CN111235228A - Method for detecting cancer marker CA125 in blood based on polymerase chain reaction and dynamic light scattering - Google Patents
Method for detecting cancer marker CA125 in blood based on polymerase chain reaction and dynamic light scattering Download PDFInfo
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Abstract
The invention discloses a method for detecting a cancer marker CA125 in blood based on polymerase chain reaction and dynamic light scattering, wherein when the cancer marker CA125 exists, the cancer marker CA125 can be combined with an aptamer sequence thereof, so that the aptamer can not be combined and extended with partial complementary strand under the action of exo-enzyme and can not be used as a template of PCR reaction. In contrast, a PCR reaction can be initiated. The PCR product can then hybridize to DNA-modified gold nanoparticle probes, resulting in aggregation of the gold nanoparticles. And (3) measuring the change of the dynamic light scattering signal (particle size) of the gold nanoparticles, and realizing high-sensitivity quantitative analysis on the content of the cancer marker. The invention has simple operation, low detection cost, less required samples, high sensitivity and good specificity, has different responses to various cancer patients, and provides a new way for noninvasive diagnosis and postoperative monitoring of cancers.
Description
Technical Field
The invention relates to a method for detecting a cancer marker CA125 in blood, belongs to the technical field of cancer noninvasive diagnosis, and particularly relates to a method for detecting the cancer marker CA125 in blood based on polymerase chain reaction and dynamic light scattering.
Background
Ovarian cancer is a gynecological disease with the first incidence of gynecological malignancies, with over 15.1 million patients worldwide each year. The survival of cancer patients is closely related to the diagnosis and treatment stage of tumors. Therefore, a highly sensitive method for early diagnosis and post-operative monitoring of tumors is urgently needed. Cancer antigen-125 (Cancer antigen-125, CA125), also known as Mucin16(MUC 16), is a high molecular weight glycoprotein (>200kDa) encoded by the MUC16 gene, first discovered and reported by American scientists in 1981, and is widely used as a serum biomarker for diagnosis and monitoring of epithelial ovarian Cancer. Various methods for detecting CA125 have been reported, including radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), Photoluminescence (PL), chemiluminescence, electrochemiluminescence, piezoelectric biosensing, electrochemical biosensing, and the like. The enzyme-linked immunosorbent assay (ELISA) and the electrochemiluminescence method are mainly adopted to detect the CA125 clinically, the kit is monopolized by foreign companies such as Roche diagnosis and the like, and the kits produced by domestic companies lack competitiveness.
Aptamers are a series of single-stranded dna (ssdna) or RNA sequences that have been screened in vitro by SELEX (systematic evolution by exponential enrichment of ligands) for specific binding capacities to target proteins, cells, etc. In recent years, aptamers have been widely used in biosensors, and aptamers against CA125 were first reported in 2012.
Polymerase Chain Reaction (PCR) replication 10 from a nucleic acid6-109The target molecules can be detected by fluorescence, chemiluminescence, electrochemistry, colorimetry, Circular Dichroism (CD) spectroscopy and the like. Gold nanoparticles (AuNPs) are one of the most commonly used nanomaterials, and their particle size changes with changes in aggregation state, which can be detected by Dynamic Light Scattering (DLS), which measures particle sizes between 0.5nm and 6 μm in diameter. Thus, DLS can be combined with PCR, and highly sensitive detection of target molecules can be achieved.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a method for detecting a cancer marker CA125 in blood based on polymerase chain reaction and dynamic light scattering, which has the advantages of simple operation, low detection cost, less required samples, high sensitivity and good specificity.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a method for detecting a cancer marker CA125 in blood based on polymerase chain reaction and dynamic light scattering, which comprises the following steps:
