CN117625768A - Universal digital PCR detection system and application thereof - Google Patents
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Abstract
The invention belongs to the technical field of nucleic acid detection and biological medicine, and particularly relates to a universal digital PCR detection system and application thereof. The specific principle of the universal digital PCR detection system in PCR detection is as follows: the specific mediated probe binds to the target sequence, is cleaved in the extension of DNA polymerase (Taq enzyme) to generate a mediator with 1-2 specific bases, and the mediator can bind to the auxiliary target sequence and extend the universal probe cleaved and bound on the auxiliary target to realize the release of fluorescent signals. Meanwhile, the universal digital PCR detection system can realize lower multi-target quantitative detection cost and higher effect of the same universal probe.
Description
Technical Field
The invention belongs to the technical field of nucleic acid detection and biological medicine, and particularly relates to a universal digital PCR detection system and application thereof.
Background
Nucleic acid amplification is an indispensable detection tool in the field of clinical diagnosis, including the differentiation of genotypes and the accurate quantification of pathogens in patient specimens. Polymerase Chain Reaction (PCR) -based techniques provide a powerful tool for amplification of minute amounts of initial target sequences. The method has been developed from low-throughput general PCR based on gel analysis to real-time quantitative PCR based on fluorescence technology, and digital PCR (ddPCR) using thousands of microreactors, and the digital PCR has the advantage of absolute quantification, which is a third generation PCR detection technology.
Existing amplification techniques typically employ non-selective DNA dyes or target-specific fluorescent probes. DNA dyes are suitable for low cost nucleic acid detection and quantification, but are limited in multiplexing and non-specific amplification. In contrast, fluorescently labeled target-specific oligonucleotides (such as hydrolysis probes or molecular beacons) are based on Fluorescence Resonance Energy Transfer (FRET) between a fluorescent group and a quenching group and interact directly with the target sequence. Labeling the target-specific probe sequences with different fluorophores enables the detection of multiple targets with high specificity during the reaction. However, fluorescent group labeling increases the cost of analysis, and the increase in cost is proportional to the increase in the number of detection targets. It can be seen that the existing amplification techniques have the disadvantage of low efficiency or high cost.
Disclosure of Invention
The invention aims to provide a universal digital PCR detection system and application thereof, wherein the universal digital PCR detection system can realize high-efficiency, universal and low-cost qualitative and quantitative detection of nucleic acid.
The invention provides a universal digital PCR detection system, which comprises a specific mediating probe, an auxiliary target sequence, a universal probe, a target primer and a PCR amplification reagent;
the specific mediating probe consists of a complementary sequence and a protruding sequence; the complementary sequence is positioned at the 3' end of the specific mediating probe and is complementary with the target template;
the protruding sequence is positioned at the 5' end of the specific mediating probe and is not complementary with the target template;
the 3' -end of the specific mediating probe is connected with a group for preventing the sequence from extending;
the auxiliary target sequence consists of a 3 'end sequence, a spacer sequence and a 5' end sequence; the 5' end sequence is complementary to the universal probe; the spacer sequence comprises N random bases, and N is an integer greater than or equal to 1; the 3' end sequence is complementary with a mediating guide, the mediating guide is formed by enzymolysis of the specific mediating probe by DNA polymerase in the amplification process, and the mediating guide consists of a protruding sequence of the specific mediating probe and target specific bases on 1-2 complementary sequences;
the 3 '-end of the auxiliary target sequence is connected with M continuous bases, the 5' -end of the auxiliary target sequence is connected with K continuous bases, and M and K are integers greater than or equal to 2 respectively.
Preferably, the length of the complementary sequence is 15-35 bases; the length of the protruding sequence is 5-25 bases; the group preventing sequence extension comprises a phosphate modification group, an MGB modification group or a short sequence which is not complementary to the target template; the short sequence that is not complementary to the target template is 2 or more bases in length.
Preferably, the length of the 5' end sequence is 15-30 bases; the length of the 3' end sequence is 5-25 bases; the spacer sequence has a length of 1 or more bases.
