CN117448422B - Method for enriching cfDNA in urine based on biotin double probes - Google Patents
Method for enriching cfDNA in urine based on biotin double probes Download PDFInfo
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 title claims abstract description 310
- 239000000523 sample Substances 0.000 title claims abstract description 253
- 229960002685 biotin Drugs 0.000 title claims abstract description 179
- 239000011616 biotin Substances 0.000 title claims abstract description 179
- 235000020958 biotin Nutrition 0.000 title claims abstract description 155
- 238000000034 method Methods 0.000 title claims abstract description 47
- 210000002700 urine Anatomy 0.000 title claims abstract description 47
- 108010090804 Streptavidin Proteins 0.000 claims abstract description 160
- 239000011324 bead Substances 0.000 claims abstract description 153
- 238000010438 heat treatment Methods 0.000 claims abstract description 12
- 238000005406 washing Methods 0.000 claims abstract description 8
- 239000000243 solution Substances 0.000 claims description 55
- 238000002360 preparation method Methods 0.000 claims description 45
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 38
- 150000003839 salts Chemical class 0.000 claims description 26
- 239000006228 supernatant Substances 0.000 claims description 26
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 20
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 20
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 20
- 239000011780 sodium chloride Substances 0.000 claims description 19
- 230000009870 specific binding Effects 0.000 claims description 19
- 229960000789 guanidine hydrochloride Drugs 0.000 claims description 18
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 claims description 18
- 239000000872 buffer Substances 0.000 claims description 17
- 238000011534 incubation Methods 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 17
- 238000002156 mixing Methods 0.000 claims description 16
- 239000000725 suspension Substances 0.000 claims description 16
- 239000007853 buffer solution Substances 0.000 claims description 14
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 13
- 150000001615 biotins Chemical class 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 11
- 239000002904 solvent Substances 0.000 claims description 10
- 238000004925 denaturation Methods 0.000 claims description 8
- 230000036425 denaturation Effects 0.000 claims description 8
- 230000000295 complement effect Effects 0.000 claims description 6
- 238000001816 cooling Methods 0.000 claims description 4
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- 108020004414 DNA Proteins 0.000 description 14
- 238000001514 detection method Methods 0.000 description 14
- 230000000052 comparative effect Effects 0.000 description 12
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 10
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- 238000005352 clarification Methods 0.000 description 7
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- 238000003260 vortexing Methods 0.000 description 7
- 108700001666 APC Genes Proteins 0.000 description 5
- 238000010828 elution Methods 0.000 description 5
- 238000000605 extraction Methods 0.000 description 5
- 239000000377 silicon dioxide Substances 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 108020004682 Single-Stranded DNA Proteins 0.000 description 3
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- 238000001179 sorption measurement Methods 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 101710163270 Nuclease Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
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- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- -1 ferroferric oxide modified biotin Chemical class 0.000 description 2
- 238000003505 heat denaturation Methods 0.000 description 2
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- 230000008569 process Effects 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
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- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
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- 230000009977 dual effect Effects 0.000 description 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N ferrosoferric oxide Chemical compound O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
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- 238000007886 magnetic bead extraction Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000004005 microsphere Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
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- 238000011056 performance test Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
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Abstract
The application relates to the technical field of cfDNA enrichment, and particularly discloses a method for enriching cfDNA in urine based on biotin double probes. The method comprises the following steps: s1, hybridizing a target cfDNA with a biotin double-probe streptavidin magnetic bead to obtain the biotin double-probe streptavidin magnetic bead with the captured cfDNA; s2, washing the biotin double-probe streptavidin magnetic beads with the captured cfDNA; s3, heating and denaturing the biotin double-probe streptavidin magnetic beads with the target cfDNA captured, incubating, and recovering to obtain the target cfDNA; the biotin double-probe streptavidin magnetic beads comprise streptavidin magnetic beads, a double-biotin-marked probe BP1 and a double-biotin-marked probe BP2. The method provided by the application can be used for efficiently enriching the small fragment cfDNA in the urine sample.
Description
Technical Field
The application relates to the technical field of cfDNA enrichment, in particular to a method for enriching cfDNA in urine based on biotin double probes.
Background
The existing enrichment method for cfDNA comprises the following steps: 1. extracting and obtaining total cfDNA in a sample, wherein the method specifically comprises the steps of combining cfDNA in a target sample with magnetic microspheres, and then eluting to obtain purified total cfDNA; 2. and (3) performing end repair and linker ligation on the total cfDNA, hybridizing with a probe, and performing PCR amplification, purification and elution to obtain the target cfDNA.
However, the following disadvantages exist when enriching cfDNA in the above-described way: in cfDNA extraction, the way of magnetic bead extraction is limited by the reaction system, so that cfDNA extraction of a large-volume sample is not suitable, and extraction of trace cfDNA is difficult to achieve by the way.
There are also methods of extracting cfDNA based on silica among the conventional methods, but it is achieved that cfDNA extraction is achieved by adsorption of cfDNA onto silica. The forces of DNA and silica adsorption are: hydrophobic interactions caused by dehydration of silica and DNA surfaces and hydrogen bonding between silica and DNA backbone. That is, the magnitude of the force is proportional to the length of the DNA fragment, which directly results in a DNA fragment of less than 50-100bp being difficult to recover. However, since nuclease is rich in urine and DNA half-life period is short, ctDNA from tumor often exists in urine in the form of short cfDNA, so that the enrichment of the short cfDNA in urine can effectively improve the accuracy, sensitivity and other performances of subsequent detection.
In summary, there is a difficulty in achieving efficient enrichment of target cfDNA in a large volume, low cfDNA concentration sample when cfDNA is enriched with existing methods.
