CN104328181A - Primer, probe, kit and detection method for detecting UGT1A1 gene polymorphism - Google Patents
Primer, probe, kit and detection method for detecting UGT1A1 gene polymorphism Download PDFInfo
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Abstract
The invention discloses a primer, a probe, a kit and a detection method for detecting UGT1A1 gene polymorphism, belonging to the technical field of biology. The primer and probe for detecting the UGT1A1 gene polymorphism comprise at least one of a primer and probe for detecting UGT1A1*6 polymorphism of UGT1A1*6 gene polymorphism and a primer and probe for detecting UGT1A1*28 polymorphism of UGT1A1*28 gene polymorphism, wherein the primer comprises a forward primer and a reverse primer. The kit comprises the primers and the probe. The primers and probe disclosed by the invention can be used for detecting the UGT1A1 gene polymorphism with high sensitivity.
Description
Technical field
The present invention relates to biological technical field, particularly a kind of primer, probe, test kit and detection method for detecting UGT1A1 gene pleiomorphism.
Background technology
Irinotecan (irinotecan, CPT-11) be the semi-synthetic derivative of camptothecine, irinotecan is in vivo through completing acid esters enzyme (carboxylesterases, CE) hydrolysis is SN38 (7-ethyl-10-hydroxy-camptothecin, SN-38), irinotecan is one for the treatment of metastatic colorectal carcinoma (metastatic colorectal cancer, mCRC) the most effective chemotherapeutics.Irinotecan is after intravenous injection, after being converted into the stronger active metabolite SN-38 of cytotoxicity in vivo, through uridine diphosphate glucuronatetransferase family (uridine diphosphate glucuronosyltransferases, UGTs) deactivation is glucal acid product (10-O-glucuronyl-SN-38, SN-38G) after, enter intestines through bile excretion, be converted to SN-38 under the effect of intestinal bacteria GRD beta-glucuronidase after, intestinal mucosa injury and late-onset diarrhea can be caused; And the UGTs enzyme in enteron aisle can catalysis SN-38 be SN-38G removing toxic substances once again, the UGTs of the mankind is divided into UGT1 and UGT2 Liang Ge family, UGT1 gene at least comprises 13 hypotypes comprising UGT1A1, wherein the expression of UGT1A1 gene pleiomorphism and the curative effect of active and irinotecan thereof and untoward reaction all closely related.
The change in TATA box region, UGT1A1 gene promoter area, in CPT-11 treatment, the allelic existence of UGT1A1*28 causes the remarkable increase of active metabolite SN-38, thus the probability that diarrhoea or Neutrophilic granulocytopenia occur significantly increases.The genotypic detection of prompting UGT1A1 can be used for the generation of the dlinial prediction serious toxic side effect relevant to CPT-11.Simultaneously, carry out UGT1A1*6 in the patient treated (the 1st exon 2 11) heterozygous mutant G/A and homozygous mutant A/A at asian population application irinotecan significantly to increase patient and 3 ~ 4 grades of Neutrophilic granulocytopenia occur, the risk of thrombopenia and diarrhoea, patient clinical for above sudden change need reduce Irinotecan dosage, to control toxic side effect, by detecting TATA box region, hCCSP T1A1 gene promoter area and the 1st exon 2 11 nucleotide gene polymorphisms, the toxic side effect of Irinotecan to tumour patient is predicted from gene level, for the dosage of clinicians make Irinotecan provides reference.