Preferably, 1-2 target specific bases in the mediator guide are located at the 3' end of the mediator guide; the mediator is not complementary to the nucleotide sequence of any known species.
Preferably, the length of the universal probe is 15-30 bases, the 5 'end of the universal probe is modified with a luminescent group, and the 3' end and/or the middle position of the universal probe are respectively modified with a quenching group.
Preferably, the nucleotide sequence of the upstream primer of the target primer is shown as SEQ ID NO.1, SEQ ID NO.7, SEQ ID NO.13, SEQ ID NO.20 or SEQ ID NO. 25;
the nucleotide sequence of the downstream primer of the target primer is shown as SEQ ID NO.2, SEQ ID NO.8, SEQ ID NO.14, SEQ ID NO.21 or SEQ ID NO. 26;
the nucleotide sequence of the universal probe is shown as SEQ ID NO.4 or SEQ ID NO. 10;
the nucleotide sequence of the specific mediated probe is shown as SEQ ID NO.5, SEQ ID NO.11, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.23 or SEQ ID NO. 28;
the nucleotide sequence of the auxiliary target sequence is shown as SEQ ID NO.6, SEQ ID NO.12, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.24 or SEQ ID NO. 29.
Preferably, the nucleotide sequence of the universal digital PCR detection system is as set forth in one or more of combinations 1) to 6):
the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 1) are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6;
the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 2) are respectively shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO. 12;
the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 3) are respectively shown as SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.10, SEQ ID NO.16 and SEQ ID NO. 17;
the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 4) are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.10, SEQ ID NO.18 and SEQ ID NO.19;
the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 5) are respectively shown as SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.10, SEQ ID NO.23 and SEQ ID NO. 24;
the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 6) are respectively shown as SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.10, SEQ ID NO.28 and SEQ ID NO. 29.
Preferably, the concentration of the target primer in the universal digital PCR detection system is 0.3-3 mu M, the concentration of the specific mediating probe is 0.3-3 mu M, the concentration of the universal probe is 0.3-3 mu M, and the concentration of the auxiliary target sequence is 0.3-3 mu M.
The invention also provides application of the universal digital PCR detection system in gene detection and/or quantification.
Preferably, the template for detection and/or quantification of the gene comprises genomic DNA, cDNA, plasmid DNA or single stranded DNA.
The beneficial effects are that:
the invention provides a universal digital PCR detection system, which comprises a specific mediating probe, an auxiliary target sequence, a universal probe, a target primer and a PCR amplification reagent; the specific mediating probe consists of a complementary sequence and a protruding sequence; the complementary sequence is positioned at the 3' end of the specific mediating probe and is complementary with the target template; the protruding sequence is positioned at the 5' end of the specific mediating probe and is not complementary with the target template; the 3' -end of the specific mediating probe is connected with a group for preventing the sequence from extending; the auxiliary target sequence consists of a 3 'end sequence, a spacer sequence and a 5' end sequence; the 5' end sequence is complementary to the universal probe; the spacer sequence comprises N random bases, and N is an integer greater than or equal to 1; the 3' end sequence is complementary with a mediating guide, the mediating guide is formed by enzymolysis of the specific mediating probe by DNA polymerase in the amplification process, and the mediating guide consists of a protruding sequence of the specific mediating probe and target specific bases on 1-2 complementary sequences; the 3 '-end of the auxiliary target sequence is connected with M continuous bases, the 5' -end of the auxiliary target sequence is connected with K continuous bases, and M and K are integers greater than or equal to 2 respectively.
The schematic diagram of the universal digital PCR detection system in the invention is shown in figure 1, and the specific principle is as follows: the specific mediated probe binds to the target sequence, is cleaved in the extension of DNA polymerase (Taq enzyme) to generate a mediator with 1-2 specific bases, and the mediator can bind to the auxiliary target sequence and extend the universal probe cleaved and bound on the auxiliary target to realize the release of fluorescent signals. The universal probe can generate lower fluorescence background because the quenching group is close to the fluorescent group, because the auxiliary target and the probe are in a hybridization state when the digital PCR detects fluorescence, and because the distance between the quenching group and the fluorescent group at two ends of the probe is increased due to hybridization, the background is raised, so that the increased fluorescence is detected by the submerged target. Meanwhile, the universal digital PCR detection system can realize lower multi-target quantitative detection cost and higher effect of the same universal probe.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments will be briefly described below.
