CN112410415A - Kit for detecting human hyperlipemia sensitive gene - Google Patents
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Abstract
The invention discloses a kit for detecting human hyperlipemia sensitive genes, which comprises an APOE primer group, an SLCO1B1-1 primer group, an SLCO1B1-2 primer group, a COQ2 primer group and a CYP3A5 primer group; the invention takes human hyperlipemia drug sensitive genes APOE, SLCO1B1, COQ2 and CYP3A5 as detection objects, and combines the nucleic acid mass spectrum detection technology through the combination of specific primers, thereby realizing the rapid, simple and accurate detection of the variation condition of related genes.
Description
Technical Field
The invention relates to a kit for detecting a human hyperlipemia sensitive gene, belonging to the technical field of biological medicines.
Background
Pharmacogenomic studies show that the metabolism of hyperlipidemic drugs (pravastatin, atorvastatin and rosuvastatin) is closely related to mitochondrial gene MT-RNR1 in the human genome; in the process of medication, the individual with MT-RNR1 variation has obvious difference from the normal individuals in the side effects such as hearing loss, deafness, nephrotoxicity and the like caused by the individual; therefore, before the medicine is used, the mutation condition of the related gene is detected, and the medicine has important guiding significance for patients.
The measurement technology of the drug metabolism gene of hyperlipidemia in the current market is mostly carried out by adopting the technologies such as fluorescence PCR and the like; the detection flux is small, and the detection cost is high.
The invention aims to perform mutation detection on sensitive genes of the human diabetes drug by a single-base-extended nucleic acid mass spectrometry technology, thereby providing safety guidance for individual clinical drug administration.
Disclosure of Invention
In view of the above technical problems, the present invention aims to: provides a more accurate and efficient nucleic acid mass spectrum detection kit for sensitive gene mutation sites of human hyperlipidemia drugs.
The technical solution of the invention is realized as follows: a kit for detecting human hyperlipemia sensitive genes comprises an APOE primer group, an SLCO1B1-1 primer group, an SLCO1B1-2 primer group, a COQ2 primer group and a CYP3A5 primer group; wherein the content of the first and second substances,
the APOE primer group consists of a forward primer APOEF1, a reverse primer APOER1 and a single-base extension primer APOESP 1;
the SLCO1B1-1 primer group consists of a forward primer SLCO1B1F1, a reverse primer SLCO1B1R1 and a single-base extension primer SLCO1B1SP 1;
the SLCO1B1-2 primer group consists of a forward primer SLCO1B1F2, a reverse primer SLCO1B1R2 and a single-base extension primer SLCO1B1SP 2;
the COQ2 primer group consists of a forward primer COQ2F1, a reverse primer COQ2R1 and a single-base extension primer COQ2SP 1;
the CYP3A5 primer group consists of a forward primer CYP3A5F1, a reverse primer CYP3A5R1 and a single-base extension primer CYP3A5SP 1;
the nucleic acid mass spectrum detection kit comprises the following reaction systems:
first, multiplex PCR reaction:
primer set a comprising primers APOEF1, APOER1, SLCO1B1F1, SLCO1B1R1, SLCO1B1F2, SLCO1B1R2, COQ2F1, COQ2R1, CYP3A5F1 and CYP3A5R 1;
PCR master Mix；
H20；
a DNA template;
the second step is that: single base extension reaction:
the above PCR reaction solution;
extending the iPLEX mixed solution;
primer set B comprising primers APOESP1, SLCO1B1SP1, SLCO1B1SP2, COQ2SP1, SP1SP 1;
H2O。
preferably, the primer sequence comprises forward primer APOEF1, reverse primer APOER1 and single base extension primer APOESP1, such as: SEQ ID Nos: 140, SEQ ID Nos: 141, SEQ ID Nos: 150, or a sequence shown in fig.
Preferably, the forward primer SLCO1B1F1, the reverse primer SLCO1B1R1 and the single-base extension primer SLCO1B1SP1 sequentially comprise the following components: SEQ ID Nos: 142, SEQ ID Nos: 143, SEQ ID Nos: 151, respectively.
Preferably, the forward primer SLCO1B1F2, the reverse primer SLCO1B1R2 and the single-base extension primer SLCO1B1SP2 sequentially comprise the following components: SEQ ID Nos: 144, SEQ ID Nos: 145, SEQ ID Nos: 152.
