JP2008161113A - Method for discriminating bacterium helicobacter pylori - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method by which a bacterium Helicobacter pyroli can clearly be discriminated with good reproducibility and to provide a reagent therefor. <P>SOLUTION: The method includes: a nucleic acid amplification reaction is conducted by using a primer (C) containing a nucleic acid sequence specific for a nucleic acid sequence encoding a C sequence of a CagA protein of the bacterium Helicobacter pyroli and complementary to a cagA gene, a primer (A) containing a nucleic acid sequence to be a pair of primers in the nucleic acid amplification reaction together with the primer (C), a primer (D) containing a nucleic acid sequence specific for the nucleic acid sequence encoding a D sequence and homologous to the cagA gene and a primer (B) containing a nucleic acid sequence to be a pair of primers in the nucleic acid amplification reaction together with the primer (D). Thus, the presence or absence of the C sequence and/or the D sequence to be an index to the toxicity of the CagA protein of the bacterium Helicobacter pyroli to be the object and the number of repetitions of the sequence are clearly determined. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for identifying Helicobacter pylori using the polymorphism of the CagA protein C-terminal region. The present invention is particularly useful for estimating the pathogenicity and carcinogenic risk of Helicobacter pylori.

  Many of Gram-negative bacteria having pathogenicity are recognized to exhibit pathogenicity by having a foreign gene group, and this foreign gene group is called a pathogenicity island (PAI). In Helicobacter pylori, it is reported that the gene cagA of cytoplasmic toxin-related protein (CagA), which is one of the pathogenic factors, is located in this PAI, and infection with a strain containing this cagA is associated with duodenal ulcer and gastric cancer It has been.

CagA protein is phosphorylated by Src when sent by Helicobacter pylori infected with stomach epithelial cells and binds to Src homology phosphatase-2 (SHP-2), thereby causing abnormalities via Ras and ERK. Dephosphorylation of focal adhesion kinase (FAK) related to cell proliferation and cell adhesion leads to improvement of cell motility. The CagA protein has phosphorylation regions named A sequence, B sequence, C sequence, and D sequence having five common amino acid motifs of EPIYA in the C-terminal region. The A sequence, B sequence, and C sequence are It exists in Helicobacter pylori which infects many Europeans and Americans, and A sequence, B sequence, and D sequence exist in Helicobacter pylori which infects many East Asians. SHP-2 is known to bind to the region of C sequence or D sequence, and D sequence has stronger binding power to SHP-2 than C sequence, and is more toxic and more likely to cause cancer. In addition, Westerners have a stronger binding force with SHP-2 when they have multiple C sequences, and East Asians have a stronger binding force with SHP-2 when they have multiple D sequences. It is known that cancer is likely to occur (see, for example, Non-Patent Documents 1 to 4).

Helicobacter pylori CagA protein has the above A, B, and C sequences and infects many Westerners (Western type), or has A, B, and D sequences and infects many East Asians For example, there is a sequencing method for determining the nucleic acid sequence of the cagA gene as a conventional technique for determining which type (East Asian type) corresponds to. In the sequencing method, information necessary for discrimination can be obtained by directly analyzing the nucleic acid sequence. However, since sequencing nucleic acid sequence preparation, DNA polymerase reaction, polyacrylamide gel electrophoresis, analysis of nucleic acid sequence, etc. are performed, a large amount of information is required. It takes effort and time. Further, labor can be saved by using a recent automatic sequencer, but there is a problem that an expensive apparatus is required.

Recently, an attempt to classify the cagA gene into a Western type or an East Asian type using a polymerase chain reaction (PCR) method (for example, see Patent Documents 1 and 2), which is a conventionally known gene amplification method. (See Non-Patent Document 5). This is done by performing PCR separately capable of amplifying the Western CagA protein gene or the East Asian CagA protein gene, and then determining the presence or absence of amplification by the hybridization reaction with the detection probe to determine the type of CagA protein. However, since this method requires two reactions of real-time PCR per sample, it involves complicated operations, and as described above, the C and / or D sequences that can be used as indicators of toxicity in the CagA protein are repeated. No knowledge about numbers can be obtained.
Japanese Examined Patent Publication No. 4-67960 Japanese Patent Publication No. 4-67957 Asahi et al., J. Exp. Med. 191, 593-602, (2000) Higashi et al., Proc. Natl. Acad. Sci. USA 99, 14428-14433, (2002) Azuma et al., J. Infect. Dis. 189, 820-827, (2004). Azuma et al., J. Clin. Microbiol. 42, 2508-2517, (2004). Yamazaki et al., FEMS Immunol. Med. Microbiol. 44, 261-268, (2005)

  An object of the present invention is to provide a method and a reagent therefor that can resolve Helicobacter pylori clearly and reproducibly by solving the above problems.

