CN113355409A - Primer, probe composition and kit for detecting copy number variation of human DMD gene exon - Google Patents

Primer, probe composition and kit for detecting copy number variation of human DMD gene exon Download PDF

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CN113355409A
CN113355409A CN202110757565.4A CN202110757565A CN113355409A CN 113355409 A CN113355409 A CN 113355409A CN 202110757565 A CN202110757565 A CN 202110757565A CN 113355409 A CN113355409 A CN 113355409A
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primer
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吴文立
于超计
王倩玉
肖江山
赵立明
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Beijing Huarui Kangyuan Biotechnology Development Co ltd
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Abstract

The invention belongs to the technical field of biological detection, and discloses a primer, a probe composition and a kit for detecting copy number variation of human DMD gene exons. The kit can be used for rapidly detecting whether 53 # exon copy number variation exists in a DMD patient by taking a highly conserved region of a 53 # exon gene coding region of the DMD gene as a target region and designing a corresponding specific primer probe by a real-time fluorescent PCR (polymerase chain reaction) technology and taking a CFTR (circulating fluorescent tr) gene as an internal reference gene to monitor a false negative result possibly occurring in the PCR reaction process, plays an auxiliary role in diagnosis of the DMD patient by a doctor and can also provide scientific guidance data for the doctor in the aspect of medication. The application process has low requirements on detection equipment, the operation is simple, the detection can be finished on a machine only by adding three components of the sample DNA, the exon reaction solution and the PCR premix, the requirements on the skills of operators are low, and the sample confusion can be effectively prevented. Good reliability, simple operation, low equipment requirement and low reagent cost.

Description

Primer, probe composition and kit for detecting copy number variation of human DMD gene exon
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a primer, a probe composition and a kit for detecting copy number variation of human DMD gene exons.
Background
Duchenne/Beckman mucc Lar dystrophy (DMD/BMD) is an X-linked recessive inherited genetic muscle disorder characterized by progressive muscle weakness and muscle atrophy caused by dysphin protein deficiency resulting from DMD genetic variation. The clinical characteristics of the medicine comprise: muscle spasm, myalgia, quadriceps femoris myopathy, asymptomatic hypercreatinekinase blood disease, X-linked dilated cardiomyopathy, etc.
DMD is completely overt in men and is mainly characterized by progressive, symmetric muscle weakness with the proximal end heavier than the distal end. The infant usually shows late walking, easy falling and slow walking, and the abnormal gait becomes obvious when the infant reaches about 3 years old; the exercise development is still continuously improved at the beginning, but the difficulty of actions such as stair climbing, running, jumping, standing and the like is gradually shown, the signs of Gower syndrome, lumbar lordosis, pseudohypertrophy of gastrocnemius muscles on two sides and the like gradually appear, after the 4-5 years old passes a plateau period, the exercise capacity begins to reverse, the independent walking capacity is lost in the 10-12 years old, the wheelchair is required to act, and the patient usually dies due to respiratory system complications or heart failure and exhaustion before the 30 years old. The clinical manifestations of female DMD gene defect carriers are greatly different, wherein the severe patients can be typical DMD manifestations, the mild patients can be mild proximal muscle weakness, gastrocnemius pseudohypertrophy and the muscle functions can be basically normal. The incidence of DMD is approximately 1/3500 in male newborns and 1/100000-1/45000 in symptomatic female carriers.
The causative factor of duchenne/bethese muscular dystrophy is DMD genetic defect. The DMD gene is the largest gene discovered to date, located at xp21.2, comprising 79 exons, 7 tissue-specific promoters, 2200000 base pairs. The molecular weight of the Dystrophin protein coded by the DMD gene is 426kD, the Dystrophin protein consists of 3685 amino acids and is divided into 4 regions: 1. amino terminal region (14-240 amino acids) that interacts with intracellular actin (F-actin): including exons 1-8; 2. central rod-like region consisting of 24 trimeric helical repeat structures (253-3040 amino acids): including exons 9-63, homologous to alpha actin (alpha-actin) and spectrin; 3. cysteine-rich region (3080-3360 amino acids) that interacts with the glycoprotein complex on the sarcolemma: including 64-68 exons; 4. the carboxy-terminal region which interacts with the intracellular syntropphins protein (3361-3685 amino acids): including number 68-79 exons.
The Dystrophin protein encoded by the DMD gene has a function of linking the cytoskeleton and the basement membrane, and forms a Dystrophin protein complex with other proteins (dystroglycan, sarcoglycan) to maintain the stability of the cell membrane. The variation of DMD gene can destroy mRNA open reading frame, seriously affect synthesis and function of dystrophin protein, cause the destruction of dystrophin protein complex, increase fragility and decrease stability of muscle cell membrane, make muscle cell membrane rupture by strong muscle contraction, and accelerate the destruction of muscle fiber by calcium ion inflow, thus leading to the clinical manifestation of Duchenne/Behcet muscular dystrophy.
At present, the clinical treatment of Du's/Behcet's muscular dystrophy mainly aims at preserving the motor function of patients and preventing and treating complications, and comprises the following steps: glucocorticoid therapy, appropriate rehabilitation exercises, surgical orthotics, and the like. There are also therapeutic approaches by gene editing, exon crossing, PTC protein repair, etc. In 2020, Viltepso (viltolrsen) was approved by the U.S. Food and Drug Administration (FDA) for patients with Duchenne Muscular Dystrophy (DMD) who were genetically tested for the presence of mutations suitable for treatment with exon 53skipping (exon 53skipping), which accounts for approximately 8% of the total number of patients with DMD. The drug promotes the production of functional dystrophin by masking (skipping) exon 53 in the dystrophin gene, thereby delaying the progression of DMD disease.
Disclosure of Invention
In order to solve the above problems in the prior art, the present invention aims to provide a primer, a probe composition and a kit for detecting copy number variation of human DMD gene exons.
The technical scheme adopted by the invention is as follows: a primer and probe composition for detecting copy number variation of exons of a human DMD gene comprises a first specific primer and a first probe composition aiming at the No. 53 exon of the DMD gene, and a second specific primer and a second probe composition aiming at an internal reference CFTR gene;
the first specific primers comprise a first specific upstream primer and a first specific downstream primer, and the nucleotide sequences of the first specific upstream primer, the first specific downstream primer and the first probe are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3;
the second specific primers comprise a second specific upstream primer and a second specific downstream primer, and the nucleotide sequences of the second specific upstream primer, the second specific downstream primer and the second probe are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
Preferably, the 5 'end of the first probe and the 5' end of the second probe are both labeled with a fluorescent group, and the 3 'end of the first probe and the 3' end of the second probe are both labeled with a quenching group.
Preferably, the fluorescent group includes, but is not limited to, any one of FAM, Cy5, ROX, VIC, and NED;
the quenching group includes, but is not limited to, any one of QSY, MGB, and BHQ 1.
A kit for detecting the copy number variation of human DMD gene exons comprises exon reaction liquid, PCR premix liquid, a positive control substance and a negative control substance.
Preferably, the exon reaction solution comprises the first specific primer and the first probe composition, and the second specific primer and the second probe composition of claim 1.
Preferably, the loading amount of the exon reaction solution is 100-500. mu.L.
Preferably, the PCR master mix comprises PCR reaction buffer, dNTPs, Mg2+, hot start Taq enzyme, UNG enzyme, ROX fluorescent reference dye.
Preferably, the loading amount of the PCR premix is 0.2-1 mL.
Preferably, the positive control is a plasmid DNA solution containing a CFTR gene sequence;
the negative control substance is a plasmid DNA solution containing a CFTR gene sequence and a DMD gene No. 53 exon gene sequence;
the loading amount of the positive control substance is 200 mu L, and the loading amount of the negative control substance is 200 mu L.
The loading amounts refer to the volume numbers of the exon reaction solution, the PCR premix solution, the positive control and the negative control respectively loaded in the reaction holes.
Preferably, the kit is used for non-diagnostic purposes in the variation of the copy number of the 53 exon of the DMD gene.
The invention has the beneficial effects that:
the invention provides a primer, a probe composition and a kit for detecting the copy number variation of the human DMD gene exon. The kit can design a corresponding specific primer probe by taking a highly conserved region of a DMD 53 exon gene coding region as a target region through a real-time fluorescence PCR technology, and simultaneously, monitor a false negative result possibly occurring in a PCR reaction process by taking a CFTR gene as an internal reference gene, realize rapid detection on whether 53 exon copy number variation exists in a DMD patient, play an auxiliary role in diagnosis of the DMD patient by a doctor, and also provide scientific guidance data for the doctor in the aspect of medication.
The application process of the kit has low requirements on detection equipment, and the main equipment is an ABI 7500 fluorescent quantitative PCR instrument, so that the kit is convenient for detection mechanisms to purchase and use.
And (III) the application process of the kit is simple to operate, the detection can be finished on the computer only by adding three components of the sample DNA, the exon reaction solution and the PCR premix, the requirement on the skill of an operator is low, and the sample confusion can be effectively prevented.
And (IV) the reliability is good, the operation is simple, the equipment requirement is low, and the reagent cost is low.
Detailed Description
The present invention is further illustrated below with reference to specific examples. It will be appreciated by those skilled in the art that the following examples, which are set forth to illustrate the present invention, are intended to be part of the present invention, but not to be construed as limiting the scope of the present invention. The reagents used are all conventional products which are commercially available.
Example (b):
the kit (100 reaction/kit) for detecting the copy number variation of the 53 # exon of the human DMD gene by the real-time fluorescent PCR method comprises the components as shown in Table 1:
TABLE 1 kit Components
Figure BDA0003148397190000051
The nucleotide sequences of the specific primers and the probes of the 53 th exon of the human DMD gene in the exon reaction liquid are shown in SEQ ID NO.1-SEQ ID NO. 3;
the nucleotide sequences of the specific primers and the probes of the CFTR gene are shown in SEQ ID NO.4-SEQ ID NO. 6;
PCR premix solution: purchased from Nanjing Novozam Biotech, Inc., Cat No: q113-03. Comprises hot start Taq enzyme, UNG enzyme, 4 dNTPs, PCR reaction buffer solution and ROX fluorescent reference dye;
negative control: comprises a specific plasmid 1 and water, wherein the nucleotide sequence of the specific plasmid 1 is shown as SEQ ID No. 7; the specific plasmid 1 is an artificially synthesized DNA sequence of an internal reference CFTR gene and an artificially synthesized DNA sequence of a DMD gene No. 53 exon, and the final concentration of the control solution plasmid 1 is 0.06 pg/mu L;
positive control: comprises a specific plasmid 2 and water, wherein the nucleotide sequence of the specific plasmid 2 is shown as SEQ ID No. 8; specific plasmid 2 is an artificially synthesized DNA sequence of the internal reference CFTR gene, and the final concentration of plasmid 2 in the control solution is 0.04 pg/. mu.L.
Detection sites and their sequences are tabulated:
Figure BDA0003148397190000061
the non-diagnostic detection method of the kit for detecting the copy number variation of the 53 th exon of the human DMD gene comprises the following steps:
(1) obtaining a human peripheral blood cell sample, extracting human cell genome DNA from the sample, measuring the nucleic acid concentration by using a micro ultraviolet spectrophotometer, and adjusting the nucleic acid concentration to 20 ng/mu L;
(2) preparing a reference substance: dissolving the positive control substance and the negative control substance for later use;
(3) preparing a reaction system: preparing a reaction system according to the reaction number N, wherein the exon reaction solution is 5N, and the PCR premix solution is 10N;
(4) sample detection: adding the positive reference substance, the negative reference substance and the gDNA sample to be detected into the reaction hole site in sequence, wherein the sample adding volume is 5 mu L;
(5) PCR reaction procedure: 2 minutes at 50 ℃ and 10 minutes at 95 ℃; entering the following cycle: 40 cycles of 95 ℃ for 15 seconds and 60 ℃ for 1 minute, and collecting FAM and VIC fluorescence signals in real time in each cycle; wherein, FAM fluorescent signal is DMD 53 exon gene, VIC fluorescent signal is CFTR internal reference gene;
(6) and (4) analyzing results: and (3) carrying out copy number quantification on the 53 # exon of the DMD gene by adopting a relative quantification mode of a delta-delta Ct value method.
Experimental example:
the kit prepared in the above embodiment is used for detecting the exon 53 of the DMD gene of human Duchenne/Behcet muscular dystrophy disease causing gene by using human peripheral blood free DNA or gDNA as a sample:
(1) nucleic acid extraction:
whole blood samples from 2 children, 2 normal females and 2 normal males who were clinically diagnosed with DMD were obtained from a hospital. Extracting whole blood sample collected by EDTA anticoagulation tube by using Tianlong full-automatic nucleic acid extractor (NP968-3S) and Tianlong whole blood genome DNA extraction kit, and measuring nucleic acid purity and concentration, OD thereof by using micro ultraviolet spectrophotometer260/280Between 1.6 and 2.0; the genomic DNA concentration was diluted to 20 ng/. mu.L with sterile double distilled water for further use.
(2) Diluting a reference substance:
dissolving the positive control substance and the negative control substance for later use.
(3) PCR reaction system was prepared with a total reaction volume of 20. mu.L, as shown in Table 2:
TABLE 2 PCR reaction System
Component (A) Sample addition amount (μ L)
Exon reaction liquid 5
PCR premix 10
gDNA template/control 5
(4) Sample detection:
and sequentially adding the negative control substance, the positive control substance and the gDNA of the sample to be detected into the reaction hole site, wherein the sample adding volume is 5 mu L.
(5) PCR reaction procedure:
2 minutes at 50 ℃ and 10 minutes at 95 ℃; entering the following cycle: 95 ℃ for 15 seconds, 60 ℃ for 1 minute (signal acquisition), for a total of 40 cycles. The used reaction instrument is ABI 7500, FAM and VIC fluorescence signals are collected in real time in each cycle, wherein the FAM fluorescence signal is DMD 53 exon gene, and the VIC fluorescence signal is CFTR internal reference gene.
(6) And (4) analyzing results: and (3) carrying out copy number quantification on the 53 # exon of the DMD gene by adopting a relative quantification mode of a delta-delta Ct value method.
Quality control:
clause and subclause Content providing method and apparatus
1 Ct values of target gene and reference gene of negative control should be less than or equal to 30
2 The delta-delta Ct of the positive control is more than 3 or no signal exists in the FAM channel
3 Ct value of reference gene of sample to be detected is less than or equal to 33
Remarking; if any one of the above 3 contents is not satisfied, the detection result is regarded as invalid, and detection is carried out again after the reason is suggested to be checked.
The following is a method for calculating the relative expression level (RQ value):
calculation of negative control Δ Ct:
the negative control has a delta Ct value between the target gene and the reference geneFAM–CtVICIs denoted as Δ Cta。
Calculating the delta Ct value between the target gene and the reference gene of the sample to be detected, wherein the delta Ct is CtFAM–CtVICIs denoted as Δ Cts。
Target gene delta Ct of sample to be detected is equal to target gene delta Ct of sample to be detecteds-△Cta；
Relative expression quantity (RQ value) of sample to be detected is 2 relative copy number of target gene of sample to be detected-△△Ct；
And (5) judging a result: the DMD gene No. 53 exon and internal reference gene CFTR were both 2 copies as normal control samples, and the results were determined as shown in Table 3:
TABLE 3 determination of results
Figure BDA0003148397190000091
Figure BDA0003148397190000101
The experimental results meet the general requirements for data analysis as follows:
ct values of FAM and VIC fluorescence channels of the negative control products are less than or equal to 30;
the delta-delta Ct of the positive control is more than 3 or no signal exists in an FAM channel;
the Ct value of the reference gene of the sample to be detected is less than or equal to 33.
The RQ values and copy numbers of DMD gene exon 53 in 2 DMD infants, 2 normal females, and 2 normal males are shown in table 4:
TABLE 4.6 sample test results
Sample(s) CtFAM CtVIC RQ Number of copies
1 Undetermined 26.29 0 0
2 Undetermined 26.36 0 0
3 27.17 26.47 1.01 2
4 27.26 26.54 0.99 2
5 28.40 26.60 0.47 1
6 28.23 26.55 0.51 1
Note: samples 1 to 2: a child suffering from DMD; samples 3 to 4: a normal female; samples 5 to 6: a normal male.
