CN106755539A - There is the positive quality control product for sporting template with 2143 sites of helicobacter pylori 23SrRNA genes - Google Patents
There is the positive quality control product for sporting template with 2143 sites of helicobacter pylori 23SrRNA genes Download PDFInfo
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- CN106755539A CN106755539A CN201710123370.8A CN201710123370A CN106755539A CN 106755539 A CN106755539 A CN 106755539A CN 201710123370 A CN201710123370 A CN 201710123370A CN 106755539 A CN106755539 A CN 106755539A
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- 238000003908 quality control method Methods 0.000 title claims abstract description 71
- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 49
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 49
- 108090000623 proteins and genes Proteins 0.000 title abstract description 10
- 108700022487 rRNA Genes Proteins 0.000 claims abstract description 41
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 20
- 230000035772 mutation Effects 0.000 claims abstract description 20
- 239000000243 solution Substances 0.000 claims description 17
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 11
- 239000007853 buffer solution Substances 0.000 claims description 11
- 239000012634 fragment Substances 0.000 claims description 10
- 230000002068 genetic effect Effects 0.000 claims description 10
- 239000008213 purified water Substances 0.000 claims description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 241000894006 Bacteria Species 0.000 claims description 3
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 2
- 238000012360 testing method Methods 0.000 abstract description 5
- 239000000523 sample Substances 0.000 description 26
- 239000013612 plasmid Substances 0.000 description 25
- 238000010790 dilution Methods 0.000 description 24
- 239000012895 dilution Substances 0.000 description 24
- 238000001514 detection method Methods 0.000 description 16
- 238000002844 melting Methods 0.000 description 15
- 230000008018 melting Effects 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 12
- 230000009514 concussion Effects 0.000 description 12
- 238000013461 design Methods 0.000 description 8
- 238000000034 method Methods 0.000 description 7
- 238000003753 real-time PCR Methods 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 5
- 206010064571 Gene mutation Diseases 0.000 description 4
- 241000877992 Lagarosiphon madagascariensis Species 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 101150088250 matK gene Proteins 0.000 description 4
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 229960002897 heparin Drugs 0.000 description 3
- 229920000669 heparin Polymers 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 210000004877 mucosa Anatomy 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- KAESVJOAVNADME-UHFFFAOYSA-N Pyrrole Chemical compound C=1C=CNC=1 KAESVJOAVNADME-UHFFFAOYSA-N 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 238000010079 rubber tapping Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 1
- 101150005607 Mdk gene Proteins 0.000 description 1
- LIQLLTGUOSHGKY-UHFFFAOYSA-N [B].[F] Chemical compound [B].[F] LIQLLTGUOSHGKY-UHFFFAOYSA-N 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
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- 239000013600 plasmid vector Substances 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
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- 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
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- 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
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- 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
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Abstract
There is the positive quality control product for sporting template in a kind of 2143 sites with helicobacter pylori 23S rRNA genes, it is characterised in that：The positive quality control product is that have reagent of the gene order of mutation as main component, the gene order such as SEQ NO with the site of helicobacter pylori 23S rRNA genes 2143:Shown in 6.Whether whether accurately the site of helicobacter pylori 23S rRNA genes 2143 be mutated this testing result during the present invention is used to verify tested sample, solves the On Quality Examining Problems of helicobacter pylori 23S rRNA genes.
Description
Technical field
The present invention relates to technical field of molecular biology, and in particular to a kind of with helicobacter pylori 23S rRNA genes
There is the positive quality control product for sporting template in 2143 sites, and the positive quality control product is used to verify that helicobacter pylori drug resistant gene is detected
Whether kit sample when pattern detection is applied to is positive auxiliary judgment.
Background technology
Quality-control product refers to the quality control material for in-vitro diagnosis, and it has the target detection thing of concentration known content,
To enable it is believed that a certain product or service quality meet product necessary to the quality requirement of regulation.The use mesh of quality-control product
Be in order to verify that whole PCR courses of reaction have accuracy, validity and a specificity, and monitor and detection system state.Instrument
Device should be calibrated periodically, it is to avoid due to experimental bias caused by instrument reason after formal coming into operation to it.Again, receiving
After every batch of novel agent, preliminary experiment should be carried out to it first, after confirmation novel agent quality, be used further to the detection to clinical samples.
