CN117448422B - Method for enriching cfDNA in urine based on biotin double probes - Google Patents

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

A method for enriching cfDNA in urine based on biotin dual probes

Technical Field

The present application relates to the technical field of cfDNA enrichment, and more specifically, it relates to a method for enriching cfDNA in urine based on a biotin dual probe.

Background Art

The current steps of the cfDNA enrichment method are: 1. Extracting the total cfDNA in the sample, specifically including combining the cfDNA in the target sample with magnetic microspheres, and then eluting to obtain the purified total cfDNA; 2. End-repairing and connecting the adapters of the total cfDNA and then hybridizing with the probe, followed by PCR amplification, purification and elution to obtain the target cfDNA.

However, there are still the following shortcomings when enriching cfDNA using the above method: when extracting cfDNA, the magnetic bead extraction method is limited by the reaction system, so it is not suitable for extracting cfDNA from large-volume samples, and this method is difficult to extract trace amounts of cfDNA.

There are also traditional methods for extracting cfDNA based on silica, but the extraction of cfDNA is achieved by adsorbing cfDNA onto silica. The forces acting on the adsorption of DNA and silica are: hydrophobic interactions caused by dehydration of the silica and DNA surfaces and hydrogen bonds between silica and the DNA backbone. In other words, the magnitude of the forces acting between the two is proportional to the length of the DNA fragment, which will directly lead to the difficulty in recovering DNA fragments shorter than 50-100bp. However, due to the abundance of nucleases in urine and the short half-life of DNA, ctDNA from tumors often exists in urine in the form of short fragments of cfDNA, so the enrichment of short fragments of cfDNA in urine can effectively improve the accuracy, sensitivity and other performance of subsequent detection.

In summary, it is difficult to effectively enrich target cfDNA in large-volume, low-cfDNA-concentration samples using existing methods for cfDNA enrichment.

Summary of the invention

In order to improve the cfDNA enrichment effect in samples with large volume and low cfDNA concentration, the present application provides a cfDNA enrichment method in urine based on biotin dual probes.

The present application provides a method for enriching cfDNA in urine based on a biotin dual probe, which adopts the following technical scheme: A method for enriching cfDNA in urine based on a biotin dual probe, comprising the following steps:

S1, hybridizing the target cfDNA and the biotin dual-probe streptavidin magnetic beads to obtain the biotin dual-probe streptavidin magnetic beads that capture the cfDNA;

S2, washing the biotin dual-probe streptavidin magnetic beads that capture cfDNA to remove non-target cfDNA, and obtaining purified biotin dual-probe streptavidin magnetic beads that capture target cfDNA;

S3, heating and denaturing the purified biotin dual-probe streptavidin magnetic beads that capture the target cfDNA, cooling, incubating, centrifuging, and taking the supernatant to recover the target cfDNA;

The biotin dual-probe streptavidin magnetic beads include streptavidin magnetic beads, dual-biotin labeled probe BP1 connected to the streptavidin magnetic beads, and dual-biotin labeled probe BP2 connected to the streptavidin magnetic beads.

By adopting the above technical scheme, the cfDNA enrichment method of the present application introduces double biotin double probe streptavidin magnetic beads to achieve enrichment. The double biotin in the biotin double probe streptavidin magnetic beads directly improves the thermal stability of the probe and the streptavidin magnetic beads. Therefore, when the biotin double probe streptavidin magnetic beads are used for the enrichment of cfDNA, it is not necessary to first extract the total cfDNA in the sample and then capture the target cfDNA with the probe. The present application is to directly denature the biotin double probe streptavidin magnetic beads and the sample containing the target cfDNA after mixing, and capture the target cfDNA at room temperature to improve efficiency. In addition, the combination of double biotin and streptavidin is used as a bridge to stably connect the probe and the magnetic beads, so that the connection stability of the probe and the magnetic beads is higher. In addition, the two probes are stably connected to the streptavidin magnetic beads with double biotin, so that multiple probes can be distributed on the surface of the streptavidin magnetic beads with better space to reduce the steric hindrance between the probes, further improve the connection stability of the probe and the magnetic beads and increase the probe density. When the biotin dual probe streptavidin magnetic beads are used to enrich small fragments of cfDNA in large-volume, low-cfDNA concentration samples, it has the advantages of high targeting, strong specificity and high recovery rate. In addition, this enrichment method will not be limited by the amount of cfDNA in the sample and the size of the cfDNA fragments. In addition, when the biotin dual probe streptavidin magnetic beads of the present application are used for cfDNA enrichment, the probes are pre-fixed on the streptavidin magnetic beads containing biotin, which also avoids the problem of endogenous biotin in the sample competing with the probe for the streptavidin magnetic beads, thereby enabling the streptavidin magnetic beads to connect more probes, further improving the enrichment efficiency.

