CN117448447B - ITPKC gene detection primer probe combination, kit and application thereof - Google Patents
ITPKC gene detection primer probe combination, kit and application thereof Download PDFInfo
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Abstract
The invention discloses an ITPKC gene detection primer probe combination, a kit and application, and relates to the field of gene detection. The invention combines ARMS with fluorescence PCR technology, has high specificity and high sensitivity, short detection time consumption, low cost, and visual and good interpretation of results; the SNP locus rs28493229 of the ITPKC gene in the oral cavity shedding cell sample or the whole blood sample is typed.
Description
Technical Field
The invention relates to the field of gene detection, in particular to an ITPKC gene detection primer probe combination, a kit and application.
Background
Kawasaki Disease (KD) is also known as cutaneous mucosal lymph node syndrome, and commonly occurs in children under 5 years of age. Kawasaki disease is of unknown etiology, and is currently thought to be the result of complex interactions of genetic factors, infection and immunity, and may be complicated with coronary lesions (coronary artery lesions, CAL), which are the primary etiology of acquired heart disease in children in developed countries [1] . According to the related research report, 15-25% of children patients can simultaneously expand coronary arteries and cause coronary artery tumors in the early stage of Kawasaki disease onset if no active and effective intervention mode is adopted [2] 。
The diagnosis of kawasaki disease is generally based on having fever symptoms, and at least 4 clinical manifestations of: bilateral bulbar conjunctiva congestion, dry red lips, strawberry tongue, diffuse congestion of mucous membrane of oral cavity, rash, redness and swelling of hands and feet in acute stage, recovery stage nail Zhou Tuopi, non-suppurative cervical lymphadenectasis. However, some infants have fewer than 4 clinical manifestations, i.e., incomplete kawasaki disease. Diagnosis of this type of infant is difficult and the treatment period is easily missed, thereby causing CAL. Thus, detection of kawasaki disease-associated CAL-related genes contributes to prognosis and risk assessment of kawasaki disease-associated CAL.
Current studies report a number of genes associated with kawasaki disease complications CAL, suggesting prognosis and risk assessment. Wherein ITPKC acts as a kinase for inositol 1,4, 5-triphosphate (IP 3), a second messenger molecule that releases calcium from the endoplasmic and sarcoplasmic reticulum. Studies show that SNP locus rs 28493229C allele of ITPKC gene is related to susceptibility of Kawasaki disease and increases risk of concurrent CAL [3] And the risk of aneurysm formation of Kawasaki patients with SNP locus rs28493229 CC genotype of ITPKC gene is increased by 24.5 times compared with that of patients with CG/GG genotype. The research results in partial areas all show that ITPKC has obvious correlation with KD and concurrent CAL [4-5] 。
SNP locus rs28493229 of ITPKC gene in human genome belongs to polymorphism of single nucleotide locus. A Single Nucleotide Polymorphism (SNP) is known as Single Nucleotide Polymorphisms, which refers to a genetic marker formed by mutation of single nucleotide on genome alleles, including substitution, transversion, deletion and insertion, and has a large number of genetic markers and abundant polymorphism. Typical SNP analysis methods are PCR-sequencing, sanger sequencing, high throughput sequencing techniques, PCR-chip hybridization, mutation amplification systems (amplification refractory mutation system, ARMS) and the like. In the analysis methods, the direct sequencing method has a long time period and can not provide guidance for doctors in time; the chip method has high cost and certain limit on application.
In view of this, the present invention has been made.
Reference is made to:
[1] the department of science of the Chinese medical society of cardiovascular science, the department of science of the Chinese medical society of science of the Chinese medical science of the department of science of the Chinese medical science of the immune science, etc., kawasaki disease diagnosis and acute phase treatment expert consensus, J.Zhonghua pediatric, 2022, 60 (1): 6-13.
[2] SHUMAN S T, ROWLEY A H. Kawasaki disease：insightsinto pathogenesis and approaches to treatment [J]. Nat Rev Rheumatol, 2015,11(8)：475-482.
[3] Lin et al. Clinical Implication of the C Allele of the ITPKC Gene SNPrs28493229 in Kawasaki Disease. Pediatr Infect Dis J 2011;30(2): 148-152.
[4] Kuo et al. A replication study for association ofITPKC and CASP3 two-locus analysis in IVIG unresponsiveness and coronary artery lesion in Kawasaki disease. Plos one, 2013; 8(7):e69685.