FIG. 1 is a schematic diagram of the general probe digital PCR system for detecting nucleic acid according to the present invention;
FIG. 2 is a comparison result of EGFR sequence in genomic DNA detected by the universal probe system and specific probe of example 1 of the present invention;
FIG. 3 is a comparison of the detection of HPV 18 targets in genomic DNA by the universal probe system and specific probes of example 2 of the present invention;
FIG. 4 shows the comparison of the detection of different targets in cDNA by the universal probe system and specific probe in example 3 of the present invention.
Detailed Description
The invention provides a universal digital PCR detection system, which comprises a specific mediating probe, an auxiliary target sequence, a universal probe, a target primer and a PCR amplification reagent;
the specific mediating probe consists of a complementary sequence and a protruding sequence; the complementary sequence is positioned at the 3' end of the specific mediating probe and is complementary with the target template;
the protruding sequence is positioned at the 5' end of the specific mediating probe and is not complementary with the target template;
the 3' -end of the specific mediating probe is connected with a group for preventing the sequence from extending;
the auxiliary target sequence consists of a 3 'end sequence, a spacer sequence and a 5' end sequence; the 5' end sequence is complementary to the universal probe; the spacer sequence comprises N random bases, and N is an integer greater than or equal to 1; the 3' end sequence is complementary with a mediating guide, the mediating guide is formed by enzymolysis of the specific mediating probe by DNA polymerase in the amplification process, and the mediating guide consists of a protruding sequence of the specific mediating probe and target specific bases on 1-2 complementary sequences;
the 3 '-end of the auxiliary target sequence is connected with M continuous bases, the 5' -end of the auxiliary target sequence is connected with K continuous bases, and M and K are integers greater than or equal to 2 respectively.
In the present invention, the specific mediating probe consists of a complementary sequence and a protruding sequence; the complementary sequence is positioned at the 3' end of the specific mediating probe and is complementary with the target template; the protruding sequence is positioned at the 5' end of the specific mediating probe and is not complementary with the target template; the 3' -end of the specific mediating probe is connected with a group for preventing the sequence from being extended;
the length of the complementary sequence in the specific mediating probe is preferably 15-35 bases; the location where the complementary sequence is complementary to the target template is located between the upstream primer and the downstream primer of the target. The group preventing sequence extension of the present invention preferably comprises a phosphate modification group, an MGB modification group, or a short sequence that is not complementary to the target template; the length of the short sequence which is not complementary to the target template is preferably 2 or more bases, more preferably 4 to 6 bases. The length of the protruding sequence is preferably 5-25 bases; the protruding sequence is preferably an engineered sequence that is not complementary to any sequence of the target nucleic acid. The protruding sequence of the invention can be digested by DNA polymerase (Taq enzyme) and released into a reaction system in an amplification reaction, so as to generate a mediator which is combined with an auxiliary target sequence. In order to avoid complementary pairing of the mediator vector with other sequences, the mediator vector of the present invention is preferably not complementary to nucleotide sequences of any known species. The enzyme cutting site of the DNA polymerase is 1-2 bases of the 5' end of the complementary sequence, and the formed mediating guide consists of the protruding sequence of the specific mediating probe and 1-2 target specific bases. The 1-2 target specific bases are complementary with the target template, and the 1-2 target specific bases are positioned at the 3' end of the mediator. In the examples of the present invention, the specific mediating probe is preferably represented by the nucleotide sequence shown in SEQ ID No.5, SEQ ID No.11, SEQ ID No.16, SEQ ID No.18, SEQ ID No.23 or SEQ ID No.28, but is not to be construed as merely limiting the scope of the present invention.
