KR101945807B1 - Marker for prediction of responsiveness of Helicobacter pylori to macrolide antibiotics - Google Patents

Abstract

Disclosed are a composition for predicting sensitivity and tolerance to clarithromycin of Helicobacter pylori comprising a substance for detecting 23S rRNA gene monoploid in which the 2143^th and 2182^th bases of a gene encoding bacterial 23S rRNA are A and T or G and C respectively; and a method for predicting reactivity to clarithromycin using the composition. The composition and method according to the present invention can predict reactivity to clarithromycin of Helicobacter pylori with high accuracy and thus increase a success rate of a primary eradication treatment of Helicobacter pylori.

Description

{Marker for prediction of responsiveness of Helicobacter pylori to macrolide antibiotics}

The present application is related to antibiotic susceptibility and resistance markers of Helicobacter pylori.

Helicobacter pylori ( H. pylori) , which is considered to be the main cause of chronic gastritis, duodenal ulcer, and gastric ulcer, is expected to be highly resistant to antibiotics.

The worldwide method of Helicobacter pylori eradication is to use triple therapy with amoxicillin and chloromycin for 7 days in the case of positive test for campylobacter-like organism (CLO). However, the efficacy of the eradication has been remarkably decreased with the increase of H. pylori showing resistance to chloromycin, and various treatments such as secondary eradication therapy, sequential therapy, It is being tried.

However, in order to overcome antibiotic resistance, first, the eradication rate should be improved.

To this end, a marker has been discovered to determine the resistance of Helicobacter pylori to clarithromycin (Rimbara E. et al., Int J Antimicrob Agents. 2007 Sep; 30 (3): 250-4. JM et al., J Microbiol Biotechnol. 2008 Sep; 18 (9): 1584-9.). According to this document, it has been disclosed that the antimicrobial resistance patterns can vary widely depending on the combination of the isolates before administration of the antibiotic and the isolates of the patient after administration of the antibiotic, even at the same site have.

However, the clarithromycin resistance used in the triple therapy may be due to the mutation of the 23S rRNA V domain, but the reactivity to antibiotics may vary depending on the type of base at various positions and at the same position in this gene, It is necessary to find a marker specific to Koreans using a combination of various mutations at a plurality of positions.

U.S. Published Patent Application No. 2016/0017406 (published on January 21, 2016) European Registered Patent No. 01078104 (registered on October 10, 2002) Japanese Published Patent Application No. 2011-062143 (Published date: March 31, 2011)

We would like to provide a marker that can determine the susceptibility of Helicobacter pylori to Markrolide antibiotics with high accuracy.

In one embodiment, the present application refers to the nucleotide sequence of Helicobacter pylori 23S rRNA gene represented by SEQ ID NO: 25, or the nucleotide sequence of Helicobacter pylori , clarithromycin , Wherein when the unicameral combinations of the 2143rd and 2182rd bases are A and T, respectively, it is indicated that Helicobacter pylori is susceptible to clarithromycin, and that the 2143th and 2182th bases Helicobacter pylori is resistant to clarithromycin and is responsive to metronidazole-amoxicillin-bismuth-PPI combination therapy when the combination is G and C, or G and T, respectively .

In one embodiment, the detecting material contained in the composition according to the present invention is a nucleic acid amplification method, and the nucleic acid amplification method comprises a LAMP method.

In one embodiment, the LAMP method comprises the following sets of 2143 and 2182 base detection primers capable of detecting the presence of a specific base at a polymorphic site of interest: (i) a primer for base G detection of 2143: SEQ ID NOS: 1 to 4; (ii) primers for detecting base A of 2143: SEQ ID NOS: 7 to 10; (iii) Primer for detecting base C of 2182: SEQ ID NOS: 13 to 16; And (iv) primers for detecting the base T of the above 2182: SEQ ID NOS: 19 to 22.

In one embodiment, the primer comprises a combination of primers (i) to (iv) further comprising primers of SEQ ID NOs: 5 and 6, SEQ ID NOs: 11 and 12, SEQ ID NOs: 17 and 18, and SEQ ID NOs: 23 and 24, respectively can do.

In one embodiment, the composition according to the present application is applied to Koreans.

In another embodiment, the subject is a single nucleotide polymorphic site of a 23S rRNA gene, wherein, with reference to the position of the sequence of SEQ ID NO: 25, 10 to 100 consecutive nucleotides comprising a single nucleotide polymorphic site of 2143 base of the sequence A primer or probe that specifically binds to the sequence; And a primer or a probe that specifically binds to 10 to 100 contiguous nucleotide sequences comprising a single nucleotide polymorphic site in which the 2182nd nucleotide of the above sequence is T. The nucleotide sequence of Helicobacter pylori ( H. pylori) There is provided a composition for predicting the clarithromycin reactivity.

In another aspect, the subject is provided herein to provide a method for predicting the response to clarithromycin in a subject in need of Helicobacter pylori eradication,

Providing a biological sample derived from said subject; Extracting nucleic acid from the biological sample; Analyzing the 2143th base and the 2182th base with reference to the sequence of SEQ ID NO: 25 in the sample; And when the 2143th base and the 2182nd basal bases are A and T, respectively, the Helicobacter pylori isolated from the subject is judged to be susceptible to clarithromycin. As a result of the analysis, Helicobacter pylori isolated from the subject is judged to be resistant to clarithromycin and is reactive to the metronidazole-amoxicillin-bismuth-PPI antibiotic regimen, when the first cochlear base is G and C, or G and T, respectively Of Helicobacter pylori is determined to be indicative of the response marker to clarithromycin of Helicobacter pylori.

In one embodiment, the method according to the present invention is performed by a LAMP method capable of detecting the presence of a specific base at the polymorphic site, and the primer set is as mentioned above.

In one embodiment, the biological sample of the method according to the present invention is obtained from a subject who has not received a triple therapy for Helicobacter pylori eradication. Especially the samples of Koreans.

The 23S rRNA gene monoclonal markers 2143 and 2182 of the gene coding for 23S rRNA of Helicobacter pylori according to the present invention are A and T, respectively, can predict the clarithromycin sensitivity of Helicobacter pylori with high accuracy. Thus, Helicobacter pylori 1 And the success rate of the tea cell eradication treatment can be increased.

Figure 1 shows the 23S rRNA mutation of a subject participating in a clinical trial in one embodiment according to the present application and the elimination of Helicobacter pylori by antibiotic therapy.
FIGS. 2 to 5 show experimental results using a LAMP primer capable of detecting a specific base at the polymorphic site identified in the present invention and produced in one embodiment according to the present invention.

The present invention is based on the discovery that the combination of specific bases at 2143 and 2182 base (based on the sequence of SEQ ID NO: 25) of the gene coding for 23S rRNA of Helicobacter pylori isolated from Korean is related to susceptibility to clarithromycin will be.