The performance experiment of the kit verifies that:
1) positive compliance rate: when the kit prepared by the embodiment is used for detecting the positive enterprise reference substances, the results are positive, and the positive compliance rate is 100%. As shown in table 5:
TABLE 5 Positive compliance reference
Figure BDA0003148397190000111
2) Negative coincidence rate: when the kit prepared by the embodiment is used for detecting the negative enterprise reference products, the results are negative, and the negative coincidence rate is 100%. As shown in table 6:
TABLE 6 negative compliance reference
Figure BDA0003148397190000112
3) Precision: the kit prepared in the embodiment is adopted to detect 1 part of positive precision reference substance and 1 part of negative precision reference substance, each sample is repeated for 10 times, and the coefficient of variation (CV%) of the Ct value of the detection result is less than or equal to 5%. As shown in table 7:
TABLE 7 precision references
Figure BDA0003148397190000113
Figure BDA0003148397190000121
4) Minimum detection limit: the kit prepared in the embodiment is used for detecting 1 part of the positive minimum detection limit reference substance and 1 part of the negative minimum detection limit reference substance, and the positive and negative coincidence rates are both 100%. As shown in table 8:
TABLE 8 reference of minimum detection limits
Figure BDA0003148397190000122
In conclusion, the kit provided by the invention has the characteristics of good reliability, simplicity in operation, high detectable rate and high speed.
The present invention is not limited to the above alternative embodiments, and any other products in various forms can be obtained by the present invention, and the present invention is within the protection scope of the present invention. The above embodiments should not be construed as limiting the scope of the present invention, and it will be understood by those skilled in the art that modifications may be made to the technical solutions described in the above embodiments, or equivalent substitutions may be made to some or all of the technical features thereof, without departing from the scope of the present invention, and at the same time, such modifications or substitutions may not make the essence of the corresponding technical solutions depart from the scope of the embodiments of the present invention.
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Claims (10)

1. A primer and probe composition for detecting the copy number variation of the exon of the DMD gene is characterized by comprising a first specific primer and a first probe composition aiming at the No. 53 exon of the DMD gene, and a second specific primer and a second probe composition aiming at an internal reference CFTR gene;
the first specific primers comprise a first specific upstream primer and a first specific downstream primer, and the nucleotide sequences of the first specific upstream primer, the first specific downstream primer and the first probe are respectively shown as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3;
the second specific primers comprise a second specific upstream primer and a second specific downstream primer, and the nucleotide sequences of the second specific upstream primer, the second specific downstream primer and the second probe are respectively shown as SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6.
2. The primer and probe composition for detecting copy number variation of exon of human DMD gene according to claim 1, wherein the 5 'end of the first probe and the 5' end of the second probe are labeled with fluorescent groups, and the 3 'end of the first probe and the 3' end of the second probe are labeled with quenching groups.
3. The primer and probe composition for detecting copy number variation of exons of human DMD gene according to claim 2, wherein said fluorophore comprises any one of FAM, Cy5, ROX, VIC, and NED;
the quenching group comprises any one of QSY, MGB and BHQ 1.
4. A kit for detecting the copy number variation of human DMD gene exons is characterized by comprising exon reaction liquid, PCR premix liquid, a positive control substance and a negative control substance.
5. The kit for detecting the copy number variation of the exon of the DMD gene of claim 4, wherein the exon reaction solution comprises the first specific primer and the first probe composition, and the second specific primer and the second probe composition of claim 1.
6. The kit for detecting the copy number variation of the exons of human DMD gene according to claim 5, wherein the loading of the exon reaction solution is 100 μ L and 500 μ L.
7. The kit for detecting the copy number variation of the exon of the DMD gene of claim 4, wherein the PCR pre-mixed solution comprises PCR reaction buffer, dNTPs, Mg2+, hot start Taq enzyme, UNG enzyme and ROX fluorescent reference dye.
8. The kit for detecting the copy number variation of the exons of the human DMD gene of claim 7, wherein the amount of the PCR solution is 0.2-1 mL.
9. The kit for detecting the exon copy number variation of the human DMD gene according to claim 4, wherein the positive control is a plasmid DNA solution containing a CFTR gene sequence;
the negative control substance is a plasmid DNA solution containing a CFTR gene sequence and a DMD gene No. 53 exon gene sequence;
the loading amount of the positive control substance is 200 mu L, and the loading amount of the negative control substance is 200 mu L.
10. The kit for detecting the exon copy number variation of the DMD gene of claim 4, wherein the kit is used for the non-diagnostic purpose of detecting the exon copy number variation of the DMD gene at 53.
CN202110757565.4A 2021-07-05 2021-07-05 Primer, probe composition and kit for detecting copy number variation of human DMD gene exon Pending CN113355409A (en)

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Application publication date: 20210907