A bispecific probe captures magnetic beads, and a preparation method and application thereof

Abstract

The application belongs to the field of biological detection, and particularly relates to a preparation method of a capture magnetic bead. More particularly, the application relates to a preparation method of a bispecific probe capture magnetic bead. The application provides a preparation method of a bispecific probe capture magnetic bead, which comprises the following steps: coupling a capture magnetic bead with a bispecific probe, wherein a hypoxanthine is introduced into a 5' end 15bp-25bp region of the bispecific probe; and the length of the bispecific probe is 20-70 bp. The magnetic bead of the application can better capture a target fragment, ensure the stability of the capture, and has higher sensitivity. For low-concentration nucleic acids in complex sample types, the detection rate can be significantly improved, and the false capture caused by mismatches can be effectively reduced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biological detection, and particularly relates to a preparation method of a capture magnetic bead. More particularly, the present application relates to a preparation method of a bispecific probe capture magnetic bead. BACKGROUND

[0002] At present, screening techniques based on DNA of complex samples (such as fecal samples) are used as a means of screening intestinal microorganisms or cancer due to their non-invasiveness and high sensitivity. Many DNA-based colorectal cancer screening products have been approved by the National Medical Products Administration, and the detection targets mainly include Septin9, SDC2, BMP3, NDRG4, miRNA, etc.

[0003] Compared with the early cancer screening method of detecting free DNA in the plasma, urine or pleural effusion of a patient, the detection of fecal samples of a patient has higher sensitivity and accuracy. However, the components contained in the fecal sample are complex, mainly various types of bacteria and food residues, which cause great background interference and inhibition to target detection, and the content of cancer cells is low, which makes the detection rate of the target low and unstable. At present, the ordinary magnetic bead extraction kit on the market can meet the subsequent detection needs for the extraction and purification of plasma, urine or pleural effusion samples, but it is difficult to enrich, extract and purify the corresponding target fragments for complex samples such as fecal samples.

[0004] Therefore, some studies combine DNA / RNA capture probes with carboxyl groups of carboxyl magnetic beads to form capture magnetic beads to capture specific sequences in complex samples. However, the conventional magnetic bead capture probe used at present is a specific nucleotide sequence with a length of about 30-50 bp. The longer the sequence, the higher the capture specificity. However, a too long sequence may form a folded dimer or hairpin structure, which affects the subsequent magnetic bead capture efficiency. Especially when detecting targets such as methylation and non-methylation of colorectal cancer, the specific detection site is mainly the difference of CpG site. Due to the particularity of the site, there may be only 1-3 base differences between the methylation and non-methylation targets during the detection process. The ordinary capture probe can still normally bind and capture when there is a single or even three base mismatches. This non-specific capture may lead to insufficient amount of captured nucleic acid or inaccurate detection of target fragments.

[0005] Therefore, there is a need in the art for a method for specifically extracting target nucleic acid from a complex sample, which has high accuracy and good sensitivity. SUMMARY

[0006] Therefore, in a first aspect, the present application provides a preparation method of a bispecific probe capture magnetic bead, characterized in that the method comprises:

[0007] coupling the capture magnetic beads with the bi-specific probe,

[0008] wherein the 5' end of the bi-specific probe is modified with a primary amino group, and hypoxanthine is introduced at a position of 15-25 bp in the 5' end of the probe.

[0009] wherein the bi-specific probe has a length of 20-70 bp.

[0010] In some specific embodiments, the number of hypoxanthines is 1-8.

[0011] In some specific embodiments, the number of hypoxanthines is 3-6.

[0012] In some specific embodiments, the number of hypoxanthines can be 1, 2, 3, 4, 5, 6, 7, or 8.

[0013] In some specific embodiments, the capture magnetic beads are carboxyl magnetic beads, and the 5' end of the bi-specific probe is modified with a primary amino group.

