Method for replacing high-temperature denaturation to generate single chain in multiplex PCR (Polymerase Chain Reaction) detection reaction, method for improving sensitivity and accuracy of multiplex PCR product liquid phase hybridization detection and matched kit

Abstract

The invention discloses a method for replacing high-temperature denaturation to generate a single chain in multiple PCR (Polymerase Chain Reaction) detection reaction, a method for improving sensitivity and accuracy of liquid-phase hybridization detection of multiple PCR products and a matched kit. A phosphate group is designed and modified at the 5'end of a primer, so that a PCR product of which one end of one chain is phosphorylated is amplified; carrying out digestion treatment on a PCR amplification product by using Lambda exonuclease, and completely removing a DNA chain with phosphorylation at one end to obtain a single chain; the method disclosed by the invention replaces a common method for generating a single chain by high-temperature denaturation of a multiplex PCR detection reaction, eliminates the negative influence of DNA renaturation on hybridization of the probe and the target sequence, improves the hybridization efficiency, and improves the accuracy and sensitivity of a liquid chip technology platform in molecular detection application.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection, and particularly relates to a method for generating single strands by replacing high-temperature denaturation in a multiplex PCR detection reaction, a method for improving the sensitivity and accuracy of liquid-phase hybridization detection of multiplex PCR products, and a supporting kit. Background Art

[0002] Liquid-phase chip technology utilizes liquid-phase hybridization technology, in which the sample to be detected and the probe are both dissolved in a liquid for hybridization reaction. This method uses microspheres or magnetic beads with special labels or codes as reaction carriers, and then covalently cross-links each coded microsphere or magnetic bead with capture molecules such as antigens, antibodies, or nucleic acid probes specific to the target analyte. After adding the sample to be detected, under liquid-phase conditions, the target molecule specifically binds to the capture molecule cross-linked on the surface of the microsphere or magnetic bead, and up to 100 different reactions can be completed simultaneously in one reaction well. Finally, through an automatic analysis by a liquid-phase chip detector, the microsphere or magnetic bead number and its fluorescence intensity are identified, thereby completing real-time, qualitative, and quantitative analysis of the reaction.

[0003] Multiplex polymerase chain reaction (MPCR) refers to a technology that simultaneously amplifies multiple targets through a single PCR reaction and combines certain detection means to detect the amplification products, thereby achieving the diagnosis of multiple targets. Since Chamberlain first proposed this concept in 1988, due to the wide range covered by MPCR, which can simultaneously detect multiple targets, greatly improving the detection efficiency while reducing the detection cost, it has the characteristics of high efficiency, high throughput, and low cost, and has been deeply studied and widely applied in various fields such as gene mutation and deletion, gene typing and quantification, genetic detection, and companion diagnosis.

[0004] In the detection of multiplex PCR products, the prerequisite for the specific binding of the target molecule to the capture probe cross-linked on the surface of the microsphere or magnetic bead under liquid-phase conditions is to denature the amplified double-stranded DNA into single-stranded DNA. Currently, the main denaturation method is thermal denaturation, that is, by high temperature, the hydrogen bonds maintaining the double helix stability are broken, and the base stacking force is damaged to form the required single-stranded DNA. However, thermally denatured DNA usually renatures after cooling, which affects the hybridization efficiency between the probe and the target sequence and reduces the detection sensitivity and accuracy.

[0005] Lambda exonuclease is a highly processive, phosphophilic alkaline exonuclease that can continuously cleave nucleic acids into individual bases, which is incomparable to many nucleases such as exonuclease I, exonuclease III, S1 nuclease, DNase I, etc. Moreover, the cleavage rate reaches 1000 nt / s. In addition, it has very strong phosphophilia, and the cleavage efficiency of phosphorylated nucleic acid strands and non-phosphorylated nucleic acid strands differs by more than 200 times. Lambda exonuclease highly orderly digests double-stranded DNA (dsDNA), digesting more than 3000 nucleotides each time, but the efficiency of cleaving single-stranded DNA (ssDNA) is very low. There are also studies showing that it cannot initiate cleavage from nick or gap sites. These unique properties are outstanding advantages that can be utilized when establishing a Lambda exonuclease-based bioanalytical sensing method. Therefore, Lambda exonuclease plays a crucial role in nucleic acid recombination, replication, typing, gene repair, and molecular cloning, and has become one of the important tools for studying gene composition, function, and expression. Summary of the Invention

[0006] In order to solve the above technical problems, the primary object of the present invention is to provide a method for generating single strands in a multiplex PCR detection reaction instead of high-temperature denaturation, eliminating the negative impact of DNA renaturation on the hybridization of probes with target sequences, improving the hybridization efficiency, and enhancing the accuracy and sensitivity of the liquid chip technology platform in molecular detection applications.

[0007] A method for generating single strands in a multiplex PCR detection reaction instead of high-temperature denaturation, comprising the following steps:

[0008] 1) One of the primers at the 5'-end of each set of upstream and downstream primers in the multiplex PCR is modified with a phosphate group, thereby amplifying a PCR product with one end phosphorylated of one strand;

[0009] Or

[0010] Add a universal sequence to the 5'-end of the specific primers in the multiplex PCR, and then design a pair of universal primers with the 3'-end partial sequences complementary to the universal sequence. One of the universal primers is modified with a phosphate group at the 5'-end, and use the specific primers and the universal primers together to amplify a PCR product with one end phosphorylated of one strand;

[0011] 2) Digest the obtained PCR amplification product with Lambda exonuclease to completely remove the DNA strand with one end phosphorylated, obtaining single strands.

