CN113801933A

CN113801933A - Detection kit for rapid typing of human SERPINB7 gene mutation

Info

Publication number: CN113801933A
Application number: CN202111091414.6A
Authority: CN
Inventors: 李明; 李明阳; 方耀东
Original assignee: Shanghai Wuseshi Medical Technology Co ltd
Current assignee: Shanghai Wuseshi Medical Technology Co ltd
Priority date: 2021-09-17
Filing date: 2021-09-17
Publication date: 2021-12-17
Anticipated expiration: 2041-09-17
Also published as: CN113801933B

Abstract

The invention discloses a detection kit for rapid typing of human SERPINB7 gene mutation. The primer probe combination of the kit avoids mutual interference between a plurality of primer pairs and corresponding detection probes through ingenious design of amplification primer pairs and detection probes, and proves that the primer probe combination is used for detecting SERPINB7 gene mutation, so that the specificity is good, and the sensitivity is high. In addition, the kit is based on a universal fluorescent quantitative PCR platform, has low equipment cost, high universality, simple operation, easy result interpretation and no pollution in the whole closed tube process, and can detect 4 pathogenic mutation sites at one time.

Description

Detection kit for rapid typing of human SERPINB7 gene mutation

Technical Field

The invention belongs to the technical field of in-vitro nucleic acid detection, and relates to a detection kit for rapid typing of human SERPINB7 gene mutation, wherein the detection kit comprises a specific primer probe composition, a matched reagent and a detection method suitable for the kit.

Background

Long island type palmoplantar keratosis (NPPK, OMIM 615598) belongs to diffuse non-destructive palmoplantar keratosis, is an autosomal recessive hereditary palmoplantar keratosis (PPK), belongs to diffuse non-epidermolysis palmoplantar keratosis, and is the most common type of palmoplantar keratosis of Chinese Han population. The disease is reported in 1977 for the first time in Changdai, the incidence rate is relatively high, and the incidence rate is about 3.1/10000 in Chinese Han people. The causative gene of NPPK has been identified to date as serine protease inhibitor B7(serpinfamilyB, SERPINB 7).

The SERPINB7 gene is located at 18q21.3, and has 8 exons, the initiation codon is located at exon 2, and the termination codon is located at exon 8, and encodes 380 amino acids. The NPPK pathogenic mutations on SERPI NB7 gene are reported to be c.796C > T, c.218_219del AGinsTAAACTTTACCT, c.455-1G > A, c.455G > T, c.650-653del CTGT, c.522-523insT, c.336+2T > G, c.122_127del TGGTCC, c.830C > T, c.382C > T, c.635delG, c.271del C and c.1136G > A, wherein the c.796C > T mutation frequency is the highest, and the carrying frequency of c.796C > T in the normal population in China is 6/197.

NPPK is clinically characterized in that mild keratosis is caused on the basis of skin erythema, and the clinical characteristics are that erythema, pimples, plaques, keratosis and desquamation appear on parts such as palms, wrists, soles, instep, ankles, achilles tendons and the like, the hyperhidrosis of hands and feet is accompanied easily with skin superficial fungal infection, and most patients are accompanied with palmoplantar hyperhidrosis and/or palmoplantar peculiar smell^[2-3]. At present, no treatment method for radically curing NPPK (NPPK) is found, wherein the treatment methods can relieve the condition of a patient by using a retinoid medicament such as tretinoin ointment and tazarotene or a hormone ointment such as clobetasol propionate and combining a fungal infection with an antifungal medicament, but the disease is easy to relapse after the medicament is stopped^[6]。

Currently, detection of mutations in SERPINB7 gene relies mainly on sanger sequencing. The basic principle of the method is that after PCR amplification, the obtained target gene fragment is subjected to sanger sequencing, and whether the pathogenic SERPINB7 gene mutation exists or not is analyzed according to the sequencing result. The method has the disadvantages of complex experimental operation, long time, corresponding professional knowledge required for result analysis and relatively high detection cost. Therefore, the real-time fluorescence quantitative PCR with high efficiency, rapidness and simple operation becomes the main method in the field of molecular diagnosis.