In the present invention, the auxiliary target sequence consists of a 3 'end sequence, a spacer sequence and a 5' end sequence; the 5' end sequence is complementary to the universal probe; the spacer sequence comprises N random bases, and N is an integer greater than or equal to 1; the 3' end sequence is complementary to the mediating guide; the 3 '-end of the auxiliary target sequence is connected with M continuous bases, the 5' -end of the auxiliary target sequence is connected with K continuous bases, and M and K are integers greater than or equal to 2 respectively.
The length of the 5' end sequence is preferably 15-30 bases; the length of the 3' end sequence is preferably 5-25 bases; the 3' end sequence is complementarily paired with the mediator and is complementary to the 1-2 target-specific bases on the mediator. The length of the spacer sequence is preferably 1 or more. In the present invention, the purpose of ligating consecutive bases at the 3 'end and the 5' end of the auxiliary target sequence is to stabilize the double-stranded structure and prevent possible non-specific extension. The numbers of M and K are preferably the same or different; the type of the continuous base is not particularly limited, and may be optionally used in A, T, C, G. In the examples of the present invention, the auxiliary target sequence is preferably represented by the nucleotide sequence shown in SEQ ID No.6, SEQ ID No.12, SEQ ID No.17, SEQ ID No.19, SEQ ID No.24 or SEQ ID No.29, but is not to be construed as merely limiting the scope of the present invention.
In the invention, the length of the universal probe is preferably 15-30 bases, and the 5' -end of the universal probe is decorated with a luminescent group, wherein the luminescent group preferably comprises, but is not limited to, AM, VIC, ROX, JOE or HEX; the 3' end and/or the middle position of the universal probe are respectively modified with a quenching group, wherein the quenching group preferably comprises but is not limited to BHQ1, BHQ2, BHQ3, ZEN, TAO or IFBQ; the specific base position at the intermediate position is not particularly limited, and a specific position may be selected conventionally. The universal probes of the present invention preferably comprise synthetic single stranded DNA or modified DNA that alters hybridization affinity, more preferably comprise one or more of Locked Nucleic Acid (LNA), peptide Nucleic Acid (PNA), or Minor Groove Binder (MGB) of DNA. In the examples of the present invention, the general probe is preferably represented by the nucleotide sequence shown in SEQ ID NO.4 or SEQ ID NO.10 to illustrate the technical scheme of the present invention, but it should not be construed as merely limiting the scope of the present invention.
In the invention, the target primer is preferably designed according to a target template; the target primer is preferably a target specific primer. In the present invention, the upstream primer of the target primer is preferably represented by the nucleotide sequence shown in SEQ ID NO.1, SEQ ID NO.7, SEQ ID NO.13, SEQ ID NO.20 or SEQ ID NO.25, but is not to be construed as the full scope of the present invention; the downstream primer is preferably represented by the nucleotide sequence shown in SEQ ID No.2, SEQ ID No.8, SEQ ID No.14, SEQ ID No.21 or SEQ ID No.26, but is not to be construed as a full scope of the present invention.
In the present invention, the nucleotide sequence of the universal digital PCR detection system preferably includes, but is not limited to, one or more of combinations 1) to 6), which should not be construed as merely limiting to the full scope of the present invention. The amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 1) are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6; the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 2) are respectively shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO. 12; the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 3) are respectively shown as SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.10, SEQ ID NO.16 and SEQ ID NO. 17; the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 4) are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.10, SEQ ID NO.18 and SEQ ID NO.19; the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 5) are respectively shown as SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.10, SEQ ID NO.23 and SEQ ID NO. 24; the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 6) are respectively shown as SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.10, SEQ ID NO.28 and SEQ ID NO. 29.
In the invention, the concentration of the target primer in the universal digital PCR detection system is preferably 0.3-3 mu M, the concentration of the specific mediating probe is preferably 0.3-3 mu M, the concentration of the universal probe is preferably 0.3-3 mu M, and the concentration of the auxiliary target sequence is preferably 0.3-3 mu M. The PCR amplification reagents of the present invention preferably comprise digital PCR amplification reagents, further preferably include, but are not limited to, hot start polymerase, polymerase buffer, dNTPs, and MgCl 2 . The sources of the components of the PCR amplification reagent are not particularly limited, and the PCR amplification reagent is a conventional commercial product.
