CN112430645A

CN112430645A - Relative quantitative method and kit for detecting human DMD gene copy number by multiple real-time fluorescence PCR method

Info

Publication number: CN112430645A
Application number: CN202011447747.3A
Authority: CN
Inventors: 于超计; 王倩玉; 赵立明
Original assignee: Beijing Huarui Kangyuan Biotechnology Development Co ltd
Current assignee: Beijing Huarui Kangyuan Biotechnology Development Co ltd
Priority date: 2020-12-09
Filing date: 2020-12-09
Publication date: 2021-03-02

Abstract

The invention discloses a relative quantitative method and a kit for detecting the copy number of a human DMD gene by a multiplex real-time fluorescence PCR method. The invention relates to a relative quantitative method and a kit for detecting the copy number of a human DMD gene by a multiplex real-time fluorescence PCR method, which amplify exons 4, 17 and 45, 8, 50 and 52, 47, 48 and 51 of the DMD gene respectively through 3 independent PCR reactions, wherein 3 reaction solutions respectively comprise 4 fluorescence channels, thereby realizing the quantitative detection of the copy number of the exons 4, 8, 17, 45, 47, 48, 50, 51 and 52 of the DMD gene.

Description

Relative quantitative method and kit for detecting human DMD gene copy number by multiple real-time fluorescence PCR method

Technical Field

The invention relates to the technical field of biological detection, in particular to a relative quantitative method and a kit for detecting the copy number of a human DMD gene by a multiplex real-time fluorescence PCR method.

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.

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 rate of the DMD in the boy infant is about 1/3500-1/4000, the boy infant is ill more than 5-6 years old, the walking is slow, the boy infant is easy to fall down, the walking posture is abnormal, the independent walking ability is lost before 13 years old, and 20-30 years old die from heart and lung function failure. The incidence rate of BMD in men is about 1/8000-1/10000, clinical symptoms appear later than DMD, the walking ability can be kept to 16 years old, the disease progress is relatively slow, the life is long, and the life can reach 40-50 years old. Pediatric or adolescent BMD patients may present with muscle cramping myalgia or asymptomatic hypermyelinemia. At present, no universal effective treatment scheme exists for the disease.

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. Although important progress is made on treatment strategies such as gene editing, exon crossing, PTC protein repair and the like, no effective radical treatment method exists at present, and genetic block is still the best coping strategy. The clinical manifestations of duchenne/bethese muscular dystrophy are characterized by significant heterogeneity.

DMD gene variants are diverse and comprise mainly 4 classes. 1: exon Copy Number Variation (CNV), including exon deletions (deletion) and exon repeats (duplication). 2: point mutations of exons (substistition); 3: small insertions and deletions of exons (indels); 4: unknown variation (unknown): mainly intronic mutations affecting splicing. The frequency statistics of the DMD genetic variants recorded in the DMD professional database LOVD are shown in table 1:

TABLE 1 DMD Gene variation types and corresponding ratios

Type of variation	Deletion	Duplication	Substitution	Indels	Unknown
						Ratio in all variations	56％	11％	31％	0.7％	1.3％
Ratio in pathogenic variation	66％	12％	20％	0.9％	1.1％

From the above DMD genetic variation types and the corresponding ratios, it can be concluded that the pathogenic variation of DMD gene, in which the fragment deletion or the repeated variation accounts for about 78%, is the main type of variation causing duchenne/betheson muscular dystrophy.

At present, the copy number detection of the DMD gene is mainly performed by using a Multiple Ligation-dependent Probe Amplification (MLPA) detection technology produced by MRC corporation, netherlands, and a P034 Probe set and a P035 Probe set are designed in an MLPA detection kit for DMD, wherein the P034 Probe set comprises 40 pairs of probes for detecting 40 exons of DMD and 9 pairs of probes for 9 reference genes; the P035 probe set included 40 pairs of probes for detection of 40 exons of DMD and 8 pairs of probes for 8 internal reference genes. After each pair of probes is hybridized with sample DNA, the two probes in the probe pair are connected end to end and can be connected into a complete DNA template under the action of ligase. After the connection of 49 pairs of probes of P034 and 48 pairs of probes of P035 is completed, PCR amplification is performed by using universal primers, the sizes of PCR fragments generated by amplification of the connection products of each probe pair are different (the 5 'end or the 3' end of two probes in each pair of probes is provided with a universal primer sequence, the sizes of PCR products are different by using a filling sequence, the sizes of products are preset), then PCR products with different sizes are screened and distinguished by using capillary electrophoresis, DMD exon numbers represented by the amplification product fragments are judged according to the lengths (bp numbers) of the PCR products, and whether each exon of the DMD gene is lost or repeated is judged according to the abundance ratio of the DMD exon PCR products and the internal reference gene PCR products.

