PAP-Liquid Phase Array and High-Throughput, High-Specific Detection Methods for Gene Mutations

Abstract

This invention relates to a PAP-liquid phase chip and a high-throughput, high-specificity detection method for gene mutations. The chip contains primers and probes. The primers include forward and reverse primers. The 5' end of the forward primer is biotin-labeled or fluorescently labeled, and the 3' ends of both the forward and reverse primers are mutation-specific nucleotides, with the last nucleotide being a dideoxynucleotide. The 5' end of the probe has an amino group, followed by an 18-25 bp spacer arm, and then a 15-30 bp specific probe containing the mutation site. The probe is covalently linked to a magnetic sheet. The chip combines PAP technology with digital liquid phase chip technology, enabling the detection of rare mutations at a single point in a single tube, as well as the detection of multiple mutation sites in a single tube. This reduces reagent usage and significantly lowers detection costs. It is suitable for both single-sample detection and large-scale sample screening, while offering high sensitivity, high specificity, and high throughput.

Description

Technical Field

[0001] This invention belongs to the field of gene chip technology, and particularly relates to a PAP-liquid phase chip and a high-throughput, high-specificity detection method for gene mutations that combines PAP technology with digital liquid phase chip technology, utilizing the advantages of high sensitivity and high specificity of PAP technology and the high throughput of liquid phase chips. Background Technology

[0002] Gene mutations refer to the addition, deletion, or alteration of base pairs in a DNA molecule. Mutations are caused by errors in DNA replication (especially meiosis) or by incorrect repair following DNA damage (such as exposure to radiation or carcinogens). Gene mutations are often associated with diseases and are even the direct cause of some diseases.

[0003] Gene mutation detection can be used for early screening, diagnosis, and prognosis of various diseases. Currently, commonly used methods for gene mutation detection include sequencing technology and ARMS-PCR (amplification-refractory mutation system, ARMS) based on polymerase chain reaction (PCR).

[0004] Sequencing technology is widely recognized as the "gold standard" for detecting gene mutations, including first-generation sequencing based on the Sanger method and next-generation sequencing (NGS). Sanger sequencing has a low sensitivity of 20% for detecting gene mutations, while NGS sequencing has a sensitivity of 5%.

[0005] ARMS-PCR has a sensitivity of 1%, which is sufficient for detecting tumor tissue samples. However, it is not sensitive enough for detecting circulating tumor cells in plasma or blood with a content of less than 1%. It cannot detect drug resistance mutations caused by clinical drug use in a timely manner. Furthermore, it is limited by the fluorescence channel and has the disadvantages of limited detection quantity and low throughput, making it difficult to detect multiple targets in the same tube.

[0006] Digital PCR has an analytical sensitivity of 0.05%–0.1% for allele mutation detection, exhibiting high sensitivity, strong specificity, and quantitative detection capabilities. However, it is cumbersome and involves numerous steps. Therefore, there is a need for a mutation detection method that is highly sensitive, specific, simple to perform, and has high throughput.

[0007] Pyrophosphorolysis-activated polymerization (PAP) is a technique for detecting rare mutations (US11 / 772,662). The primers used in PAP are not ordinary primers, but rather primers with the last nucleotide at the 3' end blocked. These primers cannot be directly extended through polymerization, but the block can be removed by pyrophosphorolysis, allowing for further extension through polymerization and the formation of the amplified product. Pyrophosphorolysis is the reverse reaction of DNA polymerization. In the presence of pyrophosphate, the 3' nucleotide is removed from the double-stranded DNA by polymerase, producing a triphosphate nucleotide and a short nucleotide double-stranded DNA: [dNMP]n+PPi → [dNMP]n-1+dNTP (Deutscher and Kornberg, 1969). This technique combines the specificity of pyrophosphorolysis activity with the specificity of polymerization activity; therefore, the specificity can reach 1 / 10. 9 .

[0008] PAP can theoretically reach 1 / 10. 11 Its specificity is demonstrated by experiments, showing that its actual specificity can reach 1 / 10. 9 You can start from 10 9 One copy of the mutant template was detected in one copy of the wild-type template. This technique allows for electrophoretic analysis after PCR amplification (e.g., patent US11 / 772,622), but it is time-consuming and prone to cross-contamination. PAP technology can also be used for the quantitative detection of rare gene mutations (e.g., patent CN101381766A), including dye intercalation, double-stranded primers, single-stranded fluorescent quenching primers, or fluorescent probes, and includes both singleton and multiplex fluorescence quantification. However, this method has low throughput and is difficult to meet the requirements of multi-target clinical applications.

[0009] Digital liquid chips are a high-throughput detection technology based on barcode magnetic beads (BMB). This technology utilizes mature semiconductor processes to miniaturize digital barcodes by millions of times, enabling low-cost mass production of optical barcode magnetic beads. The bio-liquid wafers are magnetic, easy to clean, separate, and recycle. The barcodes on the digital magnetic beads can accurately identify target analytes, and with over 4000 different barcodes available, multiple analytes can be detected in a single test. These magnetic beads can be functionalized to couple with nucleic acids, proteins, or other probe molecules, allowing for highly complex analyses in homogeneous or heterogeneous media (e.g., US20100832972). Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a PAP-liquid phase chip and a high-throughput, high-specificity detection method for gene mutations that uses PAP technology and primers with 3' ends blocked, and combines liquid phase chip technology to detect multiple different mutated genes of the same target sequence in the same tube. This detection method is not limited by the fluorescence channel.

[0011] To achieve the above objectives, the technical solution of the present invention is implemented as follows:

[0012] A PAP-liquid phase chip contains primers and probes. The primers include a forward primer and a reverse primer. The 5' end of the forward primer is biotin-labeled, and the 3' ends of both the forward and reverse primers are mutation site-specific nucleotides, with the last nucleotide being a dideoxynucleotide. The 5' end of the probe has an amino group, followed by an 18-25 bp spacer arm, and then a 15-30 bp specific probe containing the mutation site. The probe is covalently linked to the magnetic sheet.