According to the data of NCBI dbSNP database, the kit detects 4 sites: the mutant allele frequencies of c.796C > T (rs142859678), c.522_523insT (rs672601344), c.650_653delCTGT (rs534014297), c.806_818delinsT (rs1157759655) in the normal east Asian population are respectively: 0.0119, 0.0032, 0.003, 0.0006, from which the rates of carriage of the four sites in the population were calculated to be about 3.7/100.

[1]Nagashima M.Handbook of Human Genetics[M].Tokyo:Igaku Shoin,1977:23-27.

[2]Kabashima K，Sakabe J，Yamada Y，et al.“Nagashima-type”keratosis as a novel entity in the palmoplantar keratoderma category[J].A rch Dermatol，2008，144(3):375-379.

[3]Kubo A,Shiohama A,Sasaki T,et al.Mutations in SERPINB7,encoding a member of the serine protease inhibitor superfamily,cause Nagas hima-type palmoplantar keratosis[J].Am J Hum Genet,2013,93(5):945-956.

[4]Zhang J,Zhang G,Ni C,et al.Nagashima-type palmoplantar keratosis in a Chinese Han population[J].Mol Med Rep,2016,14(5):4049-4054.

[5] Daishan, Nannan vivid, Zhao hongshan, et al, Long island type palmoplantar keratosis SERPINB7 Gene mutation site research [ J ] Chinese and Western medicine integration dermatosis journal, 2017,16(2): 108-.

[6] Mutation analysis of SERPINB7 Gene in an example of Long island Palmar keratosis [ J ] Youjiang medicine, 2018,46(1):23-25.

Disclosure of Invention

The invention mainly aims to provide a detection kit for rapid genotyping of human SERPINB7 gene mutation aiming at the defect that no product capable of being used for NPPK gene diagnosis exists at home and abroad at present. The kit comprises a specific primer probe composition and other matched reagents. Meanwhile, the invention also provides a human SERPINB7 gene mutation typing detection method. The kit provided by the invention can specifically, accurately and quickly detect the pathogenicity gene of the long-island type palmoplantar keratoderma, and is suitable for large-scale popularization and application.

In order to achieve the aim, the invention provides a detection kit for rapid typing of human SERPINB7 gene mutation on one aspect, which comprises a primer probe composition, a PCR reaction container and an external quality control container;

the primer probe composition is dry powder or solution comprising the following sequence primers and probes:

c.796C > T site: primers with sequences shown as SEQ ID No.1 and SEQ ID No.2, and probes with sequences shown as SEQ ID No.3 and SEQ ID No. 4;

c.522_523insT site: primers with sequences shown as SEQ ID No.5 and SEQ ID No.6, and probes with sequences shown as SEQ ID No.7 and SEQ ID No. 8;

c.650-653 delCTGT site: primers with sequences shown as SEQ ID No.9, SEQ ID No.10 and SEQ ID No.11, and probes with sequences shown as SEQ ID No.12 and SEQ ID No. 13;

c.806_818delinsT site: primers with sequences shown as SEQ ID No.1 and SEQ ID No.14, and probes with sequences shown as SEQ ID No.15 and/or SEQ ID No. 16;

the PCR reaction container comprises DNA polymerase, dNTP and Mg²⁺Special auxiliary reinforcing agent and PCR buffer solution;

the external quality control container comprises a mutation control and a normal control; the normal contrast is the nucleic acid composition shown as sequences SEQ ID No.17, SEQ ID No.18, SEQ ID No.19 and SEQ ID No.20, and the mutation contrast is the nucleic acid composition shown as sequences SEQ ID No.21, SEQ ID No.22, SEQ ID No.23 and SEQ ID No. 24.