Disclosure of Invention
In order to improve the cfDNA enrichment effect in a large-volume sample with low cfDNA concentration, the application provides a method for enriching cfDNA in urine based on biotin double probes.
The application provides a method for enriching cfDNA in urine based on biotin double probes, which adopts the following technical scheme: a method for enriching cfDNA in urine based on biotin double probes comprises the following steps:
S1, hybridizing a target cfDNA with a biotin double-probe streptavidin magnetic bead to obtain the biotin double-probe streptavidin magnetic bead with the captured cfDNA;
s2, washing the biotin double-probe streptavidin magnetic beads captured with cfDNA to remove non-target cfDNA, and obtaining purified biotin double-probe streptavidin magnetic beads captured with target cfDNA;
S3, heating and denaturing the purified biotin double-probe streptavidin magnetic beads with the target cfDNA captured, cooling, incubating, centrifuging, and taking a supernatant to recover the target cfDNA;
The biotin double-probe streptavidin magnetic bead comprises a streptavidin magnetic bead, a double-biotin-marked probe BP1 connected to the streptavidin magnetic bead and a double-biotin-marked probe BP2 connected to the streptavidin magnetic bead.
By adopting the technical scheme, in the cfDNA enrichment method, the double-biotin double-probe streptavidin magnetic beads are introduced to realize enrichment. The dual biotin in the biotin dual-probe streptavidin magnetic beads directly improves the thermal stability of the probe and the streptavidin magnetic beads, so that when the biotin dual-probe streptavidin magnetic beads are used for enriching cfDNA, the total cfDNA in a sample does not need to be extracted first and then the probe is used for capturing the target cfDNA. The application can mix the biotin double-probe streptavidin magnetic beads with a sample containing target cfDNA, then directly denature the mixture, and capture the target cfDNA at room temperature, thereby improving the efficiency. In addition, the combination of the bisbiotin and the streptavidin is used as a bridge for stably connecting the probe and the magnetic bead, so that the connection stability of the probe and the magnetic bead is higher. In addition, the two probes are respectively and stably connected to the streptavidin magnetic beads by the double biotin, so that the plurality of probes can be distributed on the surfaces of the streptavidin magnetic beads in a better space to reduce the steric hindrance between the probes, the connection stability of the probes and the magnetic beads is further improved, and the probe density is increased. When the biotin double-probe streptavidin magnetic beads are used for enriching small-fragment cfDNA in a large-volume sample with low cfDNA concentration, the biotin double-probe streptavidin magnetic beads have the advantages of high targeting, strong specificity and high recovery rate. Furthermore, the enrichment method will not be limited by how much cfDNA is contained in the sample and the fragment size of the cfDNA. In addition, when the biotin double-probe streptavidin magnetic beads are used for cfDNA enrichment, probes are pre-fixed on the streptavidin magnetic beads containing biotin, so that the problem that endogenous biotin in a sample competes with the probes for the streptavidin magnetic beads is avoided, more probes can be connected to the streptavidin magnetic beads, and enrichment efficiency is further improved.
Optionally, the preparation method of the biotin double-probe streptavidin magnetic beads comprises the following steps: mixing the double biotin-labeled probe BP1, the double biotin-labeled probe BP2 and the streptavidin magnetic bead suspension so that the final concentration of the double biotin-labeled probe BP1 is 45-55 mu M and the final concentration of the double biotin-labeled probe BP2 is 45-55 mu M;
incubating and removing the solution to obtain the biotin double-probe streptavidin magnetic beads.
Further alternatively, the incubation time for preparing the biotin double probe streptavidin magnetic beads is 10-15min.
Alternatively, the probe BP1 comprises polyA and a short sequence specific binding sequence A complementary to the target, and the probe BP2 comprises polyA and a short sequence specific binding sequence B complementary to the target; the short sequence specific binding sequence A is 15-20nt, the short sequence specific binding sequence B is 15-20nt, and the polyA is 15-25nt.
In the present application, the length of polyA is 15-25nt, for example: may be 15nt, 18nt, 20nt, 23nt or 25nt. The length of the short sequence specific binding sequence A is 15-20nt, for example: may be 15nt, 16nt, 17nt, 18nt, 19nt or 20nt. The length of the short sequence specific binding sequence B is 15-20nt, for example: may be 15nt, 16nt, 17nt, 18nt, 19nt or 20nt.
Further alternatively, the 3' ends of the probe BP1 and the probe BP2 are both connected with a C3 blocker.
The probe to be used in the present application is not limited to the following examples, as long as it is a short sequence that can be complementary to a target and specifically binds thereto.
Optionally, when the target cfDNA is derived from urine, the sequence of the short sequence specific binding sequence a is: CTCAACCAATAAAACCTACTCCTCC; the sequence of the short sequence specific binding sequence B is as follows: CTAACTTTAAACRCTAACAAACGC.
In the present application, the sequence of the short sequence specific binding sequence A is shown as SEQ ID NO.1, and the sequence of the short sequence specific binding sequence B is shown as SEQ ID NO.2. R in the short sequence specific binding sequence B refers to a degenerate base, specifically to A or G).
