CN109251974A - APOE2 and APOE4 genotype quick detection kit based on POCT mode - Google Patents
APOE2 and APOE4 genotype quick detection kit based on POCT mode Download PDFInfo
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Abstract
The present invention provides a kind of APOE2 and APOE4 genotype quick detection kit based on POCT mode, the kit contains the primer, probe and cell pyrolysis liquid for detecting APOE2 and APOE4 genotype, the primer of APOE2 is detected as shown in SEQ ID NO.1-2, probe is as shown in SEQ ID NO.3-4, the primer of APOE4 is detected as shown in SEQ ID NO.5-6, probe is as shown in SEQ ID NO.7-8.This kit is purified without DNA, and sample can be directly added into progress PCR reaction in reagent, be particularly suitable for quick, the accurate detection of the lower sample of DNA content, Detection accuracy can be detected accurately up to 99%, high sensitivity down to 0.1ng genomic DNA；Entire detection is time-consuming short, can provide medication foundation to doctor in first time, reduce patient medication risk.
Description
Technical field
The present invention relates to molecular biology fields, specifically, being related to a kind of APOE2 and APOE4 based on POCT mode
Genotype quick detection kit.
Background technique
APOE gene is the gene for encoding mankind's apo E (apolipoprotein E, APOE), the assignment of genes gene mapping
In chromosome 19p13, there are 4 exons and 3 intrones, encoded APOE albumen category apolipoprotein family, is blood plasma
The important component of lipoprotein is mainly expressed in liver and brain tissue, participates in the lipid generation of body through a variety of ways
It thanks, is the important internal factor for influencing blood lipid level.
Studies have shown that APOE gene has nucleotide polymorphisms, two mononucleotide polymorphism sites of one of the most common
For APOE4 T388C (rs429358) and APOE2 C526T (rs7412), 3 kinds of monoploid, respectively E2 (388T- are formed altogether
526T), three kinds of E3 (388T-526C), E4 (388C-526C) pheno types, generating includes three kinds of homozygotes (2/ ε 2 of ε, 3/ ε of ε
3,4/ ε 4 of ε) and three kinds of heterozygotes (3/ ε 2 of ε, 4/ ε 2 of ε, 4/ ε 3 of ε) including 6 kinds of APOE genotype.Wherein, 2 allele of ε
Carrier, APOE concentration is high in blood, and cholesterol level is low, has protective action to atherosclerosis；And 4 allele of ε
Carrier, APOE concentration is low in blood, and cholesterol and triacylglycerol content are high, be atherosclerosis potential danger because
Element.
Accretion rate of the APOE isomers from the compatibility of lipoprotein receptor and its in vivo is different, so as to cause individual
Between blood lipid level difference.Clinically patient can be instructed correctly to select lipid-regulation medicine and drink according to the different gene phenotype of APOE
Therapeutic intervention is eaten, reduces the disease incidence of coronary heart disease and the risk of cardiovascular disease burst to reach.Studies have shown that E2 type is length
Longevity genotype, it is sensitive to statins, therefore such patient's tune rouge can preferred statins as lipid-lowering medicine.E3 type crowd
Account for it is most of in crowd, it is normal for the effect of statins lipid-loweringing, the morbidity of cardiovascular and cerebrovascular is not obviously inclined to
Property.It is higher that early onset cardiovascular disease, the risk of senile dementia occur for E4 type crowd, and when dyslipidemia is controlled with statins
Probucol In Treatment can be selected in therapeutic effect ratio E2 type, the difference of E3 type crowd, obvious using low fat diet lipid-lowering effect effect.
The crowd for taking statins is mostly the patient for suffering from the cardiovascular diseases such as coronary heart disease, myocardial infarction, by right
APOE genotype detection can instruct doctor to carry out accurate medication within the effectively treatment time limit, improve drug and use effectively
Property, reduce toxic side effect.
Currently, the method for detection APOE genotype mainly has PCR- chip hybridization methods, PCR sequencing PCR and PCR- fluorescence probe
Method.Chip method pair used by 102010897 A of PCR sequencing PCR used by 106498036 A of publication CN and patent CN
Testing result has very high precision, but requires to rely on Large-Scale Precision Instrument and Equipment, fixed laboratory and professional operation people
Member, DNA sample preparation require height, and therefore, both detection methods are at high cost, and complex steps, time-consuming, are clinically difficult
It is promoted.Although disclosed patent CN 201510288426, which uses PCR- fluorescence probe method, reduces PCR sequencing PCR and gene
The cost of instrument and reagent in chip method, but due to the reaction system can only genomic DNA sample after amplification purification,
So must be operated using multi-step multitube, it is also undesirable on clinical expansion.
Therefore, these technologies are all difficult to solve the problems, such as the urgency of the clinical detection of statins user, are difficult to reality
It is fast and accurately required under existing POCT mode.
Summary of the invention
The object of the present invention is to provide it is a set of for detect APOE2 and APOE4 genotype fluorescence quantification PCR primer and
Probe.
It is a further object of the present invention to provide a kind of APOE2 and APOE4 genotype based on POCT mode quickly to detect examination
Agent box.