Preferably, the 5 'end of any probe is connected with a fluorescent group, and the 3' end of any probe is connected with a fluorescence quenching group; the fluorescent groups are independently selected from FAM, VIC, CY5, ROX, HEX, JOE, NED, Texas Red or CY 3; the quenching group is independently selected from MGB, BHQ-1, BHQ-2, BHQ-3 or TAMRA.

Preferably, the primer probe composition is divided into a first reactant and a second reactant;

the first reactant is dry powder or solution of a primer and probe composition containing the following sequences:

primers with sequences shown as SEQ ID No.1 and SEQ ID No.2, probes with sequences shown as SEQ ID No.3, primers with sequences shown as SEQ ID No.5 and SEQ ID No.6, probes with sequences shown as SEQ ID No.7 and SEQ ID No.8, primers with sequences shown as SEQ ID No.9 and SEQ ID No.10, and probes with sequences shown as SEQ ID No. 12;

the second reactant contains dry powder or solution of a primer and probe composition with the following sequences:

primers with sequences shown as SEQ ID No.1 and SEQ ID No.2, probes with sequences shown as SEQ ID No.4, primers with sequences shown as SEQ ID No.9 and SEQ ID No.11, probes with sequences shown as SEQ ID No.13, primers with sequences shown as SEQ ID No.1 and SEQ ID No.14, and probes with sequences shown as SEQ ID No.15 and/or SEQ ID No. 16.

Preferably, the fluorescent groups attached to either of the probes are different in the first reactant or the second reactant.

Preferably, in the first reactant, the fluorophore connected to the probe with the sequence shown in SEQ ID No.3 is FAM, the fluorophore connected to the probe with the sequence shown in SEQ ID No.7 is CY5, the fluorophore connected to the probe with the sequence shown in SEQ ID No.8 is ROX, and the fluorophore connected to the probe with the sequence shown in SEQ ID No.12 is VIC;

in the second reactant, the fluorescent group connected with the probe with the sequence shown in SEQ ID No.4 is ROX, the fluorescent group connected with the probe with the sequence shown in SEQ ID No.13 is FAM, the fluorescent group connected with the probe with the sequence shown in SEQ ID No.15 is CY5, and the fluorescent group connected with the probe with the sequence shown in SEQ ID No.16 is VIC.

In the actual detection process, the first reagent is detected separately from the second reagent, and 4 different sets of fluorescence channels are detected each time. When the detection reagents are 2 groups, the accuracy, the convenient operation degree and the like of the detection reagents are in optimal balance, and the operation times can be reduced as much as possible under the condition of avoiding mutual interference of fluorescence.

Preferably, the first reactant and the second reactant are both in solution: the final concentration of each primer is 0.2-1.2. mu.M, preferably 0.6. mu.M; the final concentration of each of the probes is 0.1-0.8. mu.M, preferably 0.35. mu.M.

Preferably, the PCR buffer solution comprises Tris, 10 mM-500 mM KCl, 10 mM-500 mM (NH4)2SO4, 1-50% of glycerol by mass, 0.001-1 mg/mL of BSA, 0.1-10% of Tween 20 by mass, 1 mM/mL-100 mM/mL of dithiothreitol, 0.1-3M of betaine and 0.1-10% of DMSO by mass.

Preferably, the normal control is an artificial DNA sequence as shown in SEQ ID No.17, SEQ ID No.18, SEQ ID No.19 and SEQ ID No. 20; the mutation control is a plasmid containing the sequences shown as SEQ ID No.21, SEQ ID No.22, SEQ ID No.23 and SEQ ID No. 24.

In certain embodiments, the above-described mutant and normal controls are used in a control assay.

Preferably, the pH value of the PCR buffer solution is 7.5-9.5 at 25 ℃.

Preferably, the detection method applicable to the kit is a TaqMan probe method; the first reactant is used for detecting a mutant type of a c.796C > T site, a wild type and a mutant type of a c.522_523insT site and a wild type of a c.650_653delCTGT site; the second reagent is used for detecting a wild type of a c.796C > T site, a mutant type of a c.650_653delCTGT site, a wild type and a mutant type of a c.806_818delinsT site.

The TaqMan probe method specifically comprises the following steps:

step 1: processing a blood sample, and processing an EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood sample by using a sample releasing agent to obtain a mixed solution containing nucleic acid to be detected;

step 2: selectively and specifically amplifying a target nucleic acid sequence by using the nucleic acid solution obtained in the step 1 as a template and a PCR amplification system containing the primer probe composition; wherein the target sequence is a nucleic acid sequence corresponding to each primer pair;

and step 3: and (3) measuring the fluorescence intensity amplified in the step (2) by using an instrument, carrying out quantitative analysis, and judging the genotypes of 4 pathogenic mutation sites on the SERPINB7 gene in the detection range of the sample to be detected.

The quantitative detection principle of the TaqMan probe method is as follows: when the probe is complete, no fluorescence can be detected, in the PCR extension process, the DNA polymerase hydrolyzes the probe and then emits a fluorescent signal, and the fluorescence increase value is in positive correlation with the amount of PCR products.

In certain embodiments, the above-described rapid sample release agent for EDTA anticoagulated whole blood sample includes, but is not limited to, erythrocyte lysate, DNA release promoter, detergent, proteinase K, guanidinium salt, and the like. The rapid sample release agent can be used for extraction-free rapid nucleic acid release.

Wherein the mutation control is a plasmid containing the 4-locus mutant gene sequence, and the normal control is normal human genome DNA.

The kit provided by the invention adopts EDTA anticoagulated whole blood as a sample, and carries out extraction-free rapid typing detection on four pathogenic mutation sites of SERPINB7 genes, namely c.796C > T, c.522_523insT, c.650_653delCTGT and c.806_818delinsT, so that the kit has the following beneficial effects:

firstly, the primer probe combination of the kit avoids mutual interference between a plurality of primer pairs and corresponding detection probes through the ingenious design of amplification primer pairs and detection probes; and proved by verification, the primer probe combination is used for detecting the gene mutation of SERPINB7, and has good specificity and high sensitivity. In addition, the kit is based on a general fluorescent quantitative PCR platform, and has the advantages of low equipment cost, high universality, simple operation, easy interpretation of results and no pollution in the whole process of tube closing. Meanwhile, the detection method does not need nucleic acid extraction, the whole process from sample treatment to detection result is completed within 1.5h, and 4 pathogenic mutation sites can be detected at one time.

Detailed Description

Embodiments of the present application will be described in detail by examples, so that how to apply technical means to solve technical problems and achieve technical effects of the present application can be fully understood and implemented.

The raw materials and equipment used in the present application are all common raw materials and equipment in the field, and are all from commercially available products, unless otherwise specified. The methods used in this application are conventional in the art unless otherwise indicated.

There are many other possible embodiments of the present invention, which are not listed here, and the embodiments claimed in the claims of the present invention can be implemented.

"comprising" or "including" is intended to mean that the compositions (e.g., media) and methods include the recited elements, but not excluding others. When used in defining compositions and methods, "consisting essentially of … …" is meant to exclude other elements having any significance to the combination of the stated objects. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or steps that do not materially affect the basic and novel characteristics of the claimed application. "consisting of … …" refers to trace elements and substantial process steps excluding other components. Embodiments defined by each of these transition terms are within the scope of the present application.

Example 1

The invention aims to provide a low-cost and high-efficiency composite PCR amplification system capable of genotyping 4 SNP sites on SERPINB7 gene causing parakeratosis palmaris et plantaris at the same time. The 4 common mutation sites on the SERPINB7 gene are c.796C > T, c.522_523insT, c.650_653delCTGT and c.806_818delinsT respectively. Wherein, the wild type gene sequences of the four sites of c.796C > T, c.522_523insT, c.650_653delCTGT and c.806_818delinsT are respectively shown as SEQ ID No.17, SEQ ID No.18, SEQ ID No.19 and SEQ ID No. 20.

The gene sequences of the primer probe composition for detecting the SERPINB7 gene mutation are shown in Table 1. The primer pair probe composition of the multiplex PCR amplification system for 4 gene loci, as well as the fluorescein modification characteristics are also labeled in table 1. Wherein, the probe corresponding to each site is obtained by connecting a fluorescent group to the 5' end.

For the c.650_653delCTGT site, only one primer is shared by the wild type and the mutant, namely SEQ ID No. 9. Specifically, the primer pair corresponding to the wild type of the c.650_653delCTGT site is as follows: the sequences are shown as SEQ ID No.9 and SEQ ID No. 10. c.650-653 delCTGT site mutant corresponds to the primer pair: the sequences are shown as SEQ ID No.9 and SEQ ID No. 11.

Table 1.4 sites of 16 PCR amplification primers and fluorescein labeling characteristics

Table 2 shows the specific genotypes of the corresponding sites to be detected by the above probes.

TABLE 2

The primer probe composition shown above can be detected by a common TaqMan probe method. Each detection can be performed only aiming at one genotype of one site, and also can be performed on a plurality of sites at one time. It should be noted that, when a plurality of sites are simultaneously detected, the excitation wavelength of the fluorophore used for each probe in one detection should be different and have high resolution.

EXAMPLE 2 detection Cartridge

In this example, the reagents required to be contained in the typing test kit for palmar-shikayasis are shown in table 3. In this example, the detection reaction solution A and the detection reaction solution B constitute the probe composition. The PCR reaction vessel comprises SER main reaction liquid, reaction A auxiliary liquid and reaction B auxiliary liquid. Wherein, the formulas of the reaction A auxiliary liquid and the reaction B auxiliary liquid are all commonly used in the field.

TABLE 3

In a specific embodiment, in the detection reaction solution A and the detection reaction solution B, the final concentration of each primer can be selected within a range of 0.2 to 1.2. mu.M, and the final concentration of each probe can be selected within a range of 0.1 to 0.8. mu.M.

In this example, the final concentration of each primer was 0.6. mu.M and the final concentration of each probe was 0.35. mu.M in the detection reaction solution A and the detection reaction solution B.

Wherein the PCR reaction buffer solution also comprises the following components: tris, KCl 10-500 mM, NH 10-500 mM₄)₂SO₄1 to 50 percent of glycerol, 0.001 to 1mg/mL of BSA, 0.1 to 10 percent of Tween 20, 1 to 100mM/mL of dithiothreitol, 0.1 to 3M of betaine and 0.1 to 10 percent of DMSO. The pH value of the PCR buffer solution is 7.5-9.5 at 25 ℃.

Specific sequences corresponding to the primer probe compositions contained in the detection reaction solutions a and B are shown in table 4.

TABLE 4

The reagent is used for detecting a sample to be detected through a fluorescent quantitative PCR instrument, and the method comprises the following specific steps:

1. sample pretreatment

The kit does not need to extract and purify sample DNA before detection, and can be used for detection only after simple treatment.

The processing steps are as follows:

a) sucking 3 times of volume of precipitation treatment buffer solution of the pretreated human anticoagulated whole blood into a 0.2mL eight-connected tube, wherein the volume of the human anticoagulated whole blood is recommended to be 25 mu L, and the minimum volume of the human anticoagulated whole blood is not less than 10 mu L;

b) adding anticoagulated whole blood to be treated into a precipitation treatment buffer solution, blowing and sucking back and forth for several times by using a pipettor to completely dissolve the blood remained on the suction head, and violently shaking for 1min (fully shaking and uniformly mixing are very important); after 5 minutes at room temperature, the mixture was inverted several times, centrifuged at 7000rpm for 5 minutes, and the supernatant was aspirated off, leaving the precipitate for further processing.

c) Adding 80 μ L of nucleic acid release buffer solution into the precipitate, shaking for 1min, mixing to obtain nucleic acid solution for detection, and storing at-20 + -5 deg.C if not detected immediately;

the volume of an EDTA (ethylene diamine tetraacetic acid) anticoagulation whole blood sample required by the kit is 25 mu L, and the treated mixed solution can be directly used for the detection of the kit according to the sample volume required by the detection.

2. Sample detection

1) Taking out the kit which is stored at minus 15 to minus 25 ℃ in a dark place, and balancing the components of the kit to room temperature;

2) taking out a corresponding number of 96-well plates or PCR reaction tubes;

3) calculating and transferring a corresponding amount of reagent according to the reaction number in the current experiment, wherein the reaction number refers to the sample inspection amount plus 1 blank control (DNA is replaced by purified water), 1 mutation control and 1 normal control, and the rest of the reagent is stored under the condition of keeping out of the sun at the temperature of between 15 ℃ below zero and 25 ℃ below zero;

4) preparing a reaction system A and a reaction system B according to the scheme in the table 5;

table 5: preparation of amplification reaction tube A/B

5) The template solution refers to the sample mixed solution, the blank reference substance, the mutation reference substance and the normal reference substance which are pretreated in the step 1;

6) sealing a 96-well plate with a film or covering a PCR reaction tube cover, oscillating uniformly, centrifuging at 2000rpm for 10 seconds, and putting the product into a fluorescent quantitative PCR instrument;

7) PCR amplification was performed according to the conditions of table 6:

table 6: amplification procedure

And after the setting is finished, saving the file and operating the reaction program.

3. Results analysis (please refer to the instruction of each apparatus, and the ABI series apparatus is used as an example in the following analysis)

After the reaction is finished, the instrument automatically stores the result, and after the image is analyzed, the threshold value of the fluorescence signal is adjusted until all fluorescence channels corresponding to the wild type in the detection result of the mutation comparison product have no signal, and all fluorescence channels corresponding to the mutant type in the detection result of the wild type comparison product have no detection signal. Taking the ABI 7500 type fluorescence quantitative PCR instrument as an example, an ideal detection result can be obtained when the fluorescence signal threshold is about 25000.

4. Quality control

The results of the mutation control, normal control and blank control tests should meet the following requirements, otherwise the test is regarded as invalid.

1) The detection results of the reaction liquid A and the reaction liquid B in the kit are shown in the following table 7:

table 7: comparison table for detecting fluorescence channel and detected gene in reaction liquid A/B

2) Blank control:

no signal exists before the Ct value of any detection channel in the detection results of the reaction liquid A and the reaction liquid B is 38. If the signal rises and the Ct value is less than or equal to 38, the experimental result is invalid, and re-experiment is recommended.

3) The mutant controls need to meet both:

a) the Ct value of a channel corresponding to any wild type in the detection results of the reaction liquid A and the reaction liquid B has no signal before 38;

b) obvious signal rising is generated in the channel corresponding to any mutant in the detection results of the reaction liquid A and the reaction liquid B, and the Ct value is less than or equal to 38.

If the signal of any channel in a) rises and the Ct value is less than or equal to 38, or no signal rises or a signal rises but the Ct value is more than 38 in any channel in b), the experimental result is invalid, and the re-experiment is recommended.

4) The normal reference substance needs to satisfy both:

c) the Ct value of a channel corresponding to any mutant in the detection results of the reaction liquid A and the reaction liquid B has no signal before 38;

d) obvious signal rising is generated in the channel corresponding to any wild type in the detection results of the reaction liquid A and the reaction liquid B, and the Ct value is less than or equal to 38.

If any channel signal in c) rises and Ct value is less than or equal to 38, or no signal rises in any channel in d), or signal rises but Ct value is more than 38, the experimental result is invalid, and re-experiment is recommended.

5. Interpretation of results

Under the condition that the quality control product is normal, the result is interpreted as follows:

if any detection channel in the sample to be detected has obvious signal rising and Ct value is less than or equal to 38, the detection result of the corresponding target gene of the detection channel is positive.

