WO2023121098A1 - Pcr-reverse blot hybridization assay-based improved diagnostic method capable of detecting and identifying both helicobacter pylori and antibiotic-resistant gene thereof, and application thereof - Google Patents

Abstract

The present invention relates to a method and a composition and kit used for the method, wherein the method, based on REBA, is capable of detecting Helicobacter pylori and at the same time determining whether or not the 23S rDNA, 16S rDNA, and gyrase genes of Helicobacter pylori, which are involved in resistance to clarithromycin, tetracycline, and fluoroquinolones used for the treatment of Helicobacter pylori, are mutated.

Description

Improved diagnostic method capable of confirming simultaneous detection of Helicobacter pylori and genes related to antibiotic resistance of the bacteria based on PCR-reverse blot hybridization assay and its application

The present invention relates to an improved diagnostic method capable of confirming simultaneous detection of Helicobacter pylori and genes related to antibiotic resistance of the bacteria based on a PCR-reverse blot hybridization assay and an application thereof.

Helicobacter pylori (H.pylori) is a spiral-shaped Gram-negative bacillus parasitic between the gastric mucosa and mucus (Owen R. J. Helicobacter―classification and identification. British Medical Bulletin. 1998;54(1):17-30. doi : 10.1093/oxfordjournals.bmb.a011667.), since it was first cultured in the gastric mucosa in 1983, it has been known to cause gastritis, gastric ulcer, chronic proximal gastritis, gastric cancer, and mucosa-as-associated lymphoid tissue (MALT) lymphoma. .

Helicobacter pylori is a spiral-shaped bacterium with several flagella. Helicobacter pylori is the only bacterium known to be able to live in the human stomach, which is a strong acidic environment. It acts as a cause of lymphoma, etc. It is found on the surface of the gastric mucosa or in the mucus of the stomach, and it is very rare that the infection penetrates the gastric mucosal cells themselves. In the strong acidic gastric environment, it has urease, which plays an essential role for bacteria to live in the gastric mucosa by creating an alkaline environment.

In case of infection with Helicobacter pylori, the risk of gastric cancer is reported to be 3.8 times higher. defined as a factor. Worldwide, the Helicobacter pylori infection rate is known to be about 50% of adults, and in developing countries, Helicobacter pylori infection rates have been reported up to 80 (Ndip R. N., Malange Takang A. E., Ojongokpoko J. E. A., et al. Tropical Medicine and International Health. 2008;13(6):848-854; Tanih N. F., Ndip L. M., Clarke A. M., Ndip R. N. African Journal of Microbiology Research. 2010;4(6):426-436.), especially in Korea, the rate of Helicobacter pylori infection in the West It is known that 65% of people infected with Helicobacter pylori have gastritis and 10~ have gastric ulcer, and 60~90~ of gastric ulcer patients have Helicobacter pylori infection. It is known that people who are infected with Helicobacter pylori are two to three times more likely to develop gastric cancer than people who are not.

Therefore, it is expected that Helicobacter pylori eradication treatment can play an important role in preventing gastric cancer. For the treatment of Helicobacter pylori infection, a combination therapy of bismuth preparations and antibiotics is used.

Currently, the most representative combination therapy is proton pump inhibitor (PPI)-based combination therapy of clarithromycin (500 mg bid) and amoxicillin (1 g bid) as the first line treatment, or antibiotics such as fluoroquinolones and tetracycline as the second line. (Lee J. W., Kim N., Kim J. M., et al. Prevalence of primary and secondary antimicrobial resistance of Helicobacter pylori in Korea from 2003 through 2012. Helicobacter. 2013;18(3):206-214.) . When the eradication treatment was performed as the primary treatment, the eradication rate is known to be 55∼, and according to a domestic study, the eradication rate of the standard primary treatment was over 90% from 1991 to 1998, which was very effective, whereas the eradication rate after 2000 There is also a report that it appears to be less than 80%, so studies are underway to increase the eradication rate. [The Korean Journal of Helicobacter and Upper Gastrointestinal Research, Vol. 11, no. 1, 26-36, June 2011]

However, the increase in antibiotic resistance of Helicobacter has become a global problem, leading to treatment failure (Mahmoudi S, J Glob Antimicrob Resist. 2017 Jul 15;10:131-135.)