In order to achieve the object of the present invention, the present invention is provided to detect the primer pair of APOE2 and APOE4 genotype, detection
The nucleotide sequence of the primer pair of APOE2 gene pleiomorphism are as follows:
Upstream primer: 5 '-CGTAAGCGGCTCCTCCG-3 '
Downstream primer: 5 '-GGATGGCGCTGAGGCC-3 '；
Detect the nucleotide sequence of the primer pair of APOE4 gene pleiomorphism are as follows:
Upstream primer: 5 '-GGAGGAGACGCGGGCAC-3 '
Downstream primer: 5 '-GCCTGCACCTCGCCG-3 '.
The present invention provides with the matching used probe combinations of the primer pair, the probe combinations are as follows:
APOE2 gene wild-type probe: 5 '-TGCAGAAG+CGCCTGG-3 '
APOE2 genic mutation type probe: 5 '-CAGAAG+TGCCTGGCAG-3 '
APOE4 gene wild-type probe: 5 '-CGGCCGC+ACACGTCCT-3 '
APOE4 genic mutation type probe: 5 '-CGGCCGC+GCACGTCCT-3 '.
Wherein, "+" indicates the base on right side for LNA modification.
The present invention provides the detection reagents or kit that contain the primer and the probe combinations.
The present invention provides a kind of PCR reaction solution for detecting APOE2 and APOE4 gene pleiomorphism, including the primer with
The probe combinations.
Further, the PCR reaction solution further includes archaeal dna polymerase, dNTPs, buffer, MgCl2And cell cracking
Liquid.
Preferably, in the PCR reaction solution each component it is final concentration of: 0.5-1.5 times of PCR reaction buffer, DNA
It is polymerase 0.04-0.1U/ μ L, dNTPs 0.1-0.5mM, 0.2-0.7 μM of upstream primer, 0.2-0.7 μM of downstream primer, wild
0.2-0.7 μM of type probe, 0.2-0.7 μM of saltant type probe, Mg2+1-2.5mM and cell pyrolysis liquid；
The cell pyrolysis liquid is made of lauryl sodium sulfate and Triton X-100；
Wherein, the concentration of lauryl sodium sulfate is 0.0005-0.015% (w/v)；Triton X-100
Concentration is 0.001-0.03% (w/v).
It is highly preferred that in the PCR reaction system each component it is final concentration of: 1.1 × PCR reaction buffer, DNA are poly-
Synthase 0.05U/ μ L, dNTPs 0.2mM, 0.4 μM of upstream primer, 0.4 μM of downstream primer, 0.3 μM of wild-type probe, saltant type
0.4 μM of probe, Mg2+2.5mM and cell pyrolysis liquid.
APOE2 the and APOE4 genotype quick detection kit based on POCT mode that the present invention provides a kind of, it is described
Kit contains PCR reaction solution of the present invention.
When mentioned reagent box of the invention carries out quantitative fluorescent PCR, working procedure are as follows: 95 DEG C of initial denaturation 5min；95℃
It is denaturalized 8s, 62 DEG C of annealing and extension 35s, is recycled 50 times.
Kit of the invention is detected applied to actual sample, the detection sample can come from oral cavity wall, tongue
The positions such as head, palm, ear, the DNA that the cast-off cells from above-mentioned position release after lysate cracks are used equally for this
The genetic test of kit, since scraping oral cavity wall Sample Method is simple and efficient, and sample size is moderate, therefore chooses oral cavity
Sample is optimal.Sampling and load procedure can be completed in 20 seconds under normal circumstances.
(1) prepare before sampling
Patient must be gargled 2 times with clear water before sampling, and no less than 5 seconds every time, swallowed 2-3 times after gargling, avoided oral cavity as far as possible
Inner wall remains saliva.Sampling person must wearing gloves, mask.After above-mentioned be ready to complete, it can be sampled.
(2) sampling and sample-adding
Sampling tool is disposable oral cavity swab.Specific sampling process is as follows:
1) reaction solution and swab are taken out from kit and swab packaging bag respectively, then pulls out reagent plug (Fig. 1-a)；
2) swab end cap is removed, should pay attention to not contacting reagent plug (Fig. 1-b) in the process；
3) make cheek wall in the nearly 90 ° of contacts oral cavity of swab end, uniformly wipe 5 times (being up and down 1 time), dynamics is micro- with cheek
It is prominent to be advisable (Fig. 1-c)；
4) immediately by swab intercalation reaction liquid after sampling, pressing makes itself and the reagent seal of tube, is protected from light temporary (such as 1-d).
Analysis of test results: after reaction by analysis system can intuitively and accurately to testing result carry out analysis and
It interprets, can produce following three kinds of results altogether.
1. Wild homozygous
The testing result has been shown as in analysis system and only green fluorescence curve is exponentially increased trend, and
Have and only (cycle threshold refers to that the fluorescence signal in each reaction tube reaches and sets green fluorescence channel generation Ct value
Recurring number experienced when fixed threshold value).(Fig. 2, Fig. 5)
2. mutated homozygous
The testing result has been shown as in analysis system and only red fluorescence curve is exponentially increased trend, and
Have and only red fluorescence channel generates Ct value.(Fig. 3, Fig. 6)
3. heterozygous
The testing result shows as green fluorescence curve in analysis system and red fluorescence curve is exponentially increased
Gesture, and green fluorescence channel and red fluorescence channel generate Ct value.(Fig. 4, Fig. 7)
The invention has the following advantages that
(1) detection immediately can be achieved, purified without DNA, sample can be directly added into progress PCR reaction in reagent, 1 is small
When the interior detection information that corresponding gene loci can be obtained；Therefore medication foundation can be provided to doctor in first time, to avoid
Patient medication mistake.