But according to the applicant understood, do not have in terms of saltant type helicobacter pylori 23S rRNA gene quality controls are detected at present
There is positive quality control product.
The content of the invention
The present invention is intended to provide a kind of 2143 sites with helicobacter pylori 23S rRNA genes have sports template
Positive quality control product, for verifying tested sample in the site of helicobacter pylori 23S rRNA genes 2143 whether be mutated this detection knot
Whether fruit is accurate, solves the On Quality Examining Problems of helicobacter pylori 23S rRNA genes.
To reach above-mentioned purpose, the technical solution adopted by the present invention is：One kind is with helicobacter pylori 23S rRNA genes
2143 sites have the positive quality control product for sporting template, the positive quality control product is a kind of examination with base sequence as main component
There are the genetic fragment of mutation, the gene piece in agent, 23S rRNA gene 2143 site of the base sequence comprising helicobacter pylori
Section is 5 '-GTCGGTTAAATACCGACCTGCATGAATGGCGTAACGAGATGGGAGCTGTCTCAACC AGAGATTCAGTGAA
ATTGTAGTGGAGGTGAAAATTCCTCCTACCCGCGGCAAGACGGAGAGACCCCGTGGACCTTTACTACAACTTAGCAC
TGCTAATGGGAATATCATGCGCAGGATAGGTGGGAGGCTTTGAAGTAAGGGCTTTGGCTCTTATGGAGCCATCCTTG
AGATACCACCCTTGAT-3 ', wherein, the base position with underscore is the 23S rRNA genes of helicobacter pylori
2143 sites, the site of the 23S rRNA genes of the helicobacter pylori for characterizing 2143 anomalies.
Relevant content in above-mentioned technical proposal is explained as follows：
1st, in such scheme, buffer reagent is also contained in the positive quality control product, the buffer reagent is Tris- that pH is 7.6
Hcl buffer solutions, purified water and pH are any one in 8.0 TE buffer solutions.
2nd, in such scheme, the site of helicobacter pylori 23S rRNA genes 2143 contained in the positive quality control product has
The gene order of mutation is to be building up on plasmid vector to preserve, and is that can obtain according to prior art, and plasmid construction process is as follows：
--- --- DNA fragmentation --- positive gram of carrier construction --- conversion culture --- is reclaimed in rubber tapping to design of primers for pcr amplifications
--- 37 DEG C of shaking table culture --- plasmid extraction --- sequencings confirm base sequence for grand identification.
Plasmid after extracting is configured to the positive quality control product again, and compound method is as follows：
（1）Prepared and diluted liquid：Add the purified water → addition Tris-Hcl buffer solutions of 50% formula ratio（pH7.6）→ add residue
Purified water（Be vortexed concussion 3 times, every time 5 seconds）→ 0.002mol/L Tris-HCl（pH 7.6）Dilution；
（2）HP plasmids（2143 site mutations）Pre-dilution：Add the dilution → addition HP plasmids of 50% formula ratio（2143 sites
Mutation）The remaining dilution of solution → addition（Be vortexed concussion 3 times, every time 5 seconds）→ obtain X copy/microlitre HP plasmids（2143
Point mutation）Solution；
（3）Obtain positive quality control product：Add the dilution → addition X copy/microlitre HP plasmids of 50% formula ratio（Dash forward in 2143 sites
Become）The remaining dilution of solution → addition（Be vortexed concussion 3 times, every time 5 seconds）→ obtain positive quality control product.