1) sequence design:
designing two oligonucleotide sequences with partially complementary parts by taking a cancer marker CA125 as a recognition target; the oligonucleotide sequences are respectively sequence 1 containing aptamer 5'-TAATACGACTCACTA TAGGGAGACAAGAATAAACGCTCAA-3' and sequence 2 containing a partial complementary strand thereof;
designing a pair of forward and reverse bidirectional PCR primers containing hairpin structures;
designing a sequence 3 and a sequence 4 which can be respectively hybridized with the forward and reverse bidirectional PCR primers after the hairpin structure is opened;
2) preparation of DNA-modified gold nanoparticles:
preparation of AuNPs: adding the freshly prepared 38.8mM sodium citrate solution into 10 times of the boiled 1mM HAuCl4 solution, changing the reaction liquid from light yellow to black, then to purple, and finally to wine red, continuously heating and refluxing, and stirring for 1-100 minutes; finally, the reaction solution is cooled to room temperature and kept stirring, then is filtered by a nylon filter membrane with the diameter of 0.1-5.0 μm, and is stored in a refrigerator with the temperature of 4 ℃ for standby;
AuNPs surface-modified DNA: thiol-modified DNA sequences 3, 4 were first treated with tris (2-chloroethyl) phosphate TCEP followed by a 200: 1, incubating at room temperature for 1-500 hours, centrifuging the reaction solution at the speed of 1-100000rpm for 2-60 minutes to remove free DNA, dissolving the obtained oily precipitate in 10mM PBS buffer (pH 7.4, 0.1M NaCl) to obtain two gold nanoparticle probes, namely AuNPs-3probe and AuNPs-4probe, and storing in a refrigerator at 4 ℃ for later use;
3) blood detection:
extending blood containing a marker to be detected, a sequence 1 and a sequence 2 under the action of exo-polymerase to obtain a new double-stranded DNA template; then adding a forward and reverse bidirectional PCR primer, Taq DNA polymerase and dNTP system for PCR reaction to obtain a PCR product;
adding two gold nanoparticle probes AuNPs-3probe and AuNPs-4probe into the PCR product, and hybridizing with the PCR product to aggregate the gold nanoparticles;
and detecting the dynamic light scattering intensity.
Further, in step 1), the forward and reverse bidirectional PCR primers are designed into a hairpin structure, and an arm between C1 and C1000 is introduced into a loop part.
Further, in step 1), two partially complementary oligonucleotide sequences are designed, wherein one of the oligonucleotide sequences comprises 5'-TAATAC GACTCACTATAGGGAGACAAGAATAAACGCTCAA-3' aptamer sequence capable of binding to CA125 and other sequences of 1-1000 bases; the other oligonucleotide sequence comprises an oligonucleotide sequence which is fully or partially complementary to a CA125 aptamer.
Further, in the step 1), the sequences of the two oligonucleotides are respectively shown as SEQ ID No.1 and SEQ ID No. 2.
Further, in step 1), the designed forward and reverse bidirectional PCR primers are respectively:
forward primer 5 '-GGGAGAGAAGAACT spacer18 AGTTCTTCTCTCCCGACAGGCCCGAAGGAATAGA-3';
the reverse primer 5 '-GAGGAA GGA AAG CT spacer18AGCTTTCCTTCCTCC TCTCTCTCCACCTTCTTCT-3'.
Further, in step 2), the thiol-modified DNA sequences 3 and 4 are first treated with tris (2-chloroethyl) phosphate TCEP, followed by treatment with 200: 1, incubating at room temperature for 1-20 hours, and adding 0.2-10M NaCl in portions during the incubation period to make the NaCl concentration reach 0.01-1.0M.
Further, in step 3), the specific steps of combining the DNA to be detected with the aptamer and extending are as follows: 0.5U exo-Polymerase, 1. mu.L dNTPs,0.2M sequence 1 (part of aptamer), 0.2M sequence 2 (part of complementary strand of aptamer) and the DNA to be tested are reacted at 10-60 deg.C for 0.1-50h, heated to 65-99 deg.C to inactivate exo-A polymerase.
Further, in the step 3), the PCR product obtained by the PCR reaction has single-stranded DNA fragments at two ends, and the single-stranded DNA fragments at two ends can be respectively hybridized with the gold nanoparticle probes AuNPs-3probe and AuNPs-4 probe.
