WO2021102646A1 - Nucleic acid capture method and probe, and use thereof - Google Patents

Abstract

Provided in the present invention is an oligonucleotide capture probe for capturing a target nucleic acid in biological samples, such as a bodily fluid and faeces. The 3 'end of the capture probe is further subjected to sealing modification on the basis of a conventional capture probe. When a target nucleic acid obtained by means of enrichment by such a modified capture probe is used in subsequent nucleic acid amplification based detection, such as PCR, fluorescent quantitative PCR, and in particular, ARMS PCR detection, the probability of false positives occurring can be reduced.

Description

Nucleic acid capture method, probe and use thereof Technical field

The invention belongs to the field of biotechnology, and specifically relates to a method for capturing target nucleic acid and a method for subsequent PCR nucleic acid detection.

Background technique

Nucleic acid detection requires the separation of nucleic acid from biological samples. In recent years, liquid biopsy technology is a hot research topic. Non-invasive methods are used to extract and isolate free DNA and cell-free DNA from peripheral blood, urine, and feces, and then perform PCR and fluorescence PCR, high-throughput sequencing and other technologies can be used to detect cancer, viruses, bacteria or pathogenic infections, drug responses, etc.

But one problem is that the concentration of nucleic acid in liquid biopsy biological samples is relatively low, and there are many non-specific background nucleic acids and inhibitors that interfere with subsequent detection.

Although the use of magnetic beads is conducive to the improvement of nucleic acid extraction efficiency, the general magnetic bead method is non-specific adsorption of nucleic acids. And for some special samples, the magnetic bead method can extract DNA from humans, heterologous cells, bacteria, etc. at the same time. If the target of detection is DNA from a specific source, the impact of this non-specific adsorption is greater.

Existing technologies for specific nucleic acid capture, for example, target sequence capture and sequencing is to customize the genomic region of interest into specific probes, hybridize with genomic DNA in a sequence capture chip (or solution), and then perform DNA fragments in the target genomic region. After the enrichment, the sequencing technology is used for research. For another example, Exact Sciences of the United States has developed a non-invasive colorectal cancer screening technology Cologuard, which targets the methylation region of the target gene and the KRAS mutation region. The probe is designed according to the target region, the 5 terminal is modified with the amino group, and the magnetic microbeam with a carboxyl group. Sphere binding, capture with sequence-specific target capture reagent, hybridize with the target after adding the capture reagent and incubate, then elution, and finally separate the required target DNA for direct capture of DNA fragments from feces.

After the nucleic acid is captured, subsequent detection technologies include PCR, NGS sequencing technology, and QuARTS detection technology. The present invention hopes to improve the specific capture method of nucleic acid, especially the capture method suitable for subsequent detection technology, so that the subsequent detection result is more optimized.

Summary of the invention

We try to combine the existing nucleic acid capture technology with the commonly used ARMS-PCR technology to achieve the purpose of detecting hotspot mutations in complex biological samples. It was found that when the nucleic acid obtained by the existing nucleic acid capture technology is detected by conventional PCR technology, especially when it is detected by ARMS-PCR technology, the problem of false positives often occurs. Especially for mutation detection, it will cause false positives in the detection system.

We tried to optimize the capture system, such as probe sequence optimization, and also tried to optimize the detection system, and found it difficult to overcome this problem. The present invention proposes a simple and convenient method that can effectively improve and reduce the occurrence of false positive events in the above detection system: when specifically extracting target nucleic acids, on the basis of conventional capture probes, further control the 3'of the capture probe The end is closed and modified. The modified capture probe is used to capture and enrich the target-specific nucleic acid, and continue to be used in subsequent PCR, fluorescent quantitative PCR, especially ARMS PCR detection, and it is found that the probability of occurrence of false positive events can be effectively reduced.

Furthermore, the modification of the 3'end of the capture probe can also block nucleic acid amplification.

Further, the modification at the 3'end can be selected from phosphorylation modification, dideoxy base modification, Spacer modification and the like.

On the one hand, in order to facilitate the subsequent separation of the probe, the 5'end of the probe can be appropriately modified, such as biotin modification, dual biotin modification, amino modification, poly A modification, etc., to facilitate the use of biotin-affinity Techniques such as the element magnetic bead separation method and the amino-carboxyl magnetic bead separation method separate the probe complexes that have captured the target nucleic acid.

More specifically, the capture probe can be modified by: 5-terminal biotin modification, 3-terminal phosphorylation modification; or 5-terminal dibiotin modification, 3-terminal phosphorylation modification; or 5-terminal amino modification, 3-terminal phosphorylation modification ; Or 5 terminal poly A modification, 3 terminal phosphorylation modification; or 5 terminal carboxyl modification, 3 terminal phosphorylation modification.

