Digital pcr detection kit for detecting multiple mutation sites of pi3kca gene

Abstract

The application provides a digital PCR detection kit for detecting PIK3CA gene multi-mutation sites, and the kit realizes detection of 11 mutation sites of the PIK3CA gene through two digital PCR reaction tubes A and B. The A tube contains detection of E545K, E545A, E545G, E545D, Q546E and Q546R sites, and the B tube contains detection of E542K, C420R, H1047R, H1047L and H1047Y sites. Each site is detected by using a respective spacer molecular beacon detection probe. The application introduces a Spacer structure into a short sequence high-specificity probe, breaks through the difficulty of background control and structure ring in the detection of complex mutations by traditional molecular beacons, and significantly improves the detection accuracy and application range.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of digital PCR detection, in particular to a digital PCR detection kit for PIK3CA gene multi-mutation site detection. BACKGROUND

[0002] PIK3CA gene encodes phosphatidylinositol 3-kinase (PI3K) p110α subunit, which is an important pathway component for regulating cell proliferation, survival and metabolism. Its gene mutation can lead to continuous activation of PI3K / AKT / mTOR signaling pathway, and then induce cell malignant transformation. In breast cancer, PIK3CA is one of the most common driver genes, and it is reported that about 30%-40% of patients have activating mutations, and the incidence is higher in Chinese population, up to 44-50%. The common mutation hotspots are mainly distributed in exon 9 (E542K, E545K) and exon 20 (H1047R, H1047L) sites.

[0003] PIK3CA mutation is not only an important marker for molecular typing and prognosis, but also closely related to the sensitivity of patients to CDK4 / 6 inhibitors, endocrine therapy and other programs. Therefore, accurate, rapid and high-sensitivity detection of PIK3CA mutation has important clinical value. With the development of "liquid biopsy" technology, mutation detection based on circulating tumor DNA (ctDNA) in plasma has become an important means for non-invasive and dynamic monitoring of patient disease status. ctDNA detection has the advantages of strong repeatability and good real-time performance, and is especially suitable for clinical scenarios where tissue sample acquisition is difficult. Current PIK3CA mutation detection methods include fluorescence quantitative PCR (qPCR), second-generation sequencing (NGS) and digital PCR (digital PCR) and the like.

[0004] NGS (second-generation sequencing) has the advantages of high throughput and whole genome coverage, but its promotion in clinical detection still faces many limitations: first, the NGS process is complex, involving multiple steps such as library construction, capture, sequencing and bioinformatics analysis; second, the operation period is long, usually 5-7 working days, which is difficult to meet the clinical demand for rapid results; in addition, its detection cost is high, and the requirements for laboratory platform and personnel operation level are high, which also limits its application in grassroots and routine scenarios to some extent.

[0005] Quantitative PCR is more commonly used in clinical practice due to its simple operation and low cost. At present, the detection of gene mutation points is mainly realized through ARMS (Amplification Refractory Mutation System) technology. However, the ARMS-qPCR method has the following limitations: limited sensitivity, usually around 0.5%-1%, which is difficult to meet the needs of low-frequency mutation detection in ctDNA; low specificity, prone to false positives, especially when background heterozygous signals interfere with interpretation; limited detection sites, current PIK3CA detection kits often only cover a few common mutation sites, which cannot meet the growing needs of clinical subtyping and companion diagnosis; to improve sensitivity, different sites / genotypes usually need to be detected separately, and under the condition of low ctDNA sample size, separate templates will further reduce the overall detection sensitivity.

[0006] In contrast, digital PCR (digital PCR, dPCR) technology realizes the absolute quantification of target mutant molecules by high-throughput micro-partitioning of samples combined with Poisson distribution statistical model, does not depend on standard curve, and can improve the detection sensitivity to 0.1% or even lower, and is more suitable for small sample detection. Therefore, dPCR is particularly suitable for precise detection of PIK3CA mutations in the environment of ctDNA liquid biopsy.

[0007] Currently, there is still a lack of a digital PCR detection kit with high coverage, high sensitivity, good repeatability, and specially designed for PIK3CA multi-mutation sites. Especially in the unified detection of multiple hotspot mutations in ctDNA samples, there is a significant technical gap. Therefore, the development of a multiplex detection kit that can simultaneously detect multiple PIK3CA gene mutation sites, has intuitive results, and can realize comprehensive evaluation, has important clinical application value. SUMMARY

[0008] In order to solve the above problems, the present application provides a digital PCR detection kit for PIK3CA gene multi-mutation site detection, which realizes the detection of 11 mutation sites of PIK3CA gene through two digital PCR reaction tubes A and B, wherein the A tube contains the detection of E545K, E545A, E545G, E545D, Q546E, Q546R sites, and the B tube contains the detection of E542K, C420R, H1047R, H1047L, H1047Y sites; wherein each site is detected by using a respective spacer molecular beacon detection probe, which comprises a 5' end stem sequence, a loop sequence, a flexible Spacer structure and a 3' end stem sequence in turn, and the whole is in a hairpin structure; a fluorescent group is labeled at the 5' end, a quencher group is labeled at the 3' end, the 5' end stem sequence and the 3' end stem sequence form a stable stem structure, the 5' end stem sequence and the loop sequence are completely complementary to the target sequence to be detected, and the loop sequence and the flexible Spacer structure form a loop structure of the molecular beacon detection probe; the flexible Spacer structure is a polyethylene glycol structure.

