CN117904281B - Primer set, kit and method for detecting UGT1A1 gene polymorphism - Google Patents
Primer set, kit and method for detecting UGT1A1 gene polymorphism Download PDFInfo
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- C12Q2600/00—Oligonucleotides characterized by their use
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Abstract
本发明涉及生物检测分析技术领域，公开一种检测UGT1A1基因多态性的引物组及试剂盒与方法，其检测UGT1A1基因多态性的方法，包括：从待测样品中提取基因组DNA；使用检测UGT1A1基因多态性的试剂盒配制荧光定量PCR预混液；取配置好的荧光定量PCR预混液加入到GS反应管中，放入荧光定量PCR仪中进行荧光定量PCR反应，检测荧光信号并判定结果。本发明提供的基于荧光PCR仪的PAP‑qPCR法的试剂盒能够更精准的分析重复序列的重复数量、性能更优，且具有操作步骤少、便捷、易于标准化的优势，适合在临床实验室进行推广。
The present invention relates to the technical field of biological detection and analysis, and discloses a primer set, a kit and a method for detecting UGT1A1 gene polymorphism. The method for detecting UGT1A1 gene polymorphism comprises: extracting genomic DNA from a sample to be tested; preparing a fluorescent quantitative PCR premix using a kit for detecting UGT1A1 gene polymorphism; taking the prepared fluorescent quantitative PCR premix and adding it to a GS reaction tube, placing it in a fluorescent quantitative PCR instrument for fluorescent quantitative PCR reaction, detecting fluorescent signals and determining the results. The kit of the PAP‑qPCR method based on a fluorescent PCR instrument provided by the present invention can more accurately analyze the number of repetitions of a repetitive sequence, has better performance, and has the advantages of fewer operating steps, convenience, and easy standardization, and is suitable for promotion in clinical laboratories.
Description
Technical Field
The invention relates to a primer group, a kit and a method for detecting UGT1A1 gene polymorphism, and belongs to the technical field of biological detection and analysis.
Background
Hereditary non-binding hyperbilirubinemia, including Crigler-Najjar syndrome type I (CN-1), crigler-Najjar syndrome type II (CN-2) and Gilbert Syndrome (GS), is caused by mutation of the bilirubin uridine diphosphate glucuronyltransferase 1A1 (UDP-GTase) gene. The coding gene UGT1A1 of UDP-GT enzyme is located at 2q37.1, the DNA thereof comprises 13027 bases, the mRNA consists of 2361 bases, and the protein is encoded by 533 amino acids. In bilirubin metabolism, unbound bilirubin (IBil) is metabolized to bound bilirubin (DBil) via UGT1A1, UGT1A1 being the primary rate-limiting metabolizing enzyme in the reaction. When the UGT1A1 gene mutation results in reduced or lost enzyme activity, unbound bilirubin accumulates in the body as it is not converted to bound bilirubin.
In normal population, about 6-12% of people have deficiency of UDP-GT enzyme, and the biochemical indexes of the human UDP-GT enzyme are that indirect bilirubin is increased, ALT and other liver function indexes are normal. Patients are shown to be fatigued, listless, etc., and the causes of the illness include infections (e.g. cold or influenza), fasting or eating low calorie diets, dehydration, strenuous exercise, stress, female menstruation, etc. UGT1A1 gene polymorphism is the molecular genetic basis of Gilbert syndrome, and can be used for assisting in diagnosing the Gilbert syndrome.
UGT1A1 gene mutation comprises various forms such as insertion, deletion, single nucleotide polymorphism and the like, thus leading to great individual differences among sequences. It has now been found that 160 mutants of this gene are identified, in international generic nomenclature, as UGT1A1 x1, and mutants are identified in turn as x2, 3, 4, 5, etc. Currently, UGT1A1 genetic polymorphisms are mainly in 3 forms: ① Transliterated mutations in the exon region, such as ：UGT1A1*6(c.211G＞A,G71R)、UGT1A1*27(c.686C>A,P229Q)、UGT1A1*63(c.1091C>T,P364L)、UGT1A1*7(c.1456T>G,Y486D), are most common with nucleotide 211 at position G > A (G71R) on exon 1. ② A TATA box TA insertion mutation of the promoter is expressed by inserting dinucleotide (TA) into the TATA box at about 25-35 bp upstream of the UGT1A1 gene promoter, so that the normal wild type A [ TA ]6TAA is mutated into A [ TA ]7TAA or A [ TA ]8TAA and other polymorphism. ③ The phenobarbital response enhancing element (phenobarbital-responsive enhancer module, PBREM) region, c. -3279T > G, which has a strong linkage to A [ TA ]7 TAA. In recent years, the combination of genetic rules based on indirect bilirubin levels and gene detection results has been an important means for diagnosing Gilbert syndrome.
