CN103820526A - Nucleic acid test strip method for detecting polymorphism of 118th codon of ERCC1 (excision repair cross complement) - Google Patents
Nucleic acid test strip method for detecting polymorphism of 118th codon of ERCC1 (excision repair cross complement) Download PDFInfo
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Abstract
The invention is a technology for performing allele specific amplification by using a labeled primer on the basis of PCR (polymerase chain reaction) amplification of a target template, and eventually detecting the 118th codon of an ERCC1 (excision repair cross complement) by a nucleic acid test strip method. The main detection principle is as follows: according to immunological specific binding of an antigen and a specific antibody thereof, a nucleic acid fragment is fixed on the nucleic acid detection test strip, and then according to color development of visual colorimetric latex particles on the test strip, a result is detected. In the technology for detecting polymorphic genotypes of the 118th codon of the ERCC1 by the PCR & AS-PCR nucleic acid test strip method, a specific PCR amplification primer for PCR amplification, an allele-specific primer with a biotin label at 5' end, and a specific probe with an FITC (fluorescein isothiocyanate) label at 3' end are mainly used.
Description
Technical field
The present invention relates to utilize nucleic acid test strip method to detect the method for Excision repair cross-completion 1 gene (excision repair cross-complementing gene 1, ERCC1) the 118th bit codon polymorphism.More particularly, be to utilize amplification technique (PCR & AS-PCR) and nucleic acid membrane chromatographic fast detecting method and Lateral Flow Strip thereof to carry out the rapid detection of ERCC1 the 118th bit codon loci polymorphism.
Background technology
The main mechanism of platinum medicine is that guanine, VITAMIN B4 and the cytosine(Cyt) on DNA is combined and formed platinum-DNA adduct, causes in the interchain linkage of DNA or chain crosslinkedly, causes DNA damage, thereby causes necrocytosis.The difference of DNA repair ability is by the individual difference that directly causes tumour cell to DNA relevant cell drug toxicity susceptibility.
eRCC1 gene the 118th codon Asn-Asn has polymorphism, nucleotide sequence sports AAT by AAC, this nonsense mutation can affect the expression level of ERCC1 albumen, and the sudden change of C → T has improved the expression level of ERCC1 mRNA, strengthens DNA repair ability and cause platinum class resistance.Studies show that, contain wild-type
eRCCthe human oophoroma cell line of 1 sequence is higher to platinum medicine sensitivity than anomaly cell strain.Meanwhile, the polymorphism of ERCC1 the 118th bit codon also may affect patient's survival rate and curative effect.A base on ERCC1 the 118th bit codon is obviously relevant to chemotherapy effect to the variation of T by C: have C/C genotype patient (54/109,50.4%) median survival time is 486 days (95%CI, 333-x), and anomaly C/T(45/109,42.1%) or T/T(8/109,7.5%) median survival time is 281 days (95%CI, 214-376).
The method detecting for ERCC1 the 118th bit codon loci polymorphism at present be basic mainly with round pcr greatly, is combined with the method such as fluorescence, mass spectroscopy, gene chip or direct Sequencing.Therefore and be not suitable for basic hospital but the most complicated operation of these methods, expensive, detection time is long, and to need large-scale plant and instrument be prerequisite.
Summary of the invention
In order to solve the most complicated operation of ERCC1 loci polymorphism detection method, expensive, detection time is long, and the problem of plant and instrument that need to be large-scale, the present invention utilizes amplification technique (PCR & AS-PCR) and nucleic acid membrane chromatographic fast detecting method and Lateral Flow Strip (patent No. CN1234567890) thereof to carry out the rapid detection of ERCC1 the 118th bit codon loci polymorphism.