[0014] In some specific embodiments, the carboxyl groups of the carboxyl magnetic beads are activated, comprising:

[0015] S1, placing the magnetic beads on a magnetic stand for adsorption;

[0016] S2, adding 2-morpholinoethanesulfonic acid and shaking for mixing, and placing on a magnetic stand for adsorption.

[0017] More preferably, the activation of the carboxyl groups of the magnetic beads comprises: repeating the step S2.

[0018] In some specific embodiments, the coupling of the carboxyl magnetic beads with the bi-specific probe comprises:

[0019] mixing and shaking the bi-specific probe with the activated magnetic beads, 2-morpholinoethanesulfonic acid, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride for incubation.

[0020] In some specific embodiments, the preparation method further comprises: blocking of the bi-specific capture magnetic beads.

[0021] In some specific embodiments, the blocking comprises: 1) after the coupling is completed, placing on a magnetic stand for adsorption, and adding PBST solution (phosphate buffered saline), containing 1% BSA (bovine serum albumin), shaking for mixing, and blocking.

[0022] In some specific embodiments, the target of the bi-specific probe is SDC2 gene, NDRG4 gene, Septin9 gene, β-actin gene, and / or E. coli.

[0023] In some specific embodiments, the target of the bispecific probe is the SDC2 gene.

[0024] Further, the probe sequence is:

[0025] 5'-CGGCGTTTATTGGTTTTCGGAGTTGIIIITCGGCGTGTAA-3' (SEQ ID NO: 1).

[0026] In some specific embodiments, the target of the bispecific probe is the NDRG4 gene.

[0027] Further, the probe sequence is:

[0028] 5'-TTTATCGGGTATTTTAGTCIIIIAGAAGGCGGAAGTTACG-3' (SEQ ID NO: 2).

[0029] In some specific embodiments, the target of the bispecific probe is the Septin9 gene.

[0030] Further, the probe sequence is:

[0031] 5'-TTAGTTATTATGTCGGATTTIIIIGTTAACGCGTAGTTGG-3' (SEQ ID NO: 3).

[0032] In some specific embodiments, the target of the bispecific probe is the β-actin gene.

[0033] Further, the probe sequence is:

[0034] 5'-TTGTAATTTTTAAGGGAGGAGIIIITTTTATTGGTT-3' (SEQ ID NO: 4).

[0035] Further, the probe can also target E. coli, the sequence of which is

[0036] 5'-AGTTTATCTGCAAGGTGAIIIITTAATTCCTCTCTTTCCT-3' (SEQ ID NO: 5).

[0037] Using the magnetic bead preparation method of the present application, the double-specificity capture magnetic beads can better capture target fragments, ensure the stability of the capture, and have higher sensitivity. For low-concentration nucleic acids in complex sample types, the detection rate can be significantly improved, and multiple sample purification steps are not required, reducing the operation steps, rapidly and efficiently extracting target nucleic acids, and effectively reducing the error capture caused by mismatches.

[0038] In a second aspect, the present application provides a double-specificity capture magnetic bead, which is prepared by the above preparation method.

[0039] In some specific embodiments, the target of the double-specificity probe is an SDC2 gene, an NDRG4 gene, a Septin9 gene, a β-actin gene, and / or an Escherichia coli.

[0040] In some specific embodiments, the target of the double-specificity probe is an SDC2 gene.

[0041] Further, the probe sequence is:

[0042] 5'-CGGCGTTTATTGGTTTTCGGAGTTGIIIITCGGCGTGTAA-3' (SEQ ID NO: 1).

[0043] In some specific embodiments, the target of the double-specificity probe is an NDRG4 gene.

[0044] Further, the probe sequence is:

[0045] 5'-TTTATCGGGTATTTTAGTCIIIIAGAAGGCGGAAGTTACG-3' (SEQ ID NO: 2).

[0046] In some specific embodiments, the target of the double-specificity probe is a Septin9 gene.

[0047] Further, the probe sequence is:

[0048] 5'-TTAGTTATTATGTCGGATTTIIIIGTTAACGCGTAGTTGG-3' (SEQ ID NO: 3).