The MLPA detection technology of the DMD gene has good accuracy, but the reagent cost of the MLPA detection technology is high, and capillary electrophoresis equipment used for fragment screening, such as ABI3130 or 3500, is expensive. In addition, the MLPA detection technology process has more links, mainly comprises four main steps of hybridization overnight, probe connection, PCR amplification and fragment screening, and more links provide higher requirements for the experimental skill and proficiency of operators, and also increase the possibility that the RQ value has larger fluctuation and the sample number is wrong. In addition, the most valuable application of the copy number detection of the DMD gene is female carrier screening, so that large-sample-size detection is required, and the MLPA detection technology has certain practical operation difficulty in the detection of large samples due to more technical links.

In view of the above-mentioned problems of the MLPA detection technique of the DMD gene, the present invention quantitatively detects exon deletion or duplication of the DMD gene using multiplex fluorescent quantitative PCR technique. The fluorescence quantitative PCR technology uses a delta Ct method in real-time amplification to carry out copy number quantification, and has better accuracy than the quantitative ratio of fragment amplification abundance of the MLPA technology; in addition, the main required equipment is only a fluorescence real-time PCR instrument, the equipment price is far lower than that of fragment screening equipment used by MLPA technology, such as ABI3130 or 3500 and the like, and the equipment is convenient for configuration and use by inspection institutions or hospitals; in addition, the operation of the fluorescent quantitative PCR detection technology is very simple, and an experimenter can operate on a computer and complete detection only by adding the sample DNA and the reaction mixed solution into a 96-well plate, so that the requirement on the operation skill of the experimenter is not high; stable detection results are easy to obtain; the large sample screening operation is easy to realize; and the cost of the reagent is low.

Although the multiplex quantitative fluorescence PCR technique has the advantages, the 427m transcript of the DMD gene has 79 exons, and if all 79 exons are subjected to quantitative fluorescence detection, the operation cannot be carried out, and all the advantages are eliminated. Although the DMD gene has 79 exons, deletion or repetitive variation of exons is not randomly distributed, but there are mutation hot spots. The method specifically comprises the following steps: the deletion of an exon has two hot spots, the proximal hot spot (located at exon 3-20) and the distal hot spot (located at exon 44-53), while the most frequent regions of exon duplication are exon3-11 and exon 21-31. In order to cover the deletion type of the DMD fragment as much as possible, 1441 cases of patients with variant DMD gene copy number, which are recorded in the UMD database, are analyzed for exon deletion or repeated regions, and frequency statistics shows that if only exon 4 is detected; 8; 17; 45, a first step of; 47; 48; 50; 51; the 52 exons 9 can cover 1306 in 1441 cases, and the detection coverage of copy number variation can reach about 86%. For the rare disease Duchenne/Behcet muscular dystrophy, the statistical result of 1441 cases should have a good representativeness, can reach the variation detection coverage of 86%, and also has a good application value. And the fluorescent quantitative PCR detection of 9 exons also has convenient operability. Thus, the invention can improve the operation convenience and simultaneously achieve the ideal copy number variation detection coverage, and is particularly suitable for the auxiliary diagnosis of male patients with fragment deletion.

Disclosure of Invention

Aiming at the technical problems in the related art, the invention provides a relative quantification method and a kit for detecting the copy number of the human DMD gene by a multiplex real-time fluorescence PCR method, which can overcome the defects in the prior art.

In order to achieve the technical purpose, the technical scheme of the invention is realized as follows:

a kit for detecting the copy number of a human DMD gene by a multiplex real-time fluorescence PCR method comprises an exon reaction solution I, an exon reaction solution II, an exon reaction solution III, a main reaction mixed solution, a positive control product, a negative control product II and a blank control product;

the first exon reaction solution is a solution containing specific primers and probes for exons 4, 17 and 45 of the DMD gene and specific primers and probes for the CFTR1 gene;

the second exon reaction solution is a solution containing specific primers and probes for exons 8, 50 and 52 of the DMD gene and specific primers and probes for the CFTR2 gene;

the third exon reaction solution is a solution containing specific primers and probes for the 47 th, 48 th and 51 th exons of the DMD gene and specific primers and probes for the CFTR1 gene;

the main reaction mixed solution comprises PCR reaction buffer solution, dNTPs and Mg²⁺Hot start Taq enzyme, UNG enzyme and ROX fluorescent reference dye;

the positive control substance 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 sequence;

the blank reference substance is deionized water subjected to sterilization treatment.

Furthermore, the loading of the exon reaction solution I, the exon reaction solution II and the exon reaction solution III are all 500 mu L, the loading of the main reaction mixed solution is 1mL, the loading of the positive control is 15 mu L, the loading of the negative control is 30 mu L, and the loading of the blank control is 1 mL.

