CN109593842A - A kind of kit and method of detection and judgement helicobacter pylori drug resistance - Google Patents
A kind of kit and method of detection and judgement helicobacter pylori drug resistance Download PDFInfo
- Publication number
- CN109593842A CN109593842A CN201811570903.8A CN201811570903A CN109593842A CN 109593842 A CN109593842 A CN 109593842A CN 201811570903 A CN201811570903 A CN 201811570903A CN 109593842 A CN109593842 A CN 109593842A
- Authority
- CN
- China
- Prior art keywords
- site
- seq
- nucleotide sequence
- mutation
- primer
- 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
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
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
-
- 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
- C12Q2600/156—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
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Pathology (AREA)
- Immunology (AREA)
- Microbiology (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)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides the kit and method of a kind of detection and judgement helicobacter pylori drug resistance, using nucleotide sequence 9 pairs of primers as shown in NO.1~18 SEQ ID for detecting including 23sRNA, PBP1, PORD, OORD, 16s rRNA, GYRA, the mutation in totally 41 sites of six kinds of genotype of rdxA, comprehensive parsing is carried out to the mutational site of six kinds of helicobacter pylori drug resistant genes of current mainstream, corresponding genotype is found out according to site, to judge to clarithromycin, Amoxicillin, furazolidone, tetracycline, lavo-ofloxacin, the drug resistance of metronidazole, sequencing reading length is up to 1000bp, accuracy is up to 99.999%, have very great significance to the personalized treatment for instructing helicobacter pylori patient.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of examination of detection and judgement helicobacter pylori drug resistant gene
Agent box and method.
Background technique
Helicobacter pylori (Helicobacter pylori, Hp) is a kind of Gram-negative bacteria, and it is viscous to be mainly distributed on stomach
It is the currently known unique microbe species that can be survived in people's stomach in membrane tissue.According to statistics, pylorus spiral shell in human population worldwide
For the infection rate of spinner handle bacterium up to 50%, the infection rate in China is up to 60%-90%.Helicobacter pylori infection can lead to chronic stomach
Inflammation, gastric ulcer, gastric cancer and with the disease of digestive tracts such as gastric lymphoma, therefore the helicobacter pylori early stage of numerous patients eradicates very
It is necessary.Current several antibiotic (clarithromycin, Amoxicillin, lavo-ofloxacin, furazolidone, tetracycline, metronidazole etc.)
It is widely used as the means for eradicating Hp3-4 connection treatment.But helicobacter pylori can generate drug resistance to antibiotic, and
Antibiotic resistance international and domestic at present generally increases year by year, and eradication rate sharp fall, is down to from initial 95%
65%, it is contemplated that the principal element for hindering Hp to eradicate comprehensively can be increasingly becomed in the following drug resistance.Therefore, expert opinion and newest
Common recognition thinks that the patient for having drug resistance sign before the treatment, should do drug-resistant test, first so as to guide the choosing of suitable drug
It selects.
Have the detection method and PCR kit for individual antibiotic (such as clarithromycin) drug resistant gene at present, but
It is still to lack detection means and kit that complete detection is carried out for the drug resistance site of all mainstream antibiotic.The present invention endeavours
In technically filling up this blank.
Summary of the invention
Technical problem to be solved by the present invention lies in, provide it is a kind of detection and judgement helicobacter pylori drug resistant gene
Method and kit, can comprehensively identify for clarithromycin, Amoxicillin, lavo-ofloxacin, furazolidone, Fourth Ring
The specific site situation of plain, the currently used 6 kinds of Helicobacter pylori infection therapeutic agents of metronidazole drug resistant gene, interprets
The drug resistance of the type bacterial strain, to instruct the personalized treatment of Helicobacter pylori infection patient.And this method scalability is strong,
Further go deep into Hp Drug Resistance genomics research, if following find new drug resistant gene or drug resistance position
Point can extend in gene and site list of the invention at any time once biological function is clear.
In order to solve the above technical problems, the present invention provides the reagent of a kind of detection and judgement helicobacter pylori drug resistance
Box, including primer mixed liquor and PCR amplification reagent；The primer mixed liquor includes:
Detect the site G1939A of clarithromycin drug-resistant gene 23sRNA and/or the forward primer 1 of T1942C site mutation
With reverse primer 1, detect the site A2142G of clarithromycin drug-resistant gene 23sRNA, the site A2143G, the site A2144G,
The site C2147G, the site C2182T, the site C2245T and/or C2289T site mutation forward primer 2 and reverse primer 2,
Detect the site 320Ala/Val, the site 366Leu/Phe, 369Ala/Thr of Amoxicillin drug resistant gene PBP1
Point, the site 374Leu/Val, the site 414Arg/Ser and/or 423Leu/Phe site mutation forward primer 3 and reverse primer
3, detect the site 556Ser/Thr of Amoxicillin drug resistant gene PBP1, the site 562Asn/Tyr, the site 593Ala/Thr and/or
The forward primer 4 and reverse primer 4 of 595Gly/Ser site mutation；
Detect the forward direction of the site G353A of furazolidone drug resistant gene PORD, the site A356G and/or C357T site mutation
Primer 5 and reverse primer 5, detect the site A41G of furazolidone drug resistant gene OORD, the site A122G, the site A335G,
The forward primer 6 and reverse primer 6 in the site C349A/G, the site C156T and/or C165T site mutation；
Detect the AGA926-928TTC/AGC/TGC/GGA/CGA site mutation of tetracycline resistance gene 16s rRNA just
To primer 7 and reverse primer 7,
Detect the site 87Asn/Lys/Ile/Thr/Tyr and/or the 91Asp/ of lavo-ofloxacin hydrochloride drug resistant gene GYRA
The forward primer 8 and reverse primer 8 of Asn/Gly/Tyr site mutation；
Detect the site A61G, the site T62C, the site A91G, the site C92A, G392A of metronidazole drug resistant gene rdxA
Point, the site A610G, the site A614C and/or G175A site mutation forward primer 9 and reverse primer 9；
Wherein, primer sequence is respectively as follows:
Forward primer 1:AGCCTCTAAGCATATCCATAG (as shown in SEQ ID NO.1)；
Reverse primer 1:AATGGCGTAACGAGATGG (as shown in SEQ ID NO.2)；
Forward primer 2:GAATTGAAGCCCGAGTAAACG (as shown in SEQ ID NO.3)；
Reverse primer 2:TCAAGCAGAGACGAAAGTCGG (as shown in SEQ ID NO.4)；
Forward primer 3:AGGCGGTTGTATTCTTTAGGC (as shown in SEQ ID NO.5)；
Reverse primer 3:CACTGACCAACAAAACGATGTCAA (as shown in SEQ ID NO.6)；
Forward primer 4:TACGGCACCATGCTCAAACC (as shown in SEQ ID NO.7)；
Reverse primer 4:ATAGGGACTTTCACATCGCTG (as shown in SEQ ID NO.8)；
Forward primer 5:CCATTACACCGAGCAAAGCTA (as shown in SEQ ID NO.9)；
Reverse primer 5:GCCTATCCTATCACCCCATCA (as shown in SEQ ID NO.10)；
Forward primer 6:CATGCTTTCAGCGCGACTTAT (as shown in SEQ ID NO.11)；
Reverse primer 6:GCGTATCTTTAGGGGCAAGC (as shown in SEQ ID NO.12)；
Forward primer 7:GCGACCTGCTGGAACATTAC (as shown in SEQ ID NO.13)；
Reverse primer 7:TCGTGTCGTGAGATGTTGGG (as shown in SEQ ID NO.14)；
Forward primer 8:GGCGTATTTTGTATGCGATGC (as shown in SEQ ID NO.15)；
Reverse primer 8:GAAAGTGCGGGCCAAAGTG (as shown in SEQ ID NO.16)；
Forward primer 9:CATGGGTTGCTGATTGTGGTT (as shown in SEQ ID NO.17)；
Reverse primer 9:GGTGTTTTCAAGCGTTTCATTAAG (as shown in SEQ ID NO.18)；
Specifically, reagent 2xHieff PCR Master Mix (With Dye) can be used in the PCR amplification reagent, it is i.e.
