Method and kit for detecting CTNNB1 gene mutation through digital PCR

Abstract

The invention provides a digital PCR (Polymerase Chain Reaction) kit for detecting CTNNB1 gene mutation, the digital PCR kit is used for simultaneously detecting mutation of 32, 33, 37 and 41 amino acids of a CTNNB1 gene in a same reaction tube digital PCR reaction system, and only three Drop-off probes and a pair of amplification primers are used when the mutation of the 32, 33, 37 and 41 amino acids of the CTNNB1 gene is detected. The invention provides a triple drop-off detection strategy, probes of three different fluorophores are utilized to target wild-type sequences of three adjacent hot spots, detection in a larger mutation range is realized, the detection flux is remarkably improved, and the detection cost is reduced.

Description

Technical Field

[0001] The present invention relates to the field of digital PCR detection, and more particularly, to a digital PCR kit for detecting CTNNB1 gene mutations and its applications. Background Art

[0002] The CTNNB1 gene is located on chromosome 3p21 and encodes β-catenin, an adhesion junction protein that supports the integrity between the layers of epithelial tissues and mediates intercellular signal transduction. The CTNNB1 gene is a proto-oncogene. The β-catenin encoded by CTNNB1 is involved in intercellular adhesion and can be used as a transcriptional activator in the nucleus. Activation of the CTNNB1 gene causes the accumulation of β-catenin in the nucleus, promoting DNA transcription and cell proliferation. Mutations in the CTNNB1 gene can prevent the phosphorylation and degradation of β-catenin, leading to the deposition of β-catenin in the nucleus and cytoplasm, further activating the WNT / β-catenin signaling pathway and causing tumorigenesis. Mutations in this gene are related to the pathogenesis of tumors such as colorectal cancer (CRC), pilomatrix carcinoma (PTR), medulloblastoma (MDB), ovarian cancer, WNT-type medulloblastoma, and ameloblastoma-type craniopharyngioma.

[0003] Currently, the techniques for detecting human ctDNA mutations are mainly based on PCR or next-generation sequencing (NGS). NGS-based detection methods can generally cover a wide range of up to hundreds of genes or gene hotspots and can improve sensitivity [detecting a minimum allele frequency (MAF) of less than 0.1%], but their limitations include high cost, long turnaround time, and the need for extensive bioinformatics analysis, which are the main obstacles to routine clinical applications. In contrast, PCR-based techniques, such as Amplification refractory mutation system (ARMS) PCR and Digital PCR (dPCR), are rapid and cost-effective tools that can detect and quantify a small number of known mutations with high sensitivity (the detection MAF of ARMS PCR is 0.1%, and the detection MAF of dPCR is less than 0.01%) and do not require specialized bioinformatics interpretation. These advantages make PCR-based detection, especially dPCR detection, very suitable for liquid biopsy analysis. In 2016, the US Food and Drug Administration approved the first ctDNA detection method (Cobas epidermal growth factor receptor mutation test v2; Roche Diagnostics) for the clinical detection of epidermal growth factor receptor mutations in non-small cell lung cancer, indicating that ctDNA will soon be used for routine analysis in clinical practice.

[0004] At present, although there are some kits for detecting CTNNB1 mutations in ctDNA by digital PCR method, for example, in CN113913514A, for each mutation site, a set of mutant detection probes and wild-type detection probes need to be designed for detection, with low detection throughput and high cost.

[0005] The Drop-off digital PCR detection method, simply speaking, the Drop-off experiment includes two TaqMan probes for the same amplicon: the Drop-off probe that is complementary to the wild-type sequence and non-complementary to the mutation site, and the reference probe that is complementary to both mutant and wild-type genes. In the presence of wild-type alleles, both the Drop-off probe and the reference probe will hybridize to the target, generating a double positive signal. On the contrary, if there are mutant alleles, even a single nucleotide mutation will prevent the Drop-off probe from hybridizing to it. Therefore, only the Reference probe anneals to the target gene, resulting in a positive signal.