[0049] In some specific embodiments, the target of the double-specificity probe is a β-actin gene.

[0050] Further, the probe sequence is:

[0051] 5'-TTGTAATTTTTAAGGGAGGAGIIIITTTTATTGGTT-3' (SEQ ID NO: 4).

[0052] Further, the probe can also target E. coli, the sequence being

[0053] 5'-AGTTTATCTGCAAGGTGAIIIITTAATTCCTCTCTTTCCT-3' (SEQ ID NO: 5).

[0054] In a third aspect, the present application provides a kit for extracting nucleic acid, characterized in that it comprises the dual-specificity capture magnetic beads as described above.

[0055] Further, the present application provides a kit for extracting nucleic acid from a fecal sample, characterized in that it comprises the dual-specificity capture magnetic beads as described above.

[0056] In some specific embodiments, the kit comprises polyethylene glycol.

[0057] Preferably, the concentration of the polyethylene glycol is 15% to 35%.

[0058] More preferably, the concentration of the polyethylene glycol is 20% to 30%.

[0059] Most preferably, the concentration of the polyethylene glycol is 20%.

[0060] The use of polyethylene glycol at a suitable concentration can increase the capture efficiency of the dual-specificity capture magnetic beads, further improving the sensitivity of the dual-specificity capture magnetic beads.

[0061] In some specific embodiments, the kit further comprises a positive control and a negative control.

[0062] In a fourth aspect, the present application provides the use of the dual-specificity capture magnetic beads as described above for preparing a kit for extracting nucleic acid.

[0063] In a fifth aspect, the present application provides a method for extracting nucleic acid, comprising using the dual-specificity capture magnetic beads as described above to extract a sample.

[0064] Further, the sample is feces, urine, plasma, or pleural effusion.

[0065] Preferably, the sample is feces. BRIEF DESCRIPTION OF DRAWINGS

[0066] Figure 1 Comparison chart of the effects of different types of SDC2 gene modified magnetic beads on the extraction of high-concentration nucleic acid from fecal samples;

[0067] Figure 2 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on low-concentration fecal samples of SDC2 gene;

[0068] Figure 3 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on high-concentration fecal samples of β-actin gene;

[0069] Figure 4 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on low-concentration fecal samples of β-actin gene;

[0070] Figure 5 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on high-concentration plasma samples of SDC2 gene;

[0071] Figure 6 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on low-concentration plasma samples of SDC2 gene;

[0072] Figure 7 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on high-concentration plasma samples of β-actin gene;

[0073] Figure 8 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on low-concentration plasma samples of β-actin gene;

[0074] Figure 9 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on high-concentration urine samples of SDC2 gene;

[0075] Figure 10 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on low-concentration urine samples of SDC2 gene;

[0076] Figure 11 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on high-concentration urine samples of β-actin gene;

[0077] Figure 12 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on low-concentration urine samples of β-actin gene;

[0078] Figure 13 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on high-concentration pleural effusion samples of SDC2 gene;

[0079] Figure 14 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on low-concentration pleural effusion samples of SDC2 gene;

[0080] Figure 15 Figure for comparison of nucleic acid extraction effect of different types of modified magnetic beads on high-concentration pleural effusion samples of β-actin gene;

[0081] Figure 16 Figure for comparison of nucleic acid extraction effect of low concentration of chest and abdominal fluid sample with different types of β-actin gene modified magnetic beads;

[0082] Figure 17 Figure for extraction effect of different concentrations of PEG;

[0083] Figure 18 Figure for nucleic acid extraction effect of different positions of SDC2 gene capture probes;

[0084] Figure 19 Figure for nucleic acid extraction effect of different positions of NDRG4 gene capture probes;

[0085] Figure 20 Figure for nucleic acid extraction effect of different positions of Septin9 gene capture probes;

[0086] Figure 21 Figure for nucleic acid extraction effect of different capture probes of Septin9 with 1 mismatched base;

[0087] Figure 22 Figure for nucleic acid extraction effect of different capture probes of Septin9 with 3 mismatched bases. DETAILED DESCRIPTION

[0088] The advantages and various effects of the present application will be more clearly presented hereinafter with specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, rather than limit the present application.