Preferably, the forward primer COQ2F1, the reverse primer COQ2R1, and the single-base extension primer COQ2SP1 sequentially include: SEQ ID Nos: 146, SEQ ID Nos: 147, SEQ ID Nos: 153, respectively.
Preferably, the forward primer CYP3A5F1, the reverse primer CYP3A5R1, and the single base extension primer CYP3A5SP1 sequentially include the following: SEQ ID Nos: 148, SEQ ID Nos: 149, SEQ ID Nos: 154.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
the invention takes the sensitive gene mutation site for human hyperlipemia as a detection object, and can realize accurate, simple and rapid detection of the sensitive gene mutation site for human hyperlipemia through the combination of specific primers and the nucleic acid mass spectrum detection technology, and has higher flux and lower cost.
Drawings
The technical scheme of the invention is further explained by combining the accompanying drawings as follows:
FIG. 1 is a diagram of a single base extension reaction according to the present invention;
FIG. 2 is a nucleic acid detection map of a first gene locus;
FIG. 3 is a nucleic acid detection profile of a second gene locus;
FIG. 4 is a nucleic acid detection map of a third gene locus;
FIG. 5 is a nucleic acid detection map of a fourth gene locus;
FIG. 6 is a nucleic acid detection map of the fifth gene locus.
Detailed Description
The invention is described below with reference to the accompanying drawings.
As shown in attached figures 1-6, the kit for detecting the human hyperlipemia sensitive gene comprises an APOE primer group, an SLCO1B1-1 primer group, an SLCO1B1-2 primer group, a COQ2 primer group and a CYP3A5 primer group; wherein the content of the first and second substances,
the APOE primer group consists of a forward primer APOEF1, a reverse primer APOER1 and a single-base extension primer APOESP1, and sequentially comprises the following components: SEQ ID Nos: 140, SEQ ID Nos: 141, SEQ ID Nos: 150;
the SLCO1B1-1 primer group consists of a forward primer SLCO1B1F1, a reverse primer SLCO1B1R1 and a single-base extension primer SLCO1B1SP1, and sequentially comprises the following components: SEQ ID Nos: 142, SEQ ID Nos: 143, SEQ ID Nos: 151, or a sequence shown in seq id no;
the SLCO1B1-2 primer group consists of a forward primer SLCO1B1F2, a reverse primer SLCO1B1R2 and a single-base extension primer SLCO1B1SP2, and sequentially comprises the following components: SEQ ID Nos: 144, SEQ ID Nos: 145, SEQ ID Nos: 152;
the COQ2 primer group consists of a forward primer COQ2F1, a reverse primer COQ2R1 and a single-base extension primer COQ2SP1, and sequentially comprises the following components: SEQ ID Nos: 146, SEQ ID Nos: 147, SEQ ID Nos: 153;
the CYP3A5 primer group consists of a forward primer CYP3A5F1, a reverse primer CYP3A5R1 and a single-base extension primer CYP3A5SP1, and sequentially comprises the following components: SEQ ID Nos: 148, SEQ ID Nos: 149, SEQ ID Nos: 154;
the nucleic acid mass spectrum detection kit comprises the following reaction systems:
first, multiplex PCR reaction:
primer set a comprising primers APOEF1, APOER1, SLCO1B1F1, SLCO1B1R1, SLCO1B1F2, SLCO1B1R2, COQ2F1, COQ2R1, CYP3A5F1 and CYP3A5R 1;
PCR master Mix；
H20；
a DNA template;
the reaction conditions of the multiplex PCR reaction are as follows: 95 ℃ for 2 minutes, 95 ℃ for 30 seconds, 56 ℃ for 20 seconds, 72 ℃ for 60 seconds, 45 cycles, 72 ℃ for 5 minutes, 4 ℃ storage.
The second step is that: single base extension reaction:
the above PCR reaction solution;
extending the iPLEX mixed solution;
primer set B comprising primers APOESP1, SLCO1B1SP1, SLCO1B1SP2, COQ2SP1, SP1SP 1;
H2O；
the reaction conditions of the extension reaction are as follows:
30 seconds at 94 ℃, 5 seconds at 52 ℃, 5 seconds at 80 ℃, 5 cycles (third and fourth steps), 40 cycles (2, 3, 4 steps), 3 minutes at 72 ℃ and 4 ℃ storage.
