CN104830842B - The primer sets and immue quantitative detection reagent box of human peripheral Circulating DNA - Google Patents
The primer sets and immue quantitative detection reagent box of human peripheral Circulating DNA Download PDFInfo
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Abstract
The present invention relates to the primer sets and immue quantitative detection reagent box of human peripheral Circulating DNA, primer sets are formed by five groups for expanding the specific primer of Alu.The planning standard product of primer sets are the plasmid purification of the conserved genetic sequences containing Alu, Alu objective gene sequences are connected on plasmid by standard items by technique for gene engineering, by identifying, expanding, extracting and purifying acquisition, and absorbance reading is carried out by 260nm, it carries out the DNA that densimeter point counting analysis obtains and accurately copies number, and pass through the standard items that dilution is prepared into various concentration.The present invention is a kind of molecular biology method based on quantitative nucleic acid amplification, real-time adjustment, by using the different fragments of the conservative gene Alu of Circulating DNA in the primer amplification human peripheral of specificity, quantitative detection carries out Circulating DNA in human peripheral by the fluorescence signal for detecting release.
Description
Technical field
The present invention relates to biotechnologys, are a kind of molecular biology methods based on quantitative nucleic acid amplification, more particularly in fact
When quantitative polumerase chain reaction (real-time PCR), by using Circulating DNA in the primer amplification human peripheral of specificity
The different fragments of conservative gene Alu carry out Circulating DNA in human peripheral quantitative detection by the fluorescence signal for detecting release.
Background technology
It is that one kind is present in that Circulating DNA (circulating DNA), which is also known as dissociative DNA (cell-free DNA, cf-DNA),
The extracellular dna of acellular state in the body fluid of animals and plants and people.1977, Leon etc. had found tumor patient blood plasma DNA first
Content significantly increases, it is proposed that effect (Leon of the monitoring peripheral blood dissociative DNA level in terms of observation curative effect, prediction recurrence
SA, Shapino B, Skaaroff DM, et al.Free DNA in the serum of cancer patients and
The effect of therapy [J] .Cancer Res.1977,37 (3)：646)." Suzuki N, Kamataki A,
Yamaki J, et al.Characterization of circulating DNA in healthy human
Plasma.Clin Chim Acta, 2008,387 (1-2)：55-58 " about exists research shows that Healthy People Circulating DNA content is atomic
5ug/L or so.Document " Mulcahy HE, Lyautey J, Lederrey C, et al.A prospective study of
K-ras gene mutation in the plasma of pancreatic cancer patients[J].Clin
Cancer Res, 1998,4 (2)：271”、“GeorgDoris Auer, Inge Gaugg, et al.Prognostic
significance of methylated RASSF1A and PITX2 genes in blood-and bone marrow
Plasma of breast cancer patients [J] .Breast Cancer Res Treat, 2011,130 (8)：109-
117 " and " Chan SL, Chan AK, Hui EP, et al.Quantitation of circulating methylated
RASSFlA in prognostication and monitoring of treatment response in
Unresectable hepatocellular carcinoma fHCC) .J Clin Onc01,2011,29 (suppl)：abstr
4058 " record, when body is in tumour, autoimmune disease and inflammatory reaction, the content meeting of body-internal-circulation DNA
It accordingly increases, but its mechanism generated is not still very clear and definite so far.The Serologic detection of Circulating DNA can't be used directly at present
Judge in the early diagnosis of disease and curative effect, but the generation of it and some diseases has close contact, therefore detect with development
Circulating DNA is simultaneously determined the specific gene of certain known diseases with important clinical meaning, and Circulating DNA would be possible to
The novel molecular marker judged as the disease early diagnosis for the prospect that has much and curative effect.
