CN112391458A - Multi-diagnostic kit - Google Patents

Multi-diagnostic kit Download PDF

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CN112391458A
CN112391458A CN201910737851.7A CN201910737851A CN112391458A CN 112391458 A CN112391458 A CN 112391458A CN 201910737851 A CN201910737851 A CN 201910737851A CN 112391458 A CN112391458 A CN 112391458A
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amplification
site
diagnostic kit
kit
cyp1b1
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张辉
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Xinkaiyuan Huicheng Wuhan Medical Technology Co ltd
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Xinkaiyuan Huicheng Wuhan Medical Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Abstract

The invention discloses a multi-diagnosis kit, which comprises five pairs of amplification primers and a multiplex PCR mixed reaction solution, wherein the amplification primers are used for amplifying CYP1B1 polymorphic sites, including an rs10012 site, an rs1056836 site, an rs1056827 site and/or an rs1056836 site, and each pair of primers respectively corresponds to an upstream region and a downstream region of one SNP site. The invention adopts a multiplex PCR amplification method to simultaneously amplify five important SNP loci of CYP1B1, can flexibly select amplified locus combination according to actual requirements, can play a role of one box with multiple purposes, and has the advantages of high primer specificity, low mismatching rate, primer length and T in a kitmThe value design is reasonable, and the amplification product is convenient to detect and separate. Not only amplify a plurality of target products at one time, but also greatly improveThe amplification efficiency is high, and the amplification cost is reduced. The detection kit can be expanded to other SNP locus detection, and has good popularization value.

Description

Multi-diagnostic kit
Technical Field
The invention relates to a multi-diagnosis kit, and belongs to the field of clinical detection kits.
Background
CYP1B1, a novel member of the CYP450 family, is expressed in both the liver and extrahepatic tissues. CYP1B1 is involved not only in the metabolism of many exogenous compounds such as polycyclic aromatic hydrocarbons, but also in the metabolism of many endogenous metabolites in the body, including the metabolism of steroid hormones, fatty acids, melatonin and vitamins. In addition, CYP1B1 interacts with many nuclear receptors such as PPARs, RAR and ER to maintain metabolic homeostasis of important physiological substances in the body. In recent years, many research results have shown that the occurrence and development of obesity, hypertension, atherosclerosis and cancer can be obviously prevented by regulating CYP1B 1. It is clear that CYP1B1 has become an important target for the treatment of metabolic diseases. CYP1B1 has a plurality of gene polymorphism sites, and site mutation has important significance for screening metabolic diseases, but the index is not detected clinically at present, and an effective rapid detection method is not provided. Aiming at the blank, the polymorphic locus of the gene is to be rapidly detected. Therefore, the design and development of a multi-diagnostic kit have important clinical use value.
Disclosure of Invention
In view of the above problems of the prior art, the present invention aims to obtain a CYP1B1 multiple diagnostic kit.
In order to achieve one of the above objects, the present invention adopts the following technical scheme of a CYP1B1 multiple diagnostic kit:
the kit comprises five pairs of amplification primers and a multiplex PCR mixed reaction solution, wherein the amplification primers are used for amplifying CYP1B1 polymorphic sites, including an rs10012 site, an rs1056836 site, an rs1056827 site, an rs1056836 site and/or an rs1800440 site, and each pair of primers respectively corresponds to an upstream region and a downstream region of one SNP site.
Preferably, the sequence of the amplification primer at the rs10012 locus is shown in a sequence table SEQ ID NO: 1 to 2.
Preferably, the amplification primer sequence of the rs1056836 site is shown as a sequence table SEQ ID NO: 3 to 4.
Preferably, the amplification primer sequence of the rs1056827 locus is shown as a sequence table SEQ ID NO: 5 to 6.
Preferably, the amplification primer sequence of the rs1056836 site is shown as a sequence table SEQ ID NO: 7 to 8.