The specific sequences of the primers are shown in the following table:
example 1
In the embodiment, five gene loci of APOE, SLCO1B1-1, SLCO1B1-2, COQ2 and CYP3A5 which are sensitive gene mutation loci for human hyperlipidemia are used as detection objects, and the APOE, the SLCO1B1-1, the SLCO1B1-2, the COQ2 and the CYP3A5 are detected accurately, simply and quickly through specific primer combinations and a nucleic acid mass spectrum technology.
The experimental gene templates were: 30 wild type blood sample and 5 variant blood sample, and finally taking the accuracy of negative and positive as a standard.
First, multiplex PCR reaction:
primer set a mix (including primers APOEF1, APOER1, SLCO1B1F1, SLCO1B1R1, SLCO1B1F2, SLCO1B1R2, COQ2F1, COQ2R1, CYP3A5F1, CYP3A5R 1) 1ul
PCR master Mix 1.2ul
H2O 0.8ul
DNA template 2ul
The reaction conditions of the multiplex PCR reaction are as follows: 95 ℃ for 2 minutes, 95 ℃ for 30 seconds, 56 ℃ for 20 seconds, 72 ℃ for 60 seconds, 45 cycles, 72 ℃ for 5 minutes, 4 ℃ storage.
The second step is that: single base extension reaction:
5ul of the above PCR reaction solution
iPLEX extension mixed liquid 2ul
Primer set B (including primers APOESP1, SLCO1B1SP1, SLCO1B1SP2, COQ2SP1, SP1SP 1) 1ul
H2O 1ul
The reaction conditions of the extension reaction are as follows:
30 seconds at 94 ℃, 5 seconds at 52 ℃, 5 seconds at 80 ℃, 5 cycles (third and fourth steps), 40 cycles (2, 3 and 4 steps), 3 minutes at 72 ℃ and 4 ℃ storage.
Example 2
In the embodiment, five gene loci of APOE, SLCO1B1-1, SLCO1B1-2, COQ2 and CYP3A5 which are sensitive gene mutation loci for human hyperlipidemia are used as detection objects, and the APOE, the SLCO1B1-1, the SLCO1B1-2, the COQ2 and the CYP3A5 are detected accurately, simply and quickly through specific primer combinations and a nucleic acid mass spectrum technology.
The experimental gene templates were: 1 wild type blood sample and 1 variant blood sample, and in addition, 20 blood samples and 10 saliva samples were simultaneously tested.
The method for detecting the drug sensitive gene mutation locus for human hyperlipemia by utilizing the multiplex PCR comprises the following steps:
(1) sample treatment and template extraction quality control:
the sample application range comprises samples such as whole blood, saliva, oral test paper and the like; for the extraction process, please refer to the specification of the commercial kit, the magnetic bead extraction is taken as an example:
centrifuging the sample in the centrifuge tube at 10000 rpm for 2 min, removing the supernatant, and leaving the precipitate for later use.
Adding 350 ul of Buffer MCL and 20 ul of protease K into the centrifuge tube, shaking and uniformly mixing, and carrying out water bath at 65 ℃ for 20 min, or mixing at intervals.
After completion of the water bath, 350. mu.l Buffer MA and 25. mu.l SanMag Beads were added to the tube, mixed by shaking or inversion, and left to stand at room temperature for 3 min, and mixed occasionally.
And (3) placing the centrifugal tube on a magnetic frame for 30 s, after the SanMag Beads are completely sucked to the tube wall, sucking and removing the supernatant, and taking out the centrifugal tube from the magnetic frame.
Adding 700 mul of 70% ethanol into the centrifuge tube, sucking or point-vibrating and mixing uniformly, placing the centrifuge tube on a magnetic frame for 30 s, sucking and removing supernatant, and taking out the centrifuge tube from the magnetic frame.
Repeating the previous step once, and uncovering and drying at room temperature for 10 min until no liquid remains in the tube.
50. mu.l of TE Buffer (pH8.0) was added to the tube, and mixed together occasionally.
And (4) taking out the centrifuge tube, placing the centrifuge tube on a magnetic frame for 30 s, and carefully sucking the supernatant into a new centrifuge tube after the SanMag Beads are completely sucked onto the tube wall, so as to obtain the genome DNA.
Sample concentrations were measured with qubit 3.0.