More specifically, the magnetic beads that bind biotin probes are streptavidin magnetic beads, the magnetic beads that bind amino modification are carboxyl magnetic beads, and the probes that bind Poly A modification are oligo-dt probes.

On the other hand, using the above-mentioned modified probe, molecular hybridization between the probe and a single-stranded nucleic acid fragment (such as formed by thermal denaturation and melting of DNA) under suitable conditions, and then separating the complex bound to the target nucleic acid, And the isolate is used in the subsequent detection steps.

Further, the separated probe-target nucleic acid complexes are detected using PCR technology, which includes conventional PCR method, fluorescent quantitative PCR method, ARMS-PCR method and the like.

Further, the detection includes the detection of the mutation of the nucleotide substitution, deletion, insertion, gene fusion or any combination of the target nucleic acid, or the detection of its methylation mutation.

Further, the target nucleic acid that is captured, separated and enriched includes DNA, RNA, microRNA, and the like.

On the other hand, the capture method of the present invention is suitable for separating and enriching target nucleic acids from biological samples such as feces, peripheral blood, plasma, serum, urine, intestinal effluent, and is also suitable for methods for extracting nucleic acids from conventional tissue samples.

In one embodiment, the capture probe and capture system can achieve multiple gene capture, and the captured product can be applied to ARMS-PCR detection to determine the mutation status of the test sample.

On the other hand, the present invention provides a nucleic acid detection method that can reduce false positives: in a detection system based on nucleic acid amplification technology, the probe in the detection object containing the probe-target nucleic acid complex is 3'-end blocked Modification to block or block the amplification and extension of the probe.

Or: Provide a nucleic acid detection method that can reduce false positives: In a detection system based on nucleic acid amplification technology, if the detection target nucleic acid is obtained by molecular hybridization capture separation and enrichment by oligonucleotide probes, then the detection step Before, especially before the capture step, the 3'end of the oligonucleotide probe that captures the target nucleic acid is blocked and modified to block or block the amplification and extension of the probe.

On the other hand, the present invention provides a nucleic acid probe whose 3'end has been blocked and modified, and this probe is used to capture and enrich a target nucleic acid in a biological sample.

Further, the use of the probe for preparing nucleic acid extraction reagents or kits is provided.

Further, the use of the probe for preparing nucleic acid detection reagents or kits is provided, and the detection includes the detection of variant forms such as gene mutations, deletions, substitutions, and copy number changes. The detection technology can be conventional PCR technology, fluorescence quantitative PCR technology, ARMS-PCR technology, DNA methylation detection technology and other detection technologies based on nucleic acid amplification.

On the other hand, the present invention provides a method and kit for more accurate detection of target gene mutation sites based on probe capture and enrichment of target genes, and the 3'end of the probe has been sealed and modified.

A more specific implementation plan includes the following steps:

(1) Processing of biological samples: The biological samples mainly include biological samples such as feces, peripheral blood, plasma, serum, urine, and intestinal effluent. After the biological sample is processed, a sample that can be used for subsequent capture is obtained. The processing steps for different samples are described as follows: (1) The specific processing steps for stool samples are as follows:

① Take 1-2g of fresh feces or 4-9ml of fecal homogenate containing fecal preservation solution, and add 1.5-2 times the volume of lysate (guanidine isothiocyanate or guanidine hydrochloride).

② Shake well, centrifuge at 4500g for 10-15min, and take the supernatant.

③Add PVPP (final concentration 30-50mg/ml), shake and mix thoroughly, centrifuge at 4500g for 10-15min, and take the supernatant.

④Add 1-2 times the volume of isopropanol, mix well, centrifuge at 120,000g for 3-5min, discard the supernatant.

⑤Add 300-500ul 70%-100% ethanol, shake well, centrifuge at 12000g for 1-2min, discard the supernatant.

⑥ Repeat step ⑤

⑦ Absorb the remaining ethanol, open the lid and place it in a heating block at 50-55°C for 4-6 minutes.

⑧Add 300-500ul 1×TE solution, shake vigorously, and place in a heating block at 60-65℃ for 8-15min.

⑨ After cooling to room temperature, centrifuge at 13000g for 3-5 min, and aspirate the supernatant to obtain a sample suitable for subsequent capture steps.

(2) The specific steps for the peripheral blood sample are as follows: the peripheral blood sample is subjected to a two-step centrifugation method at a low speed and then a high speed to separate the serum or plasma as the subsequent captured sample.

(3) Plasma, serum, urine, and intestinal effluent samples can be directly used as capture samples.

(2) The specific steps of capture probe design are as follows:

(1) Determine the detection target and find out the nucleic acid sequence corresponding to the detection target from NCBI, UCSC and other databases.