[5] Onouchi et al. ITPKC and CASP3 polymorphisms and risks for IVIGunresponsiveness and coronary artery lesion formation in Kawasaki disease. The Pharmacogenomics Journal, 2013; 13(1); 52-59。
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims to provide an ITPKC gene detection primer probe combination, a kit and application.
At present, no related human ITPKC gene polymorphism detection kit exists in the market, and the product is based on a real-time fluorescence PCR technology, adopts an amplification blocking mutation system (amplification refractory mutation system, ARMS) to realize the typing of SNP locus rs28493229 of ITPKC genes in an oral cavity shed cell sample or a whole blood sample.
The invention designs a specific locus detection primer aiming at the rs28493229 locus of the ITPKC gene. For the ITPKC gene G locus, during PCR amplification, since the base at the 3' -end of the primer GG of the ITPKC gene is completely paired with the template of the ITPKC gene G locus, the primer extends and amplifies the template of the ITPKC gene G locus, and the template of the ITPKC locus is blocked because the primer cannot be completely paired with the template of the ITPKC gene C locus, the amplification of the template of the ITPKC gene C locus is inhibited, and the amplification of the ITPKC gene C locus is reversed. Thus, the typing of ITPKC genes is realized.
And the probe of the ITPKC gene is a Cy5 fluorescent label, the endogenous internal standard gene (human housekeeping gene) is a VIC fluorescent label, and 2-fold reaction in the same tube can be realized through different fluorescent labels. The internal standard can be used for monitoring the whole process of sample collection, nucleic acid extraction and PCR detection. In addition, the product is added with an anti-pollution component UNG enzyme, and the action mechanism of the product is to selectively hydrolyze and break uracil glycosidic bonds in dU-containing double-chain or single-chain DNA, and the formed DNA chain with missing bases can be further hydrolyzed and broken under alkaline medium and high temperature, so that the DNA chain is eliminated.
The aim of the invention is achieved by the following technical scheme:
the first aspect of the present invention provides a primer probe combination, the primer comprising: selected from the nucleotide sequences shown in SEQ ID NO:1, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:2, which is used for detecting the G site of the rs28493229 site of the ITPKC gene; selected from the nucleotide sequences shown in SEQ ID NO:4, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:5, a downstream primer is used for detecting the C site of the rs28493229 site of the ITPKC gene;
the probe comprises SEQ ID NO:3 and SEQ ID NO: 6; SEQ ID NO: 3G locus for detecting rs28493229 locus of ITPKC gene; SEQ ID NO:6 is used to detect the C site of the ITPKC gene rs28493229 site.
In some embodiments, the primer further comprises an internal reference primer and the probe further comprises an internal reference probe;
in some embodiments, the internal reference primer and the internal reference probe are used to quantitatively detect expression of a nucleic acid fragment of a housekeeping gene.
In some embodiments, the nucleotide sequences of the upstream primer and the downstream primer of the inner reference primer are set forth in SEQ ID NO:7 and seq id NO: shown as 8; the nucleotide sequence of the internal reference probe is shown as SEQ ID NO: shown at 9.
In addition, it is noted that in one aspect, useful primers and probes include sequences that match SEQ ID NO: 1-9, has a nucleotide sequence that is greater than 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical. Such primer and probe modifications are also contemplated and can be prepared according to standard techniques.
The primer and probe modification may be performed by a known method. Modified versions of these primer and/or probe sequences can include, by way of non-limiting example, adding one or more nucleotides to the 5 'end, one or more nucleotides to the 3' end, one or more nucleotides to the 5 'and 3' ends, adding tails, shortening the sequence, extending the sequence, shifting the sequence several bases upstream and downstream, or any combination thereof.
Base modifications such as 3'P, 5'P, 5-nitroindole, 2-aminopurine, 8-amino-2 ' -deoxyadenosine, C-5 propynyl-deoxycytidine, C-5 propynyl-deoxyuridine, 2-amino-2 ' -deoxyadenosine-5 ' -triphosphate, 2, 6-diaminopurine (2-amino-dA), inverted dT, inverted dideoxy-T, hydroxymethyl dC, iso-dC, 5-methyl dC, aminoethyl-phenoxazine-deoxycytidine, and locked nucleic acids (LNA's) and include at least one mismatched base at one of the bases, or at least one of the bases is replaced with an RNA base, to effect, for example, an increase in nucleic acid interactions at the 3' end of the mutant-specific primer to increase Tm. The addition of double-stranded stable base modifications has a positive effect on PCR, enabling it to be performed at higher temperatures, within which Taq polymerase is known to exhibit maximum activity. The modified probe should retain the ability to distinguish between the mutation site to be detected and the wild-type site.