The universal digital PCR detection system uses the specific mediating probe, the universal probe, the auxiliary target sequence and the target primer, so that the efficient and universal nucleic acid quantitative detection is realized, and the cost is lower.
Based on the advantages, the invention also provides application of the universal digital PCR detection system in detection and/or quantification of genes, more preferably in detection and quantification of genes. Templates for detection and/or quantification of the genes of the invention preferably comprise genomic DNA, cDNA, plasmid DNA or single stranded DNA, more preferably genomic DNA or cDNA. The detection and/or quantification of the gene according to the invention is preferably a digital PCR detection (dPCR) or a fluorescent quantitative PCR detection (qPCR), more preferably a digital PCR detection (dPCR).
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
A new universal probe digital PCR system comprises the following steps:
the components of the high-efficiency and general detection system are as shown in the step 1) and the step 2):
1) Based on EGFR target, the nucleotide sequences of the upstream primer, the downstream primer, the universal probe, the mediating probe and the auxiliary target of the universal probe digital PCR detection system are respectively shown as SEQ ID NO. 1-SEQ ID NO.2 and SEQ ID NO. 4-SEQ ID NO.5 from the 5'-3' end;
SEQ ID NO.1：AACGTACTGGTGAAAACACCG；
SEQ ID NO.2：ATGGTATTCTTTCTCTTCCGCAC；
SEQ ID NO.4：FAM-TAT(BHQ1)TGCTTCCCCCATTCAGGACTTG-PO4；
SEQ ID NO.5：AAGTCTAGCTGCTGTAGCCAAGATTTTGGGCTGGCCAAACTGCTaaaa；
SEQ ID NO.6：aaCAAGTCCTGAATGGGGGAAGCAATAaaaCTTGGCTACAGCAGCTAGACTTCTaa
the nucleotide sequences of an upstream primer, a downstream primer and a specific probe of a conventional specific detection system are respectively shown as SEQ ID NO. 1-SEQ ID NO.3 from the 5'-3' end;
SEQ ID NO.3：FAM-CAGTTTGGCCAGCCCAAAATCT-BHQ1；
2) The reaction volume is 15 mu L, and contains 1 Xbuffer, 1 mu M of upstream and downstream primers, 0.3 mu M of specific fluorescent probe, 1 mu M of universal fluorescent probe, 1 mu M of mediated probe, 0.4 mu M of auxiliary target and 0.15 mu L of hot start polymerase; the cell line 293T genomic DNA template was 5. Mu.L, and the specific probe and the universal probe system were each independently piped, each with a reaction volume of 15. Mu.L.
3) Droplet generation and PCR amplification for digital PCR was performed by the following procedure: enzyme activation at 95deg.C for 10min; denaturation at 95℃for 30s and annealing at 58℃for 50s,50 cycles.
4) Droplet generation, amplification and signal acquisition were performed using pilot droplet digital PCR system D3200, the results of which are shown in fig. 2. The quantitative concentration of the universal probe digital PCR system is as follows: 211 copies/. Mu.L, the specific probe quantitative concentration is: 182 copies/. Mu.L.
The results show that the universal probe digital PCR system can realize accurate quantification.