(2) it joined cell pyrolysis liquid in reagent reaction system, Direct Pyrolysis cell and can release in nucleus
Sample is added in DNA, DNA extraction is reacted the stopped pipe in same branch Reagent Tube with PCR and carried out.
(3) this kit sample (DNA or Stomatocyte) Detection accuracy is up to 99% or more.
(4) detection method, sampling process is painless noninvasive, easy to operate, can be complete in single sampling sample-adding 20s
At.
(5) detection sensitivity is high, can accurately detect the genomic DNA down to 0.1ng.It is too long for the holding time with
And the lower sample of the DNA contents such as buccal swab can be detected accurately.
(6) this kit can at least place 72h at normal temperature, and 2-8 DEG C can at least place 15 days, and -20 DEG C can at least put
It sets 12 months.
(7) present invention employs novel sample-addings to the mode of operation of one step of result, eliminates cumbersome intermediate ring
Section can go out testing result in 1 hour, solve emergency patients and treat urgent problems.Single step, single tube reagent, stopped pipe
A possibility that operating, considerably reducing environmental pollution.
(8) present invention has also matched intellectual analysis program, can directly automatically analyze to data, obtain at once
The genotype call results and medication guide of unbiasedness are reported, laboratory constraint is got rid of, to environment and operator without spy
It is different to require.
(9) common molecular beacon can be used in the probe that the present invention uses, and can also be used while having MGB
, but Taqman probe (minorgroovebinding) and the Taqman probe of LNA (locknucleicacid) dual modification
There are greater advantages compared with molecular beacon itself, Taqman probe not only greatly reduces background signal intensity, and further mentions
The high specificity of probe, sensitivity and accuracy.
(10) present invention additionally uses thermal starting enzyme, by optimizing reagent system, can contain oral cavity impurity and ring
The risk that border temperature change is brought.
Detailed description of the invention
Fig. 1 is to carry out sampling and being loaded operational flowchart in detection process using kit of the present invention.
Fig. 2 is that the wild homozygote of APOE2 C526T of the present invention detects amplification curve.
Fig. 3 is that APOE2 C526T no mutant homozygote of the present invention detects amplification curve.
Fig. 4 is APOE2 C526T Heterozygote detation amplification curve of the present invention.
Fig. 5 is that the wild homozygote of APOE4T388C detects amplification curve
Fig. 6 is that APOE4 T388C no mutant homozygote detects amplification curve
Fig. 7 is APOE4 T388C heterozygote amplification curve.
Fig. 8 is that APOE2 adds lysate and three kinds of genotype Ct Data-Statistics histograms of lysate are not added, and " * " is indicated and compareed
Group is compared, and data difference is significant (P≤0.05).
Fig. 9 is that APOE4 adds lysate and three kinds of genotype Ct Data-Statistics histograms of lysate are not added, and " * " is indicated and compareed
Group is compared, and data difference is significant (P≤0.05).
Figure 10 is that APOE2 adds lysate and three kinds of genotype EPF Data-Statistics histograms of lysate are not added." * " indicate with it is right
It is compared according to group, this group of data difference is significant (P≤0.05), and " * * " indicates that difference is extremely significant (P≤0.01).
Figure 11 is that APOE4 adds lysate and three kinds of genotype EPF Data-Statistics histograms of lysate are not added." * " indicate with it is right
It is compared according to group, this group of data difference is significant (P≤0.05), and " * * " indicates that difference is extremely significant (P≤0.01).
Figure 12 is that APOE2 and APOE4 adds lysate and lysate test accuracy statistics histogram is not added.
Figure 13 is the Ct Data-Statistics histogram of tri- kinds of genotype of APOE2 C526T.Data are united through multiple comparative test in figure
Meter learns difference, and by the descending comparison of mean value, * indicates that difference is not significant (P≤0.05), " * * " indicate difference it is extremely significant (P≤
0.01)。
Figure 14 is the Ct Data-Statistics histogram of tri- kinds of genotype of APOE4 T388C.Data are united through multiple comparative test in figure
Meter learns difference, and by the descending comparison of mean value, * indicates that difference is not significant (P≤0.05), " * * " indicate difference it is extremely significant (P≤
0.01)。
Figure 15 is the end point fluorescence Data-Statistics histogram of tri- kinds of genotype of APOE2 C526T.* not significant (the P of difference is indicated
≤ 0.05), " * * " indicates that difference is extremely significant (P≤0.01).
Figure 16 is the end point fluorescence Data-Statistics histogram of tri- kinds of genotype of APOE4 T388C.* not significant (the P of difference is indicated
≤ 0.05), " * * " indicates that difference is extremely significant (P≤0.01).
Figure 17 is the influence data statistics figure that different detection environment detect accuracy to kit of the present invention.
Figure 18 is three kinds of genotype Ct Data-Statistics figures of APOE2 A group in embodiment 7.
Figure 19 is three kinds of genotype Ct Data-Statistics figures of APOE2 B group in embodiment 7.
Figure 20 is three kinds of genotype Ct Data-Statistics figures of APOE2 C group in embodiment 7.
Figure 21 is three kinds of genotype end point fluorescence value (EPF) statistical charts of APOE2 A group in embodiment 7.
Figure 22 is three kinds of genotype end point fluorescence value (EPF) statistical charts of APOE2 B group in embodiment 7.
Figure 23 is three kinds of genotype end point fluorescence value (EPF) statistical charts of APOE2 C group in embodiment 7.