[0089] Example 1, Preparation of bispecific probe capture magnetic beads

[0090] 1.1 Carboxyl group activation:

[0091] 1) Take 1 mL of carboxyl magnetic beads from 4℃ refrigerator and place on a magnetic stand for adsorption for 2 min, and then discard the supernatant after the solution becomes clear;

[0092] 2) After adding 1 mL of 2-morpholinoethanesulfonic acid (MES, concentration 0.05-0.1 M, pH 5.0-6.0), shake and mix for 5 min, place on a magnetic stand for adsorption for 2 min, and discard the supernatant;

[0093] 3) Repeat step 2 once;

[0094] 1.2 Coupling of carboxyl magnetic beads and primary amino modified bispecific probes:

[0095] 1) Take off the centrifuge tube from the magnetic stand, and add 10 μL of primary amine modified bispecific probe (100 μM, 5' end modified with primary amine, 20-70 bp in length), 100 μL of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (10-20 mg / mL), and 1 mL of 2-morpholinoethanesulfonic acid at a volume ratio of 1:10:100;

[0096] 2) Place the coupling mixture on a constant temperature metal shaking bath, and shake at room temperature (20-31 °C) for 1-2 hours, and then place it at 4 °C for 6-10 hours (keep the magnetic beads in suspension);

[0097] 1.3 Blocking and storage:

[0098] 1) After coupling, place the mixture on a magnetic stand for 2 min, remove the supernatant, and add 1 mL of PBST solution (phosphate buffered saline with Tween 20) at pH 7.0 containing 1% BSA (bovine serum albumin), and shake at 25 °C for 1.5 hours to block the unactivated carboxyl groups on the surface of the magnetic beads;

[0099] 2) Place the blocked magnetic beads on a magnetic stand, remove the supernatant, and add 1 mL of 1% SDS washing solution (pH 7.0-7.5) to remove the probes that failed to couple, and shake gently to mix, and then place it at room temperature for 1 min, and then place it on a magnetic stand for 2 min, and discard the supernatant;

[0100] 3) Repeat step 2 once;

[0101] 4) Add 1 mL of TE buffer to the centrifuge tube, and store it at 2-8 °C.

[0102] Example 2, Extraction of sample nucleic acids by bispecific probe-captured magnetic beads

[0103] 2.1 Cell lysis:

[0104] 1) Add 3 mL of cell lysis solution (10-20 mmol / L EDTA, 100 mmol / L Tris, pH 7.5-8.0, 0.8% (W / V) SDS) and 2-4 mL of sample (supernatant of fecal sample after centrifugation, precipitate of urine and ascites sample after centrifugation) to a 10-15 mL centrifuge tube;

[0105] 2.2 Magnetic bead capture:

[0106] 1) Heat at 95 °C for 10-15 min, add 50-100 μL of capture magnetic beads and 2 mL of 10%-40% polyethylene glycol (pH 6.0-7.0), shake to mix for 2 min, and then place it at room temperature for 1 h;

[0107] 2) Place the centrifuge tube on the magnetic stand, discard the waste liquid slowly after 5 min;

[0108] 2.3 Wash and save:

[0109] 1) Add 2 mL of washing solution 1 (2.0-3.0 M guanidine hydrochloride, isopropanol, pH = 6.5-7.5), shake and mix for 30 s, and place the centrifuge tube on the magnetic separator. Magnetize for 3 min, and completely discard the liquid;

[0110] 2) Repeat step 1 once;

[0111] 3) Add 2 mL of washing solution 2 (70%-75% ethanol), shake and mix for 30 s, and place the centrifuge tube on the magnetic separator. Magnetize for 3 min, and completely discard the liquid;

[0112] 4) Repeat step 3 once;

[0113] 5) Open the cover and stand at room temperature (20-30°C) for 5 min;

[0114] 6) Add 50 μL of TE buffer (0.05-0.1 M Tris-HCl, 50 mM EDTA, pH = 7.5-8.0) preheated to 50°C, shake and mix for 30 s, elute the magnetic beads on the wall of the centrifuge tube to the bottom of the tube, stand at room temperature for 5 min for elution, and store at 2-8°C.