[0013] Furthermore, the gene sequences of the primer and probe sets are selected from SEQ ID NO:1-3,4-6,7-9,10-12,13-15,16-18,19-21,22-24; wherein SEQ ID NO:1-2 are the upstream and downstream primer sequences, SEQ ID NO:3 is the corresponding probe sequence, SEQ ID NO:4-5 are the upstream and downstream primer sequences, SEQ ID NO:6 is the corresponding probe sequence, and so on.

[0014] Furthermore, the primer gene sequences are selected from SEQ ID NO:25-27,28-30,...,79-81; wherein SEQ ID NO:25-26 are the upstream and downstream primer sequences, SEQ ID NO:27 is the corresponding probe sequence, and so on.

[0015] This invention also provides a high-throughput, high-specificity method for detecting gene mutations. The method includes: designing a PAP-liquid phase chip using primers and probes; coupling the magnetic sheet with the probes; PAP amplification; hybridization reaction; and signal analysis. Specifically, it includes the following steps:

[0016] Step 1) Design PAP-liquid phase chip using primer and probe sets: Design a PAP-liquid phase chip according to the gene mutation to be detected. The PAP-liquid phase chip contains primer and probe sets. Prepare the corresponding primer and probe gene sequences. The primers include forward primers and reverse primers. The 5' end of the forward primer is biotin-labeled or other chemical marker. The 3' ends of both the forward and reverse primers are mutation site-specific nucleotides. The last nucleotide is a dideoxynucleotide, which cannot be extended in conventional PCR reactions. The 5' end of the probe is an amino group, followed by an 18-25 bp spacer arm, and then a 15-30 bp sequence containing the mutation site.

[0017] Step 2) Coupling of magnetic sheet and probe: Covalently link the amino-modified probe with the carboxylated magnetic sheet;

[0018] Step 3) PAP amplification: In a reaction system containing a specific DNA polymerase and pyrophosphate, the primers from step 1) are used to first undergo pyrophosphate hydrolysis to unblock the primers, and then the mutant gene is amplified by PCR.

[0019] Step 4) Hybridization reaction: Take the amplification product and add it to the well of the digital magnetic sheet with pre-coupled probes to carry out the hybridization reaction. Read and photograph the fluorescence signal using a fluorescence analyzer. Based on the digital display of the fluorescence signal on the magnetic sheet, the type of gene mutation in the sample can be determined.

[0020] Furthermore, in step 2), the specific steps for coupling the probe to the magnetic sheet are as follows:

[0021] Step 2.1) Take a centrifuge tube containing a carboxylated magnetic sheet, rotate it rapidly, and then place the centrifuge tube into a magnetic holder to remove the supernatant;

[0022] Step 2.2) Wash the carboxylated magnetic sheet twice, and place the MEST buffer in a centrifuge tube to remove the supernatant; the MEST buffer contains 10-100 mM of 2-(N-morpholino)ethanesulfonic acid, 0.05-0.1% v / v of 2-methyl-4-isothiazolin-3-one at pH 5.0 and 0.01-0.05% v / v of polyoxyethylene dehydrated sorbitan monolaurate;

[0023] Step 2.3) Take 79 μL of MEST buffer into a centrifuge tube, add 1.0 μL of 100 μM amino probe, and vortex to mix; add 20 μL of 10 mg / mL crosslinking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, vortex to mix and centrifuge; place the centrifuge tube in a shaker and shake at 200-500 rpm at room temperature for 2 hours, and remove the supernatant;

[0024] Step 2.4) Washing: Add 200 μL of 10-100 mM tris(hydroxymethyl)aminomethane buffer (pH 7.4) to a centrifuge tube and incubate the centrifuge tube in a mixer at room temperature for 10-30 minutes.

[0025] Step 2.5): Blocking: Add 200 μL of blocking solution to a centrifuge tube, vortex to mix, and remove the supernatant; add 200 μL of blocking solution to a centrifuge tube, vortex at 1200–1800 rpm at room temperature for 30–60 minutes, and remove the supernatant; the blocking solution is 10 mM Na2HPO4, 1.7 mM KH2PO4, 2.7 mM KCl, 137 mM NaCl, 1% w / v bovine serum albumin, and 1% w / v skim milk powder;

[0026] Step 2.6): Washing: Take 200 μL of PBST buffer into a centrifuge tube, gently shake to remove the supernatant; repeat this step twice; add 200 μL of PBST into the centrifuge tube.

[0027] Furthermore, in step 3), the PAP reaction system consists of: 20–50 mM tris(hydroxymethyl)aminomethane, 0–100 mM potassium chloride at pH 7.4–7.8, 1–10 mM magnesium chloride, 100–400 μM Na₄PPi, 25–50 μM of each deoxyribonucleotide, 0.04–0.2 μM of each primer, 5–200 ng of template, and 1–3 U of klentaq-s enzyme;

[0028] The PAP amplification program is as follows: 92-95℃ for 3 min pre-denaturation, then enter the cycle: 92-95℃ for 15 s, 55-64℃ for 30 s, 72℃ for 1 min, 72℃ for 1-2 min, for a total of 15-45 cycles.

[0029] Furthermore, in step 4), the specific steps of the hybridization reaction are as follows:

[0030] Step 4.1) Transfer magnetic sheets coupled with different types of probes into a large centrifuge tube, place it on a magnetic rack, and remove the supernatant;

[0031] Step 4.2) Add tetramethylammonium chloride buffer to resuspend the magnetic sheet, shake to mix, and transfer the magnetic sheet into a 96-well plate; the tetramethylammonium chloride buffer contains 3M tetramethylammonium chloride, 10-100mM tris(hydroxymethyl)aminomethane, 0.05-0.2% v / v sodium dodecylsarcosinate, 1-5mM ethylenediaminetetraacetic acid, and 0.05-0.1% v / v 2-methyl-4-isothiazolin-3-one, with a pH of 8.0;

[0032] Step 4.3): Hybridization: Add 2-5 μL of biotin-labeled PCR amplification product to a 96-well plate and incubate at 52°C for 15-30 min at 500-700 rpm.

[0033] Step 4.4) Color development: Add 10 μL of 10-12 μg / mL R-phycoerythrin-labeled streptavidin to a 96-well plate and incubate at 52°C for 5-15 min at 500-700 rpm. Wash twice with PBST.