Figure 24 is three kinds of genotype Ct Data-Statistics figures of APOE4 A group in embodiment 7.
Figure 25 is three kinds of genotype Ct Data-Statistics figures of APOE4 B group in embodiment 7.
Figure 26 is three kinds of genotype Ct Data-Statistics figures of APOE4 C group in embodiment 7.
Figure 27 is three kinds of genotype end point fluorescence value (EPF) statistical charts of APOE4 A group in embodiment 7.
Figure 28 is three kinds of genotype end point fluorescence value (EPF) statistical charts of APOE4 B group in embodiment 7.
Figure 29 is three kinds of genotype end point fluorescence value (EPF) statistical charts of APOE4 C group in embodiment 7.
Figure 30 is tri- kinds of genotype Ct Data-Statistics histograms of APOE2 that concentration one, two, three matches in embodiment 8.It is identical
Letter indicates that difference is not significant (p >=0.05), and different letters indicate significant difference (P≤0.05).With the following figure herewith caption.
Figure 31 is tri- kinds of genotype Ct Data-Statistics histograms of APOE4 that concentration one, two, three matches in embodiment 8.
Figure 32 is that tri- kinds of genotype EPF of APOE2 count histogram in embodiment 8.
Figure 33 is that tri- kinds of genotype EPF of APOE4 count histogram in embodiment 8.
Figure 34 is tri- kinds of genotype Ct Data-Statistics histograms of APOE2 in embodiment 8.Be between each group difference it is not significant (P >
0.05)。
Figure 35 is tri- kinds of genotype Ct Data-Statistics histograms of APOE4 in embodiment 8, be between each group difference it is not significant (P >
0.05)。
Figure 36 is that tri- kinds of genotype end point fluorescence values (EPF) of APOE2 count histogram in embodiment 8, and " * " indicates difference
Significantly (P >=0.05) " * * " indicates that difference is extremely significant (P≤0.01).
Figure 37 is that tri- kinds of genotype end point fluorescence values (EPF) of APOE4 count histogram in embodiment 8, and " * " indicates difference
Significantly (P >=0.05) " * * " indicates that difference is extremely significant (P≤0.01).
In above-mentioned figure, " G " indicates that green fluorescence channel, " R " indicate red fluorescence channel.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, implementing
Example is according to conventional laboratory conditions or according to the condition of manufacturer's specification suggestion.Test material as used in the following examples
Material, is to be commercially available from conventional biochemical reagent company unless otherwise specified.
Embodiment 1 is used to detect the configuration of the PCR reaction solution of APOE2 and APOE4 genotype
PCR is a kind of extremely sensitive minim DNA detection technique.Clinically painless non-invasive detection methods is main at present
Sample has buccal swab, hair, nail, saliva of buccal cavity and coelomic fluid etc., these samples are both needed to professional in specialized laboratory
It is anti-can just PCR to be added by complicated operating process by after DNA extraction purification contained therein using the instrument and equipment of profession
It answers system to be reacted, needs to expend a large amount of human and material resources, financial resources.In order to solve the problems, such as that DNA is purified, directly with mouth
Chamber mucous membrane cast-off cells replace the DNA of purification to carry out PCR；In view of expanding effect is undesirable after being directly added into cell, be not suitable for
Clinical detection, applicant attempt to joined certain density self-control cell pyrolysis liquid.And add cell pyrolysis liquid and be not added
The comparative experiments of cell pyrolysis liquid.Cell pyrolysis liquid is made of lauryl sodium sulfate and Triton X-100.
Agent prescription is shown in Table 1-1, and 3 kinds of different phenotypes respectively prepare 12 repetitions.Response procedures are 95 DEG C of initial denaturation 5min；
95 DEG C of denaturation 8s, 62 DEG C of annealing and extension 35s, recycle 50 times.
Table 1-1 difference sample tests PCR and reacts total system formula table
| Composition | Concentration one | Concentration two |
| 5×Promega Colourless Buffer | 1.1× | 1.1× |
| dNTP(10mM) | 0.2mM | 0.2mM |
| MgCl2(25mM) | 2.5mM | 2.5mM |
| Cell pyrolysis liquid | 1× | - |
| APOE2/APOE4 upstream primer (100 μM) | 0.5μM | 0.5μM |
| APOE2/APOE4 downstream primer (100 μM) | 0.5μM | 0.5μM |
| APOE2/APOE4 wild-type probe | 0.5μM | 0.5μM |
| APOE2/APOE4 saltant type probe | 0.5μM | 0.5μM |
| Archaeal dna polymerase (5U/ μ L) | 1.25U | 1.25U |
| Mucous membrane of mouth cast-off cells | + | + |
| Add ultrapure water | 23.5μL | 23.5μL |
Experimental result:
(1) Ct Data-Statistics such as following table and Fig. 8 and Fig. 9.
Table 1-2 APOE2 Ct Data-Statistics table
Table 1-3 APOE4 Ct Data-Statistics table
(2) end point fluorescence value (EPF) statistics such as following table and Figure 10, Figure 11.