The technical solution adopted for the present invention to solve the technical problems is:
1, inventive principle: the present invention is exactly on the basis of pcr amplification target template, utilize the primer of tape label to carry out allele specific amplification (Allele specific Polymerase Chain Reaction, AS-PCR), and finally utilize nucleic acid test strip to carry out the technology that ERCC1 the 118th bit codon detects.The cardinal principle of its detection is according to the immunology specific binding of antigen and its specific antibody, and nucleic acid fragment is fixed in detection of nucleic acids test strip, is then developed the color and detects result in test strip by visual colour generation latex particle.Its reaction principle and step are as Fig. 1:
Detect in ERCC1 the 118th bit codon polymorphism and mainly comprise the specific PCR amplimer for pcr amplification in PCR & AS-PCR nucleic acid test strip method,, a biotin labeled allele-specific primers of 5 ' end band, the specific probe of a 3 ' end band FITC mark.PCR & AS-PCR nucleic acid test strip method detects ERCC1 the 118th bit codon Genetic polymorphism type and mainly comprises following step:
A: sampling, examinee's peripheral vein anticoagulation (frozen) 200ul, the minim DNA rapid extraction test kit that application Yousida Biological Technology Co., Ltd., Hangzhou produces carries out nucleic acid extraction, in order to gene amplification.
B: first the target template that comprises ERCC1 the 118th bit codon pleomorphism site is increased by PCR, pcr amplification in this step is not distinguished the base type of ERCC1 the 118th bit codon polymorphism, just, in order to reach the object of enriched target sequence, the primer that simultaneously carries out pcr amplification does not carry out any mark;
It is characterized in that:
C: on the basis of step b, utilize AS-PCR to carry out specific amplification according to the base type of ERCC1 the 118th bit codon Genetic polymorphism type, in suitable Tm situation, only have when on amplified production in the allele-specific primers of 5 ' end mark antigen A and step 2, to detect containing ERCC1 the 118th bit codon polymorphic bands complete complementary time, this primer could extend under the effect of archaeal dna polymerase, and its extension products can carry out combination with the probe of 3 ' end mark antigen B in reaction solution.
In the 0.2ml of sterilizing PCR pipe, in the reaction system of 20ul, divide and add different pcr amplification primers and probe (table 1) for different genotype.The genotype that isocyatic 181PF is different with 181PR primer pair is carried out specific amplification, and the temperature that 181D5F-C/181D5G-A reacts during lower than pcr amplification due to the Tm of primer, so cannot effectively participate in the index amplification procedure of 181PF/181PR;
| Primer or probe title | Sequence information |
| 181PF (forward amplimer) | 5-AAGTGTTCAGGACCACAGG |
| 181PR (oppositely amplimer) | 5-GAGGCTTCTCATAGAACAG |
| 181D5F-C (probe 1) | 5-FITC-GTGCGCAACG |
| 181D5G-A (probe 2) | 5-FITC-GTGCGCAAAG |
| 181D3B (probe 3) | 5-AATTCCCAGGGC-BIOTIN |
table 1primer and probe that ERCC1 the 118th bit codon polymorphic detection is related
PCR & AS-PCR nucleic acid test strip method detects ERCC1 the 118th bit codon polymorphism reaction system:
Template: examinee's genomic dna
Micro-the rubbing of the each 0.2-2 of PCR primer: 181PF and 181PR
Micro-the rubbing of the each 0.1-1 of the primer of mark: 181D5F-C/181D5G-A
Specific probe: 181D3B 0.1-1 is micro-to rub
DNTP:0.2-0.5 milli rubs,
(NH
4)
2sO
410 millis rub
KCl 10 millis rub
Tris-HCl(PH 9.0) 10 milli rub
Triton-100 1.0%
MgCl
21.5-3 milli rubs
Taq archaeal dna polymerase: 1-5 unit
Cumulative volume is 20 microlitres
Of short duration centrifugal laggard performing PCR circulation.PCR loop parameter is 95 ℃ of 5min; 93-96 ℃ of 1min; 60-72 ℃ of 1min; 72 ℃ of 2min; 30-50 circulation; 93-96 ℃ of 30s; 45-55 ℃ of 1min; 2-10 circulation; 95 ℃ of 5min;
In AS-PCR amplification linear amplification process, the annealing temperature of reaction will be arrived to the temperature that is applicable to 181D5F-C and 181D5G-A extension; Only have in the time that 181D5F-C/181D5G-A mates completely with the template sequence in pcr amplification, they just can extend.And this extension products can be hybridized combination with 181D3B.
