CN113604589A - Kit for simultaneously detecting drug-resistant sites, virulence genotyping and proton pump inhibitor metabolic genotyping of helicobacter pylori - Google Patents
Kit for simultaneously detecting drug-resistant sites, virulence genotyping and proton pump inhibitor metabolic genotyping of helicobacter pylori Download PDFInfo
- Publication number
- CN113604589A CN113604589A CN202110677868.5A CN202110677868A CN113604589A CN 113604589 A CN113604589 A CN 113604589A CN 202110677868 A CN202110677868 A CN 202110677868A CN 113604589 A CN113604589 A CN 113604589A
- Authority
- CN
- China
- Prior art keywords
- seq
- primer pair
- artificial sequence
- probe
- pair group
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 44
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 44
- 229940079593 drug Drugs 0.000 title claims abstract description 40
- 239000003814 drug Substances 0.000 title claims abstract description 40
- 230000001018 virulence Effects 0.000 title abstract description 31
- 238000003205 genotyping method Methods 0.000 title abstract description 20
- 229940126409 proton pump inhibitor Drugs 0.000 title abstract description 19
- 239000000612 proton pump inhibitor Substances 0.000 title abstract description 15
- 230000002503 metabolic effect Effects 0.000 title abstract description 12
- 239000000523 sample Substances 0.000 claims abstract description 141
- 238000001514 detection method Methods 0.000 claims description 110
- 238000006243 chemical reaction Methods 0.000 claims description 35
- 150000007523 nucleic acids Chemical class 0.000 claims description 34
- 108020004707 nucleic acids Proteins 0.000 claims description 33
- 102000039446 nucleic acids Human genes 0.000 claims description 33
- 238000000034 method Methods 0.000 claims description 31
- 230000003321 amplification Effects 0.000 claims description 26
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 26
- 230000035772 mutation Effects 0.000 claims description 21
- 206010059866 Drug resistance Diseases 0.000 claims description 20
- 238000012408 PCR amplification Methods 0.000 claims description 12
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 claims description 9
- 238000001269 time-of-flight mass spectrometry Methods 0.000 claims description 9
- 206010019375 Helicobacter infections Diseases 0.000 claims description 8
- 102000002260 Alkaline Phosphatase Human genes 0.000 claims description 5
- 108020004774 Alkaline Phosphatase Proteins 0.000 claims description 5
- 241000143060 Americamysis bahia Species 0.000 claims description 5
- 238000003745 diagnosis Methods 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 61
- 102100021904 Potassium-transporting ATPase alpha chain 1 Human genes 0.000 abstract description 6
- 108010083204 Proton Pumps Proteins 0.000 abstract description 6
- 239000003242 anti bacterial agent Substances 0.000 abstract description 6
- 229940088710 antibiotic agent Drugs 0.000 abstract description 6
- 238000004949 mass spectrometry Methods 0.000 abstract description 6
- 108020004414 DNA Proteins 0.000 description 82
- 238000001819 mass spectrum Methods 0.000 description 33
- 239000000047 product Substances 0.000 description 24
- 230000036267 drug metabolism Effects 0.000 description 18
- 108010026925 Cytochrome P-450 CYP2C19 Proteins 0.000 description 17
- 238000005516 engineering process Methods 0.000 description 15
- 102100029363 Cytochrome P450 2C19 Human genes 0.000 description 14
- 101100476480 Mus musculus S100a8 gene Proteins 0.000 description 14
- 229960002626 clarithromycin Drugs 0.000 description 14
- AGOYDEPGAOXOCK-KCBOHYOISA-N clarithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@](C)([C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)OC)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 AGOYDEPGAOXOCK-KCBOHYOISA-N 0.000 description 14
- 230000035945 sensitivity Effects 0.000 description 13
- 229960003022 amoxicillin Drugs 0.000 description 12
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 12
- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 description 12
- 229960000282 metronidazole Drugs 0.000 description 12
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 12
- 230000008029 eradication Effects 0.000 description 11
- 239000004098 Tetracycline Substances 0.000 description 10
- 238000010586 diagram Methods 0.000 description 10
- PLHJDBGFXBMTGZ-WEVVVXLNSA-N furazolidone Chemical compound O1C([N+](=O)[O-])=CC=C1\C=N\N1C(=O)OCC1 PLHJDBGFXBMTGZ-WEVVVXLNSA-N 0.000 description 10
- 229960001625 furazolidone Drugs 0.000 description 10
- 229960002180 tetracycline Drugs 0.000 description 10
- 229930101283 tetracycline Natural products 0.000 description 10
- 235000019364 tetracycline Nutrition 0.000 description 10
- 150000003522 tetracyclines Chemical class 0.000 description 10
- 102000004190 Enzymes Human genes 0.000 description 9
- 108090000790 Enzymes Proteins 0.000 description 9
- 239000012634 fragment Substances 0.000 description 9
- LISFMEBWQUVKPJ-UHFFFAOYSA-N quinolin-2-ol Chemical compound C1=CC=C2NC(=O)C=CC2=C1 LISFMEBWQUVKPJ-UHFFFAOYSA-N 0.000 description 9
- 238000003753 real-time PCR Methods 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- 230000003115 biocidal effect Effects 0.000 description 8
- 208000015181 infectious disease Diseases 0.000 description 8
- 238000007403 mPCR Methods 0.000 description 8
- 239000013642 negative control Substances 0.000 description 8
- 101150043901 porD gene Proteins 0.000 description 8
- 108700028369 Alleles Proteins 0.000 description 7
- 230000008901 benefit Effects 0.000 description 7
- 238000013461 design Methods 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000000137 annealing Methods 0.000 description 6
- 239000003153 chemical reaction reagent Substances 0.000 description 6
- 108091036078 conserved sequence Proteins 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 108010054814 DNA Gyrase Proteins 0.000 description 5
- 239000000539 dimer Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000004060 metabolic process Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 206010064571 Gene mutation Diseases 0.000 description 4
- 208000005718 Stomach Neoplasms Diseases 0.000 description 4
- 230000000052 comparative effect Effects 0.000 description 4
- 206010017758 gastric cancer Diseases 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 201000011549 stomach cancer Diseases 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000004907 flux Effects 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 101150070420 gyrA gene Proteins 0.000 description 3
- 238000007837 multiplex assay Methods 0.000 description 3
- 238000007481 next generation sequencing Methods 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 150000007660 quinolones Chemical class 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 108010044467 Isoenzymes Proteins 0.000 description 2
- GSDSWSVVBLHKDQ-JTQLQIEISA-N Levofloxacin Chemical compound C([C@@H](N1C2=C(C(C(C(O)=O)=C1)=O)C=C1F)C)OC2=C1N1CCN(C)CC1 GSDSWSVVBLHKDQ-JTQLQIEISA-N 0.000 description 2
- 208000008469 Peptic Ulcer Diseases 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 101150114014 cagA gene Proteins 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 230000001351 cycling effect Effects 0.000 description 2
- 238000011033 desalting Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 210000003608 fece Anatomy 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 238000012165 high-throughput sequencing Methods 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 229960003376 levofloxacin Drugs 0.000 description 2
- 244000000010 microbial pathogen Species 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000012264 purified product Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 101150080234 vacA gene Proteins 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000589875 Campylobacter jejuni Species 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 206010068051 Chimerism Diseases 0.000 description 1
- 108010001237 Cytochrome P-450 CYP2D6 Proteins 0.000 description 1
- 102100021704 Cytochrome P450 2D6 Human genes 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 241000194031 Enterococcus faecium Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 206010025323 Lymphomas Diseases 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 241000531795 Salmonella enterica subsp. enterica serovar Paratyphi A Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 206010066901 Treatment failure Diseases 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000007844 allele-specific PCR Methods 0.000 description 1
- -1 and thus Substances 0.000 description 1
- 238000009635 antibiotic susceptibility testing Methods 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 231100000357 carcinogen Toxicity 0.000 description 1
- 239000003183 carcinogenic agent Substances 0.000 description 1
- 230000000747 cardiac effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 230000006957 competitive inhibition Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 108010012052 cytochrome P-450 CYP2C subfamily Proteins 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000001839 endoscopy Methods 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000011841 epidemiological investigation Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011337 individualized treatment Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000003563 lymphoid tissue Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 230000036457 multidrug resistance Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 208000011906 peptic ulcer disease Diseases 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000003223 protective agent Substances 0.000 description 1
- 238000001243 protein synthesis Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 238000012175 pyrosequencing Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 1
- 229960001225 rifampicin Drugs 0.000 description 1
- 238000007480 sanger sequencing Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 238000011301 standard therapy Methods 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 238000007671 third-generation sequencing Methods 0.000 description 1
- 230000014616 translation Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000000304 virulence factor Substances 0.000 description 1
- 230000007923 virulence factor Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/136—Screening for pharmacological compounds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a kit for simultaneously detecting helicobacter pylori drug-resistant sites, virulence genotyping and proton pump inhibitor metabolic genotyping, in particular to a kit for detecting 16 drug-resistant gene site variations, 5 sites of 2 virulence genes and 3 sites of 2 proton pump drug metabolic genes of 6 common helicobacter pylori antibiotics based on multiple PCR-flight time mass spectrometry, and provides a matched primer, a probe combination and a kit.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a kit for simultaneously detecting drug-resistant sites, virulence genotyping and proton pump inhibitor drug metabolism genotyping of helicobacter pylori.