[0009] In an embodiment, the loop sequence is modified with a locked nucleic acid.

[0010] In an embodiment, the number of polyethylene glycol units in the flexible Spacer structure is not less than three.

[0011] In an embodiment, the number of polyethylene glycol units in the flexible Spacer structure is 3-6.

[0012] In an embodiment, the number of polyethylene glycol units in the flexible Spacer structure is 6.

[0013] In an embodiment, the spacer molecular beacon detection probe for E545K in the A tube is SEQ ID NO. 27, the spacer molecular beacon detection probe for E545A is SEQ ID NO. 28, the spacer molecular beacon detection probe for E545G is SEQ ID NO. 29, the spacer molecular beacon detection probe for E545D is SEQ ID NO. 30, the spacer molecular beacon detection probe for Q546E is SEQ ID NO. 31, and the spacer molecular beacon detection probe for Q546R is SEQ ID NO. 32.

[0014] In an embodiment, the A tube further comprises an E545 wild-type detection probe SEQ ID NO. 33, an E542-546 upstream primer SEQ ID NO. 13 and an E542-546 downstream primer SEQ ID NO. 14.

[0015] In an embodiment, the A tube further comprises a fluorescent blocking probe B1 for blocking the spacer molecular beacon detection probe of E545K, the spacer molecular beacon detection probe of E545A and the spacer molecular beacon detection probe of E545G, the sequence of which is SEQ ID NO. 34; a fluorescent blocking probe B2 for blocking the spacer molecular beacon detection probe of Q546E, the spacer molecular beacon detection probe of Q546R and the spacer molecular beacon detection probe of E545D, the sequence of which is SEQ ID NO. 35; the sequence of the fluorescent blocking probe is complementary to the 6-base region at the 5' end of each molecular beacon detection probe described above, and the 3' end of the fluorescent blocking probe is labeled with a fluorescence quenching group, which is combined with the 5' end of the molecular beacon detection probe in the free state to form a stable secondary blocking structure.

[0016] In an embodiment, the B tube further comprises the spacer molecular beacon detection probe SEQ ID NO. 40 of H1047R, the spacer molecular beacon detection probe SEQ ID NO. 41 of H1047L, the spacer molecular beacon detection probe SEQ ID NO. 42 of H1047Y, the spacer molecular beacon detection probe SEQ ID NO. 44 of C420R, and the spacer molecular beacon detection probe SEQ ID NO. 45 of E542K.

[0017] In an embodiment, the B tube further comprises a fluorescent blocking probe B3 for blocking the spacer molecular beacon detection probe of H1047R, the spacer molecular beacon detection probe of H1047L and the spacer molecular beacon detection probe of H1047Y, the sequence of which is SEQ ID NO. 46; the sequence of the fluorescent blocking probe is complementary to the 6-base region at the 5' end of each molecular beacon detection probe described above, and the 3' end of the fluorescent blocking probe is labeled with a fluorescence quenching group, which is combined with the 5' end of the molecular beacon detection probe in the free state to form a stable secondary blocking structure.

[0018] In the present application, the digital PCR detection kit for PIK3CA gene multi-mutation site detection can simultaneously detect 11 hot spot mutation sites, covering the key mutation region commonly seen in clinical breast cancer, so that the detection result is more comprehensive and accurate. The kit has strong specificity and high sensitivity, and can detect as low as 0.3 ng / μL of nucleic acid sample, and is suitable for precise detection of ctDNA in liquid biopsy.

[0019] The application is based on the locked nucleic acid (LNA) probe technology, the specificity is enhanced by shortening the probe length, and the design of the Spacer molecular beacon probe and the fluorescence blocking probe is combined, thereby effectively reducing the background signal of the system. For sites without wild type probes, the specificity of detection is further improved by blocking probes. Common mutation sites are typed by different fluorescent labels, while rare mutation sites are not typed, and a two-tube detection strategy is used, thereby efficiently and sensitively detecting 11 mutation sites, reducing the template separation effect, and improving the overall sensitivity.

[0020] The application innovatively introduces a Spacer flexible connecting arm in the molecular beacon detection probe structure, which is located between the 3' end stem sequence of the probe and the main target recognition region to be detected. The Spacer structure has the following key advantages: 1) improving spatial flexibility: enabling short sequence probes to also efficiently form stable hairpin stem-loop structures, significantly improving the closing efficiency; 2) effectively reducing the background signal: the distance between the fluorescence and the quencher group in the closed loop state is more stable, and the fluorescence inhibition in the non-binding state is more sufficient; 3) enhancing the compatibility of probe structure design: Spacer reduces the possibility of non-specific pairing between the 3' end stem and the target, avoids interference with the probe Tm, and is beneficial to multi-probe co-detection design; 4) improving the detection ability of multiple mutations: when detecting multiple homologous mutations or multi-site mutations, the Spacer structure enables the probe to maintain more consistent structural stability and background suppression ability, thereby realizing high-throughput and high-sensitivity mutation site discrimination detection.