Justice

As used herein, the " 23S rRNA gene " is one of the components constituting ribosomes, which are intracellular scavengers involved in protein production of bacteria, and their base sequences are well known. In one embodiment, the sequence is represented by the sequence of SEQ ID NO: 25.

As used herein, " clarithromycin " is a macrolide antibiotic (CAS Number: 81103-11-9) that binds to the 23S rRNA of bacterial ribosomes and inhibits the translation process of proteins in bacteria. It is used for the eradication of Helicobacter pylori and is administered orally or intravenously.

As used herein, a "gene mutation" used as a marker for determining clarithromycin susceptibility refers to a single nucleotide polymorphism (SNP) or a point mutation, which comprises a nucleotide called a wild type. SNPs are single base mutations in DNA between individuals and are one of the most common variations at the genetic level.

In one embodiment, the gene mutation herein is a single base mutation at nucleotides 2143 and 2182 relative to the sequence of SEQ ID NO: 25, wherein the polymorphisms at positions A and T, respectively.

As used herein, " monoclonal " or " haplotype " refers to a set of two or more mutations in a gene that are in an association-unbalanced relationship inherited as a group at a statistically significant level.

Here, "Korean" refers to a group of Koreans whose ancestors are Korean, and whose ancestors over 10 generations are Koreans.

As used herein, the term " subject " refers to a human requiring treatment due to infection with Helicobacter, and in one embodiment, is a target prior to treatment with triple therapy after detection of Helicobacter.

The term " substance for detection " as used herein means a substance capable of detecting the presence or absence of a specific nucleotide at the SNP site, and includes a nucleic acid or a protein. For example, in the case of nucleic acid, a probe capable of binding (including mismatch) to the site, or a primer capable of binding to a site flanking the SNP to initiate amplification, or both, no. For example, in the case of a protein, it includes, but is not limited to, a substance capable of binding to an amino acid mutation caused by the SNP, for example, an antibody.

As used herein, the term " nucleic acid " encompasses both DNA (gDNA and cDNA) and RNA molecules. Nucleotides that are basic building blocks in nucleic acid molecules include not only natural nucleotides but also analogues (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)).

As used herein, the term " probe " refers to a linear oligomer with a natural or modified monomer or linkage comprising a deoxyribonucleotide and a ribonucleotide that can hybridize to a particular nucleotide sequence. Preferably, the probe is single stranded for maximum efficiency in hybridization. The probe is preferably a deoxyribonucleotide.

As the probe used in the present invention, a sequence complementary to the sequence including the SNP may be used, but a sequence substantially complementary to the sequence not interfering with the specific hybridization may be used. Preferably, the probe used in the present invention comprises a sequence capable of hybridizing to a sequence comprising 8-100 consecutive nucleotides comprising a SNP position according to the present invention.

In one embodiment, the 3'-end or the 5'-end of the probe has a base complementary to the SNP base. Generally, the stability of the duplex formed by hybridization tends to be determined by the agreement of terminal sequences, so that in a probe having a base complementary to the SNP base at the 3'-terminal or 5'-terminal, If the part is not hybridized, such a duplex can be disassembled under stringent conditions. Suitable conditions for hybridization include, but are not limited to, Nucleic Acid Hybridization, A Practical Approach, IRL (2001) and Haymes, BD, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Press, Washington, DC (1985). The stringent condition used for hybridization can be determined by controlling the temperature, the ionic strength (buffer concentration) and the presence of a compound such as an organic solvent, and the like. This stringent condition can be determined differently depending on the sequence to be hybridized.

As used herein, a "primer" refers to a single strand of oligonucleotides, which hybridizes under suitable conditions (presence of four different nucleoside triphosphates and polymerase such as DNA or RNA polymerase) - acting as a starting point for initiating directed DNA synthesis. The suitable length of the primer is determined by the characteristics of the primer to be used, but is usually about 15 to 30 bases in length. The primer need not be exactly complementary to the sequence of the template, but must be complementary enough to form a hybridcomplex with the template.

The methods of monoclonal determination or SNP determination or genotyping in the present invention are very diverse and can be carried out by various methods known in the art. For example, techniques applicable to the present invention include a method using an Affymetrix SNP chip, a PCR-RFLP (Restriction Fragment Length Polymorphism) method, a PCR SSCP (Single Strand Conformation Polymorphism) method, a PCR-SSO An Allele Specific Oligonucleotide (ASO) hybridization method combining a PCR-SSO and a dot hybridization method, a TaqMan method, a pyrosequencing method, an Invader method, an SNaPShot method, a DNA hybridization method, a Specification Sequence Oligonucleotide method, a dot hybridization method, PCR was performed by MassArray method, Dynamic Allele-Specific Hybridization (DASH) method, Microplate Array Diagonal Gel Electrophoresis (MADG) method, GoldenGate method, SNPlex method and BeadArray method, Amplification (LCR, Wu, DY et al., Genomics 4: 560 (1989)), the ligation reaction (Miller, HI (WO 89/06700) and Davey, C. et al. (EP 329,822) Polymerase ligation reactions (Barany, PCR Methods and Appl., 1: 5 (1992)), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), 3SR (Kwoh et al., PNAS, USA, 86: 5,130, 238), but is not limited thereto.

In one embodiment, nucleic acid amplification techniques are used, and it is important to design suitable primers and probes to identify SNP bases according to the present invention. The conditions of the amplification reaction by PCR and the reagents and enzymes used can be those conventionally known in the art. The TaqMan method utilizes the 5'-nuclease property of Taq polymerase. One TaqMan probe and one pair of PCR primers are used for the reaction. A TaqMan probe with a reporter dye at the 5'-end and a quencher dye at the 3'-end is covalently coupled to a polymorphic site and a TaqMan probe, It is a method of distinguishing the difference of mismatch by using a fluorescent substance. When a TaqMan probe is combined with both a reporter dye and a light absorbing dye, the reporter dye does not emit light by the absorbing dye. However, TaqMan probes that are completely complementary to the site in which the SNPs are located are bound in the annealing step of the PCR, and the 5'-nuclease activity of the Taq polymerase at the extension step causes the 5'- The reporter dye (FAM or VIC) separates from the absorbance dye and exhibits inherent fluorescence. If the probe is mismatched with the target SNP, the probe will fall off the template and no further reaction will occur. In addition to SNP, this method can also analyze the insertion or deletion of very small sequences. Illumina's GoldenGate Genotyping assay is a SNP genotyping method that allows multiplexing (1,536-plex) multiplexing using primer extension and amplification steps using genomic DNA as a template.