  In view of the above circumstances, the present inventors have completed the present invention as a result of intensive studies.

That is, the present invention has the following configuration.
1. A method for identifying Helicobacter pylori using the polymorphism of the C-terminal region of CagA protein,
(A) at least one primer (C) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the C sequence and complementary to the cagA gene;
(B) The at least one primer (A) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (C),
(C) at least one primer (D) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the D sequence and homologous to the cagA gene;
(D) The at least one primer (B) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (D),
(E) amplifying a nucleic acid fragment using a method characterized in that when the extension product of one primer is separated from its complement, it becomes a template for the other primer;
(F) An identification method comprising analyzing one or more amplified nucleic acid fragments obtained by the method.
2. A method for identifying Helicobacter pylori using the polymorphism of the C-terminal region of CagA protein,
(A) at least one primer (C) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the C sequence and homologous to the cagA gene;
(B) The at least one primer (A) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (C),
(C) at least one primer (D) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the D sequence and complementary to the cagA gene;
(D) The at least one primer (B) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (D),
(E) amplifying a nucleic acid fragment using a method characterized in that when the extension product of one primer is separated from its complement, it becomes a template for the other primer;
(F) An identification method comprising analyzing one or more amplified nucleic acid fragments obtained by the method.
3. An oligonucleotide comprising a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 4 or a nucleic acid sequence complementary to the sequence.
4). A method for identifying Helicobacter pylori using the polymorphism of the C-terminal region of CagA protein,
(A) a nucleic acid comprising at least 15 consecutive nucleotides among the nucleic acid sequence of SEQ ID NO: 1 as a primer (C) containing a nucleic acid sequence specific to the nucleic acid sequence encoding the C sequence and complementary to the cagA gene Using an oligonucleotide containing the sequence,
(B) As a primer (A) containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (C), a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 2 Use the oligonucleotide
(C) a nucleic acid comprising at least 15 consecutive nucleotides among the nucleic acid sequence of SEQ ID NO: 3 as a primer (D) containing a nucleic acid sequence specific to the nucleic acid sequence encoding the D sequence and homologous to the cagA gene Using an oligonucleotide containing the sequence,
(D) As a primer (B) containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (D), a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 4 Use the oligonucleotide
(E) amplifying a nucleic acid fragment using a method characterized in that when the extension product of one primer is separated from its complement, it becomes a template for the other primer;
(F) An identification method comprising analyzing one or more amplified nucleic acid fragments obtained by the method.
5. 5. The identification method according to any one of 1 to 4, wherein the Tm value of the primer (C, D) is higher than the Tm value of the primer (A, B).
6). 6. The identification method according to any one of 1 to 5, wherein the concentration when the primer (C, D) is used is higher than the concentration when the primer (A, B) is used.
7). The identification method according to any one of 1 to 6, wherein the presence or absence and the number of repetitions of the C sequence and / or the D sequence in the C-terminal region of the CagA protein are determined and used as an indicator of the toxicity of Helicobacter pylori.
8). Toxicity of Helicobacter pylori that infects many Europeans and Americans based on the number of amplified nucleic acid fragments generated by the primer (C) and the primer (A) is determined, and the amplification generated by the primer (D) and the primer (B) The identification method according to any one of 1 to 7, wherein the toxicity of Helicobacter pylori that infects many East Asians is determined based on the number of nucleic acid fragments.
9. The identification method according to any one of 1 to 8, wherein the toxicity of Helicobacter pylori is a risk of carcinogenesis for a carrier.
10. A reagent for distinguishing Helicobacter pylori using the polymorphism of the C-terminal region of CagA protein,
(A) at least one primer (C) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the C sequence and complementary to the cagA gene;
(B) The at least one primer (A) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (C),
(C) at least one primer (D) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the D sequence and homologous to the cagA gene;
(D) The at least one primer (B) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (D),
(E) An identification reagent comprising a primer, a DNA polymerase, dNTP, and a buffer that serve as a template for the other primer when the extension product of one primer is separated from its complement.
11. A reagent for distinguishing Helicobacter pylori using the polymorphism of the C-terminal region of CagA protein,
(A) at least one primer (C) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the C sequence and homologous to the cagA gene;
(B) The at least one primer (A) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (C),
(C) at least one primer (D) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the D sequence and complementary to the cagA gene;
(D) The at least one primer (B) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (D),
(E) An identification reagent comprising a primer, a DNA polymerase, dNTP, and a buffer that serve as a template for the other primer when the extension product of one primer is separated from its complement.
12. It comprises the identification reagent of 10 or 11, and estimates the risk of carcinogenesis due to Helicobacter pylori that infects many Westerners from the number of C-sequence repeats in the C-terminal region of CagA protein, and CagA protein C-terminal A reagent for estimating the risk of carcinogenesis caused by Helicobacter pylori that infects many East Asians from the number of D-sequence repeats in the region.