[0021] The application introduces a Spacer structure in a short sequence high-specificity probe, thereby breaking through the problems of background control and structure ring formation of traditional molecular beacons in complex mutation detection, significantly improving the detection accuracy and application range. In addition, a short chain auxiliary probe sequence (fluorescence blocking probe) is also introduced in the system, which is labeled with a quencher group at the 3' end and is complementary to the 5-7 nt sequence at the 5' end of the main probe, so as to further close the free probe structure that is not closed. The probe system effectively improves the specific recognition ability of the enzyme-dependent molecular beacon probe in a high homologous background, making it possible to accurately detect multiple homologous mutation sites, and is especially suitable for end-point detection platforms such as digital PCR which have extremely high signal-to-noise ratio requirements.

[0022] The application also introduces a fluorescence blocking probe. The probe is a short chain oligonucleotide, the sequence of which is specifically complementary to the 5-7 base region at the 5' end of the main probe, and the 3' end is labeled with a fluorescence quencher group. When the target is not combined, the short chain probe can preferentially combine with the 5' end region of the molecular beacon to form a stable secondary closed complex structure, further improving the fluorescence inhibition ability of the beacon in the free state. This mechanism significantly reduces non-specific fluorescence release, improves the overall signal-to-noise ratio, and helps to enhance the accuracy and sensitivity of mutation detection.

[0023] Due to the application of the probe in the present application, the detection sensitivity of the present application reaches 0.1%, which is 2 to 10 times higher than that of the conventional real-time fluorescent PCR technology. The whole detection process is simple to operate, the sample can be determined to be positive or negative through copy number quantitative analysis, and mutation rate data can be provided, which is convenient for dynamic monitoring of mutation abundance change and provides reliable support for clinical decision-making.

[0024] Clinical verification results show that 20 plasma samples detected by using the kit of the present application are completely consistent with the NGS sequencing results, proving that the kit has very high accuracy, and is particularly suitable for PIK3CA mutation detection in plasma samples. The composition has strong detection specificity, comprehensive site detection, and the detection result is intuitive and easy to judge, which can provide more comprehensive and accurate drug selection basis for clinicians, ensure the effectiveness and safety of the treatment plan, has important companion diagnostic value, especially in the precise diagnosis and treatment of breast cancer, plays an irreplaceable role. DETAILED DESCRIPTION

[0025] In order to make the person skilled in the art better understand the technical solutions in the present application, the present application will be further described below in combination with embodiments. Obviously, the described embodiments are only some of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by the person skilled in the art without creative labor should belong to the scope of protection of the present application.

[0026] Example 1: Detection scheme of PIK3CA mutation detection kit

[0027] The present application provides a digital PCR (dPCR) detection kit for 11 common mutation hotspots of PIK3CA gene and a detection method thereof. The covered mutation sites include: E545K, E545A, E545G, E545D, Q546E, Q546R, E542K, C420R, H1047R, H1047L and H1047Y. These mutations have a high frequency of occurrence in various clinical samples and have important clinical and biological significance.

[0028] In the establishment of detection system, the single tube multiplex reaction strategy was initially designed, (1) E545 series (E545K / A / G / D) and Q546 series (Q546E / R) mutation sites were detected by using the same fluorescence channel molecular beacon detection probe; (2) E542K, C420R and H1047X (H1047R / L / Y) mutation sites were designed with independent fluorescence channel molecular beacon detection probe; (3) The wild type sequence of H1047 site was also designed with corresponding fluorescence probe as internal standard gene to detect whether the amplification system was normal. The primer and probe sequences are shown in Table 1.

[0029] Tm and ΔTm were obtained by OligoAnalyzer of IDT (https: / / sg.idtdna.com / pages / tools / oligoanalyzer).

[0030] Table 1. Primer and probe sequences used in one tube detection

[0031]

[0032] Note: ΔTm is the difference in Tm between the mutant probe and the mutant template matching and mismatching with the wild type template; lowercase letters are 3' stem sequences that do not match the target; 3' horizontal line capital letters represent 3' stem sequences that match the target; the horizontal line base in the middle position represents the mutation site.

[0033] Using the primer and probe combination designed in Table 1, a multiplex digital PCR reaction system was constructed, and the specific operation process is as follows:

[0034] (1) System preparation

[0035] The basic PCR reaction system (total volume 30 µL) was prepared according to the following table.

[0036]

[0037] Among them, the final concentration of primers was 600 nM, and the final concentration of probes was 200 nM.

[0038] (2) Digital PCR workflow

[0039] Microdroplet preparation: using droplet generation chip (Xinyu Manufacturing Technology (Beijing) Co., Ltd.) and sample preparation instrument (Xinyu Manufacturing Technology (Beijing) Co., Ltd.), 30 µL PCR reaction system was added to the sample hole of the droplet generation chip, 180 µL droplet generation oil was added to the oil hole, the chip and 8-union tube were put into the preparation instrument, covered with rubber pad, and microdroplet preparation was carried out.

[0040] PCR amplification: The 8-tube array containing microdroplets was placed on the PCR instrument for amplification, and the amplification program was set as shown in the following table:

[0041]

[0042] Microdroplet detection: After PCR, the 8-tube array and the droplet detection chip (Xinyu Manufacturing Technology (Beijing) Co., Ltd.) were placed in the fixture, 430 μL and 500 μL of detection oil were added to the oil holes, respectively, a rubber pad was covered, and the chip was placed in the chip analyzer (Xinyu Manufacturing Technology (Beijing) Co., Ltd.) for droplet detection.