Optionally, the preparation method of the biotin dual probe streptavidin magnetic beads comprises the following steps: mixing the dual biotin labeled probe BP1, the dual biotin labeled probe BP2 and the streptavidin magnetic bead suspension so that the final concentration of the dual biotin labeled probe BP1 is 45-55 μM, and the final concentration of the dual biotin labeled probe BP2 is 45-55 μM;

After incubation, the solution is removed to obtain the biotin dual probe streptavidin magnetic beads.

Further optionally, the incubation time for preparing the biotin dual-probe streptavidin magnetic beads is 10-15 min.

Optionally, the probe BP1 includes polyA and a short sequence-specific binding sequence A complementary to the target, and the probe BP2 includes 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 poly A is 15-25 nt, for example, it can be 15 nt, 18 nt, 20 nt, 23 nt or 25 nt. The length of short sequence-specific binding sequence A is 15-20 nt, for example, it can be 15 nt, 16 nt, 17 nt, 18 nt, 19 nt or 20 nt. The length of short sequence-specific binding sequence B is 15-20 nt, for example, it can be 15 nt, 16 nt, 17 nt, 18 nt, 19 nt or 20 nt.

Further optionally, the 3' ends of the probe BP1 and the probe BP2 are both connected to a C3 blocker.

The probes used in the present application can be any short sequence that is complementary to the target and can specifically bind to it, and are not limited to those listed below.

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: CTAACTTTAAACRCTAACAAACGC.

In the present application, the sequence of short sequence specific binding sequence A is recorded as SEQ ID NO.1, and the sequence of short sequence specific binding sequence B is recorded as SEQ ID NO.2. R in short sequence specific binding sequence B refers to a degenerate base, specifically A or G".

Optionally, the preparation method of the biotin dual-probe streptavidin magnetic beads comprises the following steps: I, mixing the streptavidin magnetic beads and a salt buffer to obtain a streptavidin magnetic bead suspension, wherein the volume ratio of the streptavidin magnetic beads to the salt buffer is 1:(0.9-1.1);

The double biotin labeled probe BP1, the double biotin labeled probe BP2, the ferroferric oxide modified biotin and the streptavidin magnetic bead suspension are mixed, so that the final concentration of the double biotin labeled probe BP1 is 45-55 μM, the final concentration of the double biotin labeled probe BP2 is 45-55 μM, and the final concentration of the ferroferric oxide modified biotin is 15-20 μM; after incubation, the solution is removed to obtain the initial biotin double probe streptavidin magnetic beads;

II. The initial biotin dual-probe streptavidin magnetic beads are mixed with a 5-6M guanidine hydrochloride solution at pH 1.2-1.8 and stirred, and the 5-6M guanidine hydrochloride solution at pH 1.2-1.8 is replaced with a 4-5M guanidine hydrochloride solution, a 3-4M guanidine hydrochloride solution, and water in sequence. After removing the water, the biotin dual-probe streptavidin magnetic beads are obtained.

Only after mixing the double-biotin-labeled probe BP1, the double-biotin-labeled probe BP2 and the streptavidin magnetic beads, cfDNA was enriched; after the obtained cfDNA was tested, the ΔCt value of the related gene fragments was unstable, and the difference in ΔCt values between repeated samples was greater than or equal to 1; that is to say, when the cfDNA enriched by this method was tested, the stability between parallel samples was poor.

Streptavidin is a non-glycosylated tetrameric protein composed of four subunits, each of which has a biotin binding site and has a very strong affinity for biotin. After the probe labeled with double biotin is directly mixed with the streptavidin magnetic beads, it is possible that all four biotin binding sites on a streptavidin molecule are occupied. One of the most likely scenarios is that two double-biotin labeled probes are simultaneously connected to a streptavidin molecule. In this case, after the two probes capture the target cfDNA, some probes will fail to successfully capture the target cfDNA due to the relatively strong steric hindrance between the probes; while some streptavidin molecules are only bound to one probe, which has less steric hindrance and can successfully capture the target cfDNA. Therefore, when preparing biotin double-probe streptavidin magnetic beads, the results of the four subunits on streptavidin binding to the probes are uncertain. This uncertainty leads to a large difference in the test results between parallel samples and poor repeatability when the cfDNA enriched from the same source sample is used for detection.

By adopting the above technical scheme, the double biotin labeled probe BP1, the double biotin labeled probe BP2, the iron tetroxide modified biotin and the streptavidin magnetic beads are mixed together and then gradient eluted with guanidine hydrochloride solution to obtain the biotin double probe streptavidin magnetic beads. After the target cfDNA is enriched with the magnetic beads, the ΔCt value of the same sample is less than 1 when the enriched cfDNA is detected, that is, the stability between samples is good. The reason may be that in the magnetic beads obtained in this way, one probe is connected to one streptavidin molecule, occupying two biotin binding stable points of streptavidin; while the other two biotin binding sites are occupied by iron tetroxide modified biotin. Therefore, when the target cfDNA is enriched with the biotin double probe streptavidin magnetic beads, the relative steric hindrance between the probes is reduced, so that the probes can successfully capture cfDNA and enrich it.