  The present invention provides a method that can clearly and easily identify Helicobacter pylori. In addition, according to the method of the present invention, the number of C and / or D sequence repeats that can be an indicator of toxicity in the CagA protein can also be determined, and Helicobacter pylori can be identified in more detail than the conventional methods. Is possible.

Hereinafter, the present invention will be described in detail.
In the present invention, the primer is an oligonucleotide having a sequence complementary to the chromosome of Helicobacter pylori or a fragment thereof, as long as it has characteristics commonly used for nucleic acid amplification, It may be modified as necessary. The primer chain length may be 9 to 35 bases, and preferably 11 to 30 bases. The amplification method using these primers is not particularly limited. A known amplification method may be used. Known amplification methods include, for example, PCR, NASBA, LCR, SDA, RCA, TMA, LAMP, ICAN, and UCAN methods.

In the present invention, nucleic acid amplification refers to amplification of a nucleic acid to be amplified from a nucleic acid in a sample (target nucleic acid) using complementarity of its inherent sequence. As a nucleic acid amplification method applicable to the method for identifying Helicobacter pylori in the present invention, it can be basically performed by using a conventional method, and usually Helicobacter pylori denatured into a single strand. When 4 types of deoxynucleoside triphosphate (dNTP) and DNA polymerase and 2 or more types of oligonucleotides or primers are allowed to act on the chromosome or fragment thereof, the sequence between the primers using the target nucleic acid as a template is amplified. The If the target nucleic acid does not contain a sufficient amount for detection, the Helicobacter pylori chromosome or a fragment thereof can be amplified in advance by the amplification reaction shown below.

Examples of nucleic acid amplification methods include PCR, NASBA (Nucleic acid sequence-based amplification method; Nature 350, 91 (1991)), LCR (International Publication No. 89/12696, JP-A-2-2934), SDA (Strand Displacement Amplification: Nucleic Acids Res. 20, 1691 (1992)), RCA (International Publication No. 90/1069), TMA (Transcription mediated amplification method; J. Clin. Microbiol. 31) 3270 (1993)), LAMP (loop-mediated isothermal amplification method: J. Clin Microbiol. 42: 1,956 (2004)), ICAN (isothermal and chimeric primer-initiated amplification of nucleic acid). acids: Kekkaku. 78, 533 (2003)), UCAN (Japan Cancer Society (H13.9.26-H13.9.28, held in Yokohama) or Japan Science and Technology Society (H13.11.8~H13.11.9, held in Tokyo) reference), and the like.

In particular, the PCR method repeats a cycle consisting of three steps of denaturation, annealing, and extension in the presence of a sample nucleic acid, four types of deoxynucleoside triphosphates, a pair of primers, and a heat-resistant DNA polymerase. In this method, the region of the sample nucleic acid sandwiched between the primers is exponentially amplified. That is, the sample nucleic acid is denatured in the denaturation step, each primer is hybridized with a region on the single-stranded sample nucleic acid complementary to each in the subsequent annealing step, and DNA is started from each primer in the subsequent extension step. A DNA strand complementary to each single-stranded sample nucleic acid serving as a template is extended by the action of the polymerase to form double-stranded DNA. By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of primers is theoretically amplified to 2 n times. Since the amplified DNA region exists in a large amount, it can be easily detected by a method such as electrophoresis. Therefore, if a gene amplification method is used, it is possible to detect even a very small amount of sample nucleic acid that could not be detected in the past (one molecule is acceptable), which is a very widely used technique recently. .

In the present invention, the nucleic acid sequence encoding the C sequence is a nucleic acid sequence encoding the C sequence in the C-terminal region of the CagA protein of Helicobacter pylori or a nucleic acid sequence complementary to the sequence, and also encodes the D sequence. The nucleic acid sequence is a nucleic acid sequence encoding a D sequence in the C-terminal region of CagA protein of Helicobacter pylori or a nucleic acid sequence complementary to the sequence.

As a method for analyzing an amplified nucleic acid fragment applicable to the method for identifying Helicobacter pylori of the present invention, a conventionally known nucleic acid analysis method can be used, and is not particularly limited, but agarose is used from the viewpoint of simplicity. Gel electrophoresis is preferred. In the case of using agarose gel electrophoresis, as an example, a primer (C) containing a nucleic acid sequence specific to a nucleic acid sequence encoding a C sequence and complementary to the cagA gene and a nucleic acid amplification reaction together with the primer (C) An amplified nucleic acid fragment produced by a primer (A) containing a nucleic acid sequence that can be a pair of primers, and a primer (D) containing a nucleic acid sequence specific to the nucleic acid sequence encoding the D sequence and homologous to the cagA gene Each primer is designed so that the chain lengths of the amplified nucleic acid fragments produced by the primer (B) containing a nucleic acid sequence that can be a pair of primers in the nucleic acid amplification reaction together with the primer (D) are different. By mixing these primers and amplifying the nucleic acid, the number of C and / or D sequence repeats that can serve as an indicator of the toxicity of the CagA protein of Helicobacter pylori is quickly determined from the amplified nucleic acid fragment pattern. it can.