Further, when the PCR reaction is initiated in the absence of CA125, the particle size is about 100-10000 nm; when an excessive amount of CA125 was present, the PCR reaction could not be carried out, and the particle size was about 10-65 nm.
Further, the linear range of the dynamic light scattering signal was 5.0 fg. mL-1-50ng·mL-1The detection limit of the cancer marker CA125 was 1.1 fg. mL-1。
Compared with the prior art, the invention has the following beneficial effects:
the method has the advantages of simple operation, low detection cost, less required samples, high sensitivity and good specificity. Meanwhile, the kit is used for detecting blood of a plurality of cancer patients, and the result shows that the kit has different responses to the cancer patients and specific response to ovarian cancer patients, thereby providing a new way for noninvasive diagnosis and postoperative monitoring of ovarian cancer.
Drawings
FIG. 1 is a schematic diagram of a method for detecting the content of CA125 in blood based on PCR and dynamic light scattering according to the present invention;
FIGS. 2A and 2B are graphs showing particle size distributions of different samples analyzed by dynamic light scattering according to the present invention;
FIG. 3 is a graph showing the results of dynamic light scattering for detecting the content of the cancer marker CA125 in blood of different cancer patients in Experimental example 1 for the effects of the present invention;
FIG. 4 is a linear regression equation between the dynamic light scattering signal and the concentration in the test example 2 of the effect of the present invention.
Detailed Description
The present invention provides a new method for detecting the cancer marker CA125 in blood based on Polymerase Chain Reaction (PCR) and Dynamic Light Scattering (DLS).
As shown in FIG. 1, in the presence of CA125, the detection method of the present invention can bind to its aptamer sequence, so that the aptamer cannot be extended by binding to a partially complementary strand by exo-enzyme and thus cannot serve as a template for PCR reaction. In contrast, a PCR reaction can be initiated. The PCR product can then hybridize to DNA-modified gold nanoparticle (DNA-AuNP) probes, causing the gold nanoparticles to aggregate.
As shown in FIGS. 2A-2B, the change of the dynamic light scattering signal of the gold nanoparticles (the particle size is about 397.93nm when the PCR reaction is initiated in the absence of CA 125; the particle size is about 53.88nm when the PCR reaction cannot be performed in the presence of excessive CA125) is measured, and the high-sensitivity quantitative analysis of the content of CA125 is realized.
The linear range of the present invention is 5.0 fg. multidot.mL-1-50ng·mL-1With a detection limit of 1.1 fg. mL-1。
The invention utilizes the specificity recognition of CA125 on the aptamer thereof, takes gold nanoparticles as a probe, combines the PCR amplification technology with high-sensitivity Dynamic Light Scattering (DLS), and establishes the CA125 detection method.
The specific content of the method can be expressed as follows:
two partially complementary oligodeoxyribonucleotides were designed, one of which contained an aptamer sequence that binds CA 125. Extending under the action of exo-polymerase and generating a new double-stranded DNA template, further generating PCR amplification, hybridizing the PCR product with the gold nano particle probe and generating a larger gold nano particle aggregate. After the CA125 is added, the generation of a new double-stranded DNA template is interrupted, the PCR amplification is terminated, the particle size of the gold nanoparticles is reduced, and the high-sensitivity quantitative analysis of the CA125 is realized.
The method is suitable for blood detection of patients with ovarian cancer, and can be used for early diagnosis of ovarian cancer.
The technical scheme of the invention comprises the following steps:
1) preparation of DNA-modified gold nanoparticles (AuNPs)
AuNPs were prepared according to the classical sodium citrate reduction method with appropriate adjustments. 2mL of freshly prepared 38.8mM sodium citrate solution was quickly added to 20mL of boiled 1mM HAuCl4 solution, the reaction turned from pale yellow to black, then purple, and finally wine red, and heating and refluxing were continued and stirring was continued for 10 minutes. Finally, the reaction was allowed to cool to room temperature while maintaining stirring, then filtered through a 0.45 μm nylon filter and stored in a refrigerator at 4 ℃ until use.