Tri- kinds of genotype end point fluorescence value (EPF) statistical forms of table 1-4 APOE2
Tri- kinds of genotype end point fluorescence value (EPF) statistical forms of table 1-5 APOE4
(3) accuracy statistics is as follows:
Table 1-6 adds lysate and lysate test accuracy statistical form is not added
Compare plus cell pyrolysis liquid can be seen that with the Ct value that cell pyrolysis liquid is not added, the reagent sun of cell pyrolysis liquid is not added
Property channel C t value it is extremely significant be greater than be added cell pyrolysis liquid positive channel C t value (P≤0.01)；Pass through the two fluorescent value ratio
Compared with, it can be seen that be added cell pyrolysis liquid after fluorescent value it is extremely significant be higher than cell pyrolysis liquid fluorescent value (P≤0.01) is not added.Compare
Accuracy, it can be seen that add after lysate accuracy rate to be higher than and lysate is not added, and be higher by about 80%.The above results show to add
After entering cell pyrolysis liquid, cell pyrolysis liquid has cracked cell and it is made to release a large amount of DNA, so that Ct value significantly reduces, it is glimmering
Light value increases, to improve the accuracy rate of detection.
2 LNA of embodiment modifies probe and improves parting accuracy
LNA is a kind of oligonucleotide derivative, with DNA/RNA have similar structure, therefore can to DNA and RNA into
Row strong identification and combination.After LNA is for the modification of oligonucleotides, the thermal stability of primer or probe can be increased, improved
3~8 DEG C of its annealing temperature.The probe of this kit developing is modified using LNA, and through software prediction, the wild type after modification is visited
The Tm value of needle and saltant type probe in conjunction with template improves 3 DEG C or so.In order to sufficiently show LNA modification probe and without
LNA modifies the difference of probe, carries out following comparative's experiment, and PCR system is shown in Table 2-1, detects wild homozygote, heterozygote respectively
And no mutant homozygote, each genotype do three repetitions, response procedures are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 8s, 62 DEG C
Annealing and extension 35s, recycle 50 times.
The primer and probe sequence are as follows:
1)APOE2 C526T
Upstream primer SEQ No.1:5 '-CGTAAGCGGCTCCTCCG-3 '
Downstream primer SEQ No.2:5 '-GGATGGCGCTGAGGCC-3 '
Wild type LNA modifies probe SEQ No.3:5 '-FAM-TGCAGAAG+CGCCTGG- MGB-3 '
Saltant type LNA modifies probe SEQ No.4:5 '-Texas Red-CAGAAG+TGCCTGG CAG-MGB-3 '
Wild type modifies probe: 5 '-FAM-TGCAGAAGCGCCTGG-MGB-3 ' without LNA
Saltant type modifies probe: 5 '-Texas Red-CAGAAGTGCCTGGCAG-MGB-3 ' without LNA
2)APOE4 T388C
Upstream primer SEQ No.5:5 '-GGAGGAGACGCGGGCAC-3 '
Downstream primer SEQ No.6:5 '-GCCTGCACCTCGCCG-3 '
Wild-type probe SEQ No.7:5 '-FAM-CGGCCGC+ACACGTCCT-MGB-3 '
Saltant type probe SEQ No.8:5 '-Texas Red-CGGCCGC+GCACGTCCT- MGB-3 '
Wild type modifies probe: 5 '-FAM-CGGCCGCACACGTCCT-MGB-3 ' without LNA
Saltant type modifies probe: 5 '-Texas Red-CGGCCGCGCACGTCCT-MGB-3 ' without LNA
(note :+be right side base be LNA modification).
Table 2-1 PCR reaction system table
Experimental result:
Tri- kinds of genotype Ct Data-Statistics of table 2-2
Note: "-" indicates parting failure without Ct value.
The end point fluorescence value (EPF) of tri- kinds of genotype of table 2-3 counts
From table 2-1, table 2-2 and Figure 13,14 as can be seen that without LNA modification probe groups heterozygote green channel without
Ct value, thus software is determined as that parting fails, and meet the requirements through LNA modification probe groups to the Ct value of three kinds of genotype, it is soft
Part determines that result is correct.
From table 2-3 and Figure 15-16 as can be seen that through LNA modify three kinds of genotype detection fluorescent value ratios of probe groups without
The fluorescent value that LNA modifies probe groups is big, the site APOE2C526T, the EPF of wild homozygote and no mutant homozygote LNA modification group
Value significant difference (P≤0.05) compared with non-LNA modification group, the EPF value and non-LNA modification group of heterozygote LNA modification group
Compared to difference it is extremely significant (P≤0.01)；The site APOE4T388C, wild homozygote and no mutant homozygote LNA modification group
EPF value difference compared with non-LNA modification group is extremely significant (P≤0.01), and the EPF value of heterozygote LNA modification group is repaired with non-LNA
Decorations group compares significant difference (P≤0.05).The experimental results showed that being conducive to probe and target sequence when probe is after LNA is modified
The combination of column improves probe in detecting accuracy.
The test of 3 accuracy of embodiment
This kit is used as amplified sample using mucous membrane of mouth cast-off cells, due to detected personnel's living habit and a
The diversity of body gene order may cause reagent and be interfered in parting, in order to verify the accuracy of this kit parting,
This experiment is tested (the wherein genotype of tester using the mucous membrane of mouth cast-off cells of three genotype difference personnel
Confirmed by PCR sequencing PCR), operating process, total sample number are 300 to operating method referring to Fig.1, agent prescription such as table 3-1.
Table 3-1 PCR reacts total system formula table
Experimental result:
The accuracy of tri- kinds of genotype of table 3-2 counts
It can be seen that two sites APOE2 and APOE4 in respective 300 test samples from table 3-2, this kit
Detection accuracy can achieve 99%.Tentatively show in the case where being tested according to Fig. 1 operating process, reagent tool
There is certain accuracy, can achieve 99%.