D: in test strip, be that anti-A antibody is adsorbed in latex particle by specific antibody, form antibody on latex particle surface coated; Be anti-B antibody by another colourless specific antibody, be fixed on film with linear, form detection line;
E: detected result: reaction product is all dripped in the application of sample place of nucleic acid test strip, add the damping fluid of 100ul, first hybridization product with antigen A and antigen B is combined with the antibody A of particle surface, forms the coloured particle mixture of antigen B-oligonucleotide chain antigen A-particle antibody A.Coloured mixture upwards flows along tunica fibrosa by capillarity in solution.In the time that mixture runs into the antibody B lines that are fixed on film, antigen B-oligonucleotide chain antigen A-particle antibody A mixture to be checked antibody B on lines is combined, and forms the coloured particle mixture of lines antibody B-antigen B-oligonucleotide chain antigen A-particle antibody A.The particle composites of coloured lines antibody B-antigen B-oligonucleotide chain antigen A-particle antibody A is detained on line, forms macroscopic colored line; This is positive.
In the time that AS-PCR reacts, if add allele-specific primers end not mate completely with template, this primer just cannot carry out AS-PCR amplification so, also just can not form the coloured particle mixture of antigen B-oligonucleotide chain antigen A-particle antibody A.Therefore coloured particle can not be combined and cross this line by the detection line on film, can not form macroscopic colored line.This is negative.
In the time that AS-PCR reacts, add respectively corresponding from polymorphic site with markd allele-specific primers when a reaction (or add the not corresponding different allele-specific primerses of mark of the same race) according to the base type of polymorphic site, then utilize multiple corresponding antibodies (or multiple nucleic acid bar test strip) coated in test strip to detect, carry out somatotype according to the band that has or not special color on it.If only have while wherein there is a band, show that examinee is homozygote, if while there are two bands (or 2 test strip occur band, referring to accompanying drawing 1), show that it is heterozygote.
Preferred version of the present invention is that the sample of the detection ERCC1 of foregoing invention is multiple.
Preferred version of the present invention is, in above-mentioned steps c, PCR & AS-PCR nucleic acid test strip method detects in ERCC1 the 118th bit codon polymorphism reaction system, each 0.2 micro-the rubbing of PCR primer: 181PF and 181PR, each 0.1 micro-the rubbing of the primer of mark: 181D5F-C/181D5G-A, specific probe: 181D3B 0.1 is micro-to rub, MgCl
22.5 millis rub, Taq archaeal dna polymerase 2 units, and PCR response procedures is: 95 ℃, 5 points; 94 ℃, 20 seconds; 60 ℃, 20 seconds; 72 ℃, 20 seconds; 40 circulations; 94 ℃, 20 seconds; 45 ℃, 20 seconds; 5 circulations.
Beneficial effect of the present invention: on the basis of our original detection of nucleic acids test strip patent, in conjunction with polymerase chain reaction (PCR) and allele specific amplification (Allele specific Polymerase Chain Reaction, and the method for the rapid detection for ERCC1 the 118th bit codon loci polymorphism of invention AS-PCR), the present invention has adopted the genotype in PCR & AS-PCR technology bind nucleic acid ELISA test strip technology for detection SNP site, with common PCR, AS-PCR compares has higher atopic and susceptibility, for people provide a kind of simple to operate, the method that cheap and detected result can detect ERCC1 the 118th bit codon polymorphism accurately.
Accompanying drawing explanation
Fig. 1 PCR-AS-PCR detects SNP reaction principle figure
Fig. 2: nucleic acid test strip method and sequencing detect ERCC1 the 118th bit codon polymorphism result, 2A: the result that nucleic acid test strip detects: 1. TT homozygote; 2. CC homozygote.2B: corresponding sequencing result, the site of the base of redline for detecting: 1. TT homozygote; 2. CC homozygote.
Fig. 3: nucleic acid test strip fado people detects the result of ERCC1 the 118th bit codon polymorphism: 1. C/T heterozygote; 2. CC homozygote.
Embodiment
The detection of the single ERCC1 of embodiment 1. the 118th bit codon polymorphism
A sampling
Examinee's peripheral vein anticoagulation (frozen) 200ul, the minim DNA rapid extraction test kit that application Yousida Biological Technology Co., Ltd., Hangzhou produces carries out nucleic acid extraction, in order to gene amplification.
B: first the target template that comprises ERCC1 the 118th bit codon pleomorphism site is increased by PCR, the primer that simultaneously carries out pcr amplification does not carry out any mark;
It is characterized in that:
C: on the basis of step b, utilize AS-PCR to carry out specific amplification according to the base type of ERCC1 the 118th bit codon Genetic polymorphism type,
In the 0.2ml of sterilizing PCR pipe, in the reaction system of 20ul, divide and add different pcr amplification primer and probe for different genotype;
PCR & AS-PCR nucleic acid test strip method detects ERCC1 the 118th bit codon polymorphism reaction system:
Template: examinee's genomic dna,
Micro-the rubbing of the each 0.2-2 of PCR primer: 181PF and 181PR,
Micro-the rubbing of the each 0.1-1 of the primer of mark: 181D5F-C/181D5G-A,
Specific probe: 181D3B 0.1-1 is micro-to rub,
DNTP:0.2-0.5 milli rubs
(NH
4)
2sO
410 millis rub
KCl 10 millis rub
Tris-HCl(PH 9.0) 10 milli rub
Triton-100 1.0%
MgCl
21.5-3 milli rubs
Taq archaeal dna polymerase: 1-5 unit
Cumulative volume is 20 microlitres
Of short duration centrifugal laggard performing PCR circulation.PCR loop parameter is 95 ℃ of 5min; 93-96 ℃ of 1min; 60-72 ℃ of 1min; 72 ℃ of 2min; 30-50 circulation; 93-96 ℃ of 30s; 45-55 ℃ of 1min; 2-10 circulation; 95 ℃ of 5min;
In AS-PCR amplification linear amplification process, the annealing temperature of reaction will be arrived to the temperature that is applicable to 181D5F-C and 181D5G-A extension;
D: in test strip, be that anti-A antibody is adsorbed in latex particle by specific antibody, form antibody on latex particle surface coated; Be anti-B antibody by another colourless specific antibody, be fixed on film with linear, form detection line;
E: detected result: reaction product is all dripped in the application of sample place of nucleic acid test strip, add the damping fluid of 100ul, first hybridization product with antigen A and antigen B is combined with the antibody A of particle surface, forms the coloured particle mixture of antigen B-oligonucleotide chain antigen A-particle antibody A.Coloured mixture upwards flows along tunica fibrosa by capillarity in solution.In the time that mixture runs into the antibody B lines that are fixed on film, antigen B-oligonucleotide chain antigen A-particle antibody A mixture to be checked antibody B on lines is combined, and forms the coloured particle mixture of lines antibody B-antigen B-oligonucleotide chain antigen A-particle antibody A.The particle composites of coloured lines antibody B-antigen B-oligonucleotide chain antigen A-particle antibody A is detained on line, forms macroscopic colored line (Fig. 2)
2A: the result that nucleic acid test strip detects: 1. TT homozygote; 2. CC homozygote.
2B: corresponding sequencing result, the site of the base of redline for detecting: 1. TT homozygote; 2. CC homozygote.Embodiment bis-, the detection of many people ERCC1 the 118th bit codon polymorphism
A sampling
Examinee's peripheral vein anticoagulation (frozen) 200ul, the minim DNA rapid extraction test kit that application Yousida Biological Technology Co., Ltd., Hangzhou produces carries out nucleic acid extraction, in order to gene amplification.
B: first the target template that comprises ERCC1 the 118th bit codon pleomorphism site is increased by PCR, the primer that simultaneously carries out pcr amplification does not carry out any mark;
It is characterized in that:
C: on the basis of step b, utilize AS-PCR to carry out specific amplification according to the base type of ERCC1 the 118th bit codon Genetic polymorphism type,
In the 0.2ml of sterilizing PCR pipe, in the reaction system of 20ul, divide and add different pcr amplification primer and probe for different genotype;
PCR & AS-PCR nucleic acid test strip method detects ERCC1 the 118th bit codon polymorphism reaction system:
Template: examinee's genomic dna,
Micro-the rubbing of the each 0.2-2 of PCR primer: 181PF and 181PR,
Micro-the rubbing of the each 0.1-1 of the primer of mark: 181D5F-C/181D5G-A,
Specific probe: 181D3B 0.1-1 is micro-to rub,
DNTP:0.2-0.5 milli rubs
(NH
4)
2sO
410 millis rub
KCl 10 millis rub
Tris-HCl(PH 9.0) 10 milli rub
Triton-100 1.0%
MgCl
21.5-3 milli rubs
Taq archaeal dna polymerase: 1-5 unit
Cumulative volume is 20 microlitres
Of short duration centrifugal laggard performing PCR circulation.PCR loop parameter is 95 ℃ of 5min; 93-96 ℃ of 1min; 60-72 ℃ of 1min; 72 ℃ of 2min; 30-50 circulation; 93-96 ℃ of 30s; 45-55 ℃ of 1min; 2-10 circulation; 95 ℃ of 5min;
In AS-PCR amplification linear amplification process, the annealing temperature of reaction will be arrived to the temperature that is applicable to 181D5F-C and 181D5G-A extension;
D: in test strip, be that anti-A antibody is adsorbed in latex particle by specific antibody, form antibody on latex particle surface coated; Be anti-B antibody by another colourless specific antibody, be fixed on film with linear, form detection line;
E: detected result: reaction product is all dripped in the application of sample place of nucleic acid test strip, add the damping fluid of 100ul, first hybridization product with antigen A and antigen B is combined with the antibody A of particle surface, forms the coloured particle mixture of antigen B-oligonucleotide chain antigen A-particle antibody A.Coloured mixture upwards flows along tunica fibrosa by capillarity in solution.In the time that mixture runs into the antibody B lines that are fixed on film, antigen B-oligonucleotide chain antigen A-particle antibody A mixture to be checked antibody B on lines is combined, and forms the coloured particle mixture of lines antibody B-antigen B-oligonucleotide chain antigen A-particle antibody A.The particle composites of coloured lines antibody B-antigen B-oligonucleotide chain antigen A-particle antibody A is detained on line, forms macroscopic colored line inspection
Survey result: reaction product is all dripped on nucleic acid test strip, 10 minutes sentence read result.What nucleic acid test strip detected ERCC1 the 118th bit codon polymorphism the results are shown in Figure 3.
Embodiment tri-, embodiment mono-or two is further limited, in step c, PCR & AS-PCR nucleic acid test strip method detects in ERCC1 the 118th bit codon polymorphism reaction system, each 0.2 micro-the rubbing of PCR primer: 181PF and 181PR, each 0.1 micro-the rubbing of the primer of mark: 181D5F-C/181D5G-A, specific probe: 181D3B 0.1 is micro-to rub, MgCl
22.5 millis rub, Taq archaeal dna polymerase 2 units, and PCR response procedures is: 95 ℃, 5 points; 94 ℃, 20 seconds; 60 ℃, 20 seconds; 72 ℃, 20 seconds; 40 circulations; 94 ℃, 20 seconds; 45 ℃, 20 seconds; 5 circulations.
More than show and described ultimate principle of the present invention and principal character and advantage of the present invention.The technician of the industry should be appreciated that; the present invention is not restricted to the described embodiments; that in above-described embodiment and specification sheets, describes just illustrates principle of the present invention; without departing from the spirit and scope of the present invention; the present invention also has various changes and modifications; these changes and improvements all fall in the claimed scope of the invention, the claimed model of the present invention.
Claims (3)
1. nucleic acid test strip method detects ERCC1 a 118th bit codon loci polymorphism,
A, sampling, frozen examinee's peripheral vein anticoagulation 200ul, carries out nucleic acid extraction with minim DNA rapid extraction test kit;
B: first the target template that comprises ERCC1 the 118th bit codon pleomorphism site is increased by PCR, the primer that simultaneously carries out pcr amplification does not carry out any mark;
It is characterized in that:
C: on the basis of step b, utilize AS-PCR to carry out specific amplification according to the base type of ERCC1 the 118th bit codon Genetic polymorphism type,
In the 0.2ml of sterilizing PCR pipe, in the reaction system of 20ul, divide and add different pcr amplification primer and probe for different genotype;
PCR & AS-PCR nucleic acid test strip method detects ERCC1 the 118th bit codon polymorphism reaction system:
Template: examinee's genomic dna,
Micro-the rubbing of the each 0.2-2 of PCR primer: 181PF and 181PR,
Micro-the rubbing of the each 0.1-1 of the primer of mark: 181D5F-C/181D5G-A,
Specific probe: 181D3B 0.1-1 is micro-to rub,
DNTP:0.2-0.5 milli rubs
(NH
4)
2sO
410 millis rub
KCl 10 millis rub
Tris-HCl(PH 9.0) 10 milli rub
Triton-100 1.0%
MgCl
21.5-3 milli rubs
Taq archaeal dna polymerase: 1-5 unit
Cumulative volume is 20 microlitres
Of short duration centrifugal laggard performing PCR circulation.PCR loop parameter is 95 ℃ of 5min; 93-96 1min; 60-72 ℃ of 1min; 72 ℃ of 2min; 30-50 circulation; 93-96 ℃ of 30s; 45-55 ℃ of 1min; 2-10 circulation; 95 ℃ of 5min;
In AS-PCR amplification linear amplification process, the annealing temperature of reaction will be arrived to the temperature that is applicable to 181D5F-C and 181D5G-A extension;
D: in test strip, be that anti-A antibody is adsorbed in latex particle by specific antibody, form antibody on latex particle surface coated; Be anti-B antibody by another colourless specific antibody, be fixed on film with linear, form detection line;
E: detected result: reaction product is all dripped in the application of sample place of nucleic acid test strip, add the damping fluid of 100ul, first hybridization product with antigen A and antigen B is combined with the antibody A of particle surface, form the coloured particle mixture of antigen B-oligonucleotide chain antigen A-particle antibody A, coloured mixture upwards flows along tunica fibrosa by capillarity in solution, in the time that mixture runs into the antibody B lines that are fixed on film, antigen B-oligonucleotide chain antigen A-particle antibody A mixture to be checked antibody B on lines is combined, form the coloured particle mixture of lines antibody B-antigen B-oligonucleotide chain antigen A-particle antibody A, the particle composites of coloured lines antibody B-antigen B-oligonucleotide chain antigen A-particle antibody A is detained on line, form macroscopic colored line,
2A: the result that nucleic acid test strip detects: 1. TT homozygote; 2. CC homozygote.
2B: corresponding sequencing result, the site of the base of redline for detecting: 1. TT homozygote; 2. CC homozygote.
2. nucleic acid test strip method according to claim 1 detects ERCC1 the 118th bit codon loci polymorphism, it is characterized in that, the sample that detects ERCC1 is multiple.
3. nucleic acid test strip method according to claim 1 and 2 detects ERCC1 the 118th bit codon loci polymorphism, it is characterized in that, in step c, PCR & AS-PCR nucleic acid test strip method detects in ERCC1 the 118th bit codon polymorphism reaction system, each 0.2 micro-the rubbing of PCR primer: 181PF and 181PR, each 0.1 micro-the rubbing of the primer of mark: 181D5F-C/181D5G-A, specific probe: 181D3B 0.1 is micro-to rub, the dNTP 0.2 milli MgCl that rubs
22.5 millis rub, Taq archaeal dna polymerase 2 units, and PCR response procedures is: 95 ℃, 5 points; 94 ℃, 20 seconds; 60 ℃, 20 seconds; 72 ℃, 20 seconds; 40 circulations; 94 ℃, 20 seconds; 45 ℃, 20 seconds; 5 circulations.
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| CN107227363A (en) * | 2017-07-05 | 2017-10-03 | 上海赛安生物医药科技股份有限公司 | ERCC1 genetic polymorphism detections system and its kit |
| CN111621575A (en) * | 2020-06-01 | 2020-09-04 | 浙江省农业科学院 | Hu sheep MC4R gene SNP 732C → G gene SNP locus detection primer, detection test strip and application |
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