[0115] Example 3, Bispecific probe captures target nucleic acid

[0116] To verify the difference between the bispecific probe capture magnetic beads and the ordinary capture magnetic beads, capture probes were prepared for the purpose of SDC2 gene after methylation and Escherichia coli, and human-derived internal standard β-actin was added as a reference gene:

[0117] Capture probe for SDC2 gene methylation:

[0118] Capture sequence:

[0119] 5'-CGGCGTTTATTGGTTTTCGGAGTTGTTAATCGGCGTGTAA-3'(SEQ ID NO:6);

[0120] Bispecific probe for capturing SDC2 gene methylation:

[0121] Capture sequence:

[0122] 5'-CGGCGTTTATTGGTTTTCGGAGTTGIIIITCGGCGTGTAA-3'(SEQ ID NO:1);

[0123] Probes for capturing E. coli:

[0124] Capture sequence:

[0125] 5'-AGTTTATCTGCAAGGTGATTCCTTAATTCCTCTCTTTCCT-3' (SEQ ID NO: 7);

[0126] Bispecific probes for capturing E. coli:

[0127] Capture sequence:

[0128] 5'-AGTTTATCTGCAAGGTGAIIIITTAATTCCTCTCTTTCCT-3' (SEQ ID NO: 5).

[0129] Probes for capturing methylation of β-actin gene:

[0130] Capture sequence:

[0131] 5'-TTGTAATTTTTAAGGGAGGAGTAGGTTTTATTGGTT-3' (SEQ ID NO: 8);

[0132] Bispecific probes for capturing methylation of β-actin gene:

[0133] Capture sequence: 5'-TTGTAATTTTTAAGGGAGGAGIIIITTTTATTGGTT-3' (SEQ ID NO: 4).

[0134] To verify the extraction effect of specific capture probes on methylation positive samples, two concentrations of nucleic acid samples were tested. Since the amount of nucleic acid cannot be controlled during extraction, the specific methylation fragments were artificially quantified and added to the samples. The methylation fragments were human genomic methylation standards. The bisulfite-converted methylation standards were added to negative fecal supernatant ( Figures 1-4 ), plasma ( Figures 5-8 ), urine ( Figures 9-12 ), or pleural effusion ( Figures 13-16 ) to prepare simulated samples containing exogenous interferents. The high concentration of methylation fragments in the simulated samples was 100 ng / μL, and the low concentration of methylation fragments was 0.1 ng / μL. Unmethylated nucleic acid fragments were also added as background signals, and the content of unmethylated fragments was 100 ng / μL.

[0135] Specific capture:

[0136] The high and low concentration simulation samples were extracted by the bispecific probe captured magnetic beads, the common probe captured magnetic beads and the carboxyl magnetic beads prepared in Example 1, the extraction steps were performed according to the operation of Example 2, and finally the nucleic acid was eluted with 50 μL TE buffer.

[0137] Sulfite conversion:

[0138] The nucleic acid extracted in Example 2 was subjected to sulfite conversion, the conversion kit was EZ DNA Methylation-lingthting Kit, and the conversion steps were strictly performed according to the operation manual, and finally the converted nucleic acid was eluted with 50 μL TE buffer.

[0139] PCR amplification detection:

[0140] The extracted nucleic acid was detected by fluorescent PCR, and the methylation primer probe was designed in the SDC2 promoter region for testing and verification. In order to ensure the accuracy of the detection, human-derived internal standard β-actin was designed as an internal reference gene.