Background
Helicobacter pylori (h. pylori) is a microaerophilic gram-negative bacterium that colonizes gastric epithelial cells, infects about 50% to 70% of the world's population, and in some developing countries, has an infection rate even as high as over 80%, is one of the most common pathogens worldwide, and can cause gastrointestinal diseases including peptic ulcer, gastric marginal zone/mucosa-associated lymphoid tissue (MALT) lymphoma, and gastric cancer. Helicobacter pylori has been identified as a class I carcinogen by the world health organization and the international agency for research on cancer (IARC). Statistically, more than 6% of all worldwide cancers and about 90% of cases of non-cardiac gastric cancer are due to H.pylori infection. According to the World Health Organization (WHO) data, cancer caused by infection in 2018 accounted for 24.2%, of which 45% were attributed to HP infection. Eradication of helicobacter pylori will significantly reduce the incidence of gastric cancer and peptic ulcers, and reduce the costs associated with controlling these incidences, especially in highly prevalent populations, where eradication of helicobacter pylori is a significant cost benefit. Currently, eradication regimens for helicobacter pylori include primarily Proton Pump Inhibitors (PPIs), gastric mucosa protective agents and one or two antibiotics, constituting triple or quadruple therapy. The antibiotics for eradicating helicobacter pylori include clarithromycin, metronidazole, quinolones (levofloxacin), amoxicillin, tetracycline, rifampicin, etc. In the early 1990 s, the rate of eradication of H.pylori exceeded 80%. However, in recent years, with the increasing bacterial resistance of helicobacter pylori strains to the most commonly used antibiotics worldwide, antibiotic resistance has exceeded the 15-20% threshold in many countries over the past 20 years, with drug resistance levels of clarithromycin, amoxicillin and metronidazole reaching as high as 50%, 30% and 95%, respectively. The emergence of multidrug resistance has significantly affected the efficacy of standard therapy for eradication of helicobacter pylori, resulting in a worldwide decline in the eradication rate of helicobacter pylori. To best optimize management of H.pylori infection, treatment of H.pylori should be based on local and individual patterns of antibiotic resistance, and tailoring effective antibiotic treatment strategies to specific patient sizes may greatly reduce treatment failure and reduce antibiotic resistance.
On the other hand, not all H.pylori-positive patients recommend eradication therapy, and over-treatment results in waste of medical resources, an increase in the drug resistance rate of the population, and the risk of disturbance of the intestinal flora that can result from antibiotic use. A large number of researches show that CagA and VacA are main virulence factors of helicobacter pylori, CagA is divided into positive and negative, wherein the positive virulence is larger than the negative, the VacA gene can be divided into four types of S1/M1, S1/M2, S2/M1 and S2/M2 according to the difference of gene sequences of a signal region (S region) and a middle region (M region), and the virulence degree can be weakened sequentially according to the sequence of S1/M1> S1/M2> S2/M1> S2/M2. Epidemiological investigation showed that the type vacA s1/m1 was significantly associated with cagA positivity, and the strains positive to vacA s1/m1 and cagA were significantly associated with the development of gastric ulcer and gastric cancer. Therefore, whether to carry out HP eradication treatment or not can be determined according to the detection result of the virulence genes and the clinical symptoms and the suggestion of doctors, and powerful judgment basis is provided for the doctors.
Proton Pump Inhibitors (PPIs) are used as the medicines with the strongest acid inhibition effect at present, have high specificity and long duration, and are widely applied to treatment of digestive system diseases. But the lack of awareness of adverse effects of PPIs has led to current worldwide PPI abuse. The use of PPIs is restricted due to the large inter-individual variation of medication. The bioavailability and metabolism of PPIs is mainly influenced by the drug metabolizing enzyme CYP2C 19. The mutation of the CYP2C19 encoding gene can cause the change of the metabolic activity of CYP2C19 enzyme, so that the difference of blood concentration in vivo appears after different patients take medicines taking CYP2C19 as a key metabolic enzyme, and even different clinical reactions are generated. At present, more than 38 CYP2C19 alleles are found, but the main functional genes in drug metabolism are CYP2C19 x 2 and CYP2C19 x 3, which are all loss-of-function alleles. In addition, CYP2C19 x 17 also plays a minor role in PPI metabolism, with minimal probability of mutations in other alleles. The CYP2C19 isozyme is divided into four different metabolic types: ultrafast Metabolizers (UM) (. 1/. 17,. 17/. 17), may cause significant increase in enzyme activity due to abnormal replication or abnormal amplification of functional region genes; rapid metabolic (EM) (. 1/. 1), normal expression of functional region genes, expression of normally active enzymes, EM is also the metabolic phenotype of normal population; intermediate metabolic forms (IM) (. 1/. 2,. 1/. 3,. 17/. 2,. 17/. 3), which may carry a functionally deficient allele or a non-functional allele, express drug metabolizing enzymes with slightly reduced activity compared to normal; the slow metabolizing form (PM) (. 2/. 2,. 2/. 3,. 3/. 3) may carry two null alleles, resulting in a significant reduction in the activity of the expressed drug metabolizing enzyme. The PM isozyme gene is mutated to generate a termination code, so that the protein synthesis is terminated early, and then the inactive CYP2C19 enzyme is generated, and the hydroxylation metabolic capability of the substance is lost. Therefore, the CYP2C19 can be subjected to genotyping detection, so that a doctor can be instructed to accurately dose PPI medicines.
In the context of drug resistance detection, endoscopy-guided helicobacter pylori culture and phenotypic Drug Susceptibility Testing (DST) are the gold standard techniques for detecting drug resistance. However, the need for invasive endoscopy to obtain gastric biopsy specimens from patients, and the need for strict conditions for helicobacter pylori transport and culture, which is difficult and time-consuming, takes at least 10 days to complete bacterial culture and antibiotic susceptibility testing, and is time-consuming and expensive, does not suggest a complete phenotypic DST prior to first-line treatment. With the continuous development and improvement of molecular detection technology, the molecular detection is increasingly widely applied to the diagnosis of pathogenic microorganism infection and the monitoring of treatment, and can be used as an effective alternative method for a culture method drug sensitivity test. The currently common pathogenic microorganism molecular detection method mainly comprises the following steps: PCR-based electrophoresis, real-time fluorescent quantitative PCR (qPCR), gene chip technology, sequencing technology (Sanger sequencing technology, pyrosequencing technology, high-throughput NGS sequencing technology), and the like. PCR is a molecular biology technology for amplifying and amplifying specific DNA fragments, has the characteristics of high sensitivity, strong specificity, simplicity, convenience, rapidness, low requirement on the purity of a sample and the like, and is the most widely applied etiology detection technology at present. Current molecular detection methods for determining helicobacter pylori resistance mainly comprise restriction fragment length polymorphism (PCR-RFLP), Fluorescence In Situ Hybridization (FISH), real-time fluorescence quantitative PCR, allele-specific PCR and the like, detection samples comprise biopsy specimens, gastric juice, colonies and even feces, and the methods show good sensitivity and specificity in the aspect of detecting 23S rRNA and GyrA gene mutations to predict drug resistance of clarithromycin and levofloxacin. Chinese patent CN109797203A, "helicobacter pylori detection system and detection kit and application" and CN111334592A "disclose a nucleic acid composition for detecting helicobacter pylori drug-resistant gene, kit and application thereof, respectively, which are fluorescent quantitative PCR amplification systems for identifying and typing helicobacter pylori and antibiotic drug resistance, but the current fluorescent quantitative PCR method has technical limitations, one detection reaction can only realize simultaneous detection of a plurality of sites at most, the detection flux is limited, and the detection is limited when more gene mutation sites need to be detected. On the other hand, the requirement for rapid detection of large sample volumes may not be met using conventional fluorescent quantitative PCR methods. Chinese patent CN105368825A, "helicobacter pylori antibiotic resistance analysis kit and resistance detection method", and CN105506160A, "helicobacter pylori quantitative and virulence multiple gene detection system, kit and application" both disclose an analysis of helicobacter pylori common antibiotic resistance in the same reaction system based on a multiple PCR-capillary electrophoresis method, but the capillary electrophoresis method can only carry out typing according to the size of the fragment, and has very high requirements on the uniformity and specificity of multiple PCR amplification. NGS high-throughput sequencing technology and chip technology are used as high-throughput high-precision nucleic acid detection technology in recent years, and pathogen separation and culture are not needed, so that the limitation of traditional microbial detection is broken, and the wide application prospect is shown in the field of clinical microbiology research. Chinese patent CN103060455B discloses a detection type gene chip for helicobacter pylori infection individualized treatment and application thereof, which can simultaneously detect human genome CYP4502C19 x 2 and CYP4502C19 x 3 polymorphism and helicobacter pylori clarithromycin and quinolone drug-resistant sites. However, the chip detection and high-throughput sequencing (next generation and third generation sequencing) are complex and expensive in equipment and process, high in detection cost and long in time consumption, and are mainly used for correlation research between genotype and phenotypic drug resistance at present and rarely used in clinical molecular detection of helicobacter pylori. Therefore, how to effectively utilize the means of nucleic acid molecule detection, and rapidly, efficiently, with high throughput, and accurately judge the helicobacter pylori resistance situation is an urgent problem to be solved.
