JPH10286099A - Primer for detecting helicobacter pylori bacterium and examination of clarithromycin resistance of helicobacter pylori bacterium with the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject new primer comprising an oligonucleotide having a specific base sequence, capable of specifically and highly sensitively amplifying the gene of Helicobacter pylori which is a bacterium causing stomach ulcer, and useful for testing the resistance of the Helicobacter pylori against clarithromycin, etc. SOLUTION: This new forward side primer for detecting Helicobacter pylori comprises an oligonucleotide having a base sequence comprising the continuous 15-38 bases of the base sequence of formula I, and the new reverse side primer comprises an oligonucleotide having a base sequence comprising the continuous 15-38 bases of the base sequence of formula II. The primers specifically and highly sensitively amplify the fragment of the<23> SrRNA of the Helicobacter pylori, and are used for the test of the Helicobacter pylori against clarithromycin. The primers are obtained by selecting the forward side and reverse side base sequences capable of amplifying the clarithromycin resistance- expressing mutated portion of the<23> SrRNA gene of the Helicobacter pylori.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to H. pylori (Helico).
The present invention relates to a primer for detecting bacter pylori) and a method for testing clarithromycin resistance of H. pylori using the same.

[0002]

2. Description of the Related Art H. pylori is a causative agent of gastric ulcer and has recently been suggested to be associated with gastric cancer. Antibiotics are used as eradication treatments for H. pylori. Among them, clarithromycin (hereinafter sometimes referred to as "CAM") is the most effective in eliminating bacteria and is the most used drug in Japan. is there.

However, recently, CAM-resistant H. pylori has been reported, and this drug resistance has been reported at position 2143 of the 23S rRNA gene of H. pylori (205 in the position of the corresponding gene in E. coli).
It has also been reported that adenine at position 8) is mutated to guanine, or that adenine at position 2144 adjacent thereto is mutated to guanine (JAMES VERSALOVIC et al., ANTIMICROBIAL AGEN).
TS AND CHEMOTHERAPY, Vol.40, No.2, Feb.1996, p.477-
480). In CAM resistant bacteria, the therapeutic effect of CAM is significantly reduced. Therefore, a method for testing whether or not H. pylori is resistant to CAM with high sensitivity is desired.

[0004]

In order to examine whether or not H. pylori is resistant to CAM, a fragment containing the site where the above mutation occurs in the 23S rRNA gene is amplified by a nucleic acid amplification method, and the site of the site in the amplification product is amplified. It is conceivable to examine the presence or absence of the mutation. In this case, what is amplified by the nucleic acid amplification method must be only a fragment of the 23S rRNA gene of H. pylori, and other genes of H. pylori and genes of other microorganisms must not be amplified. Furthermore, in order to enhance the sensitivity of the test, it is necessary to efficiently amplify the nucleic acid. However, no primer that satisfies such specificity and sensitivity of nucleic acid amplification is known.

[0005] Accordingly, an object of the present invention is to provide a method for controlling H. pylori
An object of the present invention is to provide a primer for detecting H. pylori, which is capable of specifically and highly sensitively amplifying a fragment of an SrRNA gene by a nucleic acid amplification method. The object of the present invention is
An object of the present invention is to provide a method for testing whether or not H. pylori is CAM-resistant using the primer of the present invention.

[0006]

Means for Solving the Problems As a result of intensive studies, the present inventors have found a primer capable of specifically and highly sensitively amplifying a 23S rRNA gene fragment of H. pylori by a nucleic acid amplification method, and have achieved the present invention. completed.