(2) The principle of designing capture probes based on the target detection target is as follows: the capture probe needs to cover or be close to the target detection target; the probe length is 50-100bp; for points with higher GC content or lower capture efficiency proved by subsequent experiments, you can Use multiple probes to capture at the same time; when multiple probes are captured at the same time, the probes can be in the same chain or complementary chain.

(3) Modification of the probe: 5'end biotin modification, 3'end phosphorylation modification; or 5'end dibiotin modification, 3'end phosphorylation modification; or 5'end amino group modification, 3'end phosphorylation Modification; or 5'end poly A modification, 3'end phosphorylation modification; or 5'end carboxyl group modification, 3'end phosphorylation modification, etc. The modification of the 3'end of the capture probe can also be other modifications that have the property of blocking nucleic acid amplification. For example, the modification of the 3'end can be selected from phosphorylation modification, dideoxy base modification, Spacer modification and the like. The specific steps of sample hybridization capture are as follows:

(1) Add 1/5 volume of 20×SSC buffer and 1ul capture probe with a concentration of 10nM to the processed sample. Shake and mix the supernatant-hybridization solution-capture probe, centrifuge briefly at low speed, and place in a water bath or heat block at 90-95°C for 5-10 minutes.

(2) Centrifuge briefly and immediately place it in a 60-65℃ water bath or heating block for 25min-35min (can be centrifuged briefly at low speed every 6-8min).

(3) Magnetic bead processing: Take an appropriate amount of magnetic beads (50-100ul/sample), place them on the magnetic stand for about 2-3 minutes, and discard the supernatant (discard as much as possible). Add 500ul-1000ul 0.5×-1×SSC buffer, mix well, place on the magnetic stand for about 2-3 minutes, and discard the supernatant. Add 500ul-1000ul 0.5×-1×SSC buffer and repeat once more. Finally, add an appropriate amount of 0.5×-1×SSC buffer, resuspend the magnetic beads, and place them at room temperature for later use.

(4) After the hybridization solution in the EP tube is cooled to room temperature, centrifuge for a short time. Add 50-100ul streptavidin magnetic beads processed in step 4 to each sample, and place at room temperature for 30-40min, every 5-8min. The magnetic beads need to be resuspended.

(5) Place the EP tube on the magnetic stand, let it stand for 2-3 minutes, and discard the supernatant.

(6) Add 300ul-500ul 0.1×-0.5×SSC buffer solution and mix well to resuspend the magnetic beads.

(7) Put it on a magnetic stand, let it stand for 2-3 minutes, and aspirate and discard the supernatant. Repeat step (7).

(8) Put it on a magnetic stand, let it stand for 2-3 minutes, and thoroughly suck up all the liquid.

(9) Add 50ul-100ul purified water or 1×TE solution, vortex to fully suspend the magnetic beads, and place in a 60-65℃ water bath or heating block for 5-10min.

(10) After cooling to room temperature, after instant centrifugation, place it on a magnetic stand and let it stand for 2-3 min. The supernatant is sucked to obtain the target DNA.

The capture method and capture probe of the present invention can reduce subsequent PCR detection steps, especially false positives in ARMS-PCR detection, ensure the accuracy and stability of the results, and the method is convenient and simple, and easy to operate.

Detailed ways

Some terms or definitions:

Target nucleic acid: refers to the target nucleic acid sequence fragment that is captured, enriched, or detected, also called target sequence.

Hybridization: Refers to the base-pairing interaction between two nucleic acids, leading to the formation of duplexes. It is not required that the two nucleic acids have 100% complementary pairing over their entire length.

Probes: small nucleic acid molecules that are about 15 to 3000 bases in length of DNA, RNA, and other nucleic acid derivatives (including but not limited to LNA, etc.). These small molecules are usually connected to some functional groups (including but not limited to other groups such as biotin). The probe will bind to the target DNA fragment in a form of complete or partial complementarity, and the functional group on the probe will have a strong affinity with other functional groups (including but not limited to streptavidin). Streptavidin, avidin, etc.) or specific antibodies. Other functional groups or antibodies that are usually combined with the functional groups on the probe are connected to consumables such as magnetic beads and absorbent materials, and the combination of the target fragment and the probe is removed from the reaction solution by physical means Extract it to achieve the purpose of capturing the target segment.

The preferred embodiments of the present invention will be described in detail below. The experimental methods that do not specify specific conditions in the examples usually follow conventional conditions, such as the conditions described in the Molecular Cloning Experiment Guide (third edition, J. Sambrook et al.), or the conditions recommended by the manufacturer.

The instruments used in the experiment are as follows: Lightcycler 480, centrifuge, magnetic stand, water bath or heating block.