[0006] Singleplex dPCR detection drop-off designs have been reported, using a fluorescent reference probe targeting a conserved region near the mutation hot spot and another drop-off probe targeting the wild-type (WT) sequence of the hot spot region with a different fluorophore. Therefore, the presence of wild-type alleles is manifested as a double positive fluorescence signal, while mutant alleles in the hot spot show a lower fluorescence amplitude of the drop-off probe due to loss or suboptimal binding, resulting in a distinct separation of droplets between mutant alleles and wild-type alleles. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a digital PCR kit for detecting CTNNB1 gene mutations. The digital PCR kit simultaneously detects mutations at amino acid positions 32, 33, 37, and 41 of the CTNNB1 gene in the digital PCR reaction system of the same reaction tube. Only three Drop-off probes and a pair of amplification primers are used to detect mutations at amino acid positions 32, 33, 37, and 41 of the CTNNB1 gene. The pair of primers is used to amplify the target sequence containing mutations at amino acid positions 32, 33, 37, and 41 of the CTNNB1 gene. Among the three Drop-off probes, the first Drop-off probe is a Drop-off probe for detecting mutations at amino acid positions 32 and 33 of the CTNNB1 gene, the second Drop-off probe is a Drop-off probe for detecting the mutation at amino acid position 37 of the CTNNB1 gene, and the third Drop-off probe is a Drop-off probe for detecting the mutation at amino acid position 41 of the CTNNB1 gene. When detecting mutations at amino acid positions 32 and 33 of the CTNNB1 gene, the second Drop-off probe and / or the third Drop-off probe serves as a reference probe. When detecting the mutation at amino acid position 37 of the CTNNB1 gene, the first Drop-off probe and / or the third Drop-off probe serves as a reference probe. And when detecting the mutation at amino acid position 37 of the CTNNB1 gene, the first Drop-off probe and / or the third Drop-off probe serves as a reference probe. And the first Drop-off probe and the third Drop-off probe are used to detect one sequence in the double-stranded target sequence, and the second Drop-off probe is used to detect the other sequence in the double-stranded target sequence.

[0008] In one embodiment, the mutation positions of amino acid positions 32 and 33 of the CTNNB1 gene are located in the middle region of the first Drop-off probe, the mutation position of amino acid position 37 of the CTNNB1 gene is located in the middle region of the second Drop-off probe, and the mutation position of amino acid position 41 of the CTNNB1 gene is located in the middle region of the third Drop-off probe.

[0009] In one embodiment, the first Drop-off probe is SEQ ID NO: 3

[0010] CTTACCTG+GA+CT+CTGGAATCC; the second Drop-off probe is SEQ ID NO: 4 TGGCACCA+G+AATGGATTCCA, and the third Drop-off probe is SEQ ID NO: 6 CCACT+A+C+CACAGCTCCT.

[0011] In one embodiment, the kit is used to detect mutations at the following sites:

[0012] Locus number Amino acid change Base change 1 D32Y 94G>T 2 D32H 94G>C 3 S33P 97T>C 4 S33C 98C>G 5 S37F 110C>T 6 S37C 110C>G 7 T41I 122C>T 8 T41A 121A>G

[0013] In one embodiment, the use of the above kit is provided for preparing a diagnostic product for evaluating whether a subject is suitable for immunotherapy or for pre - evaluating the effect of an immunotherapy drug on the subject.

[0014] In the present invention, probes with two different fluorescent groups target the wild - type sequences of two adjacent hotspots to detect mutations at two adjacent mutation hotspots spanned by a single amplified fragment. Therefore, each probe is a reporting probe for one mutation hotspot and a reference probe for the other mutation hotspot. Amplification of the wild - type allele results in a double - positive fluorescence signal, while a single - positive fluorescence signal indicates a mutation in the region covered by the unbound probe.

[0015] The present invention provides a triple drop - off detection strategy. Probes with three different fluorescent groups target the WT sequences of three adjacent hotspots to detect mutations at three adjacent mutation hotspots spanned by a single amplified fragment. Each probe is both a reporting probe for a mutation hotspot and a reference probe for other mutation hotspots, achieving detection of a larger mutation range, significantly improving the detection throughput. In addition, the present invention can use one probe to detect multiple mutations at one site, also greatly broadening the detection range, significantly improving the detection throughput, significantly reducing the detection cost, and improving the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] To more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments recorded in the present application. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0017] Figure 1 is a schematic diagram of the detection principle of the present invention;