Preferably, the amplification primer sequence of the rs1800440 site is as shown in a sequence table SEQ ID NO: 9 to 10.
Preferably, the mixed reaction solution for multiplex PCR comprises DNA polymerase and Mg2+And dNTPs. More preferably, the multiplex PCR mixture further comprises a PCR stabilizer and an enhancer.
Preferably, in the amplification system, the addition amount of the amplification primer and the template at each SNP site is the same.
The amplification kit of the invention generally adopts a treated DNA sample as an amplification template, the sample is generally derived from human whole blood or tissues, and the treatment of the sample can reduce the interference of immunoglobulin G, hemoglobin and lactoferrin in the blood on a PCR reaction system.
Preferably, the amplification kit further comprises a whole blood sample PCR buffer solution, wherein the buffer solution contains 100mmol/L Tris-HCl, 50mmol/L KCl and has pH of 9.3-9.5. Whole blood sample PCR buffer is a preferred embodiment of the present invention, and other types of whole blood PCR reagents can be used without limitation to the buffer and buffer formulations of the present invention.
The invention also provides a CYP1B1 polymorphism detection method, which sequentially comprises the following steps: multiplex PCR, amplification product verification and amplification product detection.
Preferably, the amplification system at least comprises a combination of two or more of rs10012, rs1056836, rs1056827, rs1056836 and/or rs1800440, wherein the addition amount of the amplification primer and the template at each SNP site is the same.
Preferably, the amplification system for amplifying the CYP1B1 genes rs1056836, rs10012, rs1056827, rs1056836 and rs1800440 is a 50 mu L PCR system, and the system simultaneously contains amplification primers of rs1056836, rs10012, rs1056827, rs1056836 and rs 1800440.
The amplification system is specifically as follows:
2×Platinum Multiplex PCR Master Mix 25μL；
2 mu L of template;
rs1056836, rs10012, rs1056827, rs1056836 and/or 1800440 at least two pairs of upstream and downstream primers (10. mu. mol/L) each having a concentration of 1. mu.L;
sterile double distilled water is filled to 50 mu L.
The multiple PCR reaction conditions recommended by the kit are as follows: pre-denaturation at 95 ℃ for 10 min; then the mixture is denatured at 96 ℃ for 30s, annealed at 60 ℃ for 90s, and extended at 72 ℃ for 60s, and after 35 cycles, the mixture is extended again at 72 ℃ for 10min and then restored to 4 ℃.
The kit generally adopts a purified DNA sample for amplification detection. However, the kit also comprises a whole blood amplification buffer solution, so that the collected human whole blood sample can be directly amplified.
The invention also aims to provide application of the multi-diagnosis kit and application of the detection kit to CYP1B1 gene polymorphism.
The third object of the present invention is to provide the use of another multiple diagnostic kit for detecting the risk of a disease caused by the polymorphism of CYP1B1 gene.
Compared with the prior art, the invention adopts the multiplex PCR amplification method to simultaneously amplify five important SNP sites of CYP1B1, can flexibly select the amplified site combination according to the actual requirement, can play a role of one box for multiple purposes, and has the advantages of high primer specificity, low mismatching rate, primer length and TmThe value design is reasonable, and the amplification product is convenient to detect and separate. Multiple target products are amplified at one time, so that the amplification efficiency is greatly improved, and the amplification cost is reduced. The detection kit can be expanded to other SNP locus detection, and has good popularization value.
Drawings
FIG. 1 is an electrophoretogram of PCR amplification products provided by the present invention.
Detailed Description
The CYP1B1 multi-diagnostic kit provided by the invention is further detailed and completely described below by referring to examples. The following examples are illustrative only and are not to be construed as limiting the invention.
The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were all commercially available unless otherwise specified.