The patent with publication number CN112592970A discloses a kit for detecting the multi-site mutation of the UGT1A1 of the Gilbert syndrome, which comprises a primer pair and a probe for detecting the HRM of a non-labeled probe aiming at four mutation sites p.G71R, p.P229Q, p.Y 4816D and A [ TA ]7TAA of the UGT1A1 respectively, wherein the method has quite high requirements on the temperature resolution and the temperature uniformity of an instrument.
Disclosure of Invention
The invention aims to solve the problems of incomplete variety, complicated operation and long time for detecting UGT1A1 gene polymorphism product typing in the prior art, and provides a primer group for detecting UGT1A1 gene polymorphism, which comprises the following components:
a primer for detecting UGT1A1 x 6 gene, comprising:
Upstream primer for 211A typing U-6A-F1:5'-CGTTGTACATCAGAGACa-3' the process of the preparation of the pharmaceutical composition,
Upstream primer for 211G typing U-6G-F1:5'-CGTTGTACATCAGAGACg-3' the process of the preparation of the pharmaceutical composition,
211G/a typing common downstream primer UGT 6-R1:5'-CCGAGACTAACAAAAGACTCT-3';
A primer for detecting UGT1A1 x 28 gene, comprising:
c. -53TA6/TA7 typing common upstream primer U-286-F1:5'-CCTGCTACCTTTGTGGACTG-3' the process of the preparation of the pharmaceutical composition,
C. Downstream primer U-7R-R21 for typing-53 TA 7: 5'-GCCCTCTCCTACTTATATATATATATAaGG-3', c. -53TA6 typing downstream primer U-6R-R21:5'-GCCCTCTCCTACTTATATATATATATATAaGG-3';
A primer for detecting UGT1A1 x 60 gene, comprising:
c. -3279T typing upstream primer U-3279T-22F1:5'-CCAAGGGTAGAGTTCAGTt-3' the process of the preparation of the pharmaceutical composition,
C. Upstream primer for typing 3279G U-3279G-22F1:5'-CCAAGGGTAGAGTTCAGTg-3' the process of the preparation of the pharmaceutical composition,
C. -3279T/G typing sharing downstream primer U-3279-22R1:5'-atCAAACTTcCTTTGATGTTCt-3';
a primer for detecting UGT1A1 x 27 gene, comprising:
upstream primer U-P229-F1 for typing 686C: 5'-GACGTGGTTTATTCCCc-3' the process of the preparation of the pharmaceutical composition,
Upstream primer U-P229Q-F1 for typing 686A: 5'-GACGTGGTTTATTCCCa-3' the process of the preparation of the pharmaceutical composition,
C.686C/A typing shares the downstream primer U-P229-R2:5'-agaGGACAGTCACCTCTCTCTGAA-3';
A primer for detecting UGT1A1 x 63 gene, comprising:
1091C/T typing common upstream primer U-P364-F11:5'-gacACCTAGATGTGTCCAGC-3' the process of the preparation of the pharmaceutical composition,
Downstream primer U-P364-R1 for 1091C typing: 5'-AGGCACGGGTCATCGg-3' the process of the preparation of the pharmaceutical composition,
Downstream primer U-P364L-R102 for 1091T typing: 5'-AAAGGCACGGGTCATCa-3';
A primer for detecting UGT1A1 x 7 gene, comprising:
1456T typing upstream primer U-Y486-F1:5'-CCTCACCTGGTACCAGt-3' the process of the preparation of the pharmaceutical composition,
1456G typing upstream primer U-Y486D-F4:5'-GACCTCAaCTGGTACCAGg-3' the process of the preparation of the pharmaceutical composition,
1456T/G typing shares the downstream primer U-Y486-R1:5'-gtttCCAAGAGGAAACCAATCAC-3'.
