CN103710452B - Kit and oligonucleotides for detecting free DNA (deoxyribonucleic acid) of peripheral blood - Google Patents

Kit and oligonucleotides for detecting free DNA (deoxyribonucleic acid) of peripheral blood Download PDF

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CN103710452B
CN103710452B CN201310741282.6A CN201310741282A CN103710452B CN 103710452 B CN103710452 B CN 103710452B CN 201310741282 A CN201310741282 A CN 201310741282A CN 103710452 B CN103710452 B CN 103710452B
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朱运峰
张爱群
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Kang Yuantai Valley (shenzhen) Gene Technology Co Ltd
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Abstract

The invention discloses a kit and oligonucleotides for detecting free DNA (deoxyribonucleic acid) of peripheral blood, and particularly relates to a group of oligonucleotides for detecting free DNA of peripheral blood, a kit containing these oligonucleotides and a detection method. The sequences of the oligonucleotides are as shown in SEQ ID NO.1 to SEQ ID NO.4 in a sequence table. The kit disclosed by the invention is high in sensitivity and specificity, low in cost and simple to operate.

Description

Detect test kit and the oligonucleotide of peripheral blood dissociative DNA
Technical field
The present invention relates to a kind of test kit utilizing peripheral blood dissociative DNA to carry out tumor screening, curative effect evaluation and prognosis prediction.Belong to biological technical field.
Background technology
Early diagnosis, early treatment are the keys improving clinical tumor curative ratio and improve prognosis, and the qualification of tumor markers is the key factor determining early discovery and curative effect evaluation with application.The most of tumor markerses used now are all the glycoprotein that metabolism is relevant, normally undertaken detecting by immunoassay, clinical data shows, the protein tumor markers of current clinical application not all shows exception on the one hand in all tumours, in the tumour patient not having these mark exceptions, cannot be detected by easy peripheral blood the validity of its treatment and evaluate, can only be undertaken checking (bearing x radiation x) by iconography means, be difficult to carry out real-time curative effect evaluation; The usual susceptibility of protein tumor markers conventional is on the other hand lower, and in some tumours, these marks lag behind the generation of tumour usually, need to carry out multi objective combine detection, the time of the detection of increase and expense; Thus very big restriction is received when the examination that cancer is early stage, so just in the urgent need to finding more responsive mark.
Peripheral blood dissociative DNA is by the general name being discharged into extracellular DNA in blood after necrocytosis, research finds, many tumour patient dissociative DNAs have very big-difference compared with normal people, in normal human peripheral blood, dissociative DNA is mainly from the cell of apoptosis, be made up of nucleus DNA and Mitochondrial DNA, its content is extremely faint, usually every milliliter is only nanogram levels, and free DNA level is significantly higher than normal people in the circulation of tumour patient blood, current discovery, it is a kind of universal phenomenon in all kinds of Tumor Growth that peripheral blood dissociative DNA content increases; Simultaneously in normal people, the mode normally apoptosis of Normal cell death, be discharged into the DNA fragmentation general shorter (below 200bp) in peripheral blood, and in tumour patient, the normal death of tumour cell is mainly downright bad, and non-viable non-apoptotic cell the DNA be discharged in peripheral blood be usually expressed as different length, like this relative to normal people, in blood of cancer patients dissociative DNA, the content of DNA long fragment also will increase; In addition, in the DNA originated with normal cell in tumour cell source, the epigenetics of some target gene modifies (methylating) significant difference.Especially it should be noted that plasma DNA accretion rate in vivo quickly, only several minutes transformation period, the situation of patient can be reflected in real time.Detection accordingly by peripheral blood DNA is assessed tumor efficiency and prognosis.It also has great importance at the screening (early diagnosis of tumor) to high-risk individuality, staging, is a kind of convenient, fast, responsive, special detection means.Detect non-invasive based on Circulating DNA simultaneously, avoid the difficulty that conventional sense needs to gather cancerous tissue sample, be applicable to very much the dynamic observation to curative effect and assessment, in a word, the detection of peripheral blood dissociative DNA is just valued by the people gradually with its irreplaceable advantage, and is becoming the focus of oncologic application research.
Because peripheral blood dissociative DNA is very micro-relative to tissue samples, need the DNA sequence dna of high copy in Select gene group so on the one hand, also need to select highly sensitive detection method on the other hand.Alu sequence and LINE-1 sequence are the tumor-necrosis factor glycoproteinss of high copy in genome, current research display, LINE-1 only activates under tumour cell and cellular stress state, and in normal cell, be in silence state, this change mainly usually epigenetics modify and to realize, namely the activation of LINE-1 is embodied in the demethylation of its promotor, and silence is then embodied in the supermethylation of its promotor.So the detection by changing the methylation level of its promoter region, can be assessed it and whether activate.