Reagents used in the experiment: capture probe (Suzhou Jinweizhi), streptavidin magnetic beads (Baimag), DNA polymerase (Roche), 10×PCR Buffer (Roche), MgCl ₂ (Roche) Company), dNTP (TaKaRa), purified water.

Probes used in the experiment: All probes should be as pure as electrophoresis (PAGE) or HPLC, and contain no contaminants.

Example 1

This embodiment takes the KRAS gene capture probe as an example to illustrate the basic optimization process of the probe. The magnetic beads used are streptavidin magnetic beads.

(1) KRAS gene capture probe design and capture verification

Genome capture verification: KRAS gene quality control is H1975 lung cancer cells without KRAS gene mutation. The sequencing results of the KRAS hotspot mutation region are as follows:

Plasmid capture verification: The KRAS gene quality control is a synthetic plasmid with the wild-type sequence of the KRAS gene. The sequencing results are as follows:

(2) Probe sequence acquisition: According to the obtained genome sequence, KRAS gene capture probe design is carried out. Design four capture probes, the sequence is as follows:

Probe 1: di-biotin-TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGT

Probe 2: biotin-TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGT

Probe 3: biotin-GAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGTAGGCAAGA

Probe 4: biotin-TTGGAGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAAT

(3) KRAS gene capture: the specific steps are as follows:

① Inject H1975 cells or KRAS-negative plasmids into purified water and configure them as simulated samples. The input amount is 500ul purified water and 20ul cells or negative plasmids (copy number: 1000copy/ul).

②Put the simulated sample volume 1/2-1/5 volume of 20×SSC buffer, 1-5ul KRAS capture probe (probe concentration: 10nm)

③ Shake and mix the prepared supernatant-hybridization solution-capture probe, centrifuge briefly at low speed, and place it in a water bath or heat block at 90-95°C for 5-10 minutes.

④Centrifuge briefly and immediately place it in a 60-65℃ water bath or heating block for 25min-35min (can be centrifuged briefly at low speed every 6-8min).

⑤ Treatment of magnetic beads: Take an appropriate amount of magnetic beads (50-100ul/sample), place them on the magnetic stand for about 2-3 minutes, and discard the supernatant (discard as much as possible). Add 500ul-1000ul 0.5×-1×SSC buffer, mix well, place on the magnetic stand for about 2-3 minutes, and discard the supernatant. Add 500ul-1000ul 0.5×-1×SSC buffer and repeat once more. Finally, add an appropriate amount of 0.5×-1×SSC buffer, resuspend the magnetic beads, and place them at room temperature for later use.

⑥ After the hybridization solution in the EP tube is cooled to room temperature, centrifuge for a short time. Add 50-100ul of streptavidin magnetic beads processed in step 4 to each sample, and leave it at room temperature for 30-40min, every 5-8min. The magnetic beads are resuspended.

⑦Place the EP tube on the magnetic stand, let it stand for 2-3 minutes, and discard the supernatant.

⑧Add 300ul-500ul 0.1×-0.5×SSC buffer solution and mix well to resuspend the magnetic beads.

⑨ Put it on the magnetic stand, let it stand for 2-3 minutes, and aspirate and discard the supernatant. Repeat step seven.

⑩ Put it on a magnetic stand, let it stand for 2-3 minutes, and thoroughly suck up all the liquid.

11 Add 50ul-100ul purified water or 1×TE solution, vortex to fully suspend the magnetic beads, and place in a 60-65℃ water bath or heating block for 5-10min.

12 After cooling to room temperature, after a brief centrifugation, place it on a magnetic stand and let it stand for 2-3 min. Aspirate the supernatant to obtain the target DNA.

(4) Verification of the capture results:

The detection of the capture fluid was carried out by using Jiangsu for real human KRAS gene mutation detection kit (fluorescence PCR method). The test results are as follows:

Table 1: Comparison of the results of capturing positive cells/plasmids by the four probes in a purified water environment

Site

Genome capture

Genome capture

Genome capture

Genome capture

Uncaptured original

Plasmid capture

Plasmid capture

Plasmid capture

Plasmid capture

Uncaptured original

To	Get-probe 1	Get-probe 2	Get-probe 3	Get-probe 4	liquid	-Probe 1	-Probe 2	-Probe 3	-Probe 4	liquid
G13D	Negative	Negative	Negative	Negative	Negative	Positive	Negative	Negative	Negative	Negative
G12D	Negative	Positive	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
G12A	Positive	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Positive	Negative
G12V	Negative	Negative	Positive	Positive	Negative	Negative	Positive	Negative	Positive	Negative
G12S	Negative	Negative	Negative	Negative	Negative	Negative	Positive	Negative	Positive	Negative
G12R	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative	Negative
G12C	Negative	Negative	Positive	Positive	Negative	Negative	Positive	Positive	Negative	Negative
Internal control	27.08	27.15	28.15	28.02	26.00	27.05	27.58	28.32	28.68	26.68

From the above results, it can be seen that after the capture of probe 1, the detected CP value is closest to the uncaptured stock solution, and the capture effect is the best. Therefore, the sequence of probe 1 is the preferred sequence in the subsequent examples. The comparison result of probe 1 and probe 2 also shows that the capture efficiency of dual biotin modification is higher than that of single biotin. Simultaneous testing showed that the four capture probes all had false positives after being captured in water, and the false positive sites were different, irregular, and the capture system was unstable.

