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 invention relates to biotechnology, in particular to a molecular biological method based on quantitative nucleic acid amplification, and particularly relates to real-time quantitative polymerase chain reaction (real-time PCR), which amplifies different fragments of a conserved gene Alu of circulating DNA in human peripheral blood by using specific primers and quantitatively detects the circulating DNA in the human peripheral blood by detecting released fluorescent signals.
Background
Circulating DNA (circulating DNA), also called free DNA (cf-DNA), is an extracellular DNA in a cell-free state that is present in body fluids of animals, plants and humans. In 1977, Leon et al first discovered that plasma DNA levels in tumor patients were significantly elevated and suggested a role in monitoring peripheral blood free DNA levels in the observation of therapeutic efficacy and prediction of recurrence (LeonsA, Shapino B, Skaaroff DM, et al. free DNA in the serum of cancer patients and of therapy [ J]Cancer res.1977, 37 (3): 646). "Suzuki N, Kamataki A, Yamaki J, et al, Characterisation of circulating DNA in heathy humanplasma. Clin Chim Acta, 2008, 387 (1-2): 55-58' study showed that the circulating DNA content of healthy people is very small, about 5 ug/L. The document "Mulcahy HE, Lyautey J, Lederrey C, et al. A.Prospectral study of K-ras gene mutation in the plasmid of secretory cancer genes [ J].ClinCancer Res，1998，4(2)：271”、“GeorgDoris Auer，Inge Gaugg，et al.Prognosticsignificance of methylated RASSF1A and PITX2 genes in blood-and bone marrowplasma of breast cancer patients[J]Breast Cancer Res Treat, 2011, 130 (8): 109-: abstr4058 "describes that when a body is in a tumor, an autoimmune disease, an inflammatory reaction, or the like, the content of circulating DNA in the body is increased accordingly, but the mechanism of its production has not yet been completely clarified. Eyes of a userThe serological detection of the pre-circulating DNA cannot be directly used for early diagnosis and curative effect judgment of diseases, but is closely related to the generation and development of some diseases, so that the detection of the circulating DNA and the determination of specific genes of certain known disease species have important clinical significance, and the circulating DNA can possibly become a novel molecular marker with good prospect for early diagnosis and curative effect judgment of diseases.
At present, the more quantitative detection methods applied at home and abroad can be divided into two major categories, namely amplification methods based on Polymerase Chain Reaction (PCR), such as methylation-specific PCR (MSP) and real-time fluorescence quantitative PCR (RTFQ-PCR), and detection methods based on signal amplification technology, such as branched DNA (bDNA) detection technology, researchers studying circulating DNA concentration in the past years mostly adopt analysis methods based on quantitative PCR, such as "Ellinger J, M ü iller DC, M ü ller SC, ethyl circulating biochemical DNA in serum: A indirect biological assay for transporting primers with high specificity and molecular amplification technology, and the detection methods can be applied to high-specificity PCR (18. the detection method is also applied to high-specificity PCR), and the detection methods can be applied to high-specificity PCR, such as PCR, U.S. Urol Oncol, 30 (4): 509 and" 11. and "Huntingbiological assay".
With the rapid development of molecular biology, molecular testing technology has become the hottest technical method in clinical testing technology and pathological diagnosis technology, such as the application of Real-time PCR technology in hepatitis B, hepatitis C, Human Papilloma Virus (HPV), breast cancer and lung cancer, and provides a solid theoretical basis for individualized diagnosis and treatment schemes.
Disclosure of Invention
The invention utilizes molecular biology Real-time PCR technology, based on the characteristic of Alu gene sequence which can be used as a specific mark of human DNA fragment (Alu is a medium repetitive sequence in human genome, about 300bp long, about 1Alu sequence every 6kb on average, richest in mammal and highest in homology, so it can be used as a specific mark of human DNA fragment), designs primers of Alu gene with different length products, selects 5 pairs of upstream and downstream Real-time PCR primers with high specificity, amplifies Alu fragments with different lengths by adopting fluorescent quantitative PCR method, and establishes standard of Alu at the same time, can quantitatively detect copy number of different Alu fragments, thereby reflecting the number of human circulating DNA, the method has high specificity and high sensitivity, can establish good and uniform molecular quality control standard, can make up for the defects of lack of comparability between different detection results and the like, and is a good circulating DNA detection method.