One of the important disclosures of the present invention is that a primer (C) containing a nucleic acid sequence specific to the nucleic acid sequence encoding the C sequence and complementary to the cagA gene, and a pair in the nucleic acid amplification reaction together with the primer (C). A primer (A) containing a nucleic acid sequence that can serve as a primer for D, a primer (D) containing a nucleic acid sequence specific to the nucleic acid sequence encoding the D sequence and homologous to the cagA gene, and a nucleic acid together with the primer (D) For example, a primer (B) containing a nucleic acid sequence that can be a pair of primers in an amplification reaction is set as shown in FIG. 1 as an example, and nucleic acid amplification reaction is performed to determine the target CagA protein of Helicobacter pylori. The presence or absence of the C sequence and / or D sequence serving as an index of the identification and the number of repetitions of the sequence can be clearly determined. It lies in the discovery of an effect.

For example, when there is one C sequence in the CagA protein of Helicobacter pylori, as shown in FIG. 1 (A), a nucleic acid sequence specific for the C sequence and complementary to the cagA gene is used. One kind of amplified nucleic acid fragment is generated by the primer (A) containing the primer (C) and the primer (A) containing the primer (C) and a nucleic acid sequence that can be a pair of primers in the nucleic acid amplification reaction. In addition, when there are two C sequences in the CagA protein of Helicobacter pylori, as shown in FIG. 1 (B), a nucleic acid sequence specific for the C sequence and complementary to the cagA gene is used. Two kinds of amplified nucleic acid fragments having different chain lengths are generated by the primer (A) containing the primer (C) and the primer (A) containing a nucleic acid sequence that can be a pair of primers in the nucleic acid amplification reaction. Furthermore, when there is one D sequence in the CagA protein of Helicobacter pylori, as shown in FIG. 1 (C), a nucleic acid sequence specific to the nucleic acid sequence encoding the D sequence and homologous to the cagA gene is used. One kind of amplified nucleic acid fragment is generated by the primer (B) containing the primer (D) and the primer (B) containing a nucleic acid sequence that can be a pair of primers in the nucleic acid amplification reaction. In addition, when there are two D sequences in the CagA protein of Helicobacter pylori, as shown in FIG. 1 (D), a nucleic acid sequence specific to the nucleic acid sequence encoding the D sequence and homologous to the cagA gene is used. Two kinds of amplified nucleic acid fragments having different chain lengths are generated by the primer (B) containing the primer (D) and the primer (B) containing a nucleic acid sequence that can be a pair of primers in the nucleic acid amplification reaction.

The primers (A to D) used in the present invention need only have properties generally used for nucleic acid amplification, and may be modified as necessary. The modification is not particularly limited, and modifications such as biotinylation, digoxigenin binding, or fluorescent dye binding may be performed. According to the above configuration, the nucleic acid fragment amplified by the primer can be detected with high sensitivity. For example, if a primer is biotinylated, a marker linked with avidin or an anti-biotin antibody can be bound to a nucleic acid fragment amplified by the primer. Further, when digoxigenin is bound to a primer, a marker linked with an anti-digoxigenin antibody can be bound to the nucleic acid fragment amplified by the primer. And by detecting the marker couple | bonded with the amplified nucleic acid fragment, while the presence or absence of the nucleic acid fragment amplified by a primer can be detected, the quantity of the nucleic acid fragment amplified can be estimated. When a fluorescent dye is bound to the primer, the presence or absence of the fluorescent dye in the nucleic acid fragment to be amplified and the intensity of the fluorescence emitted from the fluorescent dye may be detected.

At this time, the marker is not particularly limited, and a known marker can be used as appropriate. For example, the marker is preferably an enzyme. More specifically, the marker is preferably alkaline phosphatase or peroxidase. When alkaline phosphatase is used as the marker, paranitrophenyl phosphate or CDP-star may be used as a substrate. When peroxidase is used as the marker, TMB, Lumi-Light (Roche Diagnostics), or SAT-1 (Dojin Chemical) may be used as a substrate. If the reaction between the enzyme and the substrate is detected, the presence or absence of the nucleic acid fragment amplified by the primer can be detected, and the amount of the nucleic acid fragment amplified can be estimated.

