CN108070636A - A kind of processing method and kit of fluorescent PCR amplified sample - Google Patents
A kind of processing method and kit of fluorescent PCR amplified sample Download PDFInfo
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Abstract
The present invention provides a kind of processing method and kit of fluorescent PCR amplified sample, including original material and sample treatment solution, the sample treatment solution is mixed with the original material, processing product is obtained after mixing, the processing product is directly used in fluorescent PCR detection, the original material be blood sample or mouth epithelial cells sample, wherein, contain following component in the sample treatment solution：Tris HCl, NaOH, Trixon X 100, sodium diethyldithiocarbamate, sodium lauryl sarcosinate, NP 40, DMSO, glycine betaine, BSA, sample treatment solution is included in kit, the present invention overcomes existing conventional method when carrying out fluorescent PCR detection, it needs to carry out DNA extraction processs in advance, so as to cause DNA loss amounts it is big, cumbersome the problems such as, it is detected suitable for the fluorescent PCR for various purposes of the trace samples such as blood, mouth epithelial cells, convenient for promoting.
Description
Technical field
The invention belongs to biomedical sector, more particularly, to a kind of processing method and reagent of fluorescent PCR amplified sample
Box.
Background technology
In recent years, fluorescent PCR with it accurately and fast, the characteristics of facilitating, in clinical and inspection and quarantine every field
It has been more and more widely used.Particularly to infectious diseases and Analysis of Genetic Background, fluorescent PCR as it is a kind of accurately and reliably
Detection method, have become the important technical of molecule diagnosis, clinical examination, clinical treatment etc..Human genome
DNA is a kind of important analysis sample in clinical fluorescent PCR detection.And as the blood group in genomic DNA primary biological source
It knits or oral epithelium tissue, due to containing substantial amounts of PCR mortifiers, such as ferroheme, anti-coagulants and various foreign proteins etc., because
This needs first extracts purification process to genomic DNA.Conventional genome DNA extracting method has at present：Phenol-chloroform extracts
Method, post separation extraction method and Beads enrichment extraction method.These methods have simultaneously for the purpose of the DNA for obtaining higher degree
The drawbacks such as big, cumbersome, the easy cross contamination of DNA loss amounts tend not to meet high-throughput in some clinics or micro sample
This detection demand at present to solve the problems, such as these, there is many improved methods, be on the one hand by change PCR reaction conditions or
Change archaeal dna polymerase used in PCR, realize that fluorescent PCR expands by enhancing PCR system to the tolerance of mortifier,
Although these methods need not move through the extraction process of DNA, can complete fluorescent PCR detection, but these methods are that PCR is reacted
The improvement of system in practical application, can not coordinate existing commercialization fluorescence PCR detection reagent kit to be used in combination, therefore unfavorable
In promoting the use of, on the other hand, it is the improvement to existing extraction genomic DNA method, reaches quick, high-throughput extraction genome
The purpose of DNA, these methods have reached the mesh of rapid extraction genomic DNA by the improvement of reagent and simplifying for operating procedure
, but extracts reagent used can influence the effect of fluorescent PCR, therefore the multi-pass operations such as still need to centrifuge, shift and could complete,
It operates relative complex.
The content of the invention
An object of the present invention be to provide it is a kind of it is easy to operate, DNA loss amounts are small, can be combined, be convenient for kit
The processing method of the fluorescent PCR amplified sample of popularization.
The second object of the present invention is to provide a kind of kit that can quickly handle initial sample, obtain product and can use
It is detected in the fluorescent PCR of various purposes.
In order to solve the above technical problems, the technical solution adopted by the present invention is：A kind of processing side of fluorescent PCR amplified sample
Method including original material and sample treatment solution, the sample treatment solution with the original material is mixed, is handled after mixing
Product, the processing product are directly used in fluorescent PCR detection, and the original material is blood sample or mouth epithelial cells sample
This.