Example 2
The components of the high-efficiency and general probe detection system are as shown in the step 1) and the step 2):
1) Based on HPV 18 target, the nucleotide sequences of the upstream primer, the downstream primer, the universal double-quenching probe, the mediation probe and the auxiliary target of the universal probe digital PCR detection system are sequentially shown as SEQ ID NO. 7-SEQ ID NO.8 and SEQ ID NO. 10-SEQ ID NO.12 from the 5'-3' end;
SEQ ID NO.7：AGGAACATTTTGTGAACAGGCA；
SEQ ID NO.8：ACATGCAACACTTGTGCATC；
SEQ ID NO.10：VIC-GGACT(ZEN)ATGTCCGGGAACACAAAGA-IFBQ；
SEQ ID NO.11：AGATCAGCAGACTGTGACGGCATTGTTCCATGCGCAGGAGGTaata；
SEQ ID NO.12：aaaaTCTTTGTGTTCCCGGACATAGTCCaaaGCCGTCACAGTCTGCTGATCTaaaaaa；
the nucleotide sequences of an upstream primer, a downstream primer and a specific probe of a conventional specific detection system are respectively shown as SEQ ID NO. 7-SEQ ID NO.9 from the 5'-3' end;
SEQ ID NO.9：FAM-CATTGTTCCATGCGCAGGAGGT-BHQ1；
2) The reaction volume is 15 mu L, containing 1 Xbuffer, 1 mu M upstream and downstream primer, 0.3 mu M specific fluorescent probe, 0.6 mu M universal fluorescent probe, 1 mu M mediating probe, 0.6 mu M auxiliary target and 0.15 mu L hot start polymerase; the cell line Heal genomic DNA template was 5. Mu.L, and the specific probe and the universal probe system were each independently piped, each with a reaction volume of 15. Mu.L.
3) Droplet generation and PCR amplification for digital PCR was performed by the following procedure: enzyme activation at 95deg.C for 10min; denaturation at 95℃for 30s and annealing at 58℃for 50s,50 cycles.
4) Digital PCR amplification and signal acquisition were performed using a pilot system, and the results are shown in fig. 3, with a clear distinction between negative and positive droplets.
The quantitative concentration of the universal probe digital PCR system is as follows: 84.1 copies/. Mu.L, the specific probe quantitative concentration is: 95.2 copies/. Mu.L.
The results show that the universal probe digital PCR system can realize accurate quantification.
Example 3
The high-efficiency and general probe detection system is used for quantifying different target components and specific digital PCR components, and the steps are shown in the steps 1) and 2):
1) All universal digital PCR, using the same universal probe, nucleotide sequence from 5'-3' end to SEQ ID No.10: VIC-GGACT (ZEN) ATGTCCGGGAACACAAAGA-IFBQ;
based on the ACTB target, the nucleotide sequences of the upstream primer, the downstream primer, the mediating probe and the auxiliary target of the universal probe digital PCR detection system are sequentially shown as SEQ ID NO. 13-SEQ ID NO.14 and SEQ ID NO. 16-SEQ ID NO.17 from the 5'-3' end;
SEQ ID NO.13：AGGCATCCTCACCCTGAAG；
SEQ ID NO.14：CATTGTAGAAGGTGTGGTGCC；
SEQ ID NO.16：AGATCAGCAGACTGTGACGGGCATCGTCACCAACTGGGACGACAaaat；
SEQ ID NO.17：aaaaTCTTTGTGTTCCCGGACATAGTCCaaaCCCGTCACAGTCTGCTGATCTaaaaaa；
the nucleotide sequences of an upstream primer, a downstream primer and a specific probe of a conventional specific detection system are respectively shown as SEQ ID NO. 13-SEQ ID NO.15 from the 5'-3' end;
SEQ ID NO.15：FAM-CGGCATCGTCACCAACTGGGACG-BHQ1；
based on EGFR target, the nucleotide sequences of the upstream primer, the downstream primer, the mediating probe and the auxiliary target are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO. 18-SEQ ID NO.19;
SEQ ID NO.18：AGATCAGCAGACTGTGACGAGATTTTGGGCTGGCCAAACTGCTaaaa；
SEQ ID NO.19：aaaaTCTTTGTGTTCCCGGACATAGTCCaaaCTCGTCACAGTCTGCTGATCTaaaaaa；
the nucleotide sequences of an upstream primer, a downstream primer and a specific probe of a conventional specific detection system are respectively shown as SEQ ID NO. 1-SEQ ID NO.3 from the 5'-3' end;
based on the BRAF target, the nucleotide sequences of the upstream primer, the downstream primer, the mediating probe and the auxiliary target are sequentially shown as SEQ ID NO. 20-SEQ ID NO.21 and SEQ ID NO. 23-SEQ ID NO.24 from the 5'-3' end;
SEQ ID NO.20：TTCTTCATGAAGACCTCACAG；
SEQ ID NO.21：CTGTTCAAACTGATGGGACC；