[0141] Primer for detecting SDC2 gene methylation:

[0142] Forward primer: 5'-CGTAGGAGTTTTGGTTTGTCG-3'(SEQ ID NO: 9)

[0143] Reverse primer: 5'-ACAATATAACTCCCAAATAAACCCG-3'(SEQ ID NO: 10)

[0144] Probe for detecting SDC2 gene methylation:

[0145] Fluorescent probe: 5'-TTCGGAGTTGTTAATCGGCGTG-3'(SEQ ID NO: 11)

[0146] Primer for detecting E. coli:

[0147] Forward primer: 5'-TGTACAAGTCCACAAGGAAAGTAAAGAT-3'(SEQ ID NO: 12)

[0148] Reverse primer: 5'-TGTTTCGATGAGTTTATCTGCAAGGT-3'(SEQ ID NO: 13)

[0149] Probe for detecting E. coli:

[0150] Fluorescent probe: 5'-TCTAACTAGGACCGCAGAGGAAAGAGAGGAATT-3'(SEQ ID NO: 14)

[0151] Primers for detecting methylation of β-actin gene:

[0152] Forward primer: 5'-GTGTGTTGGGTGGTGGTTATTT-3' (SEQ ID NO: 15)

[0153] Reverse primer: 5'-CCAAAAAAAAAACTACTTATTCCAATTCAC-3' (SEQ ID NO: 16)

[0154] Probes for detecting methylation of β-actin gene:

[0155] Fluorescent probe: 5'-TTAAGGGAGGAGTAGGTT-3' (SEQ ID NO: 17).

[0156] PCR reaction system and amplification system are shown in Table 1 below:

[0157] Table 1, PCR reaction system table

[0158]

[0159]

[0160] Table 2, PCR fluorescence amplification procedure table

[0161]

[0162] The effects of different types of magnetic beads on extracting nucleic acids from different samples are shown in Tables 3-6 below.

[0163] Table 3, Comparison table of Ct values of different types of modified magnetic beads amplification (fecal sample)

[0164]

[0165] Table 4, Comparison table of Ct values of different types of modified magnetic beads amplification (plasma sample)

[0166]

[0167] Table 5, Comparison table of Ct values of different types of modified magnetic beads amplification (urine sample)

[0168]

[0169] Table 6, Comparison table of Ct values of different types of modified magnetic beads amplification (ascites sample)

[0170]

[0171] For fecal samples, the target nucleic acid in the dual-specificity capture magnetic beads is higher than that in the common probe capture magnetic beads and carboxyl magnetic beads, and the Ct value is earlier. At the same time, compared with the carboxyl magnetic beads, the specific capture magnetic beads are less affected by inhibitors in complex sample types, and have higher capture efficiency, especially for low-concentration nucleic acid samples. The modified capture magnetic beads can effectively bind the target fragments and exclude the influence of interfering substances, so the dual-specificity capture magnetic beads and the common capture magnetic beads can effectively distinguish target fragments from interfering substances. The carboxyl magnetic beads have the worst extraction effect, because the presence of unmethylated nucleic acid fragments interferes with the selective binding of target fragments by carboxyl magnetic beads, and the extraction concentration of target fragments is low in fecal, urine, plasma or ascites samples.

[0172] In urine, plasma and ascites samples, there are fewer exogenous interferents, and the sample environment is significantly purer than fecal samples, so there is no significant difference in capture efficiency between the dual-specificity capture magnetic beads and the common probe capture magnetic beads, and both can capture specific target fragments.

[0173] For the capture of E. coli, the results are shown in Table 7.

[0174] Table 7. Comparison of amplification Ct values of different types of modified magnetic beads (fecal samples)

[0175]

[0176] As can be seen from the table, the dual-specificity probe capture magnetic beads have better capture effect on E. coli.