[0043] Data analysis: The tens of thousands of microdroplets prepared by the droplet generation chip and the sample preparation instrument are each an independent PCR reactor. Most of the microdroplets do not contain the target gene to be tested or contain at least one target gene to be tested. After PCR amplification, the chip analyzer detects the fluorescence signal of each microdroplet, records the peak height of the microdroplet signal, and the droplet containing the target gene to be tested will be detected corresponding fluorescence signal. The fluorescence intensity in the microdroplet is digitized by a fluorescence classification threshold, the microdroplet with strong fluorescence is judged as "1" (positive), and the microdroplet with weak fluorescence is judged as "0" (negative). The number of "1" and "0" is counted, and the total copy number of each fluorescence-labeled target gene in the input template can be calculated by correcting the Poisson distribution model.

[0044] The mutant template constructed by plasmid and fragmented human wild-type genomic DNA were mixed in proportion to prepare a simulated sample with a mutation frequency of 10% (the copy numbers of mutant and wild-type templates were 50 copies / μL and 500 copies / μL, respectively, and 2 μL of mixed template was added to each reaction system). At the same time, pure wild-type genomic DNA without mutation was used as a control template to preliminarily evaluate the system performance. The detection results are shown in Table 2.

[0045] Table 2. Detection results

[0046]

[0047] Note: Non-specific detection signal is indicated by bold italic numbers.

[0048] In the preliminary detection process, it was found that some systems had a problem of high background signal, especially under wild-type template conditions, there was obvious non-specific fluorescence signal interference.

[0049] Specifically, except for E545G and Q546R, the rest of the E545X / Q546X series mutation sites can detect the positive FAM signal corresponding to the mutation template, indicating that the binding efficiency of the probe to the mutant template is good. However, in the wild-type template, the probes of these mutation sites still appear FAM positive signal, suggesting that there is a certain degree of non-specific hybridization. Further analysis found that this problem mainly comes from the low melting temperature difference (ΔTm) of some probes (<5℃), which leads to the probe still having a certain compatible binding ability on the wild-type template, making it difficult to effectively distinguish.

[0050] Similarly, in the H1047X series mutation detection, although all mutant templates can detect positive signals, the background value is relatively high, and obvious non-specific signals are observed in the wild-type template. Especially for the H1047Y site, its ΔTm is low, which becomes the main source of non-specificity.

[0051] The C420R probe performs well, and can stably detect signals in the mutant template, and has no obvious background in the wild-type template. The E542K site probe can also accurately identify the mutant template, but it also has a certain degree of non-specific hybridization signal in the wild-type template.

[0052] In order to improve the overall detection specificity and reduce the system background value, the present application proposes the following optimization strategies for the above probes:

[0053] Moderately shorten the length of the probe sequence to improve the sensitivity to base mismatches;

[0054] Introduce LNA (Locked Nucleic Acid) modified bases to enhance the overall binding stability of the probe and increase the Tm value (target Tm>65℃), so as to ensure that the probe only forms stable binding with the completely matched mutant template under strict hybridization conditions, effectively improving the detection specificity.

[0055] In addition, it is observed during the construction of the system that the probes for E545G and Q546R have abnormal detection, and no FAM fluorescence signal is detected. After in-depth analysis, it is found that the abnormal phenomenon is mainly caused by the high sequence overlap between the E542K mutation probe and the E545X / Q546X series of mutation templates, so that competitive binding interference occurs between the probes. The E542K probe is originally used to identify the specific mutation of the 542th site of the PIK3CA gene, but because its recognition region is highly overlapped with the E545 / Q546 mutation site in the nucleotide sequence, when the E542K probe hybridizes with these non-target mutation templates, the mismatched base is located in the 5' end region of the probe (the probe is a reverse probe). The tolerance for the mismatch of the 5' end of the probe in the PCR amplification system is high, so that the probe can still form a partially stable binding with these non-target templates, thereby "occupying" the binding site of the mutation template in the reaction system. Because the binding structure has a mismatch at the 5' end, the recognition and cutting of the Taq enzyme on the probe-template complex are affected, so that the fluorescence group is not released, which shows that there is no signal output in the detection channel, that is, the mutation template exists but is missed by the system, resulting in false negative.

[0056] To effectively solve the above-mentioned mutual interference between the probes, the present application proposes the following optimization strategies:

[0057] The E542K probe and the E545 / Q546 series of mutation probes are physically separated and placed in the same reaction tube with the C420R and H1047X series of probes for detection. The E542K probe has a high sequence overlap with the E545 / Q546 template, and is prone to competitive binding interference. Independent detection can effectively avoid cross interference and ensure the accuracy of the signal output of the mutation site.

[0058] The E545 and Q546 series of mutation probes can be placed in one tube for multiplex detection. The mismatch sites of such probes are mainly located in the middle region of the probe, and the structure has good specificity and good recognition ability for the mutation site. As long as no obvious non-specific signal appears in the wild type template, stable multiplex detection can be realized.

[0059] Example 2: Preferred experiment of probe detection in two tubes

[0060] According to Example 1, the present application divides 11 sites into two reaction tubes for detection:

[0061] A tube: contains E545K, E545A, E545G, E545D, Q546E, Q546R six sites, which are concentrated in the 10th exon of PIK3CA gene, with strong aggregation. The present application distinguishes the main mutation site E545K by designing different fluorescent labels, and the remaining sites use the same fluorescent label for joint detection without further typing, thereby simplifying the system and reducing cross interference.