AuNPs surface-modified DNA: thiol-modified DNA was first treated with tris (2-chloroethyl) phosphate TCEP, followed by treatment with 200: 1, incubated at room temperature for 100 hours, and added with 0.2-10M NaCl in portions to make the NaCl concentration reach 0.01-1.0M. Finally, the reaction solution was centrifuged at 13800rpm for 30 minutes to remove free DNA, and the resulting oily precipitate was dissolved in 10mM PBS buffer (pH 7.4, 0.1M NaCl) and stored in a refrigerator at 4 ℃ until use.
2) Carrying out PCR reaction
0.5U of exo-polymerase, 1. mu.L of dNTPs, 0.2M oligo1 (aptamer is part of the composition), 0.2M oligo2 and various concentrations of CA125 were reacted at 37 ℃ for 2h and heated to 95 ℃ to inactivate the exo-polymerase. The inactivated mixture is used as a PCR template for subsequent PCR amplification.
3) Mixing the PCR product with DNA-AuNP probe, and detecting the particle size change
Adding a proper amount of DNA-AuNP probe into the PCR product, aggregating the gold nanoparticles through DNA hybridization reaction, and measuring the corresponding particle size change by adopting dynamic light scattering.
The invention relates to a method for detecting a cancer marker CA125 in blood based on PCR and dynamic light scattering.
The method specifically comprises the following steps:
① PCR template extension Oligo 15' -GACAGGCCCGAAGGAATAGATAATACGACTCACTATAGGGAGA CAAGAATAAACGCTCAA-3 ' is complementary to Oligo 25 ' -CTCTCTCTCCACCTTCTTCTTTGAGCGTTTATTCTT GTCT-3 ' and is in exo-Extension by enzyme, and when CA125 exists, extension reaction is hindered to different degrees;
② PCR reaction, mixing the extension product with forward primer 5 '-GGGAGAGAAGAACT spacer18 AGTTCTTCTCTCCCGACAGGCCCGAAGGAATAGA-3' and reverse primer 5 '-GAGGAA GGA AAG CTspacer18AGCTTTCCTTCCTCC TCTCTCTCCACCTTC TTCT-3' to initiate PCR reaction;
③ hybridization, preparing two DNA-AuNP probes, adding them into PCR product, and making gold nanometer particle aggregate by DNA hybridization;
④ dynamic light scattering analysis, namely measuring the change of the dynamic light scattering signal (particle size) of the gold nanoparticles, and realizing the analysis of the content of CA125 and the early diagnosis and postoperative monitoring of ovarian cancer.
The present invention will now be described in further detail with reference to the accompanying drawings and specific examples, but the present invention is not limited to the following examples.
Example (sequence design and preparation of gold nanoparticle probes)
1. Sequence design
Two partially complementary oligonucleotide sequences (Oligo 1 containing an aptamer sequence and Oligo2 containing a sequence partially complementary to an aptamer sequence) were designed with CA125 as the recognition target, and a new double-stranded DNA template was generated after the exo-polymerase was added.
Oligo 1：5’-GACAGGCCCGAAGGAATAGATAATACGACTCACTA TAGGGAGACAAGAATAAACGCTCAA-3’；
Oligo 2：5’-CTCTCTCTCCACCTTCTTCTTTGA GCGTTTATTCTT GTCT-3’；
Designing a pair of forward and reverse bidirectional PCR primers containing specific structures corresponding to the new double-stranded DNA template: designed as a hairpin structure with a loop leading into the arms between C1-C1000.
Forward primer 5 '-GGGAGAGAAGAACT spacer18 AGTTCTTCTCTCCCGACAGGCCCGAAGGAATAGA-3';
the reverse primer 5 '-GAGGAA GGA AAG CT spacer18AGCTTTCCTTCCTCC TCTCTCTCCACCTTCTTCT-3'.