The features of the present invention and beneficial effect are：The positive quality control product be substantially in advance design, as known
The sample of concentration and base sequence, and other tested samples（Such as mucosa tissue, gastric juice or bacterial plaque）Together using correlation
Helicobacter pylori 23S rRNA gene detecting kits detected, so as to verify the accurate of tested pattern detection result
Property.That is, in sample tested using the detection of helicobacter pylori 23S rRNA gene detecting kits, sun of the invention
Property control product are the equal of a kind of positive sample of concentration known, for verifying that helicobacter pylori 23S rRNA genetic tests are tried
The testing result of agent box（2143rd Mutation this testing result i.e. on helicobacter pylori 23S rRNA genes）Whether true,
Effectively, the On Quality Examining Problems of helicobacter pylori 23S rRNA genes are solved.
Brief description of the drawings
Accompanying drawing 1 is positive helicobacter pylori flora and 23S rRNA genes 2142 and 2143 sites without fluorescence when being mutated
Quantitative pcr amplification melting curve figure；
Accompanying drawing 2 is that helicobacter pylori flora is positive and the site of 23S rRNA genes 2142 has quantitative fluorescent PCR during mutation to expand
Increase melting curve figure；
Accompanying drawing 3 is that helicobacter pylori flora is positive and the site of 23S rRNA genes 2143 has quantitative fluorescent PCR during mutation to expand
Increase melting curve figure；
Fluorescent quantitative PCR melting curve figure when accompanying drawing 4 is negative helicobacter pylori flora；
Accompanying drawing 5 is the fluorescent quantitative PCR melting curve figure of inner quality control product.
Specific embodiment
Below in conjunction with the accompanying drawings and embodiment the invention will be further described：
Embodiment：There is the positive quality control product for sporting template in a kind of 2143 sites with helicobacter pylori 23S rRNA genes
Positive quality control product 1 is a kind of reagent with base sequence as main component, and the base sequence includes wild type H spiral
The 23S rRNA genetic fragments of bacillus, the 23S rRNA genetic fragments of the wild type H pylori for 5 '-
GTCGGTTAAATACCGACCTGCATGAATGGCGTAACGAGATGGGAGCTGTCTCAACCAGAGATTCAGTGAAATTGTAG
TGGAGGTGAAAATTCCTCCTACCCGCGGCAAGACGGAAAGACCCCGTGGACCTTTACTACAACTTAGCACTGCTAAT
GGGAATATCATGCGCAGGATAGGTGGGAGGCTTTGAAGTAAGGGCTTTGGCTCTTATGGAGCCATCCTTGAGATACC
ACCCTTGAT-3’（Such as SEQ NO:Shown in 4）, wherein, the base position with underscore is respectively from left to right that wild type is imprisoned
2142 and 2143 sites of the 23S rRNA genes of door pylori, described 2142 or/and 2143 sites are used to characterize wild type
The 23S rRNA genes of helicobacter pylori can sport the site of the 23S rRNA genes of anomaly helicobacter pylori.It is described
Also contain buffer reagent in positive quality control product 1, the buffer reagent is Tris-HCl buffer solutions that pH is 7.6.
Positive quality control product 2 is a kind of reagent with base sequence as main component, and the base sequence includes H. pylori
There is a genetic fragment of mutation in the site of 23S rRNA genes 2142 of bacterium, and the genetic fragment is 5 '-
GTCGGTTAAATACCGACCTGCATGAATGGCGTAACGAGATGGGAGCTGTCTCAACCAGAGATTCAGTGAAATTGTAG
TGGAGGTGAAAATTCCTCCTACCCGCGGCAAGACGGGAAGACCCCGTGGACCTTTACTACAACTTAGCACTGCTAAT
GGGAATATCATGCGCAGGATAGGTGGGAGGCTTTGAAGTAAGGGCTTTGGCTCTTATGGAGCCATCCTTGAGATACC
ACCCTTGAT-3’（Such as SEQ NO:Shown in 5）, wherein, the base position with underscore is the 23S of helicobacter pylori
2142 sites of rRNA genes, the site of the 23S rRNA genes of the helicobacter pylori for characterizing 2142 anomalies.It is described
Also contain buffer reagent in positive quality control product 2, the buffer reagent is Tris-HCl buffer solutions that pH is 7.6.