Further, the specific primers and probe sequences of exons 4, 17 and 45 of the DMD gene in the first exon reaction solution are as follows:

upstream primer sequence of exon 4: 5'-AAGGCACTGCGGGTTTTG-3', as shown in SEQ ID NO.1,

downstream primer sequence of exon 4: 5'-GTCACAGCATCCAGACCTTGTC-3', as shown in SEQ ID NO.2,

probe sequence for exon 4:

5 '-FAM-AGAACAATAATGTAAGTAGTACCC-3' -MGB-NFQ, as shown in SEQ ID NO.3,

upstream primer sequence of exon 17: 5'-TGACCTCTGTTTCAATACTTCTCACA-3', as shown in SEQ ID NO.4,

downstream primer sequence of exon 17: 5'-GTCACCGTAGTTACTGTTTCCATTACA-3', as shown in SEQ ID NO.5,

probe sequence of exon 17:

5 '-ABY-ACCACCACTCAGCCATCACTAACACAGACA-3' -QSY, as shown in SEQ ID NO.6,

upstream primer sequence of exon 45: 5'-TCTTCCCCAGTTGCATTCAAT-3', as shown in SEQ ID NO.7,

downstream primer sequence of exon 45: 5'-CAGGAACTCCAGGATGGCATT-3', as shown in SEQ ID NO.8,

probe sequence of exon 45:

5 '-VIC-TTCTGACAACAGTTTGCCGCTGCC-3' -QSY, shown in SEQ ID NO. 9;

the specific primer and probe sequence of the CFTR1 gene in the exon reaction liquid I is as follows:

the sequence of the upstream primer is as follows: 5'-TTGTGCCTGTTGCAGCTTCT-3', as shown in SEQ ID NO.10,

the sequence of the downstream primer is as follows: 5'-TGGAGTTACAGAAAGGCCTCATG-3', as shown in SEQ ID NO.11,

the probe sequence is as follows: 5 '-Cy 5-CGAATGGCACCACCTTCTCGGTGT-3' -QSY as shown in SEQ ID NO. 12;

the concentrations of the specific upstream primers and the specific downstream primers of the exons 4, 17 and 45 of the DMD gene in the exon reaction solution I are both 100-800 nmol/L, preferably 400nmol/L, and the concentrations of the specific probes of the exons 4, 17 and 45 are 50-300 nmol/L, preferably 200 nmol/L; the concentrations of an upstream primer and a downstream primer of an internal reference CFTR1 gene in the exon reaction liquid I are both 100-800 nmol/L, preferably 400nmol/L, and the concentration of a probe of an internal reference CFTR1 gene is 50-300 nmol/L, preferably 200 nmol/L.

Further, the specific primers and probe sequences of exons 8, 50 and 52 of the DMD gene in the exon reaction solution two are as follows:

upstream primer sequence of exon 8:

5'-TGTACATCACATCACTCTTCCAAGTTT-3', as shown in SEQ ID NO.13,

downstream primer sequence of exon 8: 5'-CCTTGGCAACATTTCCACTTC-3', as shown in SEQ ID NO.14,

probe sequence of exon 8:

5 '-FAM-CTCAACAAGTGAGCATTGAAGCCATCCA-3' -QSY, as shown in SEQ ID NO.15,

upstream primer sequence of exon 50: 5'-GCTGGCTGGATCAGGAATACA-3', as shown in SEQ ID NO.16,

downstream primer sequence of exon 50: 5'-CGCAGGAGCCCTACATCTG-3', as shown in SEQ ID NO.17,

probe sequence of exon 50:

5 '-ABY-AGGCCCACTGCCTGCAATTCAGG-3' -QSY, as shown in SEQ ID NO.18,

upstream primer sequence of exon 52: 5'-GCGGTAATGAGTTCTTCCAACTG-3', as shown in SEQ ID NO.19,

downstream primer sequence of exon 52: 5'-CAACGCTGAAGAACCCTGATACT-3', as shown in SEQ ID NO.20,

probe sequence of exon 52:

5 '-VIC-CGCCTCTGTTCCAAATCCTGCATTG-3' -QSY, shown in SEQ ID NO. 21;

the specific primer and probe sequence of the CFTR2 gene in the exon reaction liquid II is as follows:

the sequence of the upstream primer is as follows: 5'-TGTTTGTACAGCCCAGGGAAA-3', as shown in SEQ ID NO.22,

the sequence of the downstream primer is as follows: 5'-CACCATCTCATTCTGCATTGTTC-3', as shown in SEQ ID NO.23,

the probe sequence is as follows: 5 '-Cy 5-CCGAGTGACCGCCATGCGC-3' -QSY as shown in SEQ ID NO. 24;

the concentrations of the specific upstream primers and the specific downstream primers of the 8 th, 50 th and 52 th exons of the DMD gene in the exon reaction liquid II are both 100-800 nmol/L, preferably 400nmol/L, and the concentrations of the specific probes of the 8 th, 50 th and 52 th exons are 50-300 nmol/L, preferably 200 nmol/L; the concentrations of an upstream primer and a downstream primer of the internal reference CFTR2 gene in the exon reaction liquid II are both 100-800 nmol/L, preferably 400nmol/L, and the concentration of a probe of the internal reference CFTR2 gene is 50-300 nmol/L, preferably 200 nmol/L.

Furthermore, the specific primer and probe sequences of the 47 th, 48 th and 51 th exons of the DMD gene in the third exon reaction solution are as follows:

upstream primer sequence of exon 47: 5'-AACGTTGTTGCATTTGTCTGTTTC-3', as shown in SEQ ID NO.25,

downstream primer sequence of exon 47: 5'-CGGGTCCTCCAGTTTCATTTAA-3', as shown in SEQ ID NO.26,

probe sequence of exon 47:

5 '-FAM-CCTGCGCCAGGGAATTCTCAAACA-3' -QSY, as shown in SEQ ID NO.27,

upstream primer sequence of exon 48: 5'-CTATAAATTCCTACTTCCACTGTGCTGTA-3', as shown in SEQ ID NO.28,

downstream primer sequence of exon 48: 5'-GGGACCACTGCAATGGAGTATT-3', as shown in SEQ ID NO.29,

probe sequence of exon 48:

5 '-ABY-TTGAGCCCAATCTCTCTCTATCCAACCTCC-3' -QSY, as shown in SEQ ID NO.30,

upstream primer sequence of exon 51: 5'-GAAATGCCATCTTCCTTGATGTT-3', as shown in SEQ ID NO.31,

downstream primer sequence of exon 51: 5'-AGAAAGCCAGTCGGTAAGTTCTGT-3', as shown in SEQ ID NO.32,

probe sequence of exon 51:

t5 '-VIC-ACCTGCTCTGGCAGATTTCAACCGG-3' -QSY as shown in SEQ ID NO. 33;

the specific primer and probe sequence of the CFTR1 gene in the exon reaction liquid III is as follows:

the concentrations of the specific upstream primers and the specific downstream primers of the 47 th, 48 th and 51 th exons of the DMD gene in the exon reaction liquid III are all 100-800 nmol/L, preferably 400nmol/L, and the concentrations of the specific probes of the 47 th, 48 th and 51 th exons are 50-300 nmol/L, preferably 200 nmol/L; the concentrations of an upstream primer and a downstream primer of an internal reference CFTR1 gene in the exon reaction liquid III are both 100-800 nmol/L, and the concentration of a probe of an internal reference CFTR1 gene is 50-300 nmol/L, preferably 200 nmol/L.

Further, the plasmid sequence in the negative control is shown as SEQ ID NO.34, and the plasmid sequence in the positive control is shown as SEQ ID NO. 35.

According to another aspect of the present invention, there is provided a method for detecting the copy number of the human DMD gene using the above-described kit, the method comprising the steps of:

(1) extracting human cell genome DNA, measuring the nucleic acid concentration by a micro ultraviolet spectrophotometer and adjusting to 20 ng/mu L;

(2) preparing a reference substance: dissolving the positive control, the negative control and the blank control for later use; preparing a multiple PCR reaction system, wherein the total reaction volume is 20 mu L;

(3) sample detection: adding the positive control substance, the negative control substance, the blank control substance and the gDNA sample to be detected into the reaction hole site in sequence, wherein the sample adding volume is 5 mu L;

(4) 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 real-time collecting FAM, VIC, ABY and Cy5 fluorescence signals in each cycle; wherein, the gene target represented by the FAM, VIC and ABY fluorescent signals is each exon of the DMD gene, and the gene target represented by the Cy5 fluorescent signal is a reference gene of CFTR;

(5) and (4) analyzing results: and (3) performing copy number quantification on 9 exons of the DMD gene by adopting a relative quantification mode of a delta-delta Ct value method.

The invention has the beneficial effects that: the relative quantitative method and the kit for detecting the copy number of the human DMD gene by the multiplex real-time fluorescence PCR method have the following advantages:

(1) the rapid result reference can be provided for clinical screening, and the detection result can realize the differentiation of DMD carriers, DMD patients and normal persons;

(2) the detection result is reliable and the repeatability is good: the result of the large sample verification shows that the mean value (AV) of the RQ values of 9 exons of a normal female is 0.99-1.03, and the variance (RQ) is less than or equal to 0.05; the Average Value (AV) of the RQ values of the 9 exons of the normal male is 0.47-0.51, the variance (RQ) is less than or equal to 0.03, the average values of the RQ values of the male and the female are respectively close to 0.5 and 1.0, and the fluctuation is small;

(3) the detection method has low requirements on detection equipment, and the main equipment is an ABI 7500 fluorescent quantitative PCR instrument, so that the detection mechanism can be conveniently purchased and used;

(4) the detection method is simple to operate, can complete detection on a computer only by adding three components of the sample DNA, the reaction solution and the PCR mixed solution, has low requirement on the skill of an operator, and can effectively prevent the sample from being mixed;

(5) the reliability is good, the operation is simple, the equipment requirement is low, the reagent cost is low, and the method is particularly suitable for screening DMD female carriers with multiple centers and high flux, and the screening of the female carriers is the central importance of DMD gene detection;

(6) copy number variation in DMD patients covering about 86% can be achieved by detecting only 9 exons of the DMD gene, achieving a good balance between detection coverage and detection exon count.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.

FIG. 1 shows the result of detecting a pair of positive controls in an exon reaction solution according to an embodiment of the present invention;

FIG. 2 shows the result of detection of a negative control according to an embodiment of the present invention;

FIG. 3 shows the result of the blank detection according to the embodiment of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments that can be derived by one of ordinary skill in the art from the embodiments given herein are intended to be within the scope of the present invention.

Example 1

The kit (100 reactions/kit) for detecting the copy number of the human DMD gene by the multiplex real-time fluorescent PCR method comprises the following components as shown in Table 2:

TABLE 2 kit Components

The first exon reaction solution is a relative quantitative detection primer probe aiming at copy numbers of exons 4, 17 and 45 of the DMD gene:

D4F:5’-AAGGCACTGCGGGTTTTG-3’，

D4R:5’-GTCACAGCATCCAGACCTTGTC-3’，

D4P:5’-FAM-AGAACAATAATGTAAGTAGTACCC-3’-MGB-NFQ，

D17F:5’-TGACCTCTGTTTCAATACTTCTCACA-3’，

D17R:5’-GTCACCGTAGTTACTGTTTCCATTACA-3’，

D17P:5’-ABY-ACCACCACTCAGCCATCACTAACACAGACA-3’-QSY，

D45 F:5’-TCTTCCCCAGTTGCATTCAAT-3’，

D45 R:5’-CAGGAACTCCAGGATGGCATT-3’，

D45 P:5’-VIC-TTCTGACAACAGTTTGCCGCTGCC-3’–QSY，

CFTR1 F:5’-TTGTGCCTGTTGCAGCTTCT-3’，

CFTR1 R:5’-TGGAGTTACAGAAAGGCCTCATG-3’，

CFTR1 P:5’-Cy5-CGAATGGCACCACCTTCTCGGTGT-3’-QSY，

in which CFTR was used as an internal reference gene. The No.4 exon detection probe adopts a taqman MGB-NFQ probe, and the other probes adopt taqman QSY probes.

And the second exon reaction solution is a primer probe for relative quantitative detection of 8 th, 50 th and 52 th exon copy numbers of the DMD gene:

D8 F:5’-TGTACATCACATCACTCTTCCAAGTTT-3’，

D8 R:5’-CCTTGGCAACATTTCCACTTC-3’，

D8 P:5’-FAM-CTCAACAAGTGAGCATTGAAGCCATCCA-3’-QSY，

D50 F:5’-GCTGGCTGGATCAGGAATACA-3’，

D50 R:5’-CGCAGGAGCCCTACATCTG-3’，

D50 P:5’-ABY-AGGCCCACTGCCTGCAATTCAGG-3’-QSY，

D52 F:5’-GCGGTAATGAGTTCTTCCAACTG-3’，

D52 R:5’-CAACGCTGAAGAACCCTGATACT-3’，

D52 P:5’-VIC-CGCCTCTGTTCCAAATCCTGCATTG-3’-QSY，

CFTR2 F:5’-TGTTTGTACAGCCCAGGGAAA-3’，

CFTR2 R:5’-CACCATCTCATTCTGCATTGTTC-3’，

CFTR2 P:5’-Cy5-CCGAGTGACCGCCATGCGC-3’-QSY，

in which CFTR was used as an internal reference gene. Reaction wells all probes were used with taqman QSY probes.

And the third exon reaction solution is a relative quantitative detection primer and a probe aiming at the copy numbers of 47 th, 48 th and 51 th exons of the DMD gene:

D47 F:5’-AACGTTGTTGCATTTGTCTGTTTC-3’，

D47 R:5’-CGGGTCCTCCAGTTTCATTTAA-3’，

D47 P:5’-FAM-CCTGCGCCAGGGAATTCTCAAACA-3’-QSY，

D48 F:5’-CTATAAATTCCTACTTCCACTGTGCTGTA-3’，

D48 R:5’-GGGACCACTGCAATGGAGTATT-3’，

D48 P:5’-ABY-TTGAGCCCAATCTCTCTCTATCCAACCTCC-3’-QSY，

D51 F:5’-GAAATGCCATCTTCCTTGATGTT-3’，

D51 R:5’-AGAAAGCCAGTCGGTAAGTTCTGT-3’，

D51 P:T5’-VIC-ACCTGCTCTGGCAGATTTCAACCGG-3’-QSY，

CFTR1 F:5’-TTGTGCCTGTTGCAGCTTCT-3’，

CFTR1 R:5’-TGGAGTTACAGAAAGGCCTCATG-3’，

CFTR1 P:5’-Cy5-CGAATGGCACCACCTTCTCGGTGT-3’-QSY，

wherein, the reaction hole takes CFTR as an internal reference gene. All probes were used with taqman QSY probes.

Main reaction mixed liquid: 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 (shown as a nucleotide sequence in SEQ ID No. 34) and water. Specific plasmid 1 is an artificially synthesized DNA sequence of the internal reference CFTR gene, and the final concentration of plasmid 1 in the control solution is 0.04 pg/. mu.L.

Positive control: comprises a specific plasmid 2 (shown as a nucleotide sequence in SEQ ID No. 35) and water. The specific plasmid 2 is an artificially synthesized DNA sequence of an internal reference CFTR gene and a DMD gene 4; 8; 17; 45, a first step of; 47; 48; 50; 51; 52 total 9 exons and the final plasmid concentration in the control solution was 0.06 pg/. mu.L.

The blank control was deionized water.

The formula of each component is as follows:

exon reaction solution one (first reaction solution) formulation: the concentration of the specific upstream primer and the specific downstream primer of exons 4, 17 and 45 of the DMD gene is 100-800 nmol/L, and preferably 400 nmol/L. The concentration of the exon-specific probes in the 4 th, 17 th and 45 th genes is 50-300 nmol/L, and preferably 200 nmol/L. The concentration of the upstream primer and the downstream primer of the internal reference CFTR1 is 100-800 nmol/L, preferably 400 nmol/L. The concentration of the internal reference probe is 50-300 nmol/L, preferably 200 nmol/L.

And (3) preparing a second exon reaction solution (second reaction solution): the concentration of the specific upstream primer and the specific downstream primer of 8 th exon, 50 th exon and 52 th exon of the DMD gene is 100-800 nmol/L, and preferably 400 nmol/L. The concentration of the 8 th, 50 th and 52 th exon-specific probes is 50-300 nmol/L, preferably 200 nmol/L. The concentration of the upstream primer and the downstream primer of the internal reference CFTR2 is 100-800 nmol/L, preferably 400 nmol/L. The concentration of the internal reference probe is 50-300 nmol/L, preferably 200 nmol/L.

And the third exon reaction solution (third reaction solution) formula: the concentration of the specific upstream primer and the specific downstream primer of the 47 th exon, 48 th exon and 51 th exon of the DMD gene is 100-800 nmol/L, and preferably 400 nmol/L. The concentration of the exon-specific probes at 47, 48 and 51 is 50-300 nmol/L, preferably 200 nmol/L. The concentration of the upstream primer and the downstream primer of the internal reference CFTR1 is 100-800 nmol/L, preferably 400 nmol/L. The concentration of the internal reference probe is 50-300 nmol/L, preferably 200 nmol/L.

Example 2

Quantitative molecular detection of DMD copy number variation of human Duchenne/Behcet muscular dystrophy causative gene using human peripheral blood free DNA or gDNA using the kit described in example 1:

(1) nucleic acid extraction:

in addition, the clinical diagnosis of DMD is 1 child, and the clinical diagnosis of DMD is 2 female carriers who were born 2 women who were born 2 normal men and 3 normal women who were born 2 normal men. 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 spectrophotometer_260/280Between 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, the negative control and the blank control for later use.

(3) PCR reaction system was prepared with a total reaction volume of 20. mu.L, as shown in Table 3:

TABLE 3 PCR reaction System

(4) Sample detection:

and adding the negative control substance, the positive control substance, the blank control substance and the gDNA of the 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: 95 ℃ for 15 seconds, 60 ℃ for 1 minute (signal acquisition), for a total of 40 cycles. The used instrument of the reaction is ABI 7500, FAM, VIC, ABY and Cy5 fluorescent signals are collected in real time in each cycle, gene targets represented by the FAM, VIC and ABY fluorescent signals are each exon of DMD genes under each reaction, and gene targets represented by the Cy5 fluorescent signals are CFTR reference genes.

The detection results of the pair of positive controls in the exon reaction solution are shown in FIG. 1, the detection results of the negative controls are shown in FIG. 2, and the detection results of the blank controls are shown in FIG. 3.

(6) And (4) analyzing results: and (3) performing copy number quantification on 9 exons of the DMD gene by adopting a relative quantification mode of a delta-delta Ct value method.

The quality control requirements of 4 PCR reactions in copy number quantification are as follows, the first 3 quality control indexes are unqualified, the experiment failure is judged, the experiment needs to be carried out again, the 4 th index is unqualified, the experiment failure of the sample is judged, and the sample needs to be detected again:

the Ct values of the FAM, VIC and ABY fluorescence channels of the positive control are less than or equal to 33, and the Ct value of the Cy5 fluorescence channel is less than or equal to 31;

the negative control products FAM, VIC and ABY fluorescence channels have no signals, and the Ct value of the Cy5 fluorescence channel is less than or equal to 31;

the blank FAM, VIC, ABY, Cy5 fluorescence channels should all be signal-free.

The Ct value of the Cy5 fluorescence channel of the sample to be detected is less than or equal to 31.

The following is a method for calculating the relative expression level (RQ value):

calculation of the positive control Δ Ct:

positive control in exon 4; 8; 17; 45, a first step of; 47; 48; 50; 51; the value of Delta Ct between the target gene and the reference gene in the 52 reaction_FAM/VIC/ABY–Ct_Cy5. MeterCalculating the average value of delta Ct of three concentration gradients of the normal control in each reaction, and recording the average value as delta Ct_a。

Respectively calculating the delta Ct values between 9 target genes of the sample to be detected and the reference genes in the corresponding reaction holes, wherein the delta Ct is Ct_FAM/VIC/ABY–Ct_Cy5Is denoted as Δ Ct_s。

Target gene delta Ct of sample to be detected is equal to target gene delta Ct of sample to be detected_s-△Ct_a；

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 results of the normal control samples, which were 2 copies of each exon of the DMD gene and the CFTR of the internal reference gene, are shown in table 4.

TABLE 4 determination of results

The experimental results meet the general requirements for data analysis as follows:

the Ct value of the positive control FAM, VIC and ABY fluorescence channels is less than or equal to 33; ct value of the Cy5 fluorescence channel is less than or equal to 31;

the blank controls, FAM, VIC, ABY, Cy5 fluorescence channels, had no significant amplification signal.

DMD gene No.4 in 1 child with DMD, 2 female carriers, 3 normal females, 2 normal males; 8; 17; 45, a first step of; 47; 48; 50; 51; the RQ values, copy numbers of 52 are shown in table 5.

TABLE 5.8 sample test results

Note: FC 1: female carrier 1; FC 2: female carrier 2; MF 1: male patient 1; FN 1-3: 1-3 of normal women; MN 1-2: normal males 1-2. The values in the table are RQ values/copy number.

(7) And (4) analyzing and verifying the result: the results of parallel tests on 1 male infant patient and 2 female carriers were performed using P034 and P035 DMD probemix 100rxn test kits of MRC, Netherlands, and are shown in Table 6.

TABLE 6 parallel test results

For 1 male infant patient and 2 female carrier samples, the copy number of 9 exons detected by the kit and the detection result of MLPA are 100%.

Example 3

And (3) reagent specificity verification: pathogen cross-reactions are common in clinical settings.

(1) Experimental sample

The specificity of the reagent was verified by taking 4 specific samples of hepatitis B virus (10)⁷copies/ml), hepatitis C virus (10)⁵copies/ml), human cytomegalovirus (10)⁴copies/ml), enterovirus type 71 (10)⁴copies/ml)。

(2) Procedure of experiment

And (3) respectively detecting the above 4 specific samples by using the first reaction solution to the third reaction solution, analyzing the detection result and verifying the specificity of the reagent.

(3) Results of the experiment

The 3 reaction solutions detected 4 specific samples were all underwent, indicating that the specificity was good and there was no cross reaction, and the specific results are shown in table 7.

TABLE 7.3 Cross-reactivity results with clinically common pathogens (UD ═ established)

In summary, according to the above technical solution of the present invention, the exons 4, 17, and 45 of DMD gene are separately subjected to 3 independent PCR reactions (reaction solution one); 8. exons 50 and 52 (reaction mixture two); 47. 48, 51 exons (reaction third) were amplified. The 3 reaction solutions all contain 4 fluorescence channels (wherein 3 fluorescence channels are target genes, and the last fluorescence channel is an internal reference gene), so that quantitative detection of copy numbers of exons 4, 8, 17, 45, 47, 48, 50, 51 and 52 of the DMD gene is realized.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> Beijing Huaruikang Yuan Biotechnology development Co., Ltd

<120> relative quantitative method and kit for detecting human DMD gene copy number by multiplex real-time fluorescence PCR method

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Claims

1. A kit for detecting the copy number of a human DMD gene by a multiplex real-time fluorescence PCR method is characterized by comprising an exon reaction solution I, an exon reaction solution II, an exon reaction solution III, a main reaction mixed solution, a positive reference substance, a negative reference substance and a blank reference substance;

the first exon reaction solution is a solution containing specific primers and probes for exons 4, 17 and 45 of the DMD gene and specific primers and probes for the CFTR gene;

the second exon reaction solution is a solution containing specific primers and probes for exons 8, 50 and 52 of the DMD gene and specific primers and probes for the CFTR gene;

the third exon reaction solution is a solution containing specific primers and probes for 47 th, 48 th and 51 th exons of the DMD gene and specific primers and probes for the CFTR gene;

the main reaction mixed solution comprises PCR reaction buffer solution, dNTPs and Mg²⁺Hot startTaqEnzyme, UNG enzyme, ROX fluorescent reference dye;

the negative control substance is a plasmid DNA solution containing a CFTR gene sequence;

the positive control substance is a plasmid DNA solution containing a CFTR gene sequence and a DMD gene sequence;

2. The kit for detecting the copy number of the human DMD gene by the multiplex real-time fluorescence PCR method according to claim 1, wherein the loading amounts of the first exon reaction solution, the second exon reaction solution and the third exon reaction solution are all 500 μ L, the loading amount of the main reaction mixture is 1mL, the loading amount of the negative control is 15 μ L, the loading amount of the positive control is 30 μ L, and the loading amount of the blank control is 1 mL.