The reason for the decrease in the eradication rate in antibiotic therapy is that Helicobacter pylori is present on the surface of the gastric mucosa or in mucus, so the antibiotic active ingredient often does not sufficiently reach the place where Helicobacter pylori is located. If there is an enemy, there is also a cause that resistance to the drug is not easy to treat. In addition, since the drugs used are quite toxic, there is a problem that the next treatment due to antibiotic resistance may be difficult if the antibiotic is arbitrarily stopped. Since Helicobacter pylori is a bacterium that grows slowly and requires a long time for culture, it is difficult to culture it, so it is difficult to confirm resistance to antibiotics. Although there are diagnostic products that can confirm resistance to clarithromycin in some cases, it is difficult to confirm overall resistance to the first-line drug, so there is a need for diagnostic products that can confirm resistance to antibiotic resistance used.

[Prior Patent Literature]

Republic of Korea Patent Publication No. 1020080028188

The present invention has been made in response to the above needs, and an object of the present invention is to provide a method for simultaneously identifying a novel Helicobacter pylori strain and a drug resistance gene of the Helicobacter pylori strain.

Another object of the present invention is to provide primers, probes, compositions thereof, and kits capable of simultaneously identifying novel Helicobacter pylori bacteria and drug resistance genes of Helicobacter pylori bacteria.

In order to achieve the above object, the present invention provides a step of separating DNA from a specimen sample;

b) A urease gene for Helicobacter identification using each primer from the DNA, a gene for determining whether Helicobacter pylori is resistant to antibiotics, 23S rDNA for clarithromycin, tetracycline PCR amplification of 16S rDNA for fluoroquinolones and gyrase A gene fragments for fluoroquinolones; and

c) 23S rDNA for clarithromycin, 16S rDNA for tetracycline, and fluoro For fluoroquinolones, a PCR-reverse blot hybridization step of hybridizing the PCR amplification product obtained in step b) with the solid support to which the oligomer probe for detecting the gyrase A gene is attached; Provided is an information providing method for simultaneous detection of a characteristic Helicobacter pylori and a gene related to antibiotic resistance of the bacterium.

In one embodiment of the present invention, primers for the urease C gene are SEQ ID NOs: 1 to 2, primers for the 23S rDNA gene are SEQ ID NOs: 3 to 4, and primers for the 16S rDNA gene are SEQ ID NOs: 5 to 6, primers for the gyrase A gene are preferably primers of SEQ ID NOs: 7 to 8, but are not limited thereto.

By attaching biotin to the 5' end of the primer, a color signal can be displayed in the reverse blot hybridization step.

In another embodiment of the present invention, the probe for the urease C gene is SEQ ID NO: 9, the probe for the 23S rDNA gene is SEQ ID NOS: 10 to 13, and the probe for the 16S rDNA gene is SEQ ID NO: 14 to 14 15, the probe for the gyrase A gene is preferably SEQ ID NOs: 16 to 23, but is not limited thereto.

In another embodiment of the present invention, the amplification product is 161 bp for the Helicobacter gene, 123 bp for the clarithromycin gene, 154 bp for the tetracycline gene and fluoroquinolone-based (fluoroquinolones) genes are preferably 142 bp in size, but are not limited thereto.

In addition, the present invention provides primers capable of simultaneously identifying the Helicobacter pylori bacteria and the drug resistance gene of the Helicobacter pylori bacteria consisting of the primers of SEQ ID NO: 1 to SEQ ID NO: 8.