Example 2

In this example, KRAS gene capture probes are used to show the detection results of DNA obtained using conventional capture probes.

(1) The above-mentioned optimized probe 1 and capture system were used to capture the KRAS gene three times in the stool samples of 3 healthy people.

(2) The capture of stool samples mainly includes stool pre-processing and multi-gene capture. The specific steps are as follows:

Step 1: Pre-treatment of feces

① Take 1-2g of fresh feces or 4-9ml of fecal homogenate containing fecal preservation solution, and add 1.5-2 times the volume of lysate (guanidine isothiocyanate or guanidine hydrochloride).

② Shake well, centrifuge at 4500g for 10-15min, and take the supernatant.

③Add PVPP (final concentration 30-50mg/ml), shake and mix thoroughly, centrifuge at 4500g for 10-15min, and take the supernatant.

④Add 1-2 times the volume of isopropanol, mix well, centrifuge at 120,000g for 3-5min, discard the supernatant.

⑤Add 300-500ul 70%-100% ethanol, shake well, centrifuge at 12000g for 1-2min, discard the supernatant.

⑥ Repeat step ⑤

⑦ Absorb the remaining ethanol, open the lid and place it in a heating block at 50-55°C for 4-6 minutes.

⑧Add 300-500ul 1×TE solution, shake vigorously, and place in a heating block at 60-65℃ for 8-15min.

⑨ After cooling to room temperature, centrifuge at 13000g for 3-5 min, and aspirate the supernatant.

Step 2: Capture the target DNA in the stool sample

⑩Add 1/5 volume of 20×SSC buffer to the supernatant of the stool sample, and 1ul of capture probe with a concentration of 10nM. Shake and mix the supernatant-hybridization solution-capture probe, centrifuge briefly at low speed, and place in a water bath or heat block at 90-95°C for 5-10 minutes.

11. Centrifuge briefly and immediately place it in a 60-65℃ water bath or heating block for 25min-35min (can be centrifuged briefly at low speed every 6-8min).

12 Magnetic bead processing: Take an appropriate amount of magnetic beads (50-100ul/sample), place them on the magnetic stand for about 2-3 minutes, and discard the supernatant (discard as much as possible). Add 500ul-1000ul 0.5×-1×SSC buffer, mix well, place on the magnetic stand for about 2-3 minutes, and discard the supernatant. Add 500ul-1000ul 0.5×-1×SSC buffer and repeat once more. Finally, add an appropriate amount of 0.5×-1×SSC buffer, resuspend the magnetic beads, and place them at room temperature for later use.

13 After the hybridization solution in the EP tube is cooled to room temperature, centrifuge for a short time. Add 50-100ul of streptavidin magnetic beads processed in step 4 to each sample, and leave it at room temperature for 30-40min, every 5-8min, The magnetic beads are resuspended.

14 Put the EP tube on the magnetic stand, let it stand for 2-3 minutes, and discard the supernatant.

15 Add 300ul-500ul 0.1×-0.5×SSC buffer and mix well to resuspend the magnetic beads.

16 Put it on a magnetic stand, let it stand for 2-3 minutes, and aspirate and discard the supernatant. Repeat step seven.

17 Put it on a magnetic stand, let it stand for 2-3 minutes, and thoroughly suck up all the liquid.

18 Add 50ul-100ul purified water or 1×TE solution, vortex to fully suspend the magnetic beads, and place in a 60-65℃ water bath or heating block for 5-10min.

19 After cooling to room temperature, after centrifugation momentarily, place it on a magnetic stand and let it stand for 2-3 min. Aspirate the supernatant to obtain the target DNA.

(3) Verification of capture results:

①The detection of the capture fluid was carried out with the Jiangsu real human KRAS gene mutation detection kit (fluorescence PCR method). The test results are as follows.

Table 2: Comparison of results of capturing stool samples by probe 1-1

The results showed that the sample captured by probe 1 was false-positive when tested with the human KRAS gene mutation detection kit (fluorescent PCR) method, and the test results were unstable, and the repeatability of the three tests was not good.

②Select the human KRAS gene mutation detection kit (Taqman-ARMS method) to detect the captured samples. The results are shown in the table below.