[0012] Furthermore, the enzyme digestion system in step 2) is 25 - 50 μL; the dosage of the Lambda exonuclease for digestion treatment is 0.5 - 2 μL, and the enzyme digestion time is 25 - 30 min.

[0013] More preferably, the digestion system in step 2) is 25 μL; the dosage of the Lambda exonuclease for digestion treatment is 1 μL, and the digestion time is 30 min.

[0014] The second object of the present invention is to provide a method for improving the sensitivity and accuracy of liquid-phase hybridization detection of multiplex PCR products. The single strand obtained by the aforementioned method is used during the liquid-phase hybridization detection.

[0015] Furthermore, it includes the following steps:

[0016] (1) The single strand obtained by the aforementioned method;

[0017] (2) Design corresponding specific probes according to the multiplex PCR target sequence and the primer amplification region. The sequence of the specific probe is within the amplification range of the upstream and downstream primers, and the 5' end is modified with NH 2 modification;

[0018] (3) Couple the specific probe to the magnetic beads with unique encoding;

[0019] (4) Mix the single strand with the magnetic beads coupled with the corresponding encoded specific probe for hybridization reaction;

[0020] (5) Analyze the detection results.

[0021] Furthermore, it also includes the following steps:

[0022] After the hybridization reaction, add a staining reagent for staining reaction and then wash; then use a fluorescence quantitative analyzer to scan and identify the magnetic bead encoding and fluorescence; finally, analyze the detection results according to the fluorescence intensity of different magnetic beads.

[0023] Furthermore,

[0024] The 5' end of the specific probe described in step (2) has the sequence TTTTTTTTTATTTTTTTTT.

[0025] The length of the specific probe is 25 - 35 bp and is complementary to the single strand sequence in step 1).

[0026] The Tm value is 55 - 65 °C.

[0027] The specific probe is modified with locked nucleic acid.

[0028] One specific probe corresponds to one magnetic bead encoding, and different specific probes correspond to different magnetic bead encodings.

[0029] The amount of the single-strand (the single-strand is the multiplex PCR product participating in the hybridization reaction) described in step (4) is 5 - 10 μL; the amount of magnetic beads is 120 - 300 beads, the volume range of the whole hybridization reaction system is 45 - 50 μL, the hybridization reaction temperature is determined according to the Tm value of the probe to be 40 - 50 °C, and further, the hybridization temperature is 45 °C; the hybridization reaction time is 20 - 30 min.

[0030] The staining reagent is SA-PE, with a concentration of 2 - 5 μg / mL, and the staining reaction time is 15 - 20 min; the washing solution is 1xPBS-T, and the washing time for each time is 30 - 60 s; wash 2 - 3 times.

[0031] The method for generating single-strands by replacing high-temperature denaturation in the multiplex PCR detection reaction of the present invention, and the method for improving the sensitivity and accuracy of the multiplex PCR product liquid-phase hybridization detection are both applied in non-diagnostic or therapeutic purposes.

[0032] The method or kit of the present invention can be applied in SNP genotyping detection or gene point mutation detection; mainly to improve the detection accuracy and sensitivity.

[0033] The third aspect of the present invention aims to provide a multiplex PCR product liquid-phase hybridization detection kit, including reagents used in conjunction with the method for improving the sensitivity and accuracy of the multiplex PCR product liquid-phase hybridization detection described above.

[0034] In the present invention, one of the primers of the multiplex PCR is modified at the 5'-end with a phosphate group (P), so as to amplify a PCR product with one end phosphorylated of one strand, and then the PCR amplification product is digested with Lambda exonuclease to obtain single-stranded DNA. This method is innovatively applied to the multiplex PCR reaction for the first time, which can eliminate the problem of cooling and renaturation after traditional high-temperature thermal denaturation, and the product can be directly used for subsequent liquid-phase hybridization detection. In short, the method of the present invention for generating single-strands by replacing high-temperature denaturation in the ordinary multiplex PCR reaction eliminates the negative impact of DNA renaturation on the hybridization of the probe with the target sequence, improves the hybridization efficiency, and also improves the accuracy and sensitivity of the liquid chip technology platform in molecular detection applications. Description of the Drawings

[0035] Figure 1 : Line graph of the average fluorescence difference value of traditional multiplex PCR and multiplex PCR liquid-phase hybridization detection in Example 4. Detailed Embodiments

[0036] The following embodiments are intended to further illustrate the present invention, rather than limiting the present invention.

[0037] Example 1: Magnetic Bead Coupled Probe

[0038] 1) Add 100 μL of 1xPBS-T to a 2 mL microcentrifuge tube. Add 20,000 required encoded magnetic beads to the 2 mL microcentrifuge tube, place it on a magnetic stand for 2 minutes, and remove the supernatant.

[0039] 2) Add 300 μL of Methanol solution, shake for about 15 seconds and then centrifuge quickly, place it on the magnetic stand for 2 minutes again, and remove the supernatant. Repeat 2 times.