Moreover, it is preferable that the Tm value of a primer (C, D) is higher than the Tm value of a primer (A, B). The Tm value of the oligonucleotide primer is a temperature at which 50% of the double-stranded DNA molecules forming the hybrid are separated from each other, and the calculation method is not particularly limited as long as it is a known method, but the Neighbor Neighbor method, It is obtained by either the Wallace method or the GC% method and needs to satisfy the characteristics of the present invention. Note that a particularly preferable Tm value is calculated by the Nearest neighbor method. The difference in Tm value between the primer (C, D) and the primer (A, B) is not particularly limited as long as it is 0.5 ° C or higher, but is preferably 1 ° C or higher.

In order to clearly discriminate the number of C and / or D sequence repeats in the CagA protein C-terminal region of Helicobacter pylori, the concentration when using the primer (C, D) is the use of the primer (A, B). It is preferably higher than the concentration at the time. As an example, in the state where the concentration of the primer (C, D) is higher than the concentration of the primer (A, B), by appropriately adjusting the concentration of the primer (A) and / or primer (B), the C sequence and / or The accuracy in determining the number of repetitions of the D array can be improved. The concentration of the oligonucleotide primer at the time of use is not particularly limited, but it is preferably used in the range of 1 to 1500 nM, particularly preferably 50 to 500 nM. Within this range, it is preferable that the concentration of the primer (C, D) is higher than the concentration of the primer (A, B. The difference in concentration is not particularly limited, and is higher than 1 times by 100 depending on the sequence of the oligonucleotide primer. It may be appropriately selected within a range lower than twice.

To explain one of the above methods, it is a method for identifying Helicobacter pylori using the polymorphism of the C-terminal region of the CagA protein, including the following steps (a) to (b).
(A) A step of performing nucleic acid amplification using the following primers (i) to (iv):
(I) an oligonucleotide in which at least one primer (C) contains a nucleic acid sequence specific for the nucleic acid sequence encoding the C sequence and complementary to the cagA gene (ii) and at least one primer (A) An oligonucleotide containing a nucleic acid sequence that can serve as a pair of primers in a nucleic acid amplification reaction together with primer (C) (iii) at least one primer (D) is specific for the nucleic acid sequence encoding D sequence and homologous to the cagA gene An oligonucleotide containing a typical nucleic acid sequence,
(Iv) The oligonucleotide (b) one or more obtained in step (a), wherein at least one primer (B) contains a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (D) The step of analyzing the presence and type of the amplified nucleic acid fragment obtained from the primer (C), or the presence and type of the amplified nucleic acid fragment obtained from one or more primers (D), or the following (a) to It is a method for identifying Helicobacter pylori using the polymorphism of the C-terminal region of CagA protein, including the step (b).
(A) A step of performing nucleic acid amplification using the following primers (i) to (iv):
(I) an oligonucleotide in which at least one primer (C) contains a nucleic acid sequence specific for the nucleic acid sequence encoding the C sequence and homologous to the cagA gene; (ii) and at least one primer (A) , An oligonucleotide containing a nucleic acid sequence that can serve as a pair of primers in a nucleic acid amplification reaction together with primer (C) (iii) at least one primer (D) is specific for the nucleic acid sequence encoding D sequence and complementary to the cagA gene Oligonucleotide (iv) containing a typical nucleic acid sequence (iv) In addition, the oligonucleotide (b) step (a) wherein the at least one primer (B) contains a nucleic acid sequence that can serve as a pair of primers in a nucleic acid amplification reaction together with the primer (D) Amplified nucleic acid obtained from one or more primers (C) obtained in Presence and type of piece, or the step of analyzing the one or absence and type of amplified nucleic acid fragments obtained from more primers (D)

The oligonucleotide of the present invention is for amplifying the chromosome of Helicobacter pylori or a fragment thereof.

An oligonucleotide containing a sequence having 15 to 23 consecutive bases of the base sequence shown in SEQ ID NO: 1 in the sequence listing is a primer containing a nucleic acid sequence specific for a nucleic acid sequence encoding a C sequence and complementary to a cagA gene (C) used in combination with a primer (A) containing a nucleic acid sequence that can serve as a pair of primers in a nucleic acid amplification reaction together with the primer (C), and the presence or absence of the C sequence in the CagA protein of Helicobacter pylori Used to determine the number of repetitions.

An oligonucleotide containing a sequence having 15 to 27 bases in the base sequence shown in SEQ ID NO: 2 in the sequence listing is a primer (A) containing a nucleic acid sequence that can serve as a pair of primers in a nucleic acid amplification reaction together with the primer (C) In combination with a primer (C) containing a nucleic acid sequence that is specific for the nucleic acid sequence encoding the C sequence and complementary to the cagA gene, and the presence or absence of the C sequence in the CagA protein of Helicobacter pylori Used to determine the number of repetitions.