At present, the more quantitative detecting method of domestic and international application can be divided into two major class：First, with polymerase chain reaction (PCR)
Based on amplification method, such as methylation status of PTEN promoter method (methylation specific PCR, MSP) and real-time fluorescence
Quantitative PCR method (real-time fluorescence quantitative PCR, RTFQ-PCR)；Second is that signal amplification technique
Detection method, such as branch chain DNA (branched DNA, bDNA) detection technique.The research of Circulating DNA concentration is studied in past few years
Person is mostly using the analysis method based on quantitative PCR, such as " Ellinger J, M ü iller DC, M ü ller SC, et
al.Circulating mitochondrial DNA in serum：A universal diagnostic biomarker
For patients with urological malignancies.Urol Oncol, 2012,30 (4)：509-515 " and
" Huang Z, Hua D, Hu Y, etal.Quantitation of plasma circulating DNA using
quantitative PCR for the detection of hepatocellular carcinoma.Pathol Oncol
Res, 2012,18 (2)：271-276 ", the real-time PCR method of high sensitivity can be by the DNA signals of specific amplification only to measure
Condition is provided, the DNA of pik (pg) grade can be detected.It certainly can be more there are also improved technology or use in conjunction technology
The content of Circulating DNA is accurately measured, but there is no unified standard.
With the rapid development of molecular biology, molecule inspection technology has become clinical testing techniques and pathological diagnosis technology
In most popular technical method, for example Real-time round pcrs are in hepatitis B, hepatitis and human papilloma virus (HPV) and breast
Application in gland cancer, lung cancer provides solid theoretical foundation for individuation diagnosis and treatment project.
Invention content
The present invention using molecular biology Real-time round pcrs, based on Alu can be as human DNA segment
(Alu is a kind of moderately repetitive sequence in human genome to the gene order feature of specific marker, is about 300bp, about flat
Just there is 1 Alu sequence every 6kb or so, content is most abundant in mammal, homology highest, therefore it can conduct
The specific marker of human DNA segment), devise the primers of Alu gene different length products, therefrom choose specificity it is high 5
To the primer of upstream and downstream Real-time PCR, the Alu segments of different length are expanded using the method for quantitative fluorescent PCR, simultaneously
The standard items of Alu are established, the copy number for detecting different Alu segments can be quantified, so as to reflect the quantity of human circulation DNA,
This method has high specific, hypersensitivity, can establish good, unified molecule quality control standard, can make up different inspections
The shortcomings of lacking comparativity between survey result, be a good Circulating DNA detection method.
First purpose of the present invention is the shortcomings of overcoming the standardization of existing detection method and not perfect Quality Control means.
Second object of the present invention is to provide the conserved sequence of Alu as detection human peripheral Circulating DNA.
Third object of the present invention is to provide the Alu high specifics primer and Alu standards of 5 pairs of different length PCR products
Product.
Fourth object of the present invention is to provide the kit that a kind of human peripheral Circulating DNA of simplicity quantitatively detects.
Technical scheme is as follows：
One group of specific primer for being used to expand Alu, respectively for the different length segment of Alu conserved sequences.5 pairs of primers
It is Alu1-F (upstream), R (downstream) respectively, Alu2-F (upstream), R (downstream), Alu3-F (upstream), R (downstream), Alu4-F
(upstream), R (downstream), Alu5-F (upstream), R (downstream).The gene order of Alu is provided, as the target dna fragment of detection, tool
There is the conservative of height, while the nucleic acid sequence of 5 pairs of primers is provided.
The primer sets of human peripheral Circulating DNA are formed by five Duis for expanding the specific primer of Alu, respectively for Alu
The different length segment of conserved sequence；Primer is respectively：
First couple of sense primer Alu1-F：5 '-aga cca tcc tgg cta aca cg-3 ',
First couple of downstream primer Alu1-R：5’-aga cgg agt ctc gct ctg tc-3’；
Second couple of sense primer Alu2-F：5 '-cta aca cgg tga aac ccc g-3 ',
Second couple of downstream primer Alu2-R：5’-cgc cca ggc tgg agt g-3’；
Third is to sense primer Alu3-F：5 '-gtg aaa ccc cgt ctc tac taa aaa-3 ',
Third is to downstream primer Alu3-R：5’-gag tgc agt ggc gcg at-3’；
4th couple of sense primer Alu4-F：5 '-tac aaa aaa tta gcc ggg cg-3 ',
4th couple of downstream primer Alu4-R：5’-gat ctc ggc tca ctg caa g-3’；
5th couple of sense primer Alu5-F：5 '-aaa att agc cgg gcg tg-3 ',
5th couple of downstream primer Alu5-R：5’-gtt cac gcc att ctc ctg c-3’.