[0007] That is, the present invention relates to SEQ ID NO: 1
A forward primer for detecting H. pylori, comprising an oligonucleotide having a nucleotide sequence consisting of 15 to 38 consecutive nucleotides of the following nucleotide sequence: The present invention also provides a reverse primer for detecting H. pylori, comprising an oligonucleotide having a nucleotide sequence consisting of 15 to 32 consecutive nucleotides of the nucleotide sequence shown by SEQ ID NO: 4 in the sequence listing. Further, the present invention provides a reverse primer for detecting H. pylori, comprising an oligonucleotide having a nucleotide sequence consisting of 15 to 38 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO: 7 in the sequence listing. Further, the present invention provides a method for amplifying a fragment of H. pylori 23S rRNA gene by a nucleic acid amplification method using the above-mentioned forward primer and reverse primer of the present invention.
Provided is a method for testing clarithromycin resistance of H. pylori, which comprises examining the presence or absence of a mutation in which adenine is mutated to guanine at positions 2143 and / or 2144 of an RNA gene.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The forward primer of the present invention used for amplifying a 23S rRNA gene fragment containing the above mutant portion of H. pylori is as described above.
15 consecutive nucleotides represented by SEQ ID NO: 1 in the sequence listing
It consists of an oligonucleotide having a base sequence consisting of bases to 38 bases. Among these, a primer comprising an oligonucleotide having a base sequence consisting of 15 to 22 consecutive bases of the base sequence shown by SEQ ID NO: 2 in the sequence listing is preferable, and further having a base sequence shown by SEQ ID NO: 3 A primer consisting of 18 bases (hereinafter, this primer may be referred to as “SF-4”)
Is preferred.

[0009] 23S containing the above mutant portion of H. pylori
The reverse primer of the present invention used for amplifying an rRNA gene fragment is roughly classified into two types.
As described above, the first reverse-side primer is composed of an oligonucleotide having a base sequence consisting of 15 to 32 consecutive bases of the base sequence represented by SEQ ID NO: 4 in the sequence listing. Among these, a primer consisting of an oligonucleotide having a base sequence consisting of 15 to 23 bases consecutive to the base sequence shown by SEQ ID NO: 5 in the sequence listing is preferable, and further having a base sequence shown by SEQ ID NO: 6 A primer consisting of 19 bases (hereinafter, this primer may be referred to as “R-1”) is preferred.

[0010] The second reverse-side primer is composed of an oligonucleotide having a base sequence consisting of 15 to 38 consecutive bases of the base sequence represented by SEQ ID NO: 7 in the sequence listing. Among these, a primer comprising an oligonucleotide having a base sequence consisting of 15 to 22 consecutive bases of the base sequence represented by SEQ ID NO: 8 in the sequence listing is preferable, and further having a base sequence represented by SEQ ID NO: 9 A primer consisting of 19 bases (hereinafter, this primer may be referred to as “R-3”) is preferred.

The base sequence near the amplification region of the 23S rRNA gene of H. pylori and the above-mentioned forward primer SF-4, reverse primers R-1 and R-3
Are shown in FIG. In addition, A at position 102 and A at position 103 in FIG. 1 (underlined in the figure) correspond to position 214 of the 23S rRNA gene of H. pylori.
No. 3 and No. 2144, and these A (adenine)
Are mutated to G (guanine) to confer CAM resistance. The base sequence shown at the top of each row in FIG. 1 is a previously reported base sequence, and the base sequence shown below is the same as that of H. pylori isolated by the present inventors. This is the base sequence of the region.

[0012] As described above, the present invention also provides a method for testing whether or not H. pylori is CAM-resistant using the forward primer and the first and / or second reverse primer of the present invention. I do. Hereinafter, this method will be described.

The specimen to be used in the method of the present invention may be any specimen for which it is desired to test the CAM resistance of H. pylori, such as gastric juice or gastric biopsy. Is not limited to these.
Extraction of DNA from these specimens can be easily performed by a well-known method using a commercially available kit or the like.

In the method of the present invention, first, the 23S rRNA gene of H. pylori in a sample is used as a template by a nucleic acid amplification method using the forward primer of the present invention and the first or second reverse primer of the present invention. Amplify the gene fragment. As the nucleic acid amplification method, any method for amplifying a nucleic acid using a primer can be adopted, and a typical method is PCR. PCR is a well-known method in this field, and kits and devices for the PCR are commercially available. Therefore, PCR can be easily performed using such commercially available kits and devices.

Amplification can be carried out using either a combination of the forward primer and the first reverse primer or a combination of the forward primer and the second reverse primer. However, in order to further improve the specificity and sensitivity, amplification is first performed using the forward primer and the first reverse primer, and then the obtained amplification product is used as a template, and the first reverse primer is used. And amplification using the second forward primer.
By the nucleic acid amplification, a region sandwiched between the used forward primer and reverse primer is amplified.