In one embodiment, the template DNA is labeled with biotin for SNP genotyping and purified from surplus hybridization solution and streptavidin conjugated paramagnetic particles (SP-PMPs). Three oligonucleotides (oligos) are added to the purified DNA template. Two oligos are ASO (Allele-Specific oligos) specific for each allele of the SNP region and the other oligo is a base pair LSO (Locus-Specific Oligo) hybridizes downstream. After oligo hybridization, the unhybridized oligo is removed and an allele-specific extension post-ligation reaction is performed. The ligation products are PCR amplified with fluorescently labeled universal PCR primers P1 (Cy3), P2 (Cy5) and P3, followed by single stranding. Single stranded DNA is hybridized to a complementary bed type (bead type, Array Matrix or BeadChip) through a unique address sequence, and the signal of the fluorescent material is read to determine the genotype automatically. GoldenGate analysis is capable of multiplexing analysis of up to 1,500 SNPs at the same time, but typing is possible, but not all SNP regions are analyzed.

For example, the Affymetrix GeneChip assay was recently developed by Affimetrix as a method for chip-based, simultaneous genotyping of hundreds of thousands of SNPs. According to a specific embodiment of this method, the genomic DNA is first cleaved with restriction enzymes (StyI and NspI) and all fragments generated are bound to an adapter recognizing the 4 bp overhang, regardless of size do. Adapted DNA fragments are amplified with a primer that recognizes the base sequence of the adapter, and the PCR amplification conditions are optimized to selectively amplify fragments 200-2,000 bp in size. The amplified DNA is then labeled by fragmentation and then hybridized to the GeneChip array. In the array, 25-mer probes corresponding to the A alleles and perfect match of the B allele were synthesized. To determine the specificity of the 25-mer probes, a single base pair mismatch A single base pair mismatch is also included to check the accuracy of genotyping.

When Zino typing is completed, association analysis is performed. In the present invention, there are a wide variety of methods for analyzing the SNP association, and various methods known in the art can be applied. or A so-called genome-wide association study (GWAS) method of examining a large amount of SNP widely distributed in a human genome using a SNP chip can be used, but is not limited thereto. A " multi-stage approach " method can be utilized to prevent random errors that may occur in the case of the above method.

The "LAMP (Loop Mediated Isothermal Amplification)" method herein is a nucleic acid amplification method (Notomi, T. et al. 2000. Loop-Mediated Isothermal Amplification of DNA. Nucleic acid amplification method capable of amplifying a target nucleic acid with high sensitivity and specificity under isothermal conditions Acids Res 28, E63) and at least four primer sets specially designed at various sites of the target nucleic acid (refer to Korean Patent No. 612551, Korean Patent No. 1739876, etc. (Nagamine, K. et al., 2002. Accelerated reaction by Loop mediated isothermal amplification using loop primers, see Mol Cell Probes 16, 223-9, and the like) using as a standard method up to six primer sets. At least four primers are required for the LAMP reaction, which include two inner primers FIP (forward inner primer) and BIP (backward inner primer) and two outer primers F3 (forward outer primer) and B3 do. For accelerated LAMP, a total of six primers can be used, including LF (Loop F) and LB (Loop B). The primer sets used for LAMP are designed for six distinct regions (B3 region, B2 region, B1 region, F1c region, F2c region and F3c) on the target nucleic acid, F3 and B3 are two outer primers, BIP is two internal primers, two internal primers are hybrid primers containing sense and antisense sequences, FIP consists of F1c region sequence and F2 region sequence, and BIP consists of B1c region sequence and B2 region sequence.

In one embodiment, the detecting material according to the present invention comprises a set of primers capable of detecting a polymorphic site using a LAMP reaction. Such a primer set can be produced based on that disclosed in Korean Patent No. 1739876 by the present applicant.

Helicobacter pylori Clarithromycin  For susceptibility prediction Sunspot  Single base hemp Big

The present invention is based on the discovery that the combination of specific bases at 2143 and 2182 base (based on the sequence of SEQ ID NO: 25) of the gene coding for 23S rRNA of Helicobacter pylori isolated from Korean is related to susceptibility to clarithromycin will be.

In particular, it has previously been known that G at position 2143 of 23S rRNA of Helicobacter pylori is associated with resistance to clarithromycin (Rimbara et al., &Quot; Mutations in the 23S rRNA gene of clarithromycin-resistant Helicobacter pylori from Japan & "Int J Antimicrob Agents. 2007 Sep; 30 (3): 250-4. Epub 2007 Jun 21).

However, it has been surprisingly found herein that Helicobacter pylori can be judged with high accuracy for clarithromycin when base A at position 2143 of 23S rRNA and base T at base 2182 are in haploid form. It was also found that Helicobacter pylori can be determined to be resistant to clarithromycin when the combination of monoclonal combinations of the 2143th and 2182th bases is G and C, respectively.

In one embodiment, if bases at 2143 and 2182 of 23S rRNA are A and T, respectively, susceptibility to clarithromycin can be predicted with an accuracy of 95.7%.

In another embodiment, Helicobacter pylori is resistant to clarithromycin when the union combination of 2143th and 2182th bases of 23S rRNA is G and C, respectively.

These susceptibility and tolerance are directly related to the success or failure rate of the sterilization using triple therapy. The susceptibility can predict the sterilization success rate and the resistance can predict the sterilization failure rate.

Here, the loci of 2143 and 2182 th SNPs of the 23S rRNA gene are shown based on the sequence of SEQ ID NO: 25. However, the above sequence is for indicating a position, and is not limited to the above sequence.

In the 2143th and 2182th bases based on the sequence of SEQ ID NO: 25 identified herein, A and T are haplotypes which are all present in one molecule of the 23S rRNA gene.

These haplotypes provide more accurate and reliable genetic information than individual single nucleotide polymorphisms. There is a strong association between the single nucleotide polymorphisms that make up the haplotype, and it can be used as a continuous biomarker because it guarantees continuity between generations and is kept constant without changing the genotype. There is also an advantage in that a single nucleotide polymorphism combination can analyze results that are difficult to identify with individual single nucleotide polymorphism information through a haplotype.

According to one embodiment according to the present application, in the case of A and T at 2143th and 2182th bases, the susceptibility of Helicobacter pylori to clarithromycin can be predicted with a high level of accuracy of 95.7%.

In one embodiment, the haplotype marker according to the present invention is Korean specific. It is already known that genetic polymorphism can be specific and different depending on race and geographical environment, and it is important to find a Korean specific marker.

Helicobacter pylori Clarithromycin  For susceptibility prediction Sunspot Marker  Usage

Detection of the marker is required to determine whether Helicobacter pylori isolated from a subject using the haplotype marker according to the present application is responsive to clarithromycin, i.e., susceptible or resistant.

