JPH0965899A - Oligonucleotide, primer for detecting helicobacter pylori consisting thereof and detection of helicobacter pylori using the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new oligonucleotide having a specific base sequence, and useful as e.g. a primer for PCR process capable of detecting in high sensitivity Helicobacter pylori, a pathogen causing digestive organ-related diseases such as gastritis. SOLUTION: This new oligonucleotide has a base sequence of formula I, II, III, IV, V, VI or VII, being useful as e.g. a primer for PCR process, capable of detecting in high sensitivity Helicobacter pylori, a pathogen causing digestive organ-related diseases such as gastritis. Any one of these new oligonucleotides, which has a base sequence subject to hybridization with the domain in the genome of Helicobacter pylori, part of the base sequences of which has been known, is obtained by chemical synthesis, using a DNA synthesizer, of the above base sequence subject to hybridization with the known base sequence. A sample DNA is amplified by PCR process using this oligonucleotide as primer to detect the corresponding microbes in high sensitivity.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a novel oligonucleotide useful for detecting Helicobacter pylori, a PCR primer comprising the same, and a method for detecting Helicobacter pylori using the same.

[0002]

2. Description of the Related Art Since Helicobacter pylori is a pathogen that causes gastrointestinal diseases such as gastritis, it may be necessary to examine whether or not Helicobacter pylori is present in the body. Conventionally, there is known a method of amplifying a part of the gene of Helicobacter pylori by PCR and detecting the amplified product to test whether or not Helicobacter pylori is present. In the conventional method, a set of oligonucleotides having the nucleotide sequences represented by SEQ ID NOs: 8 and 9 in the sequence listing is used as a primer.

[0003]

Needless to say, the higher the detection sensitivity in the detection of Helicobacter pylori, the better.

Therefore, the object of the present invention is to be used as a primer for the detection of Helicobacter pylori by PCR, which makes it possible to detect Helicobacter pylori with higher sensitivity than the conventional methods for detecting Helicobacter pylori. A novel oligonucleotide, a primer comprising the same, and a method for detecting Helicobacter pylori using the same are provided.

[0005]

Means for Solving the Problems As a result of earnest research, the inventors of the present invention found an oligonucleotide capable of detecting Helicobacter pylori with a higher sensitivity than the conventional method for detecting Helicobacter pylori by PCR,
The present invention has been completed.

[0006] That is, the present invention relates to SEQ ID NO: 1 in the sequence listing.
Provided are novel oligonucleotides having the nucleotide sequences represented by 1 to 7. Furthermore, the present invention provides a primer comprising the above-mentioned oligonucleotide of the present invention for detecting the gene of Helicobacter pylori by PCR. Furthermore, the present invention provides (1) an oligonucleotide having a base sequence represented by SEQ ID NO: 1 (hereinafter referred to as “A
-2R ") and an oligonucleotide having a base sequence represented by SEQ ID NO: 2 (hereinafter referred to as" A-2F ").
1)), a set of (2) A-2R and an oligonucleotide having the nucleotide sequence shown by SEQ ID NO: 3 (hereinafter sometimes referred to as “A-2F2”), (3) A −
Oligonucleotide having 2R and the base sequence shown by SEQ ID NO: 4 (hereinafter sometimes referred to as "A-2F3")
(4) An oligonucleotide having a base sequence represented by SEQ ID NO: 5 (hereinafter sometimes referred to as “B-1F”) and an oligonucleotide having a base sequence represented by SEQ ID NO: 6 (hereinafter referred to as “B -1R1 ")
Or (5) B-1F and an oligonucleotide having a base sequence represented by SEQ ID NO: 7 (hereinafter, "B-1R2")
PC) using a pair of
A method for detecting Helicobacter pylori, which comprises amplifying a part of the gene of Helicobacter pylori in a sample by R and detecting the amplified gene fragment.

The present invention will be described in detail below.

As described above, the oligonucleotide of the present invention has the nucleotide sequences shown by SEQ ID NOs: 1 to 7 in the sequence listing. These oligonucleotides are
Each hybridizes to a region in the genome of Helicobacter pylori. Part of the nucleotide sequence of the genome of Helicobacter Pairori are known, J. Bacteriol. 173
1920-1931 (1991) "Shuttle cloning and nucleotide se
quence of Helicobacter pylori gene responsible for
The sequence number and name of the oligonucleotide of the present invention and the region on the genome of Helicobacter pylori to which they hybridize are shown in Table 1 below.

[0009]

[Table 1]

Since the nucleotide sequence of the above-mentioned oligonucleotide of the present invention has been clarified, it can be easily produced using a commercially available DNA synthesizer.

