WO2022067743A1 - 一种用于dna单核苷酸变异检测的方法、探针及试剂盒及其应用 - Google Patents

一种用于dna单核苷酸变异检测的方法、探针及试剂盒及其应用 Download PDF

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WO2022067743A1
WO2022067743A1 PCT/CN2020/119612 CN2020119612W WO2022067743A1 WO 2022067743 A1 WO2022067743 A1 WO 2022067743A1 CN 2020119612 W CN2020119612 W CN 2020119612W WO 2022067743 A1 WO2022067743 A1 WO 2022067743A1
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reporter
dep
nucleotide sequence
seq
sequence shown
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PCT/CN2020/119612
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李峰
王冠
许骏鹏
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李峰
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Priority to PCT/CN2020/119612 priority Critical patent/WO2022067743A1/zh
Priority to CN202080105561.1A priority patent/CN116724124A/zh
Publication of WO2022067743A1 publication Critical patent/WO2022067743A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the invention relates to the technical field of biological detection, in particular to a method, a probe and a kit for DNA single nucleotide variation detection and applications thereof.
  • SNVs Single nucleotide variants
  • SNVs Single nucleotide variants
  • SNV as a genetic marker has the following advantages: (1) SNV is biallelic in the population, and its allele frequency can be estimated in any population; (2) it is widely distributed in the genome; ( 3) Compared with tandemly repeated microsatellite loci, SNVs are highly stable, especially those located in the coding region, and the high mutation rate of the former can easily cause difficulties in genetic analysis of the population; (4) Some of them are located within genes The SNVs may directly affect the structure of the product protein or the level of gene expression, therefore, they themselves may be candidate sites of alteration in the genetic mechanism of the disease; (5) It is easy to perform automated analysis and shorten the research time. Due to the above advantages of SNV, it has important application value in molecular diagnosis, clinical testing, forensic science, pathogen detection, drug efficacy evaluation, new drug development and population evolution.
  • Restriction endonucleases are a class of enzymes that recognize specific sites in DNA and cut them at specific sites.
  • the premise of this technique is that both sides of the SNP site to be detected should contain restriction endonuclease recognition sequences.
  • This method is simple and fast, and can be used for analysis of a large number of samples.
  • the disadvantage is that it cannot directly analyze the sequence of DNA, and nearly half of the SNP sites do not lead to changes in the enzyme cleavage site.
  • the reaction principle is the principle of complementary pairing of bases.
  • probes are designed for the variant site and the flanking sequence. Usually, there is only one base difference between the probes, which corresponds to different alleles.
  • All sequence-specific oligonucleotide probes are processed and spotted on solid-phase or liquid-phase carriers such as nylon membranes, glass slides or silicon wafers, and hybridized with the DNA template to be detected.
  • Types of SNVs Including DNA chip detection technology, TaqMan probe technology, allele-specific oligonucleotide hybridization and molecular beacon technology.
  • This method has high specificity and sensitivity, and can be used for genetic diagnosis and screening of genetic diseases with gene mutations, and has important value in the screening of fixed point mutations, such as p53 gene, human breast cancer genes 1 and 2 Screening of mutation hotspots.
  • fixed point mutations such as p53 gene, human breast cancer genes 1 and 2 Screening of mutation hotspots.
  • the reaction principle of this method is to first amplify a piece of DNA containing a variation site, and then anneal an oligonucleotide primer directly upstream or downstream of the base to be detected. A few bases are extended, and site information is determined based on the signal from the extension reaction.
  • the advantage of this method is that the detection sensitivity and resolution are high, but its disadvantage is that a multi-color fluorescence system and a corresponding detection system must be used, and the excitation light and emission fluorescence spectra of multiple dyes often have a large part overlap, thereby interfering with the measurement accuracy of the fluorescence intensity.
  • oligonucleotide sequences anneal to the template, and only when they completely match the template at the junction will they be joined together under the action of a ligase, so the allele-specific oligonucleotide sequence ligation technology Can probe the nature of single base variation sites.