The first purpose of the invention is to overcome the defects of the existing detection method such as incomplete standardization and quality control means.
The second objective of the invention is to provide a conserved sequence of Alu as a DNA for detecting human peripheral blood circulation.
The third purpose of the invention is to provide 5Alu high specificity primers and Alu standard for PCR products with different lengths.
The fourth purpose of the invention is to provide a simple and convenient kit for quantitative detection of human peripheral blood circulation DNA.
The technical scheme of the invention is as follows:
a group of specific primers for amplifying Alu respectively aim at different length segments of Alu conserved sequence. The 5 primer pairs were 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), respectively. The Alu gene sequence is highly conserved as a target DNA fragment for detection, and the nucleic acid sequences of 5 pairs of primers are provided.
The primer group of human peripheral blood circulation DNA consists of five pairs of specific primers for amplifying Alu, and is respectively directed at different length segments of Alu conserved sequences; the primers are respectively as follows:
the first pair of upstream primers Alu 1-F: 5'-aga cca tcc tgg cta aca cg-3' the flow of the air in the air conditioner,
the first downstream primer pair Alu 1-R: 5'-aga cgg agt ctc gct ctg tc-3', respectively;
the second pair of upstream primers Alu 2-F: 5'-cta aca cgg tga aac ccc g-3' the flow of the air in the air conditioner,
the second pair of downstream primers Alu 2-R: 5'-cgc cca ggc tgg agt g-3', respectively;
the third pair of upstream primers Alu 3-F: 5'-gtg aaa ccc cgt ctc tac taa aaa-3' the flow of the air in the air conditioner,
the third pair of downstream primers Alu 3-R: 5'-gag tgc agt ggc gcg at-3', respectively;
the fourth pair of upstream primers Alu 4-F: 5'-tac aaa aaa tta gcc ggg cg-3' the flow of the air in the air conditioner,
the fourth pair of downstream primers Alu 4-R: 5'-gat ctc ggc tca ctg caa g-3', respectively;
the fifth pair of upstream primers Alu 5-F: 5'-aaa att agc cgg gcg tg-3' the flow of the air in the air conditioner,
the fifth pair of downstream primers Alu 5-R: 5'-gtt cac gcc att ctc ctg c-3' are provided.
A kit for detecting human peripheral blood circulation DNA:
reagent A1-A5: 5 solutions consisting of 5 pairs of primers for different length PCR products of Alu.
The A1-A5 solutions contained 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 A1: dissolving a first pair of upstream primers Alu1-F (5. mu.M) and a first pair of downstream primers Alu1-R (5. mu.M) in water;
reagent A2: dissolving a second pair of upstream primers Alu2-F (5. mu.M) and a second pair of downstream primers Alu2-R (5. mu.M) in water;
reagent A3: dissolving the third pair of upstream primer Alu3-F (5. mu.M) and the third pair of downstream primer Alu3-R (5. mu.M) in water;
reagent A4: dissolving the fourth pair of upstream primer Alu4-F (5. mu.M) and the fourth pair of downstream primer Alu4-R (5. mu.M) in water;
reagent A5: dissolving a fifth pair of upstream primers Alu5-F (5. mu.M) and a fifth pair of downstream primers Alu5-R (5. mu.M) in water;
and (3) reagent B: 2 XSSYBR Green qPCR Mix comprising 400. mu.M dNTPs including dATP, dCTP, dGTP and dTTP, 100mM KCl, 4mM MgCl2, 0.2U/. mu.l Taq DNA polymerase and 2 XSSYBR Green;
and (3) reagent C: ROX (50X) or ROXII (50X), stored protected from light;
and (3) reagent D: the prepared standard substance is diluted to 1 × 10 by gradient10、1×109、1×108、1×107And 1X 106copy/mL, used for standard curve formulation;
and (3) reagent E: negative control (normal human serum);
reagent G: positive control (1X 10 used)7The standard of (4).