Optionally, the preparation method of the biotin double-probe streptavidin magnetic beads comprises the following steps: I. mixing the streptavidin magnetic beads with a salt buffer solution to obtain a streptavidin magnetic bead suspension, wherein the volume ratio of the streptavidin magnetic beads to the salt buffer solution is 1 (0.9-1.1);
Mixing the double biotin-labeled probe BP1, the double biotin-labeled probe BP2, the ferroferric oxide-modified biotin and the streptavidin magnetic bead suspension so that the final concentration of the double biotin-labeled probe BP1 is 45-55 mu M, the final concentration of the double biotin-labeled probe BP2 is 45-55 mu M, and the final concentration of the ferroferric oxide-modified biotin is 15-20 mu M; after incubation, removing the solution to obtain initial biotin double-probe streptavidin magnetic beads;
II. Mixing and stirring the initial biotin double-probe streptavidin magnetic beads and guanidine hydrochloride solution with the pH of 1.2-1.8-6M, and sequentially replacing the guanidine hydrochloride solution with the pH of 1.2-1.8-6M, guanidine hydrochloride solution with the pH of 4-5M, guanidine hydrochloride solution with the pH of 3-4M and water, so as to obtain the biotin double-probe streptavidin magnetic beads after water removal.
Only mixing the double biotin-marked probe BP1, the double biotin-marked probe BP2 and the streptavidin magnetic beads, and then enriching cfDNA; after the cfDNA is detected, the delta Ct value of the obtained related gene fragment is unstable, and the difference of the delta Ct values between repeated samples is more than or equal to 1; that is, the stability between parallel samples is poor when cfDNA enriched by this method is detected.
Streptavidin is a nonglycosylated tetrameric protein consisting of four subunits, each with a binding site for biotin, with very strong affinity for biotin. After mixing the double biotin-labeled probe directly with streptavidin magnetic beads, it is possible that all four biotin binding sites on one streptavidin molecule are occupied, one of the most likely cases being: two double biotin labelled probes are simultaneously attached to one streptavidin molecule. In this case, two probes may fail to capture the target cfDNA after capturing the target cfDNA due to the relatively strong steric hindrance between the probes; and some streptavidin molecules are only combined with one probe, so that the steric hindrance is small, and the target cfDNA can be captured successfully. Therefore, when preparing biotin double-probe streptavidin magnetic beads, the results of four subunit binding probes on streptavidin are uncertain, and the uncertainty leads to large difference of detection results between parallel samples and poor repeatability when cfDNA enriched from samples of the same source is used for detection.
By adopting the technical scheme, the biotin double-probe streptavidin magnetic beads are obtained by mixing the double-biotin-labeled probe BP1, the double-biotin-labeled probe BP2 and the ferroferric oxide-modified biotin together and then performing gradient elution by using a guanidine hydrochloride solution. After the magnetic beads are used for enriching the target cfDNA, the delta Ct value of the same sample is smaller than 1 when the enriched cfDNA is detected, namely the stability among the samples is good. The reason for this may be that the magnetic beads obtained in this way, one streptavidin molecule is linked to one probe, occupying two biotin binding sites of streptavidin; while the other two biotin binding sites are occupied by ferroferric oxide modified biotin. Therefore, when the biotin double-probe streptavidin magnetic beads are used for enriching the target cfDNA, the relative steric hindrance between the probes is reduced, so that the probes can successfully capture the cfDNA and enrich.
And after three biotin markers, namely a double biotin-marked probe BP1, a double biotin-marked probe BP2 and ferroferric oxide-modified biotin, are contacted with the streptavidin magnetic beads together, one double biotin-marked probe and one ferroferric oxide-modified biotin are combined on each streptavidin molecule, so that the combination of the two probes is avoided. The advantages are that: 1. the problem of capture failure caused by large steric hindrance between probes after two probes are combined on one streptavidin molecule is avoided; 2. the binding mode of the biotin marker and the streptavidin is relatively consistent, namely, one probe and one ferroferric oxide are combined on one streptavidin molecule to modify biotin. So that a relatively stable detection result between repeated samples can be obtained finally. And the process of gradient elution by guanidine hydrochloride solution further elutes the combined unstable double biotin labeled probe and the ferroferric oxide modified biotin so as to ensure the stability of the final detection result.
Optionally, the initial biotin double probe streptavidin magnetic beads are mixed and stirred with guanidine hydrochloride solution with pH of 1.2-1.8-6M in a volume ratio of 0.8-1.2:1.
By adopting the technical scheme, the initial biotin double-probe streptavidin magnetic beads and guanidine hydrochloride solution with the pH of 1.2-1.8-6M are mixed in a proper proportion, so that a better elution effect is achieved, and meanwhile, the combination stability of the initial biotin double-probe streptavidin magnetic beads is not affected.
Optionally, the composition of the salt buffer comprises: 0.8-1.2M NaCl,8-13mM Tris-HCl,0.03-0.07wt% Tween-20, and water as solvent.
In the hydrochloric acid buffer of the present application, the concentration of NaCl is 0.8-1.2M, for example: may be 0.8M, 0.9M, 1.0M, 1.1M or 1.2M. Tris-HCl concentration is 8-13mM, for example: may be 8mM, 10mM, 11mM or 13mM. Tween-20 is present at a concentration of 0.03 to 0.07wt%, for example: may be 0.03wt%, 0.04wt%, 0.05wt%, 0.06wt% or 0.07wt%.
Optionally, the step S1 specifically includes the following steps: mixing the target cfDNA, the incubation solution A and the biotin double-probe streptavidin magnetic beads, and heating and denaturing for 12-18min at 88-92 ℃; and mixing for 25-35min at room temperature, and centrifuging to obtain the biotin double-probe streptavidin magnetic beads with cfDNA captured.
In the present application, the temperature of the denaturation by heating in S1 is 88 to 92℃and may be, for example, 88℃89.5℃91℃or 92 ℃; the time for heat denaturation is 12-18min, and may be, for example, 12min, 13.5min, 15min, 16.5min or 18min.
Further optionally, the components of the incubation liquid a include: 4-6M NaCl solution and 8-12wt% Tween-20 solution; wherein the volume ratio of the NaCl solution to the Tween-20 solution is (18.5-20.5): 1.