[0062] B tube: contains E542K, C420R, H1047R, H1047L, H1047Y five sites. In order to avoid signal interference, E542K is independent of E545 / Q546 sites, and is separately included in B tube for detection in the present application. At the same time, since the C420R, H1047 series of mutation sites are distributed in different exons, there is no amplification interference between them, which can be detected together with E542K, thereby improving the detection efficiency.

[0063] (I) Selection of A tube reaction conditions

[0064] The primer and probe used in A tube (covering E545K, E545A, E545G, E545D, Q546E, Q546R sites) are shown in Table 1. Tm and ΔTm are obtained by OligoAnalyzer of IDT (https: / / sg.idtdna.com / pages / tools / oligoanalyzer).

[0065] As shown in Table 1 above, A tube is mainly used for detecting various mutation types of PIK3CA gene 545-546 sites. The results in Example 1 show that the ordinary molecular beacon detection probe has high background signal when detecting homologous site mutations, and some mutant probes have low ΔTm, which has non-specific signal to wild type. Therefore, in order to improve the efficiency and specificity of mutation site detection, LNA probe is used to improve specificity, by shortening the probe sequence to 14-25 bp, while improving the base pairing stability, so that the overall Tm value is maintained above 64℃, and the ΔTm is effectively improved to above 5℃, which enhances the mismatch recognition ability. The primer and probe information used are shown in Table 3 below.

[0066] Table 3. A tube primer and probe information

[0067]

[0068] Note: ΔTm is the difference in Tm between the mutant probe and the mutant template matching and mismatching with the wild type template; + represents locked nucleic acid LNA; lowercase letters are 3' stem sequence that does not match the target; 3' horizontal line capital letters represent 3' stem sequence that matches the target; the horizontal line base in the middle position represents the mutation site.

[0069] The reaction system preparation and digital PCR detection operation steps are described in Example 1. The mutant plasmid and fragmented wild-type genomic DNA were used to prepare a simulated sample with a mutation rate of 10% (mutant and wild-type concentrations were 50 and 500 copies / µL, respectively, and 2 µL was used for each system), and wild-type genome was used as a template for comparative testing of ordinary molecular beacon detection probes and LNA molecular beacon detection probes. The detection results of the probes are shown in Tables 4-5. The ordinary molecular beacon detection probe has a high background signal (Table 4), and the wild-type template has a signal on the mutant probe, and E545K also has a signal on other mutant probes, which cannot distinguish wild-type and mutant templates, and cannot distinguish E545K and other mutants; the LNA molecular beacon detection probe can detect 6 mutant sites, the copy number meets the expectation, and the specificity is good, which can distinguish mutant and wild-type, and can distinguish E545K, but the background signal of the FAM channel is high, and the signal-to-noise ratio is low.

[0070] Table 4. Detection results of ordinary molecular beacon detection probes

[0071]

[0072] Note: Non-specific detection signal is indicated by bold italic numbers.

[0073] Table 5. Detection results of LNA molecular beacon detection probes

[0074]

[0075] Further optimization of the LNA molecular beacon detection probe system reduces the background signal of the reaction system and improves the signal-to-noise ratio. The LNA modified molecular beacon detection probe is used, and a flexible Spacer 18 group is introduced. The Spacer structure is placed in the loop region of the probe, which can block the potential non-specific complementary pairing between the 3' end stem and the target, avoid interference with Tm, and improve the stability and specificity of the hairpin structure. The primer probe sequences are shown in Table 6.

[0076] Table 6. Primer probe sequences

[0077]

[0078] Note: ΔTm is the difference in Tm between the mutant probe and the mutant template matching and mismatching with the wild-type template; + represents the locked nucleic acid LNA; lowercase letters are 3' end stem sequences that do not match the target; 3' end capital letters represent 3' end stem sequences that match the target; Spacer 18 is 6 polyethylene glycol (PEG) units; the dashed base in the middle position represents the mutation site.

[0079] The reaction system preparation and digital PCR detection operation steps are described in Example 1. Mutant plasmids and fragmented wild-type genomic DNA were used to prepare 10% mutation rate simulation samples (mutant and wild-type concentrations were 50 and 500 copies / µL, respectively, and 2 µL of each system was used), and wild-type genomes were used as templates for probe testing. The detection results of the probes are shown in Table 7. After using the Spacer probe, the background signal of the system was further reduced, the signal-to-noise ratio was improved, and each mutant template and wild-type template was specifically detected, and the copy number was consistent with the expected results.

[0080] Table 7. Detection results of Spacer probes

[0081]

[0082] To further reduce the background signal of the amplification reaction system, the present application also designs a fluorescence blocking probe. The probe is a small molecule probe with a short length and a 5' end sequence complementary to the beacon probe, and the 3' end is labeled with a high-efficiency quenching group (such as BHQ1) without carrying a fluorescent group. Its mechanism of action is: when the molecular beacon detection probe is in a free and unclosed state, the fluorescence blocking probe binds to the 5' end sequence through complementarity, effectively achieving fluorescence quenching, thereby further blocking the non-specific background signal generated by the free and unclosed probe in the system. The sequence is shown in Table 8.