For the detection of H. pylori markers infected by a subject, the markers according to the invention can be used in the form of a kit, form or method comprising a substance capable of detecting it.

Accordingly, in one embodiment, the present invention provides a composition for predicting the reactivity of Helicobacter pylori to clarithromycin, which comprises the 2143th and 2182nd bases detection substance of the above sequence, with reference to the nucleic acid sequence represented by SEQ ID NO: 25 , And when the combinations of the 2143th and 2182th bases are A and T, respectively, the Helicobacter pylori is susceptible to clarithromycin, and the combinations of 2143th and 2182th bases are G and C , Helicobacter pylori is resistant to clarithromycin. The present invention also relates to a composition for predicting the clarithromycin reactivity of Helicobacter pylori.

In one embodiment, if bases at 2143 and 2182 of 23S rRNA are A and T, respectively, susceptibility to clarithromycin can be predicted with an accuracy of 95.7%.

In another embodiment, Helicobacter pylori is resistant to clarithromycin when the union combination of 2143th and 2182th bases of 23S rRNA is G and C, respectively.

These susceptibility and tolerance are directly related to the success or failure rate of the sterilization using triple therapy. The susceptibility can predict the sterilization success rate and the resistance can predict the sterilization failure rate.

The substance and method for detection contained in the composition can be referred to above.

In one embodiment, the detecting material is selected from the group consisting of nucleic acid amplification methods, polymorphic gene-specific oligonucleotide hybridization, sequencing, hybridization, 5'-nuclease digestion, single stranded polymorphism, primer-specific elongation, It is a substance used in analytical methods.

In another embodiment, the detecting material is a primer and / or a probe. Such primers or probes are oligonucleotides specific nucleic acid amplification methods, or PCR and genetic polymorphisms, such as PCR (Polymerase Chain Reaction) the nucleotide hybridization mix and TaqMan ^ⓡ The same method is used.

In another embodiment, the detection material is a set of four primers used in a LAMP reaction. In the present invention, a set of primers capable of detecting the presence or absence of a specific base at a specific position based on LAMP is amplified. Primers for detecting base G at position 2143 of the present application include SEQ ID NOS: 1 to 4. The primer for detecting base A at position 2143 of the present application comprises SEQ ID NOS: 7 to 10. Primers for detecting base C at position 2182 of the present application include SEQ ID NOS: 13 to 16. Primers for detection of base T at position 2182 of the present invention include SEQ ID NOS: 19 to 22.

In another embodiment, the detection material is a set of six primers used in a LAMP reaction. The combination of each of the above primers may further include primers of SEQ ID NOs: 5 and 6, SEQ ID NOs: 11 and 12, SEQ ID NOs: 17 and 18, and SEQ ID NOs: 23 and 24, respectively.

In this aspect, the present disclosure provides, in another aspect, a single nucleotide polymorphic site of a 23S rRNA gene, wherein, with reference to the position of the sequence of SEQ ID NO: 25, 10 to 100 nucleotide polymorphisms Primers or probes that specifically bind to consecutive nucleotide sequences; And Helicobacter pylori ( H. pylori) comprising a primer or probe that specifically binds to 10 to 100 contiguous nucleotide sequences comprising a 23S single nucleotide polymorphism site in which the 2182st base of the sequence is T, A composition for predicting the clarithromycin reactivity.

In another embodiment, the single nucleotide polymorphic site of the 23S rRNA gene is a polynucleotide sequence that is specific for 10 to 100 contiguous nucleotide sequences comprising a single nucleotide polymorphic site in which the 2143th base of the sequence is G, with reference to the position of the sequence of SEQ ID NO: A primer or a probe that binds to the target; And Helicobacter pylori ( H. pylori) comprising a primer or probe that specifically binds to 10 to 100 consecutive nucleotide sequences comprising a 23S single nucleotide polymorphism site in which the 2182st base of the sequence is C, A composition for predicting the clarithromycin reactivity.

In another aspect, the present invention provides a method for detecting Helicobacter pylori eradication comprising: providing a biological sample from the subject to provide information for predicting susceptibility to clarithromycin of a subject in need of Helicobacter pylori eradication; Extracting nucleic acid from the sample; Analyzing the sequence of the 2143th base and the 2182th base in the sample with reference to the sequence of SEQ ID NO: 25; And when the 2143th base and the 2182nd basal bases are A and T, respectively, the Helicobacter pylori isolated from the subject is judged to be susceptible to clarithromycin. As a result of the analysis, Wherein said Helicobacter pylori isolated from said subject is judged to be resistant to clarithromycin when said polynucleotide is G and C, respectively, .

In one embodiment according to the present disclosure, the subject includes a mammal, particularly a human, that is Helicobacter pylori infected and needs to be killed. In one embodiment, the subject is a subject prior to receiving the first-line therapy.

Biological samples used in the method according to the present invention include, but are not limited to, gastric biopsy specimens, and various samples requiring eradication due to Helicobacter pylori infection, such as serum or feces.

Nucleic acids such as DNA contained in a biological sample in the method according to the present invention can be extracted and analyzed as described above or with reference to methods described herein.

In one embodiment, a nucleic acid can be directly extracted from a sample derived from a subject and used as a template to determine the base at the position according to the present invention.

In one embodiment, the markers according to the present invention are detected in Primary isolates. The primary isolate means a strain isolated from the subject before receiving the triple therapy. The secondary isolate is a strain isolated from the subject after receiving the triple therapy, and is different from the one used for the primary isolate because the gene can be mutated due to the use of antibiotics.

In one embodiment, it may be carried out by a nucleic acid amplification method, mutant gene-specific oligonucleotide hybridization, sequencing, hybridization, 5'-nuclease digestion, single stranded polymorphism, primer-specific elongation, or oligorylation assays , And the above method can be referred to the above.

In one embodiment, the detection of the 2143th base and the 2182th base of the method according to the present invention is carried out in a LAMP-based manner, as described above.

When the base is identified as A and T as a result of analyzing the 2143th base and the 2182th base in the biological sample derived from the subject, it is judged that Helicobacter pylori infected with the subject has been subjected to clarithromycin, Clarithromycin can be administered for eradication.

When the base is identified as G and C as a result of analysis of 2143th base and 2182th base in a biological sample derived from a subject, it is judged that Helicobacter pylori infected to the subject is resistant to clarithromycin, and based on this, Other methods other than prescription of clarithromycin should be considered for eradication.

As mentioned earlier, the method according to the present invention can be applied specifically to Koreans.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Experimental Method

1. Subject and method

(1) Patient information

This study was conducted as a prospective, open clinical trial at the Fatima Hospital in Daegu from June 2016 to September 2017. The study was approved by IRB, Fatima Hospital, Daegu (Study Number: DFH16DRIS026). Patients 18 years of age or older, who had undergone endoscopy, were enrolled for clinical trials. Gastrectomy, pregnant women, and breastfeeding patients were excluded because they had complications or disabilities and were unable to obtain consent. Consent was obtained from all patients prior to enrollment in the trial. Patients were enrolled from June 2016 to May 2017 and the last follow-up visit was November 2017.