[0018] Figure 2 is a schematic diagram of the sequence of the CTNNB1 gene mutation hotspot region detected by the present invention, where WT - F represents the forward wild - type sequence of the CTNNB1 gene mutation hotspot region, and WT - R represents the reverse wild - type sequence of the CTNNB1 gene mutation hotspot region;

[0019] Figure 3 is a schematic diagram showing that all the drop - off probes for three hotspot regions of the CTNNB1 gene detected by the present invention are forward; The sequence at this position is the drop-off probe for the first mutation hotspot region, The sequence at this position is the drop-off probe for the second mutation hotspot region, The sequence at this position is the drop-off probe for the first mutation hotspot region. The blue sequence is the position of the amplification primer, where the blue sequence is the primer region corresponding to primer amplification;

[0020] Figure 4 It is a schematic diagram of the drop-off probes for three hotspot regions of the CTNNB1 gene detected by the present invention. The drop-off probe for the 37th amino acid is designed to be reverse, and the rest of the probes are forward. The sequence at this position is the drop-off probe for the first mutation hotspot region, The sequence at this position is the drop-off probe for the second mutation hotspot region, The sequence at this position is the drop-off probe for the third mutation hotspot region. The blue sequence is the position of the amplification primer, where the blue sequence is the primer region corresponding to primer amplification;

[0021] Figure 5 It is a FAM-VIC 2D diagram for detecting the D32Y mutant type of the present invention;

[0022] Figure 6 It is a FAM-VIC 2D diagram for detecting the D32H mutant type of the present invention;

[0023] Figure 7 It is a FAM-VIC 2D diagram for detecting the S33P mutant type of the present invention;

[0024] Figure 8 It is a FAM-VIC 2D diagram for detecting the S33C mutant type of the present invention;

[0025] Figure 9 It is a FAM-VIC 2D diagram for detecting the S37F mutant type detected by one drop-off probe of the present invention;

[0026] Figure 10 It is a FAM-VIC 2D diagram for detecting the S37F mutant type detected by another drop-off probe of the present invention;

[0027] Figure 11 It is a FAM-VIC 2D diagram for detecting the S37C mutant type of the present invention;

[0028] Figure 12 It is a FAM-ROX 2D diagram for detecting the T41A mutant type of the present invention;

[0029] Figure 13It is the FAM-ROX 2D graph for detecting the T41I mutant detected by the present invention. Detailed implementation manners

[0030] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below in conjunction with embodiments. Obviously, the described embodiments are only a part of the embodiments of this application, rather than all the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of this application. In the following embodiments, unless otherwise specified, they are all conventional methods in the art.

[0031] The present invention provides a digital PCR kit for detecting human CTNNB1 gene mutations. The digital PCR kit simultaneously detects mutations at three adjacent mutation hotspots in the digital PCR reaction system of the same reaction tube. For example, Figure 1 as shown, where the 32nd amino acid hotspot uses a HEX-labeled drop-off probe, the 37th amino acid hotspot uses a FAM-labeled drop-off probe, and the 41st amino acid hotspot uses a ROX-labeled drop-off probe. A pair of upstream and downstream primers are used, and the single amplification fragment generated by them spans the above-mentioned 32nd, 37th, and 41st amino acid hotspots. This kit realizes simultaneous detection of multiple targets in a single tube, significantly improving the detection throughput.

[0032] Taking the detection of CTNNB1 gene mutations in cerebrospinal fluid as an example, CTNNB1 gene mutations are concentrated at the 32nd, 33rd, 37th, and 41st amino acid positions. For example, Figure 2 as shown, the green highlights are the mutation positions of the 32nd and 33rd amino acids, the blue highlight is the mutation position of the 37th amino acid, and the red highlight is the mutation position of the 41st amino acid.

[0033] The base sequences of these three mutation hotspot regions (the 32nd / 33rd amino acids are one mutation hotspot region) are relatively close, and the specificity of the drop-off probe is the best when the mutation position is located in the middle region of the drop-off probe. The drop-off probes for detecting these three hotspot regions are all forward, as follows Figure 3 shown. Figure 3 In, the sequence at is the drop-off probe for the first mutation hotspot region, the sequence at is the drop-off probe for the second mutation hotspot region, the sequence at is the drop-off probe for the first mutation hotspot region. The blue sequence is the position of the amplification primer.