This test kit is by comprising the target sequence (action site of the restriction enzyme containing methyl-sensitive) on CG island in design LINE-1 promoter region, in territory, the high conserved region design PCR primer of its both sides, choose Alu sequence as internal reference simultaneously, the action site of the restriction enzyme of above-mentioned methyl-sensitive is not included in selected zone, design PCR primer, two kinds of amplified productions confirm through sequential analysis.The LINE-1 promoter sequence in normal cell source is due to hyper-methylation so in theory, the restriction enzyme of methyl-sensitive then can not cut, show as the Ct value that enzyme cuts front and back pcr amplification and there is no obvious difference, and owing to there is demethylation in the LINE-1 promoter sequence in tumour cell source, the restriction enzyme of methyl-sensitive then can cut, and showing as the Ct value that enzyme cuts front and back pcr amplification has obvious increase.Simultaneously due to design LINE-1 amplified fragments (256bp) and Alu(112bp) expanding fragment length difference is comparatively large, its Ct value difference value can as the important indicator of assessment peripheral blood dissociative DNA length variations.In addition Alu sequence due to the copy number in genome very high, the change of its concentration has also reflected the change of plasma DNA total concn.So by can be responsive to the detection of above three kinds of indexs reflect that tumour for information about, to the application of the examination of tumour early warning, curative effect evaluation and prognosis prediction, there is great directive significance.
Summary of the invention
The technical problem to be solved in the present invention is to provide strong, the highly sensitive oligonucleotide for detecting peripheral blood dissociative DNA of a group-specific.
Another technical problem that the present invention will solve is to provide a kind of simple to operate, test kit that result detects peripheral blood dissociative DNA accurately.
For achieving the above object, the present invention is by the following technical solutions:
The invention provides the oligonucleotide that a group is detected peripheral blood dissociative DNA, be made up of the oligonucleotide of base sequence shown in sequence table SEQ ID No.1 to sequence table SEQ ID No.4;
Wherein, sequence SEQ ID No.1 and SEQ ID No.2 is respectively upstream primer and the downstream primer of amplification LINE-1 gene, and sequence SEQ ID No.3 and SEQ ID No.4 is respectively upstream primer and the downstream primer of amplification Alu gene, and concrete sequence is as follows:
LINE-1 upstream primer (L1-256-U): 5 '-GAGAGGAGCCAAGATGGC-3 ' (SEQ ID NO.1)
LINE-1 downstream primer (L1-256-D): 5 '-TCYGTCACCCCTTTCTTTGA-3 ' (SEQ ID NO.2)
Alu upstream primer (ALU-112-U): 5 '-GTGGCTCACGCCTGTAATC-3 ' (SEQID NO.3)
Alu downstream primer (ALU-112-D): 5 '-TTTAGTAGAGACGGGGTTTCAC-3 ' (SEQ ID NO.4)
In above-mentioned primer sequence, Y represents C or T.
The present invention also provides a kind of test kit detecting peripheral blood dissociative DNA, comprises following reagent:
1) endonuclease reaction liquid, it comprises: HpaII enzyme, 10 × buffer;
2) qPCR reaction solution, it comprises: the upstream primer of amplification LINE-1 gene and downstream primer, the upstream primer of amplification Alu gene and downstream primer, and the PCR reaction solution containing SYBR Green;
Wherein, upstream primer and the downstream primer of amplification LINE-1 gene are the nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2 respectively, and upstream primer and the downstream primer of amplification Alu gene are the nucleotide sequence shown in SEQ ID No.3 and SEQ ID No.4 respectively;
Further, the usual commodity in use reagent of PCR reaction solution containing SYBR Green, such as, 2 × SuperReal PreMix Plus(is purchased from Tian Gen biochemical technology company limited), or DRR041ASYBR Premix Ex Taq TM(is purchased from TaKaRa Bio Inc);
3) for the preparation of the DNA standard substance of typical curve;
4) negative control DNA sample: the change that enzyme cuts the △ △ Ct of front and back quantitative fluorescent PCR is less than threshold value;
5) positive control dna sample: the change that enzyme cuts the △ △ Ct of front and back quantitative fluorescent PCR is greater than threshold value;
6）ddH 2O。
Present invention also offers a kind of using method detecting the test kit of peripheral blood dissociative DNA:
1) extracting peripheral blood dissociative DNA;
2) use HpaII enzyme human peripheral blood dissociative DNA to carry out enzyme to cut, the peripheral blood dissociative DNA simultaneously cut by non-enzyme as a control group; Enzyme cuts system in table 1:
Table 1 enzyme cuts system
Enzyme tangent condition is: 37 DEG C of 2h.