Disclosure of Invention
The invention aims to provide a method for simultaneously detecting helicobacter pylori drug resistance genes (a 23S gene related to drug resistance of clarithromycin, a gyrA gene related to drug resistance of quinolones, a RdxA gene related to drug resistance of metronidazole, a PBP1A related to drug resistance of amoxicillin, a oorD gene and a porD gene related to drug resistance of furazolidone), virulence genes (a CagA gene and a VacA gene) and a CYP2C19 typing site of a drug metabolism gene of a proton pump inhibitor, a matched primer/probe combination and a kit, and application of the detection method in clinical HP eradication therapy medication guidance. The detection result of the virulence gene can be used for assisting a doctor to evaluate the significance of HP eradication treatment, the drug resistance gene detection is used for determining and selecting the type of antibiotics, and the detection of the metabolism gene of the proton pump inhibitor can accurately guide the dosage of PPI drugs.
In a first aspect of the invention, a kit for diagnosing helicobacter pylori infection by using multiplex PCR-time-of-flight mass spectrometry is provided, the kit comprises a first primer pair group, and the first primer pair group comprises primers with sequences shown as SEQ ID No.1 to SEQ ID No. 14.
In another preferred embodiment, the kit further comprises a second primer pair group, and the second primer pair group comprises primers with sequences shown as SEQ ID NO.15 to SEQ ID NO. 30.
In another preferred embodiment, the kit further comprises a third primer pair group, wherein the third primer pair group comprises primers with sequences shown as SEQ ID NO.31 to SEQ ID NO. 48.
In another preferred embodiment, the kit further comprises a first probe set comprising probes having sequences shown in SEQ ID No.49 to SEQ ID No. 55.
In another preferred embodiment, the kit further comprises a second probe set comprising probes having sequences shown in SEQ ID No.56 to SEQ ID No. 63.
In another preferred embodiment, the kit further comprises a third probe set, wherein the third probe set comprises probes shown in SEQ ID No.64 to SEQ ID No. 72.
In another preferred embodiment, the kit comprises a first container containing the first primer pair group.
In another preferred embodiment, the kit comprises a second container, and the second primer pair group is contained in the second container.
In another preferred embodiment, the kit comprises a third container, and the third primer pair group is contained in the third container.
In another preferred embodiment, the kit comprises a fourth container, and the first probe set is contained in the fourth container.
In another preferred embodiment, the kit comprises a fifth container, and the second probe set is contained in the fifth container.
In another preferred embodiment, the kit comprises a sixth container, and the third probe set is contained in the sixth container.
In another preferred embodiment, the kit comprises a seventh container, and the seventh container contains a PCR premix; preferably, the PCR premix solution mainly comprises hot start Taq enzyme, dNTPs and Mg2+。
In another preferred embodiment, the kit comprises an eighth container containing Shrimp Alkaline Phosphatase (SAP).
In another preferred embodiment, the kit comprises a ninth container, wherein the ninth container contains an elongase.
In another preferred embodiment, the kit comprises a tenth container containing the ddNTP.
In another preferred embodiment, the kit comprises an eleventh container containing an extension reaction buffer.
In another preferred embodiment, the kit comprises a twelfth container containing purified water.
In a second aspect of the present invention, there is provided a method for diagnosing helicobacter pylori infection using multiplex PCR-time-of-flight mass spectrometry, the method comprising the steps of:
(1) providing a sample to be detected, and carrying out PCR amplification on nucleic acid in the sample to be detected to obtain a target sequence amplification product in the sample to be detected;
(2) treating the amplification product obtained in step (1) with Shrimp Alkaline Phosphatase (SAP);
(3) performing single-base extension reaction on the amplification product processed in the step (2) by using an extension probe to obtain an extension product;
(4) purifying the extension product;
(5) adopting a matrix-assisted laser desorption ionization time of flight MASS spectrometry (MALDI-TOF-MASS) system to carry out molecular weight detection on the purified extension product, and determining whether the sample to be detected has certain site drug resistance mutation or not according to molecular weight markers;
wherein, in the step (1), PCR amplification is carried out by respectively using a first primer pair group, a second primer pair group and a third primer pair group;
preferably, the first primer pair group comprises primers having sequences shown as SEQ ID No.1 to SEQ ID No. 14;
the second primer pair group comprises primers with sequences shown as SEQ ID NO.15 to SEQ ID NO. 30;
the third primer pair group comprises primers with sequences shown as SEQ ID NO.31 to SEQ ID NO. 48.
In another preferred example, in the step (3), the single base extension reaction is performed using the first probe set, the second probe set, and the third probe set, respectively;
preferably, the first probe set comprises probes having sequences shown as SEQ ID No.49 to SEQ ID No. 55;
the second probe group comprises probes with sequences shown as SEQ ID NO.56 to SEQ ID NO. 63;
the third probe group comprises probes with sequences shown as SEQ ID NO.64 to SEQ ID NO. 72.
In another preferred embodiment, the method is for non-diagnostic purposes. For example, environmental samples can be tested to identify drug-resistant helicobacter pylori in the environmental sample.
In a third aspect of the invention, the use of a primer pair group for the preparation of a detection kit for the diagnosis of helicobacter pylori infection is provided;
the primer pair group is one or more of a first primer pair group, a second primer pair group and a third primer pair group; wherein,
the first primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 14;
the second primer pair group comprises primers with sequences shown as SEQ ID NO.15 to SEQ ID NO. 30;
the third primer pair group comprises primers with sequences shown as SEQ ID NO.31 to SEQ ID NO. 48.
In a fourth aspect of the invention, the use of a probe set for the preparation of a detection kit for the diagnosis of helicobacter pylori infection;
the probe set is one or more of a first probe set, a second probe set, and a third probe set;
the first probe set comprises probes with sequences shown as SEQ ID NO.49 to SEQ ID NO. 55;
the second probe group comprises probes with sequences shown as SEQ ID NO.56 to SEQ ID NO. 63;
the third probe group comprises probes with sequences shown as SEQ ID NO.64 to SEQ ID NO. 72.
It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.
Drawings
FIG. 1 is a nucleic acid mass spectrum of a typical 23S _ A2143G-C mutation in clarithromycin resistance;
FIG. 1-1 is a nucleic acid mass spectrum plot of the lowest limit of detection of 1000 copies/mL of a typical 23S _ A2143G-C mutation in clarithromycin resistance;
FIGS. 1-2 are nucleic acid mass spectra peak plots of the 23S _ A2143G-C detection negative control;
FIG. 2 is a nucleic acid mass spectrum of a typical quinolone-resistant gyrA _ T-C261A-G mutation;
FIG. 2-1 is a nucleic acid mass spectrum peak plot of the minimum detection limit of 1000 copies/mL for a typical quinolone resistant gyrA _ T-C261A-G mutation;
FIG. 2-2 is a nucleic acid mass spectrum peak diagram of a negative control for the detection of GyrA _ T-C261A-G;
FIG. 3 is a nucleic acid mass spectrum peak diagram of typical amoxicillin resistant PBP1A _ CT1242AG mutation;
FIG. 3-1 is a nucleic acid mass spectrum peak diagram of the minimum detection limit of 1000 copies/mL of amoxicillin resistant typical PBP1A _ CT1242AG mutation;
FIG. 3-2 is a nucleic acid mass spectrum peak diagram of a PBP1A _ CT1242AG detection negative control;
FIG. 4 is a nucleic acid mass spectrum peak of typical Rdx _ G616A mutation in metronidazole resistance;
FIG. 4-1 is a nucleic acid mass spectrum peak plot of the minimum detection limit of 1000 copies/mL for a typical metronidazole resistance Rdx _ G616A mutation;
FIG. 4-2 is a nucleic acid mass spectrum peak diagram of Rdx _ G616A detection negative control;
FIG. 5 is a nucleic acid mass spectrum peak of the typical 16S-926-928 TTC mutation of tetracycline resistance;
FIG. 5-1 is a nucleic acid mass spectrum peak plot of the minimum detection limit of 1000 copies/mL for the typical 16S-926-928 TTC mutation for tetracycline resistance;
FIG. 5-2 is a nucleic acid mass spectrum peak diagram of the negative control for the 16S-926 and 928TTC detection.