  • the oligonucleotide ligation analysis was established by Samiotaki et al. in 1994. The principle is to denature the PCR product into a single strand, and then add two probes A and B (about 20 nucleotides long), the sequences of A and B They are complementary to the sequences on both sides of the variant site in the target DNA, and the 5' end of A is adjacent to the 3' end of B. If the two adjacent probes are completely complementary to the target DNA single strand, under the action of DNA ligase, the 5'-terminal phosphate group and the 3' hydroxyl group of the two probes form a phosphodiester bond and are connected.
  • the ligation reaction will not occur.
  • the ligation product is denatured, it can be used as the template of the primer.
  • the signal is measured according to a special measurement method to determine whether the ligation reaction occurs, and use this as the template DNA single-strand whether there is a base or not. basis for change.
  • the advantage of oligonucleotide ligation analysis technology is that it only needs to use 1/10 of the commonly used DNA samples to evaluate the internal sequence of DNA, and the detection results are not affected by non-specific products in the PCR amplification reaction.
  • the results can be directly transferred to the computer for storage and statistical analysis, automatic detection, which greatly improves the detection efficiency.
  • the disadvantage is that the location of the variation site must be known before the probe can be synthesized in a targeted manner, and when there are multiple sites, multiple probes need to be synthesized for detection.
  • the above-mentioned endonuclease digestion technology, allele-specific hybridization, primer extension and oligonucleotide ligation technology have disadvantages such as complicated operation procedures, insufficient sensitivity, low detection throughput or large demand for DNA samples.
  • the ideal detection method should have the characteristics of high accuracy, simple operation, high throughput and low cost. Therefore, it is still necessary to explore and establish efficient and low-cost detection methods for SNVs.
  • the present invention provides a method for DNA single nucleotide variation detection, comprising:
  • the DNA to be tested is mixed with the reaction system, heated to unwind, and then rapidly cooled for detection.
  • the two strands of the DNA to be tested are denoted as the A chain and the B chain respectively, and the tested strand is the A chain.
  • the above-mentioned heating derotation temperature is 80-99°C (specifically, such as 80, 82, 84, 86, 88, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99°C); In one embodiment of the invention, the temperature is 95°C.
  • the above-mentioned heating time is 1-10 minutes (specifically, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 minutes); in one embodiment of the present invention, the time is 5 minutes .
  • the above rapid cooling may be cooling to 0-4°C (eg, 0, 1, 2, 3, 4°C) within 1-5 minutes (eg, within 1, 2, 3, 4 or 5 minutes); In one embodiment of the invention, the above-mentioned rapid cooling is cooling to 4°C within 2 minutes.
  • the above detection is performed within 2 hours (eg, within 2 hours, within 1 hour, within 30 minutes, within 15 minutes, within 5 minutes) after rapid cooling.
  • DNA equalizer probes DNA equalizer probe, DEP:
  • n is an integer greater than 1 (eg 2, 3, 4, 5, 6, 7, 8, 9, 10);
  • n 2.
  • n 2
  • the molar ratio of DEP to the DNA to be tested is 1-500:1 (eg 1:1, 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50:1 , 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1).
  • the above reaction system may further comprise Mg 2+ , especially 0-10 mM (eg 0, 1.25, 2.5, 5, 10 mM) of Mg 2+ .
  • the above reaction system also includes a nucleotide protective agent, such as Tween 20, to prevent the potential loss of DNA oligonucleotides during dilution and pipetting; wherein, the content of the nucleotide protective agent can be 0.1%- 0.5% (v/v).
  • a nucleotide protective agent such as Tween 20
  • the content of the nucleotide protective agent can be 0.1%- 0.5% (v/v).
  • the above reaction system also includes a buffer system, such as Tris buffer.
  • a buffer system such as Tris buffer.