Positive quality control product 3 is a kind of reagent with base sequence as main component, and the base sequence includes H. pylori
There is a genetic fragment of mutation in the site of 23S rRNA genes 2143 of bacterium, and the genetic fragment is 5 '-
GTCGGTTAAATACCGACCTGCATGAATGGCGTAACGAGATGGGAGCTGTCTCAACCAGAGATTCAGTGAAATTGTAG
TGGAGGTGAAAATTCCTCCTACCCGCGGCAAGACGGAGAGACCCCGTGGACCTTTACTACAACTTAGCACTGCTAAT
GGGAATATCATGCGCAGGATAGGTGGGAGGCTTTGAAGTAAGGGCTTTGGCTCTTATGGAGCCATCCTTGAGATACC
ACCCTTGAT-3’（Such as SEQ NO:Shown in 6）, wherein, the base position with underscore is the 23S of helicobacter pylori
2143 sites of rRNA genes, the site of the 23S rRNA genes of the helicobacter pylori for characterizing 2143 anomalies.It is described
Also contain buffer reagent in positive quality control product 3, the buffer reagent is Tris-HCl buffer solutions that pH is 7.6.
The positive quality control product 1, positive quality control product 2, positive quality control product 3 are examined as helicobacter pylori 23S rRNA genes
A part in test agent box.
In the present embodiment, helicobacter pylori and its drug-tolerant gene mutation detection kit are polymerized including KOD DNA
Enzyme, primer HPC-F, primer HPC-R, selectively targeted fluorescence probe, the positive quality control product 1, positive quality control product 2 and the positive
Quality-control product 3；
The primer HPC-F is the forward primer for the design of helicobacter pylori 23S rRNA genes, the sequence of the forward primer
It is classified as 5 '-GTGGAGGTGAAAATTCCTCCTACCC-3 '（Such as SEQ NO:Shown in 1）；
The primer HPC-R is the reverse primer for the design of helicobacter pylori 23S rRNA genes, the sequence of the reverse primer
It is classified as 5 '-GGCTCCATAAGAGCCAAAGCCCTTAC-3 '（Such as SEQ NO:Shown in 2）；
The selectively targeted fluorescence probe is the spy with helicobacter pylori 23S rRNA wild type gene groups as stencil design
Pin, its base sequence is 5 '-CAAGACGGAAAGACCC -3 '（Such as SEQ NO:Shown in 3）, fluorescent dye is the pyrrole of fluorine boron two
Cough up；
The kit also includes inner quality control primer I C-F, inner quality control primer I C-R, inner quality control probe and internal matter
Control template；
The inner quality control primer I C-F is being directed to the matK genes design of Lagarosiphon madagascariensis just
To primer, the sequence of the forward primer is 5 '-CCCGGTTATTGTAGAAATTCCTTTCTCCCGTC-3 '（Such as SEQ NO:7 institutes
Show）；
The inner quality control primer I C-R is directed to the anti-of the matK genes design of Lagarosiphon madagascariensis
To primer, the sequence of the reverse primer is 5 '-CCCCATCCAGGATTGTAGAATTTGAATCAAG-3 '（Such as SEQ NO:8 institutes
Show）；
The inner quality control probe is designed as template with the matK genes of Lagarosiphon madagascariensis
Probe, the sequence of the probe is 5 '-GATCTATTCATTCGATATTCC-3 '（Such as SEQ NO:Shown in 9）；
The inner quality control template is a fragment of the matK genes of Lagarosiphon madagascariensis, internal
The sequence of mass controlled template is 5 '-GCGGTTATTGTAGAAATTCCTTTCTCCCGTCCATTTTTTCTTGAAGAAAAAAAAGA AA
TACCAAAATATCAAAATTTACGATCTATTCATTCGATATTCCCTTTTTTAGAGGACAAATTTTTACATTTAAATTAT
GTGTCTGATATAGTAATACCTTATCCTATTCATCTCGAAATCTTGATTCAAATTCTACAATCCTGGAT-3’（Such as SEQ
NO:Shown in 10）.
