CN1245521C - Synchronous PCR amplification and hybridization process - Google Patents
Synchronous PCR amplification and hybridization process Download PDFInfo
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- CN1245521C CN1245521C CN 01123507 CN01123507A CN1245521C CN 1245521 C CN1245521 C CN 1245521C CN 01123507 CN01123507 CN 01123507 CN 01123507 A CN01123507 A CN 01123507A CN 1245521 C CN1245521 C CN 1245521C
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Abstract
The present invention discloses a method for combining the amplification of PCR to nucleic acid (DNA or RNA) and the detection of amplification products into one step. The method comprises three steps of solidifying the PCR reaction technology of primers, washing plates and measuring color development (or fluorescence) terminals, and greatly decreases reagent consumption and detection time. The whole detection can be finished within three to four hours; the sensitivity also reaches 1000 copy/ml; expensive devices are not needed, and thus, the use cost is greatly reduced. The present invention brings revolutionary change to the detection in the aspects of clinic detection, blood safety, drug resistance, single nucleotide polymorphism (SNP), etc.
Description
The present invention relates to a kind of round pcr, relate in particular to and a kind ofly can detect the method that two steps be combined into a step to the amplification of PCR and product.
Employed similar Taqman on Chinese market at present
RTechnology is a kind of pcr amplification with detected for two steps and be combined into one method, this method operation is easier, but its sensitivity is 104copy/ml only, the PCR instrument that again must use can do " instant method ", to spend 600,000 Renminbi to buy this kind equipment approximately, its supporting easy consumption article are also relatively more expensive, do not have autonomous intellecture property again.Can be more and more widely for round pcr is used at coml, develop a kind of more economical, primary demand that effective means will adapt to China's numerous people, and covering spreads to all parts of the country, reach the target of cheap charge, good service.
The purpose of this invention is to provide and a kind ofly can detect the method that two steps be combined into a step to pcr amplification and product.Because the present invention has that cost is lower, highly sensitive, high specificity, characteristics such as easy and simple to handle, will bring revolutionary variation to the mensuration of clinical detection, blood use safety, resistance and single nucleotide polymorphism aspects such as (SNP).
The object of the present invention is achieved like this:
A kind of pcr amplification, hybridize same footwork, it is selected from one of following method: universal capture assay method or solid phase primer assay method.
Wherein the universal capture assay method comprises:
A) with universal capture probe bag by to microwell plate;
B) in above-mentioned batten, every hole adds 2 * PCR main reaction liquid of 1 volume, and the sample liquid that adds 1 volume is again carried out conventional PCR reaction;
C) after PCR finishes, add the sex change liquid 5-10 μ l that contains linkers;
D) continue to add hybridization solution 100 μ l in same hole, 37 ℃ are incubated 1 hour;
E) wash plate, enzyme connection, wash plate, colour developing, mensuration for the second time;
F) interpretation.
Wherein the molecule of joint liquid described in the step C is that joint liquid molecule is an a) hepatitis B virus joint liquid molecule; Or b) hepatitis C virus joint liquid molecule; Or c) gamma-globulin joint liquid molecule; Or d) HIV (human immunodeficiency virus) joint liquid molecule; Or e) tubercule bacillus joint liquid molecule; Or f) chlamydia trachomatis joint liquid molecule; Or other pathogenic agent joint liquid molecule, the structure of its joint liquid molecule is the oligonucleotide of a strand, respectively by the general part of growing 25 bases, long 5 to 4 millimicrons " space " part and the specificity of about 22-26 base partly constitute, its specificity partly for can only with have B-mode liver plays hybridization for the virus PCR product hepatitis b virus specificity order, or with have the hepatitis C virus specific order that hepatitis C virus PCR product plays hybridization, or with have the gamma-globulin specificity order that gamma-globulin PCR product plays hybridization, or with have the HIV (human immunodeficiency virus) specificity order that HIV (human immunodeficiency virus) PCR product plays hybridization, or with have the tubercule bacillus specificity order that tubercule bacillus PCR product plays hybridization, with have chlamydia trachomatis PCR product rise hybridization chlamydia trachomatis specific order or with any specificity to be measured order;
The structure of above-mentioned linkers is the oligonucleotide of a strand, and its length by 392 type dna synthesizers of PE company, adopts solid phase tricresyl phosphite fat method to carry out DNA and synthesizes about 50 to 60 base pair.It comprises three integral parts:
(1). common segment: long altogether 25 bases of common segment, with the general probe complementation that is fixed on magnetic or the micro plate, can hybridize with general probe soon just;
(2). " space ": between common segment and special part, give hybridization chain of two molecule clevis core types in order to reserve " space ", our " space " length is 5 to 4nm;
(2). the specificity part: the specificity part is " space " and then, is about 22-26 base.Its length is the G/C% by itself, whether Tm cooperates with common segment and to be determined, be decided by also whether it self has secondary level structure, its order is to select the product at PCR for use, order in the middle of a pair of primer, thereby can only be with the hybridization of the product of PCR and reactionless with biotin labeled primer, so guaranteed the specificity of this part hybridization;
The molecular structure of joint liquid:
1) long 25 base pairs of common segment wherein, its order or with (dA) of artificial arrangement
25Or 5 '-ACg, Acg, gAA, cgA, AAc ggA, Acg, Acgg-3 ' or from pBlue KS-plasmid sequence, choose one section order.PBlue KS
-Plasmid sequence is published in: Short, J.et al (1988) NucleicAcids Research 16 (15) 7583
Concrete order is
3’-CCg，AgA，TAg，ggT，TgA，TTg，TTCC-5’
2) space is made up of two space molecules (spacer-phosphoramidite), and each space is that of 18 carbon atoms compositions is long-armed, can join on dna synthesizer in the linkers;
Concrete structure is
(be published in: Nelson, P, et, (1989) Nucleic Acids Res.17：7187)
3) specificity part: all be selected from the complete sequence of each pathogen nucleic acid to be measured, HBV, HCV, HIV, TB, CT, β-globin etc. can find out in " gene library ".Their concrete structure of example is:
(1)HBV 5’-TAAAGAGAGGTGCGCCCCGTGGTC-3’
(2)HCV 5’-AGCCATAGTGGTCTGCGAAC-3’
(3)HIV 5’-GTTAAAAGAGACCATCAATGAGGAA-3’
(4)TB 5’-GCGCTTTAGCGGTGTGGGATGA-3’
(5)CT 5’-GATGAAAGACAGGAAATACGCAGA-3’
(6) gamma-globulin 5 '-ACTTTCTTGCCATGAGCCTTCACCT-3 '
Wherein the described universal capture probe of step a is its common segment.
The PCR main reaction liquid of step b) wherein: Repone K 200mM, Tutofusin tris 40mM, Taq archaeal dna polymerase 0.5/10ul, uridylic glycosylase (UNG) 1U/10ul, dNTP0.5mM, primer 40 μ g/ml;
The sample liquid preparation process is as follows:
Get 50 μ l lysates and add 50 μ l test serums, 37 ℃ are incubated 1 hour, dilute 5 times with sample diluting liquid (main component divalence Mg ion and milt dna vector molecule) behind adding 100ul sample neutralizer (main component Tris) mixing.
Step C wherein) sex change liquid is 0.5M NaOH solution, and the hybridization solution of steps d is the 0.9MNaCL citrate buffer solution.
Step C wherein) measuring available visible light, fluorescence or chemoluminescence in detects.
Wherein solid phase primer assay method comprises:
A) be specific to the dna primer (oligonucleotide, length are 20 to 30 Nucleotide) of virosome to be measured at heat-resisting and transparent reacting hole pan coating;
B) in above-mentioned reacting hole, add the 2 * PCR main reaction liquid of 1 volume (10ul-20ul) and the sample liquid of 1 volume, carry out the reaction of solid phase primer PCR;
C) after PCR finishes, wash plate, enzyme connection, wash plate for the second time, chromogenic assay, measuring method can be with visible, fluorescence or chemiluminescence detection;
D) interpretation.
Wherein the PCR main reaction liquid of step b) is:
KCL 200mM, Tris 40mM, Taq DNA polymeric acid 0.5 unit/10ulHK-UNG 0.1 unit/10ul,
DUTP 0.5mM, biotin labeled primer 400ng/10ul;
Being prepared as follows of sample liquid:
Get the 50ul lysate, add the 50ul test serum, 37 ℃ are incubated 1 hour, dilute 5 times with sample diluting liquid behind adding 100ul sample neutralizer (main component Tris) mixing.
Pcr amplification/hybridization can select for use one of following two kinds of modes to realize with footwork:
One. general trapping method
A) on heat-resisting (can be heated to 120 ℃ of not modification) and transparent reacting hole surface (8 * 12,96 hole micro plates, raw material can with nano pipe/polyhenylethylene or polycarbonate etc.) bag by the universal capture probe.
B) main reaction liquid and the 1 volume sample liquid that adds 1 volume (10ul to 20ul), 2 * PCR in above-mentioned reacting hole carries out conventional PCR reaction;
C) after PCR reaction was finished, adding the sex change liquid 5-10ul that contains linkers, to make the sex change of PCR product double chain DNA molecule simultaneously be single chain molecule,
D) continue to add hybridization solution 100ul in same hole, 27 ℃ are incubated 1 hour,
E) wash plate, enzyme connection, wash plate, colour developing, mensuration for the second time.Measure employed method except that visible light, also applicable to fluorescence or chemiluminescence detection.
At above-mentioned steps c) center tap liquid molecule is a) hepatitis B virus joint liquid molecule; Or b) hepatitis C virus joint liquid molecule; Or c) gamma-globulin joint liquid molecule; Or d) HIV (human immunodeficiency virus) joint liquid molecule; Or e) tubercule bacillus joint liquid molecule; Or f) chlamydia trachomatis joint liquid molecule; Or other pathogenic agent joint liquid molecule, the structure of its joint liquid molecule is the oligonucleotide of a strand, respectively by the general part of growing 25 bases, long 5 to 4 millimicrons " space " part and the specificity of about 22-26 base partly constitute, its specificity partly for can only with have B-mode liver plays hybridization for the virus PCR product hepatitis b virus specificity order, or with have the hepatitis C virus specific order that hepatitis C virus PCR product plays hybridization, or with have the gamma-globulin specificity order that gamma-globulin PCR product plays hybridization, or with have the HIV (human immunodeficiency virus) specificity order that HIV (human immunodeficiency virus) PCR product plays hybridization, or with have the tubercule bacillus specificity order that tubercule bacillus PCR product plays hybridization, with have chlamydia trachomatis PCR product rise hybridization chlamydia trachomatis specific order or with any specificity to be measured order;
The structure of above-mentioned linkers is the oligonucleotide of a strand, and its length by 392 type dna synthesizers of PE company, adopts solid phase tricresyl phosphite fat method to carry out DNA and synthesizes about 50 to 60 base pair.It comprises three integral parts:
1. common segment: long altogether 25 bases of common segment, just and be fixed on general probe complementation on magnetic or the micro plate, can hybridize with general probe soon.
2. " space ": between common segment and special part, give hybridization chain of two molecule clevis core types in order to reserve " space ", our " space " length is 5 to 4nm.
3. specificity part: the specificity part is " space " and then, is about 22-26 base.Its length is the G/C% by itself, whether Tm cooperates with common segment and to be determined, be decided by also whether it self has secondary level structure, its order is to select the product at PCR for use, order in the middle of a pair of primer, thereby can only be with the hybridization of the product of PCR and reactionless with biotin labeled primer, so guaranteed the specificity of this part hybridization.
The molecular structure of joint liquid:
1) long 25 base pairs of common segment wherein, its order or with (dA) of artificial arrangement
25Or 5 '-ACg, Acg, gAA, cgA, AAe ggA, Acg, Acgg-3 ' or from pBlue KS-plasmid sequence, choose one section order.PBlue KS-plasmid sequence is published in: Short, J.et al (1988) NueleicAcids Research 16 (15) 7583
Concrete order is
3’CCg，AgA，TAg，ggT，TgA，TTg，TTCC-5’
2) space is made up of two space molecules (spacer-phosphoramidite), and each space is that of 18 carbon atoms compositions is long-armed, can join in the linkers on dna synthesizer.
Concrete structure is
(be published in: Nelson, P, et, (1989) Nucleic Acids Res.17：7187)
3) specificity part: all be selected from the complete sequence of each pathogen nucleic acid to be measured, HBV, HCV, HIV, TB, CT, β-globin etc. can find out in " gene library ".Their concrete structure of example is:
(1)HBV 5’-TAAAGAGAGGTGCGCCCCGTGGTC-3’
(2)HCV 5’-AGCCATAGTGGTCTGCGAAC-3’
(3)HIV 5’-GTTAAAAGAGACCATCAATGAGGAA-3’
(4)TB 5’-GCGCTTTAGCGGTGTGGGATGA-3’
(5)CT 5’-GATGAAAGACAGGAAATACGCAGA-3’
(6) gamma-globulin 5 '-ACTTTCTTGCCATGAGCCTTCACCT-3 '
The universal capture hole is the polystyrene micropore plate with active group, and its active group connects down solid with the complementary oligonucleotide covalent linkage ground of the common segment (being the universal capture probe) of joint liquid molecule;
Hybridization solution is a 0.9M NaCl citrate buffer solution, the pH value that makes PCR product after the sex change or linkers liquid add back hybridization in the hand-hole maintains in the suitableeest scope, its ionic concn and keep temperature to cooperate with the Tm (melting point temperature) of two kinds of hybridization chains (being general hybridization chain and specific hybrid chain) of formation just and hybridization efficiency is maintained on the stable level;
Sex change liquid is 0.2M NaOH solution.
Universal capture probe in the step a) is the common segment of linkers.
Solid phase primer method
A) the heat-resisting and transparent specific dna primer of reacting hole pan coating (oligonucleotide, length are 20 to 30 Nucleotide).Utilize this immobilised primer to carry out pcr amplification reaction, a chain in the PCR product is directly fixed on the reaction hole wall.This chain just can be used as the template that is amplified another complementary strand.Under with the condition after the sex change of PCR product, can be used as the capture probe that is amplified another complementary strand again,
Example hepatitis B downstream primer 5 '-ccaataccacatcatccac-3 '
B) in above-mentioned reacting hole, add the 2 * PCR main reaction liquid of 1 volume (10ul-20ul) and the sample liquid of 1 volume, carry out solid phase primer PCR reaction (primer of another band vitamin H is present in the PCR main reaction liquid),
C) after PCR finishes, wash plate, enzyme connection, wash plate, chromogenic assay is described identical with method one, but mensuration also is applicable to fluorescence or chemiluminescence detection with method except that visible light.
The invention will be further described below in conjunction with embodiment.
Embodiment 1 general trapping method is to HBV PCR qualitative test
1) bag is by on universal capture probe to 8 * 12 microwell plates
2) in above-mentioned plate, every hole adds 10ul HBV2 * PCR main reaction liquid:
KCl200mM, Tris 40mM, Taq archaeal dna polymerase 0.5 unit/10ul, HK-UNG 0.1 unit/10ul, dUTP 0.5mM, every kind of 40ug/ml of HBV primer.
Add the 10ul sample liquid again, the sample liquid preparation process is as follows:
Get 50ul lysate (major ingredient NaOH) and add the 50ul test serum, 37 ℃ are incubated 1 hour, dilute 5 times with sample diluting liquid (major ingredient divalence Mg ion and milt dna vector molecule) after adding 100ul sample neutralizer (major ingredient Tris) mixing, be the sample liquid that preparation is finished.
Being prepared as follows of lysate wherein: take by weighing a certain amount of sodium hydroxide (NaOH) and sodium azide (NaNa), be dissolved in ultrafiltration water, the whole solubility of sodium hydroxide is 0.5Mol/L, and the whole solubility of sodium azide is 0.05%.
Being prepared as follows of the prescription of sample neutralizer: take by weighing a certain amount of Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tirs.HCL) and sodium azide (NaN
3), being dissolved in ultrafiltration water, the whole solubility of Tri(Hydroxymethyl) Amino Methane Hydrochloride is 0.5Mol/L, the whole solubility of sodium azide is 0.05%.
Being prepared as follows of the prescription of sample diluting liquid: take by weighing a certain amount of Tri(Hydroxymethyl) Amino Methane Hydrochloride (Tirs.HCL) and sodium azide (NaNs), dissolving back amount is gone into a certain amount of DNA, magnesium chloride (MgCL
2), the whole solubility 10mMol/L of Tri(Hydroxymethyl) Amino Methane Hydrochloride, the whole solubility of sodium azide is 0.05%.The whole solubility of DNA is 2 mcg/ml, and the whole solubility of magnesium chloride is 2.5mMol/L.
3) add the HBV joint liquid 5ul (including HBV linkers 20ng) that dissolves in the sex change liquid (composition 0.5M NaOH solution) after PCR finishes, add 37 ℃ of insulations of hybridization solution 100ul (0.9M MaCL citrate buffer solution) 1 hour again.
4) wash plate, enzyme connection, wash plate and colour developing for the second time, measure, measuring method is also applicable fluorescence or chemoluminescence method except that visible light.
5) interpretation: deduct background from the testing sample optical density readings, differentiate detected result.
In the step (4)
Wash plate: with washings reaction product in the hole is washed 5 times, each consumption 300 microlitres discard washings then;
The enzyme connection: enzyme connection liquid 100 microlitres of getting 50 millimicro grams per milliliters join in the above-mentioned hole, and integrated enzyme reaction was carried out in 37 ℃ of insulations in 15 minutes;
For the second time wash plate: with washings reaction product in the hole is washed 5 times, each consumption 300 microlitres discard washings then;
Colour developing: add colour developing liquid 100 microlitres in the hole, 25 ℃ keep adding after 10 minutes stop bath 100 microlitres;
Measure: above-mentioned sample moves to and measure the optical density(OD) number on the microplate reader of 450 millimicrons of wavelength.
Differentiate: record the optical density(OD) number with internal reference thing optical density(OD) is counted reference, the differentiation detected result.
Wherein washings is a 0.15MNaCL solution, and its salt concn has played the provide protection nonspecific absorption of flush away effectively to safeguarding the hybridization chain, has guaranteed the specificity of hybridization;
Enzyme connection liquid is the cross-linking agent of peroxidase and avidin;
Colour developing liquid is 1: 1 mixed solution of weight ratio of tetramethyl benzidine and hydrogen peroxide.
Stop bath is 5% sulphuric acid soln.
Reagent described in the present invention, instrument provide by domestic production producer, give birth to worker's thing engineering corporation, Shanghai Hao Yuan biotechnology art company limited and chemical industry chemical reagent shop as Shanghai.
The mensuration of embodiment 2 solid phase primer pattern HBV YMDD variants
1) wraps by PCR downstream primer 5 '-ccaataccacatcatccac-3 ' to 8 * 12 microwell plates.
2) in above-mentioned batten, every hole adds 10ul YMDD2 * PCR main reaction liquid:
(KCL200mM, Tris 40mM, TaqDNA polysaccharase 0.5 unit/10ul, HK-UNG0.1 unit/10ul, dUTP 0.5mM, the upstream primer 400ng/10ul of biotin labeled YMDD)
17 bases of the upstream primer of YMDD: 5 '-CCTCAGTCCGTTTCTCA-3 '
Add the 10ul sample liquid again.
The sample liquid preparation process is as follows:
Get 50ul lysate (with embodiment 1) and add the 50ul test serum, 37 ℃ are incubated 1 hour, with 5 times of sample diluting liquid (with embodiment 1) dilutions, are the sample liquid that preparation is finished behind adding 100ul sample neutralizer (with the embodiment 1) mixing.
Carry out conventional PCR after the mixing.
3) can directly wash plate after PCR finishes, carry out enzyme connection again, wash plate and colour developing for the second time, mensuration, it is described identical with embodiment 1 that each reaches solution formula step by step suddenly, but measuring method applicable fluorescence or chemoluminescence except that visible light.
4) interpretation: deduct background from the testing sample optical density readings, but when this numerical value during greater than 2.5 times of backgrounds interpretation be HBV YMDD variant positive findings.
Now the foregoing description data are listed in the following table:
| Embodiment 1 | The universal capture pattern | Biological sample pathogenic agent PCR product | Optical density(OD) | Interpretation | |
| Background | Sample | ||||
| A450 | A450 | ||||
| Contrast | Two primers are all in PCR solution | The HBV wild-type | 0.063 | 0.143 | - |
| The YMDD variation | Two primers are all in PCR solution | HBV YMDD product | 0.097 | 0.457 | + there is the YMDD variant to exist |
| The YMDD variation | Two primers are all in PCR solution | HBV YMDD product | 0.119 | 1.275 | + there is the YMDD variant to exist |
| Embodiment 2 | Solid phase primer pattern | Biological sample pathogenic agent PCR product | Optical density(OD) | Interpretation | |
| Background | Sample | ||||
| A450 | A450 | ||||
| Contrast | 1 primer only in the PCR solution, another primer immobilization | The HBV wild-type | 0.073 | 0.078 | - |
| The YMDD variation | 1 primer only in the PCR solution, another primer immobilization | HBV YMDD product | 0.120 | 3.294 | + there is the YMDD variant to exist |
| The YMDD variation | 1 primer only in the PCR solution, another primer immobilization | HBV YMDD product | 0.105 | 2.438 | + there is the YMDD variant to exist |
Reference:
1) Cai Jianping, Yang Zhenhua
*The research of human genome single nucleotide polymorphism is to the influence of following laboratory medicine
*2000 the 4th phase page 3 in Clinical Laboratory information Leader.
2)Newton et.al.
*Analysis of any point mutation in DNA
*TheAmplification Refractroy Mutation Systen(ARMS)Nueleic AcidsRes.1989 17(7)2503-2516.
3)Allen et.al.
*Identofication and Characterization ofMutations in Hepatitis B Virus Resistant toLamivudine
*Hepatology 1998 27(6)1670-1677.
Claims (2)
- A pcr amplification, hybridize same footwork, it comprises:A) be specific to the dna primer of virosome to be measured at heat-resisting and transparent reacting hole pan coating, described primer is that length is the oligonucleotide of 20 to 30 Nucleotide;B) in above-mentioned reacting hole, add the 2 * PCR main reaction liquid of 1 volume and the sample liquid of 1 volume, carry out the reaction of solid phase primer PCR, wherein PCR main reaction liquid is: KCl 200mM, Tris 40mM, Taq archaeal dna polymerase 0.5 unit/10ul, HK-UNG 0.1 unit/10ul, dUTP 0.5mM and biotin labeled primer 400ng/10ul;C) after PCR finishes, wash plate, enzyme connection, wash plate for the second time, chromogenic assay, described measuring method be as seen, fluorescence or chemiluminescence detection;D) interpretation.
- 2. method according to claim 1, wherein 1 volume of step b) is 10ul-20ul.
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| CN 01123507 CN1245521C (en) | 2001-07-26 | 2001-07-26 | Synchronous PCR amplification and hybridization process |
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| CN 01123507 CN1245521C (en) | 2001-07-26 | 2001-07-26 | Synchronous PCR amplification and hybridization process |
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| CN1245521C true CN1245521C (en) | 2006-03-15 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7939837B2 (en) | 2003-10-09 | 2011-05-10 | Permlight Products, Inc. | LED luminaire |
| US8013335B2 (en) | 2002-10-18 | 2011-09-06 | Semiconductor Energy Laboratory Co., Ltd. | Semiconductor apparatus and fabrication method of the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4592060B2 (en) * | 2004-04-26 | 2010-12-01 | キヤノン株式会社 | PCR amplification reaction apparatus and PCR amplification reaction method using the apparatus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8013335B2 (en) | 2002-10-18 | 2011-09-06 | Semiconductor Energy Laboratory Co., Ltd. | Semiconductor apparatus and fabrication method of the same |
| US7939837B2 (en) | 2003-10-09 | 2011-05-10 | Permlight Products, Inc. | LED luminaire |
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