[0040] 3) Add 300 μL of MES-T buffer, shake for about 15 seconds and then centrifuge quickly, place it on the magnetic stand for 2 minutes, and remove the supernatant.

[0041] 4) Add 300 μL of MES buffer, shake for about 15 seconds and then centrifuge quickly, place it on the magnetic stand for 2 minutes, and remove the supernatant.

[0042] 5) Add 180 μL of MES buffer, add 20 μL of the specific probe to be coupled (concentration is 100 μM), and shake for more than 15 seconds immediately after adding. After quick centrifugation, use a microcentrifuge tube shaker at room temperature and shake at 1600 rpm for 10 minutes.

[0043] 6) Add 50 μL of freshly prepared 20 mg / mL EDC coupling solution to each magnetic bead tube, and shake for more than 15 seconds immediately. After quick centrifugation, shake at 1600 rpm overnight at room temperature, about 15 hours.

[0044] 7) After the coupling reaction is completed, centrifuge quickly, place the 2 mL microcentrifuge tube on the magnetic stand for 2 minutes, and remove the supernatant.

[0045] 8) Add 300 μL of Tris-HCl solution (50 mM, pH 7.4) and use a microcentrifuge tube shaker to shake at 1600 rpm for 15 minutes at room temperature to terminate the coupling reaction.

[0046] 9) Centrifuge the 2 mL microcentrifuge tube after the termination reaction procedure quickly, place it on the magnetic stand for 2 minutes, remove the supernatant, then add 300 μL of Blocking buffer (1% BSA in 1xPBS buffer) solution, shake for about 15 seconds and then centrifuge quickly, place it on the magnetic stand for 2 minutes again, and remove the supernatant.

[0047] 10) Add 300 μL of Blocking buffer solution, place it on a microcentrifuge tube shaker, and shake at 1600 rpm for 60 minutes at room temperature.

[0048] 11) Centrifuge the 2 mL microcentrifuge tube after the above blocking procedure quickly, place it on the magnetic stand for 2 minutes, and remove the supernatant.

[0049] 12) Add 300 μL of PBS-T solution (0.05% Tween-20 in 1x PBS), shake for about 15 seconds, then centrifuge quickly, place on a magnetic stand for 2 minutes, and remove the supernatant. Repeat 2 times.

[0050] 13) Reconstitute with 100 μL of PBS-T solution, homogenize, and then take a sample for counting.

[0051] 14) The magnetic bead solution that has completed probe coupling is stored at 2 - 8 °C and can be directly used for sample detection.

[0052] Example 2. Testing of the reaction conditions of Lambda exonuclease

[0053] Although the manufacturer of Lambda exonuclease (New England Biolabs) has provided the reaction conditions (50 μL reaction system, 1 μL of enzyme, reaction time 30 min), Lambda exonuclease can also slowly degrade single-stranded DNA and non-phosphorylated substrates. In order to explore the optimal reaction conditions in the method of the present invention to achieve the best digestion effect, according to the reaction conditions provided by the manufacturer and the actual situation of the method of the present invention, an orthogonal experiment was designed using SPSS software to obtain the optimal reaction conditions. The test sample was a 10 ng 2% EGFR (E746_A750del) mutant sample. The orthogonal experiment designed by SPSS software was 9 groups, and the manufacturer's reaction conditions (50 μL system, 37 °C, 30 min) were used as a control, for a total of 10 groups of experiments.

[0054] Table 1 Design and synthesis of primers and hybridization probes modified with a phosphate group (P) at the 5' end:

[0055]

[0056] The primer purity is HPLC grade.

[0057] 1. PCR amplification reaction:

[0058] Reaction system: 10 μL of 2x PCR mix, 1 μL each of 10 μM upstream and downstream primers, 1 μL of 10 ng / μL DNA, 7 μL of Nuclease-Free Water;

[0059] 2. Amplification reaction program: 95 °C for 10 min, 1 cycle; 95 °C for denaturation for 30 sec, 60 °C for annealing for 30 sec, 72 °C for extension for 30 sec, 35 cycles; 72 °C for extension for 7 min.

[0060] 3. The orthogonal experiment scheme and test data are as follows:

[0061] Table 2

[0062]

[0063] The mean analysis of the 10 groups of detection results was carried out using SPSS software to obtain the optimal reaction conditions. The analysis results are as follows:

[0064] Table 3 Enzyme amount

[0065]

[0066] Table 4 Reaction time

[0067]

[0068] Table 5 Reaction volume

[0069]

[0070] It can be seen from the analysis results that the reaction conditions with the largest detection mean are an enzyme amount of 1 μL, a reaction time of 30 min, and a reaction volume of 25 μL. Therefore, these conditions were set as the restriction enzyme digestion reaction conditions.

[0071] Example 3, SNP genotyping detection

[0072] The rs2279744 locus is the 309th (T>G) mutation in the promoter region of the MDM2 gene, and its global minor allele frequency G = 36.66%. Some studies have shown that the rs2279744 polymorphism of the MDM2 gene is involved in the pathogenesis of hepatocellular carcinoma and affects the prognosis of hepatocellular carcinoma, and it is an important genetic susceptibility factor for hepatocellular carcinoma.