Further, the sample treatment solution contains following component：Tris-HCl, 10~50mM；NaOH, 1~10mM；
Trixon x-100,0.1%~0.5%；Sodium diethyldithiocarbamate, 0.05%~0.1%；Cocoyl sarcosine
Sodium, 0.01%~0.05%；NP-4,0 0.1%~0.5%；DMSO, 1%~10%；Glycine betaine, 1~5M；BSA, 2~8 μ g/
μ L, wherein Trixon X-100 and NP40 are non-ionic detergent, sodium diethyldithiocarbamate and cocoyl sarcosine
Sodium is anion denaturant, by reasonably preparing, reaches mild lytic conditions, promotes releasing for DNA in sample cell
The function of archaeal dna polymerase is not destroyed while putting, DMSO, glycine betaine and BSA is added in, is on the one hand played by reducing annealing temperature
Enhance the effect of PCR, on the one hand by protecting archaeal dna polymerase, improve the resistance to suppressed influences of PCR so that fluorescent PCR can be directly
Using blood sample or mouth epithelial cells sample as template, efficiently expand with special fluorescent PCR, Tris-HCl is buffering
Solution provides stable pH environment for treatment fluid, and NaOH can both play the purpose of cell lysis, while high ph-values environment can make
The mortifiers such as ferroheme are denatured, and help to enhance tolerance of the PCR system to mortifier.
Further, the blood sample be fresh blood, EDTA anticoagulations, blood card or with blood filter paper.
Further, specifically, the mouth epithelial cells sample is saliva or buccal swab.
Further, the fluorescent PCR detection includes dye method and sonde method.
A kind of fluorescent PCR amplified sample kit is further included, kit includes the processing method of fluorescent PCR amplified sample
In sample treatment solution.
Compared with prior art, the present invention has the advantage that is with advantageous effect：
1st, the invention belongs to the improvement to blood sample or mouth epithelial cells sample preprocessing method, therefore not to fluorescence
The ingredient of PCR reaction solution and the species of archaeal dna polymerase are limited, applied widely, can be with the fluorescent PCR reagent of each purposes
Box is used cooperatively；
2nd, original material and sample treatment solution are directly sufficiently mixed by the present invention, and the processing product obtained after mixing is directly used
In the detection of fluorescent PCR, process is isolated and purified without DNA, reduces the loss of pilot process.
Description of the drawings
Fig. 1 is IL28b gene by fluorescence quantitative PCR system canonical plottings, for sample process side provided by the present invention
Method product in DNA quantified.
Fig. 2 is that same volume blood sample extraction effect compares figure, for sample processing method provided by the invention with special
The comparison of the poba gene group DNA extraction kit extraction effect of door.
Fig. 3 is the knot that sample processing method provided by the invention detects IL28b gene rs12979860 loci polymorphisms
Fruit schematic diagram.(a) detected for pure and mild IL28b genes rs12979860 loci polymorphisms；(b) for heterozygosis IL28b genes
Rs12979860 loci polymorphisms detect.
Fig. 4 is that sample processing method provided by the invention is more to 20 blood sample IL28b gene rs12979860 sites
State property testing result.
Fig. 5 is sample processing method provided by the invention to 12 blood sample ApoE genetic polymorphism detection results.(a)
ApoE gene rs429358 loci polymorphisms are detected；(b) ApoE gene rs7412 loci polymorphisms are detected.
Fig. 6 is different blood ratios results contrast figures, for evaluating sample processing method provided by the invention to different blood
The treatment effect of liquid sample dosage compares；
Fig. 7 is sample processing method provided by the invention, and to the filter paper sample with blood and buccal swab sample, that treated is glimmering
Light PCR schemes；
Specific embodiment
It elaborates below in conjunction with the accompanying drawings to the specific embodiment of the present invention.
Embodiment 1：Method using the present invention handles blood sample
(1) experiment material：EDTA anticoagulations
(2) experimental method：
A. by after EDTA anticoagulations gently mixing, 1~10 μ L is taken to be placed in 1.5mL centrifuge tube tube bottom；
B. 200 μ L sample treatment solutions are added in；
C. it is vortexed after concussion mixing, of short duration centrifugation；
D. centrifuge tube shelf is placed in, it is spare.
Embodiment 2：Treatment effect evaluation is carried out to the blood sample handled using the method for the present invention
(1) experiment material：20 parts of EDTA anticoagulations
(2) experimental method：
A.20 part EDTA anticoagulations sample respectively takes 200 μ L, and base is extracted using domestic poba gene group DNA extraction kit
Because of a group DNA, and pass through spectrophotometric determination DNA concentration；
B. identical 20EDTA anticoagulation samples, respectively take 5 μ L, are mixed respectively with 200 μ L sample treatment solutions provided by the present invention
It closes；
C. take kit extract genomic DNA and the method for the present invention processing after each 2 μ L of sample, be separately added into PCR pipe；
D. IL28b Fluorescence PCR liquid is added in, wherein reaction solution includes the following component of following concentration or content：10mM
Tris-HCl (pH8.3), 50mM KCl, 1.5mM MgCl2, 200 μM of dNTPs, 200nM upstream and downstream primers, 200nM probes,
0.5U Taq archaeal dna polymerases.