[0034] The primer-probe combinations of the reaction system of this digital PCR kit are shown in Table 1 below. The three drop-off probes are labeled with different fluorophores respectively, covering three detection targets; a pair of upstream and downstream primers are used, and the single amplification fragment generated by them spans three detection targets.

[0035] Table 1

[0036]

[0037] Note: The “+” before the base in the sequence indicates the locked nucleic acid modification. For example, “+G” represents locked nucleic acid G, and the same applies hereinafter.

[0038] First, prepare the PCR amplification system. The PCR amplification reaction mixture includes: 4×SuperMix premix (Xinyi), 400 nM of each primer, 300 nM of each probe, 10 - 200 ng of template DNA (CTNNB1 standard), and make up the volume to 30 μl with water. Mix the reagents well. Use a sample preparation instrument (Drop Maker M1) to prepare microdroplets according to the instructions. Then place the 8-well strip containing microdroplets on a PCR instrument for amplification, and the amplification conditions are set as shown in Table 2 below.

[0039] Table 2

[0040]

[0041] After PCR amplification, use a Chip Reader R1 biochip analyzer (Xinyi) to perform droplet detection and data analysis according to the instrument operation manual. When using this protocol to detect the CTNNB1 standard, since the three drop-off probes are all in the forward direction, the length of each probe is short, and the Tm value of the probe is too low, resulting in very weak signals and unable to detect mutations.

[0042] On this basis, design the 37th amino acid drop-off probe to be in the reverse direction, and the rest of the probes to be in the forward direction, as shown Figure 4 below. Although there is sequence complementarity between the 37th amino acid drop-off probe and the drop-off probes in the other two regions in the red sequence (TGGAATCCA and CCA) part, the highest Tm value of the complementary part is about 27 °C, while the Tm value of the drop-off probe annealing with the target region reaches about 70 °C. Therefore, the complementarity between drop-off probes to this extent is very weak and does not affect the probe operation. Design primers outside the three drop-off probes, as shown in the blue sequence in the figure below, to form a triple drop-off detection system.

[0043] The primer-probe combinations of the digital PCR kit reaction system are shown in Table 3 below. The three drop-off probes are labeled with different fluorescent dyes, covering three detection targets; a pair of upstream and downstream primers are used, and the single amplified fragment generated by them spans three detection targets. Among them, 2 drop-off probes were designed for the second mutation hot spot region (drop-off probe B in the table below), and the better one was selected from them.

[0044] Table 3

[0045]

[0046] First, prepare the PCR amplification system. The PCR amplification reaction mixture includes: 4×SuperMix premix (Xinyi), 400 nM each of the primers, 300 nM each of the probes, 10 - 200 ng of template DNA (CTNNB1 standard), and make up the volume to 30 μl with water. Mix the reagents well. Use a sample preparation instrument (Drop Maker M1) to prepare microdroplets according to the instruction manual. Then place the 8-strip tube containing the microdroplets on the PCR instrument for amplification, and set the amplification conditions as shown in Table 4 below.

[0047] Table 4

[0048]

[0049] After PCR amplification, use the Chip Reader R1 biochip analyzer (Xinyi) to perform droplet detection and data analysis according to the instrument instruction manual. Detect the CTNNB1 gene mutation through fluorescence signals and directly perform absolute quantification on it.

[0050] Detection of D32Y mutant FAM-VIC 2D is as Figure 5 shown, where the red dots are wild-type signal points and the blue dots are mutant signal points. The mutant signal and the wild-type signal are significantly separable and far apart, and the detection result is good.

[0051] Detection of D32H mutant FAM-VIC 2D is as Figure 6 shown, where the red dots are wild-type signal points and the blue dots are mutant signal points. The mutant signal and the wild-type signal are significantly separable and far apart, and the detection result is good.

[0052] Detection of S33P mutant FAM-VIC 2D is as Figure 7 shown, where the red dots are wild-type signal points and the blue dots are mutant signal points. The mutant signal and the wild-type signal are significantly separable and far apart, and the detection result is good.

[0053] Detection of S33C mutant FAM-VIC 2D is as Figure 8As shown, where the red dots are wild-type signal points and the blue dots are mutant signal points. The mutant signal is significantly separable from the wild-type signal and is at a relatively large distance, and the detection result is good.

[0054] The detection of the S37F mutant for FAM-VIC 2D is as Figure 9 and 10 shown, where the red dots are wild-type signal points and the green dots are mutant signal points. Figure 9 In [reference], the mutant signal of the drop-off probe B SEQ ID NO: 4 is significantly separable from the wild-type signal and is at a relatively large distance; while Figure 10 in [reference], the mutant signal of the drop-off probe B SEQ ID NO: 5 is at a relatively close distance to the wild-type signal, and the aggregation degree of the wild-type signal is poor. Therefore, the better drop-off probe B SEQ ID NO: 4 is selected.

[0055] The detection of the S37C mutant for FAM-VIC 2D is as Figure 11 shown, where the red dots are wild-type signal points and the green dots are mutant signal points. The mutant signal is significantly separable from the wild-type signal and is at a relatively large distance, and the detection result is good.

[0056] The detection of the T41A mutant for FAM-ROX 2D Figure 12 is as follows, where the red dots are wild-type signal points and the blue dots are mutant signal points. The mutant signal is significantly separable from the wild-type signal and is at a relatively large distance, and the detection result is good.

[0057] The detection of the T41I mutant for FAM-ROX 2D is as Figure 13 follows, where the red dots are wild-type signal points and the blue dots are mutant signal points. The mutant signal is significantly separable from the wild-type signal and is at a relatively large distance, and the detection result is good.

[0058] Therefore, the finally selected primer-probe combination is as shown in Table 5 below.

[0059] Table 5

[0060]

[0061]

[0062] The verified detectable sites are as shown in Table 6 below.

[0063] Table 6

[0064]

[0065] It should be understood that the disclosed invention is not limited to the particular methods, schemes, and materials described, as these may vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0066] Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. These equivalents are also encompassed by the appended claims.

Claims

1. A digital PCR kit for detecting CTNNB1 gene mutations, characterized in that, The digital PCR kit simultaneously detects mutations at amino acid positions 32, 33, 37, and 41 of the CTNNB1 gene in the digital PCR reaction system of the same reaction tube, and only three Drop-off probes and a pair of amplification primers are used for detecting mutations at amino acid positions 32, 33, 37, and 41 of the CTNNB1 gene; The pair of primers is used to amplify a target sequence containing mutations at amino acid positions 32, 33, 37, and 41 of the CTNNB1 gene; Among the three Drop-off probes, the first Drop-off probe is a Drop-off probe for detecting amino acid positions 32 and 33 of the CTNNB1 gene, the second Drop-off probe is a Drop-off probe for detecting amino acid position 37 of the CTNNB1 gene, and the third Drop-off probe is a Drop-off probe for detecting amino acid position 41 of the CTNNB1 gene; when detecting mutations at amino acid positions 32 and 33 of the CTNNB1 gene, the second Drop-off probe and / or the third Drop-off probe serves as a reference probe, and when detecting mutations at amino acid position 37 of the CTNNB1 gene, the first Drop-off probe and / or the third Drop-off probe serves as a reference probe; and when detecting mutations at amino acid position 37 of the CTNNB1 gene, the first Drop-off probe and / or the third Drop-off probe serves as a reference probe; The first Drop-off probe and the third Drop-off probe are used to detect one sequence in the double strand of the target sequence, and the second Drop-off probe is used to detect the other sequence in the double strand of the target sequence.

2. The digital PCR kit according to claim 1, wherein The mutation positions of amino acid positions 32 and 33 of the CTNNB1 gene are located in the middle region of the first Drop-off probe, the mutation position of amino acid position 37 of the CTNNB1 gene is located in the middle region of the second Drop-off probe, and the mutation position of amino acid position 41 of the CTNNB1 gene is located in the middle region of the third Drop-off probe.

3. The digital PCR kit according to claim 2, wherein The first Drop-off probe is SEQ ID NO: 3 CTTACCTG+GA+CT+CTGGAATCC; the second Drop-off probe is SEQ ID NO: 4 TGGCACCA+G+AATGGATTCCA, and the third Drop-off probe is SEQ ID NO: 6 CCACT+A+C+CACAGCTCCT.

4. The digital PCR kit according to claim 1, wherein The kit is used to detect mutations at the following sites:

5. Use of the kit according to any one of claims 1-4, characterized in that, For preparing a diagnostic product, the diagnostic product is used to judge whether an object is suitable for immunotherapy or to pre-evaluate the effect of an immunotherapy drug on the object.

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