Influence test of 4 environment of embodiment to detection kit
Since kit of the present invention is intended to apply to clinical detection, examination is quickly detected in order to verify APOE genotype
Whether the testing result of agent box is influenced by environmental factor, and in routine office work area, (gross area is 200 square metres, altogether respectively for we
50 people are accommodated, do not limit their activity (arbitrarily can walk about or speak), environment temperature is 27 DEG C, humidity 65%) and it is empty
Gas cleaniliness classs is that 1,000,000 grades of toilet is sampled multiple personnel (its genotype is confirmed through PCR sequencing PCR), each
Personnel carry out repeating to test twice respectively in two regions, and agent prescription is referring to table 3-1, testing result statistics such as following table 4-1.
The genotype call results accuracy of two sampling areas of table 4-1 counts
As can be seen from the above results, Office Area and clean area sampling detection accuracy be not significantly different (P >=
0.05), show that environmental factor does not have larger impact to the result of detection.This time test is application convenience of the kit in hospital
Property provides data, to provide strong data supporting in the popularization of hospital and use later.
The detection of 5 reagent anti-interference ability of embodiment
In order to verify the anti-interference ability of APOE genotype quick detection kit, we require known APOE2 and
The person of being sampled of APOE4 genotype proceed as follows, it is specified that they respectively in fasting, eat sweet food, drink alkaline tea, eat acidity
Food, spicy food are drunk after Chinese medicine etc. and to be detected at once, and is compared to testing result, detection method with above-mentioned, with
Upper detection every time is at least repeated more than twice, and testing result is as shown in the table:
Table 5-1 APOE2/APOE4 reagent anti-interference capability testing result
Result above can be seen that be compared with the detection accuracy of fasting sampling, is eaten sweet food, is drunk alkaline tea, eats acid food
Object, spicy food and drink the later accuracy of Chinese medicine and have reduction, illustrate to eat after the above food have to PCR reaction it is certain
Inhibiting effect.Show to eat up larger to the interference of detection after these foods, should be carried out after 30min after eating up these food
It gargles sampling.Since this experiment sampling is limited, inspection should be still sampled after fasting 30min when carrying out clinical detection
It surveys.
6 sensitivity test of embodiment
For the sensitivity for verifying APOE2/E4 genotype quick detection kit, we are with 0.1,0.5,1ng/ μ L concentration
The wild type DNA of APOE2/E4, mutant DNA, heterozygous DNA be that template carries out routine test, each DNA concentration, every kind
Genotype is surveyed 12 times.Its result is as shown in following table 6-1:
Table 6-1 detects the testing result of different sample concentrations with APOE2 and APOE4 genotype quick detection kit
By it is upper the results show that DNA concentration be 0.1ng/ μ L, APOE2 and APOE4 genotype detection kit detected,
Its accuracy rate reaches 100%, therefore may determine that APOE2 and APOE4 genotype quick detection kit of the invention has
Very high sensitivity.
7 kit of embodiment is in room temperature, 2-8 DEG C, -20 DEG C of shelf-stability detections
Normal PCR reagent needs cold chain transportation, -20 DEG C of storages.
This kit can at least place 72h at normal temperature, and 2~8 DEG C can at least place 15 days, and -20 DEG C can at least place 12
A month.
This kit is based on POCT mode development, and the detection of hospital bed side, low-temperature storage and low-temperature operation are difficult to reality
It is existing, so after needing detection reagent to place certain time length at normal temperature, if adverse effect can be generated to testing result.It prepares big
Batch reagent is randomly divided into 3 temperature condition groups totally 24 processing groups, and grouping situation is shown in Table 7-1, wherein control group are as follows: with postponing
Upper machine testing (2-8 DEG C of preparation, the interior sampling loading coded program for completing 108 samples of 30min) immediately, each processing group is total
108 reagents, every kind genotype detection 36.After reagent processing to be done, is operated according to SOP, acquire the person's of being sampled (its
APOE2 and APOE4 genotype is confirmed through PCR sequencing PCR) buccal sample, wild homozygote, no mutant homozygote, heterozygote respectively take
36.
Table 7-1 APOE2/E4 reagent, which places condition and places duration, is grouped situation table
1, to APOE2 reagent: Ct value and end point fluorescence value (EPF) Data-Statistics and t are examined
Tri- kinds of genotyping accuracy statistical forms of table 7-2
By Figure 18-23 it can be seen that the Ct value of different disposal group, EPF value and control group respectively compared with, respectively place item
The time of part, processing is longer, and it is more the case where significant difference occur.When proving room temperature, 2-8 DEG C of placement, -20 DEG C of placements
Between Ct value and EPF value can be had an impact too long.But it can be seen that each 108 number of cases evidence of processing group from table 7-2, A/B/C tri-
1~7 component type accuracy of temperature condition is 100%, when to the 8th group of tri- temperature conditions of A/B, parting mistake occurs
Situation.Illustrate to place reagent at least 7 days under normal temperature condition, will not influence the genotyping result of reagent；Examination is placed under the conditions of 2-8 DEG C
Agent at least 15 days, it will not influence the genotyping result of reagent；It is placed reagent at least 12 months under the conditions of -20 DEG C, will not influence reagent
Genotyping result.In conclusion the APOE2 reagent of provable kit has good stability, carrying out detection in hospital makes
The non-cryogenic environment of used time, of short duration (in room temperature 5 days) are placed, and be will not influence parting accuracy, be can satisfy POCT mould completely
Formula.