[0073] rs1801394 is an A / G polymorphism at the second exon of the MTRR gene located between 5p15.3 - p15.2, and it can form three genotypes: A / A, A / G, and G / G. The rs1801394 locus is the main mutation on MTRR, which causes methylvitamin deficiency and is related to diseases such as spina bifida, Down syndrome, neural tube defects, and leukemia.

[0074] In this example, two SNP loci, rs2279744 and rs1801394, were used as target loci to investigate the beneficial effects that can be achieved by the present invention in SNP duplex detection.

[0075] Sample source: Human whole blood DNA with known SNP genotypes. The specific genotyping conditions are as follows in the table:

[0076] Table 6

[0077] RS number rs2279744 rs1801394 Sample 1 TG AA Sample 2 TG AG Sample 3 TG GG Sample 4 TT AA Sample 5 TG AG Sample 6 GG AG

[0078] 1. Design and synthesis of primers and hybridization probes modified with a phosphate group (P) at the 5' end:

[0079] Table 7

[0080]

[0081]

[0082] The primer purity is HPLC grade. / iLNA_N / indicates locked nucleic acid modification.

[0083] 2. PCR amplification reaction:

[0084] Reaction system: 2xPCR mix 10 μL, 0.6 μL of 10 μM upstream and downstream primers of rs2279744, 1 μL of 10 μM upstream and downstream primers of rs1801394, 1 μL of 10 ng / μL DNA, 7.4 μL of Nuclease-Free Water;

[0085] Amplification reaction procedure: 95°C for 10 min, 1 cycle; 95°C denaturation for 30 sec, 60°C annealing for 30 sec, 72°C extension for 30 sec, 35 cycles; 72°C extension for 7 min.

[0086] 3. After the PCR reaction, the PCR product is digested with Lambda Exonuclease.

[0087] Digestion reaction system:

[0088] Table 8

[0089] Reagent Volume μL Previous-step PCR product 20 Lambda Exonuclease buffer 2.5 Lambda Exonuclease 1 Nuclease-Free Water 1.5 Total 25

[0090] Digestion reaction procedure;

[0091] Table 9

[0092] Temperature Time Number of cycles 37℃ 30 min 1 4℃ Hold 1

[0093] 4. Sample hybridization reaction

[0094] 1) Mix two kinds of magnetic beads corresponding to rs2279744T / G and rs1801394A / G respectively and blank magnetic beads (without coupling any probes), and supplement the hybridization buffer to 45 μL per reaction tube. Place it on a super thermostatic mixer and react at a rotation speed of 1600 rpm at the hybridization temperature for 5 minutes;

[0095] 2) Then add 5 μL of the nucleic acid sample to be detected after digestion into the reaction tube one by one, and perform a hybridization reaction at a rotation speed of 1600 rpm at the hybridization temperature for 20 minutes on a super thermostatic mixer.

[0096] 3) After the hybridization reaction is completed, place the reaction tube on the magnetic rack. After standing for 2 minutes, slowly remove the supernatant, add 50 μL of 5 μg / ml SA-PE solution to the reaction tube, and place it on the super thermostatic mixer to stain at room temperature at 1600 rpm for 15 minutes.

[0097] 4) After the staining reaction, place the reaction tube on the magnetic rack for 2 minutes to fix the magnetic beads. After aspirating the supernatant, add 150 uL of 1xPBS-T, and react on the super thermostatic mixer at room temperature at 1600 rpm for 30 seconds for washing (repeat twice).

[0098] 5) Add 200 μL of detection buffer to each reaction tube, pipette at least 10 times repeatedly to make the magnetic beads evenly distributed, and use a fluorescence quantitative analyzer to perform magnetic bead identification and fluorescence detection.

[0099] 6) Data processing: Subtract the fluorescence value of the magnetic beads corresponding to NTC from the fluorescence value of each magnetic bead in the test sample;

[0100] 7) Perform difference / ratio operations on the fluorescence values of the magnetic beads corresponding to the two probes according to the result interpretation standard, and interpret the results. The final result is consistent with the first-generation sequencing genotyping result.

[0101] The method of the present invention:

[0102] Table 10

[0103]

[0104]

[0105] Traditional comparison method: The PCR product is not digested with enzymes and is denatured at 95 °C.

[0106] Table 11

[0107]

[0108] Statistically analyze the fluorescence differences between the A probe and the G probe at the rs1801394 locus and the fluorescence differences between the T probe and the G probe of the rs2279744 probe for 6 samples respectively. The results are as follows in the table:

[0109] The method of the present invention:

[0110] Table 12

[0111] Locus Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Mean rs1801394 - A 4084 2045 441 4094 2212 1982 2476.33 rs1801394 - G 230 3724 6789 306 3633 4013 3115.83 rs2279744 - T 3573 3855 3537 6057 3533 313 3478.00 rs2279744 - G 6044 5832 6245 591 5820 10839 5895.17

[0112] Traditional comparison method:

[0113] Table 13

[0114] Locus Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 Sample 6 Mean rs1801394 - A 1258 960 106 1636 1079 1289 1054.67 rs1801394 - G 222 1216 3016 115 2137 2403 1518.17 rs2279744 - T 1738 1342 1152 2654 1152 275 1385.50 rs2279744 - G 2546 2123 3505 304 2116 4453 2507.83

[0115] The experimental results show that both the method of the present invention and the traditional high-temperature denaturation method can simultaneously detect the results of two SNP sites in the same sample in one tube. However, compared with hybridization after high-temperature denaturation, the fluorescence difference value after hybridization with enzyme digestion is 2-3 times that of the traditional high-temperature denaturation method. The fluorescence value obtained after hybridization with enzyme digestion is higher, and the fluorescence difference ratio corresponding to the homozygote at the same locus for the two probes is greater, all above 10 times, with stronger discrimination. The method of the present invention can effectively improve the accuracy and precision of genotyping detection.