The first objective of this study was to clarify the effect of the 23S rRNA point mutation of Helicobacter pylori on the failure of eradication of the clarithromycin-based triple therapy. The second objective is to determine whether the efficacy of the metronidazole - based prescription for second - line treatment and the effect of point mutations on second - line outbreak rates.

(2) Helicobacter Pyorrhoid  Detection Sterilization  cure

Helicobacter pylori infection was assessed by rapid uricase assay (CLO test) using Proton Dry® NEW kit from stomach biopsy tissue. Patients who showed positive results in the rapid urease test were prescribed to take a triple therapy twice daily for 7 days, including 500 mg of clarithromycin, 1000 mg of amoxicillin, and 30 mg of lansoprazole. After the treatment, the presence of H. pylori was confirmed by urea breath test (UBT) using HUBT-20, a Helicobacter pylori breath analysis system, six weeks after treatment. When positive for elemental efficacy was measured, the patient was treated with metronidazole 250 mg, bismuth subcitrate 300 mg, tetracycline 500 mg four times daily, and omeprazole 20 mg twice a day for 14 days after metronidazole-based quadruple therapy Later on, the urea breath test was used to confirm the eradication of Helicobacter pylori.

2. Nucleic Acid Extraction and Helicobacter Pyrogenic  23S rRNA  Gene sequencing

DNA was extracted from the gastric biopsy specimens of benign cases using the QIAamp DNA mini kit when positive for the rapid urease test (CLO test). The concentration of extracted DNA is measured by a Nanodrop spectrophotometer and DNA samples measured at 50 ng / ml or less are excluded from the sequencing. To measure the nucleotide sequence of the V domain of 23S rRNA of Helicobacter pylori, approximately 239 bp was amplified using the PCR method. The primer F (5'-TGA ATG TAA CGA GAT G-3 ', corresponding to H. pylori 23S rRNA position 20522070) used in the PCR and the R primer (5'-AGG TGG GAG GCT TTG AA- 2291) above. The amplified product was analyzed by Sequent (Korea) for sequencing.

3. Clinical sample counting and statistical analysis method

References (Lim SH, et al. BMC Gastroenterol. 2013; 13: 104 .; And Lee JW et al. Helicobacter. 2013; 18: 206-14.), We assumed that the incidence of Helicobacter pylori was 45% and that the success rate of the clarithromycin-based triple therapy was 80%. The frequencies of 2143G and 2182T of 23S rRNA by reference to the literature were approximately 14% and 12%, respectively. Based on these estimates, a total of 750 patients were enrolled to cover 65 patients with 2143G or 2182T point mutations, taking a 15% dropout rate during eradication therapy and urea breath test. Data were analyzed using SPSS software (version 18.0). The baseline measurement data are expressed as mean and standard deviation, and the categorical data are expressed as number and percent. Student t test was used for comparison of measurements and categorical data were performed by Chi-squared test. Binary logistic regression analysis was calculated by the independent correlation risk and 95% confidence interval for Helicobacter pylori eradication failure in clarithromycin-based triple therapy. In all tests p-value <0.05 indicates statistical significance.

Example  1. Screening and clinical data

The screening and dropout rates of the subjects are shown in the flowchart of FIG. A total of 755 patients with upper gastrointestinal endoscopy were enrolled and tested for rapid urease test (CLO, urease activity test for H. pylori; The Diagnosis of Helicobacter pylori infection. The Korean Journal of Helicobacter and Upper Gastrointestinal Research, 2014; 14 (4): 233-236).

The CLO test confirms the presence or absence of Helicobacter pylori in gastric endoscopy in patients suspected of gastric ulcer, duodenal ulcer, dyspepsia, peptic ulcer, and early gastric cancer, which are important factors in Helicobacter pylori infection Is a primary diagnostic method.

In the CLO test, 299 were positive. Four of 299 patients (39.6%) had DNA levels lower than 50 ng / ml, and only 295 patients were sequenced. The first-line therapy, clarithromycin-based Helicobacter pylori eradication therapy, was prescribed to 253 patients. The follow - up period was performed in 201 patients after 6 weeks and 42 patients (20.9%) showed UBT positive results, indicating the failure of eradication. A second-line metronidazole-based antibiotic regimen was prescribed to 32 patients and a second urogenital test was performed 6 weeks later, with only 1 patient failing (Fig. 1).

The primary clinical data were older patients with rapid urease test positive results (p = 0.027) (Table 1). Age, sex, body mass index, gastric ulcer, duodenal ulcer, and history of H. pylori eradication did not differ between CLO positive and negative groups.

Example  2. CLO  Helicobacter pylori 23S in benign patients rRNA  Gene sequencing

As in Example 1, it was confirmed whether 5 point mutations were substituted in the 295 Helicobacter pylori 23S rRNA gene domain V of Korean subjects who were positive for CLO analysis (Table 2).

As shown in Table 2, the 2143A> G point mutation was most frequently found in about 25% of patients, followed by 2182C> T point mutations in 11.5% of patients. The other three mutations were found in 2142A> G 1.7%, 2190T> C 1.4% and 2195C> T 1.4%, respectively. Patients with mutations in 2115, 2144, 2711, 2223, 2244 sites reported to exhibit clarithromycin resistance in Helicobacter pylori were not found in the subjects of this study. The patients with 2143A-2182C in the haplotype structure were the most frequent with 64.5%, followed by 23.6% with 2143G-2182C and 10.5% with 2143A-2182T. 2143G-2182T was the lowest at 1.0% (Table 2).

Example  3. Helicobacter Pyorrhoid  23S rRNA  The polymorphic site Claris Based on Rome Sterilization Success  Correlation analysis

The failure rates of the 2143A / G and 2182C / T polymorphisms were analyzed (Table 3) to examine the role of the 23S rRNA gene polymorphism in clarithromycin-based triple therapy failure. The overall eradication failure rate of the clarithromycin-based therapy was 20.9% (42/201). 2143G-2182C and 2143G-2182T, the presence of the 2143G allele, indicative of a high-risk allele of eradication failure in the clarithromycin-based therapy, increased the rate of eradication failure by 57.7%. Interestingly, the presence of the 2182T allele lacking 2143G attenuated the treatment failure rate to about 4%, indicating that the 2143A-2182T monoclonal strain is susceptible to clarithromycin and thus predicts the success of the eradication. The most common 2143A-2182C hoplot type exhibits an 8.7% eradication failure rate.