First, design primer
Primer design is carried out according to polymorphic sites published in NCBI, and rs10012 site is 'C/G' base mutation and is expressed by 'S'. The specific sequence is as follows:
CCTCTTCAGA TGGATTATTA CAGGTAGCGG GTGGCGTGGT AGGTACTTTA AAGGAAATCA AGCGCCACCG CCTCGATGCC CGCAGCGTTG TCCCCAGATT GCAGGAACCG TTACGCGCCT TGCGGGGAGG GGAAGGGTTT GGCGCTGGGT TACAGCGAGG TGGAAACACG CCCCTTCTCT TCTCCAAGGG AGAGTGGGTT GGGGATGGGA AGGGGCGTCT TCGGCCATTT CTCCAGAGAG TCAGCTCCGA CCTCTCCACC CAACGGCACT CAGTCCCCAG AGGCTGGGGT AGGGGCGTGG GGCGCCCGCT CCTGTCTCTG CACCCCTGAG TGTCACGCCT TCTCCTCTCT GTCCCCAGCA TGGGCACCAG CCTCAGCCCG AACGACCCTT GGCCGCTAAA CCCGCTGTCC ATCCAGCAGA CCACGCTCCT GCTACTCCTG TCGGTGCTGG CCACTGTGCA TGTGGGCCAG CGGCTGCTGA GGCAACGGAG GCGGCAGCTC
S
GGTCCGCGCC CCCGGGCCCG TTTGCGTGGC CACTGATCGG AAACGCGGCG GCGGTGGGCC AGGCGGCTCA CCTCTCGTTC GCTCGCCTGG CGCGGCGCTA CGGCGACGTT TTCCAGATCC GCCTGGGCAG CTGCCCCATA GTGGTGCTGA ATGGCGAGCG CGCCATCCAC CAGGCCCTGG TGCAGCAGGG CTCGGCCTTC GCCGACCGGC CGGCCTTCGC CTCCTTCCGT GTGGTGTCCG GCGGCCGCAG CATGGCTTTC GGCCACTACT CGGAGCACTG GAAGGTGCAG CGGCGCGCAG CCCACAGCAT GATGCGCAAC TTCTTCACGC GCCAGCCGCG CAGCCGCCAA GTCCTCGAGG GCCACGTGCT GAGCGAGGCG CGCGAGCTGG TGGCGCTGCT GGTGCGCGGC AGCGCGGACG GCGCCTTCCT CGACCCGAGG CCGCTGACCG TCGTGGCCGT GGCCAACGTC ATGAGTGCCG TGTGTTTCGG CTGCCGCTAC
the rs1056836 locus recorded in NCBI is a base mutation of 'C/G', is expressed by 'S', and has the following specific sequence:
TATAATGGGA AAGACAGCAT TAGTCATGCA AGGCCTATTA CAGGAAATAT AATTCTTAAA GTCCATCTTG TAATTTAGTG AGAAATTAGG AAGCTGTTTT AGATTTTTTT CCCAGAAATA TTAATTTAGT CACTGAGCTA GATAGCCTAT TTAAGAAAAA GTGGAATTAA AATAAATTAT AATGTGCTTT CTAGATGAAA TAAGAATTTT GCTCACTTGC TTTTCTCTCT CCACATTAAA CACCAAACAG GTATCCTGAT GTGCAGACTC GAGTGCAGGC AGAATTGGAT CAGGTCGTGG GGAGGGACCG TCTGCCTTGT ATGGGTGACC AGCCCAACCT GCCCTATGTC CTGGCCTTCC TTTATGAAGC CATGCGCTTC TCCAGCTTTG TGCCTGTCAC TATTCCTCAT GCCACCACTG CCAACACCTC TGTCTTGGGC TACCACATTC CCAAGGACAC TGTGGTTTTT GTCAACCAGT GGTCTGTGAA TCATGACCCA
S
TGAAGTGGCC TAACCCGGAG AACTTTGATC CAGCTCGATT CTTGGACAAG GATGGCCTCA TCAACAAGGA CCTGACCAGC AGAGTGATGA TTTTTTCAGT GGGCAAAAGG CGGTGCATTG GCGAAGAACT TTCTAAGATG CAGCTTTTTC TCTTCATCTC CATCCTGGCT CACCAGTGCG ATTTCAGGGC CAACCCAAAT GAGCCTGCGA AAATGAATTT CAGTTATGGT CTAACCATTA AACCCAAGTC ATTTAAAGTC AATGTCACTC TCAGAGAGTC CATGGAGCTC CTTGATAGTG CTGTCCAAAA TTTACAAGCC AAGGAAACTT GCCAATAAGA AGCAAGAGGC AAGCTGAAAT TTTAGAAATA TTCACATCTT CGGAGATGAG GAGTAAAATT CAGTTTTTTT CCAGTTCCTC TTTTGTGCTG CTTCTCAATT AGCGTTTAAG GTGAGCATAA ATCAACTGTC CATCAGGTGA