The inner quality control primer I C-F, inner quality control primer I C-R, inner quality control probe and inner quality control template are used
In verifying whether the kit is true, effective when helicobacter pylori drug-tolerant gene mutation is detected, referring to shown in accompanying drawing 5.
In actual production, the helicobacter pylori and its drug-tolerant gene mutation detection kit can be made and include down
The kit of row component：
（1）Polymerization enzymatic reagent [KOD Mix]：Be made up of KOD DNA polymerases and dNTP, specification be 140 μ L × 1,
140 μ L × 2,140 μ L × 3 or 140 μ L × 6；
（2）Primed probe reagent [HPC Mix]：Visited by primer HPC-F, primer HPC-R and selectively targeted fluorescence
The mix reagent of pin composition, specification is 140 μ L × 1,140 μ L × 2,140 μ L × 3 or 140 μ L × 6；
（3）Positive quality control product 1 [HPC PC1]：Specification is 300 μ L × 1；
（4）Positive quality control product 2 [HPC PC2]：Specification is 300 μ L × 1；
（5）Positive quality control product 3 [HPC PC2]：Specification is 300 μ L × 1；
（6）Negative quality-control product [HPC NC]：The buffer solution of DNA is not contained as, and specification is 300 μ L × 1；
The gene order contained in positive quality control product 1, positive quality control product 2, positive quality control product 3 is to be building up on vector plasmid to protect
Deposit.Plasmid construction process is as follows：
--- --- DNA fragmentation --- positive gram of carrier construction --- conversion culture --- is reclaimed in rubber tapping to design of primers for pcr amplifications
--- 37 DEG C of shaking table culture --- plasmid extraction --- sequencings confirm base sequence for grand identification.
Plasmid after extracting is configured to the positive quality control product 1 again, and compound method is as follows：
（1）Prepared and diluted liquid：Add the purified water → addition Tris-Hcl buffer solutions of 50% formula ratio（pH7.6）→ add residue
Purified water（Be vortexed concussion 3 times, every time 5 seconds）→ 0.002mol/L Tris-HCl（pH 7.6）Dilution；
（2）HP wild plasmid pre-dilution：Add the dilution → addition HP wild plasmids solution of 50% formula ratio → plus
Enter the remaining dilution（Be vortexed concussion 3 times, every time 5 seconds）→ obtain X copy/microlitre HP wild plasmid solution（X is represented
What）；
（3）Obtain positive quality control product 1：Add the dilution → addition X copy/microlitre HP wild plasmid solution of 50% formula ratio
→ add remaining dilution（Be vortexed concussion 3 times, every time 5 seconds）→ obtain positive quality control product.
Plasmid after extracting is configured to the positive quality control product 2 again, and compound method is as follows：
（1）Prepared and diluted liquid：Add the purified water → addition Tris-Hcl buffer solutions of 50% formula ratio（pH7.6）→ purified water is fixed
Hold（Be vortexed concussion 3 times, every time 5 seconds）The Tris-Hcl buffer solutions of → addition 0.002mol/L（pH 7.6）, obtain dilution；
（2）HP plasmids（2142 site mutations）Pre-dilution：Add the dilution → addition HP plasmids of 50% formula ratio（2142 sites
Mutation）The remaining dilution of solution → addition（Be vortexed concussion 3 times, every time 5 seconds）→ obtain X copy/microlitre HP plasmids（2142
Point mutation）Solution；
（3）Obtain positive quality control product 2：Add the dilution → addition X copy/microlitre HP plasmids of 50% formula ratio（Dash forward in 2142 sites
Become）The remaining dilution of solution → addition（Be vortexed concussion 3 times, every time 5 seconds）→ obtain positive quality control product 2.