Followed by a risk analysis of eradication failure in clarithromycin-based therapies (Table 4). In univariate analysis, older age was associated with eradication failure (p = 0.040), whereas men, gastric ulcer, duodenal ulcer, and eradication history did not show statistical significance. The presence of the 2143G allele and the 2143G-2182C haplotype in the 23S rRNA were significantly associated with the failure of the first-line treatment (p <0.001) (2143A-2182T haplotype) (P = 0.038), respectively. To assess whether point mutations in Helicobacter pylori 23S rRNA are independent elements of eradication failure, we performed logistic regression analysis. We believe that the interaction of the 2143G allele and the 2143G-2182C haplotype presence with other risk factors such as age, sex, and eradication history may contribute to 21-fold and 18-fold increases in the risk of eradication failure in multivariate models 1 and 2 (Table 4). However, Haplotype 2143A-2182T did not play an independent role in the success of the eradication. This may be due to the low statistical value associated with the low incidence of 2182T mutations (multivariate model 3, Table 4). When analyzed for factors related to the presence of 2143G, previous history of eradication and women were independent risk factors for the presence of 2143G, the risk allele of eradication failure (Table 5).

These results indicate that 2143G-2182C type was significantly associated with eradication failure and 2143A-2143T type was associated with successful eradication.

Example  4. Helicobacter Pyorrhoid  23S rRNA  Genetic polymorphism sites and metronidazole-based antibiotic therapy Success  Correlation analysis

For patients who can not be treated with triple therapy, a treatment guide that prescribes bismuth-containing prophylactic treatment may be used. There is a wide variety of combinations of prophylactic therapies. In this example, patients with 2143G bases in four drug combinations metronidazole-amoxicillin-bismuth-PPI (Proton pump inhibitor) Specifically, 34 of the 42 patients who were positive in the urea test were treated with metronidazole-based prophylactic treatment, a second-line treatment for 7 days. Surprisingly, in the second urea breath test, which was evaluated at 6 weeks, only 1 patient had failed the sterilization. This result suggests that the use of metronidazole-amoxicillin-bismuth-PPI therapy in 2143G-positive patients can significantly reduce the rate of eradication failure (FIG. 1).

This indicates that treatment with metronidazole-amoxicillin-bismuth-PPI (proton pump inhibitor) is efficacious in patients with 2143G and 2182 patients with no A / T combination.

[Table 1] Demographic data according to CLO test result (n = 755)

The CLO (Campylobacter-like organism) test was positive in 39.6% of the subjects.

[Table 2] Analysis of gene polymorphism of domain V of 23s RNA of H. pylori

[Table 3] 23s RNA of H. pylori 2143A> G and 2182 T> C eradication failure rate based on genotype

[Table 4] Univariate and multivariate analysis of factors affecting the failure of H. pylori eradication

[Table 5] Logistic regression analysis of factors related to the presence of 2143G in the 23s RNA gene of H. pylori

Example  5: 23s using LAMP method rRNA  Base crystals at positions 2143 and 2182

In this Example, a primer was designed to detect the base identified in the present invention by the LAMP method.

The reference sequence of Helicobacter pylori used for this purpose is as follows.

[Table 6] Reference sequence of Helicobacter pylori

In this embodiment, the method described in Korean Patent No. 1739876 by the present applicant was used to identify the base at the above position. The present invention relates to a method for detecting a single base change in a LAMP based on an allele or mutation specific primer of a target nucleic acid, wherein the 5 'ends of two inner primers FIP and BIP among the four primers used in LAMP The second base from each 5 'end is designed to have a base different from the base of the target nucleic acid, and using such a primer, the presence of a specific allele at a specific position LAMP reaction can be confirmed by amplification.

(One) primer  design

As described in Korean Patent No. 1739876, the 5 'ends of two inner primers FIP and BIP among the four primers used for LAMP have bases corresponding to the allelic variant to be detected, and the 5' ends The second base was designed to have a base different from the base of the target nucleic acid, and its sequence is shown in Tables 7 to 10 below. In the above tables, bases marked with red correspond to polymorphic sites.

Prior to the detection of the polymorphic base, a Urease C primer was prepared and used to detect Helicobacter pylori itself. The experimental results of this example show that polymorphic base analysis was performed only on samples detected positively in Helicobacter pylori. The detection results using Urease C primers are not described.

[Table 7]

[Table 8]

[Table 9]

[Table 10]

The genomic DNA of the bacteria having monosaccharide bases of 23s rRNA shown in Table 6 was extracted and subjected to isothermal amplification under the following conditions using each of the primer sets prepared using the genomic DNA as a template, The detection specificity and sensitivity of 2143 and 2182 bases were detected through color change.

Specifically, the above-mentioned genomic DNA was prepared at 500 pg / μl each. The reaction for LAMP analysis consisted of 0.2 μM of each F3 and B3 primer, 1.6 μM of each FIP and BIP primer, 0.8 μM of each LF and LB primer, 0.4 M betaine, 10 mM MgSO ₄ , 1.4 mM dNTPs, 1 × ThermoPol reaction buffer (New England Biolabs), 8 U Bst DNA polymerase (New England Biolabs), and 120 μM HNB (Sigma). The concentration of the dielectric is as follows. In the first photograph of FIG. 2A, 20 ng of DNA was used and the second photograph was started at 2 pg and diluted to 1/10 of the original number, and 2 pg to 2 fg was used. In Fig. 2b, the concentration was 2 ng to 2 fg starting with 2 ng and diluted to 1/10 the number of times. In FIGS. 3 to 5, the first photograph of a was used for 20 ng of DNA, the second photograph was used for 2 pg to 2 fg, and b was used for 2 ng to 2 fg. The dielectric was included in a volume of 2 [mu] l. The mixture was subjected to isothermal amplification at 63 DEG C for 30 minutes, followed by reaction at 80 DEG C for 5 minutes to terminate the reaction.

Amplification was performed by color analysis as follows. Color analysis was performed by adding HNB (hydroxy naphthol blue) dye to the reaction solution. HNB acts as an indicator (indicator) of Mg ^{2 +} ion concentration. When there is no Mg ²⁺ ion, it appears to be light blue. When Mg ²⁺ ion is present, it is purple. Therefore, before the reaction, it is present in purple, and as the DNA synthesis reaction occurs, the concentration of Mg ²⁺ ions decreases and thus the color changes from purple to blue. The change in color is a positive reaction indicating that the nucleic acid has been amplified and qualitative and quantitative analysis is possible by measuring the absorbance at a wavelength of about 650 nm. The method according to the present invention is advantageous in qualitative and quantitative analysis when absorbance is measured at a wavelength of 650 nm using a spectrophotometer.

The results are shown in Figs. 2-5.