  • the above detection includes the following steps: heating the rapidly cooled mixture at 35-40°C (eg 35, 36, 37, 38, 39, 40°C) for 1-10 minutes (eg 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 minutes), adding a molecular probe for detecting single-stranded DNA to trigger the detection reaction; in one embodiment of the present invention, the above-mentioned detection comprises: transferring the rapidly cooled mixture to a microplate and heated in a microplate reader set at 37°C for 5 minutes, adding molecular probes that detect single-stranded DNA.
  • the detection probe may be fluorescently labeled, and the above detection further includes the step of collecting fluorescence data when the detection reaction reaches equilibrium.
  • the above-mentioned detection probes include: 2m reporter probes (reporters):
  • n is an integer greater than or equal to 1 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);
  • each reporter-F is composed of the sequences F-S1 and F-S2, wherein the nucleotide sequence of F-S1 is complementary to the partial sequence of the single-stranded DNA to be tested, and F-S2 is the same as that of the single-stranded DNA to be tested. Any sequence unrelated to DNA (which is not complementary to the single-stranded DNA to be tested, and can be 1-10 nucleotides in length);
  • each reporter-Q is complementary to the nucleotide sequence of the corresponding reporter-F, and the length of the nucleotide sequence of the reporter-Q is less than the length of the nucleotide sequence of the corresponding reporter-F;
  • each F-S1 sequence is different and does not overlap.
  • sequences complementary to each F-S1 sequence in the single-stranded DNA to be tested are separated by at least 1 (eg, 1, 2, 3, 4, 5) nucleotides.
  • At least one of the above-mentioned F-S1 sequences is related to the site to be detected in the single-stranded DNA to be tested (according to published information, single nucleotide variation may occur and cause concerns (such as drug resistance, disease-causing site) complementation.
  • each F-S1 sequence is independently 15-30 nucleotides in length (eg, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 , 29, 30 nucleotides).
  • each F-S2 sequence is independently 1-10 nucleotides in length (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides), eg, TGTAC , CGCTT.
  • each F-S2 sequence is independent, and may be the same or different.
  • each reporter-Q is independently 15-25 nucleotides (eg, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 nucleotides).
  • sequence length of each reporter-Q differs from the sequence length of reporter-Q by 5-10 nucleotides (eg, 5, 6, 7, 8, 9, 10 nucleotides).
  • each of the above-mentioned reporters is fluorescently labeled; wherein each reporter-F is labeled with a fluorescent reporter group, and each reporter-Q is labeled with a fluorescent quenching group, for example, the 5' end of reporter-F is labeled with a fluorescent reporter group , the 3' end of the corresponding reporter-Q is labeled with a fluorescent quenching group; or the 3' end of the reporter-F is labeled with a fluorescent reporter group, and the 5' end of the corresponding reporter-Q is labeled with a fluorescent quenching group group.
  • the above-mentioned fluorescent reporter group can be, for example, FAM, Texas Red, ROX, TET, VIC, JOE, HEX, Cy3, Cy3.5, Cy5, Cy5.5, LC RED640, LC RED705 and the like.
  • the above-mentioned fluorescence quenching group can be, for example, Iowa Black, TAMRA, DABCYL, ECLIPSE, BHQ1, BHQ2, BHQ3 and the like.
  • the fluorescent reporter groups labeled on each of the above reporter-Fs are different.
  • n 1
  • the molar ratio of each of the above-mentioned reporters to the DNA to be tested is 1-500:1 (for example, 1:1, 2:1, 5:1, 10:1, 20:1, 30:1, 40:1, 50 :1, 60:1, 70:1, 80:1, 90:1, 100:1, 200:1, 300:1, 400:1, 500:1).
  • the above-mentioned method may further include the step of amplifying the DNA to be tested by a nucleic acid amplification technique (eg, polymerase chain reaction (PCR)).
  • a nucleic acid amplification technique eg, polymerase chain reaction (PCR)
  • the above method also includes the step of detecting the standard (sequence is the same as the DNA to be tested without variation, which can be artificially synthesized) (the same detection step as the DNA to be tested).