The term "probe" as used herein refers to any of a variety of signaling molecules that are indicative of amplification. For example, SYBR Green and other DNA binding dyes are detection probes. May be a sequence-based detection probe, such as a 5' nuclease probe. Some detection probes are known in the art, such as Taqman probes, stem-loop molecular beacons, MGB probes, scorpion probes, locked Nucleic Acid (LNA) probes, peptide Nucleic Acid (PNA) probes, and the like.
In some embodiments, the probe is a self-quenching probe.
In some embodiments, the fluorescent emitting groups of each probe are independently selected from any of AMCA, pacific Blue, atto 425, BODIPYFL, FAM, alexa Fluor 488, TET, JOE, yakima Yellow, VIC, HEX, quasar 570, cy3, NED, TAMRA, ROX, aqua Fluor 593, texasRed, atto 590, cy5, quasar 670, and Cy 5.5.
In some embodiments, the fluorescent emission group signal of the internal reference probe is distinguishable from the detection probe.
The invention can be carried out by adopting multiple channels at the same time, and only separate probes are needed to be adopted in different channels. In some preferred embodiments, the fluorescent emitting groups on the detection probe and the internal reference probe can be distinguished under the same reaction system.
In a specific embodiment, SEQ ID NO: 3. 6, the fluorescence emitting groups on the probe are Cy5; SEQ ID NO:9 is VIC.
In some embodiments, the quenching group of each probe is independently selected from any of BHQ1, BHQ2, BHQ3, dabcyl, eclipse, and MGB, preferably BHQ3 or BHQ1.
The second aspect of the invention provides a kit comprising the primer probe combination.
The term "kit" refers to any article of manufacture (e.g., package or container) comprising at least one device, which may further comprise instructions, supplemental reagents, and/or components or assemblies for use in the methods described herein or steps thereof.
In some embodiments, the kit further comprises an amplification buffer, dNTPs, mg 2+ At least one of UNG enzyme, DNA polymerase, positive quality control, negative quality control, preservative, and water.
Suitable polymerases for the practice of the present invention are well known in the art and can be obtained from a variety of sources. Thermostable DNA polymerases are available from a variety of commercial sources using methods well known to those skilled in the art. Preferred thermostable DNA polymerases can include, but are not limited to: taqDNA polymerase or a mutant, derivative or fragment thereof.
Preferably, the nucleic acid components of the kit, such as primers, probes, positive controls, and negative controls, are stored in the kit in dry powder form. The positive control and the negative control may also be present as plasmids.
The components are preferably realized in lyophilized form, for example in the form of one or more so-called lyophilized beads. Lyophilization beads are generally understood to mean lyophilisates which are pressed into spheres after manufacture, after which the substance is usually present as a powder. Thus, the components required for a PCR batch, in particular the DNA polymerase, the nucleic acid components and the reaction buffer components, can be provided, for example, in lyophilized form. In this way, the PCR process can be started directly in a very user-friendly manner by adding the sample to be quantified and optionally other desired components. In particular, the provision of a lyophilized form is very advantageous for automated applications.
The third aspect of the invention provides an application of the primer probe combination in preparing a kit for SNP locus rs28493229 typing of ITPKC genes;
sources of nucleic acid samples in the disclosed combination and/or kit include, but are not limited to, cells, such as circulating blood, cultured cells, tumor cells. Sources of samples such as one or more of pharyngeal swabs, nasal swabs, sputum, respiratory aspirates, bronchial lavages, alveolar lavages, conjunctival swabs, saliva samples, stool specimens, anticoagulants, and serum specimens. The DNA may be DNA in the genome or in a plasmid or other vector. The invention is useful for detecting mutations in genomic DNA, which may be primate (especially human) as well as any other animal or other known or unknown having a mutation at the rs28493229 locus (G > C) of the ITPKC gene. In some embodiments, the template sequence or nucleic acid sample may be gDNA. In some embodiments, the template sequence or nucleic acid sample may be an oral shed cell sample DNA or a whole blood sample DNA. The DNA template sequence or nucleic acid sample may be any type of tissue including, for example, a formalin fixed paraffin embedded tissue sample. Template nucleic acids may also be chemically synthesized using techniques known in the art.