With the PCR aqueous premix of type, primer and template, which need to only be added, can carry out expanding the operating procedure for simplifying experiment, improve height
The reproducibility of throughput and experimental result.
In order to solve the above technical problems, the present invention also provides the sides of a kind of detection and judgement helicobacter pylori drug resistance
Method, comprising steps of
1) it extracts and expands: extracting sample DNA, carry out PCR amplification using the primer and PCR amplification reagent of design；
2) it purifies: pcr amplification product is purified, it is quantitative that sample after purification carries out fluorescent dye；
3) it is sequenced: establishing reaction document, sequencing information corresponding to the every hole of orifice plate is fabricated to table, input computer and turn
Turn to the format of sequenator input；The sample of addition needed for the every hole of orifice plate, primer are subjected to control addition according to the information of table,
MIX enzyme is added, seals up PCR film, is put into PCR instrument and carries out PCR amplification；Pcr amplification product is handled through EDTA, centrifugation；Again plus wine
Precision processing, centrifugation；The processing of height deionized formamide, centrifugation is added；Carry out PCR denaturation treatment, upper sequenator detection；Sequencing knot
Fruit and each gene mutation refer to positive strain sequence alignment, according to corresponding mutation point come its genotype of interpretation；
In the step 1), institute's primer includes:
Detect the site G1939A of clarithromycin drug-resistant gene 23sRNA and/or the forward primer 1 of T1942C site mutation
(as shown in SEQ ID NO.1) and reverse primer 1 (as shown in SEQ ID NO.2)；Detect clarithromycin drug-resistant gene 23sRNA
The site A2142G, the site A2143G, the site A2144G, the site C2147G, the site C2182T, the site C2245T and/or
The forward primer 2 (as shown in SEQ ID NO.3) and reverse primer 2 of C2289T site mutation (as shown in SEQ ID NO.4)；
Detect the site 320Ala/Val, the site 366Leu/Phe, 369Ala/Thr of Amoxicillin drug resistant gene PBP1
Point, the site 374Leu/Val, the site 414Arg/Ser and/or 423Leu/Phe site mutation (such as SEQ ID of forward primer 3
Shown in NO.5) and reverse primer 3 (as shown in SEQ ID NO.6), detect the 556Ser/Thr of Amoxicillin drug resistant gene PBP1
Site, the site 562Asn/Tyr, the site 593Ala/Thr and/or 595Gly/Ser site mutation (such as SEQ ID of forward primer 4
Shown in NO.7) and reverse primer 4 (as shown in SEQ ID NO.8)；
Detect the forward direction of the site G353A of furazolidone drug resistant gene PORD, the site A356G and/or C357T site mutation
Primer 5 (as shown in SEQ ID NO.9) and reverse primer 5 (as shown in SEQ ID NO.10) detect furazolidone drug resistant gene
The site A41G of OORD, the site A122G, the site A335G, the site C349A/G, the site C156T and/or C165T site mutation
Forward primer 6 (as shown in SEQ ID NO.11) and reverse primer 6 (as shown in SEQ ID NO.12)；
Detect the AGA926-928TTC/AGC/TGC/GGA/CGA site mutation of tetracycline resistance gene 16s rRNA just
To primer 7 (as shown in SEQ ID NO.13) and reverse primer 7 (as shown in SEQ ID NO.14)；
Detect the site 87Asn/Lys/Ile/Thr/Tyr and/or the 91Asp/ of lavo-ofloxacin hydrochloride drug resistant gene GYRA
The forward primer 8 (as shown in SEQ ID NO.15) and (such as SEQ ID NO.16 of reverse primer 8 of Asn/Gly/Tyr site mutation
It is shown)；
Detect the site A61G, the site T62C, the site A91G, the site C92A, G392A of metronidazole drug resistant gene rdxA
Point, the site A610G, the forward primer 9 (as shown in SEQ ID NO.17) of the site A614C and/or G175A site mutation and reversed
Primer 9 (as shown in SEQ ID NO.18).
Specifically, samples sources are to cut out antibiotic one month or more before being examined, proton pump suppression in the step 1)
Preparation two weeks or more, Chinese medicine two weeks or more, and positive helicobacter pylori positive patient is verified as through urea breath test
Gastric mucosa sample.The gastric mucosa sample first passes through strain isolation extraction, carries out plate training to the H. pylori strain of extraction
It supports (micro- aerobic) 4-5 days, monoclonal colonies is selected by microscopy and urease experiment after inoculation and are cultivated again to enough amounts.
Specifically, extracting sample DNA in the step 1) and using Magen HiPure Bacterial DNA Kits.
Specifically, reagent 2xHieff PCR Master Mix (With can be used in the PCR amplification reagent in step 1)
It Dye), is the PCR aqueous premix of instant, primer and template, which need to only be added, can carry out expanding the operation step for simplifying experiment
Suddenly, the reproducibility of high-throughput operation and experimental result is improved.
Specifically, in step 1), the reaction system of PCR amplification are as follows: the DNA constant volume of 5ng total amount in the ddH2O of 21ul,
It is added 25ul 2xHieff PCR Master Mix (With Dye), detects the 2ul forward primer (concentration of certain individual gene
10uM) with 2ul reverse primer (concentration 10uM), constant volume is in 50ul system in total.
Specifically, in step 1), the program of PCR amplification are as follows: 94 DEG C of initial denaturation 5min, 1 circulation；94 DEG C of denaturation 30sec,
55 DEG C of annealing 30sec, 72 DEG C of extension 30sec, 35 recycle；72 DEG C extend 30sec, 1 circulation eventually；Then 4 DEG C of terminations.PCR
After amplified reaction, PCR product is put into 4 DEG C of refrigerators and is saved.
Specifically, being purified in step 2) using paramagnetic particle method.
Specifically, in step 3), the program of PCR amplification are as follows: 96 DEG C of initial denaturation 2min, 1 circulation；96 DEG C of denaturation 10sec,
50 DEG C of annealing 10sec, 60 DEG C of extension 100sec, 30 recycle；Then 4 DEG C of terminations, 16 DEG C of heat preservations.
Specifically, EDTA processing is to take out PCR product 2.6ul EDTA solution is added, and 19ul freezes in -20 in step 3)
100% ethyl alcohol (AR) for spending refrigerator seals up PCR film, shakes 2 minutes.