Designing a sequence Oligo3 and a sequence Oligo 4 which can be respectively hybridized with the forward bidirectional PCR primer and the reverse bidirectional PCR primer after the hairpin structure is opened;
Oligo 3：5`-TTCTTCTCTCCC-C6-SH-3`；
Oligo 4：5`-CTTTCCTTCCTC-C6-SH-3`。
preparation of DNA-modified gold nanoparticles (AuNPs)
Preparation of AuNPs: 2mL of freshly prepared 38.8mM sodium citrate solution was added rapidly to 20mL of boiled 1mM HAuCl chloroaurate4In the solution, the reaction solution changed from light yellow to black, then changed to purple, and finally changed to wine red, and the solution was continuously heated under reflux and stirred for 10 minutes. Finally, the reaction was allowed to cool to room temperature while maintaining stirring, then filtered through a 0.45 μm nylon filter and stored in a refrigerator at 4 ℃ until use.
AuNPs surface-modified DNA: thiol-modified DNAs Oligo3, Oligo 4 were treated with tris (2-chloroethyl) phosphate TCEP followed by treatment with 200: 1 was added to the previously prepared AuNPs solution and incubated at room temperature for 16 hours. Finally, the reaction solution was centrifuged at 13800rpm for 30 minutes to remove free DNA, and the obtained oily precipitate was dissolved in 10mM PBS buffer (pH 7.4, 0.1M NaCl) to obtain two gold nanoparticle probes, AuNPs-Oligo3probe and AuNPs-Oligo 4probe, which were stored in a refrigerator at 4 ℃ for further use.
Effect test example 1
Method for detecting cancer marker CA125 in blood based on Polymerase Chain Reaction (PCR) and Dynamic Light Scattering (DLS):
1. extending under the action of DNA to be detected, an aptamer sequence Oligo1, a partial complementary strand sequence Oligo2 and exo-polymerase to obtain a new double-stranded DNA template; then adding a forward and reverse bidirectional PCR primer, Taq DNA polymerase and dNTP system to carry out PCR reaction, and obtaining a PCR product with single-stranded DNA fragments at two ends.
Extension: 0.5U of exo-polymerase, 1. mu.L of dNTPs, 0.2M oligo1 (aptamer is part of the composition), 0.2 Mooligo 2 and various concentrations of CA125 were reacted at 37 ℃ for 2h and heated to 95 ℃ to inactivate the exo-polymerase. The inactivated mixture is used as a PCR template for subsequent PCR amplification.
And (3) PCR reaction:
forward primer 5 '-GGGAGAGAAGAACT spacer18 AGTTCTTCTCTCCCGACAGGCCCGAAGGAATAGA-3'
Reverse primer 5 '-GAGGAA GGA AAG CT spacer18AGCTTTCCTTCCTCC TCTCTCTCCACCTTCTTCT-3'
And (3) PCR reaction conditions: initial denaturation at 94 ℃ for 3 min, (20 cycles) 94 ℃ for 15s, 52 ℃ for 20s, 72 ℃ for 30s, and final extension at 72 ℃ for 3 min.
2. Two gold nanoparticle probes, namely AuNPs-Oligo3probe and AuNPs-Oligo 4probe, are added into a PCR product and respectively hybridized with single-stranded DNAs at two ends of the PCR product, so that the gold nanoparticles are aggregated, and a strong dynamic light scattering signal (larger particle size) is generated.
CA125 can be combined with oligonucleotide chain Oligo1 containing CA125 aptamer sequence, so as to interrupt the generation of new double-stranded DNA template, further terminate PCR amplification reaction, reduce dynamic light scattering signal (particle size), and carry out high-sensitivity quantitative analysis on CA125 according to the particle size of gold nanoparticles. The change of the gold nanoparticle dynamic light scattering signal (particle size) was measured, and the dynamic light scattering intensity (particle size) decreased with the increase of the concentration of CA 125. The dynamic light scattering intensity (particle size) has a linear relation with the concentration of CA125, and can be used for the quantitative analysis of CA 125.