After the three biotin markers, the double biotin labeled probe BP1, the double biotin labeled probe BP2 and the iron tetroxide modified biotin, are contacted with the streptavidin magnetic beads together, each streptavidin molecule is bound to a double biotin labeled probe and an iron tetroxide modified biotin, avoiding the combination of two probes. Its advantages are: 1. Avoiding the problem of capture failure caused by the large steric hindrance between probes after binding two probes to one streptavidin molecule; 2. Making the binding mode of the biotin marker and streptavidin relatively consistent, that is, one probe and one iron tetroxide modified biotin are bound to one streptavidin molecule. Therefore, relatively stable detection results between repeated samples can be obtained in the end. The process of gradient elution with guanidine hydrochloride solution further elutes the unstable double biotin labeled probe and iron tetroxide modified biotin to ensure the stability of the final detection results.

Optionally, the initial biotin dual probe streptavidin magnetic beads are mixed with a 5-6M guanidine hydrochloride solution at a volume ratio of 0.8-1.2:1 and stirred.

By adopting the above technical scheme, the initial biotin dual probe streptavidin magnetic beads and the guanidine hydrochloride solution of pH 1.2-1.8 5-6M are mixed in an appropriate ratio to achieve a better elution effect without affecting the binding stability of the two.

Optionally, the components of the salt buffer include: 0.8-1.2M NaCl, 8-13mM Tris-HCl, 0.03-0.07wt% Tween-20, with water as solvent.

In the hydrochloric acid buffer of the present application, the concentration of NaCl is 0.8-1.2 M, for example, it can be 0.8 M, 0.9 M, 1.0 M, 1.1 M or 1.2 M. The concentration of Tris-HCl is 8-13 mM, for example, it can be 8 mM, 10 mM, 11 mM or 13 mM. The concentration of Tween-20 is 0.03-0.07 wt%, for example, it can be 0.03 wt%, 0.04 wt%, 0.05 wt%, 0.06 wt% or 0.07 wt%.

Optionally, S1 specifically includes the following steps: mixing the target cfDNA, incubation solution A and biotin dual-probe streptavidin magnetic beads, and heating them for denaturation at 88-92°C for 12-18 minutes; then mixing at room temperature for 25-35 minutes, and obtaining biotin dual-probe streptavidin magnetic beads that capture cfDNA after centrifugation.

In the present application, the temperature for heating denaturation in S1 is 88-92°C, for example, it can be 88°C, 89.5°C, 91°C or 92°C; the time for heating denaturation is 12-18min, for example, it can be 12min, 13.5min, 15min, 16.5min or 18min.

Further optionally, the components of incubation solution 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.

The meaning of “8-12 wt % Tween-20 solution” is that the Tween-20 solution is prepared such that the mass percentage of Tween-20 in the Tween-20 solution is 8-12%.

The volume ratio of the components of the incubation solution A of the present application, NaCl solution and Tween-20 solution, is (18.5-20.5):1. For example, the volume ratio of the NaCl solution and Tween-20 solution can be 18.5:1, 19:1, 19.5:1, 20:1 or 20.5:1.

Further optionally, the volume ratio of the incubation solution A to the biotin dual probe streptavidin magnetic beads is (48-56):1.

The volume ratio of the incubation solution A of the present application to the biotin dual 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 following steps:

The double-biotin double-probe streptavidin magnetic beads capturing cfDNA are washed with the salt buffer and then washed with a low-salt buffer; the components of the low-salt buffer include: 12-18mM NaCl, 7-14mM Tris-HCl, and the solvent is water.

The concentration of NaCl in the low-salt buffer of the present application is 12-18 mM, for example, the concentration can be 12 mM, 14 mM, 16 mM or 18 mM; the concentration of Tris-HCl is 7-14 mM, for example, the concentration can be 7 mM, 9 mM, 11 mM or 14 mM.

Optionally, the temperature of the heating denaturation in S3 is 92-98° C., and the time of the heating denaturation is 1-4 min.

In the present application, the temperature for heating denaturation in S3 is 92-98°C, for example, it can be 92°C, 94°C, 96°C or 98°C; the time for heating denaturation is 1-4min, for example, it can be 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 low temperature environment after cooling.

The method of the present application is suitable for enriching cfDNA, especially for enriching small fragments of cfDNA (e.g., 50-100bp or even 20-50bp cfDNA fragments) with low cfDNA content and large sample volume. The sample can be a urine sample but is not limited to only a urine sample. The enriched cfDNA obtained from the urine sample can be used for the detection and identification of diseases such as prostate cancer and urothelial carcinoma, but it is not limited to the detection and identification of the above diseases.

In summary, this application has the following beneficial effects:

1. Due to the introduction of double biotin in the biotin dual probe streptavidin magnetic beads of the present application, the thermal stability of the probe and the streptavidin magnetic beads is directly improved. Therefore, the magnetic beads can be directly added to the sample for denaturation and hybridization with the target cfDNA, which directly saves the time of cfDNA extraction, and thus the enrichment process is more efficient.