Specifically, ethanol postincubation is to add alcohol (70%AR) 50ul in step 3), sealing plate film is covered, into centrifuge.
Specifically, the processing of height deionized formamide is before upper machine to every hole degree of the increasing deionization of orifice plate in step 3)
Formamide (HiDi Formanide), seals up PCR film, upper centrifuge.
Specifically, PCR denaturation treatment is denaturalized 2 minutes at 96 DEG C, is taken out after finishing, and cooling, 500 in a twinkle in step 3)
From 5 seconds, sample was sequenced in being finally available on the machine for obtaining.
Specifically, each gene mutation refers to positive strain sequence are as follows: 23sRNA gene reference positive strain sequence in step 3)
Column are as shown in NO.19~20 SEQ ID；PBP1 gene reference positive strain sequence is as shown in NO.21~22 SEQ ID；PORD
Gene reference positive strain sequence is as shown in SEQ ID NO.23；OORD gene reference positive strain sequence such as SEQ ID NO.24
It is shown；16s rRNA gene reference positive strain sequence is as shown in SEQ ID NO.25；GYRA gene reference positive strain sequence
As shown in SEQ ID NO.26；RdxA gene reference positive strain sequence is as shown in SEQ ID NO.27.
Method provided by the invention can be to associated gene 23sRNA (9 sites), PBP1 (10 sites), (3 positions PORD
Point), OORD (6 sites), 16s rRNA (3 sites), GYRA (2 sites), totally 41 sites carry out rdxA (8 sites)
Mutational site is detected and compares, for determining helicobacter pylori drug resistance.Its judgment basis are as follows: if 23sRNA mutates,
Then the bacterial strain is strong to clarithromycin sensibility, to such antibiotics resistance；If PBP1 mutates, then the bacterial strain is to Amoxicillin
Sensibility is strong, to such antibiotics resistance；If PORD and/or OORD mutates, then the bacterial strain is strong to furazolidone sensibility,
To such antibiotics resistance；If 16s rRNA mutates, then the bacterial strain is strong to Tetracycline-sensitive, resistance to such antibiotic
Medicine；If GYRA mutates, then the bacterial strain is strong to lavo-ofloxacin hydrochloride sensibility, to such antibiotics resistance；As rdxA is sent out
Raw mutation, then the bacterial strain is strong to metronidazole sensitivity, to such antibiotics resistance.
Method provided by the invention can be completed at the same time the abrupt climatic change to 41 sites of 7 genes using 9 pairs of primers, survey
Sequence reads long reachable 1000bp, and accuracy is up to 99.999%.
Method provided by the invention is carried out for the mutational site of six kinds of helicobacter pylori drug resistant genes of current mainstream
Comprehensive parsing finds out corresponding genotype according to site, to judge to which kind of antibiotic medicine drug resistance, to instructing pylorus spiral shell
The personalized treatment of spinner handle bacterium patient has very great significance.
Specific embodiment
Clear, complete description will be carried out to technical solution of the present invention below, it is clear that described embodiment is this hair
Bright a part of the embodiment, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art exist
Every other embodiment obtained under the premise of creative work is not made, shall fall within the protection scope of the present invention.
1 H. pylori strain vitro extraction of embodiment and culture
1. sampling
1) confirmation patient cuts out antibiotic one month or more before being examined, and proton pump inhibitor two weeks or more, Chinese medicine two
More than week, and the positive is verified as through urea breath test (UBT).
2) the gastric mucosa tissue sample of confirmation sampling is fresh pollution-free, and no swill is left.
3) antrum and body of stomach diseased region double sample are taken as far as possible when gastroscopic biopsy samples, each two pieces, totally four pieces of samples
It is respectively put into two transporting culture medium test tubes, and confirms that tissue samples immerse in the liquid of transporting culture medium, tighten test tube cap,
2-8 degree Refrigerated Transport in 48 hours.
4) just control patient helicobacter pylori more be distributed in antrum away from pylorus 2-5cm at, the pylorus of rescue therapy patient
Pylori can be transferred to body of stomach stomach function regulating bottom from antrum, therefore acquire sample at stomach bottom and body of stomach simultaneously to the latter.
5) the multiple patient of eradication therapy failure, it is proposed that terminate at least three moon away from last treatment or even longer (common recognition is built
View 2 months).
2. vitro extraction and culture
1) mating disposable loop-carrier being managed using preservation, stomach lining sample is directly seeded in two pieces of Colombia's blood respectively
On agar medium, and it is identified antrum and body of stomach, when inoculation directly contacts in gastric mucus face with culture medium, and small area is by stomach
Mucous membrane smoothens, and pours into a little preservation liquid, carries out rotation and smoothens, then stomach lining sample is taken out and is added to urease pipe, into
Row urea enzyme reaction, observation reagent color change the time, if becoming pink as sun from normally faint yellow in for 24 hours
Property, it indicates helicobacter pylori presence, is otherwise feminine gender.
2) it after Columbia Blood Agar flat plate cover upper flat plate lid, is inverted into three gas incubators, in micro- aerobic environment
(5%O2, 10%CO2, 85%N2) temperature 37 DEG C cultivate 3-4 days.Bacterium then pass through morphologic observation (Gram's staining) and
Biochemical reaction such as urease, oxidizing ferment and catalase etc. are identified.Plate is observed in culture after 4 days, oxidase test is carried out, with oxidation
Enzyme filter paper item dips flat-plate bacterial colony respectively, and filter paper top is then gradually deepened as occurred purple at once, it was demonstrated that the bacterium colony is pylorus
Pylori, it is negative non-discolouring.Picking is full and clearly to be forwarded to another piece of Columbia Blood Agar flat for helicobacter pylori
Two are carried out in plate in three gas incubators to be commissioned to train feeding 2-3 days, by all bacterium colonies for being grown in two generation of Columbia Blood Agar plate
Addition is collected to preservation liquid is frozen, some progress urease experiments is chosen and is applied on slide and carry out Gram's staining, really
Recognize its helicobacter pylori growth conditions and form, bacterial strain is frozen and is saved in -80 DEG C of refrigerators, in case sequencing makes below
With.
The drug resistant gene of 2 helicobacter pylori of embodiment detects
1.DNA is extracted
DNA of bacteria extracting is carried out using Magen HiPure Bacterial DNA Kits.
1) 500ul bacterial strain frozen stock solution is drawn into 1.5ml centrifuge tube, and 10,000x g are centrifuged 1 minute collection bacterium, slowly
400ul supernatant is drawn to abandon.
2) 200 μ l Buffer STE and 10 μ l Lysozyme are added into 1.5ml centrifuge tube, it is thin that resuspension fullys shake
Bacterium reacts 10 minutes at 37 DEG C.
3) 220 μ l Buffer DL and 10 μ l Proteinase K are added into 1.5ml centrifuge tube, mixing fullys shake,
65 DEG C digestion reaction 30 minutes.
4) 5 μ l RNase Solution are added into 1.5ml centrifuge tube, mixing fullys shake, 25 DEG C of room temperature are reacted 30 points
Clock.
5) 220 μ l dehydrated alcohols are added into 1.5ml centrifuge tube, mixing 30 seconds fullys shake.