The detection principle is as follows: the Taqman probe method works on the principle that the 5 '→ 3' exonuclease activity of Taq enzyme is utilized. As the 5 'end of the probe is labeled with a fluorescence reporter group (such as FAM) and the fluorescence quenching group MGB is labeled close to the 3' end, an energy transfer structure is formed between the two. Therefore, when the probe is kept intact, the fluorescence signal excited by the 5 'end fluorescence reporter group is absorbed or inhibited by the 3' end quenching group, and no change of the fluorescence signal occurs. When the target gene exists in the PCR reaction system, a specific nucleic acid fragment is amplified, the fluorescent probe hybridizes with the target gene according to the base pairing principle, when the PCR enters an extension (replication) stage, Taq enzyme extends from the 3 ' end of the primer along a DNA template along with a new strand, and when the Taq enzyme extends to a position where the probe is combined, the probe is cut off under the action of exonuclease activity at the 5 ' → 3 ' end of the Taq enzyme. At this time, the energy transfer structure between the fluorescent reporter group and the quencher group is broken, the quenching effect of the quencher group is released, and the fluorescent signal of the fluorescent reporter group is released. For each replication of a specific nucleic acid fragment in the PCR reaction, one probe is cleaved with the release of a fluorescent signal. Along with the accumulation of the fluorescence signal, the fluorescence signal can be detected by a fluorescence PCR instrument. In contrast, when the probe sequence is not matched with the target gene sequence, the probe cannot be combined with the target sequence, the specific probe cannot be cut off in the PCR extension process, and a corresponding fluorescent signal cannot occur naturally.

After two SERPINB7 gene mutation samples (the specific genotypes are shown in Table 8) are treated, the nucleic acid solution is repeatedly detected for 10 times by using the kit, the detection result is positive and accords with the corresponding genotype, and the precision of the Ct value is less than or equal to 5.0%, which indicates that the kit has good repeatability.

Table 8: 2 examples of Gene mutation samples corresponding to genotypes

The nucleic acid solutions of 6 clinically diagnosed SERPINB7 gene mutation samples (the specific genotypes are shown in Table 9) are respectively detected by the kit, and the detection results are consistent with the clinical results, which shows that the kit has better accuracy and specificity.

Table 9: 6 examples of Gene mutation samples corresponding to genotypes

Sample numbering	Gene locus	Genotype(s)
			SER-01	c.796C>T	Homozygous mutations
SER-02	c.522_523insT	Homozygous mutations
			SER-03	c.796C>T	Heterozygous mutations
SER-04	c.522_523insT	Heterozygous mutations
			SER-05	c.806_818delinsT	Heterozygous mutations
SER-06	c.650_653delCTGT	Heterozygous mutations

The details not described in the specification of the present application belong to the common general knowledge of those skilled in the art.

In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, and a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect.

It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a commodity or system that includes the element.

The foregoing description shows and describes several preferred embodiments of the present application, but as aforementioned, it is to be understood that the application is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the application, which is to be protected by the claims appended hereto.

Sequence listing

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ttacagattg aaaacaaacg tacctttcag aatctaatgg aatggaccta tccaaggcga 60

