the increase in fluorescence intensity derived from the specific nucleic acid of the amplified Chlamydia trachomatis 23S rRNA over time by coexisting with the amplified nucleic acid probe. It became possible to do.

The detection reagent for Chlamydia trachomatis using the detection method of the present invention can detect Chlamydia trachomatis contained in a sample with high sensitivity, and is overwhelmingly faster than the conventional technique. It has the characteristics. Therefore, it becomes possible to present a test result to a doctor promptly, and it is considered that the present invention contributes to prevention of spread of infection and generation of resistant bacteria by administration of an appropriate drug.

The structure of the intercalating fluorescent dye-labeled nucleic acid probe produced in Example 2. B1, B2, B3 and B4 represent bases. A probe in which an intercalating fluorescent dye (oxazole yellow) is bound to a phosphodiester moiety via a linker according to the method of Ishiguro et al. (Non-patent Document 5). In order to prevent an extension reaction from the hydroxyl group at the 3 'end, the hydroxyl group at the 3' end is modified with glycolic acid. The base sequence of the standard RNA used in the present invention and GenBank No. It is a correspondence diagram of the base sequence of the 23S rRNA gene region (8778039 to 880902) of NC — 000117.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited by these Examples.

Example 1 Preparation of Standard RNA Chlamydia trachomatis 23S rRNA (hereinafter referred to as standard RNA) used in Examples described later was prepared by the method shown in (1) to (2).
(1) Using an RNA extract of Chlamydia trachomatis UW-3 / Cx strain (ATCC No. VR-885), a double-stranded DNA of the 23S rRNA nucleotide sequence region was prepared (note that the 5 ′ end side of the DNA) Is added with SP6 promoter).
(2) The double-stranded DNA prepared in (1) was inserted into the multicloning site of the pUC19 plasmid according to a conventional method, and Escherichia coli JM109 strain was transformed.
(3) A plasmid was extracted from the transformed E. coli culture of (2), and the 3 ′ end of the inserted DNA was digested with restriction enzymes to prepare linear DNA.
(4) Using the DNA prepared in (3) as a template, in vitro transcription was performed using SP6 RNA polymerase. Subsequently, the double-stranded DNA was completely digested by DNase I treatment, RNA was purified, and standard RNA was Prepared. The RNA was quantified by measuring the absorbance at 260 nm.

  The standard RNA includes the base sequence of Chlamydia trachomatis 23S rRNA and has a total length of about 3000 bases (8 bases derived from the SP6 promoter are added to the 5 'end of the RNA).

  As a result of analyzing the base sequence of the standard RNA prepared this time, GenBank No. It was confirmed that the nucleotide sequence of the 23S rRNA gene region of NC — 000117 (8778039 to 880902) was completely identical.

Example 2 Preparation of nucleic acid probe labeled with intercalating fluorescent dye A nucleic acid probe labeled with an intercalating fluorescent dye was prepared. In the method described in Non-Patent Document 5,
Between the 10th A and the 11th T from the 5 ′ end of the sequence shown in SEQ ID NO: 20,
Between the 10th C and the 11th G from the 5 ′ end of the sequence shown in SEQ ID NO: 21,
An oxazole yellow labeled nucleic acid probe in which oxazole yellow was bound to each of the phosphodiester moieties of each via a linker was prepared (FIG. 1).

Example 3 Examination of Chlamydia trachomatis 23S rRNA detection primer set Nucleic acid probe labeled with the first primer, the second primer, and the intercalating fluorescent dye in the combinations shown in Table 1 (hereinafter referred to as INAF probe) Using the cleavage oligonucleotide, standard RNA was measured by the methods shown in (1) to (4), and the detection performance, specificity and the like were evaluated.

(1) The standard RNA becomes 10 ⁴ copies / 5 μL using an RNA diluent (10 mM Tris-HCl buffer (pH 8.0), 1 mM EDTA, 0.25 U / μL ribonuclease inhibitor, 5.0 mM DTT). These were used as RNA samples.
(2) 20 μL of the reaction solution having the following composition was dispensed into a 0.5 mL capacity PCR tube (Individual Dome Cap PCR Tube, manufactured by SSI), and 5 μL of the RNA sample was added thereto.