Table 3 Comparison of results of capturing stool samples by probe 1-2

The results showed that the sample captured by probe 1 was false-positive in the detection of the human KRAS gene mutation detection kit (Taqman-ARMS method), and the detection result was unstable, and the repeatability of the three detections was not good.

Based on the above results, it can be seen that by using different detection kits to detect the captured samples, false positives have occurred, and the detection results are unstable. It shows that the optimization of the PCR detection system (detection primers, probes, components) cannot eliminate the false positive problem of the detection system.

Example 3

This embodiment takes KRAS and BRAF gene capture probes as examples to illustrate the relevant optimization process of the present invention.

To optimize the capture probe, it is mainly to select the reverse complementary probe of probe 1 for detection, and design the capture probe of the BRAF gene covering the V600E detection site in the forward and reverse directions for detection. Further verify whether it is because the capture probe sequence is different or the interaction between the probe and the primer causes the false positive of the detection system.

(1) Design of capture probe:

KRAS: Probe 5: di-biotin-ACGCCACCAGCTCCAACTACCACAAGTTTATATTCAGTCATTTTCA

BRAF: Probe 6: di-biotin-GGTGATTTTGGTCTAGCTACAGTGAAATCTCGATGGAGTGGG

BRAF: Probe 7: di-biotin-CCCACTCCATCGAGATTTCACTGTAGCTAGACCAAAATCACC

(2) Capture of stool samples

The above-mentioned genes were captured on 3 fecal samples of healthy people, and the capture steps are shown in Example 2.

(3) Capture sample detection

Use Jiangsu for real human KRAS gene mutation detection kit (fluorescent PCR method) and Jiangsu for real human BRAF gene mutation detection kit (fluorescent PCR method) for detection. The test results are as follows.

Table 5: Detection results of probe 5 capture sample

Table 6: Probe 6 and Probe 7 capture sample detection results

The results show that whether it is for the KRAS gene or the BRAF gene, the false positives still exist after the capture probe is used to capture, indicating that the sequence optimization design of the capture probe still cannot effectively solve the false positive problem of the capture system.

Example 4

In this embodiment, the KRAS gene capture probe is taken as an example to illustrate the relevant optimization process of the present invention.

Modify the 3'end of the capture probe to block or block the extension function of the 3'end of the probe. Choose 3 kinds of modification methods: phosphorylation modification, dideoxy base modification, Spacer modification.

(1) Phosphorylation modification, dideoxy base modification and Spacer modification to the 3'end of probe 1:

Phosphorylation modification:

KRAS-probe

8: di-biotin-TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGT-PO ₄

Dideoxy base modification:

KRAS-probe

9: di-biotin-TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGT-ddC

Spacer modification:

KRAS-probe

10: di-biotin-TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGT-spacerC3

(2) The modified probe is used to capture healthy human feces samples, and the capture steps are shown in Example 2.

(3) Verification of the capture results: The detection of the capture liquid was carried out using the Jiangsu real human KRAS gene mutation detection kit (fluorescence PCR method). The test results are as follows:

Table 7: Sample detection results after the capture of phosphorylated probe 8

The results show that the probe 8 modified with 3'end phosphorylation does not have a false positive problem when it is captured in a stool sample of a healthy person.

Table 8: Sample detection results after capture of dideoxybase modified probe 9

The results show that the probe 9 modified with the 3'end dideoxy base does not have a false positive problem when captured in a stool sample of healthy people.

Table 9: Test results of samples after the capture of Spacer modified probe 10

The results show that there is no false positive problem when using the 3'-end Spacer modified probe 10 to capture in a healthy human stool sample.

As a result, we unexpectedly found that when the 3'end of the capture probe was modified to block or block the 3 end extension function of the probe, the false positives in the subsequent PCR detection were resolved, and the results were reproducible.

Example 5

In this embodiment, the KRAS gene capture probe is taken as an example to compare the effect of subsequent PCR detection when the 3'end of the capture probe is blocked and unblocked. This is achieved by administering G12D mutation-positive cells in the stool samples and water of healthy people and capturing them.

The specific sequence of G12D is as follows:

(1) Capture with a probe that is phosphorylated at the 3 end (Probe 8) and unmodified (Probe 1). The capture steps mainly include: pretreatment of stool samples, extracellular delivery of G12D mutant cells, and stool capture. Specific steps are as follows:

Step 1: The stool sample is subjected to fecal DNA pretreatment with reference to step one in Example 2.

Step 2: Refer to Example 1① for external administration of mutation-positive cells.

Step 3: Refer to Step 2 of Example 2 for sample capture.

The test results captured by externally administered mutation-positive cells are as follows:

Table 10: Test results of phosphorylated modified and unmodified probes on positive cell samples

The results show that the probe 8 with 3'end phosphorylation modification can accurately capture G12D-positive samples when captured in healthy human feces samples and purified water, and there is no false positive problem at other sites.