In addition, the present invention provides a probe capable of simultaneously identifying Helicobacter pylori and a drug resistance gene of Helicobacter pylori consisting of the oligomer probes of SEQ ID NOs: 9 to 23.

In addition, the present invention provides a kit for simultaneously identifying Helicobacter pylori bacteria and a drug resistance gene of Helicobacter pylori bacteria consisting of primers of SEQ ID NOs: 1 to 8 and oligomer probes of SEQ ID NOs: 9 to 23.

In one embodiment of the present invention, the drug is preferably a drug selected from the group consisting of clarithromycin, tetracycline, and fluoroquinolones, but is not limited thereto.

In another embodiment of the present invention, the kit preferably further includes, but is not limited to, DNA polymerase, dNTPs, and a buffer as reagents for performing the PCR amplification reaction.

In addition, the present invention provides a composition capable of simultaneously identifying Helicobacter pylori and a drug resistance gene of Helicobacter pylori, including primers of SEQ ID NOs: 1 to 8 and oligomer probes of SEQ ID NOs: 9 to 23.

The primers of the present invention can be chemically synthesized using the phosphoramidite solid support method, or other well-known methods. Such nucleic acid sequences can also be modified using a number of means known in the art. Non-limiting examples of such modifications include methylation, "capping", substitution of one or more homologues of a natural nucleotide, and modifications between nucleotides, such as uncharged linkages such as methyl phosphonates, phosphotriesters, phosphotriesters, phosphoramidates, carbamates, etc.) or to charged linkages (eg phosphorothioates, phosphorodithioates, etc.).

A nucleic acid may contain one or more additional covalently linked moieties, such as proteins (eg, nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), intercalants (eg, acridine, psoralen, etc.). ), chelating agents (eg, metals, radioactive metals, iron, oxidizing metals, etc.), and alkylating agents. A nucleic acid sequence of the present invention may also be modified with a label capable of providing, directly or indirectly, a detectable signal. Examples of labels include radioactive isotopes, fluorescent molecules, and biotin.

The term "real-time PCR" of the present invention refers to amplifying a target using a target probe including a target primer and a label using DNA as a template, and at the same time adding the target probe to the amplified target. It is a molecular biological polymerization method that quantitatively detects signals generated from labels.

The present invention is a PCR-reverse blot hybrid-based molecule that can confirm resistance of 23S rDNA, 16S rDNA, and gyrase A genes involved in clarithromycin, tetracycline, and fluoroquinolones of primary drugs used together with the identification of Helicobacter pylori. A diagnostic test method was developed and its usefulness was evaluated.

As can be seen through the present invention, the present invention is based on REBA-based identification of Helicobacter pylori and the resistance of 23S rDNA, 16S rDNA, and gyrase genes involved in clarithromycin, tetracycline and fluoroquinolones of the primary drugs used together simultaneously. In addition, a color development method was used that unified the reaction temperature to improve the experimental process without any hassle and was easier to read than the existing light emission method.

1 to 4 are photographs verifying the REBA HP-DR probe with the ATCC standard strain,

A (FIG. 1) - ureC gene, B (FIG. 2) - 23S rDNA gene, C (FIG. 3) - 16S rDNA gene, D (FIG. 4) - GyrA gene. W1: ATCC 49503, W2: ATCC 700392, NC: Negative control.

Figure 5 is a photograph verifying the REBA HP-DR probe with plasmid DNA,

1: ureC clone, 2: 23S rDNA wild-type clone, 3: 23S rDNA A2142G mutant clone, 4: 23S rDNA A2142C mutant clone, 5: 23S rDNA A2143G mutant clone, 6: 16S rDNA wild-type clone, 7: 16S rDNA mutant clone, 8: GyrA87 wild-type 1 & GyrA91 wild-type clone, 9: GyrA87 wild-type 2 clone, 10: GyrA87 C261A mutant clone, 11: GyrA87 C261G mutant clone, 12: GyrA91 G271A mutant clone, 13: GyrA91 G271T mutant clone, 14: GyrA91 A272G mutant clone, 15: Negative control;

6 to 9 are the sensitivity test results of the PCR-REBA HP-DR assay, A (Fig. 6) - ureC gene, B (Fig. 7) - 23S rDNA gene, C (Fig. 8) - 16S rDNA gene, D (Fig. 9) - GyrA gene.