Composition of reaction solution: concentration is final concentration after addition of enzyme solution (in 30 μL) 60 mM Tris-HCl buffer (pH 8.6)
17 mM magnesium chloride 100 mM potassium chloride 1 mM DTT
0.25 mM dATP, dCTP, dGTP, dTTP each
Each 3.0 mM ATP, CTP, UTP, GTP
3.6 mM ITP
Each 0.5 μM first primer: the primer has a T7 promoter sequence (SEQ ID NO: 23) added to the 5 ′ end of the base sequence described in each SEQ ID NO: 0.5 μM second primer 25 nM INAF probe: Probes prepared in Example 2 0.08 μM each oligonucleotide for cleavage: hydroxyl group at the 3 ′ end of the oligonucleotide is modified with an amino group 6U ribonuclease inhibitor (manufactured by Takara Bio Inc.)
13.0% DMSO
(3) After the above reaction solution was kept at 43 ° C. for 5 minutes, 5 μL of an enzyme solution having the following composition and kept at 43 ° C. for 2 minutes in advance was added.

Composition of enzyme solution: final concentration during reaction (in 30 μL) 2.0% sorbitol 6.4 U AMV reverse transcriptase (Life Science)
142U T7 RNA polymerase (Invitrogen)
3.6 μg bovine serum albumin (4) Subsequently, using a fluorescence spectrophotometer with a temperature control function capable of directly measuring a PCR tube, the reaction solution was reacted at 43 ° C. and at the same time the fluorescence intensity of the reaction solution (excitation wavelength: 470 nm, fluorescence wavelength: 520 nm) Measurement was performed for 30 minutes over time.

  The time when the enzyme is added is defined as positive when the fluorescence intensity ratio of the reaction solution (the value obtained by dividing the fluorescence intensity value of the predetermined time by the fluorescence intensity ratio of the background) exceeds 1.2. Tables 1 to 7 show the results of the detection time.

  Tables 1, 2 and 3 show standard RNAs, Table 5 shows parrot disease Chlamydia standard RNA (GenBank No. U68447) and pneumonia chlamydia (TW-183 strain) nucleic acid extracts, and Table 6 shows Neisseria spp. used.

RNeasy mini kit (manufactured by Qiagen) was used for nucleic acid extraction of Chlamydiaceae and Neisseria.
“Nega” in the table refers to a reaction obtained by adding distilled water for injection instead of the target nucleic acid. The “determination” in the table is a comprehensive judgment from the detection time of standard RNA, the measurement result of Nega, the fluorescence intensity ratio, etc., and “◎”, “○”, “△”, “ X ".

  Table 3 shows 30 copies / 5 μL of standard RNA in order to further select G, P, AC, AE, AK, AS oligonucleotide combinations judged to have good performance among the combinations in Table 1. The result when measured was shown.

Table 4, in the combination of Table 3, good performance G is determined that the, P, for further selecting a combination of oligonucleotides AK, measured standard RNA of 10,30,10 ² copies / 5 [mu] L The result was shown.

Table 5 shows that 10 ¹⁰ copies / 5 μL of parrot disease Chlamydia standard RNA and pneumonia for further selecting G, P, AK combination oligonucleotides judged to have good performance among the combinations in Table 3. The results of measuring Chlamydia nucleic acid extract are shown.

  Table 6 shows the best combination of oligonucleotides selected in the examinations in Tables 1 to 5.

Table 7 shows the use of oligonucleotides in combination with P, as well as Chlamydia trachomatis, a common gonococcus as a causative bacterium of sexually transmitted diseases, and has no pathogenicity, but is known as a resident bacteria in the pharynx and oral cavity Tested for cross-reactivity against Neisseria spp. The concentration of Neisseria gonorrhoeae and Neisseria was adjusted so as to be 10 ⁷ CFU / test.
In all the tables, N.I. D. Means a sample having a fluorescence intensity ratio of less than 1.2 (negative determination) 30 minutes after the enzyme was added. Nega is measured using sterilized distilled water as a sample. In addition, the number of + in gel electrophoresis indicates the degree of darkness of the band derived from the amplification product. The larger the number of +, the more the amount of amplification product is generated. Indicates that it was not generated.

10, 30, 10 ^ 2 copies / test

From the examination results shown in Tables 1 to 5, an oligonucleotide sequence suitable for detecting Chlamydia trachomatis 23S rRNA has been clarified, and an oligo that can be detected quickly, with high sensitivity and with high specificity. We have found a combination of nucleotides.

