CN107955833B

CN107955833B - Heterozygous mutant dry blood spot positive quality control product suitable for deafness mutant gene detection

Info

Publication number: CN107955833B
Application number: CN201711169917.4A
Authority: CN
Inventors: 李明; 吴英松; 杨学习; 杨琳艳; 吴龙; 杨旭
Original assignee: Guangzhou Darui Biotechnology Co ltd
Current assignee: Guangzhou Darui Biotechnology Co ltd
Priority date: 2017-11-20
Filing date: 2017-11-20
Publication date: 2021-08-24
Anticipated expiration: 2037-11-20
Also published as: CN107955833A

Abstract

The invention discloses a heterozygous mutant dry blood spot positive quality control product suitable for deafness mutant gene detection, which is prepared by mixing a DNA fragment containing a mutant site or a plasmid containing the DNA fragment with whole blood; the gene mutation site comprises: 11 mutation sites of four genes of GJB2, GJB3, 12SrRNA and SLC26A 4. The heterozygous mutant dry blood spot positive quality control product suitable for deafness mutant gene detection constructed by the invention is close to a clinical sample, has less required blood volume and convenient sample collection, can maintain the biological stability of the sample, reduces the biological risk and is convenient to transport and store. Can be normally used after being stored for 2 years at 4 ℃, and does not influence the detection result.

Description

Heterozygous mutant dry blood spot positive quality control product suitable for deafness mutant gene detection

Technical Field

The invention relates to the technical field of deafness gene heterozygous mutant site detection, in particular to a heterozygous mutant dry blood spot positive quality control product suitable for deafness mutant gene detection.

Background

2780 million people in the hearing-impaired population in China account for more than one third of the total population for 8269 thousands of disabled people in China. The proportion of the genetic deafness-causing genes carried by normal people is about 6 percent, about 3 ten thousand hearing-impaired children are born each year, and more than 6 ten thousand hearing-impaired children are newly increased every year in China by the late deafness patients. 50% of people with hearing impairment belong to delayed deafness, and the traditional hearing screening cannot find that hearing is permanently lost due to the stimulation of acquired environmental factors, so that the tragedy with hearing impairment can be completely avoided by deafness gene screening. Most pediatric hearing impairment is found after the age of 2 years, resulting in irreparable impairment of hearing and speech function. If the early detection is realized, the early medical intervention can avoid the dumb caused by deafness and can basically participate in normal social life. The proportion of hereditary hearing loss in birth can be up to 50%, statistics show that the incidence rate of deafness in newborn is 1 ‰, wherein about half of deafness is related to hereditary factors, and the genes affecting it mainly include GJB2, GJB3 and SLC26A 4.

Deafness gene detection significance: in the social aspect, the financial expenditure can be saved, the birth defect prevention and treatment level can be improved, and the birth control and health service level can be promoted; the epidemiological distribution of the deafness gene in the local area can be analyzed in the hospital, and the individual who finds the drug-sensitive deafness and the delayed deafness can improve the diagnosis efficiency and reduce the rate of missed diagnosis and the like; for a family, the aims of marriage and bearing, prenatal and postnatal care can be achieved, early discovery and early intervention can be achieved, and at least deafness but not dumb can be achieved. And the result of deafness gene detection has good guiding significance for deafness prevention, auxiliary diagnosis and rehabilitation treatment.

In the process of detecting common deafness related gene mutation by using various methods and deafness gene detection kits, factors influencing the experimental process are more, such as random errors in the determination operation of experimental personnel, temperature difference of a PCR amplification instrument, residue of an inhibitor after the extraction of sample nucleic acid, concentration of target nucleic acid to be amplified, reagent problems and the like, and the factors can cause deviation of results. Therefore, strict quality control measures, that is, specific gene detection standards or quality control products, are required for detection. In the detection of hereditary diseases such as hereditary deafness and the like, a quality control substance is a sample which is obtained manually and contains known gene mutation, and can be used for verifying the effectiveness of a newly developed detection method (whether the newly developed detection method can effectively detect mutation in a standard substance), positive control and negative control in detection, preventing false positive and false negative results, replacing a real sample when a detection operator is trained, avoiding ethical problems and simultaneously ensuring the quality of operation training.

The positive quality control product can be a confirmed known positive sample, or a verified disease-related cell line or a plasmid containing a known mutant gene constructed in vitro and the like, and the preferred principle is that the characteristics of a control sample are as close as possible to a clinical sample. Although the known positive sample is real human genome DNA, the positive quality control product is continuously prepared and used for a large amount of scientific research and clinical experiments in consideration of the difficulty of sample collection, biological safety and ethical problemsBut is not suitable. The traditional in vitro construction of plasmids containing known mutant genes has two methods: 1. directly diluting the recombinant plasmid to 10 degrees with a single PCR product positive in mutation³The copies/. mu.L is used as a positive quality control material, the quality control material prepared by the method can only cover the insert containing the specific mutation site, but can not cover other regions of the genome DNA; 2. mixing two or more PCR product recombinant plasmids with positive mutation in proportion and diluting to 10³The copies/mu L is used as a positive quality control product, and the method can simulate the detection of two or more mutation sites, but cannot cover other regions of the genome DNA; the quality control products prepared by the two methods cannot truly simulate human genome DNA.

At present, a positive quality control product which is easy to prepare, good in stability and repeatability, free from biological infectivity, capable of monitoring the whole detection process and reliable in result and can be used for deafness gene heterozygous mutation is lacked

Disclosure of Invention

The first purpose of the invention is to overcome the defects of the prior art and provide a hybrid mutant dry blood spot positive quality control product suitable for deafness mutant gene detection. Aiming at 11 common mutation types of deafness related genes GJB2, GJB3, SLC26A4 and 12S rRNA, a heterozygous mutation positive quality control product is provided for carrying out quality control on multiple detection processes of deafness gene mutation.

The second purpose of the invention is to provide a preparation method of the heterozygous mutant dry blood spot positive quality control product suitable for deafness mutant gene detection.

The third purpose of the invention is to provide the application of the heterozygous mutant dry blood spot positive quality control material suitable for deafness mutant gene detection in fluorescence quantitative PCR, Sanger sequencing, semiconductor sequencing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and microarray chip platform detection of deafness mutant genes.

In order to achieve the purpose, the invention is realized by the following technical scheme:

a heterozygous mutant dry blood spot positive quality control material suitable for deafness mutant gene detection is a dry blood spot prepared by mixing a DNA fragment containing a mutant site or a plasmid containing the DNA fragment with whole blood; the gene mutation site comprises: 11 mutation sites of four genes of GJB2, GJB3, 12SrRNA and SLC26A 4; wherein, the mutation sites of GJB2 are: GJB 2: 35delG, GJB 2: 167delT, GJB 2: 176_191del16, GJB 2: 235delC, GJB 2: 299 — 300delAT, the mutation sites of GJB3 are: GJB 3: 538C > T, GJB 3: 547G & gtA, and the mutation sites of 12SrRNA are as follows: 12 SrRNA: 1494C > T, 12 SrRNA: 1555A is more than G, and the mutation sites of the SLC26A4 are as follows: SLC26a 4: IVS7-2A > G; SLC26a 4: 2168A > G.

Preferably, there are 5 DNA fragments, and the sequences are shown in SEQ ID NO: 1 to 5.

Specifically, the distribution of the 11 mutation sites over 5 fragments is as follows:

wherein, the DNA fragment 1 is shown as SEQ ID NO: 1, comprising 5 mutations: GJB 2: 35delG, GJB 2: 167delT, GJB 2: 176_191del16, GJB 2: 235delC, GJB 2: 299 — 300 delAT;

DNA fragment 2 is shown as SEQ ID NO: 2, 2 mutation sites are included: GJB 3: 538C > T, GJB 3: 547G > A;

DNA fragment 3 is shown in SEQ ID NO: 3, 2 mutation sites are included: 12 SrRNA: 1494C > T, 12 SrRNA: 1555A is greater than G;

DNA fragment 4 is shown in SEQ ID NO: 4, comprises 1 mutation site: SLC26a 4: IVS7-2A > G;

DNA fragment 5 is shown in SEQ ID NO: 5, comprises 1 mutation site: SLC26a 4: 2168A > G.

Preferably, the DNA fragment or the plasmid containing the DNA fragment is used in a ratio of 2:1 in terms of its DNA copy number to whole blood.

Preferably, the plasmid is constructed by the following method: the DNA fragment containing the mutation site is ligated to a cloning vector.

Preferably, the cloning vector is pUC 57.

The preparation method of the heterozygous mutant dry blood spot positive quality control product suitable for deafness mutant gene detection is characterized by comprising the following steps:

s1, artificially synthesizing 5 DNA fragments containing mutation sites, wherein the sequences are respectively shown as SEQ ID NO: 1 to 5;

s2, respectively connecting 5 DNAs containing the mutation sites into cloning vectors to obtain 5 recombinant plasmids.

S3, mixing 5 DNA fragments containing mutation sites or 5 plasmids containing the DNA fragments with wild type human peripheral whole blood according to the DNA copy number ratio;

and S4, dropping the mixed blood sample on a carrier, and drying to obtain the deafness gene dried blood spot positive quality control product.

Preferably, step S3 is: diluting the DNA fragment containing the mutation site or the plasmid containing the DNA fragment to 10⁶，10⁵，10⁴，10³，10²Copy/mu L, and wild type human peripheral whole blood total DNA, respectively carrying out fluorescent quantitative PCR detection; establishing a standard curve by taking the copy number of DNA contained in each microliter as an abscissa and the measured CT value as an ordinate, and obtaining a formula of the standard curve; calculating the DNA copy number of the total DNA of the wild type human peripheral whole blood according to the standard curve; mixing DNA fragments containing mutation sites or plasmids containing the DNA fragments with wild type human peripheral whole blood according to the DNA copy number ratio;

preferably, in step S3, the primer PR1-F and the primer PR1-R amplify a DNA sequence containing GJB 2: 235delC, GJB 2: 167delT, GJB 2: 176_191del16 DNA fragment 1 of the 3 mutation sites or plasmid containing the DNA fragment 1, or DNA fragment corresponding to the 3 mutation sites of the total DNA of wild type human peripheral whole blood; and a primer PR2-F and a primer PR2-R are adopted, and the amplification product contains GJB 2: 35delG DNA fragment 1 of the mutation site or plasmid containing the DNA fragment 1, or DNA fragment corresponding to the mutation site of the total DNA of wild type human peripheral whole blood; and a primer PR3-F and a primer PR3-R are adopted, and the amplification product contains GJB 2: 299_300delAT DNA segment 1 of the mutation site or plasmid containing the DNA segment 1, or wild type human peripheral whole blood total DNA of the corresponding DNA segment of the mutation site; and a primer PR4-F and a primer PR4-R are adopted, and the amplification product contains GJB 2: 538C > T, GJB 2: 547G & gtA or a plasmid containing the DNA fragment 2 or DNA fragments corresponding to the 2 mutation sites of the total DNA of wild type human peripheral whole blood; primer PR5-F and primer PR5-R, amplified a mixture containing 12 SrRNA: 1494C > T, 12 SrRNA: 1555A & gtG or a plasmid containing the DNA fragment 3 or DNA fragments corresponding to 2 mutation sites of the total DNA of wild type human peripheral whole blood; primer PR6-F and primer PR6-R, amplified SLC26A 4: DNA fragment 4 of IVS7-2A > G mutation site or plasmid containing said DNA fragment 4, or DNA fragment corresponding to wild type human peripheral whole blood total DNA mutation site; primer PR7-F and primer PR7-R, amplified SLC26A 4: 2168A > G or a plasmid containing the DNA fragment 5, or a DNA fragment corresponding to the mutation site of the total DNA of wild type human peripheral whole blood;

wherein, the primer PR1-F is shown as SEQ ID NO: 6, the primer PR1-R is shown as SEQ ID NO: 7, the primer PR2-F is shown as SEQ ID NO: 8, the primer PR2-R is shown as SEQ ID NO: 9, the primer PR3-F is shown as SEQ ID NO: 10, the primer PR3-R is shown as SEQ ID NO: 11, primer PR4-F is shown as SEQ ID NO: 12, the primer PR4-R is shown as SEQ ID NO: 13, the primer PR5-F is shown as SEQ ID NO: 14, the primer PR5-R is shown as SEQ ID NO: 15, the primer PR6-F is shown as SEQ ID NO: 16, and the primer PR6-R is shown as SEQ ID NO: 17, the primer PR7-F is shown as SEQ ID NO: 18, and a primer PR7-R is shown as SEQ ID NO: 19, respectively.

Preferably, in step S3, the dosage ratio of the DNA fragment containing the mutation site or the plasmid containing the DNA fragment to the whole blood is: the ratio of the DNA copy number to the total DNA copy number is 2: 1.

Preferably, in step S4, the carrier is a filter paper sheet.

Preferably, in step S4, the filter paper sheet is printed with circles.

Preferably, in step S4, the mixed blood sample is dropped onto the filter paper sheet at the center of the circle.

Preferably, 50 μ L of blood sample is dropped into the center of each circle in step S4.

Preferably, in step S4, the drying condition is 4 hours at room temperature or 24 hours in humid climate.

Most preferably, the preparation method of the hybrid mutant dry blood spot positive quality control product suitable for deafness mutant gene detection is characterized by comprising the following steps:

s2, respectively connecting the artificially synthesized 5 DNAs containing the mutation sites into a cloning vector to obtain the recombinant plasmid containing the DNA fragment.

S3, respectively diluting the DNA fragment containing the mutation site or the plasmid containing the DNA fragment to 10⁶，10⁵，10⁴，10³，10²Copy/mu L, and wild type human peripheral whole blood total DNA, respectively carrying out fluorescent quantitative PCR detection; establishing a standard curve by taking the copy number of DNA contained in each microliter as an abscissa and the measured CT value as an ordinate, and obtaining a formula of the standard curve; calculating the DNA copy number of the total DNA of the wild type human peripheral whole blood according to the standard curve; mixing DNA fragments containing mutation sites or plasmids containing the DNA fragments with wild type human peripheral whole blood according to the DNA copy number ratio;

s4, dropwise adding the mixed blood sample to the center of a filter paper sheet ring, wherein each ring needs 50 mu L of blood sample, drying for 4 hours at room temperature or 24 hours in a humid climate, and naturally drying to obtain the deafness gene dry blood spot positive quality control product.

The heterozygous mutant dry blood spot positive quality control product obtained by the preparation method is suitable for deafness mutant gene detection.

The dry blood spot positive quality control substance is applied to the detection of deafness gene mutation.

Preferably, the method for detecting the mutation of the deafness gene comprises the following steps: fluorescent quantitative PCR, Sanger sequencing, semiconductor sequencing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and microarray chip platform detection.

The DNA copy number refers to the copy number of a DNA fragment containing four gene mutation sites of GJB2, GJB3, 12SrRNA and SLC26A 4.

Compared with the prior art, the invention has the following beneficial effects:

the invention takes the dry blood spot sample prepared by mixing the plasmid containing the known mutant gene constructed in vitro and the human wild peripheral whole blood according to the proportion of 2:1 as a quality control product, is close to a clinical sample, and has the advantages of less blood volume, convenient sample collection, capability of keeping the biological stability of the sample, reduction of biological risk, convenience in transportation and storage and the like. Can still be normally used after being stored for 2 years at 4 ℃, and does not influence the detection result. The quality control product has the following characteristics: (1) the preparation is easy; (2) the product has good stability and repeatability in the process of storage and use; (3) no biological infectivity; (4) the whole detection process can be monitored, and the result is reliable.

Drawings

FIG. 1 is a site schematic diagram of DNA fragment 1 mutation.

FIG. 2 is a schematic diagram of the site of mutation of DNA fragment 2.

FIG. 3 is a schematic diagram of the site of mutation of DNA fragment 3.

FIG. 4 is a schematic diagram of the site of mutation of DNA fragment 4.

FIG. 5 is a schematic diagram of the site of mutation of DNA fragment 5.

FIG. 6 shows the restriction enzyme digestion verification result of the mutation-positive plasmid; a is positive recombinant plasmid P1; b is positive recombinant plasmid P2; c is positive recombinant plasmid P3; d is positive recombinant plasmid P4; e is positive recombinant plasmid P5.

FIG. 7 is a fluorescent quantitative PCR standard curve; FIG. 7A is a standard curve ST 1; FIG. 7B is a standard curve ST 2; FIG. 7C is a standard curve ST 3; FIG. 7D is a standard curve ST 4; FIG. 7E is a standard curve ST 5; FIG. 7F is a standard curve ST 6; fig. 7G is a standard curve ST 7.

FIG. 8 shows the finished product of the positive quality control product of dried blood spots.

FIG. 9 shows Sanger sequencing validation results; a is GJB 3: 538C > T and GJB 3: 547G > A mutation; b is SLC26A 4: IVS7-2A > G mutation; c is SLC26A 4: 2168A > G; d is GJB 2: 299 — 300delAT mutation; e is GJB 2: 235delC, GJB 2: 176_191del16 and GJB 2: 167 delT; f is GJB 2: a 35delG mutation; g is 12 SrRNA: m.1494C > T and 12 SrRNA: m.1555A > G mutation.

FIG. 10 shows the verification result of matrix-assisted laser desorption ionization time-of-flight mass spectrometry: a is GJB 2: 176-191del16 heterozygous mutations; b is GJB 2: 299 — 300delAT heterozygous mutation; c is SLC26A 4: IVS7-2A > G heterozygous mutation; d is GJB 3: 547G > A heterozygous mutation; e is GJB 3: 538C > T heterozygous mutation; f is GJB 2: 35delG heterozygous mutation; g is GJB 2: 167delT heterozygous mutation; h is GJB 2: 235delC heterozygous mutation; i is heterogeneous mutation of 12S rRNAm.1555A & gtG; j is 12S rRNAm.1494C > T heterogeneous mutation; k is SLC26a 4: 2168A > G heterozygous mutation.

FIG. 11 shows that Sanger sequencing method verifies dried blood spot positive quality control PC-1; a is GJB 3: 538C > T and GJB 3: 547G > A mutation; b is SLC26A 4: IVS7-2A > G mutation; c is SLC26A 4: 2168A > G mutation; d is GJB 2: 299 — 300 delAT; e is GJB 2: 235delC, GJB 2: 176_191del16 and GJB 2: a 167delT mutation; f is GJB 2: a 35delG mutation; g is 12 SrRNA: m.1494C > T and 12 SrRNA: m.1555A > G mutation.

FIG. 12 is a Sanger sequencing method for verifying a dried blood spot positive quality control product PC-2; a is GJB 3: 538C > T and GJB 3: 547G > A mutation; b is SLC26A 4: IVS7-2A > G mutation; c is SLC26A 4: 2168A > G mutation; d is GJB 2: 299 — 300 delAT; e is GJB 2: 235delC, GJB 2: 176_191del16 and GJB 2: a 167delT mutation; f is GJB 2: a 35delG mutation; g is 12 SrRNA: m.1494C > T and 12 SrRNA: m.1555A > G mutation.

FIG. 13 is a diagram showing the Sanger sequencing method for verifying the dried blood spot positive quality control PC-3; a is GJB 3: 538C > T and GJB 3: 547G > A mutation; b is SLC26A 4: IVS7-2A > G mutation; c is SLC26A 4: 2168A > G mutation; d is GJB 2: 299 — 300 delAT; e is GJB 2: 235delC, GJB 2: 176_191del16 and GJB 2: a 167delT mutation; f is GJB 2: a 35delG mutation; g is 12 SrRNA: m.1494C > T and 12 SrRNA: m.1555A > G mutation.

FIG. 14 is a diagram showing the Sanger sequencing method for verifying the dried blood spot positive quality control PC-4; a is GJB 3: 538C > T and GJB 3: 547G > A mutation; b is SLC26A 4: IVS7-2A > G mutation; c is SLC26A 4: 2168A > G mutation; d is GJB 2: 299 — 300 delAT; e is GJB 2: 235delC, GJB 2: 176_191del16 and GJB 2: a 167delT mutation; f is GJB 2: a 35delG mutation; g is 12 SrRNA: m.1494C > T and 12 SrRNA: m.1555A > G mutation.

FIG. 15 shows a Sanger sequencing method for verifying dried blood spot positive quality control PC-5; a is GJB 3: 538C > T and GJB 3: 547G > A mutation; b is SLC26A 4: IVS7-2A > G mutation; c is SLC26A 4: 2168A > G mutation; d is GJB 2: 299 — 300 delAT; e is GJB 2: 235delC, GJB 2: 176_191del16 and GJB 2: a 167delT mutation; f is GJB 2: a 35delG mutation; g is 12 SrRNA: m.1494C > T and 12 SrRNA: m.1555A > G mutation.

Detailed Description

The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

Example 1

The preparation of the heterozygous mutant dry blood spot positive quality control product suitable for deafness mutant gene detection comprises the following steps:

1. obtaining total DNA (including genomic DNA and mitochondrial DNA) of wild-type human peripheral whole blood: mu.L of wild-type human peripheral whole Blood was collected, and 100. mu.L of DNA was extracted according to the QIAamp DNA Blood Mini Kit (QIAGEN, 69504) protocol and stored at-20 ℃.

2. Obtaining of recombinant plasmid containing DNA fragment of mutation site:

5 DNA sequences containing 11 mutation sites related to deafness gene were designed and synthesized by Biotechnology (Shanghai) GmbH. The 5 DNA sequences are respectively shown as SEQ ID NO: 1-5, the mutated sites are shown in figures 1-5, the bases with bold underlining are the bases after mutation, and the bases in bold brackets are the bases existing before deletion.

DNA fragment 1: contains GJB 2: 35delG, GJB 2: 167delT, GJB 2: 176_191del16, GJB 2: 235delC, GJB 2: 299 — 300delAT five mutations.

DNA fragment 2 contains GJB 3: 538C > T, GJB 3: 547G & gtA two kinds of mutation.

DNA fragment 3 comprises 12 SrRNA: 1494C > T, 12 SrRNA: 1555A and G.

DNA fragment 4 contains SLC26a 4: IVS7-2A > G mutation.

DNA fragment 5 contains SLC26a 4: 2168A > G mutation.

The pUC57 was used as vector to construct positive recombinant plasmids P1, P2, P3, P4 and P5 containing the above 5 DNA sequences, respectively, and FIG. 6 shows the results of restriction enzyme digestion verification of recombinant plasmids.

The 5 recombinant plasmids are respectively transformed into a top10 escherichia coli strain to obtain five positive recombinant escherichia coli strains of B1, B2, B3, B4 and B5. Coli were cultured and recombinant plasmids P1, P2, P3, P4 and P5 were extracted, respectively.

3. Fluorescent quantitative PCR detection of DNA copy number

(1) Primer design and Synthesis

Relates to a primer amplification recombinant plasmid DNA fragment containing a mutation site or a DNA fragment corresponding to the total DNA of wild human peripheral whole blood, because GJB 2: 235delC, GJB 2: 167delT and GJB 2: the three mutation sites 176 — 191del16 are located relatively close to each other on the genome, so the same pair of primers is used for amplification; the same GJB 2: 538C > T and GJB 2: 547G & gtA, the two mutation sites are amplified by using the same pair of primers; 12 SrRNA: 1494C > T and 12 SrRNA: the two mutation sites 1555A & gtG were amplified using the same pair of primers. The specific primer sequences and the corresponding relationship are shown in the following table:

(2) fluorescent quantitative PCR system and reaction conditions

Five recombinant plasmids P1, P2, P3, P4 and P5 were diluted to 10 respectively⁶，10⁵，10⁴，10³， 10²(copy/. mu.L) 5 gradients were combined with total DNA of wild-type human peripheral whole blood for fluorescent quantitative PCR detection, and PR1, PR2, PR3, PR4, PR5, PR6, and PR7 were used as primers for fluorescent quantitative PCR amplification, and two replicates were performed for 84PThe CR reaction, the reaction system is shown in the following table:

the reaction conditions are shown in the following table:

4. construction of Standard Curve (Standard, ST)

Taking the copy number of the recombinant plasmid DNA per microliter as an abscissa X, measuring each CT value as an ordinate Y, establishing a standard curve, and respectively establishing 7 standard curves according to the primer pairs: ST1 corresponds to primer PR1-F and primer PR1-R (FIG. 7A); ST2 corresponds to primer PR2-F and primer PR2-R (FIG. 7B); ST3 corresponds to primer PR3-F and primer PR3-R (FIG. 7C); ST4 corresponds to primer PR4-F and primer PR4-R (FIG. 7D); ST5 corresponds to primer PR5-F and primer PR5-R (FIG. 7E), ST6 corresponds to primer PR6-F and primer PR6-R (FIG. 7F); ST7 corresponds to primer PR7-F and primer PR7-R (FIG. 7G), resulting in a linear regression equation:

ST1：Y＝-3.257X+36.52(R²＝0.999)；

ST2：Y＝-3.223X+35.58(R²＝0.999)；

ST3：Y＝-3.379X+36.224(R²＝1)；

ST4：Y＝-3.511X+36.82(R²＝0.999)；

ST5：Y＝-3.695X+37.049(R²＝1)。

ST6：Y＝-3.559X+37.95(R²＝0.999)

ST7：Y＝-3.251X+37.468(R²＝0.999)。

performing fluorescent quantitative PCR detection on the total DNA of the wild type human peripheral whole blood to obtain CT values, and calculating according to a linear regression equation to obtain 7 amplifications of the total DNA of the wild type human peripheral whole bloodThe content of the fragments is respectively A₁：1.81×10⁴、A₂：1.43×10⁴、A₃：1.68×10⁴、A₄：3.13×10⁴、A₅：1.91×10⁶、A₆： 1.54×10⁴、A₇：1.56×10⁴(copy/. mu.L) in which A₁、A₂、A₃The average value A was obtained by calculating the DNA concentration using a standard curve constructed using the recombinant plasmid P1 as a template_MFinally according to A_MTo calculate the amount of recombinant plasmid P1 in the sample. A. the_MIs 1.64X 10⁴(copies/. mu.L), coefficient of variation was 1.17%.

5. Determining the volume of the mixed sample:

will 10⁷Copies/. mu.L concentrations of recombinant plasmids P1, P2, P4, P5 and 10⁹The recombinant plasmid P3 with copy/microliter concentration and wild type human peripheral whole blood total DNA are mixed according to the copy number of 2:1, and the volume of the mixed plasmid is calculated according to the formula:

V_P(μL)＝(2×G_DNA/G_P)×V_DNA；

wherein, V_PFor incorporation into recombinant plasmid volumes; c_DNACopy number of DNA fragment containing mutation site in wild type human peripheral whole blood total DNA per microliter; c_PCopy number of DNA fragment containing mutation site in one microliter of recombinant plasmid; v_DNAIs the volume of total DNA of peripheral whole blood of the wild type human to be mixed in.

The mixing conditions were as follows:

6. and (3) preparing a dried blood spot quality control product by dripping blood spots:

diluting the corresponding volume to 10⁷P1, P2, P4, P5 and 10 at copies/μ L concentration⁹Mixing copies/. mu.L concentration P3 into 500. mu.L wild type human peripheral whole blood total DNA, vortex mixing for 2min, instant centrifuging for 5S, and dripping the mixed blood sample into a filter paper circle (half-circle) by using a micropipetteDiameter 8mm), each circle needs about 50 mul blood sample, each set of dried blood spot quality control product has 3 blood spots (figure 8), and the dried blood spot quality control product is naturally dried for at least 4 hours at room temperature (at least 24 hours under humid climate), thus obtaining the deafness gene dried blood spot positive quality control product.

Application example 1 Sanger sequencing method to verify dry blood spot positive quality control product of deafness gene heterozygous mutation

1. The dry blood spot positive quality control prepared in example 1 was subjected to DNA extraction using a dry blood spot genomic DNA extraction kit. PCR amplification was performed using the fluorescent quantitative PCR primers of example 1. The primers were synthesized by Biotechnology engineering (Shanghai) Inc.

The PCR amplification system is as follows:

the procedure in the PCR instrument was as follows:

after the PCR reaction, the PCR product was sent to the Biotechnology engineering (Shanghai) Co., Ltd for Sanger sequencing verification. The results are shown in FIG. 9.

2. The results show that: the DNA extracted from the dry blood spot positive quality control material is used for PCR amplification, and the PCR product is subjected to Sanger sequencing verification, and the result shows that: 11 sites related to deafness genes are all positive for heterozygosity mutation of corresponding types (namely GJB 3: 538C > T, GJB 3: 547G > A, SLC26A 4: IVS7-2A > G, SLC26A 4: 2168A > G, GJB 2: 299-300 delAT, GJB 2: 235delC, GJB 2: 176-191del16, GJB 2: 167delT, GJB 2: 35delG, 12 SrRNA: m.149C > T and 12 SrRNA: m.1555A > G). The positive quality control product of heterozygous mutant dry blood spots suitable for the deafness mutant gene detection prepared by the method is reliable.

Application example 2 semiconductor sequencing method for verifying dry blood spot positive quality control product of deafness gene heterozygous mutation

1. The dry blood spot positive quality control prepared in example 1 was subjected to DNA extraction using a dry blood spot genomic DNA extraction kit, and two replicates of PC1 and PC2 were performed. According to the IonTorrent semiconductor sequencing standard program, two repeated PC1 and PC2 on the extracted dry blood spot positive quality control DNA are respectively subjected to library construction, library quantification and computer sequencing, and the results are as follows after data analysis:

2. the results show that: two libraries are constructed by using DNA extracted from the dry blood spot positive quality control product, and the on-machine sequencing result is analyzed, the sequencing depth values corresponding to 11 mutation sites are all more than or equal to 100, the mutation frequency is between 40% and 60%, namely about 50%, and the results accord with the human genome DNA sequencing result of the heterozygous mutant type, which shows that the heterozygous mutant dry blood spot positive quality control product which is prepared by the method and is suitable for deafness mutant gene detection is reliable.

Dry blood spot positive quality control product for verifying deafness gene heterozygous mutation by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

1. Deafness gene detection (SLC26a4, GJB2, GJB3, 12S rRNA) based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry was performed using the dry blood spot positive quality control prepared in example 1. The procedure was followed in accordance with standard procedures based on matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Meanwhile, according to the sequences of 5 DNAs and mutation sites to be detected, an amplification primer and a single-base extension primer are designed, wherein the positive control quality control product comprises: GJB 2: 235delC hybrid human genome DNA, negative control quality control products are: wild-type human genomic DNA.

2. The results show (fig. 10): the 11 sites related to deafness genes are all heterozygosity mutation positive of corresponding types (namely GJB 3: 538C > T, GJB 3: 547G > A, SLC26A 4: IVS7-2A > G, SLC26A 4: 2168A > G, GJB 2: 299-300 delAT, GJB 2: 235delC, GJB 2: 176-191del16, GJB 2: 167delT, GJB 2: 35delG, 12 SrRNA: m.149C > T and 12 SrRNA: m.1555A > G). The positive quality control product of heterozygous mutant dry blood spots suitable for the deafness mutant gene detection prepared by the method is reliable.

Application example 4 microarray chip method for verifying dry blood spot positive quality control product of deafness gene heterozygous mutation

1. The dry blood spot positive quality control prepared in example 1 was subjected to DNA extraction using a dry blood spot genomic DNA extraction kit, and two replicates of PC1 and PC2 were performed according to the crystal core

Fifteen genetic deafness related gene detection kits (microarray chip method) standard operation flow for microarray chip verification.

2. The results are as follows:

the DNA (PC1, PC2) extracted by the dry blood spot positive quality control product is verified by fifteen genetic deafness related gene detection kits (microarray chip method), and the result shows that: the 9 sites in PC1 and PC2 contained in the detection range of the kit are all positive for the corresponding type of heterozygous mutation (i.e. GJB 3: 538C > T, SLC26A 4: IVS7-2A > G, SLC26A 4: 2168A > G, GJB 2: 299-300 delAT, GJB 2: 235delC, GJB 2: 176-191del16, GJB 2: 35delG, 12 SrRNA: m.1494C > T and 12 SrRNA: m.1555A > G). The hybrid mutation dry blood spot positive quality control product which is prepared by the method and is suitable for deafness mutation gene detection is reliable.

Application example 5 semiconductor sequencing method for verifying repeatability of preparation process of dry blood spot positive quality control product

1. Using the recombinant plasmids P1, P2, P3, P4 and P5 prepared in example 1 and 5 parts of wild-type human peripheral whole blood, 5 parts of deaf gene heterozygous mutant dry plaque positive quality controls PC-1, PC-2, PC-3, PC-4 and PC-5 were prepared according to the method of example 1. And verifying the repeatability of the 5 deafness gene heterozygous mutant dry blood spot positive quality control products according to the semiconductor sequencing method of application example 2.

2. The results are as follows:

the results show that: the sequencing depth values corresponding to 11 mutation sites of the above 5 deaf gene heterozygous mutation dry blood spot positive quality control products are all more than or equal to 500, the mutation frequency is 40-60%, and the mutation frequency variation coefficient value of 11 mutation types of PC-1, PC-2, PC-3, PC-4 and PC-5 is less than 5% through the mutation frequency variation coefficient analysis, which indicates that the preparation method of the deaf gene heterozygous mutation dry blood spot positive quality control products is stable.

Application example 6 Sanger sequencing method for verifying repeatability of dry blood spot positive quality control product preparation process

1. Using the recombinant plasmids P1, P2, P3, P4 and P5 prepared in example 1 and 5 wild-type human peripheral whole blood, 5 deaf gene heterozygous dry plaque positive quality controls PC-1, PC-2, PC-3, PC-4 and PC-5 were prepared according to the method of example 1, and Sanger sequencing was performed, the results of which are shown in FIGS. 11 to 15.

2. The results show that: the PC-1, PC-2, PC-3, PC-4 and PC-5 deafness gene heterozygous mutant dry blood spot positive quality control products are heterozygous mutant positive of 11 sites related to deafness genes (namely GJB 3: 538C > T, GJB 3: 547G > A, SLC26A 4: IVS7-2A > G, SLC26A 4: 2168A > G, GJB 2: 299_300delAT, GJB 2: 235delC, GJB 2: 176_191del16, GJB 2: 167delT, GJB 2: 35delG, 12 SrRNA: m.1494C > T and 12 SrRNA: m.1555A > G). Further indicates that the preparation method of the deafness gene heterozygous mutant dry blood spot positive quality control product is more stable.

Application example 7 semiconductor sequencing method to verify repeatability of deafness gene heterozygous mutant dry blood spot positive quality control product

1. 5 parts of deafness gene heterozygous mutant dry blood spot positive quality control products PC-1, PC-2, PC-3, PC-4 and PC-1 and PC-3 in PC-5 are prepared by the method of application example 5, semiconductor sequencing is carried out, and the repeatability is verified by 5 times of repetition.

2. Data analysis was performed using bioinformatics correlation techniques, with the following results:

the results show that: the sequencing depth values corresponding to 5-time on-machine sequencing mutation sites are all more than or equal to 100, the mutation frequency is 40-60%, and the analysis of the mutation frequency of each mutation type shows that the mutation frequency coefficient of the 11 mutation types of PC-1 and PC-3 is less than 5%, which indicates that the prepared deafness gene heterozygous mutation dry blood spot positive quality control product is relatively stable.

Application of 8 deafness gene heterozygous mutant dry blood spot positive quality control product in detecting hereditary deafness mutant gene by semiconductor sequencing method

1. The deafness gene heterozygous mutant dry blood spot positive quality control PC and negative quality control NC (wild type human genome DNA) prepared according to the method of the example 1 are used for detecting deafness samples to be detected HL1-HL20 with known mutation types, and the experimental operation is carried out according to the example 2.

the results show that: the sequencing result of the positive quality control product PC is GJB 3: 538C > T, GJB 3: 547G > A, SLC26A 4: IVS7-2A > G, SLC26A 4: 2168A > G, GJB 2: 299_300delAT, GJB 2: 235delC, GJB 2: 176_191del16, GJB 2: 167delT, GJB 2: 35delG, 12 SrRNA: m.1494C > T, 12 SrRNA: m.1555A is more than G heterozygous mutation type, and the negative quality control product is wild type; the sequencing results of 20 samples to be detected show that the samples are all positive in corresponding deafness gene mutation type, which indicates that the deafness gene heterozygous mutation dry blood spot positive quality control product prepared by the method can be used as a quality control substance for detecting hereditary deafness mutant genes by a semiconductor sequencing method.

Application of 9 deafness gene heterozygous mutant dry blood spot positive quality control product in detection of hereditary deafness by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

1. The deafness gene heterozygous mutant dry blood spot positive quality control PC and negative quality control NC (wild type human genome DNA) prepared according to the method of the example 1 are used for detecting the deafness of the known mutant type and normal samples HL21-HL40, and the experimental operation is carried out according to the application example 3.

the results show that: the sequencing result of the positive quality control product PC is GJB 3: 538C > T, GJB 3: 547G > A, SLC26A 4: IVS7-2A > G, SLC26A 4: 2168A > G, GJB 2: 299_300delAT, GJB 2: 235delC, GJB 2: 176_191del16, GJB 2: 167delT, GJB 2: 35delG, 12 SrRNA: m.1494C > T, 12 SrRNA: m.1555A is more than G heterozygous mutation type, and the negative quality control product is wild type; the detection results of 20 samples to be detected show that HL36, HL37, HL38 and HL40 are all wild types, and the rest are all corresponding mutation types with positive, which indicates that the deafness gene heterozygous mutation dry blood spot positive quality control product prepared by the method can be used as a matrix-assisted laser desorption ionization time-of-flight mass spectrometry to detect genetic deafness positive quality control substances.

Sequence listing

<110> Darriy Biotechnology Ltd, Guangzhou City

<120> a heterozygous mutant dry blood spot positive quality control product suitable for deafness mutant gene detection

<160> 19

<170> SIPOSequenceListing 1.0

<210> 1

<211> 977

<212> DNA

<213> human (Homo sapiens)

<400> 1

ctccccagca cagcaaattt ttatgatgtg tttaaagatt gggtgaatta ctcaggtgaa 60

caagctactt tttatcagag aacacctaaa aacacgttca agagggtttg ggaactatac 120

atttaatcct atgacaaact aagttggttc tgtcttcacc tgttttggtg aggttgtgta 180

agagttggtg tttgctcagg aagagattta agcatgcttg cttacccaga ctcagagaag 240

tctccctgtt ctgtcctagc tagtgattcc tgtgttgtgt gcattcgtct tttccagagc 300

aaaccgccca gagtagaaga tggattgggg cacgctgcag acgatcctgg gggtgtgaac 360

aaacactctc accagcattg gaaagatctg gctcaccgtc ctcttcattt ttcgcattat 420

gatcctcgtt gtggctgcaa aggaggtgtg gggagatgag caggccgact ttgtctgcaa 480

cacccgcagc cagctacgat cactacttcc ccatctccca catccggcta tgggcctgca 540

gctgatcttc gtgtccacgc cagcgctcct agtggccatg cacgtggcct accggagacg 600

agaagaagag gaagttcatc aagggggaga taaagagtga atttaaggac atcgaggaga 660

tcaaaaccca gaaggtccgc atcgaaggct ccctgtggtg gacctacaca agcagcatct 720

tcttccgggt catcttcgaa gccgccttca tgtacgtctt ctatgtcatg tacgacggct 780

tctccatgca gcggctggtg aagtgcaacg cctggccttg tcccaacact gtggactgct 840

ttgtgtcccg gcccacggag aagactgtct tcacagtgtt catgattgca gtgtctggaa 900

tttgcatcct gctgaatgtc actgaattgt gttatttgct aattagatat tgttctggga 960

agtcaaaaaa gccagtt 977

<210> 2

<211> 1001

<212> DNA

<213> human (Homo sapiens)

<400> 2

caagtactcc acagcgttcg ggcgcatctg gctgtccgtg gtgttcgtct tccgggtgct 60

ggtatacgtg gtggctgcag agcgcgtgtg gggggatgag cagaaggact ttgactgcaa 120

caccaagcag cccggctgca ccaacgtctg ctacgacaac tacttcccca tctccaacat 180

ccgcctctgg gccctgcagc tcatcttcgt cacatgcccc tcgctgctgg tcatcctgca 240

cgtggcctac cgtgaggagc gggagcgccg gcaccgccag aaacacgggg accagtgcgc 300

caagctgtac gacaacgcag gcaagaagca cggaggcctg tggtggacct acctgttcag 360

cctcatcttc aagctcatca ttgagttcct cttcctctac ctgctgcaca ctctctggca 420

tggcttcaat atgccgcgcc tggtgcagtg tgccaacgtg gccccctgcc ccaacatcgt 480

ggactgctac attgccttga cctaccaaga agaaaatctt cacctacttc atggtgggcg 540

cctccgccgt ctgcatcgta ctcaccatct gtgagctctg ctacctcatc tgccacaggg 600

tcctgcgagg cctgcacaag gacaagcctc gagggggttg cagcccctcg tcctccgcca 660

gccgagcttc cacctgccgc tgccaccaca agctggtgga ggctggggag gtggatccag 720

acccaggcaa taacaagctg caggcttcag cacccaacct gacccccatc tgaccacagg 780

gcaggggtgg ggcaacatgc gggctgccaa tgggacatgc agggcggtgt ggcaggtgga 840

gaggtcctac aggggctgag tgaccccact ctgagttcac taagttatgc aactttcgtt 900

ttggcagata ttttttgaca ctgggaactg ggctgtctag ccgggtatag gtaacccaca 960

ggcccagtgc cagccctcaa aggacataga ctttgaaaca a 1001

<210> 3

<211> 1159

<212> DNA

<213> human (Homo sapiens)

<400> 3

aaactcacct gagttgtaaa aaactccagt tgacacaaaa tagactacga aagtggcttt 60

aacatatctg aacacacaat agctaagacc caaactggga ttagataccc cactatgctt 120

agccctaaac ctcaacagtt aaatcaacaa aactgctcgc cagaacacta cgagccacag 180

cttaaaactc aaaggacctg gcggtgcttc atatccctct agaggagcct gttctgtaat 240

cgataaaccc cgatcaacct caccacctct tgctcagcct atataccgcc atcttcagca 300

aaccctgatg aaggctacaa agtaagcgca agtacccacg taaagacgtt aggtcaaggt 360

gtagcccatg aggtggcaag aaatgggcta cattttctac cccagaaaac tacgatagcc 420

cttatgaaac ttaagggtcg aaggtggatt tagcagtaaa ctaagagtag agtgcttagt 480

tgaacagggc cctgaagcgc gtacacaccg cccgtcactc tcctcaagta tacttcaaag 540

gacatttaac taaaacccct acgcatttat atagaggagg caagtcgtaa catggtaagt 600

gtactggaaa gtgcacttgg acgaaccaga gtgtagctta acacaaagca cccaacttac 660

acttaggaga tttcaactta acttgaccgc tctgagctaa acctagcccc aaacccactc 720

caccttacta ccagacaacc ttagccaaac catttaccca aataaagtat aggcgataga 780

aattgaaacc tggcgcaata gatatagtac cgcaagggaa agatgaaaaa ttataaccaa 840

gcataatata gcaaggacta acccctatac cttctgcata atgaattaac tagaaataac 900

tttgcaagga gagccaaagc taagaccccc gaaaccagac gagctaccta agaacagcta 960

aaagagcaca cccgtctatg tagcaaaata gtgggaagat ttataggtag aggcgacaaa 1020

cctaccgagc ctggtgatag ctggttgtcc aagatagaat cttagttcaa ctttaaattt 1080

gcccacagaa ccctctaaat ccccttgtaa atttaactgt tagtccaaag aggaacagct 1140

ctttggacac taggaaaaa 1159

<210> 4

<211> 265

<212> DNA

<213> human (Homo sapiens)

<400> 4

ctgctggatt gctcaccatt gtcgtctgta tggcagttaa ggaattaaat gatcggttta 60

gacacaaaat cccagtccct attcctatag aagtaattgt ggtaagtaga atatgtagtt 120

agaaagttca gcattatttg gttgacaaac aaggaattat taaaaccaat ggagttttta 180

acatcttttg ttttatttcg gacgataatt gctactgcca tttcatatgg agccaacctg 240

gaaaaaaatt acaatgctgg cattg 265

<210> 5

<211> 232

<212> DNA

<213> human (Homo sapiens)

<400> 5

tggagcaatg cgggttcttt gacgacaaca ttagaaagga cacattcttt ttgacggtcc 60

gtgatgctat actctatcta cagaaccaag tgaaatctca agagggtcaa ggttccattt 120

tagaaacggt aaatattcaa cctttctaca gatgtatctt ttctaaacta tcatgatttc 180

tataaatggc aaacattaca caagtctagt ctagctgttg aattttaagc ta 232

<210> 6

<211> 24

<212> DNA

<213> human (Homo sapiens)

<400> 6

tcttcttctc atgtctccgg tagg 24

<210> 7

<211> 27

<212> DNA

<213> human (Homo sapiens)

<400> 7

catttttcgc attatgatcc tcgttgt 27

<210> 8

<211> 21

<212> DNA

<213> human (Homo sapiens)

<400> 8

catctcccca cacctccttt g 21

<210> 9

<211> 22

<212> DNA

<213> human (Homo sapiens)

<400> 9

agaagtctcc ctgttctgtc ct 22

<210> 10

<211> 22

<212> DNA

<213> human (Homo sapiens)

<400> 10

ttcgaagatg acccggaaga ag 22

<210> 11

<211> 20

<212> DNA

<213> human (Homo sapiens)

<400> 11

cctgcagctg atcttcgtgt 20

<210> 12

<211> 24

<212> DNA

<213> human (Homo sapiens)

<400> 12

agcctcatct tcaagctcat catt 24

<210> 13

<211> 21

<212> DNA

<213> human (Homo sapiens)

<400> 13

gtagcagagc tcacagatgg t 21

<210> 14

<211> 23

<212> DNA

<213> human (Homo sapiens)

<400> 14

ccatcttcag caaaccctga tga 23

<210> 15

<211> 25

<212> DNA

<213> human (Homo sapiens)

<400> 15

ggtgctttgt gttaagctac actct 25

<210> 16

<211> 23

<212> DNA

<213> human (Homo sapiens)

<400> 16

gtcgtctgta tggcagttaa gga 23

<210> 17

<211> 25

<212> DNA

<213> human (Homo sapiens)

<400> 17

ttttccaggt tggctccata tgaaa 25

<210> 18

<211> 27

<212> DNA

<213> human (Homo sapiens)

<400> 18

gacgacaaca ttagaaagga cacattc 27

<210> 19

<211> 28

<212> DNA

<213> human (Homo sapiens)

<400> 19

agactagact tgtgtaatgt ttgccatt 28

Claims

1. The application of the dry blood spot positive quality control product in the preparation of the reagent for detecting the deafness gene mutation is characterized in that the blood spot positive quality control product is prepared by the following steps:

s1, artificially synthesizing 5 DNA fragments containing mutation sites, wherein the sequences are respectively shown as SEQ ID NO: 1-5;

s2, respectively connecting the 5 DNAs containing the mutation sites with a cloning vector to obtain 5 recombinant plasmids;

s3, mixing 5 DNA fragments containing mutation sites or 5 recombinant plasmids containing the DNA fragments with wild type human peripheral anticoagulated whole blood according to the DNA copy number ratio, specifically:

to a concentration of 10⁹Copy/. mu.L of the sequence is shown in SEQ ID NO: 3 or a plasmid containing the DNA fragment thereof, and a concentration of 10⁷Copy/. mu.L of the sequence is shown in SEQ ID NO: 1. 2, 4 and 5 or plasmids containing the DNA fragments thereof, and dry blood spots prepared by mixing the DNA fragments with wild type human peripheral anticoagulated whole blood;

the dosage ratio of the DNA fragment or the plasmid containing the DNA fragment to the whole blood is 2:1 according to the DNA copy number;

s4, dripping the mixed blood sample on a carrier, and drying to obtain the positive control product of the deafness gene dried blood spot.

2. Use according to claim 1, characterized in that the method for detecting mutations in the deafness gene is: fluorescent quantitative PCR, Sanger sequencing, semiconductor sequencing, matrix-assisted laser desorption ionization time-of-flight mass spectrometry and microarray chip platform detection.