SEQ ID NO.23：AGATCAGCAGACTGTGACGCATCGAGATTTCACTGTAGCTAGACCAttta；
SEQ ID NO.24：aaaaTCTTTGTGTTCCCGGACATAGTCCaaaTGCGTCACAGTCTGCTGATCTaaaaaa；
the nucleotide sequences of an upstream primer, a downstream primer and a specific probe of a conventional specific detection system are respectively shown as SEQ ID NO. 20-SEQ ID NO.22 from the 5'-3' end;
SEQ ID NO.22：FAM-CATCGAGATTTCACTGTAGCTAGAC-BHQ1；
based on the PIK3CA target, the nucleotide sequences of the upstream primer, the downstream primer, the mediation probe and the auxiliary target are sequentially shown as SEQ ID NO. 25-SEQ ID NO.26 and SEQ ID NO. 28-SEQ ID NO.29 from the 5'-3' end;
SEQ ID NO.25：GGAATCCAGAGTGAGCTTTC；
SEQ ID NO.26：ATGAAACAAATGAATGATGCAC；
SEQ ID NO.28：AGATCAGCAGACTGTGACGTGCTGTTTAATTGTGTGGAAGATCCAAaaat；
SEQ ID NO.29；aaaaTCTTTGTGTTCCCGGACATAGTCCaaaCACGTCACAGTCTGCTGATCTaaaaaa；
the nucleotide sequences of an upstream primer, a downstream primer and a specific probe of a conventional specific detection system are respectively shown as SEQ ID NO. 25-SEQ ID NO.27 from the 5'-3' end;
SEQ ID NO.27：FAM-TGCTGTTTAATTGTGTGGAAGATCC-BHQ1；
2) The reaction volume was 15. Mu.L, containing 1 Xbuffer, 1. Mu.M upstream and downstream primer, 0.3. Mu.M specific fluorescent probe, 0.6. Mu.M universal fluorescent probe, 1.3. Mu.M mediating probe, 0.5. Mu.M auxiliary target, 0.15. Mu.L hot start polymerase; mu.L of cDNA obtained by reverse transcription of U87 RNA of the cell line (used after 100-fold dilution), the specific probe and the universal probe system were each independently piped, and the reaction volume of each piped was 15. Mu.L.
3) Droplet generation and PCR amplification for digital PCR was performed by the following procedure: enzyme activation at 95deg.C for 10min; denaturation at 95℃for 30s and annealing at 58℃for 50s,50 cycles.
4) The results of digital PCR amplification and signal acquisition using a pilot system are shown in FIG. 4, where A is a graph of the results of detection of different targets using a universal probe; b is a comparison result of quantifying different targets using universal digital PCR and specific digital PCR; c is a microdroplet map using universal digital PCR to quantify different targets; d is a microdroplet plot of specific probe digital PCR quantification of different targets.
From fig. 4, it is evident that the negative and positive droplets are distinguished, and that the quantitative results of other targets are very consistent, except for ACTB targets, which are detected differently due to the extremely high concentration.
Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.
Claims (10)
1. A universal digital PCR detection system, comprising a specific mediating probe, an auxiliary target sequence, a universal probe, a target primer and a PCR amplification reagent;
the specific mediating probe consists of a complementary sequence and a protruding sequence; the complementary sequence is positioned at the 3' end of the specific mediating probe and is complementary with the target template;
the protruding sequence is positioned at the 5' end of the specific mediating probe and is not complementary with the target template;
the 3' -end of the specific mediating probe is connected with a group for preventing the sequence from extending;
the auxiliary target sequence consists of a 3 'end sequence, a spacer sequence and a 5' end sequence; the 5' end sequence is complementary to the universal probe; the spacer sequence comprises N random bases, and N is an integer greater than or equal to 1; the 3' end sequence is complementary with a mediating guide, the mediating guide is formed by enzymolysis of the specific mediating probe by DNA polymerase in the amplification process, and the mediating guide consists of a protruding sequence of the specific mediating probe and target specific bases on 1-2 complementary sequences;
the 3 '-end of the auxiliary target sequence is connected with M continuous bases, the 5' -end of the auxiliary target sequence is connected with K continuous bases, and M and K are integers greater than or equal to 2 respectively.