Compared with the prior art, the invention has the following advantages and effects:
the invention combines ARMS with fluorescence PCR technology, has high specificity and high sensitivity, short detection time consumption, low cost, and visual and good interpretation of results.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph showing the genotyping results for C3 ITPKC from a1 ng/. Mu.L whole blood sample in example 4; wherein, a, ITPKC GG PCR reaction system detection results; b. ITPKC CC PCR reaction system detection results; c. ITPKC gene generation sequencing results.
FIG. 2 is a graph showing the genotyping results for the B2 ITPKC sample of 0.3 ng/. Mu.L of oral shed cells in example 4; wherein, a, ITPKC GG PCR reaction system detection results; b. ITPKC CC PCR reaction system detection results; c. ITPKC gene generation sequencing results.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto. Unless specifically stated otherwise, reagents, equipment, methods, etc. used in the present invention are well known to those skilled in the art and will not be described in detail herein.
Unless otherwise defined, all terms (including technical and scientific terms) used to describe the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are used to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.
The terms "comprising," "including," and "comprising," as used herein, are synonymous, inclusive or open-ended, and do not exclude additional, unrecited members, elements, or method steps.
Example 1: primer and probe screening
Step 1: ITPKC gene primer and probe design and selection
Searching sequences near the rs28493229 locus of the ITPKC gene: searching ITPKC gene rs28493229 site sequence information (NG_ 012970.1) on NCBI, intercepting 300-500 bp sequence at the upstream and downstream of rs28493229 site for primer probe design, and marking as underlined[G/C]Namely ITPKC gene rs28493229 locus (G>C）。
CCTGTGCCCCGCCTCATCATTACCCCTGAGACCCCTGAGCCTGAGGCCCAGCCAGTGGGACCCCCCTCCCGGGTTGAGGGGGGCAGCGGCGGCTTCTCCTCTGCCTCTTCTTTCGACGAGTCTGAGGATGACGTGGTGGCCGGGGGCGGAGGTGCCAGCGATCCCGAGGACAGGTCTGGGGTGAGTGG[G/C]ACCCATCCTGCCCTTGAGCCACATCACGCAAAACTCCTTATTCCTCCGCCTTTGCTTAGGAAGTTCTCTACCCATTTACTGTTAGTTGCCCACCAGCAATTTCATCTCCGGGAACCTCTTCCATCCACTGACCTCCTCCCTCTGACAGCCAGGTTACTAATTCACTCCAGGCACCCCTTCTTAATTCTCTCCTTTCCCCTTCCTGCACTGTCTCCACTCCTGGGGGTCTACAATGGAGGGCCACTGACCGACTTTATGGGGCCCAGGAACCCCTGAAATTGTAGGACTAAGGAGCCTGCCGTGC
Primer and probe sequences were designed with the aid of Primer and probe design software oligo 7 and Primer Premier 5.0 software. 3 upstream/downstream primers and 2 sets of downstream/upstream primers and probes are designed by taking the base of the rs28493229 locus of the ITPKC gene as the last base of the 3' -end of the upstream/downstream primer, as shown in Table 1.
And (3) screening a blank reference substance (NTC), wild sample DNA and mutant sample DNA to-be-detected point primer and probe sequences after combining the designed upstream and downstream primers and probes. As can be seen from Table 2, the NTC detection results of the 12 pairs of primer and probe combinations at each site are all qualified and can be used for the next amplification efficiency and specificity detection. As can be seen from Table 3, the optimal combinations were selected by screening for amplification efficiency and specificity for the different combinations (Table 4).
TABLE 1 ITPKC Gene rs28493229 site primer and probe sequence information Table
Note that: the lower case labeled primers in the tables are mismatched bases that are introduced by humans.
TABLE 2 Single primer Probe NTC screening test results
TABLE 3 ITPKC Gene primer, probe specificity and amplification efficiency screening results
TABLE 4 optimal site primer and probe sequence information after screening
And (3) injection: the lower case labeled primers in the tables are mismatched bases that are introduced by humans.