" gold standard " of current detection in Gene Mutation remains sequencing, but traditional sequence measurement complex steps, operating time is long, simultaneously, the sensitivity that sequencing detects is low, can not accurately detect UGT1A1 gene polynorphisms, user replaces Sanger sequencing in the urgent need to the highly sensitive detection method of one
Summary of the invention
In order to the problem that the sensitivity solving in prior art the method detecting UGT1A1 gene pleiomorphism is low, embodiments provide a kind of primer, probe, test kit and detection method for detecting UGT1A1 gene pleiomorphism.Described technical scheme is as follows:
On the one hand, embodiments provide a kind of primer for detecting UGT1A1 gene pleiomorphism and probe, described primer and probe for detecting UGT1A1 gene pleiomorphism comprises the primer of the UGT1A1*6 polymorphism for detecting described UGT1A1*6 gene pleiomorphism and probe and at least one in the primer of the UGT1A1*28 polymorphism that detects described UGT1A1*28 gene pleiomorphism and probe, described primer comprises: forward primer and reverse primer, wherein
The forward primer of UGT1A1*6 polymorphism is as shown in SEQ ID NO.1 in sequence table;
The reverse primer of UGT1A1*6 polymorphism is as shown in SEQ ID NO.2 in sequence table;
The probe of UGT1A1*6 polymorphism is as shown in SEQ ID NO.3 in sequence table;
The forward primer of UGT1A1*28 polymorphism is as shown in SEQ ID NO.4 in sequence table;
The reverse primer of UGT1A1*28 polymorphism is as shown in SEQ ID NO.5 in sequence table;
The probe of UGT1A1*28 polymorphism is as shown in SEQ ID NO.6 in sequence table;
5 ' end of described probe is all connected with FAM fluorophor, and 3 ' end of described probe is all connected with BHQ quenching group.
On the other hand, embodiments provide a kind of test kit for detecting UGT1A1 gene pleiomorphism, described test kit comprises: above-mentioned primer and probe.
Particularly, described test kit also comprises: PCR reaction solution, positive quality control product, negative quality control product, sequencing reaction liquid, Digest enzyme and order-checking PCR primer purified reagent.
Particularly, described PCR reaction solution comprises: containing Mg
2+5 × PCR damping fluid 5 μ l, 2.5mmol/L dNTPs 1.5 μ l, 5U/ μ l Taq enzyme 0.2 μ l and 20ng/ μ l template DNA 2 μ l.
Particularly, described Digest enzyme comprises alkaline phosphatase and exonuclease I.
Particularly, described sequencing reaction liquid comprises: the forward primer 1 μ l of the UGT1A1*6 polymorphism of reverse primer 1 μ l and 3 μm ol/L as shown in SEQ ID NO.1 in sequence table of 0.3 μ l 5 × Bigdye, 0.45 μ l 2.5 × Buffer, the UGT1A1*28 polymorphism of 3 μm of ol/L as shown in SEQ ID NO.5 in sequence table.
Particularly, described order-checking PCR primer purified reagent comprises 0.75mol/L NaCl and nanometer magnetic bead.
Particularly, described negative quality control product is the recombinant plasmid containing UGT1A1 wild type gene fragment.
Particularly, described positive quality control product is the recombinant plasmid containing UGT1A1 gene pleiomorphism fragment.
Another aspect, embodiments provide a kind of method detecting UGT1A1 gene pleiomorphism, adopt above-mentioned test kit, described method comprises:
Extract the genomic dna of sample;
Using the described genomic dna obtained as described template DNA, adopt described test kit to carry out pcr amplification reaction, obtain pcr amplification product;
Purifying is carried out to described pcr amplification product, obtains the pcr amplification product of purifying;
Using the pcr amplification product of described purifying as template, carry out order-checking pcr amplification, obtain the pcr amplification product that checks order;
Described order-checking pcr amplification product is carried out purifying;
By the described order-checking pcr amplification product sequencing analysis after purifying, and the wild-type corresponding with described UGT1A1 gene pleiomorphism contrasts, and judges the described UGT1A1 gene pleiomorphism in described sample.
The beneficial effect that the technical scheme that the embodiment of the present invention provides is brought is: the primer that the embodiment of the present invention provides and probe have highly sensitive, can accurately detect UGT1A1 gene pleiomorphism.Test kit provided by the invention comprises the primer and probe that the embodiment of the present invention provides, and the test kit that the embodiment of the present invention is provided has highly sensitive.The detection method that the embodiment of the present invention provides, can accurately detect UGT1A1 gene pleiomorphism, meanwhile, adopt fluorescent PCR to increase to the gene fragment of sample to be tested, amplification situation can be judged by amplification curve and Ct value, and this can significantly improve the detection sensitivity of gene pleiomorphism.Pcr amplification product adopts alkaline phosphatase and exonuclease to digest, this effectively can remove dNTP, primer and single stranded DNA etc. residual in pcr amplification product, eliminate the step of gel electrophoresis, the time that not only saves also avoid pollution, in addition, the embodiment of the present invention adopts paramagnetic particle method to carry out purifying to order-checking PCR primer, simple and quick compared with traditional ethanol/sodium-acetate method, equipment requirements simple (only needing a magnetic frame), purification result are stablized, the order-checking peak figure obtained totally clear, without dyestuff peak.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in the embodiment of the present invention, below the accompanying drawing used required in describing embodiment is briefly described, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the order-checking peak figure of the UGT1A1*6 polymorphism that the embodiment of the present invention three provides;
Fig. 2 is the order-checking peak figure of the UGT1A1*28 polymorphism that the embodiment of the present invention three provides;
Fig. 3 is the amplification curve diagram that the embodiment of the present invention three provides.
Embodiment
For making the object, technical solutions and advantages of the present invention clearly, below embodiment of the present invention is described further in detail.
Embodiment one
The invention provides a kind of primer for detecting UGT1A1 gene pleiomorphism and probe, primer and probe for detecting UGT1A1 gene pleiomorphism comprise the primer of the UGT1A1*6 polymorphism for detecting UGT1A1*6 gene pleiomorphism and probe and at least one in the primer of the UGT1A1*28 polymorphism that detects UGT1A1*28 gene pleiomorphism and probe, primer comprises: forward primer and reverse primer, wherein
The forward primer of UGT1A1*6 polymorphism is as shown in SEQ ID NO.1 in sequence table;
The reverse primer of UGT1A1*6 polymorphism is as shown in SEQ ID NO.2 in sequence table;
The probe of UGT1A1*6 polymorphism is as shown in SEQ ID NO.3 in sequence table;
The forward primer of UGT1A1*28 polymorphism is as shown in SEQ ID NO.4 in sequence table;
The reverse primer of UGT1A1*28 polymorphism is as shown in SEQ ID NO.5 in sequence table;
The probe of UGT1A1*28 polymorphism is as shown in SEQ ID NO.6 in sequence table;
5 ' end of probe is all connected with FAM fluorophor, and 3 ' end of probe is all connected with BHQ quenching group.
The primer that the embodiment of the present invention provides and probe have highly sensitive, can accurately detect UGT1A1 gene pleiomorphism.
Embodiment two
Embodiments provide a kind of test kit for detecting UGT1A1 gene pleiomorphism, test kit comprises: the primer that the embodiment of the present invention one provides and probe and PCR reaction solution, positive quality control product, negative quality control product, sequencing reaction liquid, Digest enzyme and order-checking PCR primer purified reagent.
Particularly, in test kit, 10 μm of each 0.75 μ l of ol/L forward primer, 10 μm of each 0.75 μ l of ol/L reverse primer, 10 μm of each 0.5 μ l of ol/L probe.
Particularly, PCR reaction solution comprises: containing Mg
2+5 × PCR damping fluid 5 μ l, 2.5mmol/L dNTPs1.5 μ l, 5U/ μ l Taq enzyme 0.2 μ l and 20ng/ μ l template DNA 2 μ l.
Particularly, Digest enzyme comprises alkaline phosphatase and exonuclease I, and the enzyme activity of alkaline phosphatase and exonuclease I is than being 2:5.
Particularly, sequencing reaction liquid comprises: the forward primer 1 μ l of the UGT1A1*6 polymorphism of reverse primer 1 μ l and 3 μm ol/L as shown in SEQ ID NO.1 in sequence table of 0.3 μ l 5 × Bigdye, 0.45 μ l 2.5 × Buffer, the UGT1A1*28 polymorphism of 3 μm of ol/L as shown in SEQ ID NO.5 in sequence table.This sequencing reaction liquid is for detecting pcr amplification product.
Particularly, the PCR primer purified reagent that checks order comprises 0.75mol/L NaCl and nanometer magnetic bead.
Particularly, negative quality control product is the recombinant plasmid containing UGT1A1 wild type gene fragment.
Particularly, positive quality control product is the recombinant plasmid containing UGT1A1 gene pleiomorphism fragment.
Test kit for detecting UGT1A1 gene pleiomorphism provided by the invention, this test kit comprises the primer and probe that the embodiment of the present invention provides, and makes this test kit can accurately detect UGT1A1 gene pleiomorphism.
Embodiment three
The embodiment of the present invention provides a kind of method detecting UGT1A1 gene pleiomorphism, and adopt the test kit that the embodiment of the present invention two provides, concrete detection method is as follows:
1, the genomic dna of sample is extracted
Sample collection: extract person under inspection's venous blood 2ml by evacuated collection, inject sterile collection tube, wherein, this person under inspection's venous blood contains UGT1A1 gene pleiomorphism.
Sample preservation and transport: sample can immediately for detecting; If do not detected immediately, sample should be no more than 24 hours 4 DEG C of preservations, or-20 DEG C of preservations are no more than 3 months, then or-70 DEG C can preserve for a long time, and the sample multigelation being kept at-20 DEG C and-70 DEG C is no more than 5 times.The long-distance transport of sample should adopt 0 DEG C of curling stone, and haulage time is no more than 6 days.
Select commercialization poba gene group DNA extraction kit, the genomic dna of the sample of extraction adopts ultraviolet spectrophotometer to measure concentration and quality, and detect OD260/OD280 result between 1.8-2.0, the genomic dna concentration of sample is at 4ng/ more than μ l.The genomic dna extracted detects immediately, if the genomic dna extracted do not carry out detection in time should-20 DEG C of preservations.
2, pcr amplification
Prepare 2 PCR reaction tubess, in each PCR reaction tubes, add primer, probe and PCR reaction solution with micro sample adding appliance, wherein, 10 μm of each 0.75 μ l of ol/L forward primer, 10 μm of each 0.75 μ l of ol/L reverse primer, 10 μm of each 0.5 μ l of ol/L probe, PCR reaction solution comprises: containing Mg
2+5 × PCR damping fluid 5 μ l, 2.5mmol/L dNTPs 1.5 μ l, 5U/ μ l Taq enzyme 0.2 μ l and 20ng/ μ l template DNA 2 μ l, moisturizing to 25 μ l, wherein, 2 PCR reaction tubes corresponding UGT1A1*6 and UGT1A1*28 respectively, in PCR reaction tubes, add each 2 μ l of the genomic dna of the sample of extraction, negative quality control product and positive quality control product again, cover tightly PCR reaction tubes, through the 5000rpm centrifugal several seconds, obtain supernatant liquor, get this supernatant liquor and carry out pcr amplification reaction.
Fluorescent PCR instrument arranges following program:
After said procedure amplification, obtain pcr amplification product, as shown in Figure 3, this amplification curve is all S-type, and Ct value is between 20-30, and visible expanding effect is good.
3, purifying pcr amplification product
The each 5 μ l of PCR primer getting pcr amplification product, negative quality control product and positive quality control product respectively add in 4 PCR reaction tubess respectively, add 3 μ l Digest enzymes respectively and shake evenly, through brief centrifugation, product is reacted in PCR instrument, response procedures is: 37 DEG C 15 minutes, 95 DEG C 2 minutes, obtain the pcr amplification product of purifying.
4, check order pcr amplification
Get the pcr amplification product 3 μ l of purifying as template, then add 3 μ l sequencing reaction liquid (getting corresponding detection site sequencing reaction liquid), carry out order-checking pcr amplification, amplification condition is as follows:
Through above-mentioned amplification condition, obtain the pcr amplification product that checks order.
5, purifying order-checking pcr amplification product
Order-checking PCR primer carries out purifying through paramagnetic particle method, and operation steps is as follows:
1) added by order-checking pcr amplification product and 10 μ l are housed check order in the reaction tubes of PCR primer purified reagent, then to add 30 μ l concentration be the ethanol of 85%, after mixing, room temperature leaves standstill 2min; Wherein, the PCR primer purified reagent that checks order comprises 0.75mol/L NaCl and nanometer magnetic bead.
2) reaction tubes to be placed on magnetic frame until magnetic bead is adsorbed onto on tube wall completely, to abandon waste liquid.
3) in reaction tubes, add 150 μ l concentration is the ethanol of 85%, abandon waste liquid, then repetitive operation once.
4) room temperature places 3 ~ 5min, makes ethanol evaporation.
5) add the elutriant (elutriant is 0.01mol/L NaCl) of 40 μ l, fully mix, incubated at room 5min.
6) reaction tubes is placed in and magnetic frame acts on 3min or until solution is limpid, keeps reaction tubes on magnetic frame, shift 35 μ l supernatant liquor to new sample hose.
7) by products therefrom at 95 DEG C of sex change 4min, and rapidly at quenching 4min on ice, obtain the order-checking PCR primer of purifying.
6, interpretation of result
By the order-checking pcr amplification product sequencing analysis after purifying, and wild-type corresponding with UGT1A1*6 polymorphism and UGT1A1*28 gene pleiomorphism respectively contrasts, and judges UGT1A1 gene pleiomorphism in sample, specific as follows:
The order-checking PCR primer obtaining purifying is carried out sequencing analysis on ABI serial analysis instrument.
UGT1A1*6 polymorphism is: whether the 211st Nucleotide changes.
UGT1A1*6 polymorphism analysis: use Chromas program to open the sequencing result of UGT1A1*6 polymorphism, " Reverse+Complement " is clicked under " Edit " menu, press Ctrl+F key fast finding sequence A TCAGAGAC, contrast UGT1A1*6 wild-type sequence, see Fig. 1, wherein, UGT1A1*6 wild-type base sequence: TTG TAC ATC AGA GAC GGA.Changed at the 211st Nucleotide by comparative analysis sample, this result conforms to sample results, and as can be seen here, the method for the test kit that the embodiment of the present invention provides and detection UGT1A1*6 polymorphism can accurately detect UGT1A1*6 polymorphism.
UGT1A1*28 polymorphism is: the multiplicity of TAAGTAGGA sequence front end " TA " is inconsistent.
UGT1A1*28 polymorphism analysis: use Chromas program to open the sequencing result of UGT1A1*28 polymorphism, " Reverse+Complement " is clicked under " Edit " menu, press Ctrl+F key fast finding sequence TAAGTAGGA, contrast UGT1A1*28 wild-type sequence, see Fig. 2, wherein, UGT1A1*28 wild-type sequence is: GAT TGG TTT TTG CCA
tATATATATATAtAA.More than once in the multiplicity of TAAGTAGGA sequence front end " TA " by comparative analysis sample, this result conforms to sample results, as can be seen here, the test kit that the embodiment of the present invention provides can accurately detect UGT1A1*28 polymorphism with the method detecting UGT1A1*28 polymorphism.
The detection method that the embodiment of the present invention provides, can accurately detect UGT1A1 gene pleiomorphism, meanwhile, adopt fluorescent PCR to increase to the gene fragment of sample to be tested, amplification situation can be judged by amplification curve and Ct value, and this can significantly improve the detection sensitivity of gene pleiomorphism.Simultaneously, pcr amplification product adopts alkaline phosphatase and exonuclease to digest, this effectively can remove dNTP, primer and single stranded DNA etc. residual in pcr amplification product, eliminate the step of gel electrophoresis, the time that not only saves also avoid pollution, in addition, the embodiment of the present invention adopts paramagnetic particle method to carry out purifying to order-checking PCR primer, simple and quick compared with traditional ethanol/sodium-acetate method, equipment requirements simple (only needing a magnetic frame), purification result are stablized, the order-checking peak figure obtained totally clear, without dyestuff peak.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. one kind for detecting primer and the probe of UGT1A1 gene pleiomorphism, it is characterized in that, described primer and probe for detecting UGT1A1 gene pleiomorphism comprises the primer of the UGT1A1*6 polymorphism for detecting described UGT1A1*6 gene pleiomorphism and probe and at least one in the primer of the UGT1A1*28 polymorphism that detects described UGT1A1*28 gene pleiomorphism and probe, described primer comprises: forward primer and reverse primer, wherein
The forward primer of UGT1A1*6 polymorphism is as shown in SEQ ID NO.1 in sequence table;
The reverse primer of UGT1A1*6 polymorphism is as shown in SEQ ID NO.2 in sequence table;
The probe of UGT1A1*6 polymorphism is as shown in SEQ ID NO.3 in sequence table;
The forward primer of UGT1A1*28 polymorphism is as shown in SEQ ID NO.4 in sequence table;
The reverse primer of UGT1A1*28 polymorphism is as shown in SEQ ID NO.5 in sequence table;
The probe of UGT1A1*28 polymorphism is as shown in SEQ ID NO.6 in sequence table;
5 ' end of described probe is all connected with FAM fluorophor, and 3 ' end of described probe is all connected with BHQ quenching group.
2. for detecting a test kit for UGT1A1 gene pleiomorphism, it is characterized in that, described test kit comprises: primer as claimed in claim 1 and probe.
3. test kit according to claim 2, is characterized in that, described test kit also comprises: PCR reaction solution, positive quality control product, negative quality control product, sequencing reaction liquid, Digest enzyme and order-checking PCR primer purified reagent.
4. test kit according to claim 3, is characterized in that, described PCR reaction solution comprises: containing Mg
2+5 × PCR damping fluid 5 μ l, 2.5mmol/L dNTPs1.5 μ l, 5U/ μ l Taq enzyme 0.2 μ l and 20ng/ μ l template DNA 2 μ l.
5. test kit according to claim 4, is characterized in that, described Digest enzyme comprises alkaline phosphatase and exonuclease I.
6. test kit according to claim 4, it is characterized in that, described sequencing reaction liquid comprises: the forward primer 1 μ l of the UGT1A1*6 polymorphism of reverse primer 1 μ l and 3 μm ol/L as shown in SEQ ID NO.1 in sequence table of 0.3 μ l5 × Bigdye, 0.45 μ l2.5 × Buffer, the UGT1A1*28 polymorphism of 3 μm of ol/L as shown in SEQ ID NO.5 in sequence table.
7. test kit according to claim 4, is characterized in that, described order-checking PCR primer purified reagent comprises 0.75mol/L NaCl and nanometer magnetic bead.
8. test kit according to claim 4, is characterized in that, described negative quality control product is the recombinant plasmid containing UGT1A1 wild type gene fragment.
9. test kit according to claim 4, is characterized in that, described positive quality control product is the recombinant plasmid containing UGT1A1 gene pleiomorphism fragment.
10. detect a method for UGT1A1 gene pleiomorphism, adopt the test kit described in any one of claim 3-9, it is characterized in that, described method comprises:
Extract the genomic dna of sample;
Using the described genomic dna obtained as described template DNA, adopt described test kit to carry out pcr amplification reaction, obtain pcr amplification product;
Purifying is carried out to described pcr amplification product, obtains the pcr amplification product of purifying;
Using the pcr amplification product of described purifying as template, carry out order-checking pcr amplification, obtain the pcr amplification product that checks order;
Described order-checking pcr amplification product is carried out purifying;
By the described order-checking pcr amplification product sequencing analysis after purifying, and the wild-type corresponding with described UGT1A1 gene pleiomorphism contrasts, and judges the described UGT1A1 gene pleiomorphism in described sample.
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| CN104946784A (en) * | 2015-07-20 | 2015-09-30 | 武汉友芝友医疗科技有限公司 | Specific primer and kit for human UGT1A1 gene investigation of polymorphism |
| CN105671199A (en) * | 2016-04-22 | 2016-06-15 | 浙江中迪生物科技有限公司 | SNP (single nucleotide polymorphism) detection kit for colon cancer chemotherapeutic irinotecan sensitivity related gene UGTiA1 and use method of SNP detection kit |
| CN108570499A (en) * | 2018-01-15 | 2018-09-25 | 新开源禄西（南京）生物科技有限公司 | A kind of the rapid amplifying kit and amplification method of UGT1A1 genes |
| CN108570499B (en) * | 2018-01-15 | 2020-08-28 | 新开源禄西（南京）生物科技有限公司 | Rapid amplification kit and amplification method for UGT1A1 gene |
| CN109371127A (en) * | 2018-10-22 | 2019-02-22 | 江苏美因康生物科技有限公司 | The kit and method of a kind of while quick detection UGT1A1*6 type and UGT1A1*28 type gene pleiomorphism |
| CN111534591A (en) * | 2020-04-30 | 2020-08-14 | 北京和合医学诊断技术股份有限公司 | Method for synchronously detecting gene polymorphism of two SNP sites of UGT1A1 gene |
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