A standard product matched with 5 pairs of primers is a purified plasmid containing an Alu conserved gene sequence, the standard product is obtained by connecting an Alu target gene sequence to the plasmid through a genetic engineering technology, identifying, amplifying, extracting and purifying, reading absorbance at 260nm, calculating and analyzing the concentration to obtain the accurate copy number of DNA, and diluting to prepare the standard products with different concentrations.
The method specifically comprises the following steps:
a purified plasmid containing an Alu gene fragment SEQ ID NO. 13; in the preparation process, a target fragment is obtained by amplifying Alu and is connected with a PUC57 carrier; primer SEQ ID NO.1 and primer SEQ ID NO.2 were used to amplify Alu at 95 ℃ for 2 min; 35 cycles of 94 ℃ 20sec, 55 ℃ 30sec, 72 ℃ 30sec under conditions comprising 400. mu.M dNTPs (dATP, dCTP, dGTP, dTTP), 100mM KCl, 4mM MgCl2, 0.2U/. mu.l Taq DNA polymerase, to obtain a fragment of Alu interest by the above method; the Alu target fragment and PUC57 plasmid are subjected to endonuclease EcoRI and HindIII at 37 ℃ for more than 2 hours (AluPCR product or PUC57 plasmid 10uL, 10 xR Buffer 2uL, EcoRI enzyme 1uL, HindIIII enzyme luL and water 6uL), and the enzyme digestion product is purified by a PCR product purification column to obtain 20uL enzyme digestion product; the enzyme digestion product is reacted for 12 hours at 22 ℃ by T4 ligase (Alu enzyme digestion product 5uL, PUC57 plasmid 2uL, T4 ligase 1uL, 10 xbuffer 1uL and water 1uL), then screening, amplifying and purifying by standard gene engineering technology, and confirming that Alu is connected to the plasmid PUC57 vector by DNA sequencing of the plasmid; the purified plasmid was subjected to an absorbance measurement of 260nM and the copy number of the plasmid was calculated.
Advantageous effects
The invention utilizes the conservation and the stability of an Alu gene sequence to design 5 pairs of primers with high specificity to detect human peripheral blood circulation DNA. By optimizing the experimental reaction conditions and the reaction system, a simple, quick, sensitive and specific detection kit is formed, and can be used for tracking and dynamically analyzing the copy number of the human peripheral blood circulation DNA.
Drawings
Fig. 1Alu1 test standard curve (R1 ═ 0.997771)
Fig. 2Alu2 test standard curve (R2 ═ 0.998359)
Fig. 3Alu3 test standard curve (R3 ═ 0.998340)
Fig. 4Alu4 test standard curve (R4 ═ 0.998747)
Fig. 5Alu5 test standard curve (R5 ═ 0.999025)
FIG. 6 amplification curves for Alu1 assay standards and positive specimens
FIG. 7 amplification curves for Alu2 assay standards and positive specimens
FIG. 8 amplification curves for Alu3 assay standards and positive specimens
FIG. 9 amplification curves for Alu4 assay standards and positive specimens
FIG. 10 amplification curves for Alu5 assay standards and positive specimens
FIG. 11 dissolution curves for Alu1 amplification
FIG. 12 dissolution curves for Alu2 amplification
FIG. 13 dissolution curves for Alu3 amplification
FIG. 14 dissolution curves of Alu4 during amplification
FIG. 15 dissolution curves for Alu5 amplification
Detailed Description
The invention will be further illustrated with reference to specific embodiments. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.
Example 1
The 5 pairs of Alu primers mainly comprise 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).
Example 2
A kit for detecting human peripheral blood circulation DNA:
reagent A1-A5: 5 solutions consisting of 5 pairs of primers. The A1-A5 solutions contained 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), respectively.
And (3) reagent B: 2 XSSYBR Green qPCR Mix containing 400. mu.M dNTPs including dATP, dCTP, dGTP and dTTP, 100mM KCl, 4mM MgCl2, 0.2U/. mu.l Taq DNA polymerase and 2 XSSYBR Green.
And (3) reagent C: ROX (50X) or ROXII (50X), protected from light.
And (3) reagent D: the prepared standard substance is diluted to 1 × 10 by gradient10、1×109、1×108、1×107And 1X 106copy/mL, used for standard curve formulation;
and (3) reagent E: negative control (normal human serum);
reagent G: positive control (1X 10 used)7The standard of (4).