[0116] Example 4, Detection of Hotspot Mutations in the EGFR Gene

[0117] Epidermal growth factor receptor (EGFR) is one of the main targets for targeted therapy of colorectal cancer and lung cancer. The main forms of EGFR gene mutations found in tumor cells currently are: (1) Point mutations: mainly concentrated in the EGFR-TK region, resulting in amino acid variations after translation or premature termination of translation; (2) Gene amplification: referring to an increase in the overall copy number of the gene; (3) Gene fragment insertion or deletion, mainly seen in three situations. One is a small fragment insertion in exon 20; the second is a short sequence deletion-inside-frame deletion in exon 19; the third is the emergence of EGFR mutant variant III, which is due to gene rearrangement or alternative mRNA splicing resulting in the deletion of exons 2-7 in the extracellular ligand-binding region of the cell membrane. The incomplete proteins generated by mutations based on the above mutation forms can activate TK by themselves and trigger the activation of downstream signaling pathways. This example detects the EGFR gene hotspot mutation sites (L858R in exon 21, L861Q in exon 21, E746_A750del in exon 19, c.2310_2311insGGT in exon 20, G719A in exon 18) to investigate the effectiveness of the method of the present invention.

[0118] Sample source:

[0119] Negative control A: Human leukocyte DNA

[0120] Positive sample B: A mixture of human leukocyte DNA and EGFR E746_A750del, EGFR L861Q, EGFR InS2-M4, EGFRL858R, EGFR G719A mutant plasmids, with a mutation frequency of 10 ng 10%

[0121] 1. Design and synthesis of primers and hybridization probes with a phosphate group (P) modification at the 5' end:

[0122] Table 14

[0123]

[0124]

[0125] The primer purity is HPLC grade.

[0126] 2. PCR amplification reaction:

[0127] Reaction system: 2xPCR mix 10 μL, 10 μM upstream and downstream primers for EGFR E746_A750del

[0128] 0.1 μL, 1.2 μL of 10 μM upstream and downstream primers for EGFR L861Q, 0.4 μL of 10 μM upstream and downstream primers for EGFR InS2-M4, 0.3 μL of 10 μM upstream and downstream primers for EGFR L858R, 0.6 μL of 10 μM upstream and downstream primers for EGFR G719A, 0.2 μL of 10 μM upstream and downstream primers for EGFR internal reference, 1 μL of sample, 5.2 μL of Nuclease-Free Water;

[0129] Amplification reaction procedure: 95°C for 5 min, 1 cycle; 95°C denaturation for 15 sec, 60°C annealing for 40 sec,

[0130] 50 cycles.

[0131] 3. After the reaction, the product is digested with Lambda Exonuclease.

[0132] Digestion reaction system:

[0133] Table 15

[0134] Reagent Volume μL Previous-step PCR product 20 Lambda Exonuclease buffer 2.5 Lambda Exonuclease 1 Nuclease-Free Water 1.5 Total 25

[0135] Digestion reaction procedure;

[0136] Table 16

[0137] Temperature Time Number of cycles 37℃ 30 min 1 4℃ Hold 1

[0138] 4. Sample hybridization reaction

[0139] 1) Mix the magnetic beads corresponding to EGFR E746_A750del, EGFR L861Q, EGFR InS2-M4, EGFR

[0140] L858R, EGFR G719A and EGFR internal reference with blank magnetic beads (not coupled with any probes), make up the hybridization buffer to 45 μL per reaction tube, place it on a super thermostatic mixer, and react at a rotation speed of 1600 rpm at the hybridization temperature for 5 minutes;

[0141] 2) Then, add 5 μL of the nucleic acid sample to be detected after digestion into the reaction tube one by one, and perform a hybridization reaction at the hybridization temperature with a rotation speed of 1600 rpm on a super constant temperature mixer for 20 minutes.

[0142] 3) After the hybridization reaction is completed, place the reaction tube on the magnetic rack. After standing for 2 minutes, slowly remove the supernatant, add 50 μL of 5 μg / mL SA-PE solution into the reaction tube, and place it on the super constant temperature mixer.

[0143] React at room temperature with a rotation speed of 1600 rpm for 15 minutes for the staining reaction.

[0144] 4) After the staining reaction, place the reaction tube on the magnetic rack for 2 minutes. After aspirating the supernatant, add

[0145] 150 μL of 1x PBS-T, and react on the super constant temperature mixer at room temperature with a rotation speed of 1600 rpm for 30 seconds for washing (repeat twice).