[0034] Step 4.5) Analysis: Add 200 μL of 2xSSC resuspended magnetic beads to each well; read and photograph the fluorescence signal using a fluorescence analyzer, and determine the type of gene mutation in the sample based on the digital display of the fluorescence signal on the magnetic sheet.

[0035] The present invention also provides the use of the PAP-liquid phase chip for detecting gene mutations.

[0036] Compared with the prior art, the present invention has the following outstanding advantages:

[0037] 1. Combining PAP technology with digital liquid phase chip technology inherits the advantages of PAP technology, such as high sensitivity and high specificity, while also possessing the high throughput characteristics of liquid phase chips.

[0038] 2. It can not only detect rare mutations at a single point in a single tube, but also detect multiple mutation sites in a single tube, reducing the use of reagents and significantly lowering the detection cost.

[0039] 3. It is suitable for both single sample testing and screening of large numbers of samples. Attached Figure Description

[0040] Figure 1 This is a schematic diagram illustrating the working principle of PAP-microchip technology.

[0041] Figure 2 This is a bright-field plot of the hybridization reaction results of KRAS G12D in non-small cell lung cancer obtained in Example 1.

[0042] Figure 3 This is a fluorescence image of the hybridization reaction results of KRAS G12D in non-small cell lung cancer obtained in Example 1.

[0043] Figure 4 This is a chip result display diagram when G12D mutation occurs in Example 1.

[0044] Figure 5 This is a chip result display diagram when G12A mutation occurs in Example 1.

[0045] Figure 6This is a chip result display diagram when G12V mutates in Example 1.

[0046] Figure 7 This is a chip result display diagram when G12R mutates in Example 1.

[0047] Figure 8 This is a chip result display diagram when G12C mutation occurs in Example 1.

[0048] Figure 9 This is a chip result display diagram when G12S mutation occurs in Example 1.

[0049] Figure 10 This is a chip result display diagram when G13D mutation occurs in Example 1.

[0050] Figure 11 This is a chip result display diagram when G13C mutation occurs in Example 1.

[0051] Figure 12 This is a bright-field plot of the hybridization reaction results of EGFR T790M non-small cell lung cancer obtained in Example 2.

[0052] Figure 13 This is a fluorescence image of the hybridization reaction results of EGFR T790M in non-small cell lung cancer obtained in Example 2.

[0053] Figure 14 This is a chip result display diagram when T790M mutates in Example 2.

[0054] Figure 15 This is a chip result display diagram when E746_A750del(2) mutates in Example 2.

[0055] Figure 16 This is a chip result display diagram when L747_P753>S mutates in Example 2.

[0056] Figure 17 This is a chip result display diagram when E746_T751>I mutates in Example 2.

[0057] Figure 18 This is a chip result display diagram when E746_T751del mutates in Example 2.

[0058] Figure 19 This is a chip result display diagram when E746_S752>V mutates in Example 2.

[0059] Figure 20 This is a chip result display diagram when L747_T751>Q mutation occurs in Example 2.

[0060] Figure 21This is a chip result display diagram when L747_E749del mutates in Example 2.

[0061] Figure 22 This is a chip result display diagram when L747_S752del mutates in Example 2.

[0062] Figure 23 This is a chip result display diagram when L747_A750>P mutates in Example 2.

[0063] Figure 24 This is a chip result display diagram when L747_P753>Q mutation occurs in Example 2.

[0064] Figure 25 This is a chip result display diagram when L747_T751del mutates in Example 2.

[0065] Figure 26 This is a chip result display diagram when L747_T751>P mutates in Example 2.

[0066] Figure 27 This is a chip result display diagram when S768I mutates in Example 2.

[0067] Figure 28 This is a chip result display diagram when L858R mutates in Example 2.

[0068] Figure 29 This is a chip result display diagram when G719A mutates in Example 2.

[0069] Figure 30 This is a chip result display diagram when G719C mutates in Example 2.

[0070] Figure 31 This is a chip result display diagram when E746_A750del(1) mutates in Example 2.

[0071] Figure 32 This is a chip result display diagram when L861Q mutates in Example 2. Detailed Implementation

[0072] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0073] This invention provides a PAP-liquid phase chip containing primers and probes. The primers include a forward primer and a reverse primer. The 5' end of the forward primer is labeled with biotin or other chemical markers, and the 3' ends of both the forward and reverse primers are mutation site-specific nucleotides, with the last nucleotide being a dideoxynucleotide. The 5' end of the probe consists of an amino group, followed by an 18-25 bp spacer arm, and then a 15-30 bp specific probe containing the mutation site. The probe is covalently linked to the magnetic sheet.

[0074] Primer and probe design: Design and prepare corresponding primer and probe sequences according to the gene mutations to be detected. The 5' end of the forward primer is labeled with biotin, and the 3' ends of both the forward and reverse primers are mutation site-specific nucleotides, with the last nucleotide being a dideoxynucleotide (ddNTP), which cannot be extended in conventional PCR reactions.

[0075] The gene sequences of the primers and probes are selected from SEQ ID NO:1-3,4-6,7-9,10-12,13-15,16-18,19-21,22-24; wherein SEQ ID NO:1-2 are the upstream and downstream primer sequences, SEQ ID NO:3 is the corresponding probe sequence, SEQ ID NO:4-5 are the upstream and downstream primer sequences, SEQ ID NO:6 is the corresponding probe sequence, and so on.

[0076] The primer gene sequences are selected from SEQ ID NO:25-27,28-30,...,79-81; wherein SEQ ID NO:25-26 are the upstream and downstream primer sequences, SEQ ID NO:27 is the corresponding probe sequence, and so on.

[0077] Magnetic sheet-probe coupling: An amino-modified probe is covalently linked to a carboxylated magnetic sheet.

[0078] This invention also provides a high-throughput, high-specificity method for detecting gene mutations. The method includes: designing a PAP-liquid phase chip using primers and probes; coupling the magnetic sheet with the probes; PAP amplification; hybridization reaction; and signal analysis. Specifically, it includes the following steps:

[0079] Step 1) Design PAP-liquid phase chip using primer and probe sets: Design a PAP-liquid phase chip according to the gene mutation to be detected. The PAP-liquid phase chip contains primer and probe sets. Prepare the corresponding primer and probe gene sequences. The primers include forward primers and reverse primers. The 5' end of the forward primer is biotinylated or other chemical markers. The 3' ends of both the forward and reverse primers are mutation site-specific nucleotides. The last nucleotide is dideoxynucleotide, which cannot be extended in conventional PCR reactions. The 5' end of the probe is an amino group, followed by an 18-25 bp spacer arm, and then a 15-30 bp sequence containing the mutation site.