The 3'-end unphosphorylated probe 1 can capture G12D positive samples when captured in healthy human feces samples and purified water, but there are also false positive problems at other sites, and the results are unstable and reproducible.

Example 6

In this embodiment, the KRAS gene capture probe is taken as an example to verify the influence of different modification modes at the 5'end of the capture probe. The probes and magnetic beads used include (1) the 5'end amino group modification of the probe, and carboxyl magnetic beads. (2) Modified with carboxyl group at the 5'end of the probe, amino magnetic beads. (3) PolyA modification at the 5'end of the probe, oligo dT magnetic beads. The specific sequence is shown in the following table:

Table 11: Other modification methods and sequences at the 5'end

Probe number	Detailed Description
Probe 11	NH2-TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGT-PO 4
Probe 12	NH2-TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGT
Probe 13	COOH-TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGT-PO 4
Probe 14	COOH-TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGT
Probe 15	Poly A-TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGT-PO 4
Probe 16	Poly A-TGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGCTGGTGGCGT

Stool samples of healthy people and water were injected with G12D mutation-positive cells in and out of the water for capture.

(1) Using the above-mentioned probes and magnetic beads to perform sample processing respectively, refer to Example 2 for sample processing and extracellular administration. The subsequent hybridization capture step is performed according to the product specification of the magnetic beads. The carboxyl magnetic beads used in this embodiment are Magnosphere ^TM MX100/Carboxyl&MX200/Carboxyl carboxyl magnetic beads from Beijing Bormai Company. The amino magnetic beads and oligo dT magnetic beads used are BioMag's amination modified magnetic beads and BioMag-Oligo (dT) coated magnetic beads. The test results captured by externally administered mutant positive cells are as follows:

Table 12: Sample capture results of probe 5'modified carboxyl magnetic beads

Table 13: The results of capturing samples with carboxyl modified amino magnetic beads at the 5'end of the probe

Table 14: Results of sample capture by polyA modified oligo dT magnetic beads at the 5'end of the probe

The results showed that the probe 10, probe 12, and probe 14 modified with 3'end phosphorylation can accurately capture G12D positive samples when captured in healthy human stool samples and purified water, and there are no false positives at other sites problem.

The 3'-end unphosphorylated probe 11, probe 13, and probe 15 can capture G12D positive samples when they are captured in healthy human feces samples and purified water, but there are also false positive problems at other sites, and the results are not Poor stability and repeatability.

It shows that the capture probe modified by phosphorylation at the 3'end can also solve the false positive problem of amino-carboxyl capture and ploA-oligo dT capture.

Example 7

This example verifies the effect of simultaneously capturing multiple target nucleic acids. Use the probes with 3'end phosphorylation modification and 3'end unphosphorylation modification for multi-gene combined capture. Specific genes include: KRAS/BRAF/APC/CTNNB1/SMAD4/NRAS, etc. The specific capture sites and probes involved are shown in the table below. The design of the probe refers to the NCBI and COSMIC database.

Table 15: Design of capture probe sequence for multi-gene joint detection

G12D/Q61L/R1450*/Q61K/R361H/S45F/V600E site-positive plasmids were separately injected into the stool samples of healthy people and captured according to the method of Example 2, using the Jiangsu Real Human Colorectal Cancer Multi-Gene Joint Detection Kit ( Fluorescence PCR detection) The detection results are as follows.

Table 16: Combined capture results of multiple genes in stool samples of probes with 3'end phosphorylation modification and 3'end unphosphorylation modification

The results showed that the phosphorylation-modified probe was used to externally deliver positive plasmids in human fecal samples to capture multiple genes, and the results were stable. The positive can be detected stably, and there are no cross-reactions and false positives at other sites; the use of unphosphorylated probes to deliver positive plasmids in human stool samples for multi-gene capture can detect positives, but the rest There will be a false positive problem at the site. The result is unstable.

Example 8

G12D//R1450*/Q61K/R361H/S45F/V600E site-positive plasmids were separately administered to urine and plasma samples of healthy people, and phosphorylated probes were used to capture the detection results according to the method of Example 2. The following table:

Table 17: Results of combined capture and detection of 3'phosphorylated probes in urine/plasma samples

The results showed that the phosphorylated probe was used to externally deliver positive plasmids in human urine and plasma samples to capture multiple genes, and the PCR detection results were stable. The positive can be stably detected, and there is no cross-reaction and false positive problems at other sites.