[0083] Table 8. Sequence of fluorescence blocking probe

[0084]

[0085] The reaction system preparation and digital PCR detection operation steps are described in Example 1. Mutant plasmids and fragmented wild-type genomic DNA were used to prepare 10% mutation rate simulation samples (mutant and wild-type concentrations were 50 and 500 copies / µL, respectively, and 2 µL of each system was used), and wild-type genomes were used as templates for probe testing. The detection results of the probes are shown in Table 9. After using the fluorescence blocking probe, the FAM channel background signal of the system was reduced again, further improving the signal-to-noise ratio, and each mutant template and wild-type template was specifically detected, and the copy number was consistent with the expected results.

[0086] Table 9. Detection results of fluorescence blocking probes

[0087]

[0088] (B) Selection of B tube reaction conditions

[0089] The primer and probe used in B tube (covering E542K, C420R, H1047R, H1047L, H1047Y sites) are shown in Table 5. Similarly, Tm and ΔTm are obtained by OligoAnalyzer of IDT (https: / / sg.idtdna.com / pages / tools / oligoanalyzer).

[0090] As shown in Table 10, B tube is mainly used to detect various mutation types of PIK3CA gene 542, 420 and 1047 sites, and different sites are genotyped. H1047R is genotyped because it is the most common site in clinic, and H1047L and H1047Y are not genotyped. Based on Example 1, the ΔTm of the common molecular beacon detection probe for 1047Y is low, resulting in non-specific signal to the wild type template. Therefore, LNA modification is added to the probe to shorten the length and increase the ΔTm, thereby improving the specificity of the probe. The primer and probe information used in B tube is shown in Table 10.

[0091] Table 10. Primer and probe information of B tube

[0092]

[0093] Note: ΔTm is the difference in Tm between the mutant probe and the mutant template and the mismatch with the wild type template; + represents locked nucleic acid LNA; lowercase letters are 3' stem sequences that do not match the target; the horizontal line base in the middle position represents the mutation site.

[0094] The system preparation and digital PCR operation are described in the optimization experiment of Example 1 above.

[0095] Using mutant plasmid and fragmented wild type genomic DNA to prepare a simulated sample with a mutation rate of 10% (mutant and wild type concentrations are 50 and 500 copies / µL respectively, 2 µL is used for each system), and wild type genome as a template for probe screening, the detection results are shown in Tables 11-12. Among them, the common molecular beacon detection probe has high background signal (Table 11), and the wild type template has signal on the mutant probe, which cannot distinguish the wild type from the mutant template; the LNA molecular beacon detection probe can detect the 5 mutation sites, the copy number meets the expectation, and the specificity is good, which can distinguish the mutant from the wild type, but the background signal of ROX channel is high, and the signal-to-noise ratio is low.

[0096] Table 11. Detection results of common molecular beacon detection probe

[0097]

[0098] Note: Non-specific detection signal is indicated by bold italic numbers.

[0099] Table 12. LNA molecular beacon detection probe detection results

[0100]

[0101] Further optimization of the LNA molecular beacon detection probe system, reduce the background signal of the reaction system, improve the signal-to-noise ratio, using LNA modified molecular beacon detection probe, and the introduction of flexible Spacer18group. Spacer structure is placed in the probe ring area, which can block the potential non-specific complementary pairing between the 3' end stem and the target, avoid interference with Tm, and improve the stability and specificity of the hairpin structure. The primer probe sequence is shown in Table 13.

[0102] Table 13. B tube Spacer probe sequence

[0103]

[0104] Note: ΔTmis the difference in Tmof mutant probes and mutant template matching and mismatch with wild-type template; + represents locked nucleic acid LNA; lowercase letters are 3' end stem sequences that do not match the target; Spacer18is 6 polyethylene glycol (PEG) units; the horizontal line base in the middle position represents the mutation site.

[0105] The reaction system preparation and digital PCR detection operation steps are described in Example 1. Using mutant plasmids and fragmented wild-type genomic DNA to prepare 10% mutation rate of simulated samples (mutant and wild-type concentrations are 50 and 500 copies / µL, respectively, 2 µL is used for each system), and wild-type genome as a template for probe testing, the detection results of the probe are shown in Table 14. After using Spacer probe, the background signal of the system is further reduced, the signal-to-noise ratio is improved, and each mutant template and wild-type template is specifically detected, and the copy number meets the expected results.

[0106] Table 14. Spacer probe detection results

[0107]

[0108] To further reduce the background signal of the amplification reaction system, the present application also designs a fluorescence blocking probe. The probe is a small molecule probe with a short length and a 5' end sequence complementary to the beacon probe, with a high-efficiency quenching group (such as BHQ1) labeled at the 3' end, and does not carry a fluorescent group. Its mechanism of action is: when the molecular beacon detection probe is in a free and unclosed state, the fluorescence blocking probe binds to the 5' end sequence by complementation, effectively quenching the fluorescence, thereby further blocking the non-specific background signal generated by the free and unclosed probe in the system. The sequence is: SEQ ID NO. 46: gcatca (3' end BHQ1 label). After adding the fluorescence blocking probe, the ROX, HEX, CY5 background signals of the reaction system are further reduced.

