CN1295348C - Helicobacter pylori clarithromycin drug-resistant gene polymorphism detecting chip and its preparation method and uses - Google Patents
Helicobacter pylori clarithromycin drug-resistant gene polymorphism detecting chip and its preparation method and uses Download PDFInfo
- Publication number
- CN1295348C CN1295348C CN 200410073454 CN200410073454A CN1295348C CN 1295348 C CN1295348 C CN 1295348C CN 200410073454 CN200410073454 CN 200410073454 CN 200410073454 A CN200410073454 A CN 200410073454A CN 1295348 C CN1295348 C CN 1295348C
- Authority
- CN
- China
- Prior art keywords
- probe
- primer
- helicobacter pylori
- drug
- gene
- 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.)
- Expired - Fee Related
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 37
- AGOYDEPGAOXOCK-KCBOHYOISA-N clarithromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@](C)([C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)OC)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 AGOYDEPGAOXOCK-KCBOHYOISA-N 0.000 title claims abstract description 22
- 229960002626 clarithromycin Drugs 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 14
- 241000590002 Helicobacter pylori Species 0.000 title claims abstract description 12
- 229940037467 helicobacter pylori Drugs 0.000 title claims abstract description 12
- 229940079593 drug Drugs 0.000 title claims description 16
- 239000003814 drug Substances 0.000 title claims description 16
- 239000000523 sample Substances 0.000 claims abstract description 53
- 238000001514 detection method Methods 0.000 claims abstract description 13
- 108700022487 rRNA Genes Proteins 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 11
- 238000013461 design Methods 0.000 claims description 11
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 10
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 10
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 9
- 239000001632 sodium acetate Substances 0.000 claims description 9
- 235000017281 sodium acetate Nutrition 0.000 claims description 9
- 239000006210 lotion Substances 0.000 claims description 8
- SJECZPVISLOESU-UHFFFAOYSA-N 3-trimethoxysilylpropan-1-amine Chemical compound CO[Si](OC)(OC)CCCN SJECZPVISLOESU-UHFFFAOYSA-N 0.000 claims description 5
- 101100441878 Caenorhabditis elegans cyn-3 gene Proteins 0.000 claims description 5
- 229920001917 Ficoll Polymers 0.000 claims description 5
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 5
- 101100464856 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cyp-3 gene Proteins 0.000 claims description 5
- 229960000583 acetic acid Drugs 0.000 claims description 5
- 150000001299 aldehydes Chemical class 0.000 claims description 5
- 235000011114 ammonium hydroxide Nutrition 0.000 claims description 5
- KRVSOGSZCMJSLX-UHFFFAOYSA-L chromic acid Substances O[Cr](O)(=O)=O KRVSOGSZCMJSLX-UHFFFAOYSA-L 0.000 claims description 5
- 238000010511 deprotection reaction Methods 0.000 claims description 5
- 235000021185 dessert Nutrition 0.000 claims description 5
- 239000012895 dilution Substances 0.000 claims description 5
- 238000010790 dilution Methods 0.000 claims description 5
- AWJWCTOOIBYHON-UHFFFAOYSA-N furo[3,4-b]pyrazine-5,7-dione Chemical compound C1=CN=C2C(=O)OC(=O)C2=N1 AWJWCTOOIBYHON-UHFFFAOYSA-N 0.000 claims description 5
- 239000012362 glacial acetic acid Substances 0.000 claims description 5
- 235000011187 glycerol Nutrition 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- 238000004321 preservation Methods 0.000 claims description 5
- 238000012545 processing Methods 0.000 claims description 5
- 238000000746 purification Methods 0.000 claims description 5
- 238000005070 sampling Methods 0.000 claims description 5
- 238000004506 ultrasonic cleaning Methods 0.000 claims description 5
- 230000003321 amplification Effects 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- 230000014509 gene expression Effects 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims 1
- 230000001580 bacterial effect Effects 0.000 abstract description 4
- 206010059866 Drug resistance Diseases 0.000 abstract description 2
- 230000004907 flux Effects 0.000 abstract 1
- 230000002496 gastric effect Effects 0.000 abstract 1
- 150000008300 phosphoramidites Chemical class 0.000 description 12
- -1 polyoxyethylene Polymers 0.000 description 12
- 108091034117 Oligonucleotide Proteins 0.000 description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 7
- 239000002751 oligonucleotide probe Substances 0.000 description 7
- 210000002784 stomach Anatomy 0.000 description 5
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 4
- 125000003368 amide group Chemical group 0.000 description 4
- 238000004176 ammonification Methods 0.000 description 4
- 238000001311 chemical methods and process Methods 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N ethylene glycol Natural products OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 4
- 210000004400 mucous membrane Anatomy 0.000 description 4
- 239000002773 nucleotide Substances 0.000 description 4
- 125000003729 nucleotide group Chemical group 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 125000006850 spacer group Chemical group 0.000 description 4
- 238000010561 standard procedure Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 3
- 238000001574 biopsy Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 230000007170 pathology Effects 0.000 description 3
- 230000011664 signaling Effects 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 208000025865 Ulcer Diseases 0.000 description 2
- 238000007846 asymmetric PCR Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 230000008029 eradication Effects 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 241000972773 Aulopiformes Species 0.000 description 1
- 208000023275 Autoimmune disease Diseases 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 238000000018 DNA microarray Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 1
- 208000007882 Gastritis Diseases 0.000 description 1
- 208000018522 Gastrointestinal disease Diseases 0.000 description 1
- 101100489867 Mus musculus Got2 gene Proteins 0.000 description 1
- 208000005718 Stomach Neoplasms Diseases 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 229910052797 bismuth Inorganic materials 0.000 description 1
- JCXGWMGPZLAOME-UHFFFAOYSA-N bismuth atom Chemical compound [Bi] JCXGWMGPZLAOME-UHFFFAOYSA-N 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 208000023652 chronic gastritis Diseases 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
- 208000022602 disease susceptibility Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 210000001156 gastric mucosa Anatomy 0.000 description 1
- 208000017215 gastric mucosa-associated lymphoid tissue lymphoma Diseases 0.000 description 1
- 208000018685 gastrointestinal system disease Diseases 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000009413 insulation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229960000282 metronidazole Drugs 0.000 description 1
- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000005457 optimization Methods 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 229940126409 proton pump inhibitor Drugs 0.000 description 1
- 239000000612 proton pump inhibitor Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000007894 restriction fragment length polymorphism technique Methods 0.000 description 1
- 235000019515 salmon Nutrition 0.000 description 1
- 239000003620 semiochemical Substances 0.000 description 1
- 238000003201 single nucleotide polymorphism genotyping Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 201000000498 stomach carcinoma Diseases 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 150000004684 trihydrates Chemical class 0.000 description 1
- 231100000397 ulcer Toxicity 0.000 description 1
- 230000036269 ulceration Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention discloses a helicobacter pylori clarithromycin drug-resistance gene detecting chip and a preparation method thereof and applications. The following probes are arranged on a carrier. The present invention realizes quick and simple detection with high flux on gene polymorphism of 23SrRNA2142, 2143 and 2182 sites, the apparatus can directly detect gastric mucosae, and bacterial culture is not needed.
Description
Technical field
The present invention relates to a kind of helicobacter pylori clarithromycin drug-resistant gene polymorphism detecting chip and its production and use.
Background technology
(Helicobacter pylori, research Hp) is gastrointestinal disorder worker's heat subject to Hp always, receives digesting boundary and microbiologist's very big concern about Hp resistance problem at present.Many data show that Hp not only can cause chronic gastritis, peptide ulceration, and with relevant [1,2] such as cancer of the stomach, gastric MALT lymphoma and some cardiovascular disordeies, hemopathy, autoimmune disorder, tetter.Hp the infected merges the ulcer patient must accept eradication therapy [3], and it is that proton pump inhibitor is or/and two kinds of microbiotic such as bismuth agent associating clarithromycin, amoxycilline Trihydrate bp, metronidazole/tinidazoles that routine clinical application three or quadruple chemotherapy are eradicated Hp.And the appearance of Resistant strain brings very big difficulty for the elimination of Hp, and clarithromycin is treated its Resistant strain and often indicated the possibility very big [4] of eradicating failure.Therefore, the understanding drug-resistance of bacteria is most important before eradication therapy.And because microbiotic abuse and use lack of standardizationly, the appearance of Resistant strain is increasing, causes the Hp resistant rate to raise day by day.Studies show that: Hp clarithromycin resistance and 23S rRNA gene A 2142G, A2143G[5] and C2182T[6] point mutation is relevant.
Being used for SNPs, to do genetic analysis be to refer in particular to detect a large amount of single nucleotide variations that exist of human genome, these relevant with the poisonous substance susceptibility with disease susceptibility [7].SNPs is not only the mark of human race and individual difference, also is the mark of decision different crowd race and individual difference, can become the searching disease related gene, carries out the basis of medical diagnosis on disease, prevention and drug screening.Being used for the ordinary method that SNPs detects has allele specific oligonucleotide (ASO) hybridization, restriction enzyme enzyme process (RFLP), locus specificity primer PCR (PCR-SSP) and directly check order etc.The detection method that develops into new SNPs of biochip technology provides possibility [8].The oligonucleotide chip technology is a kind of technique of gene detection that newly-developed gets up, it can be arranged in a large amount of oligonucleotide probes on the slide regularly, these probes can show that the complementary sequence of extension increasing sequence of the sample DNA of material mark combines with signal, by semiochemicals is detected, results of hybridization is carried out the software processing analysis, thereby obtain intensity of hybridization signal and distribution pattern figure thereof.
Compare with traditional method, oligonucleotide chip can directly carry out the bacterial strain variation to the stomach mucous membrane biopsy specimen and detect, and is suitable equally when hybrid bacterial strain is infected, and is difficult to distinguish the mixing strain with the method for microbial culture, and wastes time and energy, and cultivates quite difficulty.Order-checking is a kind of classic methods, but often can only detect the sequence of dominant strain; Just detect easily when perhaps the quantity of wild strain and mutant strain is suitable; And oligonucleotide chip can detect a small amount of dissociant.This method can allow the doctor accurately judge patient's resistance situation at short notice.Therefore, oligonucleotide chip bacterial detection resistance has its superiority.
Reference:
[1]Gunn M,Stephens J,Thompson J,Rathbone B,Samani N.Significantassociation of CagA positive Helicobacter pylori strains with risk ofpremature myocardial infarction.Heart 2000;84:267-271.
[2]Zentilin P,Seriolo B,Dulbecco P,Caratto E,Liritano E,FascioloD,Bilardi C,Mansi C,Testa E,Savarino V.Eradication of Helicobacterpylori may reduce disease severity in rheumatoid arthritis.AlimentPharmacol Ther.2002,16:1291-1299
[3]NIH Consensus Conference.Hel icobacter pylori in peptic ulcer disease.NIH Consensus Development Panel on Helicobacter pylori in peptic ulcerdisease.J Am Med Assoc 1994;272:65-9
[4]Loren L,Richard H,Hala EZ,Bich N,Michael O,Jean S.Bismuth-basedquadruple therapy using a single capsule of Bismuth biskalcitrate,metronidazole and tetracycline given with omeprazole versus omeprazole,amoxicillin and clarithromycin for eradication of Helicobacter pyloriin duodenal ulcer patients:a prospective,randomized,multicenter,North American trial.The American Journal of Gastrienterology,2003,98(3):562-7
[5]Versalovic JD,Shortridge K,Kibler MV,Griffy J,Beyer RK,Flam SK,Tanaka DY,Graham,MF Go.Mutation in 23S rRNA are associated withclarithromycin resistance in Helicobacter pylori.Antimicrob AgentsChemther.1996,40:477-80
[6]Matsuoka M,Yoshida Y,Hayakawa K,Fukuchi,S,Sugano K.Simultaneouscolonization of Helicobacter pylori with and without mutations in the23S rRNA gene in patients with no history of clarithromycin exposure.Gut 1999;45:503-7
[7]Khner M K,Beerli P,Yamato J,et al.Usefulness of single nucleatidepolymorphism data for estimating populationparameters.Genetics,2000,156:439-447
[8]Hirschlhorn J N,Sklar P,Lindblad-Toh K,et al.SBE-TAGS:an array-basedmethod for efficient single-nucleotide polymorphism genotyping.ProcNatl Acad Sci USA,2000,97:12164-12169
Summary of the invention
The purpose of this invention is to provide a kind of helicobacter pylori clarithromycin drug-resistant gene polymorphism detecting chip and its production and use.
Hp clarithromycin drug-resistant gene polymorphism detecting chip: on carrier, be provided with following probe:
Hp clarithromycin drug-resistant gene polymorphism detecting chip preparation method step is:
1) processing of chip carrier: slide chromic acid lotion soaked overnight, washing, spend the night in 24~26% ammoniacal liquor, washing, slide immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates PH to 4~4.5,95% ethanol ultrasonic cleaning with Glacial acetic acid, dried 3~5 hours for 150~160 ℃, 5~10% glutaraldehyde are processed into aldehyde radicalization;
2) primer and probe is synthetic: at 23S rRNA gene A 2142G, A2142C, A2142T, A2143G, A2143C, A2143T, C2182T, C2182A and the described probe of C2182G pleomorphism site design claim 1 of Hp, its length range is in 15-17 base (table 1), at the synthetic 1 pair of primer of the 23S rRNA gene design of Hp, 21 bases of length (table 2) have a Cyp-3 signaling molecule mark in every primer.All Oligonucleolide primers or probe all adopt standard phosphoramidite chemical process synthetic on the ABi8089DNA automatic DNA synthesizer DNA, the inferior phosphinylidyne ammonification of the synthetic employing standard method of oligonucleotide, stationary probe is held with amido modified 3 ', link to each other with spacerarm (spacer, polyoxyethylene glycol phosphoramidite reagent) between amino and the probe sequence.Fluorescent dye primer directly carries out fluorescent mark with standard phosphoramidite chemical synthesis with Cy3 phosphoramidite reagent at 5 ' end of general primer sequence.Cut 13~17 hours with 50~60 ℃ of deprotections of strong aqua synthetic back, the OPC column purification, and the quantitative final vacuum of ultraviolet is dry to be concentrated, and-20~-80 ℃ of preservations are standby;
3) gene chip preparation and aftertreatment: with probe to final concentration is 100 μ mol/L; Use 750mmol/LNaCl, the 75mmol/L sodium acetate, 5% glycerine, 1% ficoll and 0.1%SDS synthesize sampling liquid; Aforementioned both dilutions in 1: 1, and get 10 μ L to 96 orifice plates, to put to the aldehyde radical slide with the chip point sample instrument, every some volume is about 0.5nL, and the dessert spacing is 500 μ m, and spot diameter is about 200 μ m, and every probe repeats a little 2 times.The arrangement of oligonucleotide probe on slide is as follows.After point sample finished, room temperature was placed after 24 hours standby.
*1 | *1 | *2 | *2 | *3 | *3 | *4 | *4 |
*5 | *5 | *6 | *6 | *7 | *7 | *8 | *8 |
*9 | *9 | *10 | *10 | *11 | *11 | *12 | *12 |
*13 | *13 | *14 | *14 | *15 | *15 | *16 | *16 |
*17 | *17 | *18 | *18 | *19 | *19 | *20 | *20 |
The Hp clarithromycin drug-resistant gene polymorphism detecting chip is used to detect Hp clarithromycin drug-fast 23s rRNA gene pleiomorphism and somatotype.
The present invention will be through the probe point sample selected on slide, hybridize respectively with through the 23S of pcr amplification rRNA gene fragment, the polymorphic situation in each site of disposable acquisition, thereby realize that 23S rRNA related gene polymorphism to Hp carries out quick, simple and direct, high throughput testing and somatotype, has important society and economic benefit.
Embodiment
Immobilised probe is an oligonucleotide probe (table 2) on the slide of the present invention, and its sequence is according to Hp 23S rRNA gene A 2142G, A2142C, A2142T, A2143G, A2143C, A2143T, C2182T, C2182A and the design of C2182G pleomorphism site.
Said primer is the gene expression characteristics design design synthetic primer (table 2) at the 23S rRNA of Hp, is used for the amplification of said gene.
The signaling molecule mark is the primer at the gene order amplification of the 23S rRNA of Hp.The signaling molecule mark is a fluorescence molecule.
The probe of table 1:Hp clarithromycin drug-resistant gene polymorphism detecting chip
Table 2: the primer of alcohol metabolism relative enzyme gene polymorphic test chip
Gene | Forward primer HPF | Reverse primer (or fluorescent mark reverse primer) HPR |
23S rRNA gene | 5’-CTGCATGAATGGCGTAACGAG-3’ | 5’-CAAGGATGACTCCATAAGAGC -3’ |
Embodiment 1: the preparation of nucleotide gene chip:
1) processing of chip carrier: slide chromic acid lotion soaked overnight, washing is spent the night in 25% ammoniacal liquor, washing.Slide immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates PH to 4.5 with Glacial acetic acid, and 95% ethanol ultrasonic cleaning was dried 4 hours for 160 ℃, and 10% glutaraldehyde is processed into aldehyde radicalization.
2) primer and probe is synthetic: the gene polymorphism sites that the present invention is directed to 23S rRNA A2142G, A2142C, A2142T, A2143G, A2143C, A2143T, C2182T, C2182A and the C2182G of Hp has designed one group of probe (seeing Table 1).2 primers (seeing Table 2) have been synthesized at the 23S rRNA gene design of Hp.A Cyp-3 fluorescent mark is arranged in every primer.All Oligonucleolide primers or probe all adopt standard phosphoramidite chemical process synthetic on ABi 8089DNA automatic DNA synthesizer DNA, the inferior phosphinylidyne ammonification of the synthetic employing standard method of oligonucleotide, stationary probe is held with amido modified 3 ', link to each other with spacerarm (spacer, polyoxyethylene glycol phosphoramidite reagent) between amino and the probe sequence.Fluorescent dye primer directly carries out fluorescent mark with standard phosphoramidite chemical synthesis with Cy3 phosphoramidite reagent at 5 ' end of general primer sequence.Synthetic back was cut the OPC column purification 15 hours with 55 ℃ of deprotections of strong aqua.The quantitative final vacuum of ultraviolet is dry to be concentrated, and-80 ℃ of preservations are standby.
3) gene chip preparation and aftertreatment: with probe to final concentration is 100 μ mol/L; Use 750mmol/LNaCl, the 75mmol/L sodium acetate, 5% glycerine, 1% ficoll and 0.1%SDS synthesize sampling liquid; Aforementioned both dilutions in 1: 1, and get 10 μ L to 96 orifice plates, to put to the aldehyde radical slide with the chip point sample instrument, every some volume is about 0.5nL, and the dessert spacing is 500 μ m, and spot diameter is about 200 μ m, and every probe repeats a little 2 times.Oligonucleotide probe arrangement such as Fig. 1 on slide.After point sample finished, room temperature was placed 24 hours.
Embodiment 2: the preparation of nucleotide gene chip:
1) processing of chip carrier: slide chromic acid lotion soaked overnight, washing, spend the night in 24% ammoniacal liquor, washing, slide immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates PH to 4.0,95% ethanol ultrasonic cleaning with Glacial acetic acid, dried 3 hours for 150 ℃, 9% glutaraldehyde is processed into aldehyde radicalization;
2) primer and probe is synthetic: the gene polymorphism sites at 23S rRNA A2142G, A2142C, A2142T, A2143G, A2143C, A2143T, C2182T, C2182A and the C2182G of Hp has designed one group of probe (seeing Table 1).2 primers (seeing Table 2) have been synthesized at the 23S rRNA gene design of Hp.A Cyp-3 fluorescent mark is arranged in every primer.All Oligonucleolide primers or probe all adopt standard phosphoramidite chemical process synthetic on ABi 8089DNA automatic DNA synthesizer DNA, the inferior phosphinylidyne ammonification of the synthetic employing standard method of oligonucleotide, stationary probe is held with amido modified 3 ', link to each other with spacerarm (spacer, polyoxyethylene glycol phosphoramidite reagent) between amino and the probe sequence.Fluorescent dye primer directly carries out fluorescent mark with standard phosphoramidite chemical synthesis with Cy3 phosphoramidite reagent at 5 ' end of general primer sequence.Cut 13 hours with 50 ℃ of deprotections of strong aqua synthetic back, the OPC column purification, and the quantitative final vacuum of ultraviolet is dry to be concentrated, and-20 ℃ of preservations are standby;
3) gene chip preparation and aftertreatment: with probe to final concentration is 100 μ mol/L; Use 750mmol/LNaCl, the 75mmol/L sodium acetate, 5% glycerine, 1% ficoll and 0.1%SDS synthesize sampling liquid; Aforementioned both dilutions in 1: 1, and get 10 μ L to 96 orifice plates, to put to the aldehyde radical slide with the chip point sample instrument, every some volume is about 0.5nL, and the dessert spacing is 500 μ m, and spot diameter is about 200 μ m, and every probe repeats a little 2 times.Oligonucleotide probe arrangement such as Fig. 1 on slide.After point sample finished, room temperature was placed 24 hours.The preparation of embodiment 3 nucleotide gene chips:
1) processing of chip carrier: slide chromic acid lotion soaked overnight, washing, spend the night in 26% ammoniacal liquor, washing, slide immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates PH to 4.5,95% ethanol ultrasonic cleaning with Glacial acetic acid, dried 5 hours for 160 ℃, 11% glutaraldehyde is processed into aldehyde radicalization;
2) primer and probe is synthetic: the gene polymorphism sites at 23S rRNA A2142G, A2142C, A2142T, A2143G, A2143C, A2143T, C2182T, C2182A and the C2182G of Hp has designed one group of probe (seeing Table 1).2 primers (seeing Table 2) have been synthesized at the 23S rRNA gene design of Hp.A Cyp-3 fluorescent mark is arranged in every primer.All Oligonucleolide primers or probe all adopt standard phosphoramidite chemical process synthetic on ABi 8089DNA automatic DNA synthesizer DNA, the inferior phosphinylidyne ammonification of the synthetic employing standard method of oligonucleotide, stationary probe is held with amido modified 3 ', link to each other with spacerarm (spacer, polyoxyethylene glycol phosphoramidite reagent) between amino and the probe sequence.Fluorescent dye primer directly carries out fluorescent mark with standard phosphoramidite chemical synthesis with Cy3 phosphoramidite reagent at 5 ' end of general primer sequence.Cut 17 hours with 60 ℃ of deprotections of strong aqua synthetic back, the OPC column purification, and the quantitative final vacuum of ultraviolet is dry to be concentrated, and-80 ℃ of preservations are standby;
3) gene chip preparation and aftertreatment: with probe to final concentration is 100 μ mol/L; Use 750mmol/LNaCl, the 75mmol/L sodium acetate, 5% glycerine, 1% ficoll and 0.1%SDS synthesize sampling liquid; Aforementioned both dilutions in 1: 1, and get 10 μ L to 96 orifice plates, to put to the aldehyde radical slide with the chip point sample instrument, every some volume is about 0.5nL, and the dessert spacing is 500 μ m, and spot diameter is about 200 μ m, and every probe repeats a little 2 times.Oligonucleotide probe arrangement such as Fig. 1 on slide.After point sample finished, room temperature was placed 24 hours.
The using method of Hp clarithromycin drug-resistant gene detection chip is:
1) slide that is loaded with oligonucleotide probe uses preceding 0.2%SDS and clear water respectively to wash twice, and is stand-by after the dry air, finishes probe covalency immobilization in surface of glass slide.
2) adopt the method for asymmetric PCR amplification for the target fragment that produces strand, the ratio optimization of forward primer HPF (5 '-CTGCATGAATGGCGTAACGAG-3 ') and fluorescent mark reverse primer HPR (5 '-CAAGGATGACTCCATAAGAGC-3 ') is 1: 10.Contain 1.5mM MgCl in the 20 μ L reaction systems
2, each 200 μ mol/L of dNTP, forward primer 0.1 μ mol, reverse primer 1 μ mol, Hp DNA50ng, 1 * PCR damping fluid and 1U Taq enzyme.The pcr amplification condition is: 94 ℃ of pre-sex change 5 minutes; 94 ℃ of sex change 30 seconds, anneal 55 ℃ 30 seconds, extend 72 ℃ 30 seconds, totally 30 circulations; Extend at last 72 ℃ 5 minutes.The PCR product is with 2% agarose gel electrophoresis analysis.
3) the asymmetric PCR product of Cy3-mark and hybridization solution (750mmol/L NaCl, 75mmol/L sodium acetate, 0.1%SDS, 1ug/ml salmon sperm DNA, 2.5% formyl ammonium) mixed by 2: 8,10 μ L mixed solutions were transferred to the hybridization zone of chip.Chip places hybridizing box 42 ℃ of water-bath insulations 1 hour.Chip is taken out in the hybridization back, and (150mmol/L NaCl, 15mmol/L sodium acetate 0.2%SDS), respectively washed 1 minute among washing lotion B (30mmol/L NaCl, 3mmol/L sodium acetate) and the washing lotion C (15mmol/L NaCl, 1.5mmol/L sodium acetate) at washing lotion A successively.Wait to do the back chip and use laser confocal scanning instrument GenePix 4000 (Axon Instrument) at excitation wavelength 532nm, emission wavelength 570nm (Cy3) scanning produces and analysis precision is 16 TIFF images of 10 μ m.
Hp clarithromycin drug-resistant gene detection chip use-case:
The stomach mucous membrane biopsy specimen is taken from the gastroscopic patient of Gastroenterology dept. of Zhejiang University Medical College The First Affiliated Hospital row, and totally 54 routine patients are selected, the male sex's 30 examples wherein, women's 24 examples, 44.3 years old mean age (25-86 year).All selected patient's pathology of gastric mucosa and PCR check all positive.Stomach hole and body of stomach are respectively got 2 mucosal tissues, and a copy of it places 10% formalin fixed, are used for pathology HE dyeing, and another part is used to extract DNA.The patient accepts the stomach mucous membrane biopsy and all signs Informed Consent Form.Extracting DNA routinely detects with aforesaid method.The result is: turn out to be Hp male 54 routine samples through pathology and PCR, results of hybridization shows that 2142 sites are wild-type (54/54); The A2143G mutation rate is 11.11% (6/54), does not find the sudden change of A2143C and A2143T as yet; The C2182T mutation rate is 12.96% (7/54), does not find the sudden change of C2182A and C2182G as yet, and all the other are wild-type.
Sequence verification:
CEQ is adopted in order-checking
TMTDCS test kit (BECKMAN COULTER
TM, USA), at CE
TM2000XL sequenator (BECKMAN COULTER
TM, carry out on USA).Oligonucleotide microarray technique is in full accord with order-checking detection Hp 23S rRNA gene pleiomorphism result as a result.
Claims (6)
2, a kind of helicobacter pylori clarithromycin drug-resistant gene detection chip according to claim 1, it is characterized in that: said carrier is a slide.
3, a kind of preparation method of helicobacter pylori clarithromycin drug-resistant gene detection chip as claimed in claim 1 is characterized in that: method steps is:
1) processing of chip carrier: wave plate chromic acid lotion soaked overnight, washing, spend the night in 24~26% ammoniacal liquor, washing, slide immerses in 95% ethanolic soln of aminopropyl trimethoxysilane, regulates PH to 4~4.5,95% ethanol ultrasonic cleaning with Glacial acetic acid, dried 3~5 hours for 150~160 ℃, 5~10% glutaraldehyde are processed into aldehyde radicalization;
2) primer and probe is synthetic: at the 23S rRNA gene A 2142G of Hp, A2142C, A2142T, A2143G, A2143C, A2143T, C2182T, C2182A and the described probe of C2182G pleomorphism site design claim 1, its length range is 15-17 base, at the synthetic 1 pair of primer of the 23S rRNA gene design of Hp, 21 bases of length, downstream primer Cyp-3 fluorescent mark, synthetic back 50~60 ℃ of deprotections of strong aqua, cut 13~17 hours, the OPC column purification, the quantitative final vacuum of ultraviolet is dry to be concentrated, and-20~-80 ℃ of preservations are standby;
3) gene chip preparation and aftertreatment: with probe to final concentration is 100 μ mol/L, uses 750mmol/LNaCl, the 75mmol/L sodium acetate, and 5% glycerine, 1% ficoll and 0.1%SDS synthesize sampling liquid; Aforementioned both dilutions in 1: 1, and get 10 μ L to 96 orifice plates, put to the aldehyde radical slide with the chip point sample instrument, every some volume is 0.5nL, and the dessert spacing is 500 μ m, and spot diameter is 200 μ m, every probe repeats a little 2 times, and after point sample finished, room temperature was placed and got final product in 24 hours.
4, the preparation method of a kind of helicobacter pylori clarithromycin drug-resistant gene detection chip according to claim 3 is characterized in that: the described probe of said claim 1 is:
5, the preparation method of a kind of helicobacter pylori clarithromycin drug-resistant gene detection chip according to claim 3, it is characterized in that: said primer is the 23S rRNA gene expression characteristics design according to Hp, be used for the amplification of said gene, said primer is: forward primer HPF5 '-CTGCATGAATGGCGTAACGAG-3 ' and reverse primer HPR 5 '-CAAGGATGACTCCATAAGAGC-3 '.
6, a kind of purposes of helicobacter pylori clarithromycin drug-resistant gene detection chip as claimed in claim 1 is characterized in that being used to detect the gene pleiomorphism in drug-fast 23S rRNA 2142,2143 of Hp clarithromycin and 2182 sites.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 200410073454 CN1295348C (en) | 2004-12-17 | 2004-12-17 | Helicobacter pylori clarithromycin drug-resistant gene polymorphism detecting chip and its preparation method and uses |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN 200410073454 CN1295348C (en) | 2004-12-17 | 2004-12-17 | Helicobacter pylori clarithromycin drug-resistant gene polymorphism detecting chip and its preparation method and uses |
Publications (2)
Publication Number | Publication Date |
---|---|
CN1730667A CN1730667A (en) | 2006-02-08 |
CN1295348C true CN1295348C (en) | 2007-01-17 |
Family
ID=35963088
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN 200410073454 Expired - Fee Related CN1295348C (en) | 2004-12-17 | 2004-12-17 | Helicobacter pylori clarithromycin drug-resistant gene polymorphism detecting chip and its preparation method and uses |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN1295348C (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101899512A (en) * | 2010-07-09 | 2010-12-01 | 北京九州泰康生物科技有限责任公司 | Non-enzymatic amplification detection gene chip based on rRNA genospecies specific sequence, detection system and detection method |
CN106636447A (en) * | 2017-03-03 | 2017-05-10 | 踏石生物科技(苏州)有限公司 | Helicobacter pylori, drug resistance gene mutation detection kit and detection method |
-
2004
- 2004-12-17 CN CN 200410073454 patent/CN1295348C/en not_active Expired - Fee Related
Also Published As
Publication number | Publication date |
---|---|
CN1730667A (en) | 2006-02-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10407718B2 (en) | Optimization of gene expression analysis using immobilized capture probes | |
US6458584B1 (en) | Customized oligonucleotide microchips that convert multiple genetic information to simple patterns, are portable and reusable | |
EP1921162B1 (en) | Probe, probe set, probe-immobilized carrier, and genetic testing method | |
JP4766750B2 (en) | Detection of nucleic acid sequence differences using a ligase detection reaction with a positionable array | |
KR101038519B1 (en) | Human infectious diseases-related pathogen differential diagnosis and simultaneous antibiotics resistance analysis, multiplex kit and chip comprising same | |
WO2017028760A1 (en) | Single molecule sequencing method, device, system, and application | |
WO1998028444A2 (en) | Customized oligonucleotide microchips as multiple biosensors | |
JP2003530116A (en) | Identification of biological (micro) organisms by detection of homologous nucleotide sequences on arrays | |
JP2009171969A (en) | Method for assaying microarray hybridization | |
WO2007056680A2 (en) | Methods and arrays for identifying human microflora | |
KR101953176B1 (en) | Methods for pcr and hla typing using raw blood | |
CN1195070C (en) | Detection type gene chip for detecting various infectious desease and use thereof | |
CA2372909A1 (en) | Single nucleotide polymorphic discrimination by electronic dot blot assay on semiconductor microchips | |
CN1814794A (en) | Leptin and leptin receptor gene polymorphism detecting chip, and its preparing method and use | |
CN1295348C (en) | Helicobacter pylori clarithromycin drug-resistant gene polymorphism detecting chip and its preparation method and uses | |
JP5681217B2 (en) | Nucleic acid detection method through promotion of formation of branched DNA complex | |
EP1164201A1 (en) | Reverse detection for identification and/or quantification of nucleotide target sequences on biochips | |
JP4301941B2 (en) | Nucleic acid probe immobilization substrate | |
CN1268769C (en) | Polymorphism detection chip for gene of enzyme relevant to ethanol metabolism, preparation method of usage | |
JP2007534331A (en) | K-ras oligonucleotide microarray and method for detecting K-ras mutations using the same | |
JP2005505287A (en) | β2 adrenergic polymorphism detection | |
WO2020161651A1 (en) | Early detection of multiple resistances to anti-bacterial treatment | |
CN1746318A (en) | Detection of DNA binding protein with exonuclease protective DNA probe and hybrid DNA microarray chip | |
JP2001327290A (en) | Method for detecting and assaying methanogen | |
CN115427567A (en) | Method for determining single-base mutation of erm (41) gene of acid-fast bacterium belonging to Mycobacterium abscessus complex, and primer set and probe used in the method |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
C17 | Cessation of patent right | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20070117 Termination date: 20121217 |