2. The biotin dual probe streptavidin magnetic beads of the present application overcome the problem of low probe density caused by steric hindrance and electrostatic repulsion of conventional probes, and significantly improve the probe density.

3. The biotin dual-probe streptavidin magnetic bead dual-probe system of the present application is compatible with double-stranded DNA capture, overcoming the technical limitation that conventional probes can only recover single-stranded DNA, that is, only half of the available molecules can be recovered and each complementary chain of the target cannot be recovered.

4. The probes on the biotin dual-probe streptavidin magnetic beads of the present application are pre-fixed on the streptavidin magnetic beads through dual biotin before hybridization with the target cfDNA. This method can avoid the competition between the biotin-linked probes and endogenous biotin during hybridization, thereby reducing the consumption of streptavidin magnetic beads and expanding the scale of the reaction system.

5. For urine, a type of sample that can obtain a large volume of sample but has a low cfDNA concentration, the dual-biotin dual-probe streptavidin magnetic beads of the present application can target, specifically, and capture a large amount of short-sequence, low-concentration target cfDNA in the sample, and the cfDNA recovery rate is high and the copy number is large.

6. The present application allows the double-biotin-labeled probe BP1, the double-biotin-labeled probe BP2, the ferroferric oxide-modified biotin and the streptavidin magnetic beads to contact each other. By occupying the biotin binding site in the streptavidin with the ferroferric oxide-modified biotin, the situation where two probes are bound to one streptavidin molecule is avoided to a certain extent, so that the binding result of streptavidin and the probe is more unique and definite, so as to further reduce the steric hindrance between the probes, ensure that the probes can effectively capture cfDNA, and ultimately improve the stability and accuracy of the detection results between parallel samples of the same sample.

DETAILED DESCRIPTION

The present application is further described in detail below in conjunction with the examples. It is particularly noted that if no specific conditions are specified in the following examples, the experiments are carried out according to conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, the raw materials used in the following examples can be obtained from common commercial sources.

Preparation Example 1

The biotin dual-probe streptavidin magnetic beads in this preparation example are used to capture the target regions of the GSTP1 gene (GRCh37, chr11: 67118615-67118704) and the APC gene (chr15: 112737761-112737851) in the urine supernatant.

The preparation method of biotin dual probe streptavidin magnetic beads comprises the following specific steps:

1) Raw material preparation: 1-1) Probe design:

A double biotin-labeled probe BP1 was designed, and its structure was: 5' double biotin modification-polyA-short sequence specific binding sequence A-C3 blocker-3'. The probe was used to capture the target region of the GSTP1 gene (GRCh37, chr11: 67118615-67118704), wherein the sequence of the short sequence specific binding sequence A was as shown in SEQ ID NO.1, and the specific sequence was: CTCAACCAATAAAACCTACTCCTCC, that is, the structure of the double biotin-labeled probe BP1 was: 5' double biotin modification-polyA-CTCAACCAATAAAACCTACTCCTCC-C3 blocker-3'. Design a double biotin labeled probe BP2, whose structure is: 5' double biotin modification-polyA-short sequence specific binding sequence B-C3 blocker-3', the probe is used to capture the target region of the APC gene (chr15: 112737761-112737851), wherein the sequence of the short sequence specific binding sequence B is shown in SEQ ID NO.1, and the specific sequence is: CTAACTTTAAACRCTAACAAACGC, that is, the structure of the double biotin labeled probe BP1 is: 5' double 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 commissioned to IDT (Integrated DNA Technologies, Inc.) for synthesis.

1-2) Preparation of streptavidin magnetic bead suspension:

A. Equilibrate the streptavidin magnetic beads at room temperature for 30 min. Then, vortex the streptavidin magnetic beads for 15 s and dispense 50 μL of streptavidin magnetic beads into 1.5 mL centrifuge tubes. That is, the experimental amount of streptavidin magnetic beads is 50 μL/reaction.

B. Then add 50 μL of salt buffer to the centrifuge tube, vortex for 10 seconds, put the centrifuge tube back into the magnetic rack, adsorb to the solution and discard the supernatant after clarification; wherein the components of the salt buffer are: 1M NaCl, 10mM Tris-HCl, 0.05wt% Tween-20, and water is the solvent.

C. Repeat "B" twice, for a total of three washes.

D. Respin the streptavidin magnetic beads with 50 μL of salt buffer to obtain a streptavidin magnetic bead suspension.

2) Preparation of double biotin double probe streptavidin magnetic beads

E. Mix the double-biotin-labeled probe BP1, double-biotin-labeled probe BP2 and streptavidin magnetic bead suspension to make the final concentration of the double-biotin-labeled probe BP1 to be 50 μM, and the final concentration of the double-biotin-labeled probe BP2 to be 50 μM, then immediately vortex for 5 seconds and incubate at room temperature for 15 minutes.