By "8-12wt% tween-20 solution" is meant that the tween-20 solution is formulated such that the mass percentage of tween-20 in the tween-20 solution is 8-12%.
The volume ratio of NaCl solution to Tween-20 solution in the composition of incubation liquid A of the application is (18.5-20.5): 1, for example, the volume ratio of NaCl solution to Tween-20 solution may be 18.5:1, 19:1, 19.5:1, 20:1 or 20.5:1.
Further alternatively, the volume ratio of said incubation liquid A to said biotin double probe streptavidin magnetic beads is (48-56): 1.
The volume ratio of the incubation liquid A of the application and the biotin double probe streptavidin magnetic beads is (48-56): 1, for example, the volume ratio can be 48:1, 50:1, 52:1, 54:1 or 56:1.
Optionally, the washing in S2 comprises the steps of:
Washing the double-biotin double-probe streptavidin magnetic beads captured with cfDNA with the salt buffer solution, and then washing with a low-salt buffer solution to obtain the double-biotin double-probe streptavidin magnetic beads; the components of the low-salt buffer solution comprise: 12-18mM NaCl,7-14mM Tris-HCl, and water as solvent.
The concentration of NaCl in the low-salt buffer of the present application is 12-18mM, for example, the concentration may be 12mM, 14mM, 16mM or 18mM; tris-HCl concentration is 7-14mM, for example, the concentration may be 7mM, 9mM, 11mM or 14mM.
Optionally, the temperature of heating denaturation in S3 is 92-98 ℃, and the time of heating denaturation is 1-4min.
In the present application, the temperature of the denaturation by heating in S3 is 92-98deg.C, for example, 92 ℃, 94 ℃, 96 ℃ or 98 ℃; the time for heat denaturation is 1-4min, and may be, for example, 1min, 1.8min, 2.6min, 3.2min or 4min.
Optionally, cooling in S3 refers to placing the heated denatured sample on ice to achieve a cooled low temperature environment.
The method is suitable for enriching cfDNA, especially for enriching small fragments cfDNA (such as cfDNA fragments of 50-100bp and even 20-50 bp) with low cfDNA content and large sample volume. The sample may be a urine sample but is not limited to just a urine sample. The enriched cfDNA obtained from the urine sample can be used for detecting and identifying diseases such as prostate cancer, urothelial cancer and the like, but is not limited to the detection and identification of the diseases.
In summary, the application has the following beneficial effects:
1. in the biotin double-probe streptavidin magnetic bead, the introduction of the double biotin directly improves the thermal stability of the probe and the streptavidin magnetic bead, so that the magnetic bead can be directly added into a sample for denaturation and hybridization with target cfDNA, the time for cfDNA extraction is directly saved, and the enrichment process is more efficient.
2. The biotin double-probe streptavidin magnetic bead solves the problem of low probe density caused by steric hindrance and electrostatic repulsion of a conventional probe, and obviously improves the probe density.
3. The double-probe system on the biotin double-probe streptavidin magnetic bead is compatible with double-strand DNA capture, and overcomes the technical limitation that a conventional probe can only recover single-strand DNA, namely only half of available molecules can be recovered, and each complementary strand of a target cannot be recovered.
4. The probe on the biotin double-probe streptavidin magnetic bead is pre-fixed on the streptavidin magnetic bead through the double-biotin before hybridization with the target cfDNA, and the mode can avoid competition of the probe connected with the biotin and endogenous biotin during hybridization, so that consumption of the streptavidin magnetic bead is reduced, and the scale of a reaction system is further enlarged.
5. Aiming at urine which can obtain a large-volume sample but has low cfDNA concentration, the double-biotin double-probe streptavidin magnetic beads can target, specifically and largely capture the short-sequence and low-concentration target cfDNA in the sample, and the recovery rate of the cfDNA is high and the copy number is large.
6. According to the application, the double-biotin-marked probe BP1, the double-biotin-marked probe BP2, the ferroferric oxide-modified biotin and the streptavidin magnetic beads are contacted together, the occupation of biotin binding sites in the streptavidin by the ferroferric oxide-modified biotin is avoided to a certain extent, the condition that two probes are combined on one streptavidin molecule is avoided, the combination result of the streptavidin and the probes is more uniquely determined, so that the steric hindrance between the probes is further reduced, the probes can be ensured to effectively capture cfDNA, and finally the stability and the accuracy of the detection result between parallel samples of the same sample are improved.
Detailed Description
The application is further described in detail below with reference to the following examples, which are specifically described: the following examples, in which no specific conditions are noted, are conducted under conventional conditions or conditions recommended by the manufacturer, and the raw materials used in the following examples are commercially available from ordinary sources except for the specific descriptions.
Preparation example 1
The biotin double-probe streptavidin magnetic beads of the preparation example aim at capturing a GSTP1 gene (GRCh 37, chr11: 67118615-67118704) target region and an APC gene (chr 15: 112737761-112737851) target region in urine supernatant.