2, APOE4 reagent: Ct value and end point fluorescence value (EPF) Data-Statistics and t are examined
Tri- kinds of genotyping accuracy statistical forms of table 7-3
By Figure 24-29 it can be seen that the Ct value of different disposal group, EPF value and control group respectively compared with, respectively place item
The time of part, processing is longer, and it is more the case where significant difference occur.When proving room temperature, 2-8 DEG C of placement, -20 DEG C of placements
Between Ct value and EPF value can be had an impact too long.But it can be seen that each 108 number of cases evidence of processing group from table 7-3, A/B/C tri-
1~7 component type accuracy of temperature condition is 100%, when to the 8th group of tri- temperature conditions of A/B/C, parting mistake occurs
Situation.Illustrate to place reagent at least 7 days under normal temperature condition, will not influence the genotyping result of reagent；Examination is placed under the conditions of 2-8 DEG C
Agent at least 15 days, it will not influence the genotyping result of reagent；It is placed reagent at least 12 months under the conditions of -20 DEG C, will not influence examination
The genotyping result of agent.In conclusion the APOE4 reagent of provable kit has good stability, detected in hospital
In use, the non-cryogenic environment of of short duration (in room temperature 5 days) is placed, it will not influence parting accuracy, can satisfy POCT mould completely
Formula.
Embodiment 8 is used to detect the optimization of the primer and probe of APOE2 and APOE4 genotype
The reagent of different systems is prepared according to table 8-1 system using primer and probe, carries out the survey of primer and probe concentration
Examination, using the mucous membrane of mouth cast-off cells of three kinds of genotype as amplified sample, every kind of genotype tests three weights for every group of test
It is multiple, observe parting stability.
Table 8-1 primed probe concentration determines that experiment PCR reacts total system formula table
Experimental result: (1) primer concentration grads experiment (concentration one, concentration two, concentration three)
A.Ct Data-Statistics are as follows
Table 8-2 APOE2 Ct Data-Statistics table
Table 8-3 APOE4 Ct Data-Statistics table
B.EPF Data-Statistics are as follows:
Tri- kinds of genotype end point fluorescence value (EPF) statistical forms of table 8-4 APOE2
Tri- kinds of genotype end point fluorescence value (EPF) statistical forms of table 8-5 APOE4
B. the ratio (G/Rratio) of the green tunnel end points fluorescent value of heterozygous and red tunnel end points fluorescent value
It counts as follows:
Table 8-6 APOE2 heterozygous G/Rratio
| Final concentration | G/Rratio (heterozygous) |
| Concentration one | 0.89 |
| Concentration two | 0.89 |
| Concentration three | 0.87 |
Table 8-7 APOE4 heterozygous G/Rratio
| Final concentration | G/Rratio (heterozygous) |
| Concentration one | 0.85 |
| Concentration two | 0.92 |
| Concentration three | 0.88 |
(2) concentration and probe concentration gradient experiment
In view of heterozygous G/Rratio as far as possible close to 1 (heterozygous G/Rratio closer to 1, heterozygosis parting mistake
Chance it is lower), therefore concentration and probe concentration is adjusted.
A.Ct Data-Statistics such as following table
Table 8-8 APOE2 Ct Data-Statistics table
Table 8-9 APOE4 Ct Data-Statistics table
B. end point fluorescence value (EPF) statistics is as follows:
Tri- kinds of genotype end point fluorescence value (EPF) statistical forms of table 8-10 APOE2
Tri- kinds of genotype end point fluorescence value (EPF) statistical forms of table 8-11 APOE4
C. the ratio (G/Rratio) of the green tunnel end points fluorescent value of heterozygous and red tunnel end points fluorescent value statistics is as follows:
Table 8-12 APOE2 heterozygous G/Rratio
| Final concentration | G/R ratio (heterozygous) |
| Concentration four | 1.05 |
| Concentration five | 1.32 |
8-13 APOE4 heterozygous G/Rratio
| Final concentration | G/R ratio (heterozygous) |
| Concentration four | 1.08 |
| Concentration five | 1.28 |
By three concentration gradients of primer prepare reagent C t value it was found that, concentration one and two Ct value difference of concentration it is different
Not significant (P > 0.05), three Ct value of concentration are noticeably greater than the Ct value (p is less than or equal to 0.05) of concentration one and concentration two, illustrate phase
For concentration three, concentration two and one primer amplification of concentration are more efficient, so reagent detection sensitivity is more under the system
It is high.
It can be seen that the end point fluorescence value of concentration two is significantly high by the reagent end point fluorescence value that three concentration gradients configure
In the end point fluorescence value of concentration one and three (p is less than or equal to 0.05).Illustrate under same probe concentration conditions, concentration two expands
May participate in PCR reaction effective product amount highest.Correctly matched amount is more for probe, and reagent is also more stable.