An oligonucleotide containing a sequence having 15 to 24 consecutive base sequences shown in SEQ ID NO: 3 in the sequence listing is a primer containing a nucleic acid sequence specific to a nucleic acid sequence encoding a D sequence and complementary to a cagA gene In combination with primer (B) containing a nucleic acid sequence that can be used as a pair of primers in a nucleic acid amplification reaction together with primer (D), and the presence or absence of D sequence in CagA protein of Helicobacter pylori Used to determine the number of repetitions.

An oligonucleotide containing a sequence having 15 to 23 consecutive bases of the base sequence shown in SEQ ID NO: 4 in the Sequence Listing is a primer (B) containing a nucleic acid sequence that can serve as a pair of primers in a nucleic acid amplification reaction together with the primer (D) In combination with a primer (D) containing a nucleic acid sequence specific to the nucleic acid sequence encoding the D sequence and complementary to the cagA gene, and the presence or absence of the D sequence in the CagA protein of Helicobacter pylori Used to determine the number of repetitions.

Preferred examples of the reagent composition for using the method for identifying Helicobacter pylori of the present invention include (a) an enzyme having DNA polymerase activity, (b) a primer for nucleic acid amplification, (c) dNTP, and (d) a reaction. A buffer, (e) a nucleic acid-containing sample, and the like.
(A) “Enzyme having DNA polymerase activity” includes various origins such as T4 or T7 phage, Escherichia coli, Thermococcus genus, Thermus genus, Pyrococcus genus, Bacillus genus, etc. DNA polymerases and improved variants thereof can be used without any particular limitation. However, when PCR is used as a nucleic acid amplification method, KOD DNA polymerase, Taq DNA polymerase, Tth DNA polymerase, Pfu DNA polymerase, etc. It is preferable to use the enzyme. Of these, KOD DNA polymerase or a DNA polymerase obtained by improving the enzyme is most preferable from the viewpoints of amplification amount and reaction rate. Specifically, in the case of KOD Dash DNA polymerase, the use of about 1 to 200 units / ml is preferred, and the use of about 10 to 100 units / ml is more preferred.
(B) The “primer for nucleic acid amplification” includes about 1-1500 nM. Above all, about 50 to 500 nM is more preferable in order to increase the amplification amount and specificity.
(C) “dNTP” includes about 50 to 1000 μM. About 100 to 700 μM is more preferable, and about 200 to 400 μM is more preferable.
(D) About “reaction buffer”, in order to maximize the ability of the enzyme contained in the reagent for using the method for identifying Helicobacter pylori of the present invention, the concentration and solution of buffer solution and metal salt The pH and the like can be appropriately selected. Further, for the purpose of enhancing the stability of the enzyme, an additive such as bovine serum albumin may be appropriately mixed with the reagent.
(E) A “nucleic acid-containing sample” can be prepared from any material such as lysate derived from bacteria, animal or plant tissue, or individual cells. The method for preparing the sample is not particularly limited. For example, the sample may be prepared from a patient's blood or tissue by a known method. Typical examples include phenol / chloroform extraction method (Biochimica et Biophysica acta 72, 619-629, 1963), alkaline SDS method (Nucleic Acids Res. 7, 1513-1523, 1979). ) Etc. in the liquid phase. As a system using a nucleic acid binding carrier for nucleic acid isolation, a method using glass particles and a sodium iodide solution (Proc. Natl. Acad. Sci. USA, Vol. 76-2, pages 615 to 619, 1979) and a method using hydroxyapatite (Japanese Patent Laid-Open No. 63-263093). Examples of other methods include a method using silica particles and chaotropic ions (J. Clinical Microbiology Vol. 28-3, 495-503, 1990, JP-A-2-289596). Moreover, the nucleic acid may be dissolved in the sample, or may be fixed to a solid phase.