In FIGS. 2 to 5, blue shows a positive reaction. Taking FIG. 2 as an example, the 2143G primer set showed positive reaction only in Helicobacter pylori 51 described in Table 6 having G base at 2143, and negative reaction was observed in 264695 strain having A base at 2143. Similarly, in FIGS. 3 to 5, each of the bases at the corresponding positions could be detected with a high specific sensitivity.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

<110> Mmonitor Inc. <120> Marker of prediction of responsiveness to macrolide antibiotics <130> DP201801011P <150> KR 10-2017-0109869 <151> 2017-08-30 <160> 25 <170> KoPatentin 3.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 2143G F3 <400> 1 tgaatggcgt aacgagatg 19 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 2143G B3 <400> 2 gccaaagccc ttacttca 18 <210> 3 <211> 35 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 2143G FIP (F1c / F2) <400> 3 caccgtcttg ccgcgtctca accagagatt cagtg 35 <210> 4 <211> 39 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 2143G BIP (B1c / B2) <400> 4 gtgaccccgt ggacctttac atcctgcgca tgatattcc 39 <210> 5 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 2143G LF <400> 5 taggaggaat ttcacctcc 19 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 2143G LB <400> 6 acaacttagc actgctaatg 20 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 2143A F3 <400> 7 tgaatggcgt aacgagatg 19 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 2143A B3 <400> 8 gccaaagccc ttacttca 18 <210> 9 <211> 35 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 2143A FIP (F1c / F2) <400> 9 taccgtcttg ccgcgtctca accagagatt cagtg 35 <210> 10 <211> 39 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 2143A BIP (B1c / B2) <400> 10 atgaccccgt ggacctttac atcctgcgca tgatattcc 39 <210> 11 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> 2143A LF <400> 11 taggaggaat ttcacctcc 19 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 2143A LB <400> 12 acaacttagc actgctaatg 20 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 2182C F3 <400> 13 agtgaaattg tagtggaggt g 21 <210> 14 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 2182C B3 <400> 14 caggccagtt agctaaca 18 <210> 15 <211> 41 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 2182C FIP (F1c / F2) <400> 15 ggtagcagtg ctaagttgta gattcctcct acccgcggca a 41 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 2182C BIP (B1c / B2) <400> 16 ccggaatatc atgcgcaggt ggtatctcaa ggatgactcc 40 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 2182C LF <400> 17 taaaggtcca cggggtcttt 20 <210> 18 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 2182C LB <400> 18 aggtgggagg ctttgaa 17 <210> 19 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> 2182T F3 <400> 19 agtgaaattg tagtggaggt g 21 <210> 20 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> 2182T B3 <400> 20 caggccagtt agctaaca 18 <210> 21 <211> 41 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 2182T FIP (F1c / F2) <400> 21 agtagcagtg ctaagttgta gattcctcct acccgcggca a 41 <210> 22 <211> 40 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > 2182T BIP (B1c / B2) <400> 22 tcggaatatc atgcgcaggt ggtatctcaa ggatgactcc 40 <210> 23 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 2182T LF <400> 23 taaaggtcca cggggtcttt 20 <210> 24 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> 2182T LB <400> 24 aggtgggagg ctttgaa 17 <210> 25 <211> 2967 <212> DNA <213> Helicobacter pylori <400> 25 taaggcagtg gtagcgctga agaatgttcg tgcaattgtc gttattcatt ataaaagggc 60 gggttttaaa ggatatttta aaatttaaaa caagctttta agagcagatg gcggatgcct 120 tgccaaagag aggcgatgaa ggacgtacta gactgcgata agctatgcgg agctgtcaag 180 gagctttgat gcgtagatgt ccgaatgggg caacccaact aatagagata ttagttactc 240 taacagagag cgaacctagt gaagtgaaac atctcagtaa ctagaggaaa agaaatcaac 300 gagattccct aagtagtggc gagcgaacgg ggaaaagggc aaaccgagtg cttgcattcg 360 gggttgagga ctgcaacatc caagagaacg ctttagcaga gttacctgga aaggtaagcc 420 atagaaagtg atagccttgt atgcgacaag gcgtttttag gtagcagtat ccagagtagg 480 ccaggacacg aggaatccag gttgaagccg gggagaccac tctccaactc taaatactac 540 tctttgagcg atagcgaaca agtaccgtga gggaaaggtg aaaagaaccg cagtgagcgg 600 agtgaaatag aacctgaaac catctgctta caatcattca gagccctatg atttatcagg 660 gtgatggact gccttttgca taatgatcct gcgagttgtg gtatctggca aggttaagcg 720 aatgcgaagc cgtagcgaaa cgagttctta atagggcgaa caagtcagat gctgcagacc 780 cgaagctaag tgatctatcc atggccaagt tgaaacgcgt gtaatagcgc gtggaggact 840 gaactcctac ccattgaaac gggttgggat gagctgtgga taggggtgaa aggccaaaca 900 aacttagtga tagctggttc tcttcgaaat atatttaggt atagcctcaa gtgataataa 960 aagggggtag agcgctgatt gggctagggc tgctcgccgc ggtaccaaac cctatcaaac 1020 ttcgaatacc ttttatcgta tcttgggagt caggcggtgg gtgataaaat caatcgtcaa 1080 aaggggaaca acccagacta ccaaataagg tccctaagtt ctattctgag tggaaaaaga 1140 tgtgtggcta ctaaaacaac caggaggttg gcttagaagc agccatcctt taaagaaagc 1200 gtaacagctc actggtctag tggtcatgcg ctgaaaatat aacggggcta agatagacac 1260 cgaatttgta gattgtgtta aacacagtgg tagaagagcg ttcataccag cgttgaaggt 1320 ataccggtaa ggagtgctgg agcggtatga agtgagcatg caggaatgag taacgataag 1380 atatatgaga attgtatccg ccgtaaatct aaggtttcct acgcgatggt cgtcatcgta 1440 gggttagtcg ggtcctaagc cgagtccgaa aggggtaggt gatggcaaat tggttaatat 1500 tccaataccg actgtggagc gtgatggggg gacgcatagg gttaagcgag ctagctgatg 1560 gaagcgctag tctaagggcg tagattggag ggaaggcaaa tccacctctg tatttgaaac 1620 ccaaacaggc tctttgagtc cttttaggac aaagggagaa tcgctgatac cgtcgtgcca 1680 agaaaagcct ctaagcatat ccatagtcgt ccgtaccgca aaccgacaca ggtagatgag 1740 atgagtattc taaggcgcgt gaaagaactc tggttaagga actctgcaaa ctagcaccgt 1800 aagttcgcga taaggtgtgc cacagcgatg tggtctcagc aaagagtccc tcccgactgt 1860 ttaccaaaaa cacagcactt tgccaactcg taagaggaag tataaggtgt gacgcctgcc 1920 cggtgctcga aggttaagag gatgcgtcag tcgcaagatg aagcgttgaa ttgaagcccg 1980 agtaaacggc ggccgtaact ataacggtcc taaggtagcg aaattccttg tcggttaaat 2040 accgacctgc atgaatggcg taacgagatg ggagctgtct caaccagaga ttcagtgaaa 2100 ttgtagtgga ggtgaaattc ctcctacccg cggcaagacg gaaagacccc gtggaccttt 2160 actacaactt agcactgcta atgggaatat catgcgcagg ataggtggga ggctttgaag 2220 taagggcttt ggctcttatg gagtcatcct tgagatacca cccttgatgt ttctgttagc 2280 taactggcct gtgttatcca caggcaggac aatgcttggt gggtagtttg actggggcgg 2340 tcgctcctaa aaagtaacgg aggcttgcaa aggttggctc attgcggttg gaaatcgcaa 2400 gttgagtgta atggcacaag ccagcctgac tgtaagacat acaagtcaag cagagacgaa 2460 agtcggtcat agtgatccgg tggttctgtg tggaagggcc atcgctcaaa ggataaaagg 2520 taccccgggg ataacaggct gatctccccc aagagctcac atcgacgggg aggtttggca 2580 cctcgatgtc ggctcatcgc atcctggggc tggagcaggt cccaagggta tggctgttcg 2640 ccatttaaag cggtacgcga gctgggttca gaacgtcgtg agacagttcg gtccctatct 2700 gccgtgggcg taggaaagtt gaggagagct gtccctagta cgagaggacc gggatggacg 2760 tgtcactggt gcaccagttg tctgccaaga gcatcgctgg gtagcacaca cggatgtgat 2820 aactgctgaa agcatctaag caggaaccaa ctccaagata aactttccct gaagctcgca 2880 caaagactat gtgcttgata gggtagatgt gtgagcgcag taatgcgttt agctgactac 2940 tactaataga gcgtttggct tgttttt 2967