Hereinafter, the present invention will be described in more detail through non-limiting examples. However, the following examples are intended to illustrate the present invention, and the scope of the present invention is not to be construed as being limited by the following examples.

Example 1. Materials

To verify the developed primers and probes, they were tested using two ATCC standard strains (ATCC 49503, ATCC 700392) and plasmid DNA, which was synthesized by Bioneer (Daejeon, Korea).

Example 2. DNA extraction

A method of taking a supernatant obtained by taking a portion of the isolates separated through the culture medium, adding 100 μl of DNA extraction solution, vortexing for 1 minute, heating at 100 ° C for 10 minutes, and centrifuging at 13,000 rpm for 3 minutes at room temperature. Nucleic acids were isolated using The isolated nucleic acid was used as a template for PCR to perform REBA HP-DR.

Example 3. Gene collection and analysis of REBA HP-DR

National center for biotechnology information (NCBI, http://www.ncbi.nlm.nih.gov ) PCR primers and oligonucleotide probes targeted at Genbank are urease for accurate bacterial identification of Helicobacter pylori. Genes and clarithromycin (23S rDNA), tetracycline (16S rDNA), and fluoroquinolones (gyraseA) genes, which can determine antibiotic resistance of Helicobacter pylori, were searched for and nucleotide sequences were collected. After performing multi-alignment ( http://multalin.toulouse.inra.fr/multalin ), two pairs of primers were designed in the base sequence region common to each target gene, and among them, Forward and Reverse primers In , biotin was attached to the 5' end for the PCR-REBA method. In addition, an oligonucleotide probe capable of detecting each target species was designed at the bacteria-specific site inside the two pairs of primers, and each oligonucleotide probe had an amine at the 5' end to enable peptide binding to the thin membrane. flag was attached In order to optimize the sensitivity of the probe, 10 to 15 oligo(T) were attached to the 5' end of the probe. The designed primers and oligonucleotide probes were synthesized by Bioneer (Daejeon, Korea).

Example 4. PCR

PCR was performed using the commercially available TOPsimple™PCR PreMIX-Multi HOT (2X) (enzynomics, Daejeon, Korea) using the genomic DNA of each extracted strain as a template. The composition of TOPsimple™PCR PreMIX-Multi HOT (2X) is nTaq-multi HOT DNA Polymerase 0.2 unit/μl, nTaq-multi HOT buffer (containing 4mM MgCl2), dNTP mixture (each 0.4 mM), and a stabilizer. Each composition for PCR consisted of 10 μl of TOPsimple ™ PCR PreMIX-Multi HOT (2X), 1 μl of each pair of 10 pmole primers, 6 μl of ultrapure water, and 2 μl of template DNA to make a total amount of 20 μl. The PCR reaction was 95 ° C for 10 minutes for initial denaturation, 95 ° C, 30 seconds, 65 ° C, and 30 seconds for primary amplification 45 times, and then reacted at 72 ° C for 10 minutes for complete elongation.