GGTGTGCTCC ATACCCAGCG
rs1056827 described in NCBI is a "G/T" base mutation, denoted by "K", and has the following specific sequence:
CAGCAGACCA CGCTCCTGCT ACTCCTGTCG GTGCTGGCCA CTGTGCATGT GGGCCAGCGG CTGCTGAGGC AACGGAGGCG GCAGCTCCGG TCCGCGCCCC CGGGCCCGTT TGCGTGGCCA CTGATCGGAA ACGCGGCGGC GGTGGGCCAG GCGGCTCACC TCTCGTTCGC TCGCCTGGCG CGGCGCTACG GCGACGTTTT CCAGATCCGC CTGGGCAGCT GCCCCATAGT GGTGCTGAAT GGCGAGCGCG CCATCCACCA GGCCCTGGTG CAGCAGGGCT CGGCCTTCGC CGACCGGCCG K
CCTTCGCCTC CTTCCGTGTG GTGTCCGGCG GCCGCAGCAT GGCTTTCGGC CACTACTCGG AGCACTGGAA GGTGCAGCGG CGCGCAGCCC ACAGCATGAT GCGCAACTTC TTCACGCGCC AGCCGCGCAG CCGCCAAGTC CTCGAGGGCC ACGTGCTGAG CGAGGCGCGC GAGCTGGTGG CGCTGCTGGT GCGCGGCAGC GCGGACGGCG CCTTCCTCGA CCCGAGGCCG CTGACCGTCG TGGCCGTGGC CAACGTCATG AGTGCCGTGT GTTTCGGCTG CCGCTACAGC CACGACGACC
rs1056836 described in NCBI is a base mutation, and "S" indicates that the mutant base is a "C/G" mutation, and the specific sequence is as follows:
TATAATGGGA AAGACAGCAT TAGTCATGCA AGGCCTATTA CAGGAAATAT AATTCTTAAA GTCCATCTTG TAATTTAGTG AGAAATTAGG AAGCTGTTTT AGATTTTTTT CCCAGAAATA TTAATTTAGT CACTGAGCTA GATAGCCTAT TTAAGAAAAA GTGGAATTAA AATAAATTAT AATGTGCTTT CTAGATGAAA TAAGAATTTT GCTCACTTGC TTTTCTCTCT CCACATTAAA CACCAAACAG GTATCCTGAT GTGCAGACTC GAGTGCAGGC AGAATTGGAT CAGGTCGTGG GGAGGGACCG TCTGCCTTGT ATGGGTGACC AGCCCAACCT GCCCTATGTC CTGGCCTTCC TTTATGAAGC CATGCGCTTC TCCAGCTTTG TGCCTGTCAC TATTCCTCAT GCCACCACTG CCAACACCTC TGTCTTGGGC TACCACATTC CCAAGGACAC TGTGGTTTTT GTCAACCAGT GGTCTGTGAA TCATGACCCA
S
TGAAGTGGCC TAACCCGGAG AACTTTGATC CAGCTCGATT CTTGGACAAG GATGGCCTCA TCAACAAGGA CCTGACCAGC AGAGTGATGA TTTTTTCAGT GGGCAAAAGG CGGTGCATTG GCGAAGAACT TTCTAAGATG CAGCTTTTTC TCTTCATCTC CATCCTGGCT CACCAGTGCG ATTTCAGGGC CAACCCAAAT GAGCCTGCGA AAATGAATTT CAGTTATGGT CTAACCATTA AACCCAAGTC ATTTAAAGTC AATGTCACTC TCAGAGAGTC CATGGAGCTC CTTGATAGTG CTGTCCAAAA TTTACAAGCC AAGGAAACTT GCCAATAAGA AGCAAGAGGC AAGCTGAAAT TTTAGAAATA TTCACATCTT CGGAGATGAG GAGTAAAATT CAGTTTTTTT CCAGTTCCTC TTTTGTGCTG CTTCTCAATT AGCGTTTAAG GTGAGCATAA ATCAACTGTC CATCAGGTGA GGTGTGCTCC ATACCCAGCG
rs1800440 described in NCBI is "A/C/G" base mutation, denoted by "V", and has the following specific sequence:
GTCTGCCTTG TATGGGTGAC CAGCCCAACC TGCCCTATGT CCTGGCCTTC CTTTATGAAG CCATGCGCTT CTCCAGCTTT GTGCCTGTCA CTATTCCTCA TGCCACCACT GCCAACACCT CTGTCTTGGG CTACCACATT CCCAAGGACA CTGTGGTTTT TGTCAACCAG TGGTCTGTGA ATCATGACCC ACTGAAGTGG CCTAACCCGG AGAACTTTGA TCCAGCTCGA TTCTTGGACA AGGACGGCCT CATCA
V
CAAGGACCTG ACCAGCAGAG TGATGATTTT TTCAGTGGGC AAAAGGCGGT GCATTGGCGA AGAACTTTCT AAGATGCAGC TTTTTCTCTT CATCTCCATC CTGGCTCACC AGTGCGATTT CAGGGCCAAC CCAAATGAGC CTGCGAAAAT GAATTTCAGT TATGGTCTAA CCATTAAACC CAAGTCATTT AAAGTCAATG TCACTCTCAG AGAGTCCATG GAGCTCCTTG ATAGTGCTGT CCAAAATTTACAAGC
specific amplification primers are designed according to the sequences, so that rapid and efficient amplification is facilitated. The primer sequences are as follows:
Figure RE-GDA0002195196520000061
in this example, the multiplex PCR method is used to amplify five SNP sites simultaneously or randomly.
II, kit composition
The rapid amplification kit in the embodiment is a multiplex PCR kit for simultaneously amplifying five higher SNP sites of CYP1B1, and comprises upstream and downstream amplification primers of five pairs of SNP sites, an amplification template extraction reagent and a multiplex PCR mixed reaction solution, wherein the reaction solution comprises DNA polymerase and Mg2+dNTPs, PCR stabilizers and enhancers.
The DNA template used in the kit of this embodiment is a human blood DNA template extracted after processing, and the collected human whole blood or tissue needs to be processed in advance. If the sample to be amplified is an unprocessed whole blood sample, a buffer solution for PCR of the whole blood sample is adopted, wherein the buffer solution contains 100mmol/L Tris-HCl, 50mmol/L KCl and has the pH value of 9.3-9.5.
Triple, multiplex PCR
The Multiplex PCR selects Platinum Multiplex PCR Master Mix as a reaction system, which comprises DNA polymerase and Mg2+dNTPs, PCR stabilizer and enhancer, wherein the Invitrogen Platinum Multiplex PCR Master Mix is selected in the experiment, and the PCR system is 50 mu L, wherein
2×Platinum Multiplex PCR Master Mix 25μL；
2 mu L of template;
at least two pairs of upstream and downstream primers (10 mu mol/L) of rs1056836, rs10012, rs1056827, rs1056836 and rs1800440 are respectively 1 mu L; sterile double distilled water is filled to 50 mu L.