Claims (13)

A composition for predicting the reactivity of Helicobacter pylori to clarithromycin comprising the 2143th and 2182th base detecting substances of the above sequence with reference to the nucleic acid sequence of the Helicobacter pylori 23S rRNA gene represented by SEQ ID NO: ,
The Helicobacter pylori is a primary isolate,
When the combinations of the 2143th and 2182th bases are A and T, respectively, the Helicobacter pylori is susceptible to clarithromycin,
Helicobacter pylori is resistant to clarithromycin, and metronidazole-tetracycline-bismuth-PPI (Proton Pump Inhibitor) therapy when the combination of monoclonal combinations of the 2143th and 2182th bases is G and C, or G and T, respectively Lt; RTI ID = 0.0 &gt; Of Helicobacter pylori. &Lt; / RTI &gt;
The method according to claim 1,
Such a detection substance can be used for nucleic acid amplification, allele-specific oligonucleotide hybridization, sequencing, hybridization, 5'-nuclease digestion, single strand polymorphism, primer-specific elongation, or oligo-ligation assays A composition for predicting the clarithromycin reactivity of Helicobacter pylori.
The method according to claim 1,
Wherein the detection substance is a nucleic acid amplification method, and the nucleic acid amplification method is a LAMP method, wherein the composition for predicting the clarithromycin reactivity of Helicobacter pylori.
The method of claim 3,
Wherein the detection material used in the LAMP method for 2143 and 2182 base detection comprises a combination of primers as follows: &lt; RTI ID = 0.0 &gt; Helicobacter pylori &lt; / RTI &gt;
(i) primers for detecting base G of 2143: SEQ ID NOS: 1 to 4;
(ii) primers for detecting base A of 2143: SEQ ID NOS: 7 to 10;
(iii) Primer for detecting base C of 2182: SEQ ID NOS: 13 to 16; And
(iv) primer for base T detection of the above 2182: SEQ ID NOs: 19 to 22.
5. The method of claim 4,
Wherein the combination of the primers of (i) to (iv) further comprises primers of SEQ ID NOs: 5 and 6, SEQ ID NOs: 11 and 12, SEQ ID NOs: 17 and 18, and SEQ ID NOs: 23 and 24, For the prediction of clarithromycin reactivity.
The method of claim 1,
Wherein the composition is applied to a Korean person.
delete In order to provide information for predicting the response to clarithromycin in a subject requiring Helicobacter pylori eradication,
Providing a biological sample derived from said subject;
Separating the Helicobacter pylori from the biological sample and extracting nucleic acid from the Helicobacter pylori, wherein the Helicobacter pylori is a primary isolate,
Analyzing the 2143th base and the 2182th base with reference to the sequence of SEQ ID NO: 25 in the sample; And
As a result of the analysis, when the 2143th base and the 2182nd basal bases are A and T, respectively, the Helicobacter pylori isolated from the subject is judged to be susceptible to clarithromycin,
As a result of the analysis, when the 2143th base and the 2182nd basal bases are G and C, or G and T, respectively, the Helicobacter pylori isolated from the subject is resistant to clarithromycin and metronidazole-tetracycline-bismuth- 0.0 &gt; of Helicobacter pylori, &lt; / RTI &gt; wherein said step of determining is reactive to the PPI agent therapy.
9. The method of claim 8,
The step of analyzing the sequence may be performed by a nucleic acid amplification method, an allele-specific oligonucleotide hybridization, a nucleotide sequence analysis method, a hybridization method, a 5'-nuclease decomposition method, a single strand structural polymorphism, a primer- specific elongation method, Of Helicobacter pylori. &Lt; / RTI &gt;
9. The method of claim 8,
Wherein the biological sample is obtained from a subject who has not been subjected to the triple therapy for Helicobacter pylori.
9. The method of claim 8,
Wherein the composition is applied to a Korean person.
9. The method of claim 8,
Wherein the step of analyzing the 2143th base and the 2182th base is performed by the LAMP method, and the primer used in the LAMP method is as follows: Method for detecting reactive marker for clarithromycin of Helicobacter pylori:
(i) primers for detecting base G of 2143: SEQ ID NOS: 1 to 4;
(ii) primers for detecting base A of 2143: SEQ ID NOS: 7 to 10;
(iii) Primer for detecting base C of 2182: SEQ ID NOS: 13 to 16; And
(iv) primer for base T detection of the above 2182: SEQ ID NOs: 19 to 22.
13. The method of claim 12,
Wherein the combination of the primers of (i) to (iv) further comprises primers of SEQ ID NOs: 5 and 6, SEQ ID NOs: 11 and 12, SEQ ID NOs: 17 and 18, and SEQ ID NOs: 23 and 24, A method for detecting reactive markers for clarithromycin.

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