A kind of kit for detecting human peripheral Circulating DNA：
Reagent A 1-A5：The 5 kinds of solution formed for the primer of Alu different length PCR products by 5 Duis.
A1-A5 solution includes 5uM Alu1-F (upstream), 5uM Alu1-R (downstream) respectively,
5uM Alu2-F (upstream), 5uM Alu2-R (downstream), 5uM Alu3-F (upstream),
5uM Alu3-R (downstream), 5uM Alu4-F (upstream), 5uM Alu4-R (downstream),
5uM Alu5-F (upstream), 5uM Alu5-R (downstream)；
Wherein,
Reagent A 1：By first couple of sense primer Alu1-F (5 μM), first pair of downstream primer Alu1-R (5 μM), it is dissolved in
Water；
Reagent A 2：By second couple of sense primer Alu2-F (5 μM), second pair of downstream primer Alu2-R (5 μM), it is dissolved in
Water；
Reagent A 3：Sense primer Alu3-F (5 μM), third are dissolved in downstream primer Alu3-R (5 μM) by third
Water；
Reagent A 4：By the 4th pair of sense primer Alu4-F (5 μM), the 4th pair of downstream primer Alu4-R (5 μM), it is dissolved in
Water；
Reagent A 5：By the 5th pair of sense primer Alu5-F (5 μM), the 5th pair of downstream primer Alu5-R (5 μM), it is dissolved in
Water；
Reagent B：2 × SYBR Green qPCR Mix include 400 μM of dATP, dCTP, dGTP and dTTP
DNTPs, 100mM KCl, 4mM MgCl2,0.2U/ μ l Taq archaeal dna polymerases and 2 × SYBR Green；
Reagent C：ROX (50 ×) or ROXII (50 ×), is kept in dark place；
Reagent D：The standard items of preparation reach 1 × 10 by gradient dilution10、1×109、1×108、1×107With 1 × 106
Copy/mL, for the formulation of standard curve；
Reagent E：Negative control (normal human serum)；
Reagent G：Positive control (uses 1 × 107Standard items).
A kind of standard items of mating 5 pairs of primers, are the plasmid purification of the conserved genetic sequences containing Alu, and standard items pass through base
Because Alu objective gene sequences are connected on plasmid by engineering technology, by identifying, expanding, extracting and purifying acquisition, and pass through
260nm carries out absorbance reading, carries out the DNA that densimeter point counting analysis obtains and accurately copies number, and pass through dilution and be prepared into
The standard items of various concentration.
Specially：
Plasmid purification containing Alu genetic fragment SEQ ID NO.13；Preparation process after being expanded to Alu by obtaining purpose
Segment simultaneously connects PUC57 carriers；It is expanded with SEQ ID NO.1 and SEQ ID NO.2 primer pairs Alu, program is 95 DEG C
2min；94 DEG C of 20sec, 55 DEG C of 30sec, 72 DEG C of 30sec totally 35 cycle, condition be comprising 400 μM of dNTPs (dATP,
DCTP, dGTP, dTTP), 100mM KCl, 4mM MgCl2,0.2U/ μ l Taq archaeal dna polymerases, Alu is obtained by above method
Target fragment；Alu target fragments and PUC57 plasmids by restriction endonuclease EcoRI and HindIII at 37 DEG C, 2 hours or more (Alu
PCR product or PUC57 plasmids 10uL, 10 × R Buffer 2uL, EcoRI enzyme 1uL, HindIIII enzymes luL, water 6uL), enzyme
It cuts product and obtains 20uL digestion products by PCR product purifying column purification；Digestion products are using the 22 DEG C of reactions 12 of T4 ligases
Hour (Alu digestion products 5uL, PUC57 plasmid 2uL, T4 ligases 1uL, 10 × Buffer 1uL, water 1uL), then pass through mark
Accurate technique for gene engineering, is screened, expanded and is purified, and confirms that Alu is connected by carrying out DNA sequencing to plasmid
In plasmid PUC57 carriers；The absorbance detection of 260nM is carried out to plasmid purification, calculates the copy number of plasmid.