3) real-time fluorescence PCR quantitative amplification is carried out, the primer wherein used is the amplification LINE-1 gene upstream and downstream primer shown in sequence table SEQ IDNo.1 and SEQ ID No.2, and the upstream and downstream primer of the amplification ALU gene shown in sequence table SEQ ID No.3 and SEQ ID No.4; This reaction system is respectively in table 2, table 3:
Table 2LINE-1 sequence qPCR reaction system
Table 3Alu sequence qPCR reaction system
Reaction conditions is: Pre-incubation:95 DEG C, 15min
3Step Amplification:95 DEG C, 15s; 55 DEG C, 20s; 72 DEG C, 20s; 40 circulations.
4) result judges, according to calculating as follows
(a)△Ct=∣(Ct-L1precut–Ct-Aluprecut)∣；
(b)△△Ct=∣(Ct-L1precut–Ct-Aluprecut)—(Ct-L1postcut–Ct-Alupostcut)∣；
C the absolute concentration of Alu in () plasma DNA: the DNA standard substance drawing standard curve using preparation standard curve, sets up the corresponding relation between Ct value and DNA concentration, according to the absolute concentration of Alu in standard curve determination plasma DNA.
In theory at treatments period, the validity for the treatment of should show as plasma DNA total amount to be increased (amount of non-viable non-apoptotic cell released dna increases), along with tumour cell is swept off after treatment, DNA total amount then should significantly decline, should increasing to some extent (minimizing of DNA long fragment quantity) of △ Ct value, △ △ Ct value should decline to some extent (the LINE-1DNA quantity in tumour source reduces).According to preliminary analysis of statistical data and in conjunction with related index, the critical value setting of Alu absolute concentration in peripheral blood dissociative DNA is 2.5ng/ml by we, is less than 2.5ng/ml for negative; Be 3.2 by the critical value setting of △ Ct, be greater than 3.2 for negative; Be 0.8 by △ △ Ct value critical value setting, be less than 0.8 for negative.
The present invention has following features relative to prior art:
1, hypersensitivity: peripheral blood dissociative DNA detection kit, combines judge by three kinds of indexs, drastically increases the sensitivity of detection.
2, specificity: the target gene LINE-1 selected due to us only activates (cellular stress in tumour cell, also its transient activation can be caused, as inflammation), and in normal cell, be silencer, when getting rid of inflammation, the specificity adding tumour is detected to it like this.
3, direct: conventional protein tumor mark normally detects the exception of metabolic index, and be indirect indexes, and peripheral blood dissociative DNA directly comes from cell itself, is direct index, more genuine and believable.
4, real-time: simultaneously due to the metabolism very fast (can remove at several minutes intracellular metabolite) in vivo of peripheral blood dissociative DNA, real-time evaluation can be carried out to result for the treatment of like this.
5, stability: for protein markers, the stability of DNA is better, relatively low to conditional requests such as the storage transports of sample.
6, early warning: basic medical research thinks that the basic reason of generation of tumour is genomic unstable, the silence of LINE-1 plays very important effect in the genomic stability of maintenance, so the activation detecting LINE-1 has very great significance to the risk tool that predicting tumors is fallen ill.
Accompanying drawing explanation
Fig. 1 is the qPCR amplification curve of the DNA standard substance for the preparation of typical curve;
Fig. 2 is linear relationship corresponding between DNA standard concentration with Ct value;
Fig. 3 is the qPCR Amplification Analysis figure after typical normal human peripheral blood's dissociative DNA enzyme cuts process;
Fig. 4 is the qPCR Amplification Analysis figure after a typical blood of cancer patients dissociative DNA enzyme cuts process.
Embodiment
Below in conjunction with specific embodiment, the present invention is further described.