6) HiPure gDNA Micro Column in 2ml collecting pipe, 1.5ml centrifugation liquid in pipe is all added
Enter, 10,000x g are centrifuged 1 minute.
7) it falls to abandon waste liquid, pillar is reinstalled in collecting pipe.It is added on 500 μ l Buffer GW1 to pillar.10,000x g
Centrifugation 1 minute.
8) it falls to abandon waste liquid, pillar is reinstalled in collecting pipe.650 μ l Buffer GW2 are added into pillar.10,000x g
Centrifugation 1 minute.
9) it falls to abandon waste liquid, pillar is reinstalled in collecting pipe.650 μ l Buffer GW2 are added into pillar.10,000x g
Centrifugation 1 minute.
10) it falls to abandon waste liquid, pillar is reinstalled in collecting pipe.10,000 × g is centrifuged 2 minutes, abandons 2ml collecting pipe.
11) by pillar in new 1.5ml centrifuge tube.30 μ l are added and are preheated to 65 DEG C of Buffer TE into pillar film
Centre.It places 1 minute, 10,000 × g is centrifuged 1 minute.
12) pillar is abandoned, 1.5ml centrifuge tube is stored in 4 DEG C of refrigerators.
13) Yeason dsDNA HS Assay Kit for is usedIt is enterprising in 3.0 instrument of Thermo Qubit
Row fluorescent dye is quantitative.
2. amplification
1) it takes the DNA constant volume of 5ng total amount in the ddH2O of 21ul, 25ul 2xHieff PCR Master Mix is added
(With Dye), 2ul forward primer (concentration 10uM) and 2ul reverse primer (concentration 10uM), in total constant volume 50ul system in
In 0.2ml centrifuge tube, centrifuge tube lid is covered.
| Component | Volume (ul) |
| DNA profiling | 21 |
| Forward primer (10uM) | 2 |
| Reverse primer (10uM) | 2 |
| 2xHieff PCR Master Mix(With Dye) | 25 |
| In total | 50 |
The specific corresponding addition primer situation of medicine-resistant medicine detection is as shown in the table:
2) sample is carried out using oscillator to mix 10 seconds, reuse compact centrifuge and carry out moment centrifugation, make reaction solution
It is centrifuged tube bottom, PCR instrument is expanded in preparation.
3) it opens PCR instrument and carries out the heating of plate lid, be put into 0.2ml centrifuge tube, cover plate lid, select amplification program
4) PCR product after reaction, is put into 4 DEG C of refrigerators to save.
3. purifying
1) sufficiently oscillation mixed AMPURE BEADS XP, and at incubation at room temperature 30 minutes or more.
2) it is added in the magnetic bead to PCR product of 45ul vibrated, is mixed using oscillator.
3) 5 minutes are placed at room temperature for, is then placed on magnetic frame and is adsorbed, the time is 5 minutes, abandons waste liquid.
4) 75% ethyl alcohol of 200ul is added, covers centrifuge tube lid, the slow rotating centrifugal pipe of 180 degree, make magnetic bead be adsorbed onto from
The heart pipe other side, then 180 degree is rotated to original position, so that its magnetic bead space in ethyl alcohol is shuttled, reaches cleaning function, time 30
Second.
5) waste liquid is abandoned after magnetic bead absorption, repeats step 4, and abandon waste liquid.
6) it is put into 37 degree of metal baths, took out observation magnetic bead group whether there is or not cracking state to 1-2 minutes, slightly there is cracking phenomena
, should not too dry.
7) 22ul is added and saves liquid or water, fully shake, be placed at room temperature for 1 minute, be inserted on magnetic frame.
8) 1.5ml centrifuge tube, aspiration step 7 are taken separately) sample 20ul to pipe in, be put into 4 degree of refrigerators and save for being sequenced
Sample prepares.
9) Yeason dsDNA HS Assay Kit for is usedIt is carried out on 3.0 instrument of Thermo Qubit
Fluorescent dye is quantitative.
4. sequencing
1) reaction document is established, sequencing information corresponding to the every hole of 96 orifice plates is fabricated to table, inputs computer and is converted into
The format of sequenator input.Printing is a.
2) plate hole is loaded, and the sample of addition needed for each hole, primer is carried out control addition according to the information of table, centainly
Notice that the position of orifice plate is corresponding with the position in table.
3) it is mixed in sample and primer to 96 hole PCR plates according to following table.
After adding wink from once, after concussion again wink from once, into next step PCR initial denaturation, 96 degree 2 minutes, taking-up is set
It in ice water, is added 1ul MIX enzyme (dGTP BigDye Teminator), seals up PCR film, moment is centrifuged after concussion, is put into
It is expanded in PCR instrument.
4) it opens PCR instrument and carries out the heating of plate lid, select amplification program
5) PCR product is taken out and 2.6ul EDTA solution is added, 19ul freezes in 100% ethyl alcohol (AR) of -20 degree refrigerators, envelope
Upper PCR film shakes 2 minutes.
6) 4000 turns of 10 degree of refrigerated centrifuge are put in be centrifuged 30 minutes, completes to stand 1 minute in ice water after centrifugation, temperature from
4000 turns of scheming are centrifuged 1 minute.
7) take out after uncap, be inverted on toilet paper, be put into centrifuge wink from 5 seconds 700 turns.
8) it takes out and puts just, add alcohol (70%AR) 50ul, with the alcohol splashed out on toilet paper wipe-dry board, cover sealing plate film,
Into centrifuge.
9) it is centrifuged 10 minutes for 4000 turns 10 degree.
10) take out after tear sealed membrane, be inverted on toilet paper, be put into centrifuge wink from 5 seconds 700 turns.
11) take out put just, be placed in allow within 15 minutes in air alcohol volatilization (or into PCR instrument heat 5 minutes, not cover, or
It is 2 minutes dry with vacuum oven) it has to guarantee that alcohol volatilization is clean.
12) add HiDi on before machine, 96 dried orifice plates are removed, adds the every hole HiDi Formanide 10ul, seals up
PCR film, upper centrifuge 700 is in a twinkle from 5 seconds.
13) take out laggard PCR and do reaction of degeneration (RD), 96 degree 2 minutes, take out, cooled down for a moment after finishing, 500 in a twinkle from 5 seconds,
What is obtained is finally available on the machine.
14) machine on ready-made 96 orifice plate need to be remembered this hole A12 against the interior of machine by 3730 sequenator of ABI on
Unfilled corner, the document that the importing first step is done are run into machine into program.
5. analysis determines
1) mutational site situation is obtained according to sequencing result, each gene mutation is as follows with reference to positive strain sequence:
2) according to corresponding mutation point come its genotype of interpretation
3) corresponding medicine-resistant medicine is obtained according to genotype
Mutational site and corresponding antibiotic medicine are referring to table
It is detected using gastric mucosa sample of the present invention to a certain case of Nanjing No.1 Hospital, which need to be to 6 kind
Antibiotics resistance situation is detected, and result is as follows:
Testing result explanation: mutation prompts the bacterial strain to clarithromycin, lavo-ofloxacin hydrochloride, metronidazole drug resistance.