As shown in FIG. 3, the present invention responds differently to multiple cancer patients and specifically to ovarian cancer patients. The dynamic light scattering signals generated by the blood samples of the breast cancer, the prostate cancer, the liver cancer and the bladder cancer patients are similar to those of the control sample (and the blood sample of a normal person), which indicates that the blood samples of the cancers do not generate response in the method. The dynamic light scattering signal of the blood sample of ovarian cancer decreased dramatically, indicating that the method has a specific response to ovarian cancer.
Effect test example 2
Adding Oligo1, Oligo2, Exo at the same concentration-Respectively adding the Taq polymerase, the AuNPs-Oligo3probe and the AuNPs-Oligo 4probe into the mixture, respectively adding CA125 with different concentrations, detecting the change condition of the dynamic light scattering signal along with the concentration of the CA125, and calculating a linear regression equation between the signal and the concentration.
The results are shown in fig. 4, and a linear regression equation of Y430.48-49.60 ㏒ C (Y represents the average particle size and C represents the concentration of CA125) was obtained with a linear range of 5.0fg · mL-1-50ng·mL-1The linear correlation coefficient was 0.995, and the detection limit was 1.1fg mL-1。
The present invention is not limited to the above-described embodiments, and various changes and modifications of the present invention are intended to be included within the scope of the claims and the equivalent technology of the present invention if they do not depart from the spirit and scope of the present invention.
Sequence listing
<110> Zhongshan university
<120> method for detecting cancer marker CA125 in blood based on polymerase chain reaction and dynamic light scattering
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<213> Artificial Sequence (Artificial Sequence)
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Claims (10)
1. A method for detecting a cancer marker CA125 in blood based on polymerase chain reaction and dynamic light scattering, comprising the steps of:
1) sequence design:
designing two oligonucleotide sequences with partially complementary parts by taking a cancer marker CA125 as a recognition target; the oligonucleotide sequences are respectively sequence 1 containing aptamer 5'-TAATAC GACTCACTA TAGGGAGACAAGAATAAACGCTCAA-3' and sequence 2 containing a partial complementary strand thereof;
designing a pair of forward and reverse bidirectional PCR primers containing hairpin structures;
designing a sequence 3 and a sequence 4 which can be respectively hybridized with the forward and reverse bidirectional PCR primers after the hairpin structure is opened;
2) preparation of DNA-modified gold nanoparticles:
preparation of AuNPs: adding the freshly prepared 38.8mM sodium citrate solution into 10 times of the boiled 1mM HAuCl4 solution, changing the reaction liquid from light yellow to black, then to purple, and finally to wine red, continuously heating and refluxing, and stirring for 1-100 minutes; finally, the reaction solution is cooled to room temperature and kept stirring, then is filtered by a nylon filter membrane with the diameter of 0.1-5.0 μm, and is stored in a refrigerator with the temperature of 4 ℃ for standby;
AuNPs surface-modified DNA: thiol-modified DNA sequences 3, 4 were first treated with tris (2-chloroethyl) phosphate TCEP followed by a 200: 1 is added into the AuNPs solution prepared in advance, and the incubation is carried out for 1 to 500 hours at room temperature; then, centrifuging the reaction solution at the rotating speed of 1-100000rpm for 2-60 minutes to remove free DNA, dissolving the obtained oily precipitate in 10mMPBS buffer solution (pH 7.4, 0.1M NaCl) to obtain two gold nanoparticle probes, namely AuNPs-3probe and AuNPs-4probe, and storing the two gold nanoparticle probes in a refrigerator at 4 ℃ for later use;
3) blood detection:
extending blood containing a marker to be detected, a sequence 1 and a sequence 2 under the action of exo-polymerase to obtain a new double-stranded DNA template; then adding a forward and reverse bidirectional PCR primer, Taq DNA polymerase and dNTP system for PCR reaction to obtain a PCR product;
adding two gold nanoparticle probes AuNPs-3probe and AuNPs-4probe into the PCR product, and hybridizing with the PCR product to aggregate the gold nanoparticles;
and detecting the dynamic light scattering intensity.
2. The method of claim 1, wherein: in the step 1), the forward and reverse bidirectional PCR primers are designed into a hairpin structure, and an arm between C1 and C1000 is introduced into a ring part.