Embodiment 1 design of primers
LINE-1 aligning primer method of design: the present invention is according to the LINE-1 sequence reported, increase LINE-1 promoter region sequence from different human peripheral blood cells, the sequence of conserved portions is found out by sequence alignment program, region, CG island is rich in selection, adopts DNAMAN software design primer sequence;
Alu sequence primer design method: because Alu family exists different variations, the present invention carries out sequence analysis to different members in family, finds out not containing the conservative region of methylation sensitive restriction endonuclease action site, adopts DNAMAN software design primer sequence;
Primer sequence is as follows:
LINE-1 upstream primer (L1-256-U): 5 '-GAGAGGAGCCAAGATGGC-3 ' (SEQ ID NO.1);
LINE-1 downstream primer (L1-256-D): 5 '-TCYGTCACCCCTTTCTTTGA-3 ' (SEQ ID NO.2);
Alu upstream primer (ALU-112-U): 5 '-GTGGCTCACGCCTGTAATC-3 ' (SEQID NO.3);
Alu downstream primer (ALU-112-D): 5 '-TTTAGTAGAGACGGGGTTTCAC-3 ' (SEQ ID NO.4);
Above-mentioned primer wins the synthesis of polygala root company by Beijing three.
Embodiment 2 is for detecting the composition (being stored in-20 DEG C) of tumor peripheries blood dissociative DNA test kit
1) endonuclease reaction liquid, it comprises: HpaII enzyme, and 10 × buffer(is purchased from NEB company).
2) qPCR reaction solution, it comprises: the PCR reaction solution containing SYBR Green, the upstream primer of amplification LINE-1 gene and downstream primer, the upstream primer of amplification Alu gene and downstream primer;
Wherein, upstream primer and the downstream primer of amplification LINE-1 gene are the nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2 respectively, and upstream primer and the downstream primer of amplification Alu gene are the nucleotide sequence shown in SEQ ID No.3 and SEQ ID No.4 respectively;
3) the DNA standard substance of preparation standard curve;
4) negative control DNA sample: the change that enzyme cuts the △ △ Ct of front and back quantitative fluorescent PCR is less than threshold value;
5) positive control dna sample: the change that enzyme cuts the △ △ Ct of front and back quantitative fluorescent PCR is greater than threshold value;
6）ddH 2O。
Embodiment 3 is for detecting the using method of tumor peripheries blood dissociative DNA test kit
1. the preparation of peripheral blood dissociative DNA
Adopt Qiagen company QIAamp DNA Blood Mini Kit test kit extracting plasma dna, extracting method carries out to specifications.
2.DNA enzyme is cut
1) enzyme cuts premixed liquid preparation (carrying out on ice or under 4 degree of conditions)
Enzyme cuts the preparation of premixed liquid, gets the 1.5ml centrifuge tube that autoclaving is crossed, and adds HpaII enzyme 1ul, 10 × buffer2ul, ddH 2o15ul; Mix rear of short duration centrifugal for subsequent use.
2) endonuclease reaction:
Get the centrifuge tube of 2 0.5ml, the enzyme adding 18ul respectively cuts premixed liquid and ddH 2o (non-enzyme cuts contrast), and then add the DNA sample of 2ul respectively, of short duration centrifugal mixing.Carry out hatching 2h in 37 DEG C, reaction terminates rear of short duration centrifugal, can carry out next step qPCR experiment.If downstream reaction can not be carried out in time, should-20 DEG C of preservations.
3. real-time fluorescence PCR quantitative amplification
1) primer working fluid is prepared
By primer dry powder ddH 2o dissolves, and is mixed with the conserving liquid of 100uM ,-20 DEG C of preservations; Use ddH 2o is diluted to the primer working fluid of 10uM, and carries out packing, avoids multigelation.
2) premixed liquid (carrying out on ice or under 4 degree of conditions) of the upstream and downstream primer of LINE-1 and Alu gene is prepared.
Detect sample for each, the primer working fluid of the primer working fluid of LINE-1 gene and Alu gene is mixed with the PCR reaction solution containing SYBR Green respectively, of short duration centrifugal for subsequent use.
3) real-time fluorescence PCR quantitative amplification
Premixed liquid is added respectively DNA after cutting with enzyme before enzyme is cut in 96 orifice plates, after capping, on board-like whizzer, 3000rpm, 1min are centrifugal.Place it on Roche480 quantitative real time PCR Instrument, it is as follows that working procedure is set:
Pre-incubation:95℃,15min
3Step Amplification:95 DEG C, 15s; 55 DEG C, 20s; 72 DEG C, 20s; 40 circulations.
Fig. 3, Fig. 4 are respectively the qPCR amplification figure of normal people and blood of cancer patients dissociative DNA.