The preparation method of the biotin double-probe streptavidin magnetic beads comprises the following specific steps:
1) Preparing raw materials: 1-1), probe design:
The double biotin labeling probe BP1 is designed, and the structure is as follows: the 5 '-bisbiotin modification-polyA-short sequence specific binding sequence A-C3 blocker-3', the probe is used for capturing a target region of GSTP1 gene (GRCh 37, chr11: 67118615-67118704), wherein the sequence of the short sequence specific binding sequence A is shown as SEQ ID NO.1, and the specific sequence is: CTCAACCAATAAAACCTACTCCTCC, the structure of the double biotin-labeled probe BP1, is: 5 'bis-biotin modification-polyA-CTCAACCAATAAAACCTACTCCTCC-C3 blocker-3'. The double biotin labeling probe BP2 is designed, and the structure is as follows: the 5 '-bisbiotin modification-polyA-short sequence specific binding sequence B-C3 blocker-3' is used for capturing a target region of an APC gene (chr 15: 112737761-112737851), wherein the sequence of the short sequence specific binding sequence B is shown as SEQ ID NO.1, and the specific sequence is as follows: CTAACTTTAAACRCTAACAAACGC, the structure of the double biotin-labeled probe BP1, is: 5 'bis-biotin modification-polyA-CTAACTTTAAACRCTAACAAACGC-C3 blocker-3'. The size of polyA is 20nt. Both the double biotin-labeled probe BP1 and the double biotin-labeled probe BP2 were synthesized by IDT Ed (INTEGRATED DNA Technologies, inc.).
1-2), Streptavidin magnetic bead suspension preparation:
A. Balancing streptavidin magnetic beads for 30min at room temperature; the streptavidin beads were then vortexed for 15s and 50 μl of streptavidin beads, i.e. 50 μl/reaction, was dispensed in a 1.5mL centrifuge tube.
B. Then adding 50 mu L of salt buffer solution into the centrifuge tube, swirling for 10s, putting the centrifuge tube back into a magnetic rack, adsorbing the solution to clarify, and discarding the supernatant; wherein, the salt buffer solution comprises the following components: 1M NaCl,10mM Tris-HCl,0.05wt% Tween-20, water as solvent.
C. The "B" was repeated twice for a total of three washes.
D. the streptavidin magnetic beads were re-spun with 50. Mu.L of salt buffer to give streptavidin magnetic bead suspension.
2) Preparation of double-biotin double-probe streptavidin magnetic beads
E. the double biotin-labeled probe BP1, the double biotin-labeled probe BP2 and the streptavidin magnetic bead suspension were mixed so that the final concentration of the double biotin-labeled probe BP1 was 50. Mu.M, so that the final concentration of the double biotin-labeled probe BP2 was 50. Mu.M, followed by immediately vortexing for 5s, and then incubating at room temperature for 15min.
F. Add 50. Mu.L of salt buffer (composition of salt buffer: 1M NaCl,10mM Tris-HCl,0.05wt% Tween-20, water as solvent), vortex for 10s, then place the centrifuge tube back on the magnetic rack, adsorb to the supernatant after clarification of the solution.
G. The "F" procedure was repeated twice and washed three times with a total of salt buffer.
H. Adding 50 mu L of salt buffer solution (the components of the salt buffer solution are 1M NaCl,10mM Tris-HCl,0.05wt% Tween-20 and water are taken as solvents) to obtain the double-biotin double-probe streptavidin magnetic beads, wherein the double-biotin double-probe streptavidin magnetic beads are suspended in the salt buffer solution.
Preparation example 2
The difference between the preparation example and the preparation example 1 is that the prepared double-biotin single-probe streptavidin magnetic beads are prepared, and the probe used is only the double-biotin labeled probe BP1, namely the probe of the preparation example only captures the target region of GSTP1 gene (GRCh 37, chr11: 67118615-67118704) in urine supernatant.
The preparation method of the double-biotin single-probe streptavidin magnetic beads comprises the following specific steps:
This comparative example was directed to capturing the GSTP1 gene (GRCh 37, chr11: 67118615-67118704) target region in urine supernatant.
A double-biotin single-probe streptavidin magnetic bead for cfDNA enrichment comprises the following preparation method:
step one, raw material preparation:
(1) And designing a probe.
The double biotin labeling probe BP1 is designed, and has the same structure as that of preparation example 1, and the structure is as follows: 5 'bis-biotin modification-polyA-CTCAACCAATAAAACCTACTCCTCC-C3 blocker-3'. The double biotin-labeled probe BP1 was synthesized by IDT Ed Co., INTEGRATED DNA Technologies, inc.
(2) Preparation of streptavidin magnetic bead suspension, and the specific steps are the same as those of the method 1-2 of preparation example 1).
Step two, preparation of double biotin single probe streptavidin magnetic beads
E. The double biotin-labeled probe BP1 and the streptavidin magnetic bead suspension were mixed so that the final concentration of the double biotin-labeled probe BP1 was 50. Mu.M, followed by immediate vortexing for 5s, and then incubation at room temperature for 15min.
Step F-H was performed in the same manner as in preparation example 1 to obtain a double biotin single probe streptavidin magnetic bead, which was suspended in a salt buffer.
Preparation example 3
The difference between the preparation example and the preparation example 1 is that the prepared double-biotin single-probe streptavidin magnetic beads are prepared, and the probe used is only the double-biotin labeled probe BP2.
The preparation method of the double-biotin single-probe streptavidin magnetic beads comprises the following specific steps:
this comparative example was directed to capturing the target region of the APC gene (chr 15: 112737761-112737851) in urine supernatant.
A double-biotin single-probe streptavidin magnetic bead for cfDNA enrichment comprises the following preparation method:
step one, raw material preparation:
(1) And designing a probe.
The double biotin-labeled probe BP2 was designed, and its structure was the same as that of preparation example 1, specifically: 5 'bis-biotin modification-polyA-CTAACTTTAAACRCTAACAAACGC-C3 blocker-3'. The double biotin-labeled probe BP2 was synthesized by IDT Ed Co., ltd (INTEGRATED DNATECHNOLOGIES, inc.).
(2) Preparation of streptavidin magnetic bead suspension, and the specific steps are the same as those of the method 1-2 of preparation example 1).