And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 10 min; then the mixture is denatured at 96 ℃ for 30s, annealed at 60 ℃ for 90s, and extended at 72 ℃ for 60s, and after 35 cycles, the mixture is extended again at 72 ℃ for 10min and then restored to 4 ℃.
Fifth, detection of amplification result
1. Amplification product detection
Because multiple PCR has more influence factors, reaction conditions, primer sites, amplification products and other information need to be comprehensively considered during amplification, and the amplification primer combination in the embodiment is repeatedly verified through multiple experiments, and the multiple PCR can be realized with the minimum mutual influence.
After the upstream and downstream primers in this embodiment are used for combined amplification, amplification products are verified, 2% agarose gel electrophoresis is selected for PCR products, and the electrophoresis results are shown in fig. 1, wherein bands 1 and 2 are the amplification product sequence of rs10012, bands 3 and 4 are the amplification product sequence of rs1056836, bands 5 and 6 are the amplification product sequence of rs1056827, bands 7 and 8 are the amplification product sequence of rs1056836, bands 9 and 10 are the amplification product sequence of rs1800440, and the electrophoresis band positions indicate that the amplification results are correct.
And recovering the electrophoresis result gel and then sequencing, wherein the sequencing result is consistent with the sequence of the target product.
Finally, it must be said here that: the above embodiments are only used for further detailed description of the technical solutions of the present invention, and should not be understood as limiting the scope of the present invention, and the insubstantial modifications and adaptations made by those skilled in the art according to the above descriptions of the present invention are within the scope of the present invention.

Claims (10)

1. A multi-diagnostic kit characterized by: the kit comprises five pairs of amplification primers and a multiplex PCR mixed reaction solution, wherein the amplification primers are used for amplifying CYP1B1 polymorphic sites, including an rs10012 site, an rs1056836 site, an rs1056827 site, an rs1056836 site and/or an rs1800440 site, and each pair of primers respectively corresponds to an upstream region and a downstream region of one SNP site.
2. The multiple diagnostic kit of claim 1, wherein: the sequence of the amplification primer of the rs10012 locus is shown in a sequence table SEQ ID NO: 1 to 2.
3. The multiple diagnostic kit of claim 1, wherein: the amplification primer sequence of the rs1056836 locus is shown as a sequence table SEQ ID NO: 3 to 4.
4. The multiple diagnostic kit of claim 1, wherein: the sequence of the amplification primer of the rs1056827 locus is shown as a sequence table SEQ ID NO: 5 to 6.
5. The multiple diagnostic kit of claim 1, wherein: the amplification primer sequence of the rs1056836 locus is shown as a sequence table SEQ ID NO: 7 to 8.
6. The multiple diagnostic kit of claim 1, wherein: the amplification primer sequence of the rs1800440 locus is shown as a sequence table SEQ ID NO: 9 to 10.
7. The multiple diagnostic kit of claim 1, wherein: the multiple PCR mixed reaction solution comprises DNA polymerase and Mg2+And dNTPs.
8. The multiple diagnostic kit according to claim 7, wherein the amplification system comprises at least a combination of two or more of rs10012, rs1056836, rs1056827 and/or rs1056836, wherein the amplification primers and templates are added in the same amount for each SNP site.
9. Use of a multiple diagnostic kit according to any one of claims 1 to 8, characterized in that: the detection kit is applied to detecting CYP1B1 polymorphism.
10. Use of a multiple diagnostic kit according to any one of claims 1 to 8, characterized in that: the multi-diagnosis kit is applied to detecting the risk of diseases caused by CYP1B1 gene polymorphism.
CN201910737851.7A 2019-08-12 2019-08-12 Multi-diagnostic kit Pending CN112391458A (en)

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Application publication date: 20210223