As can be seen from the above table, helicobacter pylori drug resistant gene is detected and is determined using the present invention, Ke Yiyou
The concrete condition for targetedly identifying drug resistant gene locus mutation, facilitates the drug resistance for interpreting the type bacterial strain, to guidance
The patient personalized treatment of Helicobacter pylori infection has very great significance.
In conclusion the various embodiments described above are only presently preferred embodiments of the present invention, it is not of the invention to limit
Protection scope, all within the spirits and principles of the present invention, any modification, equivalent substitution, improvement and etc. done should be all included in
In protection scope of the present invention.
Sequence table
<110>Co., Ltd, Shanghai core super medical test institute
<120>kit and method of a kind of detection and judgement helicobacter pylori drug resistance
<130> CPC-NP-18-101217
<160> 27
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 1
agcctctaag catatccata g 21
<210> 2
<211> 18
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 2
aatggcgtaa cgagatgg 18
<210> 3
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 3
gaattgaagc ccgagtaaac g 21
<210> 4
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 4
tcaagcagag acgaaagtcg g 21
<210> 5
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 5
aggcggttgt attctttagg c 21
<210> 6
<211> 24
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 6
cactgaccaa caaaacgatg tcaa 24
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 7
tacggcacca tgctcaaacc 20
<210> 8
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 8
atagggactt tcacatcgct g 21
<210> 9
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 9
ccattacacc gagcaaagct a 21
<210> 10
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 10
gcctatccta tcaccccatc a 21
<210> 11
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 11
catgctttca gcgcgactta t 21
<210> 12
<211> 20
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 12
gcgtatcttt aggggcaagc 20
<210> 13
<211> 20
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 13
gcgacctgct ggaacattac 20
<210> 14
<211> 20
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 14
tcgtgtcgtg agatgttggg 20
<210> 15
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 15
ggcgtatttt gtatgcgatg c 21
<210> 16
<211> 19
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 16
gaaagtgcgg gccaaagtg 19
<210> 17
<211> 21
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 17
catgggttgc tgattgtggt t 21
<210> 18
<211> 24
<212> DNA
<213>artificial sequence (artificial sequence)
<400> 18
ggtgttttca agcgtttcat taag 24
<210> 19
<211> 379
<212> DNA
<213>23sRNA gene mutation is with reference to positive strain sequence 1 (23sRNA gene mutation refers to positive strain sequence 1)
<400> 19
atgagtattc taaggcgcgt gaaagaactc tggttaagga actctgcaaa ctagcaccgt 60
aagttcgcga taaggtgtgc cgcagcaatg cggtctcagc aaagagtccc tcccgactgt 120
ttaccaaaaa cacagcactt tgccaactcg taagaggaag tataaggtgt gacgcctgcc 180
cggtgctcga aggttaagag gatgcgtcag tcgcaagatg aagcgttgaa ttgaagcccg 240
agtaaacggc ggccgtaact ataacggtcc taaggtagcg aaattccttg tcggttaaat 300
accgacctgc atgaatggca aacgagatgg aaaacggagg cttgcagagg ttggctcatt 360
gcggttggaa atcgcaagt 379
<210> 20
<211> 398
<212> DNA
<213>23sRNA gene mutation is with reference to positive strain sequence 2 (23sRNA gene mutation refers to positive strain sequence 2)
<400> 20
cgtaacgaga tgggagctgt ctcaaccaga gattcagtga aattgtagtg gaggtgaaaa 60
ttcctcctac ccgcggcaag acggaaagac cccgtggacc tttactacaa cttagcactg 120
ctaatgggaa tatcatgcgc aggataggtg ggaggctttg aagtaagggc tttggctctt 180
atggagccat ccttgagata ccacccttga tgtttctgtt agctaactgg cctgtgttat 240
ccacaggcag gacaatgctt ggtgggtagt ttgactgggg cggtcgcctc ctaaaaagta 300
acggaggctt gcaaaggttg gctcattgcg gttggaaatc gcaagttgag tgtaatggca 360
caagccagcc tgactgtaag acatacaagt caagcaga 398
<210> 21
<211> 785
<212> DNA
<213>PBP1 gene mutation is with reference to positive strain sequence 1 (PBP1 gene mutation refers to positive strain sequence 1)
<400> 21
tgaagtgcca atcgtctata accaaacttc cacgcaaaat atcgctccct atgtcgtgga 60
tgaagtgttg aagcaattgg atcaattaga cgggttaaaa actcaaggct ataccataaa 120
gctcacgata gatttggatt accaacgctt agcgttagag tccttgcgtt ttgggcatca 180
aaaaatctta gaaaaaatcg ctaaagaaaa gccaaaaact aacgcctctg atgaagatga 240
agacaacttg aacgccagca tgatagttac agacacgagc accggtaaga ttttagcttt 300
agtggggggg attgattata aaaaaagcgc tttcaatcgc gccacgcaag ccaaacggca 360
gtttgggagc gcgataaagc cttttgtgta tcaaatcgct tttgataatg gctattccac 420
cacttccaaa atccctgata ccgcgcgaaa ctttgaaaat ggcaattata gtaaaaacag 480
cgaacaaaac cacgcatggc accccagcaa ttattctcgc aagtttttag ggcttgtaac 540
cttgcaagaa gccttaagcc attcgttaaa tctagccacg atcaatttaa gcgatcagct 600
tggctttgaa aaaatttatc aatctttaag cgatatgggg tttaaaaacc tccctaagga 660
cttgtctatc gtgttaggga gctttgctat ctcacccatt gatgcggctg aaaagtattc 720
tttattttct aattacggca ccatgctcaa acccatgctc attgaaagca tcactgacca 780
acaac 785
<210> 22
<211> 471
<212> DNA
<213>PBP1 gene mutation is with reference to positive strain sequence 2 (PBP1 gene mutation refers to positive strain sequence 2)
<400> 22
aaaactttca cgcctatgga aaccaaaaag atcacctcca aagaacaggc ttttttaacc 60
ctttcagtgc tgataaatgc ggtagaaaac ggcacagggc gtttggctcg cactaaaggt 120
ttagaaatcg ccggtaaaac cgggacttct aacaacaata ttgacgcttg gttcattggc 180
tttaccccca ccttgcaaag cgtgatctgg tttgggagag acgataacac gcctattagc 240
aaaggagcga caggaggcgt tgtgagtgca cctgtgtatt cgtatttcat gcgtaatatt 300
ttagcgattg aaccttcttt aaaaagaaag tttgatgtcc ccaaaggctt gcgtaaagaa 360
atcgtggata aaatccccta ctactcaact cctaattcca tcacccccac ccccaaaaga 420
acagacgata gtgaggaacg cttgttgttc taatcctaca tggctatagg g 471
<210> 23
<211> 372
<212> DNA
<213>PORD gene mutation is with reference to positive strain sequence (PORD gene mutation refers to positive strain sequence)
<400> 23
ggcgcgtggc taagcctgtg cataacaata atatttgcat caattgcttt aattgttggg 60
tttattgccc agacgctgct attctttcaa gagagggtaa gttaaagggc gtggattatt 120