[0146] 5) Add 200 μL of detection buffer to each reaction tube, pipette at least 10 times repeatedly to evenly distribute the magnetic beads, and use a fluorescence quantitative analyzer to detect the results.

[0147] 6) Data processing: Subtract the fluorescence value of the magnetic beads corresponding to NTC from the fluorescence value of each magnetic bead of the detection sample, and then subtract the fluorescence value of the magnetic beads corresponding to the negative control from the fluorescence value of the magnetic beads corresponding to the target to obtain the fluorescence difference corresponding to each target;

[0148] The method of the present invention:

[0149] Table 17

[0150]

[0151] Traditional comparison method: The PCR product is not digested, denatured at 95 °C, and other processes remain unchanged.

[0152] Table 18

[0153]

[0154] The line graph of the average value of the fluorescence difference detected by the two methods is shown in Figure 1 .

[0155] The experimental results show that compared with the traditional hybridization after high-temperature denaturation, the hybridization after digestion in the present invention has a fluorescence difference that is increased by 2 - 3 times. It has a stronger discrimination from negative samples, is more conducive to the judgment of positive results, and improves the detection accuracy. And the average value of CV% is 1.14%, which is much lower than 7.05% of the traditional method, indicating that the method of the present invention can significantly improve the detection precision.

[0156] Example 5. Detection limit study of gene point mutation detection

[0157] Test samples:

[0158] Negative control A: Human leukocyte DNA

[0159] Positive sample B: Mixture of human leukocyte DNA and plasmids with EGFR E746_A750del, EGFR L861Q, EGFR InS2-M4, EGFRL858R, EGFR G719A mutations, mutation frequency 10 ng 0.1%.

[0160] Positive sample C: Mixture of human leukocyte DNA and plasmids with EGFR E746_A750del, EGFR L861Q, EGFR InS2-M4, EGFRL858R, EGFR G719A mutations, mutation frequency 10 ng 0.5%.

[0161] Positive sample D: Mixture of human leukocyte DNA and plasmids with EGFR E746_A750del, EGFR L861Q, EGFR InS2-M4, EGFRL858R, EGFR G719A mutations, mutation frequency 10 ng 1%.

[0162] Positive sample E: Mixture of human leukocyte DNA and plasmids with EGFR E746_A750del, EGFR L861Q, EGFR InS2-M4, EGFRL858R, EGFR G719A mutations, mutation frequency 10 ng 10%.

[0163] Positive sample F: Mixture of human leukocyte DNA and plasmids with EGFR E746_A750del, EGFR L861Q, EGFR InS2-M4, EGFRL858R, EGFR G719A mutations, mutation frequency 10 ng 50%. 1. Primer design is the same as in Example 4.

[0164] 2. PCR amplification reaction:

[0165] Reaction system: 2xPCR mix 10 μL, 10 μM upstream and downstream primers for EGFR E746_A750del

[0166] 0.1 μL, 10 μM upstream and downstream primers for EGFR L861Q 1.2 μL, 10 μM upstream and downstream primers for EGFR InS2-M4 0.4 μL, 10 μM upstream and downstream primers for EGFR L858R 0.3 μL, 10 μM upstream and downstream primers for EGFR G719A 0.6 μL, 10 μM upstream and downstream primers for EGFR internal reference 0.2 μL, sample 1 μL, Nuclease-

[0167] Free Water 5.2 μL;

[0168] Amplification reaction procedure: 95 °C for 5 min, 1 cycle; 95 °C for denaturation for 15 sec, 60 °C for annealing for 40 sec,

[0169] 50 cycles.

[0170] 3. After the reaction, the product is digested with Lambda Exonuclease.

[0171] Digestion reaction system:

[0172] Table 19

[0173] Reagent Volume μL Previous-step PCR product 20 Lambda Exonuclease buffer 2.5 Lambda Exonuclease 1 Nuclease-Free Water 1.5 Total 25

[0174] Digestion reaction procedure;

[0175] Table 20

[0176] Temperature Time Number of cycles 37℃ 30 min 1 4℃ Hold 1

[0177] 4. Sample hybridization reaction

[0178] 1) Mix the magnetic beads corresponding to EGFR E746_A750del, EGFR L861Q, EGFR InS2-M4, EGFR

[0179] L858R, EGFR G719A and the internal reference of EGFR with blank magnetic beads (not coupled with any probes), supplement the hybridization buffer to 45 μL per reaction tube, and place it on a super thermostatic mixer at a rotation speed of

[0180] 1600 rpm at the hybridization temperature for 5 minutes;

[0181] 2) Then add 5 μL of the nucleic acid sample to be detected after digestion to each reaction tube one by one, and perform a hybridization reaction at 1600 rpm at the hybridization temperature on a super thermostatic mixer for 20 minutes.

[0182] 3) After the hybridization reaction is completed, place the reaction tube on a magnetic rack, let it stand for 2 minutes, then slowly remove the supernatant, add 50 μL of 5 μg / ml SA-PE solution to the reaction tube, and place it on a super thermostatic mixer to react at room temperature at 1600 rpm for 15 minutes for the staining reaction.