The method for identifying Helicobacter pylori and the reagent for identification of the present invention are preferably used for predicting the risk of carcinogenesis due to Helicobacter pylori. Examples of the cancer include stomach cancer. As described above, the polymorphism of the C-terminal region of CagA protein can be detected by using the identification method and identification reagent of the present invention. More specifically, by using the identification method and identification reagent of the present invention, it is possible to detect the presence or absence of a C sequence and / or D sequence closely related to carcinogenesis, and the number of C sequences and / or D sequences. Can also be detected. The presence or absence of carcinogenesis can be predicted by the presence or absence of the C sequence and / or the D sequence, and the possibility of carcinogenesis (the C sequence is determined by the number of the C and / or D sequences). And / or the greater the D sequence, the greater the risk of carcinogenesis). As an example, for Helicobacter pylori that frequently infects Westerners, the possibility of carcinogenesis can be predicted by the presence or absence of the C sequence, and the possibility of carcinogenesis (the larger the C sequence, the more The risk of cancer is high) can be predicted by the number of C sequences. In addition, for Helicobacter pylori that infects many East Asians, the possibility of carcinogenesis can be predicted by the presence or absence of the D sequence, and the possibility of carcinogenesis (the more the D sequence, the more The risk of cancer is high) can be predicted by the number of D sequences.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1
Identification of Helicobacter pylori (1) Synthesis of oligonucleotides Oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 1 to 4 (hereinafter referred to as Oligo 1) using the DNA synthesizer type 392 manufactured by PerkinElmer, Inc. by the phosphoramidite method -4)) was synthesized. The synthesis was performed according to the manual, and the deprotection of each oligonucleotide was carried out overnight at 55 ° C. with ammonia water. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, DNA commissioned companies (Nippon Bioservice, Sigma-Aldrich Japan, Operon Biotechnology, etc.) were requested.
Oligo 1 is an antisense strand, a primer (C) containing a nucleic acid sequence that is specific to the nucleic acid sequence encoding the C sequence and complementary to the cagA gene, and is an oligonucleotide for amplification reaction in combination with oligo 2 of the sense strand Used as. Oligo 2 is a primer (A) containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with primer (C). Oligo 3 is a sense strand that is a primer (D) containing a nucleic acid sequence specific to the nucleic acid sequence encoding the D sequence and complementary to the cagA gene. Used as an oligonucleotide. Oligo 4 is a primer (B) containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with primer (D).

(2) Analysis of Helicobacter pylori cagA gene by PCR method Using the DNA solution extracted from Helicobacter pylori bacteria of sample numbers 1 to 6 in Table 1 by the phenol-chloroform method as a sample, the following reagents were added. Then, the cagA gene of Helicobacter pylori by PCR was analyzed under the following conditions.

Reagents A 25 μl solution containing the following reagents was prepared.
KOD DNA polymerase reaction solution Oligo 1 10 pmol,
5 pmol of oligo 2,
Oligo 3 10 pmol,
Oligo 4 5 pmol,
X10 buffer 2.5 μl,
2.5 μl of 2 mM dNTP,
1 μl of 25 mM MgCl ₂ ,
KOD DNA polymerase 0.5U,
Extracted DNA solution 100ng

Amplification conditions 94 ° C, 5 minutes 94 ° C, 15 seconds,
60 ° C for 30 seconds,
68 ℃, 30 seconds (35 cycles)
25 ° C for 15 minutes.

(3) Detection using agarose gel electrophoresis After 5 μl of the amplification reaction solution was electrophoresed on a 3% agarose gel and stained with ethidium bromide, fluorescence under ultraviolet irradiation was detected. A photograph obtained by agarose electrophoresis is shown in FIG. The lane numbers in FIG. 2 correspond to the sample numbers in Table 1. The electrophoresis was performed at a constant voltage of 100 V for 60 minutes. In addition to the reaction solution, a molecular weight marker was also run at the same time, which was used as a reference when comparing the chain lengths of the detected amplified nucleic acid fragments.

(4) Results As is clear from the sample numbers 1 to 6 in FIG. 2 (and Table 1), according to the method for identifying Helicobacter pylori of the present invention, the C sequence repeats more than the number of amplified nucleic acid fragments longer than 500 bp. The number of repeats of the D sequence can be determined from the number of amplified nucleic acid fragments shorter than 500 bp. These results indicate that the six strains were identified using the sequencing method, which can obtain information necessary for discrimination by directly analyzing the nucleic acid sequence, but has the disadvantages that a great deal of labor and time are required. Consistent with (Table 1).

That is, in the method for identifying Helicobacter pylori according to the present invention, the number of work steps required for the analysis is greatly reduced while realizing the same discrimination ability as the conventional method, and CagA of Helicobacter pylori can be easily and quickly. It is possible to clearly determine the presence / absence of a C sequence and / or a D sequence, which are indicators of protein toxicity, and the number of repetitions of the sequence.

According to the present invention, it is possible to clearly determine the presence or absence of the C sequence and / or D sequence and the number of repetitions of the sequence, which are indicators of the toxicity of the CagA protein of Helicobacter pylori. Therefore, it is expected to make a significant contribution to the industry because it can obtain results with good reproducibility quickly and easily.