ctcattgtaa aggctgtggc gtgtgcgtgg atgtctgccc taccaaccct aaatcgctat 180
ggatgtttga agaacaaatt gagcccgcta ccgctctcac tcaatggccc caaaaacaag 240
aaaagaaaaa atcataagga aaaaatatgg caaaaagtat tgaattgcaa gagatagaag 300
tgtgggatgg caacaccgct agttctaacg ctttaagaca ggctcaaatt gatgtcatcg 360
cagcctatcc ta 372
<210> 24
<211> 481
<212> DNA
<213>OORD gene mutation is with reference to positive strain sequence (OORD gene mutation refers to positive strain sequence)
<400> 24
gccgtttggg tgaatgaaga caggtgtaag ggttgtgata tttgcgtatc ggtatgccct 60
gctggggttc ttggcatggg gattgaaaaa gaaagggtgc ttggaaaagt ggccaaagta 120
gcctacccag agagttgtat cggttgcgtg caatgcgagt tgcactgccc ggattttgcg 180
atttatgtgg ctgacagaaa ggatttcaaa ttcgctaaag tttctaaaga agcccaagaa 240
agaagcgaaa aggttaaggc caataaatac atgctcttag aagagactat tttagaaggg 300
agaggcaaat aatgcgtgag attatttctg atgggaatga attagtcgct aaagcggcga 360
ttgaagtggg gtgtcggttt tttgggggct atcctattac gccagttcgg atattatgca 420
tgcaatgagc gtggctttgc ccaaatgcgg cggccatttt atccaaatgg aagatgaaat 480
c 481
<210> 25
<211> 302
<212> DNA
<213>16s rRNA gene mutation is with reference to positive strain sequence (16s rRNA gene mutation refers to positive strain sequence)
<400> 25
gattagatac cctggtagtc cacgccctaa acgatggatg ctagttgttg gagggcttag 60
tctctccagt aatgcagcta acgcattaag catcccgcct ggggagtacg gtcgcaagat 120
taaaactcaa aggaatagac ggggacccgc acaagcggtg gagcatgtgg tttaattcga 180
agatacacga agaaccttac ctaggcttga cattgagaga atccgctaga aatagtggag 240
tgtctagctt gctagacctt gaaaacaggt gctgcacggc tgtcgtcagc tcgtgtcgtg 300
ag 302
<210> 26
<211> 541
<212> DNA
<213>GYRA gene mutation is with reference to positive strain sequence (GYRA gene mutation refers to positive strain sequence)
<400> 26
tcgcttataa aaaaagcgct aggatcgtgg gtgatgtgat cggtaaatac cacccccatg 60
gcgataatgc ggtttatgat gcgctagtga gaatggcgca agatttttct atgcgtttgg 120
aattagtgga tgggcagggc aactttggct ctattgatgg cgataacgct gcagcgatgc 180
gttacactga agccagaatg actaaggcga gtgaagaaat tttaagggat attgataaag 240
acaccattga ttttgtgcct aattatgatg acaccttaaa agagccagat attttaccaa 300
gccgtctgcc taacctttta gtcaatgggg ctaatgggat cgctgtgggg atggcgactt 360
ctatcccccc tcataggatt gatgaaatca tagacgcttt agtgcatgtc ttagaaaacc 420
ctaacgctga attagatgaa attttggaat ttgtcaaagg gcctgatttt cccaccggtg 480
ggatcattta tggcaaggcg ggcattattg aagcctataa aacggggcga gggcgcgtga 540
a 541
<210> 27
<211> 691
<212> DNA
<213>rdxA gene mutation is with reference to positive strain sequence (rdxA gene mutation refers to positive strain sequence)
<400> 27
tgcggatttt acagagagcc agacagccaa atgggggttt attttttgaa tttgagcatg 60
gggcagattt taagcttatt tatggtaatt gtttcgttag ggattttatt gtatgctaca 120
aaaaattcta aaaaaataaa ggaaaatcaa tgaaattttt ggatcaagaa aaaagaagac 180
aattactaaa ggagcgccat tcttgcaaga tgtttgatag ccattatgag ttttctagcg 240
aggaattaga agaagtcgct gaaatcgcca ggctatcgcc aagctcttac aacacgcagc 300
catggcattt tgtgatagtt actaataagg atttaaacca cccaagcaga aatcccaaac 360
atctttagtg tttgggatga atgctgctaa tttgtagtat aatatctcca tacatttgta 420
tctagcgtag gaagtacgca aagttacgcc tttggagata tgatgtgtga gacctgtagg 480
gaatgcgttg gagatcaaac tctgtaaaat ccctatgatt agggacacaa agtgagaacc 540
aaactttccc tatgggcaac atcagccgag gaagcccaat cgctttagcg tttgggtgct 600
tcacctataa tcaaacctaa attaaagttt aaggagtggc attttgttta aaagaatggt 660
tttaatcgct cttttagggg tgttttcaag c 691
Claims (10)
1. the kit of a kind of detection and judgement helicobacter pylori drug resistance, which is characterized in that including primer mixed liquor and PCR
Amplifing reagent；The primer mixed liquor includes:
The nucleotide sequence in the site G1939A and/or T1942C site mutation of detecting clarithromycin drug-resistant gene 23sRNA is such as
Forward primer 1 shown in SEQ ID NO.1 and the nucleotide sequence reverse primer 1 as shown in SEQ ID NO.2；It is mould to detect carat
The site A2142G of plain drug resistant gene 23sRNA, the site A2143G, the site A2144G, the site C2147G, the site C2182T,
The nucleotide sequence of the site C2245T and/or C2289T site mutation forward primer 2 and nucleotide as shown in SEQ ID NO.3
Sequence reverse primer 2 as shown in SEQ ID NO.4；
Detect the site 320Ala/Val of Amoxicillin drug resistant gene PBP1, the site 366Leu/Phe, the site 369Ala/Thr,
The nucleotide sequence in the site 374Leu/Val, the site 414Arg/Ser and/or 423Leu/Phe site mutation such as SEQ ID NO.5
Shown in forward primer 3 and the nucleotide sequence reverse primer 3 as shown in SEQ ID NO.6；Detect Amoxicillin drug resistant gene
The site 556Ser/Thr of PBP1, the site 562Asn/Tyr, the site 593Ala/Thr and/or 595Gly/Ser site mutation core
Nucleotide sequence forward primer 4 as shown in SEQ ID NO.7 and the nucleotide sequence reverse primer 4 as shown in SEQ ID NO.8；
Detect the nucleotides sequence of the site G353A of furazolidone drug resistant gene PORD, the site A356G and/or C357T site mutation
Arrange the forward primer 5 as shown in SEQ ID NO.9 and the nucleotide sequence reverse primer 5 as shown in SEQ ID NO.10, detection
The site A41G of furazolidone drug resistant gene OORD, the site A122G, the site A335G, the site C349A/G, the site C156T and/or
The nucleotide sequence of C165T site mutation forward primer 6 as shown in SEQ ID NO.11 and nucleotide sequence such as SEQ ID
Reverse primer 6 shown in NO.12；
Detect the nucleotide of the AGA926-928TTC/AGC/TGC/GGA/CGA site mutation of tetracycline resistance gene 16s rRNA
Sequence forward primer 7 as shown in SEQ ID NO.13 and the nucleotide sequence reverse primer 7 as shown in SEQ ID NO.14；
Detect the site 87Asn/Lys/Ile/Thr/Tyr and/or the 91Asp/Asn/ of lavo-ofloxacin hydrochloride drug resistant gene GYRA
The nucleotide sequence of Gly/Tyr site mutation forward primer 8 as shown in SEQ ID NO.15 and nucleotide sequence such as SEQ ID
Reverse primer 8 shown in NO.16；
Detect the site A61G of metronidazole drug resistant gene rdxA, the site T62C, the site A91G, the site C92A, the site G392A,
The nucleotide sequence in the site A610G, the site A614C and/or G175A site mutation forward primer as shown in SEQ ID NO.17
9 and nucleotide sequence reverse primer 9 as shown in SEQ ID NO.18.