4. result judges, according to calculating as follows
A (Ct-L1precut – Ct-Aluprecut) ∣: Ct represents PCR cycle index to () △ Ct=∣, and L1 represents LINE-1, and Precut represents before enzyme cuts.This value represents the relative quantity in plasma DNA between long segment and short-movie section, in the dissociative DNA of being originated by tumour or inflammatory cell, DNA long fragment quantity increases, and cause the relative concentration between itself and short-movie section close, this value larger expression long segment is lower relative to the concentration of short-movie section.
B () △ △ Ct=∣ (Ct-L1precut – Ct-Aluprecut)-(Ct-L1postcut – Ct-Alupostcut) ∣: Ct represents PCR cycle index, L1 represents LINE-1, Precut represents before enzyme cuts, and Postcut represents after enzyme cuts.Because tumour cell source methylates different from the target gene that normal cell is originated (LINE-1), the difference processing and the difference of this modification is converted into pcr template amount is cut like this by enzyme, this value is larger represents that the relative quantity shared by demetalated dna is larger, and namely the dissociative DNA in tumour source is more.
C the absolute concentration of Alu in () plasma DNA: the DNA standard substance drawing standard curve using preparation standard curve, sets up the corresponding relation between Ct value and DNA concentration, according to the absolute concentration of Alu in standard curve determination plasma DNA.
5. for the preparation of the preparation method of the DNA standard substance of typical curve
Get the pcr amplification product of Alu gene, spectrophotometer carries out Concentration Testing and obtains DNA concentration values, then carry out serial dilution (10 times of gradient dilutions) as mother liquor, obtain reference concentration sample respectively, choose 4 reference concentration samples as DNA standard substance.Be that template carries out qPCR with standard substance, utilize the relation (see Fig. 1 and Fig. 2) between computed in software DNA profiling concentration and Ct value carried in equipment, its linear relationship meets linear linear function (Y=kx+b, wherein Y is cycle index, k is slope, x is the logarithm of concentration, and b is the intercept in Y-axis).
According to preliminary analysis of statistical data and in conjunction with related index, the critical value setting of Alu in peripheral blood dissociative DNA is 2.5ng/ml by we, is less than 2.5ng/ml for negative; Be 3.2 by the critical value setting of △ Ct, be greater than 3.2 for negative; Be 0.8 by △ △ Ct value critical value setting, be less than 0.8 for negative.In table 4.
The reference of table 4 peripheral blood dissociative DNA detect parameters
cfDNA Test item Negative reference value Unit
cfDNA-Alu Dissociative DNA internal reference concentration <2.50 ng/ml
cfDNA△Ct Dissociative DNA △ Ct >3.20 Ct
cfDNA△△Ct Dissociative DNA △ △ Ct <0.80 Ct
The combinatory analysis of embodiment 4 for detecting with conventional tumor markers, improves the sensitivity and specificity detected
1. material
The detection sample of the present embodiment is provided by PLA General Hospital tumor center laboratory, totally 100 examples;
Meanwhile, for exploring itself and the relation of conventional protein tumor mark, selecting the tumor markers combination that 12 kinds common, the detected result of two kinds of methods is compared;
12 kinds of protein tumor marks are respectively: CA19-9; NSE; CEA; CA242; CA72-4; β-HCG; AFP; SCC; C-PSA; CA125; CK19; CA15-3(purchased from Shanghai Ming Yuanshu health Science and Technology Ltd., the accurate font size S20020026 of traditional Chinese medicines).
2. method
Test kit of the present invention is adopted to detect detection sample; And with 12 kinds of tumor markerses, detection sample is detected as a control group.
3. result
12 kinds of tumor markerses and test kit of the present invention are respectively the coincidence rate that dissociative DNA detects:
DNA positive coincidence rate (namely 12 kinds of tumor markers any one occur positive, and dissociative DNA detects any one in three indexs and occurs positive): 80%
DNA negative match-rate: 90%
Embodiment 5 is for the analysis and assessment to tumor efficiency
1. material
The blood plasma of detection sample for gathering for 7 days with Post operation before treatment of the present embodiment, by PLA General Hospital, liver and gall surgical department provides, and totally 10 is right.
2. method
Test kit of the present invention is adopted to detect detection sample.