F. Add 50 μL of saline buffer (the components of the saline buffer are: 1 M NaCl, 10 mM Tris-HCl, 0.05 wt% Tween-20, water as solvent), vortex for 10 seconds, then put the centrifuge tube back into the magnetic rack, adsorb to the solution and discard the supernatant after clarification.

G. Repeat "F" twice, washing a total of three times with saline buffer.

H. Add 50 μL of saline buffer (the components of the saline buffer are: 1 M NaCl, 10 mM Tris-HCl, 0.05 wt% Tween-20, and water is the solvent) to obtain double-biotin double-probe streptavidin magnetic beads, which are suspended in the saline buffer.

Preparation Example 2

The difference between this preparation example and preparation example 1 is that the prepared double-biotin single-probe streptavidin magnetic beads are used, and the probe used is only the double-biotin labeled probe BP1, that is, the probe of this preparation example only captures the target region of GSTP1 gene (GRCh37, chr11: 67118615-67118704) in urine supernatant.

The preparation method of double-biotin single-probe streptavidin magnetic beads comprises the following specific steps:

This comparative example aims at capturing the target region of GSTP1 gene (GRCh37, chr11: 67118615-67118704) in urine supernatant.

A double-biotin single-probe streptavidin magnetic bead for cfDNA enrichment, wherein the preparation method thereof is as follows:

Step 1: Raw material preparation:

(1) Probe design.

Design a double biotin labeled probe BP1, the structure of which is the same as that of Preparation Example 1, and the structure is: 5' double biotin modified-polyA-CTCAACCAATAAAACCTACTCCTCC-C3 blocker-3'. The double biotin labeled probe BP1 was commissioned to IDT (Integrated DNA Technologies, Inc.) for synthesis.

(2) Preparation of streptavidin magnetic bead suspension. The specific steps are the same as steps 1-2 of Preparation Example 1).

Step 2: Preparation of double-biotin single-probe streptavidin magnetic beads

E. Mix the double-biotin-labeled probe BP1 and the streptavidin magnetic bead suspension to make the final concentration of the double-biotin-labeled probe BP1 50 μM, then immediately vortex for 5 seconds and incubate at room temperature for 15 minutes.

Steps F-H are the same as those in Preparation Example 1, to obtain dual-biotin single-probe streptavidin magnetic beads, which are suspended in a salt buffer.

Preparation Example 3

The difference between this preparation example and preparation example 1 is that the prepared beads are double-biotin single-probe streptavidin magnetic beads, and the probe used is only the double-biotin labeled probe BP2.

The preparation method of double-biotin single-probe streptavidin magnetic beads comprises the following specific steps:

This comparative example aims at capturing the target region of the APC gene (chr15: 112737761-112737851) in the urine supernatant.

A double-biotin single-probe streptavidin magnetic bead for cfDNA enrichment, wherein the preparation method thereof is as follows:

Step 1: Raw material preparation:

(1) Probe design.

A double biotin labeled probe BP2 was designed, and its structure was the same as that in Preparation Example 1, specifically: 5' double biotin modified-polyA-CTAACTTTAAACRCTAACAAACGC-C3 blocker-3'. The double biotin labeled probe BP2 was commissioned to be synthesized by IDT (Integrated DNA Technologies, Inc.).

(2) Preparation of streptavidin magnetic bead suspension. The specific steps are the same as steps 1-2 of Preparation Example 1).

Step 2: Preparation of double-biotin single-probe streptavidin magnetic beads

E. Mix the double-biotin-labeled probe BP2 and the streptavidin magnetic bead suspension to make the final concentration of the double-biotin-labeled probe BP2 50 μM, then immediately vortex for 5 seconds and incubate at room temperature for 15 minutes.

Steps F-H are the same as those in Preparation Example 1, to obtain dual-biotin single-probe streptavidin magnetic beads, which are suspended in a salt buffer.

Example 1

This example aims to capture the target region of GSTP1 gene (GRCh37, chr11: 67118615-67118704) and the target region of APC gene (chr15: 112737761-112737851) in urine supernatant.

A method for enriching cfDNA in urine based on a biotin dual probe, the specific steps are:

S1. Preparation of biotin dual-probe streptavidin magnetic beads for capturing cfDNA

The target cfDNA is hybridized with the biotin dual-probe streptavidin magnetic beads to obtain the biotin dual-probe streptavidin magnetic beads that capture the cfDNA. The specific steps are as follows:

S11. Preparation of urine cfDNA

1) Prepare a 15 mL centrifuge tube, add 500 μL 0.5 M EDTA and 100 μL 1 M Tris-HCl, and mix well. Collect urine into a sterile container; after collecting urine, add 10 mL of urine sample to the prepared 15 mL centrifuge tube and mix well by inverting.

2) Then centrifuge at 8000g for 5 min.

3) After centrifugation, transfer the supernatant to a new 15 mL centrifuge tube for experiment and store at -80°C.