Step 2: internal reference primer and probe design and selection
The internal reference gene is selected from human housekeeping Genes (GAPDH), 2 sets of primers and corresponding probes are designed according to the taqman probe design principle under the assistance of Primer and probe design software oligo 7 and Primer Premier 5.0 software, the 5 'end of each probe is marked by a fluorescence reporter group (VIC), the 3' end of each probe is marked by a non-fluorescence quenching agent (BHQ-1), so that background interference is reduced, and the design of the internal reference Primer probe is shown in Table 5.
The designed 2 sets of internal reference primer probes are used for detecting blank control, wild sample DNA and mutant sample DNA, and the effects of GAPDH-F3, GAPDH-R3 and GAPDH-P are better after analysis and screening.
Sequence information of the reference primers and probes in Table 5
Step 3: multiple system primer probe combination
Combining the above results of selecting the ITPKC gene locus detection primer probe and the internal reference primer probe, the selected sequence (see table 4) enters the combination of the primer probes of the next multiple system (see table 6 and table 7), the combined primers and probes are optimized, and the finally screened optimal amounts can be seen in table 9 and table 10. According to tables 9 and 10, corresponding primer and probe mixed solutions are prepared, and specific detection results of oral cavity exfoliated cell samples and whole blood samples with different genotypes are shown in example 2.
In summary, by screening the ITPKC gene and the internal reference gene primer probes, the preferred primer probe combinations and internal reference primer probe combinations are finally determined as shown in Table 8.
TABLE 6 ITPKC GG Combined primer and Probe sequence information
TABLE 7 ITPKC CC Combined primer and Probe sequence information
TABLE 8 most preferably primer probe combination sequence information
Example 2: optimization of reaction system and reaction program
By researching different Mix usage amounts, different primer, probe concentration and usage amounts and different DNA template usage amounts, an optimal reaction system is established, and by researching different annealing temperatures, time and cycle numbers, an optimal reaction program is established. The reaction system and the reaction procedure are shown in tables 9 to 14.
Specifically, the human ITPKC gene polymorphism detection kit (fluorescence PCR method) comprises the following steps:
s1: extracting nucleic acid;
s2: preparing a reaction solution;
s3: sample adding;
s4: qPCR amplification;
s5: and (5) data analysis.
The step S1 specifically comprises the following steps:
(1) Oral cast cell sample: vortex oscillating the centrifuge tube with the oral swab for 1 min to thoroughly wash off the oral exfoliated cells, and discarding the swab; nucleic acid extraction is performed using a nucleic acid extraction or purification reagent (record number: yueXuimechanical arm 20211439);
(2) Whole blood sample: nucleic acid extraction was performed using a nucleic acid extraction or purification reagent (accession number: yueXuimechanical arm 20230207).
The step S2 specifically comprises the following steps: and (3) subpackaging the PCR reaction liquid according to the number n of the samples to be tested (including negative quality control products and positive quality control products), and subpackaging the PCR reaction liquid into PCR reaction tubes/plates according to 23 mu l/tube. In the step S2, 3 PCR reaction solutions are provided, wherein the PCR reaction solutions comprise ITPKC GG, ITPKC CC primers and probe mixed solutions, the preparation is shown in tables 9 and 10, and 1 PCR reaction buffer solution is shown in table 11, the sequences of the primers and probes used for preparing the PCR reaction solutions are shown in table 4 (the group of detection systems are adopted unless special emphasis is given in the invention):
TABLE 9 preparation of ITPKC GG primer and Probe Mixed solution
TABLE 10 preparation of ITPKC CC primer and Probe Mixed solution
TABLE 11 preparation of PCR reaction buffers
The step S3 specifically comprises the following steps: and respectively adding the nucleic acid extracted from the sample to be detected, the nucleic acid extracted from the negative quality control product and the positive quality control product into the PCR reaction liquid, covering the tube cover or sealing the film, and carrying out instantaneous centrifugation for a plurality of seconds to concentrate the liquid to the bottom of the tube and transfer the liquid into a nucleic acid amplification region. The reaction system was prepared as shown in the following tables 12 and 13:
TABLE 12 ITPKC GG PCR reaction System
TABLE 13 ITPKC CC PCR reaction System
The step S4 specifically comprises the following steps: (1) placing a PCR reaction tube/plate into a sample groove of a quantitative PCR instrument, setting a positive quality control product, a negative quality control product and an unknown sample according to corresponding names, and setting sample names and detection target names; (2) fluorescence detection channel selection: ITPKC: cy5, GAPDH: VIC; the quenching group is selected from "none"; reference dye Passive Reference selected "none"; (3) setting a reaction program; (4) the reaction system was selected to be 25. Mu.L; (5) and after the setting is finished, storing a file and running a reaction program. The reaction procedure used in step S4 is as shown in table 14:
table 14 reaction procedure
The step S5 specifically comprises the following steps: (1) results analysis conditions were set, baseline and threshold line were adjusted: (2) judging a quality control product; (3) and judging the detection result. The quality control criteria used in step S5 are shown in table 15, and the result judgment criteria are shown in table 16:
table 15 quality control Standard
Table 16 results judging Table
Thus, the composition, package and number of human ITPKC gene polymorphism detection kit (24 parts/kit) are as shown in table 17:
table 17 composition, package and quantity of kit
Example 3: accuracy detection experiment of kit
The accuracy detection experiment of the kit provided by the invention is carried out by selecting 32 oral cavity shedding cell samples with different genotypes at the rs28493229 locus of the ITPKC gene, wherein the sample number is A1-A32. Referring to the procedure of example 2, the samples were subjected to nucleic acid extraction according to the oral cavity exfoliated cell sample extraction method of step S1, PCR reaction solutions were prepared according to tables 9, 10 and 11 of step S2, 2 reaction systems were prepared according to tables 12 and 13 of step S3, and A1-a32 DNA sample, negative quality control, and positive quality control were sequentially added. After the system preparation is completed, the PCR reaction tube/plate cover is covered, fully and uniformly mixed, and the mixture is centrifuged at 8,000 rpm for 15 seconds and then transferred to an amplification detection area. The amplification procedure is set up and the fluorescence channel is selected as shown in step S4. And (5) after the amplification is finished, performing quality control and result judgment on the result according to the step S5. The results of the 32 clinical samples are shown in Table 18.
Table 18 results of oral shed cell sample detection
As can be seen from Table 18, in the 32 samples of oral cavity shedding cells detected, the rs28493229 locus genotyping results of ITPKC genes are 32 GG types, 0 GC types and 0 CC types, and the genotyping results of the kit are completely consistent with the genotyping results verified by the first-generation sequencing.
Example 4: detection limit research experiment of kit
3 oral cavity shedding cell samples and whole blood samples with different genotypes at the rs28493229 locus of the ITPKC gene are selected for detection limit research experiments. The serial numbers of the oral cavity exfoliated cell samples are B1-B3, and the serial numbers of the whole blood samples are C1-C3. Referring to the procedure of example 2, samples B1 to B3 and C1 to C3 were subjected to nucleic acid extraction according to the oral cavity exfoliated cell sample extraction method and the whole blood sample extraction method of step S1, respectively, and after sample extraction, samples B1 to B3 were diluted to 0.3 ng/. Mu.L, 0.5 ng/. Mu.L, 0.8 ng/. Mu.L and 1 ng/. Mu.L, respectively, and samples C1 to C3 were diluted to 1 ng/. Mu.L, 1.5 ng/. Mu.L, 2 ng/. Mu.L and 2.5 ng/. Mu.L, respectively. Preparing PCR reaction solutions according to the table 9, the table 10 and the table 11 of the step S2, preparing 2 reaction systems according to the table 12 and the table 13 of the step S3, adding B1-B3, C1-C3, negative quality control products and positive quality control products in sequence, and carrying out 3 repeated experiments on each sample. After the system preparation is completed, the PCR reaction tube/plate cover is covered, fully and uniformly mixed, and the mixture is centrifuged at 8,000 rpm for 15 seconds and then transferred to an amplification detection area. The amplification procedure is set up and the fluorescence channel is selected as shown in step S4. After the amplification, the results were quality-controlled and judged according to step S5, and the detection results are shown in tables 19, 20, 21, 22 and fig. 1 and 2.
TABLE 19 Whole blood sample ITPKC GG PCR reaction System detection results
TABLE 20 detection results of whole blood sample ITPKC CC PCR reaction System
TABLE 21 detection results of oral shed cell sample ITPKC GG PCR reaction System
Table 22 detection results of oral shed cell sample ITPKC CC PCR reaction system
As can be seen from tables 19, 20, 21, 22 and FIGS. 1 and 2, the detection limit of the whole blood sample was 1 ng/. Mu.L, and the detection limit of the oral cavity exfoliated cell sample was 0.3 ng/. Mu.L. When the concentration of the whole blood sample is greater than or equal to 1 ng/. Mu.L or the concentration of the oral cavity shedding cell sample is greater than or equal to 0.3 ng/. Mu.L, the genotypes of the ITPKC gene rs28493229 loci of the 3 repeated experiments of the 3 samples detected respectively are consistent with the genotypes verified by a first-generation sequencing method.