Example 5. Performance of REBA HP-DR

If REBA HP-DR using the amplified PCR product was performed using the experimental conditions suggested by the manufacturer, the experimental method is as follows. The double-stranded PCR reaction product was denatured into a single-stranded PCR product by mixing the same amount of denaturation solution (0.2 N NaOH / 0.2 mM EDTA) and reacting at room temperature for 5 minutes. During the reaction, dilute with hybridization solution (2 X SSPE / 0.1% SDS) on the thin film REBA HP membrane to which the bacteria-specific oligonucleotide probe is attached, put it in a 10 well plate, react at 50 ° C for 30 minutes, wash solution (0.5 X SSPE / 0.5% SDS) at 50 ° C for 10 minutes, and then washed with alkaline phosphatase- diluted 1:2000 (v / v) in Conjugate Dilution solution (0.1 M Tris (pH 7.5) / 0.15 M NaCl) Labeled streptavidin conjugate (Roche, Germany) was treated and reacted at room temperature for 15 minutes. After the reaction, the membrane was washed with conjugate dilution solution (0.1 M Tris (pH 7.5) / 0.15 M NaCl) at room temperature for 1 minute, washed with distilled deionized water for 1 minute at room temperature, dried at 37 ° C for 10 minutes, and stained with a staining solution ( 0.1 M Tris (pH 9.5) / 0.1 M NaCl / 0.05 M MgCl ₂ ) was diluted 1:50 (v/v) with NBT/BCIP (Roche, Germany), a color development reagent, for 4 minutes and then evaluated.

The results of the above examples are as follows.

Verification result of REBA HP-DR probe using standard strain

PCR-REBA was performed to confirm the reaction of the bacteria-specific oligonucleotide probe prepared using the PCR product amplified through the Helicobacter pylori standard strain (ATCC 49503, ATCC 700392) (FIGS. 1 to 4). In addition, as a result of confirming the probe of REBA HP-DR using each plasmid DNA, it was confirmed that each DNA sample reacted only to the probe of the target and no other cross-reaction occurred (FIGS. 1 to 4 and 5). ).

Sensitivity evaluation of REBA HP-DR by step dilution experiments

The sensitivity of REBA HP-DR was evaluated by conducting a serial dilution experiment from 100 pg/μl to 100 ag/μl with the Helicobacter pylori standard strain (ATCC 49503). It was confirmed that ureaC was 1 fg/μl, 23S rDNA, 16S rDNA, and GyrA were 10 fg/μl LoD. (FIGS. 6 to 9)

sequence number	primer	Sequence (5'→3')	size
One	HP-urec-2F	ATA AAG TGG GYG AGA GCG TCG	161 bp
2	HP-rueC-4R*	GAC CGG AGC CAC CTT ATA AG
3	23S-2639-F*	TCT CAA CCA GAG ATT CAG TGA AAT TGT	123 bp
4	23S-2760R*	CTG CGC ATG ATA TTC CCA TTA GCA
5	HP-16s-3F	CAC AAG CGG TGG AGC ATG		154bp
6	HP-16s-2R*	TCT CAC GAC ACG AGC TGA CGA C
7	gyrA233-F*	TCG TGG GTG ATG TGA TTG GTA AAT AC	142 bp
8	gyrA374-R*	TTA TCG CCA TCA ATA GAG CCA AAG TT

Table 1 shows the primer sequences used in the present invention.

sequence number	probe	Sequence (5'→3')
9	ureC-2 (10T)	TTTTTTTTTT CAC TCT TTC CCC AAA CAT TTG AAT TT
10	23S-2694W-1	TTTTTTTTTTTTTTT AAG ACG GAA AGA CCC CGT
11	Q_23S_M1_A2142G_1	TTTTTTTTTT AAG ACG GGA AGA CCC CGT		A2142G
12	23S-A2142C-1	TTTTTTTTTTTTTTT AAG ACG GCA AGA CCC CGT		A2142C
13	23S-A2143G-1	TTTTTTTTTTTTTTT AAG ACG GAG AGA CCC CGT		A2143G
14	16S WT (10T)	TTTTTTTTTT GGT TTA ATT CGA AGA TAC ACG AAG		16S AGA
15	16S MT (10T)	TTTTTTTTTT GT TTA ATT CGA TTC TAC ACG AAG	16S TTC
16	gyrA-87W-AAC-2	TTTTTTTTTT CAT GGC GAT AAC GCV GTT TAT	87W-AAC
17	gyrA_87W2_AT_3	TTTTTTTTTT CAT GGC GAT AAT GCV GTT T	87W-AAT
18	gyrA_87M1_AA_1	TTTTTTTTTT CAT GGC GAT AAA GCV GTT TAT	87M-AAA
19	gyrA-87M-AAG-4	TTTTTTTTTT A AAT GGC GAT AAG GCV GTT TAT	87M-AAG
20	gyrA 91 WT (10T)	TTTTTTTTTT GTT TAT GAT GCR YTA GTG AGA ATG	91W-GAT
21	gyrA_91M1_AA_1	TTTTTTTTTT GTT TAT AAT GCR YTA GTG AGA ATG	91M-AAT
22	gyrA_91M2_TA_1	TTTTTTTTTT GTT TAT TAT GCR YTA GTG AGA ATG	91M-TAT
23	Q_gyrA_91M3_GG-1	TTTTTTTTTT GCG GTT TAT GGT GCR CT	91M-GGT