Preferably, the 5 'end of the primer U-6A-F1、U-6G-F1、U-P229-F1、U-P229Q-F1、U-Y486-F1、U-Y486D-F4、U-7R-R21、U-6R-R21、U-3279T-22F1、U-3279G-22F1、U-P364-R1、U-P364L-R102 is marked with a fluorescence reporting group, and the 3' end is marked with a fluorescence quenching group; preferably, differently labeled primers at the same locus are labeled with the same label.
Preferably, the fluorescent reporter group marked on the 5 'end comprises one or more of FAM, texas Red and ROX, HEX, VIC, NED, CY, and the fluorescent quenching group marked on the 3' end comprises one or more of BHQ1, BHQ2 and BHQ 3.
Preferably, the 3' end of the primer is dehydroxylated.
The invention also provides a kit for detecting UGT1A1 gene polymorphism, which comprises a PCR reaction solution and a GS reaction tube, wherein the GS reaction tube contains a primer group for detecting UGT1A1 gene polymorphism.
Preferably, the PCR reaction solution comprises 0.1-0.2U/. Mu.L Taq enzyme, 1.5-5 mM MgCl 2, 20mM Tris-HCl, 100mM KCl, 0.05-0.5 mM dNTPs, and 5mM sodium pyrophosphate.
Preferably, the concentration of the primer set is 12 pmol/tube.
The invention also provides a method for detecting UGT1A1 gene polymorphism, which comprises the following steps:
Extracting genome DNA from a sample to be detected;
Preparing fluorescent quantitative PCR premix by using a kit for detecting UGT1A1 gene polymorphism;
And adding the prepared fluorescent quantitative PCR premix into a GS reaction tube, putting the GS reaction tube into a fluorescent quantitative PCR instrument for fluorescent quantitative PCR reaction, detecting a fluorescent signal and judging a result.
Preferably, the fluorescent quantitative PCR premix comprises 44. Mu.L of PCR reaction liquid, 120-400 ng of sample DNA and water.
Preferably, the reaction procedure of the fluorescent quantitative PCR is: 95 ℃ for 10min;95 ℃,10 s,60 ℃, 45s,5 cycles; 95 ℃,10 s,60 ℃, 35s,40 cycles; 25 ℃ for 10s
The invention has the beneficial effects that:
1. The invention uses a pyrophosphorolysis-started fluorescent PCR method (PAP-qPCR) to detect UGT1A1 gene polymorphism, and the principle of the method is as follows: inorganic pyrophosphoric acid is added into a PCR reaction system, when the 3 '-end of the primer is not provided with hydroxyl, the primer is completely complementary with the template, inorganic pyrophosphoric acid is used for decomposing the 3' -end base of the primer (which can be regarded as the reverse reaction of DNA polymerization, and template DNA is needed to exist for the hydrolysis of DNA chain), and after the first base of the 3 '-end of the primer is removed, the primer for carrying out PCR reaction is formed at the 3' -end, namely the PCR reaction is started; the labeled primer is labeled with a 5 'fluorescent group-3' quenching group, which is detached from the primer when pyrophosphorolysis occurs, forming a fluorescent signal.
2. Compared with the PCR-Sanger sequencing method, the kit based on the PAP-qPCR detection method of the fluorescent PCR instrument provided by the invention is simple and convenient to operate, short in detection time and timely in report response. In addition, PAP-qPCR can more accurate analysis repeat sequence's repetition number, performance are better, and have that the operation step is few, convenient, easily standardized advantage, be fit for promoting in clinical laboratory.
Drawings
FIG. 1 is a fluorescent quantitative PCR amplification curve of sample No. 3 UGT1A1 x 6 type site.
FIG. 2 is a fluorescent quantitative PCR amplification curve of sample No. 1 UGT1A1 x 6 type site.
FIG. 3 is a fluorescent quantitative PCR amplification curve for sample No. 13 UGT1A1 x 6.
FIG. 4 is a fluorescent quantitative PCR amplification curve of sample No. 3 UGT1A1 x 28 type site.
FIG. 5 is a fluorescent quantitative PCR amplification curve for sample No. 1 UGT1A1 x 28 type sites.
FIG. 6 is a fluorescent quantitative PCR amplification curve for sample No. 6 UGT1A1 x 28 type sites.
FIG. 7 is a fluorescent quantitative PCR amplification curve for sample No. 7 UGT1A1 x 60 type sites.