FIG. 6 is a nucleic acid mass spectrum peak of a typical porD _ G353A mutation in furazolidone resistance;
FIG. 6-1 is a nucleic acid mass spectrum peak plot of 1000 copies/mL of the lowest detection limit of a typical porD _ G353A mutation in furazolidone resistance;
FIG. 6-2 is a nucleic acid mass spectrum peak plot of a negative control for the detection of porD _ G353A.
FIG. 7 is a mass spectrum generated by extending the primer with VS-ZP-F0, which is used to detect the virulence gene VacA, but does not distinguish the S1/S2 typing-specific extended primer peak.
FIG. 8 is a mass spectrum generated by extending the primer VS-ZP-F1 for detecting the S1 typing of VacA virulence gene.
FIG. 9 is a CagA virulence gene specific site CaA-1 nucleic acid mass spectrum peak diagram CagA specific site 1(CagA1) mass spectrum peak diagram
FIG. 10 is a CagA virulence gene specific site CagA-2 nucleic acid mass spectrum peak diagram.
FIG. 11 is a CagA virulence gene specific site CagA-3 nucleic acid mass spectrum peak diagram.
FIG. 12 is a nucleic acid mass spectrum peak of the combination of control primer pair 1 and control extension probe 1.
Detailed Description
Based on multiple PCR-time-of-flight mass spectrometry, the invention provides a method for simultaneously carrying out three noninvasive detections by using a stool sample: the method for detecting drug-resistant gene locus variation of 6 commonly used helicobacter pylori antibiotics (comprising clarithromycin, metronidazole, quinolones, amoxicillin, tetracycline and furazolidone), detecting 5 loci of 2 virulence genes and 3 loci of 2 proton pump drug metabolism genes and parting, and provides a matched primer/probe combination and a kit. Based on a MassARRAY matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF) system (Sequenom, Inc., San Diego, CA, USA), 5 types of virulence gene typing sites (CagA gene, S region of VacA gene, M region of VacA gene) and 3 types of SNP sites (rs4986893, rs4244285 and rs12248560) for typing of drug metabolism gene CYP2C19, 16 types of common HP drug-resistant sites, 5 types of virulence gene typing sites and 3 types of CYP2C19 can be simultaneously detected and analyzed in the same reaction system, and the method comprises five types of drug resistance: tetracycline resistance (2 sites for 16S), clarithromycin resistance (2 sites for 23S rRNA), quinolone resistance (3 sites for GyrA), amoxicillin resistance (2 sites for PBP 1A), metronidazole resistance (2 sites for RdxA), furazolidone resistance (2 sites for oorD and 3 sites for porD). The detection method provided by the invention has the advantages of high precision, high sensitivity, high flux, low cost and rapid detection.
Multiplex PCR (multiplex PCR), also called multiplex PCR or multiplex PCR, is a PCR reaction in which two or more pairs of primers are added to the same PCR reaction system to simultaneously amplify multiple nucleic acid fragments, and the reaction principle, reaction reagents and operation process are the same as those of ordinary PCR.
There are many factors that affect multiplex PCR reactions, such as:
(1) the imbalance of the reaction system causes some dominant primers and templates thereof to be rapidly amplified in the previous rounds of reactions, and a large amount of amplification products are obtained, and the amplification products are good inhibitors of DNA polymerase. Therefore, the polymerization ability of polymerase is more and more strongly inhibited with the occurrence of a large amount of amplification products, and thus, primers and templates thereof which are at a disadvantage in the early stage are more difficult to react, and finally, the amount of amplification products is so small that they cannot be detected.
(2) The primer specificity, if the primer has stronger binding force with other non-target gene fragments in the system, the ability of the target gene to bind the primer is contended, thereby leading to the reduction of the amplification efficiency.
(3) The optimal annealing temperatures are different, a plurality of pairs of primers are placed in a system for amplification, and the optimal annealing temperatures of each pair of primers are required to be close to each other because the annealing temperatures for PCR reaction are the same.
(4) Primer dimers, including dimers between primers and hairpin structures formed by the primers themselves, are third-party DNA-mediated dimers, and these dimers, like non-specific primers, interfere with the competition between primers and target binding sites, affecting amplification efficiency.
Although several factors affecting amplification efficiency are mentioned above, more are not clear. To date, there is no effective method for clearly predicting amplification efficiency.
The determination method of the application is based on multiple PCR technology and flight time mass spectrum, 24 common helicobacter pylori drug-resistant sites, virulence gene sites and drug metabolism gene sites, 24 specific site primers are designed, a specific conserved sequence is selected, PCR primers are designed, a pair of PCR primers are used, the 5' end of the primers is provided with 10 basic group (ACGTTGGATG) sequences, the total length is made to reach 29 basic groups or more, and the primers and the probes are distinguished from each other in terms of molecular weight. A single base extension probe is designed in the conserved sequence region in the amplification region, the length of the probe is 15-21 bases, and a base determined by design is allowed to extend at the 3' end of the probe to be used as the genotype specific sequence marker.
Although the multiplex PCR-time-of-flight mass spectrometry detection technology can carry out ultrahigh-flux detection, the requirement on the quality of a PCR amplification product is high. The inventor finds that the existing primers and probes capable of performing detection by a multiplex fluorescence PCR method are directly applied to multiplex PCR-time-of-flight mass spectrometry, and have many defects, such as false negative of mass spectrometry caused by incapability of performing single base extension reaction, low sensitivity and poor repeatability, which are difficult to meet clinical application. Therefore, the inventor redesigns a plurality of pairs of primers and extension probes for each detection site, performs multiple combined detection verification under the condition that single-site detection can meet the requirement, and finally obtains a multiple PCR detection system and extension probes which have high sensitivity, good specificity and stable detection result and are suitable for flight time mass spectrometry detection through a large amount of test screening.
Therefore, in a preferred embodiment, the invention provides a method, a primer and a probe combination which can simultaneously detect helicobacter pylori drug-resistant gene mutation, virulence genotyping and proton pump inhibitor drug metabolism genotyping, and also comprises a detection reagent required by detection.
In another preferred embodiment, the present invention provides a method for detecting 3 items simultaneously from a fecal sample based on multiplex PCR time-of-flight mass spectrometry, comprising the steps of:
1. and (3) PCR reaction: and obtaining a target sequence amplification product in the sample to be detected through the first round of PCR amplification.
2. Shrimp Alkaline Phosphatase (SAP) treatment: the unbound remaining nucleic acids are dephosphorylated (dNTPs) and prevented from interfering with the next base extension reaction.
3. Base extension reaction: and in the second round of amplification, extending the 3' end of the single-base extension probe to form a sequence-specific mononucleotide as a molecular weight marker, wherein the molecular weight difference between the obtained extension product and the extension probe and other extension products is not less than 16 Da.
4. Resin desalting: purifying the extension reaction product, and adsorbing Na and K ions in the system.
5. Mass spectrum detection: and (3) carrying out molecular weight detection on the purified product by adopting a matrix assisted laser desorption ionization time of flight MASS spectrometry (MALDI-TOF-MASS) system, determining whether a sample to be detected has drug resistance mutation at a certain site according to molecular weight markers, judging the type, and automatically processing a report judgment result by software.
Preferably, a negative control, which is normal human blood DNA, is added to each reaction.
Compared with other existing technologies for detecting helicobacter pylori drug resistance or the like, the technical scheme of the invention fully exerts the advantages of PCR-mass spectrometry combination for simultaneously detecting a plurality of sites, and can detect virulence gene sites and drug metabolism gene sites. The kit has the advantages of no need of fluorescent labeling, no need of washing, reaction in a micro system, little sample loading amount (the concentration is higher than 1 ng/. mu.L and can be detected), and the like, so that the cost of reagent consumables is in inverse proportion to the detection PCR multiplicity, and 16 drug-resistant gene locus variations, 5 loci of 2 virulence genes and 3 loci of 2 proton pump drug metabolism genes can be detected simultaneously by a plurality of probes in a batch of reactions. The first round of PCR amplification area is a genotype specific fragment of 50-130bp, and the second round of PCR amplification adopts a specific probe end single-point method, so that more cycles can be allowed, the detection sensitivity and specificity are improved to the maximum extent, the detection sensitivity reaches 1aM level single copy level, and the false negative problem caused by detection omission is avoided. Meanwhile, the specific fragment amplification design technology can thoroughly eliminate the problems of false positive caused by PCR product pollution and homologous sequence probe mismatching, and provides a reliable experimental method for multiple detection of respiratory pathogens.