[0183] 4) After the staining reaction, place the reaction tube on the magnetic rack for 2 minutes, aspirate the supernatant, and then add

[0184] 150 μL of 1xPBS-T, and react at room temperature at 1600 rpm on a super thermostatic mixer for 30 seconds for washing (repeat twice).

[0185] 5) Add 200 μL of detection buffer to each reaction tube, pipette at least 10 times repeatedly to evenly distribute the magnetic beads, and use a fluorescence quantitative analyzer to detect the results.

[0186] 6) Data processing: Subtract the fluorescence value of the magnetic beads corresponding to the NTC from the fluorescence value of each magnetic bead in the test sample to obtain the fluorescence difference value; perform difference calculation and result interpretation on the fluorescence values of the magnetic beads corresponding to different target probes according to the result interpretation criteria. According to the resolution of the detection platform, a fluorescence difference value greater than 5000 compared with the detection result of the negative sample is judged as positive. The detected fluorescence difference data is shown in the following table:

[0187] The method of the present invention:

[0188] Table 21

[0189]

[0190]

[0191] Traditional comparison method: The PCR product is not digested with enzymes and is denatured at 95 °C.

[0192] Table 22

[0193]

[0194]

[0195] The analysis results of the detection results are as follows:

[0196] The method of the present invention:

[0197] Table 23

[0198]

[0199] Traditional comparison method:

[0200] Table 24

[0201]

[0202]

[0203] Sample negative / positive interpretation:

[0204] According to the resolution of the detection platform, a fluorescence difference value greater than 5000 between the target and the detection result of the negative sample is judged as positive. The statistics of the negative / positive interpretation results are as follows:

[0205] Table 25

[0206]

[0207]

[0208] The experimental results show that compared with the traditional method, the lowest detection limit of the method of the present invention is

[0209] 10 ng with 0.5% mutation, while the traditional method is 10 ng with 2.0% mutation. The method of the present invention has higher sensitivity, and

[0210] the CV values detected by the method of the present invention are all within 5.00%, while the maximum CV value of the traditional method is 12.56%. The method of the present invention has higher precision in detecting low-frequency samples.

[0211] Example 6 Application of Universal Primer Sequences

[0212] Universal primers can simplify the design, improve the efficiency and reduce the cost in multiplex PCR, and are widely used in fields such as pathogen detection, genotyping and expression analysis. Introducing universal primers in the present invention can effectively reduce the number of primers that need to be modified, reduce the cost, and at the same time balance the amplification efficiency of primers at different sites, improving the experimental consistency. In this example, eight common functional genes in the genome, namely PIK3CA, NRG1, VHL, EGFR, ERBB2, PDGFRA, BRAF and CEACAM5, are taken as examples to test the feasibility and effectiveness of the application of universal primers in multiplex PCR detection.

[0213] Test samples:

[0214] Positive control: Human leukocyte DNA

[0215] Negative control: Nuclease-Free Water

[0216] 1. Primer design:

[0217] Table 26

[0218]

[0219]

[0220]

[0221] 2. PCR amplification reaction:

[0222] Reaction system: 10 μL of 2xPCR mix, 2 μL of 10 μM universal upstream and downstream primers, 0.2 μL of 100 nM EGFR upstream and downstream primers, 0.2 μL of 100 nM PIK3CA upstream and downstream primers, 0.4 μL of 100 nM NRG1 upstream and downstream primers, 0.2 μL of 100 nM VHL upstream and downstream primers, 0.2 μL of 100 nM BRAF upstream and downstream primers, 0.2 μL of 100 nM PDGFRA upstream and downstream primers, 0.1 μL of 100 nM CEACAM5 upstream and downstream primers, 0.5 μL of 100 nM ERBB2 upstream and downstream primers, 1 μL of corresponding negative / positive sample, 5 μL of Nuclease-Free Water;

[0223] Amplification reaction procedure: 5 min at 95°C, 1 cycle; denaturation at 95°C for 15 sec, annealing at 60°C for 30 min, extension at 72°C for 1 min, 3 cycles; denaturation at 95°C for 15 sec, annealing and extension at 72°C for 1 min, 38 cycles.

[0224] 3. After the reaction, the product is digested with Lambda Exonuclease.

[0225] Digestion reaction system:

[0226] Table 27

[0227] Reagent Volume μL Previous-step PCR product 20 Lambda Exonuclease buffer 2.5 Lambda Exonuclease 1 Nuclease-Free Water 1.5 Total 25

[0228] Digestion reaction procedure;

[0229] Table 28

[0230] Temperature Time Number of cycles 37℃ 30 min 1 4℃ Hold 1

[0231] 4. Sample hybridization reaction

[0232] 1) Mix the magnetic beads corresponding to EGFR, PIK3CA, NRG1, VHL, ERBB2, PDGFRA, BRAF and CEACAM5 with blank magnetic beads (not coupled with any probes), and make up the hybridization buffer to 45 μL per reaction tube. Place it on a super-constant temperature mixer and react at a rotation speed of 1600 rpm at the hybridization temperature for 5 minutes;

[0233] 2) Then add 5 μL of the nucleic acid sample to be detected after digestion to each reaction tube one by one, and perform a hybridization reaction at a rotation speed of 1600 rpm at the hybridization temperature for 20 minutes on a super-constant temperature mixer.