3. The method of claim 2, wherein: in the step 1), two partially complementary oligonucleotide sequences are designed, wherein one oligonucleotide sequence comprises 5'-TAATAC GACTCACTA TAGGGAGACAAGAATAAACGCTCAA-3' aptamer sequence capable of binding with CA125 and other sequences with 1-1000 bases;
the other oligonucleotide sequence comprises an oligonucleotide sequence which is fully or partially complementary to a CA125 aptamer.
4. The method of claim 3, wherein: step 1), the two oligonucleotide sequences are respectively shown as SEQ ID No.1 and SEQ ID No. 2.
5. The method of claim 3, wherein: step 1), designing forward and reverse bidirectional PCR primers respectively as follows:
forward primer 5 '-GGGAGAGAAGAACT spacer18 AGTTCT TCTCTCCCGACAGGCCCGA AGGAATAGA-3';
the reverse primer 5 '-GAGGAA GGA AAG CT spacer18AGCTTTCCTTCCTCC TCTCTCTCC ACCTTCTTCT-3'.
6. The method of claim 1, wherein: in step 2), thiol-modified DNA sequences 3 and 4 are first treated with tris (2-chloroethyl) phosphate TCEP, followed by treatment with 200: 1, incubating at room temperature for 1-20 hours, and adding 0.2-10M NaCl in portions during the incubation period to make the NaCl concentration reach 0.01-1.0M.
7. The method of claim 1, wherein: in step 3), the specific steps of combining the DNA to be detected with the aptamer for extension are as follows: 0.5U exo-Polymerase, 1 μ L dNTPs, 0.2M sequence 1 (aptamer is part of), 0.2M sequence 2 (part of the complementary strand of aptamer is part) and the DNA to be tested are reacted at 10-60 deg.C for 0.1-50h, heated to 65-99 deg.C to inactivate exo-A polymerase.
8. The method of claim 1, wherein: in the step 3), the PCR product obtained by the PCR reaction has single-stranded DNA fragments at two ends, and the single-stranded DNA fragments at the two ends can be respectively hybridized with the gold nanoparticle probes AuNPs-3probe and AuNPs-4 probe.
9. The method of claim 1, wherein: when CA125 does not exist, the PCR reaction is initiated, and the particle size is 100-10000 nm; when excessive CA125 exists, PCR reaction can not be carried out, and the particle size is 10-65 nm.
10. The method of claim 1, wherein: the linear range of the dynamic light scattering signal is 5.0 fg. mL-1-50ng·mL-1The detection limit of the cancer marker CA125 was 1.1 fg. mL-1。
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| CN111879738A (en) * | 2020-06-15 | 2020-11-03 | 山东师范大学 | Non-labeled aptamer probe system and detection method and application thereof |
| CN112698020A (en) * | 2020-11-12 | 2021-04-23 | 中山大学 | Multimodal coupling analysis method of cross response system based on DNA-AuNP coding |
| CN114034692A (en) * | 2021-10-26 | 2022-02-11 | 中山大学 | Formaldehyde colorimetric detection method based on metal nanoparticles and triple helix chain and application |
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| CN111879738A (en) * | 2020-06-15 | 2020-11-03 | 山东师范大学 | Non-labeled aptamer probe system and detection method and application thereof |
| CN111879738B (en) * | 2020-06-15 | 2023-03-14 | 山东师范大学 | Non-labeled aptamer probe system and detection method and application thereof |
| CN112698020A (en) * | 2020-11-12 | 2021-04-23 | 中山大学 | Multimodal coupling analysis method of cross response system based on DNA-AuNP coding |
| CN114034692A (en) * | 2021-10-26 | 2022-02-11 | 中山大学 | Formaldehyde colorimetric detection method based on metal nanoparticles and triple helix chain and application |
| CN114034692B (en) * | 2021-10-26 | 2023-05-02 | 中山大学 | Formaldehyde colorimetric detection method based on metal nano particles and triple helix chains and application thereof |
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