Table 2 shows the nucleotide sequences of the probes used in the present invention.

Claims (8)

1. a) isolating DNA from the specimen sample;

   b) urease C gene for detection of Helicobacter pylori bacteria using each PCR primer from the DNA, 23S rDNA for clarithromycin as a gene for determining whether Helicobacter pylori antibiotic resistance is present, PCR amplification of 16S rDNA for tetracycline and gyrase A gene fragments for fluoroquinolones; and

   c) urease C gene for detection of Helicobacter pylori, 23S rDNA for clarithromycin and 16S rDNA for tetracycline as a gene for determining whether Helicobacter pylori is resistant to antibiotics For , and fluoroquinolones, PCR-reverse blot hybridization step of hybridizing the PCR amplification product obtained in step b) with the solid support to which the oligomer probe for detecting gyrase A gene is attached It is characterized by including;

   Here, the primers for the urease C gene are SEQ ID NOs: 1 to 2, the primers for the 23S rDNA gene are SEQ ID NOs: 3 to 4, the primers for the 16S rDNA gene are SEQ ID NOs: 5 to 6, and the Xyrase A method for providing information for simultaneous detection of Helicobacter pylori and genes related to antibiotic resistance of the bacteria, characterized in that the primers for the gyrase A gene are primers of SEQ ID NOs: 7 to 8.
2. According to claim 1, wherein the probe for the urease (urease) C gene SEQ ID NO: 9,

   Probes for the 23S rDNA gene are SEQ ID NOs: 10 to 13;

   Probes for the 16S rDNA gene are SEQ ID NOs: 14 to 15, and

   The method characterized in that the probe for the gyrase A gene consists of the nucleotide sequences set forth in SEQ ID NOs: 16 to 23.
3. The method of claim 1, wherein the amplification product is 161 bp for the Helicobacter pylori gene, 123 bp for the clarithromycin gene, 154 bp for the tetracycline gene, and fluoroquinolones A method characterized in that the size of the gene is 142 bp.
4. A kit comprising primers of SEQ ID NOs: 1 to 8 and oligomer probes of SEQ ID NOs: 9 to 23, capable of simultaneously identifying Helicobacter pylori and a drug resistance gene of Helicobacter pylori.
5. The kit according to claim 4, wherein the drug is a drug selected from the group consisting of clarithromycin, tetracycline, and fluoroquinolones.
6. The kit according to claim 4, wherein the kit further comprises DNA polymerase, dNTPs, and a buffer as reagents for performing the PCR amplification reaction.
7. SEQ ID NO: 1 to SEQ ID NO: 8 primers and SEQ ID NOS: 9 to 23 comprising the oligomer probes Helicobacter pylori strain identification and Helicobacter pylori composition that can be identified at the same time a drug resistance gene.
8. The composition according to claim 7, wherein the drug is a drug selected from the group consisting of clarithromycin, tetracycline, and fluoroquinolones.

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