A primer and probe combination for helicobacter pylori nucleic acid detection based on multiplex PCR flight time mass spectrum is used for detecting 16 common HP drug-resistant sites, and comprises six drug-resistant types: tetracycline resistance (2 sites of 16S rRNA), clarithromycin resistance (2 sites of 23S rRNA), quinolone resistance (3 sites of GyrA), amoxicillin resistance (2 sites of PBP 1A), metronidazole resistance (2 sites of RdxA), furazolidone resistance (2 sites of oorD, 3 sites of porD); 2 virulence gene typing (including the CagA gene, and the s-fragment and m-fragment typing sites of the VacA gene); genotyping proton pump inhibitor drug metabolism (including CYP2C19 x 1, x 2, x 3, x 17).
The combination of the primer and the probe comprises,
1) primer sequence combinations as shown in table 1; the PCR primers Panel are divided into 3 combinations, which are respectively:
w1 multiplex primer combination: SEQ ID No.1 to SEQ ID No. 14;
w2 multiplex primer combination: SEQ ID No.15 to SEQ ID No. 30;
w3 multiplex primer combination: SEQ ID NO.31 to SEQ ID NO. 48.
Wherein F is a forward primer, and R is a reverse primer. During the amplification reaction, each reaction tube contains a multiplex primer combination.
2) Combinations of probe sequences, as shown in table 2; the extension primers Panel were also divided into 3 combinations:
w 1: SEQ ID No.49 to SEQ ID No. 55;
w 2: SEQ ID No.56 to SEQ ID No. 63;
w 3: SEQ ID NO.64 to SEQ ID NO. 72.
TABLE 1 primer sequences
TABLE 2 Probe sequences
The inventor designs 24 specific site primers aiming at 24 common helicobacter pylori drug-resistant sites, virulence gene sites and drug metabolism gene sites, selects a specific conserved sequence, designs a PCR primer, uses a pair of PCR primers, and has a 10 base (ACGTTGGATG) sequence at the 5' end of the primer, so that the total length reaches 29 bases or more, and distinguishes the primers from the probe in terms of molecular weight. A single base extension probe is designed in the conserved sequence region in the amplification region, the length of the probe is 15-21 bases, and a base determined by design is allowed to extend at the 3' end of the probe to be used as the genotype specific sequence marker.
The synthetic polynucleotides listed in tables 1 and 2 were synthesized by conventional polynucleotide synthesis methods. Purification was ePAGE.
Besides the above-mentioned primers and probes, the invention also provides a helicobacter pylori drug-resistant site nucleic acid detection kit, wherein the specific contents of the components in the detection kit are as follows:
TABLE 3
The main advantages of the invention are:
at present, most detection means mainly detect through fluorescence quantitative PCR, but can not realize the simultaneous detection of a plurality of drug-resistant sites, virulence genes and proton pump drug metabolism genes, and the sequencing means is also used, but the cost is higher, and the invention has the following advantages:
(1) according to the invention, through a large amount of screening and deep research, a multiple primer probe combination suitable for flight time mass spectrum detection is finally obtained, so that the detection of the helicobacter pylori drug-resistant site by multiple PCR-flight time mass spectrum becomes possible; meanwhile, three detection items of noninvasive detection can be synchronously performed by adopting one excrement sample. Therefore, the detection efficiency is greatly improved, and the detection cost is obviously reduced.
(2) 16 common HP drug-resistant sites can be detected simultaneously, and the detection method comprises six drug-resistant types: tetracycline resistance (2 sites of 16S rRNA), clarithromycin resistance (2 sites of 23S rRNA), quinolone resistance (3 sites of GyrA), amoxicillin resistance (2 sites of PBP 1A), metronidazole resistance (2 sites of RdxA), furazolidone resistance (2 sites of oorD, 3 sites of porD); 2 virulence gene typing (including the CagA gene, and the s-fragment and m-fragment typing sites of the VacA gene); genotyping proton pump inhibitor drug metabolism (including CYP2C19 x 1, x 2, x 3, x 17).
(3) The method has high sensitivity, and the detection concentration can be as low as 1000 copies/mL.
(4) The method has extremely high detection efficiency, and can carry out detection of hundreds of fluxes in the same batch, thereby greatly improving the detection efficiency.
The present invention will be described in further detail with reference to the following examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures for conditions not specified in detail in the following examples are generally carried out under conventional conditions such as those described in molecular cloning, A laboratory Manual (Huang Petang et al, Beijing: scientific Press, 2002) by Sambrook. J, USA, or under conditions recommended by the manufacturer. Unless otherwise indicated, percentages and parts are by weight. The test materials and reagents used in the following examples are commercially available without specific reference.
Example 1
The embodiment of the invention provides a method for synchronously detecting and/or identifying helicobacter pylori drug-resistant sites, virulence genes and proton pump inhibitor drug metabolism genotype, wherein the samples to be detected comprise: and (3) fluorescent quantitative detection of known positive stool samples and known negative stool samples. The method comprises the following steps:
(1) aiming at 24 common helicobacter pylori drug-resistant sites, virulence gene sites and drug metabolism gene sites, 24 specific site primers are designed, a specific conserved sequence is selected, a PCR primer is designed, a pair of PCR primers is used, the 5' end of the primer is provided with a 10 base (ACGTTGGATG) sequence, the total length reaches 29 bases or more, and the primers and the probes are distinguished from each other in terms of molecular weight. A single base extension probe is designed in the conserved sequence region in the amplification region, the length of the probe is 15-21 bases, and a base determined by design is allowed to extend at the 3' end of the probe to be used as the genotype specific sequence marker. Probes at 24 sites are shown in Table 2.
(2) And obtaining a target sequence amplification product in the sample to be detected through PCR amplification.
Selecting a sample: selecting positive samples with fluorescence quantitative results of clarithromycin resistance, quinolone resistance, amoxicillin resistance, metronidazole resistance, tetracycline resistance and furazolidone resistance respectively; a positive plasmid with partial site mutation was synthesized and diluted to 1000 copies/mL.
The PCR reaction system is prepared in Table 4.
TABLE 4
| Reagent | | Volume | 1 × (μ L) |
| 1mM PCR Primer Mix | 0.2mM | 1.00 | |
| 2.5×PCR Mix | 1Unit | 2.00 | |
| DNA Template(0.5-2ng/μL) | 1.00 | ||
| RNase-Free Water | N/A | 1.00 | |
| Total | 5.00 |
The PCR enzyme in Mix was hot-started at 95 ℃ for 10 min and then PCR-cycle amplified. The reaction conditions are 95 ℃ denaturation for 30 seconds, 56 ℃ annealing for 30 seconds and 72 ℃ extension for 1 minute, and the total time is 45 cycles; the final 72 ℃ extension for 5 minutes was thermostatted at 4 ℃ after completion.
(3) The unbound residual nucleic acids (dNTPs) are dephosphorylated and inactivated by treatment with Shrimp Alkaline Phosphatase (SAP) to prevent interference with the next base extension reaction. The SAP digestion enzyme reaction system is shown in Table 5.
TABLE 5
| | Volume | 1 × (μ L) |
| ddH2O | 1.75 | |
| SAP Enzyme | 0.25 | |
| Total | 2.00 |
Incubating for 10 minutes at 37 ℃ under the reaction condition, and removing residual dNTPs; the SAP enzyme was then inactivated at 85 ℃ for 5 minutes and thermostated at 4 ℃ after completion.
(4) Extending a sequence-specific mononucleotide at the 3' end of the single-base extension probe by base extension reaction as a molecular weight marker, wherein the molecular weight difference between the obtained extension product and the extension probe and the extension products of each type is not less than 16Da, and the extension reaction system is shown in Table 6.
TABLE 6
Concentration of extension reaction probe Mix was adjusted linearly according to the size of each type of molecular weight.
Cycling reactions were 200 short step programs, comprising two cycling chimerism, starting with denaturation at 95 ℃ for 30 seconds, followed by denaturation at 95 ℃ for 5 seconds, annealing at 52 ℃ for 5 seconds, and extension at 80 ℃ for 5 seconds, for 40 cycles, each with 5 intervening misfires and extensions; the final 72 ℃ extension for 3 minutes, after completion thermostating at 4 ℃.
(5) The extension reaction product was purified by resin desalting.