Example 5: clinical application experiment
20 cases of whole blood clinical samples are selected for clinical application experiments on the kit, and the sample numbers are 1-20. Referring to the procedure of example 2, the clinical samples were subjected to nucleic acid extraction on 20 samples according to the whole blood sample extraction method of step S1, PCR reaction solutions were prepared according to tables 9, 10 and 11 of step S2, 2 reaction systems were prepared according to tables 12 and 13 of step S3, and 1 to 20 DNA samples, negative quality control products and positive quality control products were sequentially added. After the system preparation is completed, the PCR reaction tube/plate cover is covered, fully and uniformly mixed, and the mixture is centrifuged at 8,000 rpm for 15 seconds and then transferred to an amplification detection area. The amplification procedure is set up and the fluorescence channel is selected as shown in step S4. And (5) after the amplification is finished, performing quality control and result judgment on the result according to the step S5. The results of the 20 clinical samples are shown in Table 23.
TABLE 23 Whole blood sample detection results
As can be seen from Table 23, in the 20 whole blood samples tested, the rs28493229 locus genotyping results of ITPKC genes were 19 GG types, 1 GC type and 0 CC type, and the genotyping results using the kit of the invention were completely consistent with the genotyping results verified by the first-generation sequencing.
The method is used for detecting the ITPKC gene rs28493229 locus polymorphism in the human oral cavity shedding cell sample or the whole blood sample. The extraction method of the oral cavity exfoliated cell sample is simple and quick, and can meet the product detection requirement; the product design adopts common chemical substances and common Taqman probes, the cost is low, and the detection result is reliable; the sensitivity is high, and the lowest can detect 0.3 ng/. Mu.L DNA; the detection accuracy is high, the oral cavity exfoliated cell samples and the whole blood samples with different genotypes are detected, and the detection result is completely consistent with the genotype result of the first-generation sequencing detection. Fills up the blank condition of the human ITPKC gene polymorphism detection kit (fluorescence PCR method) in the current market, and can provide prognosis and risk assessment of Kawasaki disease complicated CAL for children suffering from clinical Kawasaki disease symptoms.
The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.
The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. The scope of the invention is therefore intended to be covered by the appended claims, and the description and drawings may be interpreted in accordance with the contents of the claims.
Claims (5)
1. Primer probe combination, its characterized in that: the primer comprises: the nucleotide sequence is shown in SEQ ID NO:1, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:2, which is used for detecting the G site of the rs28493229 site of the ITPKC gene; the nucleotide sequence is shown in SEQ ID NO:4, and the nucleotide sequence of the upstream primer is shown as SEQ ID NO:5, a downstream primer is used for detecting the C site of the rs28493229 site of the ITPKC gene;
the probe comprises SEQ ID NO:3 and SEQ ID NO: 6; SEQ ID NO:3 is used for detecting G locus of rs28493229 locus of ITPKC gene; SEQ ID NO:6 is used for detecting the C site of the rs28493229 site of the ITPKC gene;
the primer further comprises an internal reference primer, and the probe further comprises an internal reference probe; the internal reference primer and the internal reference probe are used for quantitatively detecting the expression of the nucleic acid fragment of the housekeeping gene; the nucleotide sequence of the upstream primer and the downstream primer of the internal reference primer is shown as SEQ ID NO:7 and SEQ ID NO: shown as 8; the nucleotide sequence of the internal reference probe is shown as SEQ ID NO: shown at 9.
2. The primer probe combination of claim 1, wherein: the probe is a self-quenching probe.
3. Use of the primer probe combination according to any one of claims 1-2 in the preparation of a kit for the genotyping of SNP locus rs28493229 of ITPKC gene.
4. A kit comprising the primer probe combination of any one of claims 1 to 2.
5. The kit of claim 4, wherein: the kit also comprises an amplification buffer solution, dNTPs and Mg 2+ At least one of UNG enzyme, DNA polymerase, positive quality control, negative quality control, preservative, and water.
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