The concentration is qualified to be more than 3 ng/ul.
(2) The template obtained in the step (1) is amplified by using the multiplex PCR detection kit, and the reaction system is as follows:
first, multiplex PCR reaction:
primer set a mix (including primers APOEF1, APOER1, SLCO1B1F1, SLCO1B1R1, SLCO1B1F2, SLCO1B1R2, COQ2F1, COQ2R1, CYP3A5F1, CYP3A5R 1) 1ul
PCR master Mix 1.2ul
H2O 0.8ul
DNA template 2ul
The reaction conditions of the multiplex PCR reaction are as follows: 95 ℃ for 2 minutes, 95 ℃ for 30 seconds, 56 ℃ for 20 seconds, 72 ℃ for 60 seconds, 45 cycles, 72 ℃ for 5 minutes, 4 ℃ storage.
The second step is that: single base extension reaction:
5ul of the above PCR reaction solution
iPLEX extension mixed liquid 2ul
Primer set B (including primers APOESP1, SLCO1B1SP1, SLCO1B1SP2, COQ2SP1, SP1SP 1) 1ul
H2O 1ul
The reaction conditions of the extension reaction are as follows:
30 seconds at 94 ℃, 5 seconds at 52 ℃, 5 seconds at 80 ℃, 5 cycles (third and fourth steps), 40 cycles (2, 3 and 4 steps), 3 minutes at 72 ℃ and 4 ℃ storage.
(3) And (3) detection: detecting the reacted sample by using an MASSARRAY IV nucleic acid mass spectrometer; the mass spectrum detection process comprises the following steps: adding 41ul of water into each well of the sample plate with the sample and then centrifuging; putting the 96-hole plate into a mass spectrometer, opening a sample application program, and performing automatic sample application operation; opening flight mass spectrum software, setting parameters and detecting; after the detection introduction, the result is automatically generated in software and then exported.
And (3) analyzing the accuracy: in 30 cases, the wild type sample and the 5 variant sample are successfully detected, and the positive detection rate and the negative detection rate are both 100%; the kit disclosed by the invention is indicated to have high detection accuracy.
And (3) repeatability experiment: in each experiment, the wild type sample and the variant sample are used as controls and are repeated for 20 times, and each time can be successfully detected, which indicates that the repeatability is good.
The invention can detect the sensitive gene mutation gene locus of human hyperlipemia medication at the same time, can detect 96 samples in 6 hours, and has low cost; has the advantages of rapidness, accuracy and low cost.
The above-mentioned embodiments are merely illustrative of the technical idea and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the scope of the present invention, and all equivalent changes or modifications made according to the spirit of the present invention should be covered in the scope of the present invention.
Weikang (Suzhou) Gene Technology Co., Ltd.
Kit for detecting human hyperlipemia sensitive gene
15
1
30
DNA
Artificial sequences
1
ACGTTGGATGCTGTCCAAGGAGCTGCAGG 30
2
30
DNA
Artificial sequences
2
ACGTTGGATGGAGCATGGCCTGCACCTCG 30
3
17
DNA
Artificial sequences
3
CGGACATGGAGGACGTG 17
4
30
DNA
Artificial sequences
4
ACGTTGGATGACAAGTGGATAAGGTCGATG 30
5
30
DNA
Artificial sequences
5
ACGTTGGATGGATGTTCTTACAGTTACAGG 30
6
22
DNA
Artificial sequences
6
GATGTTGAATTTTCTGATGAAT 22
7
30
DNA
Artificial sequences
7
ACGTTGGATGAGTACAGACCCTTCTCTCAC 30
8
30
DNA
Artificial sequences
8
ACGTTGGATGATGCATCCTCACATTACCAC 30
9
26
DNA
Artificial sequences
9
ATGTATACATATATACACACTTTTAC 26
10
30
DNA
Artificial sequences
10
ACGTTGGATGGAAAGAACTAAAAGCAACTC 30
11
30
DNA
Artificial sequences
11
ACGTTGGATGTTCTACCACAACTTTCCCAC 30
12
20
DNA
Artificial sequences
12
13
30
DNA
Artificial sequences
13
ACGTTGGATGGTAATGTGGTCCAAACAGGG 30
14
30
DNA
Artificial sequences
14
ACGTTGGATGATGTACCACCCAGCTTAACG 30
15
19
DNA
Artificial sequences
15
TCCAAACAGGGAAGAGATA 19。
Claims (6)
1. A kit for detecting a human hyperlipemia sensitive gene is characterized in that: comprises an APOE primer group, an SLCO1B1-1 primer group, an SLCO1B1-2 primer group, a COQ2 primer group and a CYP3A5 primer group; wherein the content of the first and second substances,
the APOE primer group consists of a forward primer APOEF1, a reverse primer APOER1 and a single-base extension primer APOESP 1;
the SLCO1B1-1 primer group consists of a forward primer SLCO1B1F1, a reverse primer SLCO1B1R1 and a single-base extension primer SLCO1B1SP 1;
the SLCO1B1-2 primer group consists of a forward primer SLCO1B1F2, a reverse primer SLCO1B1R2 and a single-base extension primer SLCO1B1SP 2;
the COQ2 primer group consists of a forward primer COQ2F1, a reverse primer COQ2R1 and a single-base extension primer COQ2SP 1;
the CYP3A5 primer group consists of a forward primer CYP3A5F1, a reverse primer CYP3A5R1 and a single-base extension primer CYP3A5SP 1;
the nucleic acid mass spectrum detection kit comprises the following reaction systems:
first, multiplex PCR reaction:
primer set a comprising primers APOEF1, APOER1, SLCO1B1F1, SLCO1B1R1, SLCO1B1F2, SLCO1B1R2, COQ2F1, COQ2R1, CYP3A5F1 and CYP3A5R 1;
PCR master Mix；
H20；
a DNA template;
the second step is that: single base extension reaction:
the above PCR reaction solution;
extending the iPLEX mixed solution;
primer set B comprising primers APOESP1, SLCO1B1SP1, SLCO1B1SP2, COQ2SP1, SP1SP 1;
H2O。
2. the kit for detecting a human hyperlipemia-sensitive gene as claimed in claim 1, wherein: the primer APOEF1, the reverse primer APOER1 and the single-base extension primer APOESP1 sequentially comprise the following components: SEQ ID Nos: 140, SEQ ID Nos: 141, SEQ ID Nos: 150, or a sequence shown in fig.
3. The kit for detecting a human hyperlipemia-sensitive gene as claimed in claim 1, wherein: the forward primer SLCO1B1F1, the reverse primer SLCO1B1R1 and the single-base extension primer SLCO1B1SP1 sequentially comprise the following components: SEQ ID Nos: 142, SEQ ID Nos: 143, SEQ ID Nos: 151, respectively.
4. The kit for detecting a human hyperlipemia-sensitive gene as claimed in claim 1, wherein: the forward primer SLCO1B1F2, the reverse primer SLCO1B1R2 and the single-base extension primer SLCO1B1SP2 sequentially comprise the following components: SEQ ID Nos: 144, SEQ ID Nos: 145, SEQ ID Nos: 152.
5. The kit for detecting a human hyperlipemia-sensitive gene as claimed in claim 1, wherein: the forward primer COQ2F1, the reverse primer COQ2R1 and the single-base extension primer COQ2SP1 sequentially comprise the following components: SEQ ID Nos: 146, SEQ ID Nos: 147, SEQ ID Nos: 153, respectively.
6. The kit for detecting a human hyperlipemia-sensitive gene as claimed in claim 1, wherein: the forward primer CYP3A5F1, the reverse primer CYP3A5R1 and the single-base extension primer CYP3A5SP1 sequentially comprise: SEQ ID Nos: 148, SEQ ID Nos: 149, SEQ ID Nos: 154.
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| CN202011228757.8A CN112410415A (en) | 2020-11-06 | 2020-11-06 | Kit for detecting human hyperlipemia sensitive gene |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011228757.8A CN112410415A (en) | 2020-11-06 | 2020-11-06 | Kit for detecting human hyperlipemia sensitive gene |
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| CN112410415A true CN112410415A (en) | 2021-02-26 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852956A (en) * | 2021-03-23 | 2021-05-28 | 上海康黎诊断技术有限公司 | Kit for guiding medication of human hyperlipidemia |
-
2020
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112852956A (en) * | 2021-03-23 | 2021-05-28 | 上海康黎诊断技术有限公司 | Kit for guiding medication of human hyperlipidemia |
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Application publication date: 20210226 |