atgacctcta agtatgttga ggtatttttt cctcagttct tcatagagaa gaattatgaa 120

atgaaacaat atttgagagc cctagg 146

<210> 18

<211> 125

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

atttgaaatg gcaatttatg gtttcgcttc tggtgaaggc tgattgccac ttgcctttga 60

agtacacagc attcaccagc accattacag cagatgagct tatgccacct tcaccaatca 120

cgttc 125

<210> 19

<211> 130

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

gggaaggcag tcgccatgat gcatcaggac aggaagttca atttgtctgt tattgaggac 60

ccatcaatga agattcttga gctcagatac aatggtggca taaacatgta cgttctgctg 120

cctgagaatg 130

<210> 20

<211> 146

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

ttacagattg aaaacaaacg tacctttcag aatctaatgg aatggaccta tccaaggcga 60

atgacctcta agtatgttga ggtatttttt cctcagttct tcatagagaa gaattatgaa 120

atgaaacaat atttgagagc cctagg 146

<210> 21

<211> 146

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

ttacagattg aaaacaaact gacctttcag aatctaatgg aatggaccaa tccaaggtga 60

atgacctcta agtatgttga ggtatttttt cctcagttca agatagagaa gaattatgaa 120

atgaaacaat atttgagagc cctagg 146

<210> 22

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

atttgaaatg gcaatttatg gtttcgcttc tggtgaaggc tgattgccac ttgcctttga 60

agtacacaag cattcaccag caccattaca gcagatgagc ttatgccacc ttcaccaatc 120

acgttc 126

<210> 23

<211> 126

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

gggaaggcag tcgccatgat gcatcaggac aggaagttca atttgttatt gaggacccat 60

caatgaagat tcttgagctc agatacaatg gtggcataaa catgtacgtt ctgctgcctg 120

agaatg 126

<210> 24

<211> 134

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

ttacagattg aaaacaaacg tacctttcag aatctaatgg aatggaccta tccaaggcga 60

atgaccttgg tattttttcc tcagttcttc atagagaaga attatgaaat gaaacaatat 120

ttgagagccc tagg 134

Claims

1. A detection kit for rapid typing of human SERPINB7 gene mutation is characterized by comprising a primer probe composition, a PCR reaction container and an external quality control container;

2. The detection kit of claim 1, wherein the 5 'end of any of the probes is linked to a fluorescent group and the 3' end of any of the probes is linked to a fluorescence quenching group; the fluorescent groups are independently selected from FAM, VIC, CY5, ROX, HEX, JOE, NED, Texas Red or CY 3; the quenching group is independently selected from MGB, BHQ-1, BHQ-2, BHQ-3 or TAMRA.

3. The test kit of claim 2, wherein the primer probe composition is divided into a first reagent and a second reagent;

4. The test kit of claim 3, wherein the fluorescent group attached to either of the probes is different in the first reagent or the second reagent.

5. The test kit of claim 4, wherein:

in the first reactant, the fluorescent group connected with the probe with the sequence shown as SEQ ID No.3 is FAM, the fluorescent group connected with the probe with the sequence shown as SEQ ID No.7 is CY5, the fluorescent group connected with the probe with the sequence shown as SEQ ID No.8 is ROX, and the fluorescent group connected with the probe with the sequence shown as SEQ ID No.12 is VIC;

6. The test kit of claim 5, wherein the first and second reagents are both in solution: the final concentration of each primer is 0.2-1.2. mu.M, preferably 0.6. mu.M; the final concentration of each of the probes is 0.1-0.8. mu.M, preferably 0.35. mu.M.

7. The assay of claim 1The kit is characterized in that the PCR buffer solution comprises Tris, 10 mM-500 mM KCl and 10 mM-500 mM (NH)₄)₂SO₄1 to 50 percent of glycerol, 0.001 to 1mg/mL of BSA, 0.1 to 10 percent of Tween 20, 1 to 100mM/mL of dithiothreitol, 0.1 to 3M of betaine and 0.1 to 10 percent of DMSO.

8. The detection kit of claim 1, wherein the normal control is an artificial DNA sequence as set forth in sequences SEQ ID No.17, SEQ ID No.18, SEQ ID No.19, SEQ ID No. 20; the mutation control is a plasmid containing the sequences shown as SEQ ID No.21, SEQ ID No.22, SEQ ID No.23 and SEQ ID No. 24.

9. The detection kit according to claim 1, wherein the pH of the PCR buffer is 7.5 to 9.5 at 25 ℃.

10. The test kit of claim 1, wherein the suitable test method is TaqMan probe method; the first reactant is used for detecting a mutant type of a c.796C > T site, a wild type and a mutant type of a c.522_523insT site and a wild type of a c.650_653delCTGT site; the second reagent is used for detecting a wild type of a c.796C > T site, a mutant type of a c.650_653delCTGT site, a wild type and a mutant type of a c.806_818delinsT site.