S12. Capture of target cfDNA in urine supernatant

4) Take 10 mL of the supernatant obtained in S11 (containing the target cfDNA), add 2.5 mL of 5 M NaCl, 127 μL of 10 wt% Tween-20 and 50 μL of the biotin dual probe streptavidin magnetic beads prepared in Preparation Example 1, mix in a 15 mL centrifuge tube, and mix by inversion.

5) Then place the 15 mL centrifuge tube in a water bath and incubate at 90°C for 15 min.

6) After the end, fix the 15mL centrifuge tube on the vertical mixer and mix at room temperature for 30 minutes. Remove the centrifuge tube from the vertical mixer, centrifuge at 5000g for 5 minutes, and discard the supernatant to obtain the biotin dual-probe streptavidin magnetic beads that capture cfDNA.

S2, washing the biotin dual-probe streptavidin magnetic beads that capture cfDNA to remove non-target cfDNA, and obtaining purified biotin dual-probe streptavidin magnetic beads that capture target cfDNA. The specific steps are as follows 7) to 10):

7) Place all the biotin dual-probe streptavidin magnetic beads that captured cfDNA obtained in S1 into a 1.5 mL centrifuge tube, and then add 1 mL of high salt buffer to suspend the biotin dual-probe streptavidin magnetic beads that captured cfDNA.

8) Place the 1.5 mL centrifuge tube obtained in 7) on a magnetic stand and let it stand for 1 min. After the solution becomes clear, discard the supernatant.

9) Then, add 1 mL of high salt buffer to the 1.5 mL centrifuge tube treated in 8), pipette to mix, centrifuge instantly, and then place on a magnetic stand for 1 min. After the solution is clarified, discard the supernatant completely.

10) Finally, add 1 mL of low-salt buffer to the 1.5 mL centrifuge tube treated in 9), pipette to mix, centrifuge instantly, place on a magnetic rack and let stand for 1 min. After the solution is clarified, discard the supernatant completely.

The components of the high-salt buffer are: 1M NaCl, 10mM Tris-HCl and 0.05wt% Tween-20, and the solvent is water; the components of the low-salt buffer are: 15mM NaCl and 10mM Tris-HCl, and the solvent is water.

The biotin dual-probe streptavidin magnetic beads capturing cfDNA are washed by S2 to remove non-target cfDNA, thereby obtaining purified biotin dual-probe streptavidin magnetic beads capturing target cfDNA.

S3, heat denature the purified biotin dual-probe streptavidin magnetic beads that capture the target cfDNA, then incubate in a low temperature environment, take the supernatant to recover the target cfDNA. The specific steps are as follows 11) to 13): 11), add 20μL of nuclease-free water to the purified biotin dual-probe streptavidin magnetic beads that capture the target cfDNA finally obtained in S2, resuspend the magnetic beads, transfer the resuspended magnetic beads to a new 0.2mL PCR tube, and use a PCR instrument to denature at 95℃ for 2min.

12) After denaturation, place the 0.2mL PCR tube on an ice box and incubate for 3 minutes without cooling down.

13) After incubation, place the 0.2 mL PCR tube on a magnetic rack and let it stand for 1 minute. After the solution is clarified, transfer the supernatant to a new 1.5 mL low-adsorption centrifuge tube, which contains the target cfDNA.

S4. Perform qPCR amplification using the target cfDNA finally recovered in S3 as a template to enrich the cfDNA. The qPCR amplification can be performed using conventional methods.

Comparative Example 1 Method for enriching cfDNA in urine

A method for enriching cfDNA in urine, which differs from Example 1 in that an equal volume of the double-biotin single-probe streptavidin magnetic beads prepared in Preparation Example 2 is used to replace the biotin double-probe streptavidin magnetic beads prepared in Preparation Example 1, and the rest is the same as Example 1.

Comparative Example 2 Method for enriching cfDNA in urine

A method for enriching cfDNA in urine, which differs from Example 1 in that an equal volume of the double-biotin single-probe streptavidin magnetic beads prepared in Preparation Example 3 is used to replace the biotin double-probe streptavidin magnetic beads prepared in Preparation Example 1, and the rest is the same as Example 1.

Performance testing

1. Copy number determination

1000 copies of 50nt single-stranded DNA were added to 1mL urine sample for targeted capture, with 3 technical replicates. The copy number was quantitatively determined by qPCR, and the results showed that: Example 1 captured two strands of dsDNA through a dual probe system, and its copy number was 9950 copies, the copy number of Comparative Example 1 was 7300, and the copy number of Comparative Example 2 was 8400. The gene copy number obtained by the enrichment method of Example 1 was 1.36 times that of Comparative Example 1 and 1.18 times that of Comparative Example 2.