(6) And (3) carrying out molecular weight detection on the purified product by adopting a matrix-assisted laser desorption ionization time-of-flight mass spectrometry system, and determining whether the sample to be detected has certain site mutation according to the molecular weight marker.
(7) In FIG. 1, the abscissa represents molecular weight and the ordinate represents peak intensity. The dotted vertical line in the figure shows the same color as the molecular weight position of the genotype extension probe, and if the genotype extension probe peak does not exist, the peak is unchanged; if the copy number of the detected sample is more than 1000, the probe can be completely consumed, and the left peak disappears and turns to the right; the dotted, same color line on the right indicates the extension product molecular weight position. Reading the spectrogram by software, automatically analyzing and reporting the result, and exporting data. The data is interpreted that the extension probe of each site or reference gene has corresponding molecular weight peak at different mass positions of a mass spectrogram, a single-base extension product appears when the probe finds that the target gene works, the molecular weight peak of the probe is transferred to a product molecular peak, and the analysis result is reported to be positive. Positive results are divided into four cases of interpretation: A. the result is reliable; B. moderate reliability; C. is generally reliable; D. low degree and reliability. The first three are regarded as effective extension reactions, can confirm that the site has mutation and corresponds to certain drug resistance, and the fourth one needs artificial auxiliary judgment to observe whether the probe is consumed or not and judge the result as suspicious infection or negative result. For suspicious infection samples, a reproducibility validation test can be performed if necessary.
(8) The test performs mass spectrometric detection on positive samples with fluorescence quantitative results showing that the positive samples respectively have clarithromycin resistance, quinolone resistance, amoxicillin resistance, metronidazole resistance, tetracycline resistance and furazolidone resistance, and the results show that the detection results are consistent with the fluorescence quantitative results and can detect corresponding drug-resistant sites (as shown in figures 1, 2, 3, 4, 5 and 6); the detection results of the positive plasmids are shown in figures 1-1, 2-1, 3-1, 4-1, 5-1 and 6-1; and the negative control results in the detection are shown in FIGS. 1-2, 2-2, 3-2, 4-2, 5-2, and 6-2.
(9) The extended primer of VS-ZP-F0 can recognize the VacA gene, but does not distinguish S1/S2 typing, the extended primer of VS-ZP-F1 is used for detecting S1 type, if there is an extended peak in VS-ZP-F1, it is S1 type, if there is no extended peak in VS-ZP-F0, it is S2 type, if there is no extended peak in VS-ZP-F0 and VS-ZP-F1, it indicates that the VacA gene is not detected.
(10) CagA-1, CagA-2 and CagA-3 are specific sites at 3 different positions designed in a relatively conserved region of the CagA gene, because the sequence variability of the CagA gene is large, 3 sites are set, and if any specific site is detected, the specific site is positive for the CagA gene
(11) Positive results of both VacA gene s1 and CagA detected HP as virulent strain strongly suggest eradication therapy under the guidance of doctor.
(12) The invention carries out mass spectrum detection of proton pump inhibitor drug metabolism genotyping on feces samples with positive fluorescence quantification, and the proton pump metabolism type and frequency, and the genotyping and the frequency are found to be in accordance with the convention after being collated (see table 7); the present inventors have also found that the ratio of each type is similar to that of the known database pharmgkb (east asian) or literature (yinju et al, frequency of drug metabolism CYP2C19 and CYP2D6 genotype distribution in chinese population) (see table 8 below).
TABLE 7 proton Pump metabolism and genotyping and frequency
TABLE 8 comparison of the frequencies of CYP2C19 allele distributions (%)
And (4) conclusion: the method and the kit can simultaneously detect six drug-resistant gene mutations of helicobacter pylori, virulence genotyping and proton pump inhibitor drug metabolism genotyping, and the total number of the detection sites is 24. And the detection sensitivity is as low as 1000 copies/mL.
Example 2 specific assay
The kit provided by the invention has the advantages that the detection result of the multiple PCR-flight time mass spectrum detection kit does not have a false positive result by taking Escherichia coli, staphylococcus aureus, campylobacter jejuni, candida albicans, enterococcus faecium, streptococcus pneumoniae, salmonella paratyphi A and human genome DNA nucleic acid as templates, and the kit has good specificity.
Example 3 clinical sample testing
The method for detecting and/or identifying the helicobacter pylori drug-resistant sites with high sensitivity is used for detecting nucleic acid samples of 100 helicobacter pylori positive patients and verifying the nucleic acid samples by adopting a commercially available single fluorescent quantitative PCR detection kit.
The results show that:
in 100 positive samples, 100 positive samples are detected by the single fluorescent quantitative PCR and the multiple PCR-time-of-flight mass spectrometry detection method, and the positive detection rate is 100%. 11 tetracycline resistant cases, 35 clarithromycin resistant cases, 32 quinolone resistant cases, 3 amoxicillin resistant cases, 56 metronidazole resistant cases and 3 furazolidone resistant cases are identified, and the rest samples are infected by sensitive strains.
Comparative example 1 screening of primer set and extension Probe
The invention artificially designs a large amount of primers and extension probes through a large amount of experiments, and then optimally selects and verifies the primers and the extension probes to finally determine the sequences and the combination of the multiple PCR amplification primers and the extension probes which can be used for the flight time mass spectrometry detection.
This comparative example illustrates the 23S _ A2143G-C site, showing primers and extension probes that are partially ineffective.
23S _ A2143G-C site primer and extension probe sequences:
control primer pair 1:
F-1：ACGTTGGATGGTGAAATTGTAGTGGAGGTG(SEQ ID NO.:73)
R-1：ACGTTGGATGTTCCCATTAGCAGTGCTAAG(SEQ ID NO.:74)
control primer pair 2:
F-2：ACGTTGGATGGCTGTCTCAACCAGAGATTC(SEQ ID NO.:75)
R-2：ACGTTGGATGCGCATGATATTCCCATTAGC(SEQ ID NO.:76)
control extension probe 1:
P-1：TCATACCCGCGGCAAGACGGA(SEQ ID NO.:77)
the primer pair of the invention comprises: SEQ ID NO.9 and 10
The extension probe of the invention: SEQ ID NO.53
The specific method is the same as example 1, single PCR amplification is firstly carried out, then different extension probes are used for single base extension, and then mass spectrum detection is carried out on extension products. The detection sensitivity can only reach 1 × 10 by combining the control primer pair 1 with the control extension probe 1 and the extension probe (SEQ ID NO.53) of the invention respectively5copy/mL. The combination of control primer pair 2 and control extension probe 1 worked well in the single detection line, but in the multiplex systemA positive result cannot be obtained by mass spectrometry (as shown in fig. 12).
The combination of the primer pair (SEQ ID NO.9 and 10) of the invention and the extension probe (SEQ ID NO.53) of the invention can achieve the detection sensitivity of 1 x 103copy/mL.
Comparative example 2 construction of multiplex assay System
Due to the reasons of competitive inhibition between primers, primer specificity difference, inconsistent annealing temperature, primer dimer and the like in a multiplex reaction system, it is difficult to obtain an effective multiplex PCR amplification primer and single-base extension probe combination.
Therefore, it is necessary to optimize a multiplex detection system by multiplex combination of the candidate primer pairs selected for each mutation site and the extension probe. This comparative example illustrates a multiple detection system with partially unsatisfactory results.
Control multiplex assay system 1:
TABLE 9
| Serial number | Site of the |
| 1 | GyrA_T-C261A- |
| 2 | GyrA_G271A- |
| 3 | GyrA_A272G-T |
| 4 | 16S_926- |
| 5 | 16S_926-928AGA |
| 6 | 23S_A2142G-C |
| 7 | 23S_A2143G- |
| 8 | RdxA_G565T |
| 9 | RdxA_G616A |
Control multiplex assay system 2:
watch 10
| Serial number | Site of the |
| 1 | |
| 2 | |
| 3 | 23S_A2142G-C |
| 4 | 23S_A2143G- |
| 5 | oorD_A041G |
| 6 | oorD_A122G |
| 7 | |
| 8 | porD_A356G |
| 9 | porD_C357T |
The specific method is the same as example 1, the control multiplex detection system 1 is a system before optimization of W1 multiplex primer combination, wherein two sites, namely 16S _926-928TTC and 16S _926-928AGA, have competition inhibition phenomena, thereby causing the situation that the detection cannot be carried out.