[0177] Example 4. Dual-specificity probe capture probe combined with PEG to extract target nucleic acid

[0178] During the extraction process of the dual-specificity capture magnetic beads, we found that adding a certain concentration of polyethylene glycol can increase the complementary binding rate between the capture sequences. By adding different concentrations of polyethylene glycol during the extraction process, the results are shown in Figure 17 As can be seen from the figure, the extraction effect of 20% and 30% PEG is better than that of the remaining concentrations of PEG.

[0179] Example 5. Effect of introducing hypoxanthine at different positions of the capture probe on capture efficiency

[0180] To verify the effect of introducing hypoxanthine at different positions of the capture probe on the capture efficiency, the methylation of the target genes SDC2, NDRG4 and Septin9 was used as the capture probe, and hypoxanthine was introduced at the 5' end, 3' end and middle region of the capture sequence. The results are shown in Figures 18-20As shown in the figure, it can be seen that the introduction of hypoxanthine at the position of 15-25 bp region from the 5' end of the bispecific probe can greatly improve the sensitivity of the capture probe.

[0181] Probe for capturing methylation of SDC2 gene:

[0182] Capture sequence:

[0183] 5'-CGGCGTTTATTGGTTTTCGGAGTTGTTAATCGGCGTGTAA-3'(SEQ ID NO: 6);

[0184] Bispecific probe 1 for capturing methylation of SDC2 gene:

[0185] Capture sequence:

[0186] 5'-CGGCGTTTATTGGTTTTCGGAGTTGIIIITCGGCGTGTAA-3'(SEQ ID NO: 1);

[0187] Bispecific probe 2 for capturing methylation of SDC2 gene:

[0188] Capture sequence:

[0189] 5'-CIIIITTTATTGGTTTTCGGAGTTGTTAATCGGCGTGTAA-3'(SEQ ID NO: 18);

[0190] Probe 3 for capturing methylation of SDC2 gene:

[0191] Capture sequence:

[0192] 5'-CGGCGTTTATTGGTTTTCGGAGTTGTTAATCGGCGIIIIA-3'(SEQ ID NO: 19).

[0193] Probe for capturing methylation of NDRG4 gene:

[0194] Capture sequence:

[0195] 5'-TTTATCGGGTATTTTAGTCGCGTAGAAGGCGGAAGTTACG-3'(SEQ ID NO: 20);

[0196] Bispecific probe 1 for capturing methylation of NDRG4 gene:

[0197] Capture sequence:

[0198] 5'-TTTATCGGGTATTTTAGTCIIIIAGAAGGCGGAAGTTACG-3' (SEQ ID NO: 2);

[0199] Bispecific probe 2 that captures methylation of the NDRG4 gene:

[0200] Capture sequence:

[0201] 5'-TTIIIIGGGTATTTTAGTCGCGTAGAAGGCGGAAGTTACG-3' (SEQ ID NO: 21);

[0202] Bispecific probe 3 that captures methylation of the NDRG4 gene:

[0203] Capture sequence:

[0204] 5'-TTTATCGGGTATTTTAGTCGCGTAGAAGGCGGAAIIIICG-3' (SEQ ID NO: 22).

[0205] Probe that captures methylation of the Septin 9 gene:

[0206] Capture sequence:

[0207] 5'-TTAGTTATTATGTCGGATTTCGCGGTTAACGCGTAGTTGG-3' (SEQ ID NO: 23);

[0208] Bispecific probe 1 that captures methylation of the Septin 9 gene:

[0209] Capture sequence:

[0210] 5'-TTAGTTATTATGTCGGATTTIIIIGTTAACGCGTAGTTGG-3' (SEQ ID NO: 3);

[0211] Bispecific probe 2 that captures methylation of the Septin 9 gene:

[0212] Capture sequence:

[0213] 5'-TTIIIIATTATGTCGGATTTCGCGGTTAACGCGTAGTTGG-3' (SEQ ID NO: 24);

[0214] Bispecific probe 3 that captures methylation of the Septin 9 gene:

[0215] Capture sequence:

[0216] 5'-TTAGTTATTATGTCGGATTTCGCGGTTAACGCGTIIIIGG-3' (SEQ ID NO: 25).