2. Recovery rate determination

Targeted capture was performed on a 1mL urine sample with 1000 copies of 50nt single-stranded DNA added, and the copy numbers of cfDNA in Example 1, Comparative Example 1 and Comparative Example 2 were quantitatively determined by qPCR and the recovery rate was calculated (% recovery = number of copies obtained by qPCR quantification/initial number of copies × 100%, initial number of copies is 1000). The recovery rate of Comparative Example 1 was 73%, the recovery rate of Comparative Example 2 was 84%, and the recovery rate of Example 1 was 99.5%. It can be seen that the enrichment method of the present application has excellent enrichment and recovery effects on small fragments of cfDNA in urine samples with large volume and low cfDNA concentration.

Preparation Example 4

The biotin dual-probe streptavidin magnetic beads in this preparation example are used to capture the target regions of the GSTP1 gene (GRCh37, chr11: 67118615-67118704) and the APC gene (chr15: 112737761-112737851) in the urine supernatant.

The preparation method of biotin dual probe streptavidin magnetic beads comprises the following specific steps:

I. Mix streptavidin magnetic beads and salt buffer to obtain a streptavidin magnetic bead suspension. Mix the double biotin labeled probe BP1, the double biotin labeled probe BP2, ferric oxide modified biotin and the streptavidin magnetic bead suspension; remove the solution after incubation 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, and the details were the same as those in Example 1.

I-II), preparation of streptavidin magnetic bead suspension, the specific steps are the same as those in Example 1.

I-III) Initial preparation of biotin dual probe streptavidin magnetic beads

E. Mix the double-biotin labeled probe BP1, the double-biotin labeled probe BP2, the ferroferric oxide-modified biotin (purchased from Xi'an Qiyue Biological Co., Ltd., 95wt% purity) and the streptavidin magnetic bead suspension to make the final concentration of the double-biotin labeled probe BP1 to be 50μM, the final concentration of the double-biotin labeled probe BP2 to be 50μM, and the final concentration of the ferroferric oxide-modified biotin to be 18μM. Then immediately vortex for 5s, and then incubate at room temperature for 15min. Then put the centrifuge tube back into the magnetic stand, adsorb to the solution and discard the supernatant after clarification to obtain the initial biotin double probe streptavidin magnetic beads.

II. The initial biotin dual probe streptavidin magnetic beads are mixed with the guanidine hydrochloride solution and stirred, and gradient eluted with the guanidine hydrochloride solution to obtain the biotin dual probe streptavidin magnetic beads. The specific steps are shown in F to L.

F. Mix 50 μL of the initial biotin dual-probe streptavidin magnetic beads and 50 μL of 5.5 M guanidine hydrochloride solution at pH 1.5, and then immediately vortex for 10 seconds; put the centrifuge tube back into the magnetic rack, adsorb to the solution and discard the supernatant after clarification.

G. Add 50 μL of 4.5 M guanidine hydrochloride solution to the centrifuge tube in F and mix, then immediately vortex for 10 seconds; put the centrifuge tube back into the magnetic rack, adsorb to the solution and discard the supernatant after clarification.

H. Add 50 μL of 3.5 M guanidine hydrochloride solution to the centrifuge tube in G and mix, then immediately vortex for 10 seconds; put the centrifuge tube back into the magnetic rack, adsorb to the solution and discard the supernatant after clarification.

I. Add 50 μL of nuclease-free water to the centrifuge tube in H and mix, then immediately vortex for 10 seconds; put the centrifuge tube back into the magnetic rack, adsorb to the solution and discard the supernatant after clarification.

J. Add 50 μL of saline buffer (the components of the saline buffer are: 1 M NaCl, 10 mM Tris-HCl, 0.05 wt% Tween-20, water as solvent) to the centrifuge tube in I, and vortex for 10 seconds; put the centrifuge tube back into the magnetic rack, adsorb to the solution and discard the supernatant after clarification.

K. Repeat "J" twice, washing a total of three times with saline buffer.

L, add 50 μL of saline buffer (the components of the saline buffer are: 1M NaCl, 10mM Tris-HCl, 0.05wt% Tween-20, water is the solvent) to obtain biotin dual probe streptavidin magnetic beads, which are suspended in the saline buffer.

Example 2

This example aims to capture the target region of GSTP1 gene (GRCh37, chr11: 67118615-67118704) in urine supernatant.

A method for enriching cfDNA in urine based on a biotin dual probe. The difference between this embodiment and embodiment 1 is that the biotin dual probe streptavidin magnetic beads prepared in preparation example 1 are replaced with an equal volume of biotin dual probe streptavidin magnetic beads prepared in preparation example 4, and the rest are the same as embodiment 1.

1. 1 mL of urine sample with 1000 copies and 50 nt single-stranded DNA was added, and targeted capture and enrichment were performed by the methods of Example 1 and Example 2, respectively. Quantitative determination was performed by qPCR, and 3 parallel samples were designed for each group. The ΔCt value was calculated. The specific results are shown in Table 1.