  • the above detection may be qualitative detection or quantitative detection.
  • the above-mentioned detection is a qualitative detection, which can be performed by comparing the strength of the detection signal (eg, fluorescent signal) of the DNA sample to be tested and an equal amount of a standard.
  • the detection signal eg, fluorescent signal
  • the above method includes the step of establishing a standard curve, for example, establishing a standard curve using the amount of the standard substance and the detection signal (eg, fluorescence signal) of the corresponding standard substance.
  • the detection signal eg, fluorescence signal
  • the above-mentioned detection is a quantitative detection, and the amount of non-variant DNA in the DNA sample to be tested can be calculated by using the detected signal (such as a fluorescence signal) of the DNA sample to be tested and using a standard curve, Then, the amount of mutant DNA in the sample is obtained.
  • the detected signal such as a fluorescence signal
  • the above detection is quantitative detection
  • this method is suitable for targets of different length ranges, and the length of the DNA to be detected can be, for example, 32-87 bp.
  • the above-mentioned single nucleotide variation can be a substitution (substituting with one of A, T, C, G), insertion (inserting one of A, T, C, G) or deletion.
  • the above method can simultaneously detect one or more single nucleotide variation sites in the same DNA to be tested, and can also simultaneously detect multiple different DNAs to be tested.
  • the subjects detected by the above method can be parasites (eg Trichuris trichiura, Ascaris lumbricoides), viruses (eg HBV) or animals (eg mammals (eg humans)).
  • parasites eg Trichuris trichiura, Ascaris lumbricoides
  • viruses eg HBV
  • animals eg mammals (eg humans)
  • the present invention also provides a probe for DNA single nucleotide variation detection, comprising: 2m reporter probes:
  • n is an integer greater than or equal to 1 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);
  • each reporter-F is composed of the sequences F-S1 and F-S2, wherein the nucleotide sequence of F-S1 is complementary to the partial sequence of the single-stranded DNA to be tested, and F-S2 is the same as that of the single-stranded DNA to be tested. Any sequence unrelated to DNA (which is not complementary to the single-stranded DNA to be tested, and can be 1-10 nucleotides in length);
  • each reporter-Q is complementary to the nucleotide sequence of the corresponding reporter-F, and the length of the nucleotide sequence of the reporter-Q is less than the length of the nucleotide sequence of the corresponding reporter-F;
  • each F-S1 sequence is different and does not overlap.
  • the above-mentioned reporter probes have the above-mentioned corresponding definitions of the present invention.
  • the present invention also provides a kit for detection of DNA single nucleotide variation, which comprises the above-mentioned probe for DNA single nucleotide variation detection of the present invention.
  • the above-mentioned kit also includes: n DNA equalizer probes (DNA equalizer probes, DEP):
  • n is an integer greater than 1 (eg 2, 3, 4, 5, 6, 7, 8, 9, 10);
  • the above-mentioned DEP has the above-mentioned corresponding definitions of the present invention.
  • the above-mentioned kit may further comprise Mg 2+ , in particular 0-10 mM (eg 0, 1.25, 2.5, 5, 10 mM) of Mg 2+ .
  • the above kit also includes a nucleotide protective agent, such as Tween 20, to prevent the potential loss of DNA oligonucleotides during dilution and pipetting; wherein, the content of the nucleotide protective agent can be 0.1%- 0.5% (v/v).
  • a nucleotide protective agent such as Tween 20
  • the content of the nucleotide protective agent can be 0.1%- 0.5% (v/v).
  • the above kit also includes a buffer system, such as Tris buffer.
  • a buffer system such as Tris buffer.
  • the above detection system comprises: 1 mM Mg 2+ , 0.1% Tween 20 (v/v), 1 ⁇ Tris buffer, DEP, and reporter probe.
  • the above-mentioned kit also includes a standard, the sequence of which is the same as the DNA to be tested without variation, which can be synthesized artificially.