Step two, preparation of double biotin single probe streptavidin magnetic beads
E. The double biotin-labeled probe BP2 and the streptavidin magnetic bead suspension were mixed so that the final concentration of the double biotin-labeled probe BP2 was 50. Mu.M, followed by immediate vortexing for 5s, and then incubation at room temperature for 15min.
Step F-H was performed in the same manner as in preparation example 1 to obtain a double biotin single probe streptavidin magnetic bead, which was suspended in a salt buffer.
Example 1
This example is directed to capturing GSTP1 gene (GRCh 37, chr11: 67118615-67118704) target region and APC gene (chr 15: 112737761-112737851) target region in urine supernatant.
A method for enriching cfDNA in urine based on biotin double probes comprises the following specific steps:
S1, preparation of biotin double-probe streptavidin magnetic beads with cfDNA captured
And hybridizing the target cfDNA with the biotin double-probe streptavidin magnetic beads to obtain the biotin double-probe streptavidin magnetic beads with the captured cfDNA. The method comprises the following specific steps:
s11, preparation of urine cfDNA
1) A15 mL centrifuge tube was prepared, 500. Mu.L of 0.5M EDTA and 100. Mu.L of 1M Tris-HCl were added, and mixed well. Collecting urine into a sterile container; after urine collection, 10mL urine samples were added to a prepared 15mL centrifuge tube and mixed upside down.
2) Followed by centrifugation at 8000g for 5min.
3) After centrifugation, the supernatant was transferred to a new 15mL centrifuge tube to be tested and stored at-80 ℃.
S12, capturing target cfDNA in urine supernatant
4) 10ML of the supernatant (containing the target cfDNA) obtained in S11 was taken, 2.5mL of 5M NaCl, 127. Mu.L of 10wt% Tween-20 and 50. Mu.L of the biotin double-probe streptavidin magnetic beads prepared in preparation example 1 were added, and the mixture was mixed in a 15mL centrifuge tube, and the mixture was inverted and homogenized.
5) Subsequently, 15mL centrifuge tubes were placed in a water bath and incubated at 90℃for 15min.
6) After the completion, a 15mL centrifuge tube is fixed on a vertical mixer, and the mixture is uniformly mixed for 30min at room temperature. Taking down the centrifuge tube on a vertical mixer, centrifuging under the conditions of 5000g and 5min, and absorbing and discarding the supernatant to obtain the biotin double-probe streptavidin magnetic beads with cfDNA captured.
S2, washing the biotin double-probe streptavidin magnetic beads captured with cfDNA to remove non-target cfDNA, and obtaining the purified biotin double-probe streptavidin magnetic beads captured with target cfDNA. The specific steps are as follows 7) to 10):
7) The biotin double-probe streptavidin magnetic beads with the captured cfDNA obtained in the step S1 are all placed in a 1.5mL centrifuge tube, and then 1mL high-salt buffer solution is added to suspend the biotin double-probe streptavidin magnetic beads with the captured cfDNA.
8) And placing the 1.5mL centrifuge tube obtained in the step 7) on a magnetic rack for standing for 1min, and discarding the supernatant after the solution is clarified.
9) And then adding 1mL of high-salt buffer solution into the 1.5mL centrifuge tube subjected to the treatment of 8), blowing and sucking uniformly, centrifuging instantaneously, standing for 1min in a magnetic rack, and thoroughly discarding and sucking the supernatant after the solution is clarified.
10 And finally adding 1mL of low-salt buffer solution into the 1.5mL of centrifuge tube after 9) treatment, blowing and sucking uniformly, performing instantaneous centrifugation, standing for 1min on a magnetic rack, and thoroughly discarding and sucking the supernatant after the solution is clarified.
Wherein, the high salt buffer solution comprises the following components: 1M NaCl, 10mM Tris-HCl and 0.05wt% Tween-20, wherein the solvent is water; the low salt buffer comprises the following components: 15mM NaCl and 10mM Tris-HCl, the solvent being water.
And (3) washing the biotin double-probe streptavidin magnetic beads captured with the cfDNA through S2 to remove non-target cfDNA, so as to obtain the purified biotin double-probe streptavidin magnetic beads captured with the target cfDNA.
S3, heating and denaturing the purified biotin double-probe streptavidin magnetic beads with the target cfDNA captured therein, and then incubating in a low-temperature environment, and taking a supernatant to recover the target cfDNA. The specific steps are as follows 11) to 13): 11 Adding 20 mu L of nuclease-free water to the purified biotin double-probe streptavidin magnetic beads captured with the target cfDNA finally obtained in the step S2, re-suspending the magnetic beads, transferring the re-suspended magnetic beads into a new 0.2mL PCR tube, and denaturing for 2min at 95 ℃ by using a PCR instrument.
12 After denaturation, 0.2mL PCR tube was immediately placed on ice box for 3min incubation without cooling.
13 After incubation is completed, placing a 0.2mL PCR tube on a magnetic rack, standing for 1min, transferring the supernatant to a new 1.5mL low-adsorption centrifuge tube after the solution is clarified, wherein the centrifuge tube contains target cfDNA.
And S4, performing qPCR amplification by taking the target cfDNA finally recovered in the step S3 as a template to enrich the cfDNA, and performing qPCR amplification by a conventional method.
Method for enriching cfDNA in urine of comparative example 1
A method for enriching cfDNA in urine is different from example 1 in that the biotin double-probe streptavidin magnetic beads prepared in preparation example 1 are replaced by the double-biotin single-probe streptavidin magnetic beads prepared in preparation example 2 with equal volume, and the method is the same as that of example 1.