The above-mentioned oligonucleotide of the present invention can be used as a primer for detecting the genome of Helicobacter pylori by PCR. In this case, A-2R, B-1R1 and B-1R2 are used as reverse primers, and A-2F1 and A-2F are used.
2, A-2F3 and B-1F are used as forward primers. Table 2 below shows preferable primer combinations and names of those combinations (primer sets), and regions amplified by each combination and their sizes.

[0012]

[Table 2]

The sample to be used in the method of the present invention is not particularly limited, but includes, for example, tissues of a living body, body fluids such as blood and gastric juice,
Mention may be made of flights and the like. After suspending these in a buffer,
Boiled DNA or DNA extracted from these by a conventional method can be subjected to PCR. In this case, DN
The extraction of A can be easily performed using a commercially available nucleic acid extraction reagent.

Next, PCR is performed. The PCR method itself is well known in the art, and reagent kits and devices for performing PCR are commercially available. PCR can be performed using these using a conventional method. An example of specific conditions is described in the following example.

After PCR, the amplification product can be detected by a conventional method. For example, the amplification product is subjected to agarose gel electrophoresis by a conventional method, the gel is stained with a nucleic acid staining reagent such as ethidium bromide, and then the band is detected under ultraviolet irradiation if necessary. Alternatively, for example, the amplification product can be bound to a DNA probe complementary to a part of the amplification product and detected by an enzymatic reaction, a luminescent reaction, or the like. When Helicobacter pylori is present in the sample, the amplification band is detected, and when it is not present, the amplification band is not detected, so that the presence or absence of Helicobacter pylori in the sample can be known.

Helicobacter pylori can be detected with high sensitivity by using the primer sets shown in Table 2 above, but further amplification can be performed twice by using different primer sets. The detection sensitivity can be increased. This is the so-called ne
This is a technique known as "sted PCR", and the region in the first PCR amplification product is further amplified by the second PCR. Table 3 below shows examples of preferable combinations of the first and second primer sets in this case, together with the size of the amplification product.

[0017]

[Table 3]

[0018]

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

Example 1 The gastric mucosal tissue of a patient with a digestive disorder was DN-treated using SepaGene (manufactured by Sanko Junyaku Co., Ltd.), which is a commercially available nucleic acid extraction reagent.
A extraction was performed. The extracted DNA was dissolved in 50 μl of buffer and 10 μl was used in the PCR reaction system. The PCR reaction is
Commercial PCR kit and thermal cycler (trade name)
Using, the cycle of 94 ° C. for 1 minute, 52 ° C. for 1 minute, 72 ° C. for 1 minute was repeated 30 times. As the primer set, the primer set A shown in Table 2 above was used.
1, A2, A3, B1 and B2, respectively. In addition, 100 samples were provided for each primer set. 10 μl of the amplified product was electrophoresed on an agarose gel according to a conventional method, then stained with ethidium bromide, and a band was detected under ultraviolet irradiation. As a result, 50% or more of the samples were positive regardless of which primer set was used.

On the other hand, for comparison, detection was performed in the same manner as above using HPU (having the nucleotide sequences shown in SEQ ID NOS: 8 and 9) which is a conventional primer set. The results are shown in Tables 4 to 7 below in comparison with the case where the primer sets A1, A2 and B1 were used. In these tables, "+" indicates positive and "-" indicates negative.

[0021]

[Table 4]

[0022]

[Table 5]

[0023]

[Table 6]

[0024]

[Table 7]

As shown in Table 4 above, when the conventional primer set was used, only 38 out of 100 samples were determined to be positive, whereas according to the method of the present invention, In each case, 50 samples or more are determined to be positive. Further, when the results are examined in detail for each primer set, as shown in Tables 5 to 7, those which were determined to be positive by the conventional method despite being determined as negative by the method of the present invention There were only 1 specimen for A1, 2 specimens for B1, and 0 specimens for A2. Contrary to this, 19 specimens were A1 and B1 which were positively determined by the method of the present invention despite being negatively determined by the conventional method, and A2.
There were 24 samples. From these, it was confirmed that the method of the present invention can detect Helicobacter pylori with higher sensitivity than conventional methods.

Example 2 PCR was carried out in exactly the same manner as in Example 1. However,
The primer sets used were A1, A2 and B2, which are the first primer set shown in Table 3 above. Then, PCR was performed in the same manner as the first time using 5 μl of the obtained PCR product. However, the primer set used was the second primer set shown in Table 3, and the number of cycles was 20. The obtained amplification product was detected in the same manner as in Example 1. As a result, the number of samples determined to be positive as shown in Table 8 was increased from 5 samples to 7 samples compared to the number of samples determined to be positive as a result of the first PCR shown in Table 7. From this, it is understood that the method can further increase the sensitivity.

[0027]

[Table 8]

[0028]

According to the present invention, the detection of Helicobacter pylori can be carried out with higher sensitivity than the conventional method.