  • the above-mentioned kit may further include a negative control substance, which is a system without DNA to be tested, such as H 2 O (such as sterile double-distilled water, sterile deionized water, etc.).
  • a negative control substance which is a system without DNA to be tested, such as H 2 O (such as sterile double-distilled water, sterile deionized water, etc.).
  • the above-mentioned kit may also include DNA extraction reagents and materials for extracting DNA from the sample to be tested, and any suitable reagents and materials for DNA extraction known in the prior art may be used.
  • the above-mentioned kit may further include a pretreatment reagent for the sample to be tested, and the pretreatment reagent may be a reagent known in the prior art for pretreatment of samples for DNA extraction, for example, physiological saline and the like.
  • the present invention also provides a probe for detecting single nucleotide variation of Trichuris trichiura (for example, from the sequence shown in SEQ ID NO: 1 to the sequence shown in SEQ ID NO: 2), which Include: reporter-F and reporter-Q; wherein, reporter-F has the nucleotide sequence shown in SEQ ID NO:5 (or the nucleotide sequence of reporter-F is represented by the nucleotide sequence shown in SEQ ID NO:5 acid sequence), reporter-Q has the nucleotide sequence shown in SEQ ID NO: 6 (or the nucleotide sequence of reporter-Q consists of the nucleotide sequence shown in SEQ ID NO: 6).
  • reporter-F has the nucleotide sequence shown in SEQ ID NO:5 (or the nucleotide sequence of reporter-F is represented by the nucleotide sequence shown in SEQ ID NO:5 acid sequence
  • reporter-Q has the nucleotide sequence shown in SEQ ID NO: 6 (or the nucleotide
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group A fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group.
  • the above-mentioned reporter-F is 5'-G GAC GAA ACA TAC TGC ATA GA CATGT-FAM-3'; reporter-Q is 5'-Iowa Black FQ-ACATG TC TAT GCA GTA TGT- 3'.
  • the present invention also provides a probe for HBV single nucleotide variation detection, comprising: reporter-F and reporter-Q, wherein, reporter-F has the nucleotide sequence shown in SEQ ID NO: 10 ( or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO: 10), and reporter-Q has the nucleotide sequence shown in SEQ ID NO: 11 (or the nucleotide sequence of reporter-Q The acid sequence consists of the nucleotide sequence shown in SEQ ID NO: 11).
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group A fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group.
  • the above-mentioned reporter-F is 5'-FAM-CGCTT AGG TTG GTG AGT GATT GG AGG TT-3'; reporter-Q is 5'-A ATC ACT CAC CAA CCT AAGCG-Iowa Black FQ -3'.
  • the present invention also provides a probe for detecting single nucleotide variation of Trichuris trichiura (for example, mutated from the sequence shown in SEQ ID NO: 12 to the sequence shown in SEQ ID NO: 13), which Comprising: reporter-F1, reporter-Q1, reporter-F2, reporter-Q2, wherein, reporter-F1 has the nucleotide sequence shown in SEQ ID NO: 16 (or the nucleotide sequence of reporter-F1 is represented by SEQ ID NO: 16)
  • reporter-Q1 has the nucleotide sequence shown in SEQ ID NO: 17 (or the nucleotide sequence of reporter-Q1 consists of the core shown in SEQ ID NO: 17) nucleotide sequence
  • reporter-F2 has the nucleotide sequence shown in SEQ ID NO: 18 (or the nucleotide sequence of reporter-F2 consists of the nucleotide sequence shown in SEQ ID NO: 18)
  • reporter-F2 -Q2 has the nucleotide sequence shown
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group Fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group; and the fluorescent reporter group labeled on reporter-F1 and reporter-F2 Groups are different.
  • the above-mentioned reporter-F1 is 5'-AT GAA GC G CTT TAC GAT ATT TGT TTC CGA-Cy5-3'; reporter-Q is 5'-Iowa Black RQ-TCG GAA ACA AAT ATC GTA AAG C–3'.