Advantageous effect
The present invention is designed the primer of 5 pairs of high specifics, is detected people periphery using the conservative and stability of Alu gene orders
Blood circulation DNA.By Optimal Experimental reaction condition and reaction system, simple, quick, sensitive, special detection kit is formed,
Tracking and dynamic analysis available for human peripheral Circulating DNA copy number.
Description of the drawings
Figure 1A lu1 examination criterias curves (R1=0.997771)
Fig. 2A lu2 examination criterias curves (R2=0.998359)
Fig. 3 Alu3 examination criterias curves (R3=0.998340)
Fig. 4 Alu4 examination criterias curves (R4=0.998747)
Fig. 5 Alu5 examination criterias curves (R5=0.999025)
The amplification curve of Fig. 6 Alu1 examination criterias product and positive sample
The amplification curve of Fig. 7 Alu2 examination criterias product and positive sample
The amplification curve of Fig. 8 Alu3 examination criterias product and positive sample
The amplification curve of Fig. 9 Alu4 examination criterias product and positive sample
The amplification curve of Figure 10 Alu5 examination criterias product and positive sample
Solubility curve when Figure 11 Alu1 are expanded
Solubility curve when Figure 12 Alu2 are expanded
Solubility curve when Figure 13 Alu3 are expanded
Solubility curve when Figure 14 Alu4 are expanded
Solubility curve when Figure 15 Alu5 are expanded
Specific embodiment
The invention will be further elucidated with reference to specific embodiments.It should be understood that these embodiments are merely to illustrate this hair
It is bright rather than limit the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, art technology
Personnel can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Fixed range.
Embodiment 1
5 pairs of Alu primers, mainly including Alu1-F (upstream), R (downstream), Alu2-F (upstream), R (downstream), Alu3-F
(upstream), R (downstream), Alu4-F (upstream), R (downstream), Alu5-F (upstream), R (downstream).
Embodiment 2
A kind of kit for detecting human peripheral Circulating DNA：
Reagent A 1-A5：5 kinds of solution being made of 5 pairs of primers.A1-A5 solution respectively comprising 5uM Alu1-F (upstream),
5uM Alu1-R (downstream), 5uM Alu2-F (upstream), 5uM Alu2-R (downstream), 5uM Alu3-F (upstream), 5uM Alu3-
R (downstream), 5uM Alu4-F (upstream), 5uM Alu4-R (downstream), 5uM Alu5-F (upstream), 5uM Alu5-R (downstream).
Reagent B：2 × SYBR Green qPCR Mix include 400 μM of dATP, dCTP, dGTP and dTTP
DNTPs, 100mM KCl, 4mM MgCl2,0.2U/ μ l Taq archaeal dna polymerases and 2 × SYBR Green.
Reagent C：ROX (50 ×) or ROXII (50 ×), is kept in dark place.
Reagent D：The standard items of preparation reach 1 × 10 by gradient dilution10、1×109、1×108、1×107With 1 × 106
Copy/mL, for the formulation of standard curve；
Reagent E：Negative control (normal human serum)；
Reagent G：Positive control (uses 1 × 107Standard items).
The standard items, the plasmid purification containing Alu genetic fragment SEQ ID3.Preparation process after being expanded to Alu by obtaining
Target fragment simultaneously connects PUC57 carriers.It is expanded with SEQ ID NO.1 and SEQ ID NO.2 primer pairs Alu, program 95
℃2min；94 DEG C of 20sec, 55 DEG C of 30sec, 72 DEG C of 30sec totally 35 cycle, condition be comprising 400 μM of dNTPs (dATP,
DCTP, dGTP, dTTP), 100mM KCl, 4mM MgCl2,0.2U/ μ l Taq archaeal dna polymerases, Alu is obtained by above method
Target fragment.Alu target fragments and PUC57 plasmids by restriction endonuclease EcoRI and HindIII at 37 DEG C, 2 hours or more (Alu
PCR product or PUC57 plasmids 10uL, 10 × R Buffer 2uL, EcoRI enzyme 1uL, HindIIII enzymes 1uL, water 6uL), enzyme
It cuts product and obtains 20uL digestion products by PCR product purifying column purification.Digestion products are using the 22 DEG C of reactions 12 of T4 ligases
Hour (Alu digestion products 5uL, PUC57 plasmid 2uL, T4 ligases 1uL, 10 × Buffer 1uL, water 1uL), then pass through mark
Accurate technique for gene engineering, is screened, expanded and is purified, and confirms that Alu is connected by carrying out DNA sequencing to plasmid
In plasmid PUC57 carriers.The absorbance detection of 260nM is carried out to plasmid purification, calculates the copy number of plasmid.