[0109] The reaction system preparation and digital PCR detection operation steps are described in Example 1. Using mutant plasmids and fragmented wild-type genomic DNA, a 10% mutation rate of the simulated sample (mutant and wild-type concentrations are 50 and 500 copies / µL, respectively, and 2 µL of each system is used) is prepared, and the wild-type genome is used as a template for probe testing, and the detection results of the probe are shown in Table 15. After using the fluorescence blocking probe, the FAM channel background signal of the system is reduced again, further improving the signal-to-noise ratio, and each mutant template and wild-type template is specifically detected, and the copy number meets the expected results.

[0110] Table 15. Detection results of adding fluorescence blocking probe

[0111]

[0112] Example Three Optimization of Reaction System Components and Reaction Conditions

[0113] In the above examples, the primers and probes used for screening and combination optimization are plasmid nucleic acids and fragmented genomic DNA, and the amount of nucleic acid template added to the system is 2 µL. In high-sensitivity blood PIK3CA mutation detection, to meet the detection needs of low-abundance mutations in clinical detection, nucleic acids need to be extracted from large-volume plasma samples, and as much as possible large-volume templates need to be added to the amplification reaction to improve the detection sensitivity.

[0114] In this example, 2 mL of simulated plasma samples (containing 10 copies of each type of mutant plasmid DNA) are used for nucleic acid extraction, and large-volume simulated plasma samples are prepared and divided into small portions for standby use. During the extraction process, a small volume of 40 µL is used for elution to increase the concentration of nucleic acids. Subsequently, as much as possible large-volume nucleic acid templates are added to the amplification system to enhance the sensitivity of detection. In this example, 20 µL of nucleic acid template is added, and the system components in Example One are used as a control to optimize the system.

[0115] The optimization measures include: 1) adding BSA (bovine serum albumin) component to improve the stability of the amplification reaction. 2) To improve the dispersion of nucleic acid in the droplet and the amplification efficiency, the extracted nucleic acid is pretreated: heating at 80°C for 5 minutes to denature double-stranded nucleic acid into single-stranded nucleic acid, thereby improving the uniformity of the target copy in the droplet. Heating treatment also helps to remove potential residual PCR reaction inhibitors, improving overall detection sensitivity and reliability.

[0116] The A tube reaction system is prepared as follows:

[0117]

[0118] The B tube reaction system is prepared as follows:

[0119]

[0120] The digital PCR operation is as described in Example One. The template includes heating pretreatment and no heating pretreatment. The detection results are as follows. As can be seen, when using a large volume (2 ml) of plasma to extract nucleic acid, and the template large volume sample amount is 20 μl, the addition of BSA can stably detect low copy positive mutations, and low copy positive mutations without BSA cannot be stably detected; in addition, after heating pretreatment of the nucleic acid template, the copy number is further improved. The template sample amount of the kit of the present application is 20 μl, when the template concentration is 0.4 ng / μl, the template sample amount is 6 ng, by heating pretreatment, the wild type copy number can be obtained about 3000-5000 copies, when the mutant copy number is greater than 3 copies, the report positive result, at this time, the sensitivity reaches 0.1% requirement.

[0121]

[0122] Example Four Composition of the detection kit of the present application

[0123] The kit comprises two reaction solutions, wherein the reaction solution A detects PIK3CA gene E545K (labeled ROX fluorescence), E545A, E545G, E545D, Q546E, Q546R (all labeled FAM fluorescence, not typing), 545-546 wild type (labeled HEX fluorescence, as an internal standard gene); the reaction solution B detects PIK3CA gene E542K (labeled CY5.5 fluorescence), C420R (labeled FAM fluorescence), H1047R (labeled CY5 fluorescence), H047L and H1047Y (labeled ROX fluorescence, not typing), H1047 wild type (labeled HEX fluorescence, as an internal standard gene). Three nucleic acid reaction solutions (reagent A, reagent B and reagent C), negative control (DEPC treated water), positive control (each mutant plasmid mixture) are packaged together, and product instruction is attached, to obtain the PIK3CA gene 11 mutation hot spot kit in the application. The compositions of the premix, the detection solution, the negative control and the positive control are shown in Table 16.

[0124] Table 16. PIK3CA gene mutation detection kit components

[0125]

[0126] Example five verification of the kit on clinical samples

[0127] (1) Reagent preparation: according to the number of samples to be tested, negative control and positive control, take out the PCR reaction solution, calculate the number of each reaction solution n = sample number + positive control + negative control, according to the required sample number n, respectively add 7.5 μL PCR reagent A and 3 μL PCR reagent B (or C) into the PCR reaction tube, mix thoroughly, and centrifuge for standby.

[0128] (2) Sample processing: the sample is 2 ml of anticoagulated plasma, and the commercial extraction kit is used for plasma free DNA extraction. After extraction, the DNA is eluted with TE buffer.

[0129] To the reaction tube, 10.5 μl PCR reaction liquid B or C and 19.5 μl free DNA to be detected were added, and droplet generation, PCR amplification and droplet detection were carried out according to the operation in Example 1 described above. After the end of the PCR amplification reaction, genotype determination was carried out according to the target copy number. The principles of the result determination are as follows: ① The target copy number and the wild type copy number of the positive control are both greater than 50 copies; ② The target copy number of the negative control group is 0, and the wild type copy number is not higher than 5 copies; ③ The above two conditions are met at the same time, and the experiment is determined to be successful, and the sample to be detected is typed according to the target copy number, and the interpretation criteria are as follows: when the copy number of a certain target is greater than 3 copies, and the copy number of the internal standard gene (wild type + mutant copy number) is greater than 500 copies, the target is positive; if the copy number of all targets is not higher than 3 copies, and the copy number of the internal standard gene (wild type + mutant copy number) is greater than 500 copies, the sample is negative; if the copy number of the internal standard gene is less than 500 copies, the detection result is invalid, there may be amplification inhibition or low nucleic acid concentration, and the nucleic acid needs to be extracted and detected again.