It can be seen that by green red tunnel end points fluorescent value ratio (G/R ratio), the green red tunnel end points fluorescent value of concentration two
Ratio (G/Rratio) is closer to 1, as a result more acurrate reliable when illustrating to detect heterozygote genotype.It is dense by probe two
Spend gradient prepare reagent end point fluorescence value and green red channel fluorescence ratio (G/Rratio) it was found that, the terminal of concentration four
Fluorescent value is significantly higher than the end point fluorescence value (p is less than or equal to 0.05) of concentration five, the green red channel fluorescence ratio (G/ of concentration four
Rratio) closer to 1, kit sensitivity under the concentration is higher, and testing result is more acurrate.It can be seen that APOE genotype
Reaction system high sensitivity used in quick detection kit, stability is good, as a result accurately and reliably.
Sequence table
<110>Chongqing Jing Yin biotechnology Co., Ltd
<120>APOE2 the and APOE4 genotype quick detection kit based on POCT mode
<130> KHP181110932.6
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
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<213>artificial sequence (Artificial Sequence)
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cgtaagcggc tcctccg 17
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<213>artificial sequence (Artificial Sequence)
<400> 2
ggatggcgct gaggcc 16
<210> 3
<211> 15
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<213>artificial sequence (Artificial Sequence)
<400> 3
tgcagaagcg cctgg 15
<210> 4
<211> 16
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<213>artificial sequence (Artificial Sequence)
<400> 4
cagaagtgcc tggcag 16
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<213>artificial sequence (Artificial Sequence)
<400> 5
ggaggagacg cgggcac 17
<210> 6
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<213>artificial sequence (Artificial Sequence)
<400> 6
gcctgcacct cgccg 15
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<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
cggccgcaca cgtcct 16
<210> 8
<211> 16
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
cggccgcgca cgtcct 16
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<213>artificial sequence (Artificial Sequence)
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tgcagaagcg cctgg 15
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cagaagtgcc tggcag 16
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cggccgcaca cgtcct 16
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cggccgcgca cgtcct 16
Claims (10)
1. a kind of primer combination for detecting APOE2 and APOE4 gene pleiomorphism, which is characterized in that detection APOE2 gene pleiomorphism
Primer pair nucleotide sequence are as follows:
Upstream primer: 5 '-CGTAAGCGGCTCCTCCG-3 '
Downstream primer: 5 '-GGATGGCGCTGAGGCC-3 '；
Detect the nucleotide sequence of the primer pair of APOE4 gene pleiomorphism are as follows:
Upstream primer: 5 '-GGAGGAGACGCGGGCAC-3 '
Downstream primer: 5 '-GCCTGCACCTCGCCG-3 '.
2. with the matching used probe combinations of primer pair described in claim 1, which is characterized in that the probe combinations are as follows:
APOE2 gene wild-type probe: 5 '-TGCAGAAGCGCCTGG-3 '
APOE2 genic mutation type probe: 5 '-CAGAAGTGCCTGGCAG-3 '
APOE4 gene wild-type probe: 5 '-CGGCCGCACACGTCCT-3 '
APOE4 genic mutation type probe: 5 '-CGGCCGCGCACGTCCT-3 '.
3. probe combinations according to claim 2, which is characterized in that the probe combinations include:
APOE2 gene wild-type probe: 5 '-TGCAGAAG+CGCCTGG-3 '
APOE2 genic mutation type probe: 5 '-CAGAAG+TGCCTGGCAG-3 '
APOE4 gene wild-type probe: 5 '-CGGCCGC+ACACGTCCT-3 '
APOE4 genic mutation type probe: 5 '-CGGCCGC+GCACGTCCT-3 ',
Wherein, "+" indicates the base on right side for LNA modification.
4. detection reagent or kit containing probe combinations described in primer described in claim 1 and Claims 2 or 3.
5. a kind of PCR reaction solution for detecting APOE2 and APOE4 gene pleiomorphism, which is characterized in that the PCR reaction solution includes
Primer described in claim 1 and probe combinations described in claim 2 or 3.
6. PCR reaction solution according to claim 5, which is characterized in that the PCR reaction solution further include archaeal dna polymerase,
DNTPs, buffer, MgCl2And cell pyrolysis liquid.
7. PCR reaction solution according to claim 6, which is characterized in that the final concentration of each component in the PCR reaction solution
Are as follows: 0.5-1.5 times of PCR reaction buffer, archaeal dna polymerase 0.05-0.1U/ μ L, dNTPs 0.1-0.5mM, upstream primer
0.2-0.7 μM, 0.2-0.7 μM of downstream primer, 0.2-0.7 μM of wild-type probe, 0.2-0.7 μM of saltant type probe, Mg2+1-
2.5mM and cell pyrolysis liquid；
The cell pyrolysis liquid is made of lauryl sodium sulfate and Triton X-100；
Wherein, the concentration of lauryl sodium sulfate is 0.0005-0.015% (w/v)；The concentration of Triton X-100 is
0.001-0.03% (w/v).
8. PCR reaction solution according to claim 7, which is characterized in that the final concentration of each component in the PCR reaction system
Are as follows: 1.1 × PCR reaction buffer, archaeal dna polymerase 0.05U/ μ L, dNTPs 0.2mM, 0.4 μM of upstream primer, downstream primer 0.4
μM, 0.3 μM of wild-type probe, 0.4 μM of saltant type probe, Mg2+2.5mM and cell pyrolysis liquid.
9. APOE2 the and APOE4 genotype quick detection kit based on POCT mode, which is characterized in that the kit contains
It has the right to require any PCR reaction solution of 6-8.
10. kit according to claim 9, which is characterized in that when the kit carries out quantitative fluorescent PCR, work
Program are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 8s, 62 DEG C of annealing and extension 35s, recycle 50 times.
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