Method for enriching cfDNA in urine of comparative example 2
A method for enriching cfDNA in urine is different from example 1 in that the biotin double-probe streptavidin magnetic beads prepared in preparation example 1 are replaced by the double-biotin single-probe streptavidin magnetic beads prepared in preparation example 3 with equal volume, and the method is the same as that of example 1.
Performance test
1. Copy number determination
1ML urine sample with 1000 copies of 50nt single-stranded DNA added was targeted for capture, and 3 techniques were repeated. The copy number was quantitatively determined by qPCR, which found: example 1 both strands of dsDNA were captured by a two probe system with a copy number of 9950 copies, a copy number of 7300 for comparative example 1 and 8400 for comparative example 2. The enrichment method of example 1 gave a gene copy number 1.36 times that of comparative example 1 and 1.18 times that of comparative example 2.
2. Recovery measurement
For 1mL urine samples added with 1000 copies of 50nt single-stranded DNA, targeted capture was performed, the copy numbers of cfDNA of example 1, comparative example 1 and comparative example 2 were quantitatively determined by qPCR and the recovery rate was calculated (recovery% = copy number of qPCR quantification/initial copy number x 100%, initial copy number is 1000), recovery of comparative example 1 was 73%, recovery of comparative example 2 was 84%, and recovery of example 1 was 99.5%. Therefore, the method disclosed by the application has an excellent enrichment and recovery effect on small-fragment cfDNA in a urine sample with a large volume and a low cfDNA concentration.
Preparation example 4
The biotin double-probe streptavidin magnetic beads of the preparation example aim at capturing a GSTP1 gene (GRCh 37, chr11: 67118615-67118704) target region and an APC gene (chr 15: 112737761-112737851) target region in urine supernatant.
The preparation method of the biotin double-probe streptavidin magnetic beads comprises the following specific steps:
I. And mixing the streptavidin magnetic beads with a salt buffer solution to obtain a streptavidin magnetic bead suspension. Mixing a double biotin-labeled probe BP1, a double biotin-labeled probe BP2, ferroferric oxide-modified biotin and a streptavidin magnetic bead suspension; after incubation, the solution was removed to obtain the initial biotin double probe streptavidin magnetic beads. The specific steps are as follows I-I) to I-III).
I-I), probe design:
The double biotin-labeled probe BP1 and the double biotin-labeled probe BP2 were designed in the same manner as in example 1.
I-II), streptavidin magnetic bead suspensions were prepared, and the specific procedure was as in example 1.
I-III) preparation of initial biotin double-probe streptavidin magnetic beads
E. The double biotin-labeled probe BP1, the double biotin-labeled probe BP2, the ferroferric oxide-modified biotin (purchased from Seaman biological Co., ltd., 95% by weight purity) and the streptavidin magnetic beads suspension were mixed so that the final concentration of the double biotin-labeled probe BP1 was 50. Mu.M, so that the final concentration of the double biotin-labeled probe BP2 was 50. Mu.M, and the final concentration of the ferroferric oxide-modified biotin was 18. Mu.M, followed by immediate vortexing for 5s, followed by incubation at room temperature for 15min, followed by putting the centrifuge tube back to a magnetic frame, adsorbing to the supernatant after clarification of the solution, to obtain the initial biotin-double probe streptavidin magnetic beads.
II. Mixing and stirring the initial biotin double-probe streptavidin magnetic beads with guanidine hydrochloride solution, and performing gradient elution by using the guanidine hydrochloride solution to obtain the biotin double-probe streptavidin magnetic beads. The specific steps are shown as F to L.
F. 50. Mu.L of the initial biotin double probe streptavidin magnetic beads and 50. Mu.L of guanidine hydrochloride solution pH 1.5.5M were mixed, followed by immediate vortexing for 10s; the centrifuge tube is put back into a magnetic rack, and the centrifuge tube is adsorbed to the supernatant after solution clarification.
G. 50. Mu.L of 4.5M guanidine hydrochloride solution was added to the centrifuge tube of F and mixed, followed by immediate vortexing for 10s; the centrifuge tube is put back into a magnetic rack, and the centrifuge tube is adsorbed to the supernatant after solution clarification.
H. 50 μl of 3.5M guanidine hydrochloride solution was added to the centrifuge tube of G and mixed, followed by immediate vortexing for 10s; the centrifuge tube is put back into a magnetic rack, and the centrifuge tube is adsorbed to the supernatant after solution clarification.
I. Add 50. Mu.L of nuclease free water to the H centrifuge tube and mix, immediately vortex for 10s; the centrifuge tube is put back into a magnetic rack, and the centrifuge tube is adsorbed to the supernatant after solution clarification.
J. adding 50 mu L of salt buffer (the salt buffer comprises 1M NaCl,10mM Tris-HCl,0.05wt% Tween-20 and water as solvent) into the centrifuge tube of the I, and swirling for 10s; the centrifuge tube is put back into a magnetic rack, and the centrifuge tube is adsorbed to the supernatant after solution clarification.
K. the "J" procedure was repeated twice and the total of three washes with salt buffer.
L, 50 mu L of salt buffer (the composition of the salt buffer is 1M NaCl,10mM Tris-HCl,0.05wt% Tween-20 and water is taken as a solvent) is added to obtain biotin double-probe streptavidin magnetic beads, and the biotin double-probe streptavidin magnetic beads are suspended in the salt buffer.
Example 2
This example is directed to capturing a target region of GSTP1 gene (GRCh 37, chr11: 67118615-67118704) in urine supernatant.
The difference between this example and example 1 is that the biotin double-probe streptavidin magnetic beads prepared in preparation example 1 are replaced by the biotin double-probe streptavidin magnetic beads prepared in preparation example 1 with equal volumes of the biotin double-probe streptavidin magnetic beads prepared in preparation example 4.