2. kit as claimed in claim, which is characterized in that the PCR amplification reagent is reagent 2xHieff PCR
Master Mix。
3. a kind of method of detection and judgement helicobacter pylori drug resistance, which is characterized in that comprising steps of
1) it extracts and expands: extracting sample DNA, carry out PCR amplification using the primer and PCR amplification reagent of design；
2) it purifies: pcr amplification product is purified, it is quantitative that sample after purification carries out fluorescent dye；
3) it is sequenced: establishing reaction document, sequencing information corresponding to the every hole of orifice plate is fabricated to table, input computer and be converted into
The format of sequenator input；The sample of addition needed for the every hole of orifice plate, primer are subjected to control addition according to the information of table, are added
MIX enzyme seals up PCR film, is put into PCR instrument and carries out PCR amplification；Pcr amplification product is handled through EDTA, centrifugation；Again plus at alcohol
Reason, centrifugation；The processing of height deionized formamide, centrifugation is added；Carry out PCR denaturation treatment, upper sequenator detection；Sequencing result with
Each gene mutation refers to positive strain sequence alignment, according to corresponding mutation point come its genotype of interpretation；
In the step 1), institute's primer includes:
The nucleotide sequence in the site G1939A and/or T1942C site mutation of detecting clarithromycin drug-resistant gene 23sRNA is such as
Forward primer 1 shown in SEQ ID NO.1 and the nucleotide sequence reverse primer 1 as shown in SEQ ID NO.2；It is mould to detect carat
The site A2142G of plain drug resistant gene 23sRNA, the site A2143G, the site A2144G, the site C2147G, the site C2182T,
The nucleotide sequence of the site C2245T and/or C2289T site mutation forward primer 2 and nucleotide as shown in SEQ ID NO.3
Sequence reverse primer 2 as shown in SEQ ID NO.4；
Detect the site 320Ala/Val of Amoxicillin drug resistant gene PBP1, the site 366Leu/Phe, the site 369Ala/Thr,
The nucleotide sequence in the site 374Leu/Val, the site 414Arg/Ser and/or 423Leu/Phe site mutation such as SEQ ID NO.5
Shown in forward primer 3 and the nucleotide sequence reverse primer 3 as shown in SEQ ID NO.6；Detect Amoxicillin drug resistant gene
The site 556Ser/Thr of PBP1, the site 562Asn/Tyr, the site 593Ala/Thr and/or 595Gly/Ser site mutation core
Nucleotide sequence forward primer 4 as shown in SEQ ID NO.7 and the nucleotide sequence reverse primer 4 as shown in SEQ ID NO.8；
Detect the nucleotides sequence of the site G353A of furazolidone drug resistant gene PORD, the site A356G and/or C357T site mutation
Arrange the forward primer 5 as shown in SEQ ID NO.9 and the nucleotide sequence reverse primer 5 as shown in SEQ ID NO.10, detection
The site A41G of furazolidone drug resistant gene OORD, the site A122G, the site A335G, the site C349A/G, the site C156T and/or
The nucleotide sequence of C165T site mutation forward primer 6 as shown in SEQ ID NO.11 and nucleotide sequence such as SEQ ID
Reverse primer 6 shown in NO.12；
Detect the nucleotide of the AGA926-928TTC/AGC/TGC/GGA/CGA site mutation of tetracycline resistance gene 16s rRNA
Sequence forward primer 7 as shown in SEQ ID NO.13 and the nucleotide sequence reverse primer 7 as shown in SEQ ID NO.14；
Detect the site 87Asn/Lys/Ile/Thr/Tyr and/or the 91Asp/Asn/ of lavo-ofloxacin hydrochloride drug resistant gene GYRA
The nucleotide sequence of Gly/Tyr site mutation forward primer 8 as shown in SEQ ID NO.15 and nucleotide sequence such as SEQ ID
Reverse primer 8 shown in NO.16；
Detect the site A61G of metronidazole drug resistant gene rdxA, the site T62C, the site A91G, the site C92A, the site G392A,
The nucleotide sequence in the site A610G, the site A614C and/or G175A site mutation forward primer as shown in SEQ ID NO.17
9 and nucleotide sequence reverse primer 9 as shown in SEQ ID NO.18.
4. method as claimed in claim 3, which is characterized in that in the step 1), samples sources are to cut out before being examined
Antibiotic one month or more, proton pump inhibitor two weeks or more, Chinese medicine two weeks or more, and the positive is verified as through urea breath test
Helicobacter pylori positive patient gastric mucosa sample.
5. method as claimed in claim 3, which is characterized in that in the step 1), extract sample DNA and use Magen
HiPure Bacterial DNA Kits。
6. method as claimed in claim 3, which is characterized in that in the step 1), the program of PCR amplification are as follows: 94 DEG C of pre- changes
Property 5min, 1 circulation；94 DEG C of denaturation 30sec, 55 DEG C of annealing 30sec, 72 DEG C of extension 30sec, 35 recycle；72 DEG C extend eventually
30sec, 1 circulation；Then 4 DEG C of terminations.
7. method as claimed in claim 3, which is characterized in that in step 2), purified using paramagnetic particle method.
8. method as claimed in claim 3, which is characterized in that in step 3), the program of PCR amplification are as follows: 96 DEG C of initial denaturations
2min, 1 circulation；96 DEG C of denaturation 10sec, 50 DEG C of annealing 10sec, 60 DEG C of extension 100sec, 30 recycle；Then 4 DEG C of terminations,
16 DEG C of heat preservations.
9. method as claimed in claim 3, which is characterized in that in step 3), EDTA processing is to take out PCR product to be added
2.6ul EDTA solution, 19ul freeze in 100% ethyl alcohol (AR) of -20 degree refrigerators, seal up PCR film, shake 2 minutes；Ethanol postincubation
It is to add 70% alcohol 50ul, sealing plate film is covered, into centrifuge；The processing of height deionized formamide is before upper machine to the every hole of orifice plate
Degree of increasing deionized formamide seals up PCR film, upper centrifuge；PCR denaturation treatment is denaturalized 2 minutes at 96 DEG C, is taken after finishing
Out, cooling, 500 in a twinkle from 5 seconds, and sample is sequenced in being finally available on the machine for obtaining.