Plasmid after extracting is configured to the positive quality control product 3 again, and compound method is as follows：
（1）Prepared and diluted liquid：Add the purified water → addition Tris-Hcl buffer solutions of 50% formula ratio（pH7.6）→ add residue
Purified water（Be vortexed concussion 3 times, every time 5 seconds）→ 0.002mol/L Tris-HCl（pH 7.6）Dilution；
（2）HP plasmids（2143 site mutations）Pre-dilution：Add the dilution → addition HP plasmids of 50% formula ratio（2143 sites
Mutation）The remaining dilution of solution → addition（Be vortexed concussion 3 times, every time 5 seconds）→ obtain X copy/microlitre HP plasmids（2143
Point mutation）Solution；
（3）Obtain positive quality control product 3：Add the dilution → addition X copy/microlitre HP plasmids of 50% formula ratio（Dash forward in 2143 sites
Become）The remaining dilution of solution → addition（Be vortexed concussion 3 times, every time 5 seconds）→ obtain positive quality control product 3.
Detection method is comprised the following steps：
The first step, prepare object, using object as detection object, object be without any treatment tested sample or
Person is the helicobacter pylori nucleic acid extracted from tested sample, the mucosa tissue that the tested sample is behaved, and is detected sample
Machine can be directly gone up to be detected.Directly during use mucosa tissue, it is to avoid use contains the sample more than blood.If using blood constituent
Content is more, or the sample more than heparin content, it is noted that fully remove blood and heparin compositions.Blood constituent or heparin remaining
In the case of, can have an impact to pcr amplification reaction, it is impossible to normal detection.When needing to preserve sample, less than -80 DEG C please be stored in.
Using Long-term Cryopreservation sample when, reused after room temperature must be returned to.
Second step, is templet gene chain with the tested sample, and it is anti-to carry out fluorescent quantitative PCR using the kit
Should, 13.2 μ L reaction systems of fluorescent quantitative PCR reaction are：
Primer HPC-F 0.1umol/L, 0.52 μ l；
Primer HPC-R 0.1umol/L, 0.52 μ l；
Selectively targeted fluorescence probe 0.1umol/L, 0.4 μ l；
Inner quality control primer I C-F 0.1umol/L, 0.52 μ l；
Inner quality control primer I C-R 0.1umol/L, 0.52 μ l；
Inner quality control probe 0.1umol/L, 0.4 μ l；
Inner quality control template 4ng/ul, 1.8 μ l；
MgCl2*6H2O 0.35g/L, 0.52 μ L；
DNTPs 0.02mM, 0.4 μ L；
KOD DNA polymerases 0.02U/ul, 0.4 μ l；
KOD buffer 3.2μl；
The μ l of template 4；
The course of reaction of the fluorescent quantitative PCR reaction includes：
（1）94.0 DEG C of predegenerations 30.0 seconds,
（2）97.0 DEG C are denatured 1.0 seconds,
（3）60.0 DEG C are annealed 3.0 seconds,
（4）63.0 DEG C extend 5.0 seconds, wherein, step（2）~（4）Circulation 50 times.
3rd step, is detected with high-resolution melting curve method, and detection condition is followed successively by：
（1）94.0 DEG C, 30.0 seconds,
（2）39.0 DEG C, 30.0 seconds,
（3）40.0 ~ 75.0 DEG C, 0.09 DEG C/sec；
Whole fluorescent quantitative PCR reaction is directly in mdk gene analyzer GENECUBE（Manufacturer is TOYOBO）It is enterprising
OK.Concretely comprise the following steps：By the μ L of tested sample 4, polymerization enzymatic reagent [KOD Mix] 4 μ L, primed probe reagent [HPC Mix] 5.2 μ
After L mixing, reaction solution is modulated into.Special suction pipe draws reaction solution in special plastic capillary.Carry out amplified reaction.Examined
Go out, it is the fluorescent value of 510nm ~ 550nm and 573nm ~ 613nm to determine wavelength.The fluorescent value of measure is by dedicated analysis software solution
Analysis, is converted into representing the fluorescence differential value of change in fluorescence amount.Parsing fluorescence differential value, shows measurement result on a display screen.