[0080] Step 2) Coupling of the magnetic sheet and the probe: The amino-modified probe is covalently linked to the carboxylated magnetic sheet; the specific steps for coupling the probe to the magnetic sheet are as follows:

[0081] Step 2.1) Take a centrifuge tube containing a carboxylated magnetic sheet, rotate it rapidly, place the centrifuge tube in a magnetic holder, and remove the supernatant;

[0082] Step 2.2), Cleaning: Clean the carboxylated magnetic sheet twice, place the MEST buffer in a centrifuge tube, and remove the supernatant; the MEST buffer is prepared as follows: weigh 0.9762 g of 2-(N-morpholine)ethanesulfonic acid, add 80 mL of pure water to dissolve it completely, add 0.05 mL of 2-methyl-4-isothiazolin-3-one and 0.01 mL of polyoxyethylene dehydrated sorbitan monolaurate, adjust the pH to 5.0 with NaOH or hydrochloric acid, and add pure water to make up to 100 mL;

[0083] Step 2.3) Coupling: Take 79 μL of carboxylated magnetic sheet into a centrifuge tube, add 1.0 μL of 100 μM aminoated probe, and vortex to mix; add 20 μL of 10 mg / mL crosslinking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, vortex to mix and centrifuge; place the centrifuge tube in a shaker and shake at 500 rpm at room temperature for 2 hours, then remove the supernatant;

[0084] Step 2.4) Washing: Add 200 μL of 50 mM tris(hydroxymethyl)aminomethane buffer (pH 7.4) to a centrifuge tube and incubate the centrifuge tube at room temperature for 15 minutes in a mixer.

[0085] Step 2.5): Blocking: Add 200 μL of blocking solution to a centrifuge tube, vortex to mix, and remove the supernatant; add 200 μL of blocking solution to a centrifuge tube, vortex at 1500 rpm at room temperature for 30 minutes, and remove the supernatant; the blocking solution is prepared as follows: weigh 0.1420 g Na2HPO4, 0.0231 g KH2PO4, 0.0201 g KCl, 0.8007 g NaCl, 1 g bovine serum albumin, and 1 g skim milk powder, add pure water to a final volume of 100 mL, and dissolve and mix thoroughly;

[0086] Step 2.6): Washing: Take 200 μL of PBST buffer into a centrifuge tube, gently shake to remove the supernatant; repeat this step twice; add 200 μL of PBST into the centrifuge tube.

[0087] Step 3) PAP amplification: In a reaction system containing a specific DNA polymerase and pyrophosphate, the primers from step 1) are used to first undergo pyrophosphate hydrolysis to unblock the primers, and then the mutant gene is amplified by PCR.

[0088] The PAP reaction system consists of: 20–50 mM tris(hydroxymethyl)aminomethane, 0–100 mM potassium chloride at pH 7.4–7.8, 1–10 mM magnesium chloride, 100–400 μM Na₄PPi, 25–50 μM of each deoxyribonucleotide, 0.04–0.2 μM of each primer, 5–200 ng of template, and 1–3 U of klentaq-s enzyme;

[0089] The PAP amplification program is as follows: 95℃ for 3 min pre-denaturation, then enter the cycle: 94℃ for 15 s, 55℃ for 30 s, 72℃ for 1 min, 72℃ for 1-2 min, for a total of 15 to 45 cycles.

[0090] Step 4) Hybridization reaction: Take the polymerase chain reaction (PCR) amplification product from step 3) and add it to the wells of the digital magnetic sheet pre-coupled with probes for hybridization reaction. The specific steps are as follows:

[0091] Step 4.1) Transfer magnetic sheets coupled with different types of probes into a large centrifuge tube, place it on a magnetic rack, and remove the supernatant;

[0092] Step 4.2) Add tetramethylammonium chloride buffer to resuspend the magnetic sheet, shake to mix, and transfer the magnetic sheet into a 96-well plate;

[0093] The tetramethylammonium chloride buffer solution is prepared as follows: Weigh 32.8800g of tetramethylammonium chloride, 0.6057g of tris(hydroxymethyl)aminomethane, and 0.0372g of ethylenediaminetetraacetic acid, dissolve them thoroughly in 80mL of purified water, add 0.1mL of sodium dodecyl sarcosinate and 0.05mL of 2-methyl-4-isothiazolin-3-one, adjust the pH to 8.0 with NaOH or hydrochloric acid, and add purified water to bring the volume to 100mL.

[0094] Step 4.3): Hybridization: Add 2-5 μL of biotin-labeled PCR amplification product to a 96-well plate and incubate at 52°C for 20 min at 500 rpm.

[0095] Step 4.4): Color development: Add 10 μL of 10 μg / mL R-phycoerythrin-labeled streptavidin to a 96-well plate and incubate at 52°C for 10 min at 500 rpm; wash twice with PBST.

[0096] Step 4.4) Analysis: Add 200 μL of 2xSSC resuspended magnetic beads to each well; read and photograph the fluorescence signal using a fluorescence analyzer, and determine the type of gene mutation in the sample based on the digital display of the fluorescence signal on the magnetic sheet.

[0097] When using the chip, the PAP reaction system is as follows: 20mM tris(hydroxymethyl)aminomethane, pH 7.4, 75mM potassium chloride, 1.5mM magnesium chloride, 300μM Na4PPi, 40μM of each deoxyribonucleotide, 0.1μM forward primer, 0.04μM reverse primer, 5-200ng template, and 2.5U klentaq-s enzyme.

[0098] When using the chip, the PAP amplification program is as follows: 95℃ for 3 min pre-denaturation, then enter the cycle, 94℃ for 15 s, 55℃ for 30 s, 72℃ for 1 min, 72℃ for 1-2 min, for a total of 20 cycles.

[0099] The present invention also provides a PAP-liquid phase chip, which is used for detecting gene mutations.

[0100] Example 1: Detection of eight hotspot mutant genes in human KRAS.