10. method as claimed in claim 3, in step 3), each gene mutation is respectively as follows: 23sRNA with reference to positive strain sequence
Gene reference positive strain sequence is as shown in NO.19~20 SEQ ID；PBP1 gene reference positive strain sequence such as SEQ ID
Shown in NO.21~22；PORD gene reference positive strain sequence is as shown in SEQ ID NO.23；OORD gene reference positive strain
Sequence is as shown in SEQ ID NO.24；16s rRNA gene reference positive strain sequence is as shown in SEQ ID NO.25；GYRA base
Because reference positive strain sequence is as shown in SEQ ID NO.26；RdxA gene reference positive strain sequence such as SEQ ID NO.27 institute
Show.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811570903.8A CN109593842B (en) | 2018-12-21 | 2018-12-21 | Kit and method for detecting and judging drug resistance of helicobacter pylori |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811570903.8A CN109593842B (en) | 2018-12-21 | 2018-12-21 | Kit and method for detecting and judging drug resistance of helicobacter pylori |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109593842A true CN109593842A (en) | 2019-04-09 |
| CN109593842B CN109593842B (en) | 2021-01-01 |
Family
ID=65963183
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811570903.8A Active CN109593842B (en) | 2018-12-21 | 2018-12-21 | Kit and method for detecting and judging drug resistance of helicobacter pylori |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109593842B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111518932A (en) * | 2020-05-13 | 2020-08-11 | 南开大学 | Diagnostic method and kit for detecting and judging drug resistance of helicobacter pylori by taking feces as sample |
| 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 |
| CN112553356A (en) * | 2020-12-31 | 2021-03-26 | 江苏意诺飞生物科技有限公司 | Method for high-throughput detection and determination of drug resistance of helicobacter pylori |
| CN113186136A (en) * | 2021-06-02 | 2021-07-30 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Multiple drug-resistant helicobacter pylori and application thereof |
| 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 (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999051270A1 (en) * | 1998-04-06 | 1999-10-14 | Dalhousie University | A novel nitroreductase and therapeutic uses therefor |
| CN103898195A (en) * | 2012-12-25 | 2014-07-02 | 泰州医药城博奥邦科生物科技有限公司 | Helicobacter pylori drug resistance nucleic acid detection kit |
| 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 |
| CN105368825A (en) * | 2015-10-14 | 2016-03-02 | 华东医院 | Helicobacter pylori antibiotic drug resistance analysis kit and drug resistance detecting method |
| CN105441583A (en) * | 2016-02-02 | 2016-03-30 | 华东医院 | Multiple-gene helicobacter pylori detection system and kit and application thereof |
-
2018
- 2018-12-21 CN CN201811570903.8A patent/CN109593842B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999051270A1 (en) * | 1998-04-06 | 1999-10-14 | Dalhousie University | A novel nitroreductase and therapeutic uses therefor |
| CN103898195A (en) * | 2012-12-25 | 2014-07-02 | 泰州医药城博奥邦科生物科技有限公司 | Helicobacter pylori drug resistance nucleic acid detection kit |
| 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 |
| CN105368825A (en) * | 2015-10-14 | 2016-03-02 | 华东医院 | Helicobacter pylori antibiotic drug resistance analysis kit and drug resistance detecting method |
| CN105441583A (en) * | 2016-02-02 | 2016-03-30 | 华东医院 | Multiple-gene helicobacter pylori detection system and kit and application thereof |
Non-Patent Citations (8)
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111518932A (en) * | 2020-05-13 | 2020-08-11 | 南开大学 | Diagnostic method and kit for detecting and judging drug resistance of helicobacter pylori by taking feces as sample |
| 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 |
| CN112553356A (en) * | 2020-12-31 | 2021-03-26 | 江苏意诺飞生物科技有限公司 | Method for high-throughput detection and determination of drug resistance of helicobacter pylori |
| CN113186136A (en) * | 2021-06-02 | 2021-07-30 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Multiple drug-resistant helicobacter pylori and application thereof |
| 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 |
|---|---|
| CN109593842B (en) | 2021-01-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN109593842A (en) | A kind of kit and method of detection and judgement helicobacter pylori drug resistance | |
| Inglis et al. | Laboratory techniques used for entomopathogenic fungi: Hypocreales | |
| US7262021B1 (en) | Method for antimicrobial susceptibility testing of microorganisms | |
| CN109182564B (en) | Method for detecting and analyzing toxicity and pathogenicity of helicobacter pylori | |
| CN101665824B (en) | Biochip for detecting drug resistant genes of helicobacter pylori clarithromycin and preparation method and application thereof | |
| JPS58126784A (en) | Diagnostic test of gonorrhoea, strain and test kit for testing gonorrhoea strain | |
| Thoreson et al. | Development of a PCR-based technique for detection of Helicobacter pylori | |
| CN109536628A (en) | A kind of helicobacter pylori specific molecular marker and detection kit | |
| JP2005518203A (en) | M.M. TUBERCULOSIS deletion sequences, methods for detecting mycobacteria using these sequences, and vaccines | |
| KR100361965B1 (en) | Probes for Detecting and Identifying Helicobacter pylori | |
| Neiger et al. | Diagnosis and identification of gastric Helicobacter species by polymerase chain reaction in dogs | |
| Safaee et al. | Microbial diversity in bighorn sheep revealed by culture-independent methods | |
| CN111676160B (en) | Application of beautiful millettia root endophyte RH5 in promoting strong growth of beautiful millettia root | |
| CN114317290A (en) | Bacterial strain capable of degrading diuron and application thereof | |
| CN100402665C (en) | Method for preparing grape anthracnose disease biochip | |
| CN113388689A (en) | Primer combination, probe, kit and use method thereof | |
| Matsumoto et al. | Phylogeny of a novel “Helicobacter heilmannii” organism from a Japanese patient with chronic gastritis based on DNA sequence analysis of 16S rRNA and urease genes | |
| CN101906478A (en) | Method for quickly diagnosing bovine endometritis pathogen | |
| US20110256583A1 (en) | Method and medium for accelerating target analyte growth in a test sample | |
| Faraj et al. | Molecular and Phenotypic Analysis as A Diagnostic Tool of Candida Spp Infections Associated with COVD19 | |
| JP5138197B2 (en) | LG21 strain primer and detection method using the primer | |
| JPH11127899A (en) | Oligonucleotide probe specific in species to bacteroides group bacterium | |
| Manam et al. | ISOLATION AND IDENTIFICATION OF BIOLUMINESCENT PHOTOBACTERIUM LEIOGNATHI FROM MARINE SQUID BY GENOMIC DNA AND RESTRICTION SITE ANALYSIS | |
| RU2595422C1 (en) | MICROBIAL TEST STRAIN OF Helicobacter pylori GKPM-Obolensk V-7215 FOR INDICATION AND IDENTIFICATION OF Helicobacter pylori IN SAMPLES OF BIOPSY MATERIALS OF GASTRIC OR DUODENAL ULCER | |
| JP3950918B2 (en) | Aerofaciens primer |
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 | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20201211 Address after: 4 / F, building 5, No. 151, libing Road, Pudong New Area, Shanghai, 201203 Applicant after: SHANGHAI OUTDO BIOTECH Co.,Ltd. Address before: No.15, Lane 908, Ziping Road, Pudong New Area, Shanghai Applicant before: SHANGHAI OUTDO CLINIC Co.,Ltd. |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |