CN101671740A - Method for detecting gene polymorphism of UGT1A1 and liquid phase chip - Google Patents
Method for detecting gene polymorphism of UGT1A1 and liquid phase chip Download PDFInfo
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Abstract
The invention discloses a liquid phase chip for detecting gene polymorphism of UGT1A1 and a detection method using the liquid phase chip, the liquid phase chip comprises microspheres respectively enveloped with specific corresponding wide-type and variant-type anti-tag sequences, wherein the anti-tag sequences are selected from the group consisting of SEQ ID NO.13 and SEQ ID NO.14 directed to UGT1A1*28 gene type, SEQ ID NO.9 and SEQ ID NO. 10 directed to UGT1A1*6 gene type, and/or SEQ ID NO.11 and SEQ ID NO.12 directed to UGT1A1*93 gene type; each of the above microspheres includes different color codings, primers including variant target sequences of the UGT1A1*28gene type, the UGT1A1*6 gene type and/or the UGT1A1*93 gene type are amplified, and the primers are modified by biotin so thatthe corresponding PCR reaction product contains a biotin labeling and has a sequence capable of complementary pairing with anti-tag. The matching rate of the detection method provided by the inventionand sequencing method reaches as high as 100%. The prepared liquid phase chip for detecting gene polymorphism of UGT1A1 has quite excellent signal-to-noise ratio and, basically, no cross reaction ispresent between the designed probe and the anti-tag sequence.
Description
Technical field
The invention belongs to biology field, relate to medical science and biotechnology, the concrete UGT1A1 gene pleiomorphism that relates to detects liquid-phase chip and detection method thereof.
Background technology
(Irinotecan was a kind of by suppressing the chemotherapeutics that the DNA topoisomerase I plays antitumous effect CPT-11) to irinotecan, obtained the drugs approved by FDA listing in 1998.In the dna replication dna process, topoisomerase I reversibility ground cuts the dna single chain, and re-assemblies formation dna double chain.The active metabolite SN-38 of irinotecan combines with topoisomerase I DNA mixture, stops re-assemblying of DNA chain, causes the dna double chain break, causes necrocytosis.
Yet the minimizing of the neutrophilic granulocyte that irinotecan causes and serious intestines toxicity problem become one of key factor that limits its dosage.The active metabolite SN-38 of irinotecan is through liver uridine diphosphate glucuronate transferring enzyme (UDP-GT) deactivation (mainly by UGT1A1, UGT1A7 and UGT1A9 metabolism), thereby the cell that protects the health is avoided Cytotoxic influence.Scientific research shows that the toxic side effect of irinotecan and the gene pleiomorphism of UGT1A1 are closely related.Three kinds of common allele type UGT1A1*28, UGT1A1*6 and the UGT1A1*93 of UGT1A1 can cause the miopragia of this enzyme, thereby pharmaceutical activity metabolite residence time in blood is prolonged.
UGT1A1*28 is the sudden change of promotor TATA box.The heterozygote of UGT1A1*28 is similar with the enzymic activity of wild-type, but the enzymic activity of the sudden change homozygote correspondence of UGT1A1*28 only is 35% of a wild-type.The normal homozygote in this site is 6 multiple TA base pair A (TA)
6TAA (6/6), the sudden change homozygote is 7 multiple TA base pair A (TA)
7TAA (7/7), in the genotypic allelotrope of heterozygous mutation, one is wild-type, one is mutant (6/7).The UGT1A1 carrier of different genotype, the probability difference of generation toxic side effect when accepting irinotecan.Wild-type UGT1A1 (6/6) can not produce toxic side effect when accepting irinotecan, and the probability of mutant heterozygote (6/7) generation toxic side effect is 12.5%, and mutant homozygote (7/7) then has 50% probability generation toxic side effect.In the Aisa people, each genotypic occurrence frequency of UGT1A1 gene is respectively that 6/6 type accounts for 70.2%, 6/7 type and accounts for 27.7%, 7/7 type and account for 2.1%.Therefore, drugs approved by FDA in 2005 are upgraded the product description of irinotecan, require to indicate that in information warning user's hereditary difference will influence its reaction to this medicine, should carry out UGT1A1*28 before the prompting medication and detect.
UGT1A1*93 is that (3156G＞A), UGT1A1*6 are the point mutation (211G＞A) on the exons 1 for point mutation on the promotor.Clinical study finds, has-the genotypic patient of 3156G has only 12.5% toxic side effects to occur, and-the genotypic patient of 3156A, have 50% side effect after accepting irinotecan, to occur, and the time that toxic side effect occurs is also significantly early.The distribution frequency of UGT1A1*93 in the crowd of East Asia is about 9%.The ratio of the AUC of genotypic SN-38G of UGT1A1*6 and SN-38 (blood medicine-time lower curve area) significantly is lower than wild-type, and get nowhere survival rate and total survival rate that UGT1A1*6 genotype patient accepts after the irinotecan all significantly descend.The distribution frequency of UGT1A1*6 in the crowd of East Asia is about 13.6%.
The launch of existing a plurality of both at home and abroad at present detection UGT1A1*28 allelotypes, as Third Wave, Genelex, Genzyme, LabCorp, Shen, Shanghai friend's biology and sky, Shanghai sky biology etc.But the product that these three gene locuss of joint-detection UGT1A1 are not arranged.Product in the market mainly is based upon the detection technique based on PCR, as direct sequencing, PCR-single-strand conformation polymorphism analysis (SSCP) detects, these technology exist sensitivity low, the shortcoming that sample easily pollutes, false positive rate is high, regular-PCR method and quantitative fluorescent PCR can not satisfy clinical needs owing to detect the limitation of flux.And polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analytical technology and once can only carry out a kind of detection of sudden change, time and effort consuming based on the allelotrope discriminant analysis of TaqMan technology.
Based on the principle of chip, it is the suspension liquid-phase chip technology of carrier with the microballoon that U.S. Luminex company has developed.This technology is utilized the carrier of polystyrene microsphere as reaction, as detection platform, nucleic acid and protein and other is carried out high-throughout many indexs parallel detection with fluorescence detector.In the manufacturing processed of microballoon, mix the ruddiness and the infrared light staining agent of different ratios, thereby form the microballoon of 100 kinds of different colours codings of as many as.Different microballoon covalent attachment at the protein of difference thing to be detected or nucleic acid molecule as probe molecule, reporter molecules is with biotin labeling, and uses high-sensitive fluorescent dyeing.These microballoons and determinand, reporter molecules, fluorescent marker just form complete microballoon detection architecture and are used for reading of Luminex system.Excitated red respectively laser of Luminex reading system and green laser are used for the detection of microsphere system, and wherein red laser detects the red classification of microsphere surface intensity of fluorescence, and according to different color in the microballoon and number class, thereby determine the type of reaction; Green laser detects the fluorescence intensity of fluorescent marker in the sample, detects microballoon kind, quantity by machine and computer automatic statistical analysis laser again, thereby judges sample to be tested plurality of target tester concentration separately.Therefore, liquid-phase chip technology had both satisfied the requirement of high throughput testing, had possessed simultaneously quick and precisely, and was highly sensitive, and specificity is good, as a result advantage such as good reproducibility.We adopt the xTAG liquid-phase chip technology can detect multiple SNPs simultaneously, realize the operation of fast and convenientization of high-throughput, have improved detection efficiency greatly, maintain the leading position in similar detection technique.
Summary of the invention
One of purpose of the present invention provides the UGT1A1 gene pleiomorphism and detects liquid-phase chip.This liquid-phase chip can be used for detecting the variation of normal genotype and three kinds of common allele type UGT1A1*28, UGT1A1*6 and the UGT1A1*93 of UGT1A1 gene.
A kind of UGT1A1 gene pleiomorphism detects liquid-phase chip, mainly includes:
(1). be coated with the microballoon of the anti-tag sequence of special corresponding wild-type and anomaly respectively, described anti-tag sequence is selected from UGT1A1*28 genotypic SEQ ID NO.13 and SEQ ID NO.14, at the genotypic SEQ IDNO.9 of UGT1A1*6 and SEQ ID NO.10 and/or at UGT1A1*93 genotypic SEQ ID NO.11 and SEQ ID NO.12, and above-mentioned every kind of microballoon has the different colours coding;
(2). amplify have UGT1A1*28, the primer of UGT1A1*6 and/or the genotypic variation target sequence of UGT1A1*93, described primer has biotin modification, so that corresponding PCR reaction product contains biotin labeling, and described PCR reaction product have can with the sequence of anti-tag complementary pairing.
Preferably, described amplimer is at UGT1A1*28 and UGT1A1*6 genotypic SEQ ID NO.15 and SEQ IDNO.16, and/or at UGT1A1*93 genotypic SEQ ID NO.17 and SEQ ID NO.18, one end in above-mentioned every pair of primer has biotin modification.Preferably, the ASPE primer that described UGT1A1 gene pleiomorphism detection liquid-phase chip also includes corresponding wild-type and anomaly is right, every kind of ASPE primer being made up of the specific primer sequence and 5 ' the tag sequence of holding at the different genotype goal gene site of detecting of 3 ' end, described specific primer sequence is for respectively at UGT1A1*6 genotypic SEQ ID NO.5 and SEQ ID NO.6, and/or at UGT1A1*93 genotypic SEQ ID NO.7 and SEQ ID NO.8, described tag sequence can with corresponding anti-tag sequence complementary pairing on the microballoon.
Another object of the present invention provides the method for using above-mentioned liquid-phase chip that the UGT1A1 gene pleiomorphism is detected.
A kind of method of using above-mentioned liquid-phase chip to the detection of UGT1A1 gene pleiomorphism mainly may further comprise the steps:
(1) use amplimer, pcr amplification testing sample DNA must contain biotin labeled PCR reaction product;
(2) the PCR reaction product is carried out hybridization with bag by the microballoon of special anti-tag sequence; Perhaps the PCR reaction product is carried out enzyme with the ExoSAP-IT test kit and is cut processing, carry out primer extension reaction with described ASPE primer again, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band, the microballoon of the anti-tag sequence that being coated with of corresponding A SPE primer is special and the product behind the above-mentioned extension carry out hybridization;
(3) product behind the hybridization and Streptavidin-phycoerythrin react;
(4) detect by fluorescence detector.
Major advantage of the present invention is:
1. the identical rate of detection method provided by the present invention and sequencing is up to 100%.Prepared UGT1A1 gene pleiomorphism detects liquid-phase chip and has extraordinary signal-noise ratio, and there is not cross reaction between designed probe and the anti-tag sequence basically, can also not need ASPE type specificity primer, detection method is easier.
2. gene pleiomorphism provided by the present invention detects liquid-phase chip and detection method thereof, not only can be in one-time detection a plurality of pleomorphism sites of parallel detection, realized simultaneously in one-time detection the parallel detection of different polymorphism types has been realized the important improvement to existing liquid-phase chip technology.
3. the ASPE type specificity primer of the present invention's design has extraordinary specificity, can accurately distinguish the genotype of various types, and have better signal to noise ratio, helps the parallel detection of many indexs.
4. detection method step of the present invention is simple, three pleomorphism sites detect by a step multiplex PCR can finish two amplifications with target sequence of pleomorphism site, many uncertain factors of existing in the complex operations processes such as repeated multiple times PCR have been avoided, thereby can improve the detection accuracy rate greatly, embodied qualitative, quantitative analysis feature of accurate while.
5. the needed time of detection method provided by the present invention meets clinical needs especially well below sequencing technologies commonly used.
6. not only to have overcome traditional solid phase chip susceptibility not high in the present invention, and the defective of the repeatability difference of detected result is improved existing liquid-phase chip technology simultaneously, makes prepared microballoon can be applicable to different test items, has very strong expansion.The fluorescent signal value that detects improves greatly, thereby makes the sensitivity that detects be further enhanced, and signal to noise ratio strengthens, and detected result more accurately and reliably.
Embodiment
Embodiment 1UGT1A1 gene pleiomorphism detects liquid-phase chip, mainly includes:
One, ASPE primer
Design specific primer sequence respectively at two kinds of common allele type UGT1A1*6 of UGT1A1 gene and the variant sites of UGT1A1*93.The ASPE primer is made up of " Tag+ specific primer sequence ".The ASPE primer sequence is as shown in the table:
Table 1 ASPE primer sequence
Every the ASPE primer comprises two parts, and 5 ' end is the specificity tag sequence at anti-tag sequence on the corresponding microballoon, and 3 ' end is mutant or wild-type specific primer sequence (shown in above-mentioned table 1).All ASPE primers are synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.Every primer after synthetic is mixed with the stock solution of 100pmol/mL respectively with 10mmol/L Tris Buffer.
Two, the microballoon of anti-tag/ probe sequence bag quilt
Target sequence according to required detection, select the tag sequence, reduce between the anti-tag sequence of each microballoon to greatest extent and secondary structure that tag and target fragment to be detected may form, corresponding anti-tag/ probe sequence is as shown in table 2 on six kinds of microballoons numberings of selection and the microballoon:
Corresponding anti-tag/ probe sequence on table 2 microballoon numbering and the microballoon
6 kinds of microballoons selecting are available from U.S. Luminex company, with anti-tag/ probe sequence bag by with microballoon on.Be connected with the spacerarm preface of 5-10 T between anti-tag/ probe sequence and the microballoon, the spacerarm sequence that promptly adds the preceding paragraph 5-10 T before each anti-tag/ probe sequence, the anti-tag/ probe sequence is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.With synthetic anti-tag sequence sterilization ddH
2O is made into the stock solution of 100nmol/ml.Described spacerarm is to be used for specific probe and microsphere surface is spaced apart or specific probe is placed the sequence of hydrophilic environments.By the spacerarm sequence of suitable length is set between probe sequence and amino, can reduce sterically hinderedly, improve the efficient of hybridization and the specificity of hybridization.Common spacerarm sequence comprises poly dT, i.e. poly (dT), and oligomerization four polyoxyethylene glycol and (CH2) n spacerarm (n 〉=3) are as (CH2) 12, (CH2) 18 etc.In addition, if exist poly (dA) to disturb, can also use poly (TTG) as spacerarm.Spacerarm of the present invention is preferably 5-10 T, in China, and the synthetic technology comparative maturity of T, cost is relatively low.
The process of microballoon bag quilt is as follows:
Get 5 * 10 respectively
6The carboxylated microballoon of individual above-mentioned numbering (available from Luminex company) is suspended in the MES solution of 50ul 0.1mol/L (pH4.5), adds 10ul synthetic anti-tag molecule (100nmol/ml).EDC (N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide) (available from the Pierce Chemical company) working fluid of preparation 10ng/ml.Add the EDC working fluid of 2.5ul in the microballoon suspension, constant temperature was hatched 30 minutes, added the EDC working fluid of 2.5ul again, and constant temperature was hatched 30 minutes again.After reaction finished, the Tween-20 washing with 0.02% was once washed once with 0.1% SDS liquid again.The microballoon that is coated with the anti-tag sequence after the washing is resuspended in the Tris-EDTA solution of 100ul, and [10mmol/L Tris (pH8.0), among the 1mmol/L EDTA, 2-8 ℃ keeps in Dark Place.
Three. amplify the primer of target sequence with mutational site:
Three kinds of common allele type UGT1A1*28, UGT1A1*6 of UGT1A1 gene and UGT1A1*93.UGT1A1*28 is the polymorphism of TA base pair multiplicity, and UGT1A1*93 is point mutation, all occurs in promoter region, but apart from each other; UGT1A1*6 occurs in exons 1.Utilize Primer5.0 design two pairs of primers (seeing Table 3), amplify target sequence respectively.
Table 3 amplifies the primer of the target sequence with mutational site
Embodiment 2 utilization embodiment 1 described UGT1A1 gene pleiomorphism detects the detection of liquid-phase chip (not using the ASPE primer) to clinical sample
The prescription of described various solution is as follows:
MES damping fluid (pH5.0) prescription (250ml) of 50mM:
| Reagent | The source | Final concentration | The consumption of every 250ml |
| ??MES(2[N-Morpholino] ??ethanesulfonic?acid) | ??Sigma?M-2933 | ??0.05M | ?2.44g |
| ??5M?NaOH | ??Fisher?SS256-500 | ??--- | 5 |
2 * Tm hybridization buffer
| Reagent | The source | Final concentration | The consumption of every 250ml |
| ??1MTris-HCl，pH8.0 | ??SigmaT3038 | ??0.2M | ??50ml |
| ??5M?NaCl | ??Sigma?S5150 | ??0.4M | ??20ml |
| ??Triton?X-100 | ??Sigma?T8787 | ??0.16％ | ??0.4ml |
Be stored in 4 ℃ after the filtration.
The primer of biotin modification is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
Extract the test kit explanation in a small amount with reference to AxyPrep whole blood genome, obtain DNA. to be detected
Two, the pcr amplification of testing sample
Utilize two pairs of primers of Primer5.0 design, with primer multiplex PCR one step of containing biotin modification amplify the promoter fragment of UGT1A1 and exons 1 totally two have biotin labeled target sequence of containing of pleomorphism site, the product size is respectively 586bp and 374bp.Primer sequence (SEQ NO.15-18) is seen shown in the above-mentioned table 4.
At first prepare PCR primer working fluid: the primer stock solution 100ul that respectively gets SEQ NO.15-16 respectively mixes and is PCR primer working fluid in the 1.5ml Eppendorf tube.The PCR reaction system is as follows:
10 * damping fluid (contains Mg
2+) 5ul
DNTP (each 2.5mmol/L) 4ul
Taq enzyme (5U/ul) 0.5ul
PCR primer working fluid (each 25pmol/mL) 4ul
Template DNA (10ng/ul) 1ul
ddH
2O?????????????????????????35.5ul
?????????????????????????????????????????????
Be total to 50ul
The pcr amplification program is: 95 ℃ of 3min; 94 ℃ of 20s, 56 ℃ of 30s, 72 ℃ of 30s, 30 circulations; 72 ℃ of 10min; 4 ℃ of preservations are standby.The base sequence of the amplified production that obtains: the sequence at UGT1A1*28, UGT1A1*6 variant sites is positioned on the 379-965bp of NG_009254, is positioned on the NG_0026011568-1942bp at the sequence of the variant sites of UGT1A1*93.
Three, hybridization
1. (microballoon concentration is 2.5 * 10 to choose the above-mentioned 6 kinds of microballoons of UGT1A1*6, the UGT1A1*93, the UGT1A1*28 that are coated with special anti-tag sequence
5Individual/ml).Every kind of microballoon has the different colours coding respectively, and every kind of microsphere surface is connected with one section specific oligonucleotide sequence (anti-tag) respectively simultaneously, and these anti-tag sequences can be respectively and the corresponding PCR product specific combination that contains biotin modification;
2. the microballoon of getting every kind of numbering of 1ul respectively is in the Eppendorf tube of 1.5ml;
3. microballoon is in 〉=centrifugal the 1-2min of 10000g;
4. supernatant discarded, microballoon is resuspended in 2 * Tm hybridization buffer of 100ul, the vortex mixing;
5. get the above-mentioned microballoon suspension of 25ul in the corresponding hole of 96 hole filter plates, control wells adds the ddH of 25ul
2O;
6. the PCR reaction solution of getting 5-25ul is used ddH in corresponding hole
2O complements to 50ul;
7. encase 96 orifice plates with lucifuge with masking foil, 95 ℃ of 60s, 60 ℃ of 15min are hatched hybridization;
8. the microballoon after the hybridization is in 〉=centrifugal the 2-5min of 3000g;
9. remove supernatant, microballoon is resuspended in 1 * Tm hybridization buffer of 75ul;
10. microballoon is in 〉=centrifugal the 2-5min of 3000g;
11 are resuspended in microballoon in 1 * Tm hybridization buffer of 75ul, and adding 15ul concentration is Streptavidin-phycoerythrin (SA-PE) of 10ug/ml;
12.60 ℃ hatch 15min, on the Luminex instrument, detect.
Four, the result detects and data analysis
The reaction after product is by Luminex serial analysis instrument detecting.With the carrier of polystyrene microsphere, as detection platform, nucleic acid molecule is carried out high-throughout many indexs parallel detection with fluorescence detector as reaction.In the manufacturing processed of microballoon, mix the ruddiness and the infrared light staining agent of different ratios, thereby form the microballoon of 100 kinds of different colours codings of as many as.Different microballoon covalent attachment at the nucleic acid molecule of difference thing to be detected as probe molecule, reporter molecules is with biotin labeling, and uses high-sensitive fluorescent dyeing.These microballoons and determinand, reporter molecules, fluorescent marker just form complete microballoon detection architecture and are used for reading of Luminex system.Excitated red respectively laser of Luminex reading system and green laser are used for the detection of microsphere system, and detected result is shown in table 4 and table 5.
Fluorescent value (MFI) and data processing there are following requirement:
1. each site need have an allelotrope MFI at least greater than 300 and greater than 10 * PCR negative control MFI;
2.NET MFI=sample MFI-PCR negative control MFI (NET MFI less than 0 with 0 the expression);
3. satisfy the data of above two conditions, calculate sudden change ratio by following formula:
Sudden change ratio=mutant NET MFI ÷ (mutant NET MFI+ wild-type NET MFI)
4. rule of thumb the sudden change ratio of each detection site is determined threshold value (cut-off value), to divide wild-type homozygote, heterozygote and mutant homozygote.
Use present method to detect the UGT1A1 gene pleiomorphism of great amount of samples, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Being provided with of threshold value (cut-off value) is as follows: the sudden change ratio range is considered as the wild-type homozygote at 0%-20%; 30%-70% is considered as heterozygote; 80%-100% is considered as the anomaly homozygote.Compare with the liquid-phase chip result with the sequencing detection, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments UGT1A1 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.As seen UGT1A1 gene pleiomorphism provided by the present invention detects liquid-phase chip can detect the UGT1A1 genotype exactly, and the result is reliable and stable.
Table 4 pattern detection result (MFI)
| Sequence number NO. | ?? *28-6 | ?? *28-7 | ?211G＞A-w | ?211G＞A-m | ??-3156G＞A-w | ??-3156G＞A-m |
| Negative control | ??17 | ??5 | ?15 | ?3 | ??16 | ??3 |
| ??1 | ??668 | ??675 | ?2015 | ?48 | ??2098 | ??32 |
| ??2 | ??3395 | ??37 | ?2443 | ?38 | ??661 | ??682 |
| ??3 | ??2712 | ??23 | ?2261 | ?37 | ??2179 | ??29 |
| ??4 | ??3340 | ??13 | ?2783 | ?19 | ??2441 | ??40 |
| ??5 | ??2808 | ??49 | ?2716 | ?35 | ??2597 | ??20 |
| ??6 | ??3097 | ??49 | ?2883 | ?25 | ??2130 | ??16 |
| ??7 | ??652 | ??586 | ?2492 | ?13 | ??2104 | ??49 |
| ??8 | ??665 | ??643 | ?2697 | ?38 | ??618 | ??599 |
| ??9 | ??2654 | ??25 | ?2515 | ?17 | ??2168 | ??38 |
| ??10 | ??3337 | ??49 | ?602 | ?651 | ??2630 | ??13 |
| ??11 | ??3384 | ??28 | ?2337 | ?41 | ??2740 | ??32 |
| ??12 | ??3161 | ??24 | ?2558 | ?31 | ??2113 | ??16 |
| ??13 | ??2958 | ??46 | ?2665 | ?34 | ??2233 | ??39 |
| ??14 | ??2817 | ??21 | ?2913 | ?12 | ??2601 | ??21 |
| ??15 | ??2981 | ??39 | ?2437 | ?40 | ??35 | ??2321 |
| ??16 | ??647 | ??551 | ?2142 | ?15 | ??2259 | ??31 |
| ??17 | ??3243 | ??14 | ?2097 | ?36 | ??2955 | ??27 |
| ??18 | ??687 | ??568 | ?2902 | ?22 | ??2891 | ??39 |
| ??19 | ??2674 | ??29 | ?2607 | ?32 | ??2429 | ??46 |
| ??20 | ??2581 | ??33 | ?2752 | ?17 | ??2004 | ??12 |
Table 5 sample Polymorphism Analysis result
Embodiment 3 utilization embodiment 1 described UGT1A1 gene pleiomorphism detects the detection of liquid-phase chip (using the ASPE primer) to clinical sample
The prescription of described various solution is as described in the embodiment 1.
The ExoSAP-IT test kit is available from U.S. USB company.
Biotin labeled dCTP is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
One, the DNA extraction of sample:
Extract the test kit explanation in a small amount with reference to AxyPrep whole blood genome, obtain DNA. to be detected
Two, the pcr amplification of testing sample: as described in embodiment 1.
Three, the enzyme of PCR product is cut processing
With reference to the explanation of ExoSAP-IT test kit, detailed step is as follows:
1. get the reacted product of 7.5ul PCR, add 3ul ExoSAP-IT enzyme;
2.37 ℃ hatch 15min.Hatch 15min for 80 ℃, make unnecessary enzyme-deactivating.The product that enzyme is cut after the processing is directly used in follow-up ASPE primer extension reaction.
Four, site-specific primer extension reaction (ASPE)
Utilize the locus specificity primer of above-mentioned design to carry out primer extension reaction, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band.
At first prepare blended ASPE primer working fluid: respectively get respectively 211G＞A-w, 211G＞A-m ,-3156G＞A-w and-the corresponding ASPE primer of 3156G＞A-m stock solution 10ul is in the 1.5ml Eppendorf tube, add 10mmol/L Tris Buffer and mend, mix and be ASPE mix primer working fluid to 200ul.The system of ASPE reaction is as follows:
10 * damping fluid 2ul
MgCl
2(50mmol/L)??????????????????????0.5ul
Biotin-dCTP(400umol/L)???????????????0.25ul
DATP, dGTP, dTTP mixed solution (each 100umol/L) 1ul
Tsp enzyme (5U/ul) 0.25ul
Blended ASPE primer working fluid (each 500nmol/L) 1ul
Enzyme is cut the pcr amplification product 5ul of processing
ddH
2O????????????????????????????????10.ul
?????????????????????????????????????????????????????
Be total to 20ul
The PCR program is: 96 ℃ of 2min; 94 ℃ of 30s, 58 ℃ of 1min, 72 ℃ of 2min, 30 circulations; 4 ℃ of preservations are standby.
Five, hybridization
1. according to the ASPE primer of design, (microballoon concentration is 2.5 * 10 to select the above-mentioned 4 kinds of microballoons of UGT1A1*6, UGT1A1*93
5Individual/ml).Every kind of microballoon has the different colours coding respectively, simultaneously every kind of microsphere surface is connected with one section specific oligonucleotide sequence (anti-tag) respectively, and these anti-tag sequences can be respectively and the tag sequence or the PCR product specific combination of corresponding ASPE primer 3 ' end;
2. 4 kinds of microballoons getting the above-mentioned UGT1A1*6 of 1ul, UGT1A1*93 respectively are in the Eppendorf tube of 1.5ml;
3. microballoon is in 〉=centrifugal the 1-2min of 10000g;
4. supernatant discarded, microballoon is resuspended in 2 * Tm hybridization buffer of 100ul, the vortex mixing;
5. get the above-mentioned microballoon suspension of 25ul respectively in the corresponding hole of 96 hole filter plates, control wells adds the ddH of 25ul
2O;
6. the ASPE reaction solution of getting 5-25ul UGT1A1*6 and * 93 is respectively used ddH in corresponding hole
2O complements to 50ul;
7. encase 96 orifice plates with lucifuge with masking foil, 95 ℃ of 60s, 37 ℃ of 15min are hatched hybridization;
8. the microballoon after the hybridization is in 〉=centrifugal the 2-5min of 3000g;
9. remove supernatant, microballoon is resuspended in 1 * Tm hybridization buffer of 75ul;
10. microballoon is in 〉=centrifugal the 2-5min of 3000g;
11 are resuspended in microballoon in 1 * Tm hybridization buffer of 75ul, and adding 15ul concentration is Streptavidin-phycoerythrin (SA-PE) of 10ug/ml;
12.37 ℃ hatch 15min, on the Luminex instrument, detect.
Seven, the result detects and data analysis
Data analysis is as described in the embodiment 1.
Use present method to detect the UGT1A1 gene pleiomorphism of great amount of samples, experimental data meets above-mentioned requirements, therefore can calculate their sudden change ratio.Being provided with of threshold value (cut-off value) is as follows: the sudden change ratio range is considered as the wild-type homozygote at 0%-20%; 30%-70% is considered as heterozygote; 80%-100% is considered as the anomaly homozygote.Compare with the liquid-phase chip result with the sequencing detection, calculate the identical rate of classifying method detected result provided by the present invention.Present method detects 20 increments UGT1A1 genotype detection result and the sequencing result rate of coincideing originally and reaches 100%.As seen UGT1A1 gene pleiomorphism provided by the present invention detects liquid-phase chip can detect the UGT1A1 genotype exactly, and the result is reliable and stable.
Table 6 pattern detection result (MFI)
| Sequence number NO. | ?211G＞A-w | ?211G＞A-m | ??-3156G＞A-w | ??-3156G＞A-m |
| Negative control | ?15 | ?0 | ??15 | ??5 |
| ??1 | ?2179 | ?29 | ??2097 | ??36 |
| ??2 | ?2783 | ?19 | ??596 | ??685 |
| ??3 | ?2441 | ?40 | ??2015 | ??48 |
| ??4 | ?2716 | ?35 | ??2443 | ??38 |
| ??5 | ?2492 | ?13 | ??2597 | ??20 |
| ??6 | ?2130 | ?16 | ??2261 | ??37 |
| ??7 | ?2883 | ?25 | ??2104 | ??49 |
| ??8 | ?2697 | ?38 | ??608 | ??599 |
| ??9 | ?2515 | ?17 | ??2558 | ??31 |
| ??10 | ?563 | ?681 | ??2665 | ??34 |
| ??11 | ?2168 | ?38 | ??2913 | ??12 |
| ??12 | ?2630 | ?13 | ??2233 | ??39 |
| ??13 | ?2740 | ?32 | ??2437 | ??40 |
| ??14 | ?2113 | ?16 | ??2601 | ??21 |
| ??15 | ?2337 | ?41 | ??44 | ??2221 |
| ??16 | ?2259 | ?31 | ??2142 | ??15 |
| ??17 | ?2955 | ?27 | ??2098 | ??32 |
| ??18 | ?2891 | ?39 | ??2902 | ??22 |
| ??19 | ?2429 | ?46 | ??2607 | ??32 |
| ??20 | ?2004 | ?12 | ??2752 | ??17 |
Table 7 sample UGT1A1 Polymorphism Analysis result
More than be at the specifying of possible embodiments of the present invention, but this embodiment is not in order to limiting claim of the present invention, does not allly break away from the equivalence that skill spirit of the present invention does and implement or change, all should be contained in the claim of the present invention.
Sequence table
＜110〉Guangzhou Yishan Biotechnology Co., Ltd.
＜120〉method and the liquid-phase chip of the detection of UGT1A1 gene pleiomorphism
<160>18
<170>PatentIn?version?3.1
<210>1
<211>44
<212>DNA
＜213〉artificial sequence
<400>1
atcaaatctc?atcaattcaa?caatctcgtt?gtacatcaga?gacg?????44
<210>2
<211>44
<212>DNA
＜213〉artificial sequence
<400>2
tcatttcaat?caatcatcaa?caatctcgtt?gtacatcaga?gaca?????44
<210>3
<211>40
<212>DNA
＜213〉artificial sequence
<400>3
ctttttcaat?cactttcaat?tcatcccagc?ccacctgtcc?????40
<210>4
<211>40
<212>DNA
＜213〉artificial sequence
<400>4
ttcataacta?caatacatca?tcatcccagc?ccacctgtct?????40
<210>5
<211>20
<212>DNA
＜213〉artificial sequence
<400>5
ctcgttgtac?atcagagacg???????????????????????????20
<210>6
<211>20
<212>DNA
＜213〉artificial sequence
<400>6
ctcgttgtac?atcagagaca?????????20
<210>7
<211>16
<212>DNA
＜213〉artificial sequence
<400>7
cccagcccac?ctgtcc?????????????16
<210>8
<211>16
<212>DNA
＜213〉artificial sequence
<400>8
cccagcccac?ctgtct?????????????16
<210>9
<211>24
<212>DNA
＜213〉artificial sequence
<400>9
attgttgaat?tgatgagatt?tgat????24
<210>10
<211>24
<212>DNA
＜213〉artificial sequence
<400>10
attgttgatg?attgattgaa?atga??????24
<210>11
<211>24
<212>DNA
＜213〉artificial sequence
<400>11
atgaattgaa?agtgattgaa?aaag??????24
<210>12
<211>24
<212>DNA
＜213〉artificial sequence
<400>12
atgatgatgt?attgtagtta?tgaa??????24
<210>13
<211>26
<212>DNA
＜213〉artificial sequence
<400>13
ttgccatata?tatatatata?agtagg????26
<210>14
<211>26
<212>DNA
＜213〉artificial sequence
<400>14
gccatatata?tatatatata?agtagg????26
<210>15
<211>20
<212>DNA
＜213〉artificial sequence
<400>15
ctgctacctt?tgtggactga???????????20
<210>16
<211>20
<212>DNA
＜213〉artificial sequence
<400>16
cgtcagcatg?acatcaaagc???????????20
<210>17
<211>23
<212>DNA
＜213〉artificial sequence
<400>17
gaacattcta?acggttcata?aag????23
<210>18
<211>18
<212>DNA
＜213〉artificial sequence
<400>18
gcagcttcct?gggcacag??????????18
Claims (6)
1, a kind of UGT1A1 gene pleiomorphism detects liquid-phase chip, it is characterized in that, mainly includes:
(1). be coated with the microballoon of the anti-tag sequence of special corresponding wild-type and anomaly respectively, described anti-tag sequence is selected from UGT1A1*28 genotypic SEQ ID NO.13 and SEQ ID NO.14, at the genotypic SEQ IDNO.9 of UGT1A1*6 and SEQID NO.10 and/or at UGT1A1*93 genotypic SEQ ID NO.11 and SEQ ID NO.12, and above-mentioned every kind of microballoon has the different colours coding;
(2). amplify have UGT1A1*28, the primer of UGT1A1*6 and/or the genotypic variation target sequence of UGT1A1*93, described primer has biotin modification, so that corresponding PCR reaction product contains biotin labeling, and described PCR reaction product have can with the sequence of anti-tag complementary pairing.
2. UGT1A1 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that the base sequence of described amplimer is at UGT1A1*28 and UGT1A1*6 genotypic SEQ ID NO.15 and SEQ ID NO.16; And/or at UGT1A1*93 genotypic SEQ ID NO.17 and SEQ ID NO.18; One end in above-mentioned every pair of primer has biotin modification.
3. UGT1A1 gene pleiomorphism according to claim 1 and 2 detects liquid-phase chip, it is characterized in that, the ASPE primer that also includes corresponding wild-type and anomaly is right, every kind of ASPE primer being made up of the specific primer sequence and 5 ' the tag sequence of holding at the different genotype goal gene site of detecting of 3 ' end, described specific primer sequence is respectively at genotypic SEQID NO.5 of UGT1A1*6 and SEQ ID NO.6, and/or at UGT1A1*93 genotypic SEQ ID NO.7 and SEQ ID NO.8, described tag sequence can with corresponding anti-tag sequence complementary pairing on the microballoon.
4. UGT1A1 gene pleiomorphism according to claim 1 detects liquid-phase chip, it is characterized in that, mainly includes
(1). be coated with the microballoon of the anti-tag sequence of special corresponding wild-type and anomaly respectively, described anti-tag sequence is selected from UGT1A1*28 genotypic SEQ ID NO.13 and SEQ ID NO.14, at the genotypic SEQ IDNO.9 of UGT1A1*6 and SEQ ID NO.10 with at UGT1A1*93 genotypic SEQ ID NO.11 and SEQ ID NO.12, and above-mentioned every kind of microballoon has the different colours coding;
(2). amplify a pair of primer SEQ ID NO.15 and SEQ ID NO.16 and amplify a pair of primer SEQ ID NO.17 and SEQID NO.18 with the genotypic variation target sequence of UGT1A1*93 with UGT1A1*28 and the genotypic variation target sequence of UGT1A1*6; One end in the every pair of primer has biotin modification;
(3). the ASPE primer of corresponding wild-type and anomaly is right, and described ASPE primer is to being respectively at UGT1A1*6 genotype SEQID NO.1 and SEQ ID NO.2 with at UGT1A1*93 genotypic SEQ ID NO.3 and SEQ ID NO.4.
5. one kind is used the described liquid-phase chip of claim 1-2 to the method that the UGT1A1 gene pleiomorphism detects, and it is characterized in that, mainly may further comprise the steps:
(1) use amplimer, pcr amplification testing sample DNA must contain biotin labeled PCR reaction product;
(2) the PCR reaction product is carried out hybridization with bag by the microballoon of special anti-tag sequence;
(3) product behind the hybridization and Streptavidin-phycoerythrin react;
(4) detect by fluorescence detector.
6. one kind is used the described liquid-phase chip of claim 3-4 to the method that the UGT1A1 gene pleiomorphism detects, and it is characterized in that, mainly may further comprise the steps:
(1) use amplimer, pcr amplification testing sample DNA must contain biotin labeled PCR reaction product;
(2) the PCR reaction product is carried out enzyme with the ExoSAP-IT test kit and is cut processing, carry out primer extension reaction with described ASPE primer again, in reaction process, mix biotin labeled dCTP, thereby make a plurality of biotin labeling on the reacted product band, the microballoon of the anti-tag sequence that being coated with of corresponding A SPE primer is special and the product behind the above-mentioned extension carry out hybridization;
(3) product behind the hybridization and Streptavidin-phycoerythrin react;
(4) detect by fluorescence detector.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304566A (en) * | 2011-04-29 | 2012-01-04 | 广州益善生物技术有限公司 | Specific primers and liquid phase chip for polymorphic detection of human hedgehog interacting protein (HHIP) gene |
| CN102899406A (en) * | 2012-09-19 | 2013-01-30 | 长沙三济生物科技有限公司 | Sequencing primer for qualitative detection of genetic typing of uridinediphosphoglucuronate glucuronosyltransferase 1A1 and kit thereof |
| 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 |
| CN105177115A (en) * | 2014-11-17 | 2015-12-23 | 步迅 | UGT1A1 combined gene locus fluorescence detection kit for guiding irinotecan chemotherapeutic drug individualized treatment |
| CN106459932A (en) * | 2014-04-25 | 2017-02-22 | 吉尼松公司 | Treatment of hyperbilirubinemia |
| CN109504747A (en) * | 2017-09-15 | 2019-03-22 | 益善生物技术股份有限公司 | HCCSP T1A1 genetic polymorphism detection kit based on Taqman-MGB probe |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102304566A (en) * | 2011-04-29 | 2012-01-04 | 广州益善生物技术有限公司 | Specific primers and liquid phase chip for polymorphic detection of human hedgehog interacting protein (HHIP) gene |
| CN102899406A (en) * | 2012-09-19 | 2013-01-30 | 长沙三济生物科技有限公司 | Sequencing primer for qualitative detection of genetic typing of uridinediphosphoglucuronate glucuronosyltransferase 1A1 and kit thereof |
| CN102899406B (en) * | 2012-09-19 | 2014-06-04 | 长沙三济生物科技有限公司 | Sequencing primer for qualitative detection of genetic typing of uridinediphosphoglucuronate glucuronosyltransferase 1A1 and kit thereof |
| CN106459932A (en) * | 2014-04-25 | 2017-02-22 | 吉尼松公司 | Treatment of hyperbilirubinemia |
| CN106459932B (en) * | 2014-04-25 | 2022-01-11 | 吉尼松公司 | Treatment of hyperbilirubinemia |
| CN105177115A (en) * | 2014-11-17 | 2015-12-23 | 步迅 | UGT1A1 combined gene locus fluorescence detection kit for guiding irinotecan chemotherapeutic drug individualized treatment |
| CN105177115B (en) * | 2014-11-17 | 2018-06-15 | 步迅 | A kind of UGT1A1 Polymorphisms site fluorescence detection reagent kit for being used to instruct Irinotecan based chemotherapy drug individualized treatment |
| 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 |
| CN109504747A (en) * | 2017-09-15 | 2019-03-22 | 益善生物技术股份有限公司 | HCCSP T1A1 genetic polymorphism detection kit based on Taqman-MGB probe |
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|---|---|
| CN101671740B (en) | 2012-05-23 |
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