Next, the presence or absence of a mutation in which adenine is changed to guanine is examined at positions 2143 and / or 2144 of the 23S rRNA gene of H. pylori located in the obtained amplification product. This can be done by any method that can determine whether such mutations have occurred at positions 2143 and 2144. For example, it can be performed by determining the base sequence of the amplification product. However, the so-called RF
It is convenient and preferable to carry out by a method called LP. In this method, an amplification product is digested with a predetermined restriction enzyme, and the digestion product is subjected to electrophoresis to detect a band. Here, as the restriction enzyme, those whose cleavage occurs or not depending on the presence or absence of the mutation are selected and used. As such a restriction enzyme, MboII can be mentioned as a restriction enzyme for detecting the mutation at position 2143;
BsaI can be mentioned as a restriction enzyme for detecting a position mutation. When the amplification product is digested with these restriction enzymes, if the amplified gene is of a normal type, no cleavage occurs with any of the restriction enzymes, and only one band is observed by electrophoresis in each case. On the other hand, when A at position 2143 is mutated to G, cleavage occurs with MboII and two bands are observed, but only one band is observed without cleavage even after digestion with BsaI. When A at position 2144 is mutated to G, cleavage occurs with BsaI, and two bands are observed. However, only one band is observed without cleavage even after digestion with MboII. Therefore, by dividing the amplified product into two, digesting each with MboII or BsaI, and examining the electrophoresis pattern, normal type, 2143 mutant type and 2144 position mutant type can be distinguished.

[0017]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below more specifically based on embodiments. However, the present invention is not limited to the following examples.

The gastric juice was collected from Example gastric ulcer patients, from gastric fluid 100μl using a commercially available DNA extraction kit prepared DNA. This D
Using the NA as a template, the forward primer S
The first PCR was carried out using F-4 and reverse-side primer R-1 in a total volume of 100 μl. The PCR conditions were as follows: denaturation step: 94 ° C., 15 seconds; annealing step: 50 ° C., 30 seconds; extension step: 72 ° C., 1 minute. This cycle was repeated 30 times. The other PCR conditions were the same as those used in the conventional method (Gene Manipulation Technical Manual, published by Medical Shoin, 1995). Using 2 μl of the obtained reaction product, a second PCR was carried out using the forward primer SF-4 and the reverse primer R-3 to make the total amount 100 μl. The temperature and time of each of the denaturation, annealing, and extension steps were the same as in the first PCR, and the cycle of the steps was repeated 20 times.

The obtained PCR product is placed in two tubes of 10 μl each, and 1 unit of each is added to MboII or Bs.
aI (all manufactured by New England Biolabs) and added 37
The reaction was performed at 12 ° C. for 12 hours. 10 μl of each reaction product was taken, electrophoresed on an agarose gel containing ethidium bromide, and the band size was confirmed.

As a result, in the normal type, only one band of 135 bp was observed when digested with any of the restriction enzymes. On the other hand, those having a mutation at position 2143 showed a total of two bands near 39 bp and 96 bp when digested with MboII, but 135 bp when digested with BsaI.
Only one band was observed. In addition, those having a mutation at position 2144 have 40 bp and 95 bp when digested with BsaI.
A total of two bands were observed around bp, but only one 135 bp band was observed when digested with MboII.

[0021]

Industrial Applicability According to the present invention, 23SrRN of H. pylori
A primer capable of specifically and highly sensitively amplifying a fragment of the A gene was provided. The present invention also provides a method for accurately and simply testing whether or not H. pylori is CAM-resistant.

[0022]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 38 Sequence type: nucleic acid sequence GTGAAATTGT AGTGGAGGTG AAAATTCCTC CTACCCGC 38

SEQ ID NO: 2 Sequence length: 22 Sequence type: nucleic acid sequence GTAGTGGAGG TGAAAATTCC TC 22

SEQ ID NO: 3 Sequence length: 18 Sequence type: nucleic acid sequence AGTGGAGGTG AAAATTCC 18

SEQ ID NO: 4 Sequence length: 32 Sequence type: nucleic acid sequence CCATAAGAGC CAAAGCCCTT ACTTCAAAGC CT 32

SEQ ID NO: 5 Sequence length: 23 Sequence type: nucleic acid sequence CATAAGAGCC AAAGCCCTTA CTT 23

SEQ ID NO: 6 Sequence length: 19 Sequence type: nucleic acid sequence TAAGAGCCAA AGCCCTTAC 19

SEQ ID NO: 7 Sequence length: 38 Sequence type: nucleic acid sequence TGCGCATGAT ATTCCCATTA GCAGTGCTAA GTTGTAGT 38

SEQ ID NO: 8 Sequence length: 22 Sequence type: nucleic acid sequence ATATTCCCAT TAGCAGTGCT AA 22

SEQ ID NO: 9 Sequence length: 18 Sequence type: nucleic acid sequence ATTCCCATTA GCAGTGCT 18

[Brief description of the drawings]

FIG. 1 is a diagram showing the nucleotide sequence of the 23S rRNA gene of H. pylori amplified using the primer of the present invention, together with the region to which the primer of the present invention hybridizes.

Claims (13)

[Claims] 1. A forward primer for detecting H. pylori, comprising an oligonucleotide having a nucleotide sequence consisting of 15 to 38 consecutive nucleotides of the nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing. 2. The primer according to claim 1, comprising an oligonucleotide having a base sequence consisting of 15 to 22 consecutive bases of the base sequence represented by SEQ ID NO: 2 in the sequence listing. 3. The primer according to claim 2, comprising an oligonucleotide having a base sequence represented by SEQ ID NO: 3 in the sequence listing. 4. A reverse primer for detecting H. pylori comprising an oligonucleotide having a nucleotide sequence consisting of 15 to 32 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO: 4 in the sequence listing. 5. The primer according to claim 4, comprising an oligonucleotide having a base sequence consisting of 15 to 23 consecutive bases of the base sequence represented by SEQ ID NO: 5 in the sequence listing. 6. The primer according to claim 5, comprising an oligonucleotide having a base sequence represented by SEQ ID NO: 6 in the sequence listing. 7. A reverse primer for detecting H. pylori, comprising an oligonucleotide having a nucleotide sequence consisting of 15 to 38 consecutive nucleotides of the nucleotide sequence shown in SEQ ID NO: 7 in the sequence listing. 8. The primer according to claim 7, comprising an oligonucleotide having a nucleotide sequence consisting of 15 to 22 consecutive nucleotides of the nucleotide sequence represented by SEQ ID NO: 8 in the sequence listing. 9. The primer according to claim 8, comprising an oligonucleotide having a base sequence represented by SEQ ID NO: 9 in the sequence listing. 10. A 23S rRNA gene of H. pylori by a nucleic acid amplification method using the forward primer according to any one of claims 1 to 3 and the reverse primer according to any one of claims 4 to 6. Amplifying the fragment, at positions 2143 and / or 2144 of the 23S rRNA gene of H. pylori located in the amplified fragment;
A method for testing clarithromycin resistance of H. pylori, comprising examining the presence or absence of a mutation in which adenine is changed to guanine. 11. The 23S rRNA gene of H. pylori by a nucleic acid amplification method using the forward primer according to any one of claims 1 to 3 and the reverse primer according to any one of claims 7 to 9. Amplifying the fragment, at positions 2143 and / or 2144 of the 23S rRNA gene of H. pylori located in the amplified fragment;
A method for testing clarithromycin resistance of H. pylori, comprising examining the presence or absence of a mutation in which adenine is changed to guanine. 12. The 23S rRNA gene of H. pylori by a nucleic acid amplification method using the forward primer according to any one of claims 1 to 3 and the reverse primer according to any one of claims 4 to 6. Amplifying the fragment, and then using the resulting amplification product as a template, the forward primer according to any one of claims 1 to 3 and the reverse primer according to any one of claims 7 to 9 Using H. pylori by nucleic acid amplification method
Amplifying a fragment of the 3S rRNA gene and examining the presence or absence of a mutation in which adenine is mutated to guanine at positions 2143 and / or 2144 of the 23S rRNA gene of H. pylori located in the amplified fragment. Test method for clarithromycin resistance. 13. Examining the presence or absence of a mutation at which adenine is mutated to guanine at position 2143 and / or 2144 of the 23S rRNA gene of H. pylori,
The method according to any one of claims 10 to 12, comprising digesting the amplified product with MboII and BsaI, and subjecting the digested product to electrophoresis to detect a band.