[0101] The PAP-liquid phase microarray method was used to detect seven hotspot mutations in the KRAS gene of human lung cancer: G12A / G12D / G12V / G12R / G12C / G12S / G13D / G13C. The inventors blocked the 3' ends of the primers themselves. The specific primer sequences are shown in Table 1 of the sequence listing below.

[0102] Table 1 Primer and probe sequence list

[0103]

[0104]

[0105] Seven probes targeting different mutations were chemically coupled to liquid magnetic sheets, and then the magnetic sheets coupled with probes of different mutations were mixed in the same molar ratio for use in the hybridization reaction after PAP amplification.

[0106] Nucleic acid was extracted from paraffin-embedded tissue sections containing KRAS G12D from non-small cell lung cancer tissues according to the instructions of the Qiagen paraffin-embedded tissue genomic DNA extraction kit.

[0107] The PAP reaction system consisted of: 20 mM tris(hydroxymethyl)aminomethane, pH 7.4, 75 mM potassium chloride, 1.5 mM magnesium chloride, 300 μM Na4PPi, 40 μM of each deoxyribonucleotide, 0.1 μM forward primer, 0.04 μM reverse primer, 5-200 ng template, and 2.5 U klentaq-s enzyme.

[0108] The PAP amplification program is as follows: 95℃ for 3 min pre-denaturation, then enter the cycle: 94℃ for 15 s, 55℃ for 30 s, 72℃ for 1 min, 72℃ for 1-2 min, for a total of 20 cycles.

[0109] Experimental results are as follows Figure 2 and Figure 3 As shown, the fluorescence analyzer scans the code on the magnetic sheet under bright field. Based on the code, the probe coupled to the corresponding magnetic bead can be identified (see Table 1.1). The fluorescence analyzer scans the magnetic sheet with fluorescence and reads the fluorescence value. The magnetic sheet numbered 1011100 has a very strong fluorescence value. Therefore, it can be determined that the sample is a KRAS G12D mutation.

[0110] Table 1.1 Results of detection of seven hotspot mutant genes in KRAS

[0111]

[0112] Example 2: Detection of 19 deletion, substitution or mutation genes in human EGFR.

[0113] Nineteen deletion, substitution, or mutation genes in human EGFR were detected using the PAP-liquid phase chip method: E746_A750del(1) / E746_A750del(2) / L747_P753>S / E746_T751>I / E746_T751del / E746_S752>V / L747_T751>Q / L747_E749del / L747_S752del / L747_A750>P / L747_P753>Q / L747_T751del / L747_T751>P / S768I / L858R / G719A / G719C / T790M / L861Q. The inventors themselves blocked the 3' ends of the primers. The specific primer sequences are listed in Table 2.

[0114] Table 2 Primer and probe sequence list

[0115]

[0116]

[0117]

[0118] Seven probes targeting different mutations were chemically coupled to liquid magnetic sheets, and then the magnetic sheets coupled with probes of different mutations were mixed in the same molar ratio for use in the hybridization reaction after PAP amplification.

[0119] Take clinical plasma samples containing T790M and extract nucleic acids according to the instructions of the Qiagen Serum / Plasma Nucleic Acid Purification Kit.

[0120] The PAP reaction system consisted of: 20 mM tris(hydroxymethyl)aminomethane, pH 7.4, 75 mM potassium chloride, 1.5 mM magnesium chloride, 300 μM Na4PPi, 40 μM of each deoxyribonucleotide, 0.1 μM forward primer, 0.04 μM reverse primer, 5-200 ng template, and 2.5 U klentaq-s enzyme.

[0121] The PAP amplification program is as follows: 95℃ for 3 min pre-denaturation, then enter the cycle: 94℃ for 15 s, 55℃ for 30 s, 72℃ for 1 min, 72℃ for 1-2 min, for a total of 20 cycles.

[0122] Experimental results are as follows Figure 12 and Figure 13 As shown, the fluorescence analyzer scans the code on the magnetic sheet under bright field. Based on the code, the probe coupled to the corresponding magnetic bead can be identified (see Table 2.1). The fluorescence analyzer scans the magnetic sheet with fluorescence and reads the fluorescence value. The magnetic sheet numbered 0011111 has a very strong fluorescence value. Therefore, it can be determined that the sample has the EGFR T790M mutation.

[0123] Table 2.1 Detection table of 19 deletion, substitution or mutation genes of EGFR

[0124]

[0125]

[0126] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention without departing from the principles and spirit of the present invention.

[0127] The foregoing has shown and described the basic principles and main features of the present invention and its advantages. It will be apparent to those skilled in the art that the present invention is not limited to the details of the above exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or basic characteristics of the present invention. Therefore, the embodiments should be regarded as exemplary and non-limiting in all respects. The scope of the present invention is defined by the appended claims rather than the foregoing description. Therefore, it is intended that all changes falling within the meaning and scope of the equivalents of the claims be included within the present invention, and no reference numerals in the claims should be regarded as limiting the scope of the claims.

[0128] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0129]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139]

[0140]