  Based on the detection time of Chlamydia trachomatis standard RNA, the fluorescence intensity ratio, and the measurement result of Nega using the combination of oligonucleotides A to AV shown in Table 1, the combination of oligonucleotides G, P, AC, AE, AK and AS were judged to be good.

  As the oligonucleotide for cleavage, a sequence complementary to each of SEQ ID NOs: 1, 2, 3, and 4 can be used. Therefore, the nucleotide sequence including the corresponding sequence can be hybridized to SEQ ID NO: 5 under stringent conditions. It was found that can be used.

  Since the first primer can use a sequence complementary to each of SEQ ID NOs: 6, 7, 8, and 9, the nucleotide sequence including those corresponding to them can be hybridized to SEQ ID NO: 10 under stringent conditions. It was found that can be used.

  Since the second primer can use a sequence complementary to each of SEQ ID NOs: 15, 17, and 18, a base sequence that can be hybridized under stringent conditions to SEQ ID NO: 19 is used. I understood that I could do it.

  Since the sequences described in SEQ ID NOs: 20 and 21 can be used for the INAF probe, the bases capable of hybridizing under stringent conditions with the sequence shown in SEQ ID NO: 22 or the complementary strand thereof, including the corresponding sequences. It turns out that an array can be used.

  That is, as a cleavage oligonucleotide, using a nucleotide sequence that can hybridize to SEQ ID NO: 5 under stringent conditions, as a first primer, using a nucleotide sequence that can hybridize to SEQ ID NO: 10 under stringent conditions, As the second primer, a nucleotide sequence that can hybridize under stringent conditions to SEQ ID NO: 19 is used, and as an INAF probe, a nucleotide sequence that can hybridize under stringent conditions to SEQ ID NO: 22 or its complementary strand is used. Thus, it was found that Chlamydia trachomatis standard RNA can be rapidly amplified and detected with high specificity.

  From Table 3, it was possible to select three combinations (G, P, AK) with particularly good detection sensitivity from the six combinations selected in Table 1. In the case of the combination of AC, AE, and AS, the rise of Nega was not seen in the experiment of Table 2, but the rise of Nega was seen in the experiment of Table 3. However, it can be seen that the fluorescence intensity ratio of Nega of any combination is sufficiently smaller than the fluorescence intensity ratio of Posi (30 copies / test). Therefore, it is considered that these combinations can be used by appropriately adjusting the threshold value.

  Table 4 compares the detection sensitivity of standard RNA using the three combinations selected in Table 3. Although any combination shows a difference in detection time, any combination can be detected up to 10 copies / test, and the best combination cannot be selected.

Table 5 is a measurement of the parrot disease chlamydia standard RNA and the nucleic acid extract of cultured pneumoniae chlamydia cells using the three combinations selected in Table 3. Parrot disease chlamydia standard RNA was not detected in any combination, but pneumonia chlamydia nucleic acid extract was positive in the AK combination. The combinations G and P were negative with either one, and it was found that they have high specificity for Chlamydia trachomatis.
However, as a result of gel electrophoresis analysis of the reaction solution, it was found that there was a difference in the amount of amplification product generated between the combinations G and P.

  Table 6 lists the finally selected oligonucleotide combinations. It was found that Chlamydia trachomatis can be detected rapidly and with high sensitivity by using a detection reagent containing the oligonucleotides of the described combination.

Table 7 shows the reactivity of the Neisseria spp., Including Neisseria gonorrhoeae, to the nucleic acid extract, using the oligonucleotide combination P, negative for all nucleic acid extracts tested, and very high specificity. It was found to have sex.

Example 3 Detection reagent for Chlamydia trachomatis 23S rRNA

Using the suitable oligonucleotide combinations G and P obtained in Example 2, an attempt was made to produce a detection reagent capable of measuring Chlamydia trachomatis rapidly and with high sensitivity.
The reagent composition described in Example 2 was prepared, and two types of Chlamydia trachomatis detection reagents (oligonucleotide combinations G and P) were prepared.
Chlamydia trachomatis (UW-3 / Cx strain) was infected with Hela cells, and RNA extracted from a product in which Chlamydia trachomatis sufficiently proliferated was measured with the above-mentioned Chlamydia trachomatis detection reagent.
10 ^ -2, 10 ^ -4, 10 ^ -6, 10 ^ -8 in the table represent the degree of dilution of the extract, and the stock solution was 10 ² times, 10 ⁴ times, 10 ⁶ times, respectively. 10 ⁸ diluted.