[0130] The detection results are shown in the following table. A total of 11 clinical samples were detected, including 5 different mutant positive samples and 6 negative samples, and the results of using the kit of the application were consistent with the NGS sequencing results, indicating that the composition of the application can well detect the PIK3CA mutation in the plasma free DNA. And the kit of the application can accurately give the mutation rate of the sample, which is convenient for clinical dynamic monitoring of mutation abundance change.

[0131]

[0132] It should be understood that the disclosed application is not limited only to the specific methods, protocols and substances described, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing only specific embodiments and is not intended to limit the scope of the present application, which is limited only by the claims appended hereto.

[0133] Those skilled in the art will further appreciate that the application described herein is susceptible to a broad utility and application, having regard to the many possible embodiments of the application. Many variations and modifications will now become apparent to those skilled in the art. It is to be understood that the application includes all such variations and modifications. Therefore, the application as described and claimed should be understood to include all such variations and modifications.

Claims

1. A digital PCR detection kit for detecting a multi-mutation site of a PIK3CA gene, characterized in that, The kit detects 11 mutation sites of PIK3CA gene through two digital PCR reaction tubes A and B, wherein the A tube contains detection of E545K, E545A, E545G, E545D, Q546E, Q546R sites, and the B tube contains detection of E542K, C420R, H1047R, H1047L, H1047Y sites; wherein each site is detected using a respective spacer molecular beacon detection probe, which comprises a 5' end stem sequence, a loop sequence, a flexible Spacer structure and a 3' end stem sequence in turn, and the whole is a hairpin structure; the 5' end is labeled with a fluorescent group, and the 3' end is labeled with a quencher group, and the 5' end stem sequence and the 3' end stem sequence form a stable stem structure; the 5' end stem sequence and the loop sequence are completely complementary to the target sequence to be detected, and the loop sequence and the flexible Spacer structure form a loop structure of the molecular beacon detection probe; the flexible Spacer structure is a polyethylene glycol structure; The A tube includes the spacer molecular beacon detection probe SEQ ID NO. 27 for E545K, the spacer molecular beacon detection probe SEQ ID NO. 28 for E545A, the spacer molecular beacon detection probe SEQ ID NO. 29 for E545G, the spacer molecular beacon detection probe SEQ ID NO. 30 for E545D, the spacer molecular beacon detection probe SEQ ID NO. 31 for Q546E, and the spacer molecular beacon detection probe SEQ ID NO. 32 for Q546R; the A tube includes the E542-546 upstream primer SEQ ID NO. 13 and the E542-546 downstream primer SEQ ID NO. 14; The B tube further includes the spacer molecular beacon detection probe SEQ ID NO. 40 for H1047R, the spacer molecular beacon detection probe SEQ ID NO. 41 for H1047L, the spacer molecular beacon detection probe SEQ ID NO. 42 for H1047Y, the spacer molecular beacon detection probe SEQ ID NO. 44 for C420R, and the spacer molecular beacon detection probe SEQ ID NO. 45 for E542K; the B tube includes three groups of upstream and downstream primers, which are SEQ ID NO. 13 and SEQ ID NO. 14, SEQ ID NO. 15 and SEQ ID NO. 16, and SEQ ID NO. 17 and SEQ ID NO. 18, respectively.

2. The digital PCR detection kit according to claim 1, characterized in that, The A tube further includes the E545 wild type detection probe SEQ ID NO.

33.

3. The digital PCR detection kit according to claim 1, characterized in that, The A tube further comprises a fluorescent blocking probe B1 for closing the spacer molecular beacon detection probe of E545K, the spacer molecular beacon detection probe of E545A and the spacer molecular beacon detection probe of E545G, the sequence of which is SEQ ID NO. 34; a fluorescent blocking probe B2 for closing the spacer molecular beacon detection probe of Q546E, the spacer molecular beacon detection probe of Q546R and the spacer molecular beacon detection probe of E545D, the sequence of which is SEQ ID NO. 35; the sequence of the fluorescent blocking probe is complementary to the 6-base region at the 5' end of each molecular beacon detection probe described above, and the 3' end of the fluorescent blocking probe is labeled with a fluorescence quenching group, which is combined with the 5' end of the molecular beacon detection probe in the free state to form a stable secondary closed structure.

4. The digital PCR detection kit according to claim 1, characterized in that, The B tube further comprises a fluorescent blocking probe B3 for closing the spacer molecular beacon detection probe of H1047R, the spacer molecular beacon detection probe of H1047L and the spacer molecular beacon detection probe of H1047Y, the sequence of which is SEQ ID NO. 46; the sequence of the fluorescent blocking probe is complementary to the 6-base region at the 5' end of each molecular beacon detection probe described above, and the 3' end of the fluorescent blocking probe is labeled with a fluorescence quenching group, which is combined with the 5' end of the molecular beacon detection probe in the free state to form a stable secondary closed structure.

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