The control multiplex detection system 2 is a W3 multiplex primer combination optimization pre-system, wherein after two sites 23S _ A2143G-C and porD _ G353A are formed due to new combination, the molecular weight of the extension product of each site or the molecular weight difference between the extension probe and each extension product is larger than 16Da, and the control multiplex detection system cannot work as a multiplex detection system.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
<110> Jiangsukang was a century Biotechnology GmbH
<120> kit for simultaneously detecting drug-resistant sites, virulence genotyping and proton pump inhibitor metabolic genotyping of helicobacter pylori
<130> 035003
<160> 77
<170> SIPOSequenceListing 1.0
<210> 1
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 1
acgttggatg gattggtaaa taccaccccc 30
<210> 2
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 2
acgttggatg gcatggaaaa atcttgcgcc 30
<210> 3
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 3
acgttggatg gcatggaaaa atcttgcgcc 30
<210> 4
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 4
acgttggatg gattggtaaa taccaccccc 30
<210> 5
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 5
acgttggatg gcatggaaaa atcttgcgcc 30
<210> 6
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 6
acgttggatg gattggtaaa taccaccccc 30
<210> 7
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 7
acgttggatg tgaaaattcc tcctacccgc 30
<210> 8
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 8
acgttggatg atcctgcgca tgatattccc 30
<210> 9
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 9
acgttggatg tgaaaattcc tcctacccgc 30
<210> 10
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 10
acgttggatg atcctgcgca tgatattccc 30
<210> 11
<211> 33
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 11
acgttggatg atcaataagc ctaaaatcgc atg 33
<210> 12
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 12
acgttggatg gaaaacaccc ctaaaagagc g 31
<210> 13
<211> 33
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 13
acgttggatg atcaataagc ctaaaatcgc atg 33
<210> 14
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 14
acgttggatg gaaaacaccc ctaaaagagc g 31
<210> 15
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 15
acgttggatg gactgtaagt ggtttctcag 30
<210> 16
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 16
acgttggatg aacatcagga ttgtaagcac 30
<210> 17
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 17
acgttggatg caaatttgtg tcttctgttc 30
<210> 18
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 18
acgttggatg ggatttgagc tgaggtcttc 30
<210> 19
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 19
acgttggatg tctttcttgc ctgggatctc 30
<210> 20
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 20
acgttggatg ctctttccat tgctgaaaac g 31
<210> 21
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 21
acgttggatg acaccgcaaa atcaatcgc 29
<210> 22
<211> 33
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 22
acgttggatg ccaacaatgg ctggaatgat cac 33
<210> 23
<211> 29
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 23
acgttggatg acaccgcaaa atcaatcgc 29
<210> 24
<211> 33
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 24
acgttggatg ccaacaatgg ctggaatgat cac 33
<210> 25
<211> 33
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 25
acgttggatg ggtatcaatc cagaatggat ttc 33
<210> 26
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 26
acgttggatg ttcaaggtcg ctttttgctt g 31
<210> 27
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 27
acgttggatg ggcctrctgg tggggattgg 30
<210> 28
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 28
acgttggatg tatgtcggtg gtagtagtgg c 31
<210> 29
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 29
acgttggatg gagtggctta agctcgtgaa 30
<210> 30
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 30
acgttggatg cttggtggaa aacttgaacg 30
<210> 31
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 31
acgttggatg aacagcgaac aaaaccacgc 30
<210> 32
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 32
acgttggatg tcttgcaagg ttacaagccc 30
<210> 33
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 33
acgttggatg aaatggcaca gggagtttgg 30
<210> 34
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 34
acgttggatg taaagccaat gaaccaagcg 30
<210> 35
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 35
acgttggatg aactcaaagg aatagacggg 30
<210> 36
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 36
acgttggatg gtcaagccta ggtaaggttc 30
<210> 37
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 37
acgttggatg aactcaaagg aatagacggg 30
<210> 38
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 38
acgttggatg gtcaagccta ggtaaggttc 30
<210> 39
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 39
acgttggatg gcacaaagga gaatgaatgg 30
<210> 40
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 40
acgttggatg ccaagcaccc tttctttttc 30
<210> 41
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 41
acgttggatg gcacaaagga gaatgaatgg 30
<210> 42
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 42
acgttggatg ccaagcaccc tttctttttc 30
<210> 43
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 43
acgttggatg cgctatggat gtttgaagaa c 31
<210> 44
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 44
acgttggatg ccatcccaca cttctatctc 30
<210> 45
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 45
acgttggatg cgctatggat gtttgaagaa c 31
<210> 46
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 46
acgttggatg ccatcccaca cttctatctc 30
<210> 47
<211> 31
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 47
acgttggatg cgctatggat gtttgaagaa c 31
<210> 48
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 48
acgttggatg ccatcccaca cttctatctc 30
<210> 49
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 49
accaccccca tggcgataa 19
<210> 50
<211> 16
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 50
attctcacta gcgcat 16
<210> 51
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 51
gccattctca ctagcgca 18
<210> 52
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 52
<210> 53
<211> 16
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 53
aaggtccacg gggtct 16
<210> 54
<211> 15
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 54
<210> 55
<211> 24
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 55
tgatcaagaa aatcaaaagt tgat 24
<210> 56
<211> 16
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 56
ttggccttac ctggat 16
<210> 57
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 57
<210> 58
<211> 15
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 58
<210> 59
<211> 16
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 59
caaagtcatg ccgcct 16
<210> 60
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 60
gcctttttca caaccgtga 19
<210> 61
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 61
tgatttcaaa aatggcaa 18
<210> 62
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 62
ttaacaaaaa acaatctt 18
<210> 63
<211> 15
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 63
<210> 64
<211> 18
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 64
aaacttgcga gaataatt 18
<210> 65
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 65
<210> 66
<211> 15
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 66
<210> 67
<211> 15
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 67
<210> 68
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 68
gagcgctcca gatggggtt 19
<210> 69
<211> 16
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 69
ggttcttggc atgggg 16
<210> 70
<211> 17
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 70
aagaacaaat tgagcct 17
<210> 71
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 71
gaagaacaaa ttgagcctgc t 21
<210> 72
<211> 20
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 72
<210> 73
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 73
acgttggatg gtgaaattgt agtggaggtg 30
<210> 74
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 74
acgttggatg ttcccattag cagtgctaag 30
<210> 75
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 75
acgttggatg gctgtctcaa ccagagattc 30
<210> 76
<211> 30
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 76
acgttggatg cgcatgatat tcccattagc 30
<210> 77
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequence)
<400> 77
tcatacccgc ggcaagacgg a 21
Claims (10)
1. A kit for diagnosing helicobacter pylori infection by utilizing multiplex PCR-time-of-flight mass spectrometry is characterized by comprising a first primer pair group, wherein the first primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 14.
2. The kit of claim 1, further comprising a second primer pair group comprising primers having sequences set forth in SEQ ID No.15 to SEQ ID No. 30.
3. The kit of claim 2, further comprising a third primer pair group comprising primers having sequences set forth in SEQ ID No.31 to SEQ ID No. 48.
4. The kit of claim 3, further comprising a first probe set comprising probes having sequences set forth in SEQ ID No.49 through SEQ ID No. 55.
5. The kit of claim 4, further comprising a second probe set comprising probes having sequences set forth in SEQ ID No.56 to SEQ ID No. 63.
6. The kit of claim 5, further comprising a third probe set comprising probes having sequences set forth in SEQ ID No.64 to SEQ ID No. 72.
7. The kit of claim 6, comprising a first container, a second container, a third container, a fourth container, a fifth container, and a sixth container, wherein the first primer-set group is contained within the first container; the second container contains the second primer pair group; the third container contains the third primer pair group; the fourth container contains the first probe group; the second probe set is contained in the fifth container; the third probe set is contained in the sixth container.
8. A method for detecting drug-resistant sites of helicobacter pylori using multiplex PCR-time-of-flight mass spectrometry, the method comprising the steps of:
(1) providing a sample to be detected, and carrying out PCR amplification on nucleic acid in the sample to be detected to obtain a target sequence amplification product in the sample to be detected;
(2) treating the amplification product obtained in step (1) with Shrimp Alkaline Phosphatase (SAP);
(3) performing single-base extension reaction on the amplification product processed in the step (2) by using an extension probe to obtain an extension product;
(4) purifying the extension product;
(5) adopting a matrix-assisted laser desorption ionization time of flight MASS spectrometry (MALDI-TOF-MASS) system to carry out molecular weight detection on the purified extension product, and determining whether the sample to be detected has certain site drug resistance mutation or not according to molecular weight markers;
wherein, in the step (1), PCR amplification is carried out by respectively using a first primer pair group, a second primer pair group and a third primer pair group;
preferably, the first primer pair group comprises primers having sequences shown as SEQ ID No.1 to SEQ ID No. 14;
the second primer pair group comprises primers with sequences shown as SEQ ID NO.15 to SEQ ID NO. 30;
the third primer pair group comprises primers with sequences shown as SEQ ID NO.31 to SEQ ID NO. 48.