E. augmentation detection is carried out in the fluorescent PCR instrument of model ABI 7500, the fluorescent PCR amplification program is as follows：
The first step：60 DEG C, 30s；Xun Huan 1 time collects fluorescence；
Second step：95 DEG C, 2min；Xun Huan 1 time；
3rd step：95 DEG C, 15s；60 DEG C, 1min；Xun Huan 40 times collects fluorescence；
4th step：60 DEG C, 30s；Xun Huan 1 time collects fluorescence.
(3) experimental result：
Using sample treatment solution provided by the invention, extracted with common domestic poba gene group DNA extraction kit
Sample compares, and is detected by fluorescent PCR, and sample process effect is evaluated.It is detected by spectrophotometer, domestic blood
The DNA of genome DNA extracting reagent kit extraction, 200ul blood samples extract total amount between 2~6ug, unit sample amount
DNA yield is 10ng~30ng/ul blood.Due to the processing method provided by the present invention, final product is the crude extract containing DNA,
It is not suitable for directly detecting by spectrophotometer, thus it is almost quantitative by the standard curve progress of quantitative fluorescent PCR.As Fig. 1,
Shown in Fig. 2, IL28b quantitative fluorescent PCRs system used is linearly 0.999, and efficiency 98.137% passes through standard curve and state
The DNA concentration of production poba gene group DNA extraction kit extraction converts, sample processing method provided by the invention, 5ul blood
For DNA total amounts between 0.2~1.2ug, the DNA yield of unit sample amount is 100ng~240ng/ul blood after liquid sample process.It says
Bright sample processing method provided by the invention is compared with special poba gene group DNA extraction kit, the DNA of unit sample amount
Yield higher.
Embodiment 3：Detection to IL28b gene rs12979860 loci polymorphisms
(1) experiment material：20 parts of EDTA anticoagulations
(2) experimental method：
A.20 part EDTA anticoagulations sample respectively takes 5 μ L, is mixed respectively with 200 μ L sample treatment solutions provided by the present invention；
B. each 2 μ L of sample after handling are taken, add in PCR pipe；
C. IL28b Fluorescence PCR liquid is added in, wherein reaction solution includes the following component of following concentration or content：10mM
Tris-HCl (pH8.3), 50mM KCl, 1.5mM MgCl2, 200 μM of dNTPs, 200nM upstream and downstream primers, the double probes of 200nM
(c-type and T-shaped), 0.5U Taq archaeal dna polymerases.
D. augmentation detection is carried out in the fluorescent PCR instrument of model ABI 7500, the fluorescent PCR amplification program is as follows：
The first step：60 DEG C, 30s；Xun Huan 1 time collects fluorescence；
Second step：95 DEG C, 2min；Xun Huan 1 time；
3rd step：95 DEG C, 15s；60 DEG C, 1min；Xun Huan 40 times collects fluorescence；
4th step：60 DEG C, 30s；Xun Huan 1 time collects fluorescence.
(3) experimental result：
Using sample treatment solution provided by the invention, cooperation conventional fluorescent PCR allelic gene typings reagent (sonde method) is right
In the human blood sample of 20 parts of separate sources IL28b genes rs12979860 sites carry out fluorescent PCR detection, as a result as Fig. 3,
Shown in Fig. 4, the background signal of all reacting holes is good, and for Ct values between 28~31, amplification curve is S-type, different parting amplifications
Curve resolution is clear, and genotypic results are accurate, illustrates that sample processing method provided by the invention can be used for conventional fluorescent PCR
Detection and analysis.
Embodiment 4：Detection to ApoE gene rs429358 sites and rs7412 loci polymorphisms
(1) experiment material：20 parts of EDTA anticoagulations
(2) experimental method：
A.20 part EDTA anticoagulations sample respectively takes 5 μ L, is mixed respectively with 200 μ L sample treatment solutions provided by the present invention；
B. each 2 μ L of sample after handling are taken, add in PCR pipe；
C. ApoE Fluorescence PCR liquid is added in, wherein reaction solution includes the following component of following concentration or content：10mM
Tris-HCl (pH8.3), 50mM KCl, 1.5mM MgCl2, 200 μM of dNTPs, 200nM upstream and downstream primers, the double probes of 200nM,
0.5U Taq archaeal dna polymerases.