The standard product is a purified plasmid containing an Alu gene fragment SEQ ID 3. The preparation process comprises the steps of obtaining a target fragment after Alu amplification and connecting a PUC57 carrier. Primer SEQ ID NO.1 and primer SEQ ID NO.2 were used to amplify Alu at 95 ℃ for 2 min; 35 cycles of 94 ℃ 20sec, 55 ℃ 30sec, and 72 ℃ 30sec were carried out under conditions comprising 400. mu.M dNTPs (dATP, dCTP, dGTP, dTTP), 100mM KCl, 4mM MgCl2, and 0.2U/. mu.l Taq DNA polymerase, and the desired fragment of Alu was obtained by the above method. The Alu target fragment and PUC57 plasmid were digested with restriction enzymes EcoRI and HindIII at 37 ℃ for more than 2 hours (AluPCR product or PUC57 plasmid 10uL, 10 XR Buffer 2uL, EcoRI enzyme 1uL, HindIIII enzyme 1uL, water 6uL), and the digested product was purified with PCR product purification column to obtain 20uL digested product. The cleavage products were reacted with T4 ligase at 22 ℃ for 12 hours (Alu cleavage product 5uL, pUC57 plasmid 2uL, T4 ligase 1uL, 10 XBuffer 1uL, water 1uL), followed by screening, amplification and purification by standard genetic engineering techniques, and confirmed by DNA sequencing of the plasmid that Alu had been ligated to the plasmid PUC57 vector. The purified plasmid was subjected to an absorbance measurement of 260nM and the copy number of the plasmid was calculated.
Example 3
The kit of the invention is used for Real-time fluorescent quantitative PCR (Real-time PCR) detection. Firstly, the required amount of each reagent is calculated according to the number of samples to be tested, and the test steps are as follows:
1. in addition to the 5 standard and specimen duplicate wells, a negative control and a positive control were tested.
2. 5 tubes of reaction solution (Alu-Alu5) were prepared in the same order and amounts: 340uL of water, 40uL of reagent A, 500uL of reagent B and 20uL of reagent C are added, and experiments with 20 reaction wells can be performed per tube of reaction solution.
3. The reaction solution is mixed evenly, and 45uL of the reaction solution is sucked each time and added into a PCR reaction tube with 20 holes.
4. 5uL of standard, specimen, negative and positive controls (below) were added to each tube in order.
5. After the cover is covered, the tube is put into a fluorescent quantitative PCR instrument for detection. The program design is 95 ℃ 30s, 95 ℃ 5s, 60 ℃ 34s, total 40 cycles, the fluorescence signal value at 60 ℃ is detected, the amplification curve is shown in figure 6-10. The melting curve collection program was designed to 95 ℃ for 15s, 60 ℃ for 60s, 95 ℃ for 15s, 1 cycle, and the melting curves for Alu1-Alu5 are shown in FIGS. 11-15.
6. And (4) calculating a result: and recording Ct values of different standards and drawing a standard curve (shown in figures 1-5) by analysis software of a fluorescent quantitative PCR instrument, and calculating the copy number of Alu1-Alu5 of each sample by calculating the standard curve. The quality control of the experimental process is carried out by a negative control and a positive control.
Claims (3)
1. The primer group of human peripheral blood circulation DNA is characterized in that the primer group consists of five pairs of specific primers for amplifying Alu, and the specific primers are respectively used for different length segments of Alu conserved sequences; the primers are respectively as follows:
the first pair of upstream primers Alu 1-F: 5'-aga cca tcc tgg cta aca cg-3' the flow of the air in the air conditioner,
the first downstream primer pair Alu 1-R: 5'-aga cgg agt ctc gct ctg tc-3', respectively;
the second pair of upstream primers Alu 2-F: 5'-cta aca cgg tga aac ccc g-3' the flow of the air in the air conditioner,
the second pair of downstream primers Alu 2-R: 5'-cgc cca ggc tgg agt g-3', respectively;
the third pair of upstream primers Alu 3-F: 5'-gtg aaa ccc cgt ctc tac taa aaa-3' the flow of the air in the air conditioner,
the third pair of downstream primers Alu 3-R: 5'-gag tgc agt ggc gcg at-3', respectively;
the fourth pair of upstream primers Alu 4-F: 5'-tac aaa aaa tta gcc ggg cg-3' the flow of the air in the air conditioner,
the fourth pair of downstream primers Alu 4-R: 5'-gat ctc ggc tca ctg caa g-3', respectively;
the fifth pair of upstream primers Alu 5-F: 5'-aaa att agc cgg gcg tg-3' the flow of the air in the air conditioner,
the fifth pair of downstream primers Alu 5-R: 5'-gttcac gcc att ctc ctg c-3' are provided.