[0141] <110> Xiamen Tongling Biomedical Technology Co., Ltd. <120> PAP-liquid phase chip and high-throughput, high-specificity detection method for gene mutations <160> 81 <210> 1 <211> 34 <212> DNA <213> Artificial sequence <400> atgactgaat ataaacttgt ggtagttgga gctg 34 <210> 2 <211> twenty three <212> DNA <213> Artificial sequence <400> tcaaggcact cttgcctacg cca 23 <210> 3 <211> 36 <212> DNA <213> Artificial sequence <400> tttttttttt tttttttttt ggagctgctg gcgtag 36 <210> 4 <211> 34 <212> DNA <213> Artificial sequence <400> atgactgaat ataaacttgt ggtagttgga gctg 34 <210> 5 <211> twenty three <212> DNA <213> Artificial sequence <400> tcaaggcact cttgcctacg cca 23 <210> 6 <211> 36 <212> DNA <213> Artificial sequence <400> tttttttttt tttttttttt ggagctgatg gcgtag 36 <210> 7 <211> 34 <212> DNA <213> Artificial sequence <400> atgactgaat ataaacttgt ggtagttgga gctg 34 <210> 8 <211> twenty three <212> DNA <213> Artificial sequence <400> tcaaggcact cttgcctacg cca 23 <210> 9 <211> 36 <212> DNA <213> Artificial sequence <400> tttttttttt tttttttttt ggagctgttg gcgtag 36 <210> 10 <211> 33 <212> DNA <213> Artificial sequence <400> atgactgaat ataaacttgt ggtagttgga gct 33 <210> 11 <211> twenty two <212> DNA <213> Artificial sequence <400> aaggcactct tgcctacgcc ac 22 <210> 12 <211> 36 <212> DNA <213> Artificial sequence <400> tttttttttt tttttttttt ggagctcgtg gcgtag 36 <210> 13 <211> 33 <212> DNA <213> Artificial sequence <400> atgactgaat ataaacttgt ggtagttgga gct 33 <210> 14 <211> twenty two <212> DNA <213> Artificial sequence <400> aaggcactct tgcctacgcc ac 22 <210> 15 <211> 36 <212> DNA <213> Artificial sequence <400> tttttttttt tttttttttt ggagcttgtg gcgtag 36 <210> 16 <211> 33 <212> DNA <213> Artificial sequence <400> atgactgaat ataaacttgt ggtagttgga gct 33 <210> 17 <211> twenty two <212> DNA <213> Artificial sequence <400> aaggcactct tgcctacgcc ac 22 <210> 18 <211> 36 <212> DNA <213> Artificial sequence <400> tttttttttt tttttttttt ggagctagtg gcgtag 36 <210> 19 <211> 32 <212> DNA <213> Artificial sequence <400> tgaatataaa cttgtggtag ttggagctgg tg 32 <210> 20 <211> 25 <212> DNA <213> Artificial sequence <400> tatcgtcaag gcactcttgc ctacg 25 <210> twenty one <211> 35 <212> DNA <213> Artificial sequence <400> ttttttttttttttttttga gctggtgacg taggc 35 <210> twenty two <211> 31 <212> DNA <213> Artificial sequence <400> tgaatataaa cttgtggtag ttggagctgg t 31 <210> twenty three <211> twenty two <212> DNA <213> Artificial sequence <400> gtcaaggcac tcttgcctac gc 22 <210> twenty four <211> 35 <212> DNA <213> Artificial sequence <400> ttttttttttttttttttga gctggttgcg taggc 35 <210> 25 <211> 31 <212> DNA <213> Artificial sequence <400> gtgagaaagt taaaattccc gtcgctatca a 31 <210> 26 <211> 29 <212> DNA <213> Artificial sequence <400> ggatttcctt gttggctttc ggagatgtt 29 <210> 27 <211> 39 <212> DNA <213> Artificial sequence <400> tttttttttt ttttttttgt cgctatcaaa acatctccg 39 <210> 28 <211> 31 <212> DNA <213> Artificial sequence <400> tgagaaagtt aaaattcccg tcgctatcaa g 31 <210> 29 <211> 28 <212> DNA <213> Artificial sequence <400> ggatttcctt gttggctttc ggagatgt 28 <210> 30 <211> 40 <212> DNA <213> Artificial sequence <400> ttttttttttttttttttgt cgctatcaag acatctccga 40 <210> 31 <211> 33 <212> DNA <213> Artificial sequence <400> agaaagttaa aattcccgtc gctatcaagg aat 33 <210> 32 <211> 28 <212> DNA <213> Artificial sequence <400> tcatcgagga tttccttgtt ggctttcg 28 <210> 33 <211> 41 <212> DNA <213> Artificial sequence <400> tttttttttt ttttttttct atcaaggaat cgaaagccaa c 41 <210> 34 <211> 26 <212> DNA <213> Artificial sequence <400> agttaaaatt cccgtcgcta tcaaaa 26 <210> 35 <211> twenty three <212> DNA <213> Artificial sequence <400> tccttgttgg ctttcggaga tat 23 <210> 36 <211> 42 <212> DNA <213> Artificial sequence <400> ttttttttttttttttttgc tatcaaaata tctccgaaag cc 42 <210> 37 <211> 31 <212> DNA <213> Artificial sequence <400> tgagaaagtt aaaattcccg tcgctatcaa g 31 <210> 38 <211> 27 <212> DNA <213> Artificial sequence <400> gaggatttcc ttgttggctt tcggaga 27 <210> 39 <211> 40 <212> DNA <213> Artificial sequence <400> tttttttttt ttttttttgt cgctatcaag tctccgaaag 40 <210> 40 <211> twenty three <212> DNA <213> Artificial sequence <400> taaaattccc gtcgctatca agg 23 <210> 41 <211> twenty two <212> DNA <213> Artificial sequence <400> gatttccttg ttggctttcg ga 22 <210> 42 <211> 40 <212> DNA <213> Artificial sequence <400> tttttttttt ttttttttct atcaaggttc cgaaagccaa 40 <210> 43 <211> 18 <212> DNA <213> Artificial sequence <400> ccgtcgctat caaggagc 18 <210> 44 <211> twenty one <212> DNA <213> Artificial sequence <400> cttgttggct ttcggagatt g 21 <210> 45 <211> 40 <212> DNA <213> Artificial sequence <400> tttttttttt ttttttttat caaggagcaa tctccgaaag 40 <210> 46 <211> 32 <212> DNA <213> Artificial sequence <400> agaaagttaa aattcccgtc gctatcaagg aa 32 <210> 47 <211> 27 <212> DNA <213> Artificial sequence <400> ttccttgttg gctttcggag atgttgc 27 <210> 48 <211> 41 <212> DNA <213> Artificial sequence <400> tttttttttt ttttttttgc tatcaaggaa gcaacatctc c 41 <210> 49 <211> 32 <212> DNA <213> Artificial sequence <400> agaaagttaa aattcccgtc gctatcaagg aa 32 <210> 50 <211> 27 <212> DNA <213> Artificial sequence <400> atcgaggatt tccttgttgg ctttcgg 27 <210> 51 <211> 41 <212> DNA <213> Artificial sequence <400> tttttttttt ttttttttct atcaaggaac cgaaagccaa c 41 <210> 52 <211> 20 <212> DNA <213> Artificial sequence <400> ttcccgtcgc tatcaaggaa 20 <210> 53 <211> 20 <212> DNA <213> Artificial sequence <400> gttggctttc ggagatgttg 20 <210> 54 <211> 40 <212> DNA <213> Artificial sequence <400> ttttttttttttttttttat caaggaacca acatctccga 40 <210> 55 <211> twenty one <212> DNA <213> Artificial sequence <400> ttcccgtcgc tatcaaggaa c 21 <210> 56 <211> twenty three <212> DNA <213> Artificial sequence <400> gaggatttcc ttgttggctt tct 23 <210> 57 <211> 41 <212> DNA <213> Artificial sequence <400> tttttttttt ttttttttct atcaaggaac agaaagccaa c 41 <210> 58 <211> 33 <212> DNA <213> Artificial sequence <400> agaaagttaa aattcccgtc gctatcaagg aat 33 <210> 59 <211> 26 <212> DNA <213> Artificial sequence <400> gaggatttcc ttgttggctt tcggag 26 <210> 60 <211> 41 <212> DNA <213> Artificial sequence <400> tttttttttttttttttttc gctatcaagg aatctccgaa a 41 <210> 61 <211> 18 <212> DNA <213> Artificial sequence <400> cccgtcgcta tcaaggaa 18 <210> 62 <211> 20 <212> DNA <213> Artificial sequence <400> cttgttggct ttcggagatg 20 <210> 63 <211> 40 <212> DNA <213> Artificial sequence <400> tttttttttttttttttttc aaggaaccat ctccgaaagc 40 <210> 64 <211> twenty four <212> DNA <213> Artificial sequence <400> tcgatgaagc ctacgtgatg gcca 24 <210> 65 <211> twenty one <212> DNA <213> Artificial sequence <400> cacacgtggg ggttgtccac g 21 <210> 66 <211> 36 <212> DNA <213> Artificial sequence <400> tttttttttt ttttttttat ggccatcgtg gacaac 36 <210> 67 <211> 28 <212> DNA <213> Artificial sequence <400> cagcatgtca agatcacaga ttttgggc 28 <210> 68 <211> 20 <212> DNA <213> Artificial sequence <400> ccgcacccag cagtttggcc 20 <210> 69 <211> 39 <212> DNA <213> Artificial sequence <400> tttttttttt ttttttttat tttgggcggg ccaaactgc 39 <210> 70 <211> 34 <212> DNA <213> Artificial sequence <400> aaggaaactg aattcaaaaa gatcaaagtg ctgg 34 <210> 71 <211> 19 <212> DNA <213> Artificial sequence <400> cgtgccgaac gcaccggag 19 <210> 72 <211> 35 <212> DNA <213> Artificial sequence <400> tttttttttt ttttttttgt gctggcctcc ggtgc 35 <210> 73 <211> 34 <212> DNA <213> Artificial sequence <400> gaaggaaact gaattcaaaa agatcaaagt gctg 34 <210> 74 <211> 19 <212> DNA <213> Artificial sequence <400> gtgccgaacg caccggagc 19 <210> 75 <211> 36 <212> DNA <213> Artificial sequence <400> ttttttttttttttttttaa gtgctgtgct ccggtg 36 <210> 76 <211> twenty two <212> DNA <213> Artificial sequence <400> acctccaccg tgcagctcat ca 22 <210> 77 <211> twenty one <212> DNA <213> Artificial sequence <400> cagccgaagg gcatgagctg c 21 <210> 78 <211> 38 <212> DNA <213> Artificial sequence <400> ttttttttttttttttttgc tcatcatgca gctcatgc 38 <210> 79 <211> 27 <212> DNA <213> Artificial sequence <400> agatcacaga ttttgggctg gccaaac 27 <210> 80 <211> 25 <212> DNA <213> Artificial sequence <400> gtattctttc tcttccgcacccagc 25 <210> 81 <211> 36 <212> DNA <213> Artificial sequence <400> ttttttttttttttttttgg ccaaacagct gggtgc 36