1. For 1mL urine samples added with 1000copy and 50nt single-stranded DNA, targeted capture and enrichment were performed in the methods of example 1 and example 2, respectively, 3 replicates were designed for each group by qPCR quantification, and the ΔCt values were calculated, with specific results shown in Table 1.
Table 1 cfDNA obtained for different embodiments delta Ct values after qPCR quantification
2. 60 Urine samples were selected, targeted capture and enrichment were performed by the methods of example 1 and example 2, respectively, and quantitative determination was performed by qPCR, 3 replicates were designed for each group, and the delta Ct value of the assay was calculated. If the delta Ct value difference between any two parallel samples of the same urine sample is greater than or equal to 1, the detection results of the parallel samples of the sample are considered to have obvious differences, and the detection results are considered to be inaccurate; the delta Ct value difference between any two parallel samples of the same urine sample is smaller than 1, and the detection result between the parallel samples of the sample is considered to be accurate. The accuracy of the detection results of each sample obtained by the enrichment methods of example 1 and example 2 was counted separately, and the accuracy (%) was calculated; accuracy (%) = is the number of samples/total number of samples of the detection result accurate x 100%. The specific results are shown in Table 2.
TABLE 2 accuracy of delta Ct values after qPCR quantitative determination of cfDNA obtained by different enrichment methods
As can be seen from the data results in table 2, compared with the method of example 1, the method of example 2 can relatively accurately and completely enrich cfDNA of small fragments in urine, so as to ensure the accuracy of the detection results, and make the stability of the detection results between samples excellent.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.
Claims (6)
1. A method for enriching cfDNA in urine based on biotin double probes, which is characterized by comprising the following steps:
S1, hybridizing a target cfDNA with a biotin double-probe streptavidin magnetic bead to obtain the biotin double-probe streptavidin magnetic bead with the captured cfDNA;
s2, washing the biotin double-probe streptavidin magnetic beads captured with cfDNA to remove non-target cfDNA, and obtaining purified biotin double-probe streptavidin magnetic beads captured with target cfDNA;
S3, heating and denaturing the purified biotin double-probe streptavidin magnetic beads with the target cfDNA captured, cooling, incubating, centrifuging, and taking a supernatant to recover the target cfDNA;
the biotin double-probe streptavidin magnetic beads comprise streptavidin magnetic beads, a double-biotin-marked probe BP1 connected to the streptavidin magnetic beads and a double-biotin-marked probe BP2 connected to the streptavidin magnetic beads;
the preparation method of the biotin double-probe streptavidin magnetic beads comprises the following steps:
I. Mixing the streptavidin magnetic beads with a salt buffer solution to obtain a streptavidin magnetic bead suspension, wherein the volume ratio of the streptavidin magnetic beads to the salt buffer solution is 1 (0.9-1.1);
Mixing the double biotin-labeled probe BP1, the double biotin-labeled probe BP2, the ferroferric oxide-modified biotin and the streptavidin magnetic bead suspension so that the final concentration of the double biotin-labeled probe BP1 is 45-55 mu M, the final concentration of the double biotin-labeled probe BP2 is 45-55 mu M, and the final concentration of the ferroferric oxide-modified biotin is 15-20 mu M; after incubation, removing the solution to obtain initial biotin double-probe streptavidin magnetic beads;
wherein the probe BP1 comprises polyA and a short sequence specific binding sequence A complementary to a target, the probe BP2 comprises polyA and a short sequence specific binding sequence B complementary to the target, and the sequence of the short sequence specific binding sequence A is as follows: CTCAACCAATAAAACCTACTCCTCC, the sequence of the short sequence specific binding sequence B is: CTAACTTTAAACRCTAACAAACGC;
II. Mixing and stirring the initial biotin double-probe streptavidin magnetic beads and guanidine hydrochloride solution with the pH of 1.2-1.8-6M, and sequentially replacing the guanidine hydrochloride solution with the pH of 1.2-1.8-6M, guanidine hydrochloride solution with the pH of 4-5M, guanidine hydrochloride solution with the pH of 3-4M and water, so as to obtain the biotin double-probe streptavidin magnetic beads after water removal.
2. The method for enriching cfDNA in urine based on the biotin double probe according to claim 1, wherein the incubation time for preparing the streptavidin magnetic beads of the biotin double probe is 10-15min.
3. The method for enriching cfDNA in urine based on biotin double probes according to claim 1, wherein the initial biotin double probe streptavidin magnetic beads are mixed and stirred with guanidine hydrochloride solution with a pH of 1.2-1.8-6M in a volume ratio of 0.8-1.2:1.
4. The method for enriching cfDNA in urine based on biotin double probes according to claim 1, wherein the composition of the salt buffer comprises: 0.8-1.2M NaCl,8-13mM Tris-HCl,0.03-0.07wt% Tween-20, and water as solvent.
5. The method for enriching cfDNA in urine based on biotin double probes according to claim 1, wherein the step S1 specifically comprises the following steps: mixing target cfDNA, an incubation solution A and biotin double-probe streptavidin magnetic beads, and heating and denaturing at 88-92 ℃ for 12-18min, wherein the incubation solution A comprises the following components: 4-6M NaCl solution and 8-12wt% Tween-20 solution, wherein the volume ratio of the NaCl solution to the Tween-20 solution is (18.5-20.5): 1.
6. The method for enriching cfDNA in urine based on biotin double probes according to claim 1, wherein the heating denaturation temperature in the step S3 is 92-98 ℃ and the heating denaturation time is 1-4min.
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