Embodiment 3
Real-time fluorescence quantitative PCR (Real-time PCR) is carried out with kit of the present invention to detect.First, according to experiment
Sample's number calculates the requirement of each reagent, and experimental procedure is as follows：
1. need the multiple holes detection for carrying out 5 standard items and sample outer, it is also necessary to detect negative control and positive control.
2. prepare 5 tube reaction liquid (Alu-Alu5) according to such as sequence and quantity：Add in water 340uL, reagent A 40uL, reagent
B 500uL, reagent C 20uL can carry out the experiment of 20 reacting holes per tube reaction liquid.
3. the above-mentioned reaction solution of mixing draws the PCR reaction tubes that 45uL is separately added into 20 holes every time.
4. each pipe adds in standard items, sample, feminine gender and the positive control (as follows) of 5uL in sequence.
5. it is put into quantitative fluorescent PCR instrument after covering lid to be detected.It is programmed to 95 DEG C of 30s, 95 DEG C of 5s, 60
DEG C 34s, totally 40 cycles, fluorescence signal value detected at 60 DEG C, amplification curve is as illustrated in figures 6-10.Solubility curve collects journey
Sequence is designed as 95 DEG C of 15s, 60 DEG C of 60s, 95 DEG C of 15s, and 1 cycle, the melt curve analysis of Alu1-Alu5 is as shown in figs. 11 and 15.
6. result calculates：By the analysis software of fluorescence quantitative PCR instrument, record the Ct values of various criterion product and draw standard
Curve (as shown in Figs. 1-5), the copy number for calculating the Alu1-Alu5 of each sample by calculating standard curve.Pass through negative control
Quality control is carried out to experimentation with positive control.
Claims (3)
1. the primer sets of human peripheral Circulating DNA, it is characterized in that, the primer sets are drawn by five Duis for expanding the specificity of Alu
Object forms, respectively for the different length segment of Alu conserved sequences；Primer is respectively：
First couple of sense primer Alu1-F：5 '-aga cca tcc tgg cta aca cg-3 ',
First couple of downstream primer Alu1-R：5’-aga cgg agt ctc gct ctg tc-3’；
Second couple of sense primer Alu2-F：5 '-cta aca cgg tga aac ccc g-3 ',
Second couple of downstream primer Alu2-R：5’-cgc cca ggc tgg agt g-3’；
Third is to sense primer Alu3-F：5 '-gtg aaa ccc cgt ctc tac taa aaa-3 ',
Third is to downstream primer Alu3-R：5’-gag tgc agt ggc gcg at-3’；
4th couple of sense primer Alu4-F：5 '-tac aaa aaa tta gcc ggg cg-3 ',
4th couple of downstream primer Alu4-R：5’-gat ctc ggc tca ctg caa g-3’；
5th couple of sense primer Alu5-F：5 '-aaa att agc cgg gcg tg-3 ',
5th couple of downstream primer Alu5-R：5’-gttcac gcc att ctc ctg c-3’.