[0234] 3) After the hybridization reaction is completed, place the reaction tube on the magnetic rack. After standing for 2 minutes, slowly remove the supernatant, add 50 μL of 5 μg / ml SA-PE solution to the reaction tube, and place it on a super thermostatic mixer. React at room temperature at 1600 rpm for 15 minutes for the staining reaction.

[0235] 4) After the staining reaction, place the reaction tube on the magnetic rack for 2 minutes. After aspirating the supernatant, add 150 μL of 1xPBS-T, and react on a super thermostatic mixer at room temperature at 1600 rpm for 30 seconds for washing (repeat twice).

[0236] 5) Add 200 μL of detection buffer to each reaction tube, pipette at least 10 times repeatedly to evenly distribute the magnetic beads, and use a fluorescence quantitative analyzer to detect the results.

[0237] 6) Data processing: Subtract the fluorescence value of the magnetic beads corresponding to NTC from the fluorescence value of each magnetic bead in the test sample to obtain the fluorescence difference; perform difference calculation and result interpretation on the fluorescence values of the magnetic beads corresponding to different target probes according to the result interpretation criteria. According to the resolution of the detection platform, a fluorescence difference > 1000 from the detection result of the negative sample is judged as positive. The detected fluorescence difference data is shown in the following table:

[0238] Table 29

[0239]

[0240]

[0241] Table 30

[0242]

[0243] The results show that the 8-plex PCR amplified with the universal sequence primer can detect the presence of genomic DNA as low as 0.2 ng after digestion and hybridization.

Claims

1. A method for replacing high temperature denaturation to generate single strands in a multiplex PCR detection reaction, characterized in that: The following steps are involved: 1) The 5' end of one of the primers in each set of upstream and downstream primers of the multiplex PCR is modified with a phosphate group, thereby amplifying a PCR product with one end of one of the chains phosphorylated; or Add a universal sequence to the 5' end of the multiplex PCR specific primer, then design a pair of universal primers whose 3' end partial sequence is complementary to the universal sequence, one of the universal primers has a phosphate group modified at the 5' end, and use the specific primer and the universal primer together to amplify a PCR product with one end of one chain phosphorylated; 2) The obtained PCR amplification product is digested with Lambda exonuclease to completely remove the DNA chain with phosphorylation at one end to obtain a single strand.

2. The method according to claim 1, characterized in that The enzyme digestion system of step 2) is 25-50 μL; the amount of the Lambda exonuclease digestion enzyme is 0.5-2 μL, and the enzyme digestion time is 25-30 min.

3. A method for improving the sensitivity and accuracy of liquid phase hybridization detection of multiple PCR products, characterized in that: The single strand obtained by the method described in claim 1 or 2 is used in liquid phase hybridization detection.

4. The method according to claim 3, characterized in that The following steps are involved: (1) A single strand obtained by the method of claim 1 or 2; (2) designing corresponding specific probes according to the multiplex PCR target sequence and the primer amplification region, wherein the specific probe sequence is within the amplification range of the upstream and downstream primers and has an NH2 modification at the 5' end; (3) coupling specific probes to magnetic beads with unique codes; (4) The single strand is mixed with magnetic beads coupled to specific probes with corresponding codes to perform hybridization reaction; (5) Analyze the test results.

5. The method according to claim 4, characterized in that The following steps are also included: After the hybridization reaction, a staining reagent is added to carry out staining reaction and then washing; then a fluorescence quantitative analyzer is used to scan and identify the magnetic bead coding and fluorescence; finally, the detection results are analyzed according to the fluorescence intensity of different magnetic beads.

6. The method according to claim 4, characterized in that The 5' end of the specific probe in step (2) has a TTTTTTTTTATTTTTTTTT sequence. Furthermore, the specific probe is 25-35 bp in length, complementary to the single-stranded sequence in step (1), and has a Tm value of 55-65°C. Furthermore, the specific probe is modified with a locked nucleic acid; further, one specific probe corresponds to one magnetic bead code, and different specific probes correspond to different magnetic bead codes.

7. The method according to claim 4, characterized in that In step (4), the amount of the single strand is 5-10 μL, the amount of the magnetic beads is 120-300, the volume range of the entire hybridization reaction system is 45-50 μL, the hybridization reaction temperature is determined at 40-50°C according to the Tm value of the probe, and further, the hybridization temperature is 45°C; the hybridization reaction time is 20-30 min.

8. The method according to claim 5, characterized in that The staining reagent is SA-PE with a concentration of 2-5 μg / mL, and the staining reaction time is 15-20 min; the cleaning solution is 1xPBS-T, and each cleaning time is 30-60 s; the cleaning is performed 2-3 times.

9. The method for replacing high temperature denaturation to generate single strands in a multiplex PCR detection reaction as claimed in claim 1 or 2, and the method for improving the sensitivity and accuracy of liquid phase hybridization detection of multiplex PCR products as claimed in any one of claims 3 to 8, characterized in that: All are applications not for diagnostic or therapeutic purposes.

10. A multiplex PCR product liquid phase hybridization detection kit, characterized in that: The invention comprises reagents used in conjunction with the method for improving the sensitivity and accuracy of liquid phase hybridization detection of multiple PCR products as described in any one of claims 3 to 8.

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