Claims

1. A PAP-liquid phase chip comprising primers and probe sets, wherein the primers include forward primers and reverse primers, characterized in that, The 5' end of the forward primer is biotin-labeled, and the 3' ends of both the forward and reverse primers are mutation site-specific nucleotides, with the last nucleotide being dideoxynucleotide; the 5' end of the probe is an amino group, followed by an 18-25 bp spacer arm, and then a 15-30 bp specific probe containing the mutation site, and the probe is covalently linked to the magnetic sheet. The magnetic sheet is a carboxylated magnetic sheet, and the probe is covalently linked to the magnetic sheet via 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in MEST buffer and blocked with BSA / skim milk powder. The chip is used to perform a hybridization reaction in a buffer solution containing tetramethylammonium chloride; The gene sequences of the primers and probes are selected from SEQ ID NO:1-3,4-6,7-9,10-12,13-15,16-18,19-21,22-24; wherein SEQ ID NO:1-2 are the upstream and downstream primer sequences, SEQ ID NO:3 is the corresponding probe sequence, SEQ ID NO:4-5 are the upstream and downstream primer sequences, SEQ ID NO:6 is the corresponding probe sequence, and so on.

2. A PAP-liquid phase chip, comprising primers and probe sets, wherein the primers include forward primers and reverse primers, characterized in that, The 5' end of the forward primer is biotin-labeled, and the 3' ends of both the forward and reverse primers are mutation site-specific nucleotides, with the last nucleotide being dideoxynucleotide; the 5' end of the probe is an amino group, followed by an 18-25 bp spacer arm, and then a 15-30 bp specific probe containing the mutation site, and the probe is covalently linked to the magnetic sheet. The magnetic sheet is a carboxylated magnetic sheet, and the probe is covalently linked to the magnetic sheet via 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide in MEST buffer and blocked with BSA / skim milk powder. The chip is used to perform a hybridization reaction in a buffer solution containing tetramethylammonium chloride; The primer gene sequences are selected from SEQ ID NO:25-27,28-30,31-33,34-36,37-39,40-42,43-45,46-48,49-51,52-54,55-57,58-60,61-63,64-66,67-69,70-72,73-75,76-78,79-81; wherein SEQ ID NO:25-26 are the upstream and downstream primer sequences, SEQ ID NO:27 is the corresponding probe sequence, and so on.