[0029]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 21 Sequence type: Nucleic acid Sequence ATGGAAGTGT GAGCCGATTT G 21

SEQ ID NO: 2 Sequence length: 21 Sequence type: Nucleic acid Sequence TCACCCCAAA AGAGTTAGAT A 21

SEQ ID NO: 3 Sequence length: 22 Sequence type: Nucleic acid Sequence ATATTATGGA AGAAGCGAGA GC 22

SEQ ID NO: 4 Sequence length: 20 Sequence type: Nucleic acid Sequence CATGAAGTGG GTATTGAAGC 20

SEQ ID NO: 5 Sequence length: 23 Sequence type: Nucleic acid sequence TTCACCCCAA CAAATCCCTA CAG 23

SEQ ID NO: 6 Sequence length: 24 Sequence type: Nucleic acid sequence TTCTACACAA CCGGCTTCAT TCAA 24

SEQ ID NO: 7 Sequence length: 19 Sequence type: Nucleic acid Sequence GCCGCCAGCG CCTTCAGTG 19

SEQ ID NO: 8 Sequence length: 18 Sequence type: Nucleic acid Sequence GCCAATGGTA AATTAGTT 18

SEQ ID NO: 9 Sequence length: 18 Sequence type: Nucleic acid Sequence CTCCTTAATT GTTTTTAC 18

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Claims (16)

[Claims] 1. An oligonucleotide having a base sequence represented by SEQ ID NO: 1 in the sequence listing. 2. An oligonucleotide having a base sequence represented by SEQ ID NO: 2 in the sequence listing. 3. An oligonucleotide having a base sequence represented by SEQ ID NO: 3 in the sequence listing. 4. An oligonucleotide having a base sequence represented by SEQ ID NO: 4 in the sequence listing. 5. An oligonucleotide having a base sequence represented by SEQ ID NO: 5 in the sequence listing. 6. An oligonucleotide having a base sequence represented by SEQ ID NO: 6 in the sequence listing. 7. An oligonucleotide having a base sequence represented by SEQ ID NO: 7 in the sequence listing. 8. A primer for detecting the gene of Helicobacter pylori by PCR, which comprises the oligonucleotide according to any one of claims 1 to 7. 9. A part of the Helicobacter pylori gene in a sample is amplified by PCR using the set of the oligonucleotide according to claim 1 and the oligonucleotide according to claim 2 as a primer, and the amplified gene fragment is obtained. A method for detecting Helicobacter pylori comprising detecting. 10. A part of the Helicobacter pylori gene in a sample is amplified by PCR using the set of the oligonucleotide of claim 1 and the oligonucleotide of claim 3 as a primer, and the amplified gene fragment is obtained. A method for detecting Helicobacter pylori comprising detecting. 11. A part of the Helicobacter pylori gene in a sample is amplified by PCR using the set of the oligonucleotide according to claim 1 and the oligonucleotide according to claim 4 as a primer, and the amplified gene fragment is obtained. A method for detecting Helicobacter pylori comprising detecting. 12. A part of the Helicobacter pylori gene in a sample is amplified by PCR using the set of the oligonucleotide according to claim 5 and the oligonucleotide according to claim 6 as a primer, and the amplified gene fragment is obtained. A method for detecting Helicobacter pylori comprising detecting. 13. A part of the Helicobacter pylori gene in a sample is amplified by PCR using the set of the oligonucleotide according to claim 5 and the oligonucleotide according to claim 7 as a primer, and the amplified gene fragment is obtained. A method for detecting Helicobacter pylori comprising detecting. 14. A part of the gene of Helicobacter pylori in a sample is amplified by PCR using the set of the oligonucleotide of claim 1 and the oligonucleotide of claim 2 as a primer, and then the set of claim 1. A method for detecting Helicobacter pylori, which comprises further amplifying a part of the amplified fragment by PCR using a set of an oligonucleotide and the oligonucleotide according to claim 3 as a primer, and detecting the amplified gene fragment. 15. A part of the gene of Helicobacter pylori in a sample is amplified by PCR using the set of the oligonucleotide according to claim 1 and the set of oligonucleotides according to claim 3 as a primer, and then the set of claim 1. A method for detecting Helicobacter pylori, which comprises further amplifying a part of the amplified fragment by PCR using a set of the oligonucleotide and the oligonucleotide according to claim 4 as a primer, and detecting the amplified gene fragment. 16. A part of the gene of Helicobacter pylori in a sample is amplified by PCR using the set of the oligonucleotide according to claim 5 and the set of oligonucleotides according to claim 7 as a primer, and then the method according to claim 5. A method for detecting Helicobacter pylori, which comprises further amplifying a part of the amplified fragment by PCR using a set of the oligonucleotide and the oligonucleotide according to claim 6 as a primer, and detecting the amplified gene fragment.