3. result, detected result is in table 5:
Table 5 liver cancer patient Postoperative Peripheral Blood of Patients dissociative DNA detects numerical value
Sample * △Ct △△Ct Alu concentration
C4 4.7 1.09 2.525
C4A 5.93 0.33 5.5
C5 5.11 0.72 4.2
C5A 5.19 0.82 2.525
C9 4.88 1.01 2.65
C9A 5.15 0.98 3.95
C11 5.43 0.6 2.2825
C11A 5.83 0.34 3.05
C21 5.28 0.62 1.425
C21A 5.16 0.86 3.775
C86 6.04 0.34 3.65
C86A 6.08 0.33 8.4
C49 5.47 0.4 21.75
C49A 6.02 0.09 6.95
C42 4.65 1.21 1.4325
C42A 5.12 1.32 3.9
C29 4.29 0.86 2.145
C29A 5.35 0.55 4.425
C32 4.56 3.04 1.5525
C32A 5.13 1.12 3.85
Wherein, C+ numeral is the code name of patient; A represents sample (Post operation 7 days) after same corrective surgery;
△ Ct raises and shows that the relative concentration of DNA long fragment and short segment DNA reduces, and because DNA long fragment mainly comes from tumour cell, show the DNA density loss of tumour cell release in blood, namely treatment effectively.
△ △ Ct reduces the DNA concentration reduction showing that tumour is originated, and namely hypomethylated DNA amount reduces (L1DNA in tumour source is hypomethylation), shows that treatment is effective.
Alu concentration increases and shows there is a large amount of necrocytosiss at treatments period, and DNA is discharged in blood by the cell of these death, also accelerates relevant with Post operation cell proliferation simultaneously, and treatment is effective.
Embodiment 6 is for the analysis to tumor invasion risk early warning and alert
1. material
The detection sample of the present embodiment is provided by Hangzhou Science and Technology Ltd. of rich nations, totally 192 examples; Comprising tumour family medical history (mammary cancer and colorectal cancer) crowd 24 example, non-tumour family medical history crowd 168 example.
2. method
Tumor peripheries blood dissociative DNA detection kit of the present invention is adopted to detect sample; The difference of comparison of tumor susceptibility crowd and general population's Testing index.
3. result
Having in Family history of cancer crowd, peripheral blood dissociative DNA occurs that the number of cases of abnormal (in three kinds of indexs, any one is positive) is 4 people, and positive rate is about 16%; Without Family history of cancer and in crowd, peripheral blood dissociative DNA occurs that the number of cases of abnormal (in three kinds of indexs, any one is positive) is 15 people, and positive rate is 9%.Visible peripheral blood dissociative DNA inflammatory patients and there is Family history of cancer crowd in occur that abnormal ratio obviously increases, because crowd's number with tumour family medical history is less, so amplification detection amount will contribute to the relation illustrated between the two further.Tumorigenic early stage main manifestations is inflammatory reaction, i.e. so-called inflammation-cancer-chain, and the inflammation do not healed for a long time obviously by increasing the risk of tumor invasion, points out this peripheral blood dissociative DNA detection kit to can be used for the early warning and alert of tumor invasion.
Obviously, the above embodiment of the present invention is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here cannot give exhaustive to all embodiments.Every belong to technical scheme of the present invention the apparent change of extending out or variation be still in the row of protection scope of the present invention.

Claims (2)

1. one group is detected the oligonucleotide of peripheral blood dissociative DNA, it is characterized in that, is made up of the oligonucleotide of base sequence shown in sequence table SEQ ID No.1 to sequence table SEQ ID No.4; Wherein SEQ ID No.1 and SEQ ID No.2 is respectively the upstream and downstream primer of amplification LINE-1 gene, and SEQ ID No.3 and SEQ ID No.4 is respectively the upstream and downstream primer of amplification Alu gene.
2. detect a test kit for peripheral blood dissociative DNA, it is characterized in that, composed of the following components:
1) endonuclease reaction liquid, it comprises: HpaII enzyme, 10 × buffer;
2) qPCR reaction solution, it comprises: the upstream primer of amplification LINE-1 gene and downstream primer, the upstream primer of amplification Alu gene and downstream primer, and the PCR reaction solution containing SYBR Green;
Wherein, upstream primer and the downstream primer of amplification LINE-1 gene are the nucleotide sequence shown in SEQ ID No.1 and SEQ ID No.2 respectively, and upstream primer and the downstream primer of amplification Alu gene are the nucleotide sequence shown in SEQ ID No.3 and SEQ ID No.4 respectively;
3) the DNA standard substance of preparation standard curve;
4) negative control DNA sample;
5) positive control dna sample;
6）ddH 2O。
CN201310741282.6A 2013-12-27 2013-12-27 Kit and oligonucleotides for detecting free DNA (deoxyribonucleic acid) of peripheral blood Expired - Fee Related CN103710452B (en)

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