An example of a method for identifying Helicobacter pylori Identification results of Helicobacter pylori

Claims (12)

A method for identifying Helicobacter pylori using the polymorphism of the C-terminal region of CagA protein,
(A) at least one primer (C) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the C sequence and complementary to the cagA gene;
(B) The at least one primer (A) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (C),
(C) at least one primer (D) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the D sequence and homologous to the cagA gene;
(D) The at least one primer (B) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (D),
(E) amplifying a nucleic acid fragment using a method characterized in that when the extension product of one primer is separated from its complement, it becomes a template for the other primer;
(F) An identification method comprising analyzing one or more amplified nucleic acid fragments obtained by the method. A method for identifying Helicobacter pylori using the polymorphism of the C-terminal region of CagA protein,
(A) at least one primer (C) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the C sequence and homologous to the cagA gene;
(B) The at least one primer (A) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (C),
(C) at least one primer (D) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the D sequence and complementary to the cagA gene;
(D) The at least one primer (B) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (D),
(E) amplifying a nucleic acid fragment using a method characterized in that when the extension product of one primer is separated from its complement, it becomes a template for the other primer;
(F) An identification method comprising analyzing one or more amplified nucleic acid fragments obtained by the method. An oligonucleotide comprising a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in any one of SEQ ID NOs: 1 to 4 or a nucleic acid sequence complementary to the sequence. A method for identifying Helicobacter pylori using the polymorphism of the C-terminal region of CagA protein,
(A) a nucleic acid comprising at least 15 consecutive nucleotides among the nucleic acid sequence of SEQ ID NO: 1 as a primer (C) containing a nucleic acid sequence specific to the nucleic acid sequence encoding the C sequence and complementary to the cagA gene Using an oligonucleotide containing the sequence,
(B) As a primer (A) containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (C), a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 2 Use the oligonucleotide
(C) a nucleic acid comprising at least 15 consecutive nucleotides among the nucleic acid sequence of SEQ ID NO: 3 as a primer (D) containing a nucleic acid sequence specific to the nucleic acid sequence encoding the D sequence and homologous to the cagA gene Using an oligonucleotide containing the sequence,
(D) As a primer (B) containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (D), a nucleic acid sequence consisting of at least 15 consecutive bases of the nucleic acid sequence shown in SEQ ID NO: 4 Use the oligonucleotide
(E) amplifying a nucleic acid fragment using a method characterized in that when the extension product of one primer is separated from its complement, it becomes a template for the other primer;
(F) An identification method comprising analyzing one or more amplified nucleic acid fragments obtained by the method. The identification method according to any one of claims 1 to 4, wherein the Tm value of the primer (C, D) is higher than the Tm value of the primer (A, B). The identification method according to any one of claims 1 to 5, wherein the concentration when the primer (C, D) is used is higher than the concentration when the primer (A, B) is used. The identification according to any one of claims 1 to 6, wherein the presence or absence and the number of repetitions of the C sequence and / or D sequence in the C-terminal region of the CagA protein are determined and used as an indicator of Helicobacter pylori toxicity. Method. Toxicity of Helicobacter pylori that infects many Europeans and Americans based on the number of amplified nucleic acid fragments generated by the primer (C) and the primer (A) is determined, and the amplification generated by the primer (D) and the primer (B) The identification method according to any one of claims 1 to 7, wherein the toxicity of Helicobacter pylori that infects many East Asians is determined based on the number of nucleic acid fragments. The identification method according to claim 1, wherein toxicity of Helicobacter pylori is a risk of carcinogenesis for a carrier. A reagent for distinguishing Helicobacter pylori using the polymorphism of the C-terminal region of CagA protein,
(A) at least one primer (C) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the C sequence and complementary to the cagA gene;
(B) The at least one primer (A) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (C),
(C) at least one primer (D) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the D sequence and homologous to the cagA gene;
(D) The at least one primer (B) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (D),
(E) An identification reagent comprising a primer, a DNA polymerase, dNTP, and a buffer that serve as a template for the other primer when the extension product of one primer is separated from its complement. A reagent for distinguishing Helicobacter pylori using the polymorphism of the C-terminal region of CagA protein,
(A) at least one primer (C) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the C sequence and homologous to the cagA gene;
(B) The at least one primer (A) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (C),
(C) at least one primer (D) is an oligonucleotide containing a nucleic acid sequence specific for the nucleic acid sequence encoding the D sequence and complementary to the cagA gene;
(D) The at least one primer (B) is an oligonucleotide containing a nucleic acid sequence that can be a pair of primers in a nucleic acid amplification reaction together with the primer (D),
(E) An identification reagent comprising a primer, a DNA polymerase, dNTP, and a buffer that serve as a template for the other primer when the extension product of one primer is separated from its complement. The CagA protein comprising the reagent for identification according to claim 10 or 11, wherein the risk of carcinogenesis due to Helicobacter pylori that infects many Westerners is estimated from the number of C-sequence repeats in the C-terminal region of the CagA protein. A reagent for estimating the risk of carcinogenesis due to Helicobacter pylori that infects many East Asians from the number of D-sequence repeats in the C-terminal region.