Phenylketonuria monogenic disease mutation detection primer group, kit and method
阅读说明：本技术 一种苯丙酮尿症单基因病突变检测引物组、试剂盒及方法 (Phenylketonuria monogenic disease mutation detection primer group, kit and method ) 是由 张惠丹 戴敬 李阳 赵洪玉 于 2019-12-18 设计创作，主要内容包括：本发明提供了一种苯丙酮尿症单基因病突变检测引物组、试剂盒及方法,采用不同的引物设计软件,并结合agilent earray design探针设计系统,通过不断优化引物序列,最终将20个苯丙酮尿症单基因病突变位点的多重PCR扩增压缩至1个well中进行。本申请技术实验设计灵活,结果准确,检测速度快,无需PCR荧光标记,成本大大降低。适合批量筛查。(The invention provides a primer group, a kit and a method for detecting phenylketonuria monogenic disease mutation, which adopt different primer design software and combine with an agilent early design probe design system to finally compress the multiplex PCR amplification of 20 phenylketonuria monogenic disease mutation sites into 1 well for carrying out by continuously optimizing primer sequences. The application technology has the advantages of flexible experimental design, accurate result, high detection speed, no need of PCR fluorescent labeling and greatly reduced cost. Is suitable for batch screening.)
1. A phenylketonuria monogenic disease mutation detection primer group comprises the following paired PCR amplification primers:
2. the primer set for detecting phenylketonuria monogenic mutation according to claim 1, wherein the primer set is designed for 20 phenylketonuria monogenic mutation sites, and the mutation sites comprise: rs62508620, rs199475657, rs672601294, rs199475653, rs62507283, rs62508722, rs62508728, rs199475658, rs62642919, rs199475689, rs199475694, rs281865446, rs199475695, rs199475692, rs199475693, rs199475696, rs199475645, rs1025860114, rs199475684, rs 62507334.
3. The primer set for detecting phenylketonuria monogenic mutation according to claim 1, further comprising a single base extension primer/probe corresponding to each primer pair:
1, probe 1: CGGTCCAGGTGGTTAC, respectively;
and (3) probe 2: CCACGGTCATAGGGTAA, respectively;
and 3, probe 3: AGGGTCGCAGGTCAGC, respectively;
and 4, probe 4: ACGGCATACGCAGTAC, respectively;
and 5, probe: TCCAAGGTCATAAGTTCAT, respectively;
and 6, probe 6: CCCCAGTCAGTAGGCCA, respectively;
and (7) probe: AAGTCATCCAAAGGGTCAT, respectively;
and (3) probe 8: CATCATTCGGTAGTCGG, respectively;
and (3) probe 9: CCGGCGCCAGCCATCCC, respectively;
a probe 10: GGGGTGCCAGCACCAT, respectively;
a probe 11: ATCACCGAGTCCCAG, respectively;
the probe 12: GCATCCAACATTCAATC, respectively;
and (3) probe 13: CCGCACGAGCCAGGTC, respectively;
the probe 14: AGCATCACAGTCAGTAAAG, respectively;
and (3) probe 15: AGGACGGGTGTGAGA, respectively;
a probe 16: CACAGTACCAGTCGCGG, respectively;
a probe 17: CAGAAGAAGCAGGTAG, respectively;
the probe 18: ACCAGTACGCAGTAGG, respectively;
a probe 19: GTAGTCATCATCGGGTCA, respectively;
and (3) probe 20: GTCGGGTGGTGGGTC are provided.
4. A phenylketonuria monogenic disease mutation detection kit, comprising the phenylketonuria monogenic disease mutation detection primer set of any one of claims 1-3.
5. A method for detecting phenylketonuria monogenic disease mutation, which adopts the primer group for detecting phenylketonuria monogenic disease mutation of any one of claims 1-3, and comprises the following steps:
1) carrying out PCR amplification in a 384-well plate, wherein the total volume of each PCR amplification reaction system is 5 mu l, each 5 mu l PCR amplification reaction system contains 20-50 ng of template DNA, 0.5U of Hotstar Taq, 0.5pmol of each amplification primer and 0.1 mu l of 25 MdNTPs;
2) performing alkaline phosphatase treatment on the PCR product;
3) after the alkaline phosphatase treatment is finished, carrying out single-base extension reaction, wherein the total volume of the reaction system is 9 mu l, and for each reaction hole, the single-base extension reaction system comprises 7 mu l of PCR product after SAP treatment and 2 mu l of EXTEND Mix solution, and the EXTEND Mix solution comprises 0.94 mu l of each extension reaction primer mixture;
4) the resin purified extension product was transferred to 384-well SpectroCHIP chips;
5) and (3) carrying out mass spectrometry on the spotted SpectroCHIP chip, and typing and outputting the detection result.
6. The method for detecting phenylketonuria monogenic disease mutation according to claim 5, wherein the PCR amplification reaction conditions in step 1) are as follows: 4 minutes at 94 ℃; 94 ℃ for 20 seconds, 56 ℃ for 30 seconds, 72 ℃ for 1 minute, 45 cycles; 3 minutes at 72 ℃; keeping at 4 ℃.
7. The method for detecting phenylketonuria monogenic mutation according to claim 5, wherein in step 2), the 384-well plate is placed on a PCR instrument compatible with the 384-well plate, and the PCR reaction conditions are set as follows: 40 minutes at 37 ℃; 5 minutes at 85 ℃; the temperature was maintained at 4 ℃ and the PCR machine was started to perform alkaline phosphatase treatment.
8. The method for detecting phenylketonuria monogenic mutation according to claim 5, wherein in step 3), the single base extension reaction conditions are as follows:
。
Technical Field
The invention relates to the technical field of biological gene detection, in particular to a phenylketonuria monogenic disease mutation detection primer group, a kit and a method.
Background
Phenylketonuria (PKU) is a common autosomal recessive inherited metabolic disease that seriously compromises human health. The disease is mainly caused by mutation of phenylalanine dehydrogenase (PAH) gene, so that the activity of the PAH is reduced or lacked, the phenylalanine cannot be metabolized normally, and phenylalanine and bypass metabolites thereof in blood and tissues are accumulated, so that the central nervous system is irreversibly damaged, and symptoms such as abnormal psychobehavior, epilepsy and mental retardation appear.
PKU is a genetic disease which can be treated through diet control, early treatment is found to be the key of the treatment, but expensive treatment cost brings heavy economic burden to families and society, and the birth rate of PKU patients cannot be reduced fundamentally. Currently, an important means for solving this series of problems is to actively carry out genetic diagnosis and prenatal diagnosis.
The diagnosis of the single-gene disease is mainly based on the second-generation sequencing at present by depending on the development of scientific technology, but the operation steps are complicated, the detection time is long, the requirements on the operation and analysis capability of technical personnel are high, the detection results are easily influenced, meanwhile, the second-generation sequencing detection reagent and the analysis consumable material are expensive, the high cost brings a plurality of challenges to the application of the second-generation sequencing disease molecular diagnosis and the crowd screening, and the popularization of the application is influenced.
Disclosure of Invention
Aiming at the technical problems, the invention develops a phenylketonuria monogenic disease mutation detection primer group, a kit and a method based on the MassArray technology, and can solve the defects of the prior art in the aspects of operability, sensitivity, time saving and cost reduction.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a phenylketonuria monogenic disease mutation detection primer group, which comprises the following paired PCR amplification primers:
preferably, the primer sets are primers designed for 20 phenylketonuria monogenic disease mutation sites respectively, and the mutation sites comprise rs62508620, rs199475657, rs672601294, rs199475653, rs62507283, rs62508722, rs62508728, rs199475658, rs62642919, rs199475689, rs199475694, rs281865446, rs199475695, rs199475692, rs199475693, rs199475696, rs199475645, rs1025860114, rs199475684, rs 62501997334.
Preferably, the primer set further comprises a single base extension primer/probe corresponding to each primer pair:
1, probe 1: CGGTCCAGGTGGTTAC, respectively;
and (3) probe 2: CCACGGTCATAGGGTAA, respectively;
and 3, probe 3: AGGGTCGCAGGTCAGC, respectively;
and 4, probe 4: ACGGCATACGCAGTAC, respectively;
and 5, probe: TCCAAGGTCATAAGTTCAT, respectively;
and 6, probe 6: CCCCAGTCAGTAGGCCA, respectively;
and (7) probe: AAGTCATCCAAAGGGTCAT, respectively;
and (3) probe 8: CATCATTCGGTAGTCGG, respectively;
and (3) probe 9: CCGGCGCCAGCCATCCC, respectively;
a probe 10: GGGGTGCCAGCACCAT, respectively;
a probe 11: ATCACCGAGTCCCAG, respectively;
the probe 12: GCATCCAACATTCAATC, respectively;
and (3) probe 13: CCGCACGAGCCAGGTC, respectively;
the probe 14: AGCATCACAGTCAGTAAAG, respectively;
and (3) probe 15: AGGACGGGTGTGAGA, respectively;
a probe 16: CACAGTACCAGTCGCGG, respectively;
a probe 17: CAGAAGAAGCAGGTAG, respectively;
the probe 18: ACCAGTACGCAGTAGG, respectively;
a probe 19: GTAGTCATCATCGGGTCA, respectively;
and (3) probe 20: GTCGGGTGGTGGGTC are provided.
The invention also provides a kit containing the primer group.
The invention also provides a phenylketonuria monogenic disease mutation detection method, which adopts the primer group and comprises the following steps:
1) performing PCR amplification in a 384-well plate, wherein the total volume of each PCR amplification reaction system is 5 mu l, each 5 mu l PCR amplification reaction system contains 20-50 ng of template DNA, 0.5U of Hotstar Taq, 0.5pmol of each amplification primer and 0.1 mu l of 25 MdNTPs;
2) performing alkaline phosphatase treatment on the PCR product;
3) after the alkaline phosphatase treatment was completed, the single-base extension reaction was performed in a total volume of 9. mu.l, and the single-base extension reaction contained 7. mu.l of SAP-treated PCR product and 2. mu.l of EXTEND Mix (0.94. mu.l of each extension primer Mix) per well.
4) The resin purified extension product was transferred to 384-well SpectroCHIP chips;
5) and (3) carrying out mass spectrometry on the spotted SpectroCHIP chip, and typing and outputting the detection result.
Preferably, the PCR amplification reaction conditions in step 1) are: 4 minutes at 94 ℃; 94 ℃ for 20 seconds, 56 ℃ for 30 seconds, 72 ℃ for 1 minute, 45 cycles; 3 minutes at 72 ℃; keeping at 4 ℃.
Preferably, in step 2), the 384-well plate is placed on a 384-well plate compatible PCR instrument, and PCR reaction conditions are set: 40 minutes at 37 ℃; 5 minutes at 85 ℃; the temperature was maintained at 4 ℃ and the PCR machine was started to perform alkaline phosphatase treatment.
Preferably, in step 3), the single base extension reaction conditions are:
I
94℃
30s
II
94℃
5s
III
52℃
5s
IV
80℃
5s
V
GOTO III
4more times
VI
GOTO II
39more times
VII
72℃
3min
VIII
4℃
forever
。
the invention has the following beneficial effects:
the method has the advantages of flexible technical experiment design, accurate result, high detection speed, no need of PCR fluorescent labeling and greatly reduced cost. Is suitable for batch screening.
Detailed Description
The present invention will be further illustrated by the following specific examples, which should be understood as not limiting the scope of the invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
According to the professional genetic databases such as OMIM, clinvar and the like, the applicant consults Chinese databases such as the known network and the like to collect the common mutations of Chinese, and 20 mutation sites of the phenylketonuria monogenic disease are collected:
rs62508620、rs199475657、rs672601294、rs199475653、rs62507283、rs62508722、rs62508728、rs199475658、rs62642919、rs199475689、rs199475694、rs281865446、rs199475695、rs199475692、rs199475693、rs199475696、rs199475645、rs1025860114、rs199475684、rs62507334。
by the MassArray technique, if 20 gene mutation sites are placed in 1 reaction well, the primers need to be continuously optimized in consideration of experimental conditions. We used different primer design software, such as Primr premier, Oligo, VectorNTI Suit, Dnasis, Omiga, Dnastar, etc., and combined with the agilent early design probe design system, by continuously optimizing the primer sequence, finally compressed the 20 sites of multiplex PCR amplification into 1 well.
TABLE 1 primer and probe sequences for different detection sites
Numbering
Detection site
Positive strand primer
Minus strand primer
Probe needle
1
rs62508620
AGTCATCCATCATGTGTGT
GACCAGGTTCCAGTCATT
CGGTCCAGGTGGTTAC
2
rs199475657
AAGAAGCCAGTCATTCA
GCATTTTAGCAAGAAG
CCACGGTCATAGGGTAA
3
rs672601294
TCGAGGCCGAGACACG
AGCGGGCAGGAGGTAC
AGGGTCGCAGGTCAGC
4
rs199475653
CAACACAGTACGGCCATA
GGCCACACGAGGGCAC
ACGGCATACGCAGTAC
5
rs62507283
CAGGGATCATTACGCA
GTCATAGTTCAGGTCATCC
TCCAAGGTCATAAGTTCAT
6
rs62508722
AGGCAGAGCCCAGTC
GGTCCCCAGTCCCCAGTT
CCCCAGTCAGTAGGCCA
7
rs62508728
CCCAGCCGCAGTAGCAT
GGGAGAAAGGTCCCC
AAGTCATCCAAAGGGTCAT
8
rs199475658
ACCCCAGGTAGCCCCCAT
GAGTACCCCAGTGTC
CATCATTCGGTAGTCGG
9
rs62642919
GGGTCAGCATCACCAT
TCATCAGCCCGCGGGTG
CCGGCGCCAGCCATCCC
10
rs199475689
CAAGGGCAAAGGTGTA
GGACCAGTAGCAGCAA
GGGGTGCCAGCACCAT
11
rs199475694
GCCAGCAAGGTCATTAT
CAGTTTCGAGCAGTCAC
ATCACCGAGTCCCAG
12
rs281865446
GGTAGGGTTCATCACCA
AGGACACAATGTTACAT
GCATCCAACATTCAATC
13
rs199475695
CGGAGACCAGGTACCAT
CCATATCAGCCATTCCG
CCGCACGAGCCAGGTC
14
rs199475692
AGCCAGTAGGACACCATCC
GGGATTCAGGTACATTC
AGCATCACAGTCAGTAAAG
15
rs199475693
GGGACATCGAAGCAGGT
AGGCACCAGCAGAGG
AGGACGGGTGTGAGA
16
rs199475696
GTACGAGCGCATCGC
CATTCCAGTACGGCGTA
CACAGTACCAGTCGCGG
17
rs199475645
AGGCGCGAGTAGAAGC
AAGAGCACACGCCATTT
CAGAAGAAGCAGGTAG
18
rs1025860114
ACACAGATCCGGCAGTA
CCGGGTAACAGGTCAGT
ACCAGTACGCAGTAGG
19
rs199475684
GATCCAGAGTAGCGG
CAGCAAGTACACATCATT
GTAGTCATCATCGGGTCA
20
rs62507334
GTCATCAGTGTCGGAC
GACGCAGCCATACCAG
GTCGGGTGGTGGGTC
The above primers and probes can be used as a kit according to actual requirements.