Further, in the step (3), performing a single base extension reaction using the first probe set, the second probe set, and the third probe set, respectively;
preferably, the first probe set comprises probes having sequences shown as SEQ ID No.49 to SEQ ID No. 55;
the second probe group comprises probes with sequences shown as SEQ ID NO.56 to SEQ ID NO. 63;
the third probe group comprises probes with sequences shown as SEQ ID NO.64 to SEQ ID NO. 72.
9. The application of the primer pair group in preparing a detection kit for diagnosing helicobacter pylori infection;
the primer pair group is one or more of a first primer pair group, a second primer pair group and a third primer pair group; wherein,
the first primer pair group comprises primers with sequences shown as SEQ ID NO.1 to SEQ ID NO. 14;
the second primer pair group comprises primers with sequences shown as SEQ ID NO.15 to SEQ ID NO. 30;
the third primer pair group comprises primers with sequences shown as SEQ ID NO.31 to SEQ ID NO. 48.
10. The use of a probe set for the preparation of a detection kit for the diagnosis of helicobacter pylori infection;
the probe set is one or more of a first probe set, a second probe set, and a third probe set;
the first probe set comprises probes with sequences shown as SEQ ID NO.49 to SEQ ID NO. 55;
the second probe group comprises probes with sequences shown as SEQ ID NO.56 to SEQ ID NO. 63;
the third probe group comprises probes with sequences shown as SEQ ID NO.64 to SEQ ID NO. 72.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110677868.5A CN113604589B (en) | 2021-06-18 | 2021-06-18 | Kit for simultaneously detecting drug-resistant locus and virulence genotyping of helicobacter pylori and metabolic genotyping of proton pump inhibitor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110677868.5A CN113604589B (en) | 2021-06-18 | 2021-06-18 | Kit for simultaneously detecting drug-resistant locus and virulence genotyping of helicobacter pylori and metabolic genotyping of proton pump inhibitor |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113604589A true CN113604589A (en) | 2021-11-05 |
| CN113604589B CN113604589B (en) | 2024-03-29 |
Family
ID=78336580
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110677868.5A Active CN113604589B (en) | 2021-06-18 | 2021-06-18 | Kit for simultaneously detecting drug-resistant locus and virulence genotyping of helicobacter pylori and metabolic genotyping of proton pump inhibitor |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113604589B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114574601A (en) * | 2022-01-22 | 2022-06-03 | 北京新基永康生物科技有限公司 | Primer and probe composition for detecting helicobacter pylori 23SrRNA gene mutation, application thereof and kit |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060455A (en) * | 2013-01-14 | 2013-04-24 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Detection gene chip for helicobacter pylori infection individualized treatment and application of gene chip |
| CN104846097A (en) * | 2015-05-21 | 2015-08-19 | 杭州千基生物科技有限公司 | Helicobacter pylori (HP) type and drug-resistant mutation gene detection kit |
| CN108474042A (en) * | 2014-10-17 | 2018-08-31 | 弗雷德哈钦森癌症研究中心 | A kind of agent formulations of assessment helicobacter pylori infections, kit and method |
| CN111705148A (en) * | 2020-06-24 | 2020-09-25 | 南开大学 | Rapid molecular diagnosis method and kit for personalized medication guidance in helicobacter pylori eradication treatment |
| CN111996273A (en) * | 2020-11-02 | 2020-11-27 | 北京健为医学检验实验室有限公司 | Method and kit for detecting drug-resistant gene mutation of helicobacter pylori |
| CN112080563A (en) * | 2020-09-01 | 2020-12-15 | 美思睿(北京)生物科技有限公司 | Kit for detecting accurate medication genes of chronic diseases |
-
2021
- 2021-06-18 CN CN202110677868.5A patent/CN113604589B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103060455A (en) * | 2013-01-14 | 2013-04-24 | 中国人民解放军军事医学科学院放射与辐射医学研究所 | Detection gene chip for helicobacter pylori infection individualized treatment and application of gene chip |
| CN108474042A (en) * | 2014-10-17 | 2018-08-31 | 弗雷德哈钦森癌症研究中心 | A kind of agent formulations of assessment helicobacter pylori infections, kit and method |
| CN104846097A (en) * | 2015-05-21 | 2015-08-19 | 杭州千基生物科技有限公司 | Helicobacter pylori (HP) type and drug-resistant mutation gene detection kit |
| CN111705148A (en) * | 2020-06-24 | 2020-09-25 | 南开大学 | Rapid molecular diagnosis method and kit for personalized medication guidance in helicobacter pylori eradication treatment |
| CN112080563A (en) * | 2020-09-01 | 2020-12-15 | 美思睿(北京)生物科技有限公司 | Kit for detecting accurate medication genes of chronic diseases |
| CN111996273A (en) * | 2020-11-02 | 2020-11-27 | 北京健为医学检验实验室有限公司 | Method and kit for detecting drug-resistant gene mutation of helicobacter pylori |
Non-Patent Citations (1)
| Title |
|---|
| RAM M.R.等: "polymorphisms in the host CYP2C19 gene and antibiotic-resistance attributes of Helicobacter pylori isolates influence the outcome of triple therapy", J ANTIMICROB CHEMOTHER, vol. 74, pages 11 - 16 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114574601A (en) * | 2022-01-22 | 2022-06-03 | 北京新基永康生物科技有限公司 | Primer and probe composition for detecting helicobacter pylori 23SrRNA gene mutation, application thereof and kit |
| CN114574601B (en) * | 2022-01-22 | 2024-06-11 | 北京新基永康生物科技有限公司 | Primer and probe composition for detecting helicobacter pylori 23SrRNA gene mutation, application thereof and kit |
Also Published As
| Publication number | Publication date |
|---|---|
| CN113604589B (en) | 2024-03-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110438223B (en) | Primer and probe for detecting Kras gene point mutation, kit and detection method thereof | |
| CN107447013B (en) | Method for detecting mutation sites of codons 12 and 13 of Kras gene and kit thereof | |
| CN110484621B (en) | Early warning method for liver cancer | |
| CN113817850A (en) | Mycobacterium tuberculosis drug-resistant gene detection primer composition and application thereof | |
| CN111996273B (en) | Method and kit for detecting drug-resistant gene mutation of helicobacter pylori | |
| CN112941210A (en) | Kit and method for detecting drug-resistant mutation of mycobacterium tuberculosis rifampicin and isoniazid | |
| CN111440852B (en) | Kit and method for detecting methylation sites of DMR2 region of MGMT gene promoter through multiple probes | |
| US20220136046A1 (en) | Detection and antibiotic resistance profiling of microorganisms | |
| US6355435B1 (en) | Methods for detecting and enumerating Campylobacter jejuni in environmental samples and for identifying antibiotic-resistant strains | |
| KR102675157B1 (en) | Method and Kit for Diagnosing Bovine tuberculosis using PNA | |
| CN110592217A (en) | Kit for detecting KRAS gene mutation in free DNA of peripheral blood and application thereof | |
| US11535897B2 (en) | Composite epigenetic biomarkers for accurate screening, diagnosis and prognosis of colorectal cancer | |
| CN113604589B (en) | Kit for simultaneously detecting drug-resistant locus and virulence genotyping of helicobacter pylori and metabolic genotyping of proton pump inhibitor | |
| CN116694786B (en) | Primer probe combination for detecting helicobacter pylori drug-resistant mutant gene | |
| CN116970722A (en) | Method for simultaneously identifying helicobacter pylori infection and detecting drug-resistant gene mutation site, primer probe composition, application and kit | |
| EP2154247A1 (en) | Method of amplifying methylated nucleic acid or unmethylated nucleic acid | |
| CA2775273A1 (en) | Composition, method and kit for detecting bacteria by means of sequencing | |
| US20230227913A1 (en) | Methods, treatment, and compositions for characterizing thyroid nodule | |
| CN116004775A (en) | Primer probe composition, kit and method for quantifying copy number of human motor neurons | |
| KR102334994B1 (en) | A METHOD FOR DETECTING AND CONFIRMING GENOTYPE OF Porcine circovirus type 2 AND COMPOSITION THEREFOR | |
| CN114292935A (en) | Nucleic acid composition and kit for detecting drug resistance gene of mycobacterium tuberculosis and method for detecting drug resistance of mycobacterium tuberculosis | |
| US11542559B2 (en) | Methylation-based biomarkers in breast cancer screening, diagnosis, or prognosis | |
| KR102502129B1 (en) | Method and Kit for Identifying the serotype of Actinobacillus pleuropneumoniae causing pneumonia in Pigs using PNA probe | |
| CN112831558B (en) | Early screening method and kit for Crohn disease susceptibility genes | |
| KR102520374B1 (en) | Method and Kit for Identifying the serotype of Actinobacillus pleuropneumoniae causing pneumonia in Pigs using PNA probe |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |