CN1730667A - 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
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- CN1730667A CN1730667A CN 200410073454 CN200410073454A CN1730667A CN 1730667 A CN1730667 A CN 1730667A CN 200410073454 CN200410073454 CN 200410073454 CN 200410073454 A CN200410073454 A CN 200410073454A CN 1730667 A CN1730667 A CN 1730667A
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Abstract
The invention discloses a Helicobacter pylori Clarithromycin resisting gene detection chip, the method for preparation and use, wherein the carrier is provided with a probe. The invention realizes the fast polymorphism, simple and high-flux detection to genes at 23S rRNA 2142, 2143 and 2182 sites of Helicobacter pylori, and the gastric mucosa can be detected directly without the need of bacteria culture.
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.Helicobacter 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.Mutat ion 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: 23S rRNA gene A 2142G, A2142C, A2142T, A2143G, A2143C, A2143T, C2182T, C2182A and C2182G pleomorphism site at Hp have designed one group of probe, 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 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 13～17 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/L NACl, the 75mmol/L sodium acetate, 5%glycerol, 1%Ficoll and 0.1%SDS synthesizes 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 Hp23S 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/L NACl, the 75mmol/L sodium acetate, 5%glycerol, 1%Ficoll and 0.1%SDS synthesizes 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/L NACl, the 75mmol/L sodium acetate, 5%glycerol, 1%Ficoll and 0.1%SDS synthesizes 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/L NACl, the 75mmol/L sodium acetate, 5%glycerol, 1%Ficoll and 0.1%SDS synthesizes 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 MgCl2 in the 20 μ L reaction systems, 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: pre-94 ℃ of 5min of sex change; 94 ℃ of 30sec of sex change, the 55 ℃ of 30sec that anneal extend 72 ℃ of 30sec, totally 30 circulations; Extend 72 ℃ of 5min at last.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 sodiumacetate, 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 CEQ
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 (7)
1, a kind of helicobacter pylori clarithromycin drug-resistant gene detection chip is characterized in that: be provided with following probe on carrier:
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 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 C2182G pleomorphism site have designed one group of probe, 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/L NACl, the 75mmol/L sodium acetate, and 5%glycerol, 1%Ficoll and 0.1%SDS synthesizes 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: said probe 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 respectively applied for the amplification of said gene, said primer is: forward primer HPF (5 '-CTGCATGAATGGCGTAACGAG-3 ') and reverse primer HPR (5 '-CAAGGATGACTCCATAAGAGC-3 ').
6, the preparation method of a kind of helicobacter pylori clarithromycin drug-resistant gene detection chip according to claim 3 is characterized in that: said signaling molecule mark is a fluorescence molecule.
7, a kind of purposes of helicobacter pylori clarithromycin drug-resistant gene detection chip is characterized in that being used to detect the gene pleiomorphism in the drug-fast 23S rRNA2142 of Hp clarithromycin, 2143 and 2182 sites.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
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