D. augmentation detection is carried out in the fluorescent PCR instrument of model ABI 7500.The fluorescent PCR amplification program is as follows：
The first step：60 DEG C, 30s；Xun Huan 1 time collects fluorescence；
Second step：95 DEG C, 2min；Xun Huan 1 time；
3rd step：95 DEG C, 15s；60 DEG C, 1min；Xun Huan 40 times collects fluorescence；
4th step：60 DEG C, 30s；Xun Huan 1 time collects fluorescence.
(3) experimental result：
Using sample treatment solution provided by the invention, cooperation conventional fluorescent PCR allelic gene typings reagent (sonde method) is right
Rs429358 the and rs7412 sites of ApoE genes carry out fluorescent PCR detection in the human blood sample of 20 parts of separate sources.As a result
As shown in figure 5, all reacting hole genotypic results are accurate.Illustrate sample processing method provided by the invention for high GC content
The fluorescent PCR of (more than 80%) gene is still effective, it was demonstrated that the popularity of the sample processing method scope of application.
Embodiment 5：The treatment effect of different blood sample dosages compares
(1) experiment material：EDTA anticoagulations
(2) experimental method：
A. gradient proportion mixing, EDTA anticoagulations are carried out to EDTA anticoagulations sample with sample treatment solution provided by the present invention
Finally ratio is respectively：50%, 25%, 12.5%, 6.3%, 3.1%, 1.6%, 0.8%, 0.4%, 0.2%, 0.1%；
B. each 2 μ L of sample after mixing are taken, add in PCR pipe；
C. Fluorescence PCR liquid is added in, wherein reaction solution includes the following component of following concentration or content：10mM Tris-
HCl (pH8.3), 50mM KCl, 1.5mM MgCl2, 200 μM of dNTPs, 200nM upstream and downstream primers, 200nM probes, 0.5U
Taq archaeal dna polymerases.
D. augmentation detection is carried out in the fluorescent PCR instrument of model ABI 7500.The fluorescent PCR amplification program is as follows：
The first step：60 DEG C, 30s；Xun Huan 1 time collects fluorescence；
Second step：95 DEG C, 2min；Xun Huan 1 time；
3rd step：95 DEG C, 15s；60 DEG C, 1min；Xun Huan 40 times collects fluorescence；
4th step：60 DEG C, 30s；Xun Huan 1 time collects fluorescence.
(3) experimental result：
Use sample treatment solution provided by the invention, cooperation conventional fluorescent PCR reagent (sonde method), to different sample sizes
IL28b genes carry out fluorescent PCR detection in human blood sample.As shown in fig. 6, when concentration of specimens is 0.4%~6.3%, institute
The background signal for having reacting hole is good, and for Ct values between 28~31, amplification curve is S-type, therefore, at sample provided by the invention
In reason method, it is only necessary to which micro blood sample (0.1 μ L~10 μ L) can carry out fluorescent PCR detection.
Embodiment 6：The treatment effect of different sample types compares
(1) experiment material：Filter paper, buccal swab with blood
(2) experimental method：
A. filter paper of the fritter with blood is taken, is placed in 1.5mL centrifuge tubes, adds in 200 μ L of sample treatment solution provided by the present invention,
4 DEG C of placement more than 2h, vortex mixing, of short duration centrifugation；
B. the head of buccal swab is put into 1.5mL centrifuge tubes, adds in 200 μ of sample treatment solution provided by the present invention
L, vortex mixing, of short duration centrifugation；
C. each 2 μ L of sample after mixing are taken, add in PCR pipe；
D. Fluorescence PCR liquid is added in, wherein reaction solution includes the following component of following concentration or content：10mM Tris-
HCl (pH8.3), 50mM KCl, 1.5mM MgCl2, 200 μM of dNTPs, 200nM upstream and downstream primers, 200nM probes, 0.5U
Taq archaeal dna polymerases.