The RNA quantitative value in the table is an RNA concentration (copy / test) obtained from a standard curve prepared by measuring standard RNA having a known concentration and detecting from the detection time.
The measurement results of the above-mentioned reagents are as shown in Table 8, and Chlamydia trachomatis became positive within almost 10 minutes in all dilution series, and it was found that measurement can be performed very rapidly and with high sensitivity.

  It was found that the method shown in the present example can measure Chlamydia trachomatis with high sensitivity, high specificity, and very rapid measurement. It can be said that the method shown in this example is the most preferable system in the present invention.

Claims (3)

(1) A second primer having a sequence complementary to a part of a specific base sequence in Chlamydia trachomatis 23S rRNA is hybridized to the RNA, and the RNA is used as a template by an enzyme having RNA-dependent DNA polymerase activity. Extending the second primer, synthesizing cDNA complementary to the specific base sequence to generate an RNA-DNA duplex with the RNA,
(2) a step of generating single-stranded DNA by degrading the RNA-DNA double-stranded RNA with an enzyme having ribonuclease H (RNase H) activity;
(3) A first primer having a sequence homologous to a part of the specific base sequence is hybridized to the single-stranded DNA obtained in (2), and the DNA is obtained by an enzyme having DNA-dependent DNA polymerase activity. The template is used as a template to extend the first primer (wherein either the second primer or the first primer has an RNA polymerase promoter sequence added to its 5 ′ end), a specific nucleotide sequence or a specific sequence Generating a double-stranded DNA containing a promoter sequence capable of transcribing RNA having a sequence complementary to the base sequence;
(4) generating an RNA transcript using the double-stranded DNA as a template by an enzyme having RNA polymerase activity;
(5) using the RNA transcript as a template for cDNA synthesis in the reaction of (1) above, a step of generating an RNA transcript in a chain; and
(6) a step of measuring the amount of RNA transcript produced,
A method for amplifying and detecting a specific base sequence of Chlamydia trachomatis 23S rRNA, comprising:
The first primer is an oligonucleotide composed of a sequence complementary to the base sequence described in SEQ ID NO: 6 or 7, and the second primer is composed of a sequence complementary to the base sequence described in SEQ ID NO: 17 or 18. The method, which is an oligonucleotide. (1) A second primer comprising a sequence complementary to the base sequence set forth in SEQ ID NO: 17 or 18 is hybridized to the RNA, and the second RNA is used as a template by an enzyme having RNA-dependent DNA polymerase activity. Extending a primer and synthesizing cDNA complementary to a specific base sequence to generate an RNA-DNA duplex with the RNA,
(2) a step of generating single-stranded DNA by degrading the RNA-DNA double-stranded RNA with an enzyme having ribonuclease H (RNase H) activity;
(3) An enzyme having a DNA-dependent DNA polymerase activity by hybridizing the first primer comprising a sequence complementary to the base sequence described in SEQ ID NO: 6 or 7 to the single-stranded DNA obtained in (2) above By extending the first primer using the DNA as a template (here, a promoter sequence of RNA polymerase is added to the 5 ′ end of the first primer) and complementary to the specific base sequence or the specific base sequence Generating a double-stranded DNA comprising a promoter sequence capable of transcribing the sequence RNA;
(4) generating an RNA transcript using the double-stranded DNA as a template by an enzyme having RNA polymerase activity;
(5) using the RNA transcript as a template for cDNA synthesis in the reaction of (1) above, a step of generating an RNA transcript in a chain; and
(6) measuring the amount of the produced RNA transcript,
Here, prior to step (3), the base according to SEQ ID NO: 1 or 2 , which overlaps with the 5 ′ terminal site of the homologous region with the first primer and is complementary to the region adjacent to the 5 ′ direction from the site. Performing the step of cleaving Chlamydia trachomatis 23S rRNA at the 5 ′ end site of the specific base sequence with an oligonucleotide having a sequence complementary to the sequence and an enzyme having ribonuclease H (RNase H) activity, The method to amplify and detect the specific base sequence of Chlamydia trachomatis 3S rRNA of Claim 1 . A reagent for amplifying and detecting a specific base sequence of Chlamydia trachomatis 23S rRNA in a sample, comprising a first primer comprising a sequence complementary to the base sequence set forth in SEQ ID NO: 6 or 7, and SEQ ID NO: 17 or characterized in that it comprises a second primer having a sequence complementary to the nucleotide sequence set forth in 18, wherein the reagent.

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