Example 9

Collected 20 cases of bowel cancer patients with pathological information, adenoma patients' whole stool samples before colonoscopy and 10 healthy people's whole stool samples. The multi-gene capture system of Example 8 was used to capture and detect. All samples were tested for fecal occult blood FIT. The comparison between test results and pathological information is shown in the following table:

Table 18: Test results of colorectal cancer samples captured by multi-genes with phosphorylation modification at 3'end and unphosphorylation modification at 3'end

Table 19: 3'end phosphorylation modification and 3'end non-phosphorylation modification multi-gene capture adenoma (advanced and non-advanced) sample test results

Table 20: Test results of 3'end phosphorylation modification and 3'end unphosphorylation modification polygene healthy people/colitis samples

Sample number	Phosphorylation modification	Unphosphorylated modification	Fecal occult blood FIT test
Sample 21-healthy people	Negative	G12D, G13D,	Positive
Sample 22-healthy people	Negative	G12V, G12C	Negative

Sample 23-healthy people	Negative	Negative	Negative
Sample 24-healthy people	Negative	Negative	Negative
Sample 25-healthy people	Negative	V600E	Negative
Sample 26-Enteritis patient	Negative	Negative	Positive
Sample 27-Enteritis patient	Negative	Negative	Negative
Sample 28-Enteritis patient	Negative	Negative	Positive
Sample 29-Enteritis patient	Negative	Q1378*	Positive
Sample 30-Enteritis patient	Negative	Negative	Negative

The results showed that compared with the FIT test, among the non-advanced adenomas and healthy people, 3 non-advanced adenomas and 5 healthy people, and 5 patients with enteritis, the test results were: (1) Phosphorylated modified The specificity of the multi-gene combined capture and detection system is 100%. (2) The specificity of the multi-gene combined capture and detection system without phosphorylation modification is 69.2%. (3) The specificity of FIT detection is 53.8%. The probe modification method established by this method and the stool multi-gene combined capture multi-gene mutation detection kit for colorectal cancer have higher detection specificity than existing products and can be used as a new method for early diagnosis of colorectal cancer.

Claims (18)

1. Nucleic acid probes, including target complementary regions capable of base hybridization with target nucleic acid target regions, are characterized in that the 3'end of the probe has also been blocked and modified to block or block the extension of the 3'end of the probe.
2. The probe according to claim 1, wherein the blocking modification at the 3'end is selected from the group consisting of phosphorylation, dideoxy base modification, Spacer modification, amino modification and the like.
3. The probe according to claim 1, wherein the 5'end of the probe includes a functional group suitable for physically separating the probe, and the functional group is selected from the group consisting of biotin, amino, carboxyl or PolA.
4. A composition for targeted nucleic acid capture, which comprises the nucleic acid probe according to any one of claims 1-3.
5. The composition according to claim 4, which comprises nucleic acid probes for multiple target genes.
6. The composition according to claim 4 or 5, which is used to capture target nucleic acids in biological samples such as stool, blood, plasma, serum, urine, and intestinal fluid.
7. The probe according to any one of claims 1-3, or the composition according to any one of claims 4-6, is used for preparing reagents or kits for capturing or detecting target nucleic acids in biological samples.
8. The use according to claim 7, wherein the biological sample includes feces, blood, plasma, serum, urine, intestinal fluid and the like.
9. According to the use of claim 7, the detection includes PCR detection, fluorescence quantitative PCR detection, and ARMS-PCR detection.
10. According to the use of claim 7, the detection includes the detection of nucleotide substitution, deletion, insertion, gene fusion, or mutation of any combination of the target nucleic acid.
11. A gene detection kit comprising the nucleic acid probe according to any one of claims 1-3, or the composition according to any one of claims 4-6.
12. A method for targeted capture and enrichment of nucleic acids includes processing biological samples, hybridizing probes with single-stranded DNA formed by thermal denaturation; adding magnetic beads to form magnetic bead probe complexes, separating magnetic beads, and the like, and is characterized by: Use the probe of any one of claims 1-3, or the composition of any one of claims 4-6.
13. The method according to claim 12, wherein the biological sample includes feces, blood, plasma, serum, urine, intestinal fluid, and the like.
14. A gene detection composition, comprising a PCR amplification reagent, characterized in that the detection system includes the residual nucleic acid probe according to any one of claims 1-3, or the composition according to any one of claims 4-6.
15. Use of the composition according to claim 14 for preparing reagents or kits for the detection of target nucleic acids in biological samples.
16. The use according to claim 15, wherein the biological sample includes feces, blood, plasma, serum, urine, intestinal fluid and the like.
17. According to the use of claim 15, the detection includes detection methods such as PCR detection, fluorescence quantitative PCR detection, ARMS-PCR detection and the like.
18. The use according to claim 15, wherein the detection includes the detection of nucleotide substitution, deletion, insertion, gene fusion, or mutation of any combination of the target nucleic acid.