4th step, detection result judges that the basis for estimation of three kinds of results of positive cutoff value is by positive cutoff value：
（1）The helicobacter pylori flora positive and 23S rRNA genes 2142 and 2143 sites are without mutation：Fluorescence differential value >=10,
And the melting temperature where melting curve peak value is between 50 DEG C ~ 60 DEG C, referring to shown in accompanying drawing 1；
（2）The helicobacter pylori flora positive and 23S rRNA genes 2142 or 2143 sites have mutation：Fluorescence differential value >=10,
And the melting temperature where melting curve peak value is between 42 DEG C ~ 50 DEG C, referring to shown in accompanying drawing 2 and accompanying drawing 3；
（3）Helicobacter pylori flora is negative：Unstressed configuration differential value, inner quality control fluorescence differential value >=1.0, and its melting curve
Melting temperature where peak value between 42 DEG C ~ 68 DEG C, referring to shown in accompanying drawing 4；
The fluorescence differential value refers to that the value that varies with temperature of fluorescence intensity of the object to detecting does differential derivation, is obtained
Fluorescence differential value.
Only when inner quality control fluorescence differential value >=1.0 in the melting curve of accompanying drawing 5, and where its melting curve peak value
When melting temperature is between 42 DEG C ~ 68 DEG C, show that whole detecting system is normal, effective, be the moon especially for judged result
It is even more important during property.
The above embodiments merely illustrate the technical concept and features of the present invention, its object is to allow person skilled in the art
Scholar will appreciate that present disclosure and implement according to this that it is not intended to limit the scope of the present invention.It is all according to the present invention
The equivalent change or modification that Spirit Essence is made, should all be included within the scope of the present invention.
SEQUENCE LISTING
<110>Step on stone biotechnology（Suzhou）Co., Ltd
<120>Helicobacter pylori drug-tolerant gene mutation detection kit and its detection method
<130>
<160> 10
<170> PATENTIN VERSION 3.5
<210> 1
<211> 25
<212> DNA
<213>Artificial sequence
<400> 1
gtggaggtgaaaattcctcctaccc 25
<210> 2
<211> 26
<212> DNA
<213>Artificial sequence
<400> 2
ggctccataagagccaaagcccttac 26
<210> 3
<211> 16
<212> DNA
<213>Artificial sequence
<400> 3
caagacggaaagaccc 16
<210> 4
<211> 240
<212> DNA
<213>Artificial sequence
<400> 4
gtcggttaaataccgacctgcatgaatggcgtaacgagatgggagctgtctcaaccagagattcagtgaaatt
gtagtggaggtgaaaattcctcctacccgcggcaagacggaaagaccccgtggacctttactacaacttagcactgc
taatgggaatatcatgcgcaggataggtgggaggctttgaagtaagggctttggctcttatggagccatccttgaga
taccacccttgat
240
<210> 5
<211> 240
<212> DNA
<213>Artificial sequence
<400> 5
gtcggttaaataccgacctgcatgaatggcgtaacgagatgggagctgtctcaaccagagattcagtgaaatt
gtagtggaggtgaaaattcctcctacccgcggcaagacgggaagaccccgtggacctttactacaacttagcactgc
taatgggaatatcatgcgcaggataggtgggaggctttgaagtaagggctttggctcttatggagccatccttgaga
taccacccttgat
240
<210> 6
<211> 240
<212> DNA
<213>Artificial sequence
<400> 6
gtcggttaaataccgacctgcatgaatggcgtaacgagatgggagctgtctcaaccagagattcagtgaaatt
gtagtggaggtgaaaattcctcctacccgcggcaagacggagagaccccgtggacctttactacaacttagcactgc
taatgggaatatcatgcgcaggataggtgggaggctttgaagtaagggctttggctcttatggagccatccttgaga
taccacccttgat
240
<210> 7
<211> 32
<212> DNA
<213>Artificial sequence
<400> 7
cccggttattgtagaaattcctttctcccgtc 32
<210> 8
<211> 31
<212> DNA
<213>Artificial sequence
<400> 8
ccccatccaggattgtagaatttgaatcaag 31
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence
<400> 9
gatctattcattcgatattcc 21
<210> 10
<211> 203
<212> DNA
<213>Artificial sequence
<400> 10
gcggttattgtagaaattcctttctcccgtccattttttcttgaagaaaaaaaagaaataccaaaatatcaaa
atttacgatctattcattcgatattcccttttttagaggacaaatttttacatttaaattatgtgtctgatatagta
ataccttatcctattcatctcgaaatcttgattcaaattctacaatcctggat 203
Claims (2)
1. there is the positive quality control product for sporting template in a kind of 2143 sites with helicobacter pylori 23S rRNA genes, its feature
It is：The positive quality control product is a kind of reagent with base sequence as main component, and the base sequence includes H. pylori
There is a genetic fragment of mutation in the site of 23S rRNA genes 2143 of bacterium, and the genetic fragment is 5 '-
GTCGGTTAAATACCGACCTGCATGAATGGCGTAACGAGATGGGAGCTGTCTCAACCAGAGATTCAGTGAAATTGTAG
TGGAGGTGAAAATTCCTCCTACCCGCGGCAAGACGGAGAGACCCCGTGGACCTTTACTACAACTTAGCACTGCTAAT
GGGAATATCATGCGCAGGATAGGTGGGAGGCTTTGAAGTAAGGGCTTTGGCTCTTATGGAGCCATCCTTGAGATACC
ACCCTTGAT-3 ', wherein, the base position with underscore is 2143 of the 23S rRNA genes of helicobacter pylori
Point, the site of the 23S rRNA genes of the helicobacter pylori for characterizing 2143 anomalies.
2. there is the sun for sporting template in 2143 sites with helicobacter pylori 23S rRNA genes according to claim 1
Property quality-control product, it is characterised in that：Also contain buffer reagent in the positive quality control product, the buffer reagent is 7.6 for pH
Tris-Hcl buffer solutions, purified water and pH are any one in 8.0 TE buffer solutions.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710123370.8A CN106755539A (en) | 2017-03-03 | 2017-03-03 | There is the positive quality control product for sporting template with 2143 sites of helicobacter pylori 23SrRNA genes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710123370.8A CN106755539A (en) | 2017-03-03 | 2017-03-03 | There is the positive quality control product for sporting template with 2143 sites of helicobacter pylori 23SrRNA genes |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106755539A true CN106755539A (en) | 2017-05-31 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710123370.8A Pending CN106755539A (en) | 2017-03-03 | 2017-03-03 | There is the positive quality control product for sporting template with 2143 sites of helicobacter pylori 23SrRNA genes |
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| CN (1) | CN106755539A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103361426A (en) * | 2013-06-19 | 2013-10-23 | 中国疾病预防控制中心传染病预防控制所 | Kit for detecting drug resistance gene of clarithromycin to helicobacter pylori |
| 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 |
| CN104164491A (en) * | 2014-07-21 | 2014-11-26 | 北京新基永康生物科技有限公司 | ARMS-qPCR detection method and kit for helicobacter pylori 23S rDNA gene mutation subtype |
-
2017
- 2017-03-03 CN CN201710123370.8A patent/CN106755539A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| CN103361426A (en) * | 2013-06-19 | 2013-10-23 | 中国疾病预防控制中心传染病预防控制所 | Kit for detecting drug resistance gene of clarithromycin to helicobacter pylori |
| CN104164491A (en) * | 2014-07-21 | 2014-11-26 | 北京新基永康生物科技有限公司 | ARMS-qPCR detection method and kit for helicobacter pylori 23S rDNA gene mutation subtype |
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Effective date of registration: 20190124 Address after: Osaka Japan Applicant after: Toyo Boseki Address before: 215123 No. 99 Jinjihu Avenue, Suzhou Industrial Park, Jiangsu Province Applicant before: Stepping stone biological technology (Suzhou) Co., Ltd. |
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Application publication date: 20170531 |