2. A kit for quantitative determination of human peripheral blood circulating DNA comprising the primer set according to claim 1, characterized by comprising the following reagents:
reagent A1: dissolving the first upstream primer pair Alu1-F with the concentration of 5 μ M and the first downstream primer pair Alu1-R with the concentration of 5 μ M in water;
reagent A2: dissolving the second upstream primer pair Alu2-F with the concentration of 5 μ M and the second downstream primer pair Alu2-R with the concentration of 5 μ M in water;
reagent A3: dissolving the third upstream primer pair Alu3-F with the concentration of 5 μ M and the third downstream primer pair Alu3-R with the concentration of 5 μ M in water;
reagent A4: dissolving the 5 mu M fourth pair of upstream primers Alu4-F and 5 mu M fourth pair of downstream primers Alu4-R in water;
reagent A5: dissolving the 5 mu M fifth pair of upstream primers Alu5-F and 5 mu M fifth pair of downstream primers Alu5-R in water;
and (3) reagent B: 2 XSSYBR Green qPCR Mix containing 400. mu.M dNTPs including dATP, dCTP, dGTP and dTTP, 100mM KCl, 4mM MgCl20.2U/. mu.l Taq DNA polymerase and 2 XSSYBR Green;
and (3) reagent C: ROX 50X or ROXII 50X, protected from light;
and (3) reagent D: the prepared standard substance is diluted to 1 × 10 by gradient10、1×109、1×108、1 ×107And 1X 106copy/mL, used for standard curve formulation;
and (3) reagent E: a negative control, which is normal human serum;
reagent G: a positive control which employs 1X 107copies/mL of standard.
3. The kit for quantitatively detecting human peripheral blood circulation DNA according to claim 2, wherein the standard substance is
Diluting into the purified plasmids of the Alu gene segment SEQ ID NO.13 with different concentrations; the preparation process of the purified plasmid comprises the steps of obtaining a target fragment after Alu amplification and connecting the target fragment with a PUC57 vector; amplifying Alu by using primers released by SEQ ID NO.11 and SEQ ID NO.12 to obtain Alu target fragments; wherein the amplification program is 95 ℃ for 2 min; 35 cycles of 94 ℃ 20sec, 55 ℃ 30sec, 72 ℃ 30 sec; the amplification system contained 400. mu.M dATP, dCTP, dGTP, dTTP, 100mM KCl, 4mM MgCl20.2U/. mu.l Taq DNA polymerase; the Alu target fragment and the PUC57 plasmid are cut by restriction enzymes EcoRI and HindIII, and the cutting reaction system is as follows: alu PCR product or pUC57 plasmid 10uL, 10 XR Buffer 2uL, EcoRI enzyme 1uL, HindIIII enzyme 1uL, water 6 uL; reacting the enzyme digestion reaction system at 37 ℃ for more than 2 hours, and purifying the enzyme digestion product by using a PCR product purification column to obtain 20uL purified enzyme digestion product; and reacting the purified enzyme-cleaved product for 12 hours at 22 ℃ by using T4 ligase, and connecting the reaction system: 5uL of Alu enzyme digestion product, 2uL of PUC57 plasmid enzyme digestion product, 1uL of T4 ligase, 10 Xbuffer 1uL and 1uL of water; subsequently, screening, amplification and purification were carried out by standard genetic engineering techniques, and it was confirmed by DNA sequencing of the plasmid that Alu had been ligated to the plasmid PUC57 vector; the purified plasmid was subjected to an absorbance measurement of 260nM and the copy number of the plasmid was calculated.
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| 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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| 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 |
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