3. A high-throughput, high-specificity method for detecting gene mutations, characterized in that: The detection method includes: designing a PAP-liquid phase chip using the primer and probe set described in claim 1 or claim 2, coupling the magnetic sheet with the probe, PAP amplification, hybridization reaction, and signal analysis, specifically including the following steps: Step 1) Design a PAP-liquid chip using primers and probes: Design a PAP-liquid chip according to the gene mutations to be detected. The PAP-liquid chip contains primers and probes. Prepare the corresponding primer and probe gene sequences. The primers include forward primers and reverse primers. The 5' end of the forward primer is biotin-labeled or fluorescently labeled. The 3' ends of both the forward and reverse primers are mutation site-specific nucleotides. The last nucleotide is a dideoxynucleotide, which cannot be extended in conventional PCR reactions. The 5' end of the probe has an amino group, followed by an 18-25 bp spacer arm, and then a 15-30 bp sequence containing the mutation site. Step 2) Coupling of magnetic sheet and probe: Covalently link the amino-modified probe with the carboxylated magnetic sheet; Step 3) PAP amplification: In a reaction system containing a specific DNA polymerase and pyrophosphate, the primers from step 1) are used to first undergo pyrophosphate hydrolysis to unblock the primers, and then the mutant gene is amplified by PCR. The PAP amplification reaction system consisted of: 20–50 mM tris(hydroxymethyl)aminomethane, 0–100 mM potassium chloride at pH 7.4–7.8, 1–10 mM magnesium chloride, 100–400 µM Na⁴PPi, 25–50 µM of each deoxyribonucleotide, 0.04–0.2 µM of each primer, 5–200 ng of template, and 1–3 U of klentaq-s enzyme. The PAP amplification program is as follows: 92-95℃ for 3 min pre-denaturation, then enter the cycle: 92-95℃ for 15 s, 55-64℃ for 30 s, 72℃ for 1 min, 72℃ for 1-2 min, for a total of 15-45 cycles. Step 4) Hybridization reaction: Take the amplification product and add it to the well of the digital magnetic sheet pre-coupled with the probe for hybridization reaction. Read and photograph the fluorescence signal using a fluorescence analyzer. Based on the digital display of the fluorescence signal on the magnetic sheet, the type of gene mutation can be determined. The specific steps of the hybridization reaction are as follows: Step 4.1) Transfer magnetic sheets coupled with different types of probes into a large centrifuge tube, place it on a magnetic rack, and remove the supernatant; Step 4.2) Add tetramethylammonium chloride buffer to resuspend the magnetic sheet, shake to mix, and transfer the magnetic sheet to a 96-well plate; the tetramethylammonium chloride buffer contains 3M tetramethylammonium chloride, 10-100mM tris(hydroxymethyl)aminomethane, 0.05-0.2% v / v sodium dodecylsarcosinate, 1-5mM ethylenediaminetetraacetic acid, and 0.05-0.1% v / v 2-methyl-4-isothiazolin-3-one, with a pH of 8.0; Step 4.3): Hybridization: Add 2-5 µL of biotin-labeled PCR amplification product to a 96-well plate and incubate at 52°C for 15-30 min at 500-700 rpm. Step 4.4): Color development: Add 10 µL of 10-12 µg / mL R-phycoerythrin-labeled streptavidin to a 96-well plate and incubate at 52°C for 5-15 min at 500-700 rpm. Wash twice with PBST. Step 4.5) Analysis: Add 200µL of 2xSSC resuspended magnetic beads to each well; read and photograph the fluorescence signal using a fluorescence analyzer, and determine the gene mutation type based on the digital display of the fluorescence signal on the magnetic sheet; The detection method is neither a diagnostic nor a treatment method.

4. The high-throughput, high-specificity method for detecting gene mutations according to claim 3, characterized in that: In step 2), the specific steps for coupling the probe to the magnetic sheet are as follows: Step 2.1) Take a centrifuge tube containing a carboxylated magnetic sheet, rotate it rapidly, and then place the centrifuge tube into a magnetic holder to remove the supernatant; Step 2.2) Wash the carboxylated magnetic sheet twice, and place the MEST buffer in a centrifuge tube to remove the supernatant; the MEST buffer contains 10-100 mM of 2-(N-morpholino)ethanesulfonic acid, 0.05-0.1% v / v of 2-methyl-4-isothiazolin-3-one at pH 5.0, and 0.01-0.05% v / v of polyoxyethylene dehydrated sorbitan monolaurate; Step 2.3) Take 79 µL of carboxylated magnetic sheet into a centrifuge tube, add 1.0 µL of 100 µM amino probe, and vortex to mix; add 20 µL of 10 mg / mL crosslinking agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, vortex to mix and centrifuge; place the centrifuge tube in a shaker and shake at 200-500 rpm at room temperature for 2 hours, then remove the supernatant; Step 2.4): Washing: Add 200µL of 10-100mM tris(hydroxymethyl)aminomethane buffer (pH 7.4) to a centrifuge tube and incubate the centrifuge tube in a mixer at room temperature for 10-30 minutes. Step 2.5): Blocking: Add 200µL of blocking solution to a centrifuge tube, vortex to mix, and remove the supernatant; add 200µL of blocking solution to a centrifuge tube, vortex at 1200-1800rpm at room temperature for 30-60 minutes, and remove the supernatant; the blocking solution is 10mM Na2HPO4, 1.7mM KH2PO4, 2.7mM KCl, 137mM NaCl, 1% w / v bovine serum albumin, and 1% w / v skim milk powder; Step 2.6): Washing: Take 200µL of PBST buffer into a centrifuge tube, gently shake to remove the supernatant; repeat this step twice; add 200µL of PBST into the centrifuge tube.

5. A PAP-liquid phase chip according to claim 1 or 2, characterized in that: The PAP-liquid phase chip is used for the detection of single or multiple genes.

6. A PAP-liquid phase chip according to claim 1 or 2, characterized in that: The PAP-liquid phase chip is used to detect mutations in single or multiple genes, including mutations in different genes or different mutations in the same gene.

7. A PAP-liquid phase chip according to claim 1 or 2, characterized in that: The PAP-liquid phase chip is used to detect single or multiple high-throughput gene sequences.

Images