FIG. 8 is a fluorescent quantitative PCR amplification curve for sample No. 1 UGT1A1 x 60.
FIG. 9 is a fluorescent quantitative PCR amplification curve for sample No. 5 UGT1A1 x 60.
FIG. 10 is a fluorescent quantitative PCR amplification curve for sample No. 2 UGT1A1 x 27 type sites.
FIG. 11 is a fluorescent quantitative PCR amplification curve for sample No. 3 UGT1A1 x 27 type sites.
FIG. 12 is a fluorescent quantitative PCR amplification curve for sample No. 1 UGT1A1 x 63.
FIG. 13 is a fluorescent quantitative PCR amplification curve for sample No. 21 UGT1A1 x 63.
FIG. 14 is a fluorescent quantitative PCR amplification curve for sample No. 2 UGT1A1 x 7.
FIG. 15 is a fluorescent quantitative PCR amplification curve for sample No. 26 UGT1A1 x 7.
Detailed Description
The invention is further described below with reference to the accompanying drawings. The following examples are only for more clearly illustrating the technical aspects of the present invention, and are not intended to limit the scope of the present invention.
The invention detects UGT1A1 gene polymorphism sites by PAP-qPCR method, wherein the sites to be detected are Gilbert syndrome related sites, and the related information of the sites to be detected is shown in Table 1.
TABLE 1 sites to be tested
Examples
A method for detecting UGT1A1 gene polymorphism, comprising the steps of:
(1) Sample DNA extraction
Oral swab samples of 26 testers were collected and DNA was extracted using a commercial DNA extraction kit (beijing root DNA extraction kit, DP 322), with the following steps:
a) The oral swab sample was placed in a 2mL centrifuge tube, 400. Mu.L of buffer GA and 20. Mu.L of proteinase K solution were added, mixed well with shaking, and left at 56℃for 60min.
B) 400. Mu.L of buffer GB was added, mixed well with shaking, and left at 70℃for 10min, and the solution was transferred to a new centrifuge tube.
C) 200 mu L of absolute ethyl alcohol is added into a new centrifuge tube, the mixture is stirred and mixed uniformly, the mixture is added into an adsorption column, the mixture is centrifuged at 12000rpm for 30s, and washing liquid in a waste liquid collecting tube is poured out.
D) mu.L of buffer GD was added, and the mixture was centrifuged at 12000rpm for 30 seconds, to discard the washing liquid in the waste collection tube.
E) 600. Mu.L of the rinse PW was added and centrifuged at 12000rpm for 30s, the wash solution was poured off from the waste collection tube, and the mixture was centrifuged at 12000rpm for 2min.
F) Placing the adsorption column into a 1.5mL centrifuge tube, adding 50 mu L of eluent, centrifuging at 12000rpm for 2min to elute DNA, and collecting the solution into the centrifuge tube to obtain the solution which is the sample DNA.
(2) Fluorescent quantitative PCR detection
Fluorescent quantitative PCR premix was prepared using a kit for detecting UGT1A1 gene polymorphism, and the detailed composition of the kit is shown in tables 2,3 and 4. The PCR reaction solution in the kit for detecting UGT1A1 gene polymorphism was taken out, and a premix was prepared according to Table 5. Taking a reaction tube in a kit for detecting UGT1A1 gene polymorphism, performing instantaneous centrifugation for 30s, sequentially sucking 20 mu L of premix liquid, adding the premix liquid into a prepared GS reaction tube, closing a PCR tube cover by an upper cover, and recording sample loading and placing sequences. The PCR reaction tube was placed in an ABI 7500 fluorescence quantitative PCR apparatus, the procedure was as in Table 6, and fluorescence detection was performed at 60℃for "40 cycles" in the third step of the procedure, to detect fluorescence of FAM and HEX.