3. The kit for detecting the copy number of the human DMD gene by the multiplex real-time fluorescence PCR method according to claim 1, wherein the specific primers and probe sequences of exons 4, 17 and 45 of the DMD gene in the first exon reaction solution are as follows:

probe sequence for exon 4:

5 '-FAM-AGAACAATAATGTAAGTAGTACCC-3' -MGB-NFQ, as shown in SEQ ID NO.3,

probe sequence of exon 17: 5 '-ABY-ACCACCACTCAGCCATCACTAACACAGACA-3' -QSY, as shown in SEQ ID NO.6,

probe sequence of exon 45: 5 '-VIC-TTCTGACAACAGTTTGCCGCTGCC-3' -QSY, shown in SEQ ID NO. 9;

the probe sequence is as follows: 5 '-Cy 5-CGAATGGCACCACCTTCTCGGTGT-3' -QSY, as shown in SEQ ID NO. 12.

4. The kit for detecting the copy number of the human DMD gene by the multiplex real-time fluorescence PCR method according to claim 3, wherein the concentrations of exon-specific upstream primers and downstream primers of DMD gene 4, 17 and 45 in the first exon reaction solution are all 100-800 nmol/L, and the concentrations of exon-specific probes of 4, 17 and 45 are 50-300 nmol/L; the concentrations of an upstream primer and a downstream primer of the CFTR1 gene in the first exon reaction solution are both 100-800 nmol/L, and the concentration of a probe of the CFTR1 gene is 50-300 nmol/L.

5. The kit for detecting the copy number of the human DMD gene by the multiplex real-time fluorescence PCR method according to claim 1, wherein the specific primers and probe sequences of exons 8, 50 and 52 of the DMD gene in the exon reaction solution II are as follows:

upstream primer sequence of exon 8:

5'-TGTACATCACATCACTCTTCCAAGTTT-3', as shown in SEQ ID NO.13,

probe sequence of exon 8:

5 '-FAM-CTCAACAAGTGAGCATTGAAGCCATCCA-3' -QSY, as shown in SEQ ID NO.15,

probe sequence of exon 50: 5 '-ABY-AGGCCCACTGCCTGCAATTCAGG-3' -QSY, as shown in SEQ ID NO.18,

probe sequence of exon 52: 5 '-VIC-CGCCTCTGTTCCAAATCCTGCATTG-3' -QSY, shown in SEQ ID NO. 21;

the probe sequence is as follows: 5 '-Cy 5-CCGAGTGACCGCCATGCGC-3' -QSY, as shown in SEQ ID NO. 24.

6. The kit for detecting the copy number of the human DMD gene by the multiplex real-time fluorescence PCR method according to claim 5, wherein the concentrations of exon-specific upstream primers and downstream primers of DMD genes in the second exon reaction solution are all 100-800 nmol/L, and the concentrations of exon-specific probes of 8, 50 and 52 are 50-300 nmol/L; the concentrations of an upstream primer and a downstream primer of the CFTR2 gene in the exon reaction liquid II are both 100-800 nmol/L, and the concentration of a probe of the CFTR2 gene is 50-300 nmol/L.

7. The kit for detecting the copy number of the human DMD gene by the multiplex real-time fluorescence PCR method according to claim 1, wherein the specific primers and probe sequences of 47 th, 48 th and 51 th exons of the DMD gene in the third exon reaction solution are as follows:

probe sequence of exon 47:

5 '-FAM-CCTGCGCCAGGGAATTCTCAAACA-3' -QSY, as shown in SEQ ID NO.27,

probe sequence of exon 48: 5 '-ABY-TTGAGCCCAATCTCTCTCTATCCAACCTCC-3' -QSY, as shown in SEQ ID NO.30,

probe sequence of exon 51: t5 '-VIC-ACCTGCTCTGGCAGATTTCAACCGG-3' -QSY as shown in SEQ ID NO. 33;

8. The kit for detecting the copy number of the human DMD gene by the multiplex real-time fluorescence PCR method according to claim 7, wherein the concentrations of the specific upstream primers and the specific downstream primers of the 47 th exon, 48 th exon and 51 st exon of the DMD gene in the third exon reaction solution are all 100-800 nmol/L, and the concentrations of the specific probes of the 47 th exon, 48 th and 51 st exon are 50-300 nmol/L; the concentrations of an upstream primer and a downstream primer of the CFTR1 gene in the exon reaction liquid III are both 100-800 nmol/L, and the concentration of a probe of the CFTR1 gene is 50-300 nmol/L.

9. The kit for detecting the copy number of the DMD gene of the human being through the multiplex real-time fluorescence PCR method according to claim 1, wherein the plasmid sequence in the negative control product is shown as SEQ ID No.34, and the plasmid sequence in the positive control product is shown as SEQ ID No. 35.

10. A method for detecting the copy number of the DMD gene in a human by using the kit according to any one of claims 1 to 9, comprising the steps of:

(1) extracting human cell genome DNA, measuring the nucleic acid concentration by using a micro ultraviolet spectrophotometer and adjusting the nucleic acid concentration to 20 ng/muL;

(2) preparing a reference substance: dissolving the positive control, the negative control and the blank control for later use; preparing a multiple PCR reaction system, wherein the total reaction volume is 20 muL;

(3) sample detection: adding a positive control substance, a negative control substance, a blank control substance and a gDNA sample to be detected into the reaction hole position in sequence, wherein the sample adding volume is 5 muL;