[0217] Accuracy of capturing by different capture probes

[0218] To verify the accuracy of capturing by the capture probe of the present application in a fecal sample, the methylation of the target gene Septin9 after colorectal cancer was taken as the capture target fragment, and 1-3 mismatched bases were introduced into the synthesized methylation fragment. The fragment with the introduced mismatch was put into the negative fecal supernatant to prepare a simulated sample, and the content of the mismatched methylation fragment in the simulated sample was 100 ng / μL. At the same time, the nucleic acid extraction effects of the bispecific magnetic beads, ordinary magnetic beads and carboxyl magnetic beads were compared:

[0219] The sequence of the methylation of the Septin9 gene with 1 mismatched base introduced:

[0220] Mismatched sequence: 5'-TTAGTTATTATGT T GGATTTCGCGGTTAACGCGTAGTTGG-3' (SEQ ID NO: 26);

[0221] The sequence of the methylation of the Septin9 gene with 3 mismatched bases introduced:

[0222] Mismatched sequence: 5'-TTAGTTATTATGT T GGATTTCGCGGTTAA T G T GTAGTTGG-3' (SEQ ID NO: 27).

[0223] Table 8: Comparison table of Ct values extracted by different capture magnetic beads

[0224]

[0225] From Figures 21-22 It can be seen from Table 8 that the carboxyl magnetic beads cannot distinguish the accuracy of the nucleic acid sequence, and can bind as long as there is nucleic acid, so the detection result is the highest in nucleic acid concentration; the ordinary capture magnetic beads still have a small amount of mismatched sequences combined with the capture probe when there is only a difference of 1-3 bases in the sequence, so the detection result is positive; the bispecific capture magnetic beads have enhanced specificity of the capture sequence due to the introduction of hypoxanthine, so the detection result is negative when there is a mismatched base in the sequence that cannot normally combine with the magnetic beads, and the accuracy of capturing is the highest. Therefore, it can be seen that the accuracy of capturing by the bispecific probe of the present application is much higher than that of the ordinary probe and the carboxyl magnetic beads, and the accuracy of capturing is significantly improved when there is only 1 base mismatch.

Claims

1. A method for preparing bi-specific probe-captured magnetic beads, characterized in that, The method comprises: coupling the capture magnetic beads with the bispecific probe, wherein 4-6 consecutive hypoxanthines are introduced in the 15 bp-25 bp region at the 5' end of the bispecific probe to replace the original bases; wherein the length of the bispecific probe is 36-70 bp.

2. The method of claim 1, wherein, The target of the bispecific probe is SDC2 gene, NDRG4 gene, Septin9 gene, beta-actin gene, and / or Escherichia coli.

3. The method of claim 2, wherein, The sequence of the bispecific probe is one or more of the sequences shown in SEQ ID NO: 1-5.

4. The method according to any one of claims 1 to 3, characterized in that, The capture magnetic beads are carboxyl magnetic beads, and the 5' end of the bispecific probe is modified with primary amino groups.

5. The method of claim 4, wherein, Coupling the carboxyl magnetic beads with the bispecific probe comprises: S1, placing the magnetic beads on a magnetic stand for adsorption; S2, adding 2-morpholinoethanesulfonic acid, oscillating and mixing, placing on a magnetic stand for adsorption and activation; S3, incubating the bispecific probe with the activated carboxyl magnetic beads, 2-morpholinoethanesulfonic acid and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in step S2.

6. A bispecific probe-captured magnetic bead, characterized in that, Prepared by the preparation method of any one of claims 1-5.

7. A kit for extracting nucleic acids, characterized by comprising: The bispecific probe capture magnetic beads of claim 6.

8. The kit of claim 7, wherein The kit comprises polyethylene glycol.

9. The kit of claim 8, wherein The concentration of the polyethylene glycol is 20%-30%.

10. Use of the bispecific probe capture magnetic beads of claim 6 for preparing an extraction nucleic acid kit.

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