2. comprising primer sets described in claim 1 quantitative detection human peripheral Circulating DNA kit, it is characterized in that including with
Lower reagent：
Reagent A 1：Water is dissolved in by 5 μM first couple of sense primer Alu1-F and the first of 5 μM couple of downstream primer Alu1-R；
Reagent A 2：Water is dissolved in by 5 μM second couple of sense primer Alu2-F and the second of 5 μM couple of downstream primer Alu2-R；
Reagent A 3：Water is dissolved in downstream primer Alu3-R to sense primer Alu3-F and 5 μM of third by 5 μM of thirds；
Reagent A 4：Water is dissolved in by 5 μM the 4th couple of sense primer Alu4-F and the 4th of 5 μM the couple of downstream primer Alu4-R；
Reagent A 5：Water is dissolved in by 5 μM the 5th couple of sense primer Alu5-F and the 5th of 5 μM the couple of downstream primer Alu5-R；
Reagent B：2 × SYBR Green qPCR Mix, include dATP, dCTP, dGTP and dTTP 400 μM of dNTPs,
100mM KCl、4mM MgCl2, 0.2U/ μ l Taq archaeal dna polymerases and 2 × SYBR Green；
Reagent C：ROX 50 × or ROXII 50 ×, it is kept in dark place；
Reagent D：The standard items of preparation reach 1 × 10 by gradient dilution10、1×109、1×108、1 ×107With 1 × 106It copies
Shellfish/mL, for the formulation of standard curve；
Reagent E：Negative control is normal human serum；
Reagent G：Positive control is using 1 × 107The standard items of copy/mL.
3. the kit according to claim 2 for quantitatively detecting human peripheral Circulating DNA, which is characterized in that the standard
Product are
It is diluted to the plasmid purification of the Alu genetic fragment SEQ ID NO.13 of various concentration；The preparation process of the plasmid purification
By obtaining target fragment after being expanded to Alu and connecting PUC57 carriers；It is released with SEQ ID NO.11 and SEQ ID NO.12
Primer pair Alu is expanded, and obtains Alu target fragments；Wherein, amplification program is 95 DEG C of 2min；94 DEG C of 20sec, 55 DEG C
30sec, 72 DEG C of 30sec, totally 35 recycle；Amplification system include 400 μM of dATP, dCTP, dGTP, dTTP, 100mM KCl,
4mM MgCl2, 0.2U/ μ l Taq archaeal dna polymerases；Alu target fragments and PUC57 plasmids by restriction endonuclease EcoRI and
HindIII digestions, endonuclease reaction system are as follows：Alu PCR products or PUC57 plasmids 10uL, 10 × R Buffer 2uL,
EcoRI enzymes 1uL, HindIIII enzyme 1uL, water 6uL；Endonuclease reaction system is reacted 2 hours or more at 37 DEG C, digestion products pass through
PCR product purifying column purification obtains the digestion products of 20uL purifying；The digestion products of purifying are using the 22 DEG C of reactions of T4 ligases
12 hours, coupled reaction system：Alu digestion products 5uL, PUC57 plasmid enzyme restriction product 2uL, T4 ligases 1uL, 10 ×
Buffer 1uL, water 1uL；Then by the technique for gene engineering of standard, screened, expanded and purified, and by plasmid
Carrying out DNA sequencing confirms that Alu has been connected to plasmid PUC57 carriers；The absorbance detection of 260nM is carried out to plasmid purification,
Calculate the copy number of plasmid.
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| CN102421915A (en) * | 2009-02-25 | 2012-04-18 | 蒂雅卡特有限责任公司 | Free circulating dna bio-markers and their applications |
| CN102732631A (en) * | 2012-07-04 | 2012-10-17 | 南通大学附属医院 | Method for detecting integrity of human free DNA (deoxyribonucleic acid) |
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|---|---|---|---|---|
| US5858658A (en) * | 1994-09-26 | 1999-01-12 | Immuno Aktiengesellschaft | Method of quantitating genomic DNA |
| US7537889B2 (en) * | 2003-09-30 | 2009-05-26 | Life Genetics Lab, Llc. | Assay for quantitation of human DNA using Alu elements |
| CN102421915A (en) * | 2009-02-25 | 2012-04-18 | 蒂雅卡特有限责任公司 | Free circulating dna bio-markers and their applications |
| CN102732631A (en) * | 2012-07-04 | 2012-10-17 | 南通大学附属医院 | Method for detecting integrity of human free DNA (deoxyribonucleic acid) |
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