E. augmentation detection is carried out in the fluorescent PCR instrument of model ABI 7500, the fluorescent PCR amplification program is as follows：
The first step：60 DEG C, 30s；Xun Huan 1 time collects fluorescence；
Second step：95 DEG C, 2min；Xun Huan 1 time；
3rd step：95 DEG C, 15s；60 DEG C, 1min；Xun Huan 40 times collects fluorescence；
4th step：60 DEG C, 30s；Xun Huan 1 time collects fluorescence.
(3) experimental result：
Use sample treatment solution provided by the invention, cooperation conventional fluorescent PCR reagent (sonde method), to different types of sample
IL28b genes carry out fluorescent PCR detection in this.As shown in fig. 7, when sample type is the filter paper with blood, buccal swab, own
The background signal of reacting hole is good, and for Ct values between 28~31, amplification curve is S-type, illustrates sample process provided by the invention
Method is suitable for polytype sample.
One embodiment of the present of invention is described in detail above, but the content is only the preferable implementation of the present invention
Example, it is impossible to be construed as limiting the practical range of the present invention.All all the changes and improvements made according to the present patent application scope
Deng, should all still belong to the present invention patent covering scope within.
Claims (6)
1. a kind of processing method of fluorescent PCR amplified sample, it is characterised in that：Including original material and sample treatment solution, by institute
It states sample treatment solution to mix with the original material, processing product is obtained after mixing, the processing product is directly used in fluorescent PCR
Detection, the original material are blood sample or mouth epithelial cells sample.
2. a kind of processing method of fluorescent PCR amplified sample according to claim 1, it is characterised in that：At the sample
Reason liquid contains following component：
Tris-HCl, 10~50mM；
NaOH, 1~10mM；
Trixon x-100,0.1%~0.5%；
Sodium diethyldithiocarbamate, 0.05%~0.1%；
Sodium lauryl sarcosinate, 0.01%~0.05%；
NP-4,0 0.1%~0.5%；
DMSO, 1%~10%；
Glycine betaine, 1~5M；
BSA, 2~8 μ g/ μ L.
3. a kind of processing method of fluorescent PCR amplified sample according to claim 1, it is characterised in that：The blood sample
This is fresh blood, EDTA anticoagulations, blood card or with blood filter paper.
4. a kind of processing method of fluorescent PCR amplified sample according to claim 1, it is characterised in that：It is specifically, described
Mouth epithelial cells sample is saliva or buccal swab.
5. a kind of processing method of fluorescent PCR amplified sample according to claim 1, it is characterised in that：The fluorescent PCR
Detection includes dye method and sonde method.
6. a kind of fluorescent PCR amplified sample kit, it is characterised in that：Expand including any fluorescent PCRs of claim 1-5
Increase the sample treatment solution in the processing method of sample.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112662746A (en) * | 2019-10-15 | 2021-04-16 | 杭州百迈生物股份有限公司 | Quality control product for genotyping detection and preparation method thereof |
| CN112662743A (en) * | 2019-10-15 | 2021-04-16 | 杭州百迈生物股份有限公司 | Fluorescent PCR method, kit and reagent |
| CN112921075A (en) * | 2021-03-31 | 2021-06-08 | 武汉友芝友医疗科技股份有限公司 | Hand-taking-free reagent for blood sample fluorescence PCR and application thereof |
| CN113106148A (en) * | 2021-03-31 | 2021-07-13 | 湖南菲思特精准医疗科技有限公司 | Clopidogrel dose-related gene polymorphism detection kit and detection method and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112662746A (en) * | 2019-10-15 | 2021-04-16 | 杭州百迈生物股份有限公司 | Quality control product for genotyping detection and preparation method thereof |
| CN112662743A (en) * | 2019-10-15 | 2021-04-16 | 杭州百迈生物股份有限公司 | Fluorescent PCR method, kit and reagent |
| CN112662746B (en) * | 2019-10-15 | 2023-04-11 | 杭州百迈生物股份有限公司 | Quality control product for genotyping detection and preparation method thereof |
| CN112921075A (en) * | 2021-03-31 | 2021-06-08 | 武汉友芝友医疗科技股份有限公司 | Hand-taking-free reagent for blood sample fluorescence PCR and application thereof |
| CN113106148A (en) * | 2021-03-31 | 2021-07-13 | 湖南菲思特精准医疗科技有限公司 | Clopidogrel dose-related gene polymorphism detection kit and detection method and application thereof |
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