TABLE 2 composition of the kit
TABLE 3 primer composition in GS reaction tube
| Reaction tube number | Primer(s) |
| 1 | U-6A-F1、UGT*6-R1 |
| 2 | U-6G-F1、UGT*6-R1 |
| 3 | U-286-F1、U-7R-R21 |
| 4 | U-286-F1、U-6R-R21 |
| 5 | U-3279T-22F1、U-3279-22R1 |
| 6 | U-3279G-22F1、U-3279-22R1 |
| 7 | U-P229-F1、U-P229-R2 |
| 8 | U-P229Q-F1、U-P229-R2 |
| 9 | U-P364-F11、U-P364-R1 |
| 10 | U-P364-F11、U-P364L-R102 |
| 11 | U-Y486-F1、U-Y486-R1 |
| 12 | U-Y486-F4、U-Y486-R1 |
Table 4 primers
TABLE 5 composition of premix
TABLE 6 fluorescent quantitative PCR reaction procedure
(3) Analysis of results
And automatically storing the result after the reaction is finished, and analyzing the amplification curve. And adjusting the Start value and End value of Baseline according to the analyzed image (the user can adjust the image according to the actual situation, the Start value can be set at 3-15, the End value can be set at 5-20), the Threshold value (Rn) is set at the inflection point of fluorescence signal rise, the analysis is performed by clicking Analyze, then the Ct value of each reaction is recorded under the Plate window, and the amplification curves of part of samples are shown in figures 1-15.
Judging the genotype of the sample according to the delta Ct of the difference between the Ct value of the mutant type detection system and the Ct value of the wild type detection system, and if the delta Ct is less than or equal to-3, judging the genotype of the locus of the sample to be mutant; if delta Ct is less than 3 and minus 3, the genotype of the gene locus of the sample is heterozygous; if delta Ct is more than or equal to 3, the genotype of the gene locus of the sample is wild type, the result in the table 7 is referred to the result judgment and detection data are analyzed, and the sample result is shown in the table 8.
Table 7 results determination basis
TABLE 8 sample test results
As can be seen from Table 8, 26 samples were examined for UGT1A1 gene polymorphism by the method of example, wherein 11 wild-type cases, 13 heterozygotes and 2 mutant cases were found at UGT1A1 x 6 locus; 6 wild-type cases, 9 heterozygotes and 11 mutant cases of UGT1A1 x 28 locus are shown; 4 wild-type cases, 15 heterozygotes and 7 mutant cases of UGT1A1 x 60 locus; 23 wild-type cases and 3 heterozygous cases of UGT1A1 x 27 locus are shown; 25 wild-type cases and 1 case of heterozygous cases of UGT1A1 x 63 sites; there were 24 wild-type cases, 1 case for heterozygous cases, and 1 case for mutant cases at UGT1A1 x 7 sites. The 26 samples in this example were simultaneously genotyped using Sanger sequencing, and the measurement result was consistent with that of the detection method in this example, so the method in this example was highly accurate, and compared with the Sanger sequencing, the detection method in this example was simpler and more time-saving to operate.
The foregoing is merely a preferred embodiment of the present invention, and it should be noted that modifications and variations could be made by those skilled in the art without departing from the technical principles of the present invention, and such modifications and variations should also be regarded as being within the scope of the invention.
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| CN101381766A (en) * | 2008-06-11 | 2009-03-11 | 厦门大学 | A Quantitative Detection Method for Rare Gene Mutations |
| KR101712454B1 (en) * | 2015-12-11 | 2017-03-07 | 대한민국 | Reagent for SNP-genotyping of a gene related with anticancer drug-metabolizing enzyme and transporter, the kit comprising the same, and the method for the SNP-genotyping |
| CN106520950A (en) * | 2016-11-16 | 2017-03-22 | 武汉海吉力生物科技有限公司 | UGT1A1 gene polymorphism detection primer and probe and kit |
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| EP2071025A4 (en) * | 2006-11-30 | 2009-12-09 | Arkray Inc | Primer set for amplification of ugt1a1 gene, reagent for amplification of ugt1a1 gene comprising the same, and use of the same |
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| KR101712454B1 (en) * | 2015-12-11 | 2017-03-07 | 대한민국 | Reagent for SNP-genotyping of a gene related with anticancer drug-metabolizing enzyme and transporter, the kit comprising the same, and the method for the SNP-genotyping |
| CN106520950A (en) * | 2016-11-16 | 2017-03-22 | 武汉海吉力生物科技有限公司 | UGT1A1 gene polymorphism detection primer and probe and kit |
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| 利用多重荧光定量PCR协助诊断Crigler-Najjar综合征Ⅱ型及Gilbert综合征;刘凡 等;胃肠病学和肝病学杂志;20161231;第25卷(第12期);第1390页1． 4 引物与Tapman 探针的设计 * |
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