Table 1 ΔCt values of cfDNA obtained from different implementation schemes after qPCR quantification

2. Select 60 urine samples, perform targeted capture and enrichment using the methods of Example 1 and Example 2, and perform quantitative determination using qPCR. Design 3 parallel samples for each group, and calculate the ΔCt value of the determination results. If the ΔCt value difference between any two parallel samples of the same urine sample is greater than or equal to 1, it is considered that the test results between the parallel samples of the sample have significant differences, and the test results are counted as inaccurate; if the ΔCt value difference between any two parallel samples of the same urine sample is less than 1, it is considered that there is no significant difference between the test results between the parallel samples of the sample, and the test results are counted as accurate. The accuracy of the test results of each sample obtained by the enrichment method of Example 1 and Example 2 was counted separately, and the accuracy rate (%) was calculated; accuracy rate (%) = number of samples with accurate test results/total number of samples × 100%. See Table 2 for specific results.

Table 2 Accuracy of ΔCt values after qPCR quantitative determination of cfDNA obtained by different enrichment methods

It can be seen from the data results in Table 2 that, compared with the method in Example 1, the method in Example 2 can relatively accurately and completely enrich small fragments of cfDNA in urine to ensure the accuracy of the test results, thereby achieving excellent stability of the test results between samples.

This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims (6)

1. A method for enriching cfDNA in urine based on a biotin dual probe, comprising the following steps: S1, hybridizing the target cfDNA and the biotin dual-probe streptavidin magnetic beads to obtain the biotin dual-probe streptavidin magnetic beads that capture the cfDNA; S2, washing the biotin dual-probe streptavidin magnetic beads that capture cfDNA to remove non-target cfDNA, and obtaining purified biotin dual-probe streptavidin magnetic beads that capture target cfDNA; S3, heating and denaturing the purified biotin dual-probe streptavidin magnetic beads that capture the target cfDNA, cooling, incubating, centrifuging, and taking the supernatant to recover the target cfDNA; The biotin dual probe streptavidin magnetic beads include streptavidin magnetic beads, dual biotin labeled probe BP1 connected to the streptavidin magnetic beads, and dual biotin labeled probe BP2 connected to the streptavidin magnetic beads; The preparation method of the biotin dual-probe streptavidin magnetic beads comprises the following steps: 1. Mixing the streptavidin magnetic beads and salt buffer to obtain a streptavidin magnetic bead suspension, wherein the volume ratio of the streptavidin magnetic beads to the salt buffer is 1:(0.9-1.1); The double biotin labeled probe BP1, the double biotin labeled probe BP2, the ferroferric oxide modified biotin and the streptavidin magnetic bead suspension are mixed, so that the final concentration of the double biotin labeled probe BP1 is 45-55 μM, the final concentration of the double biotin labeled probe BP2 is 45-55 μM, and the final concentration of the ferroferric oxide modified biotin is 15-20 μM; after incubation, the solution is removed to obtain the initial biotin double probe streptavidin magnetic beads; Wherein, the probe BP1 includes polyA and a short sequence-specific binding sequence A complementary to the target, the probe BP2 includes polyA and a short sequence-specific binding sequence B complementary to the target, the sequence of the short sequence-specific binding sequence A is: CTCAACCAATAAAACCTACTCCTCC, and the sequence of the short sequence-specific binding sequence B is: CTAACTTTAAACRCTAACAAACGC; II. The initial biotin dual-probe streptavidin magnetic beads are mixed with a 5-6M guanidine hydrochloride solution at pH 1.2-1.8 and stirred, and the 5-6M guanidine hydrochloride solution at pH 1.2-1.8 is replaced with a 4-5M guanidine hydrochloride solution, a 3-4M guanidine hydrochloride solution, and water in sequence. After removing the water, the biotin dual-probe streptavidin magnetic beads are obtained. 2. According to a method for enriching cfDNA in urine based on a biotin dual probe according to claim 1, it is characterized in that the incubation time for preparing the biotin dual probe streptavidin magnetic beads is 10-15 min. 3. According to a method for enriching cfDNA in urine based on a biotin dual probe according to claim 1, it is characterized in that the initial biotin dual probe streptavidin magnetic beads are mixed and stirred with a guanidine hydrochloride solution of 5-6M at a volume ratio of 0.8-1.2:1. 4. A method for enriching cfDNA in urine based on a biotin dual probe according to claim 1, characterized in that the components of the salt buffer include: 0.8-1.2M NaCl, 8-13mM Tris-HCl, 0.03-0.07wt% Tween-20, with water as the solvent. 5. According to a method for enriching cfDNA in urine based on biotin dual probes according to claim 1, it is characterized in that S1 specifically includes the following steps: mixing the target cfDNA, incubation solution A and biotin dual probe streptavidin magnetic beads, heating and denaturing at 88-92°C for 12-18 minutes, the components of the incubation solution A include: 4-6M NaCl solution and 8-12wt% Tween-20 solution, and the volume ratio of the NaCl solution to the Tween-20 solution is (18.5-20.5):1. 6. A method for enriching cfDNA in urine based on a biotin dual probe according to claim 1, characterized in that the temperature of the heating denaturation in S3 is 92-98°C, and the time of the heating denaturation is 1-4 minutes.