2. The universal digital PCR detection system according to claim 1, wherein the complementary sequence is 15-35 bases in length; the length of the protruding sequence is 5-25 bases; the group preventing sequence extension comprises a phosphate modification group, an MGB modification group or a short sequence which is not complementary to the target template; the short sequence that is not complementary to the target template is 2 or more bases in length.
3. The universal digital PCR detection system according to claim 1, wherein the 5' end sequence of the auxiliary target sequence is 15-30 bases in length; the length of the 3' end sequence is 5-25 bases; the spacer sequence has a length of 1 or more bases.
4. The universal digital PCR detection system according to claim 1 or 3, wherein 1-2 target specific bases in the mediator guide are located at the 3' end of the mediator guide; the mediator is not complementary to the nucleotide sequence of any known species.
5. The universal digital PCR detection system according to claim 1, wherein the length of the universal probe is 15-30 bases, the 5 'end of the universal probe is modified with a luminescent group, and the 3' end and/or the middle position of the universal probe are respectively modified with a quenching group.
6. The universal digital PCR detection system according to claim 1, wherein the nucleotide sequence of the upstream primer of the target primer is set forth in SEQ ID No.1, SEQ ID No.7, SEQ ID No.13, SEQ ID No.20 or SEQ ID No. 25;
the nucleotide sequence of the downstream primer of the target primer is shown as SEQ ID NO.2, SEQ ID NO.8, SEQ ID NO.14, SEQ ID NO.21 or SEQ ID NO. 26;
the nucleotide sequence of the universal probe is shown as SEQ ID NO.4 or SEQ ID NO. 10;
the nucleotide sequence of the specific mediated probe is shown as SEQ ID NO.5, SEQ ID NO.11, SEQ ID NO.16, SEQ ID NO.18, SEQ ID NO.23 or SEQ ID NO. 28;
the nucleotide sequence of the auxiliary target sequence is shown as SEQ ID NO.6, SEQ ID NO.12, SEQ ID NO.17, SEQ ID NO.19, SEQ ID NO.24 or SEQ ID NO. 29.
7. The universal digital PCR detection system of claim 1, wherein the nucleotide sequence of the universal digital PCR detection system is as set forth in one or more of combinations 1) through 6):
the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 1) are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO. 6;
the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 2) are respectively shown as SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO. 12;
the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 3) are respectively shown as SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.10, SEQ ID NO.16 and SEQ ID NO. 17;
the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 4) are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.10, SEQ ID NO.18 and SEQ ID NO.19;
the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 5) are respectively shown as SEQ ID NO.20, SEQ ID NO.21, SEQ ID NO.10, SEQ ID NO.23 and SEQ ID NO. 24;
the amino acid sequences of the upstream primer, the downstream primer, the universal probe, the specific mediating probe and the auxiliary target sequence in the combination 6) are respectively shown as SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.10, SEQ ID NO.28 and SEQ ID NO. 29.
8. The universal digital PCR detection system according to claim 1, wherein the concentration of the target primer in the universal digital PCR detection system is 0.3-3 μΜ, the concentration of the specific mediating probe is 0.3-3 μΜ, the concentration of the universal probe is 0.3-3 μΜ, and the concentration of the auxiliary target sequence is 0.3-3 μΜ.
9. Use of a universal digital PCR detection system according to any one of claims 1 to 8 for the detection and/or quantification of genes.
10. The use according to claim 8, wherein the template for detection and/or quantification of the gene comprises genomic DNA, cDNA, plasmid DNA or single stranded DNA.
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