  • the present invention also provides a probe for detection of cancer-related gene mutation BRAF-D594G (for example, mutated from the sequence shown in SEQ ID NO:20 to the sequence shown in SEQ ID NO:21), comprising: reporter-F and reporter -Q, wherein reporter-F has the nucleotide sequence shown in SEQ ID NO:24 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:24), reporter- Q has the nucleotide sequence shown in SEQ ID NO:25 (or the nucleotide sequence of reporter-Q consists of the nucleotide sequence shown in SEQ ID NO:25).
  • reporter-F has the nucleotide sequence shown in SEQ ID NO:24 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:24)
  • reporter- Q has the nucleotide sequence shown in SEQ ID NO:25 (or the nucleotide sequence of reporter
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group A fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group.
  • the above-mentioned reporter-F is 5'-AGA CCA A AA CCA CCT ATT TTT CATGT-FAM-3'; the reporter-Q is 5'-Iowa Black FQ-ACATG AAA AAT AGG TGG TT- 3'.
  • the present invention also provides a probe for the detection of cancer-related gene mutation BRAF-V600E (for example, mutated from the sequence shown in SEQ ID NO: 26 to the sequence shown in SEQ ID NO: 27), comprising: reporter-F and reporter-F -Q, wherein reporter-F has the nucleotide sequence shown in SEQ ID NO:30 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:30), reporter- Q has the nucleotide sequence shown in SEQ ID NO:31 (or the nucleotide sequence of reporter-Q consists of the nucleotide sequence shown in SEQ ID NO:31).
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group A fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group.
  • the above-mentioned reporter-F is 5'-ATC GAG A TT TCT CTG TAG CTA CATGT-FAM-3'; reporter-Q is 5'-Iowa Black FQ-ACATG TAG CTA CAG AGA AA- 3'.
  • the present invention also provides a probe for the detection of cancer-related gene mutation EGFR-G719A (for example, from the sequence shown in SEQ ID NO:32 to the sequence shown in SEQ ID NO:33), comprising: reporter-F and reporter -Q, wherein reporter-F has the nucleotide sequence shown in SEQ ID NO:36 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:36), reporter- Q has the nucleotide sequence shown in SEQ ID NO:37 (or the nucleotide sequence of reporter-Q consists of the nucleotide sequence shown in SEQ ID NO:37).
  • reporter-F has the nucleotide sequence shown in SEQ ID NO:36 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:36)
  • reporter- Q has the nucleotide sequence shown in SEQ ID NO:37 (or the nucleotide sequence of reporter-
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group A fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group.
  • the above-mentioned reporter-F is 5'-FAM-TGTAC CGC ACC GGA GGC CA G CAC TTT-3'; reporter-Q is 5'-T GGC CTC CGG TGC G GTACA-Iowa Black FQ -3'.
  • the present invention also provides a probe for the detection of cancer-related gene mutation EGFR-L858R (for example, from the sequence shown in SEQ ID NO: 38 to the sequence shown in SEQ ID NO: 39), comprising: reporter-F and reporter-F -Q, wherein reporter-F has the nucleotide sequence shown in SEQ ID NO:42 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:42), reporter- Q has the nucleotide sequence shown in SEQ ID NO:43 (or the nucleotide sequence of reporter-Q consists of the nucleotide sequence shown in SEQ ID NO:43).
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group A fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group.
  • the above-mentioned reporter-F is 5'-ACA GAT T TT GGG CGG GCC AAA CATGT A-FAM-3'; reporter-Q is 5'-Iowa Black FQ-T ACATG T TTG GCC CGC CCA A–3'.
  • the present invention also provides a probe for detection of cancer-related gene mutation EGFR-L861Q (for example, from the sequence shown in SEQ ID NO: 44 to the sequence shown in SEQ ID NO: 45), comprising: reporter-F and reporter-F -Q, wherein reporter-F has the nucleotide sequence shown in SEQ ID NO:48 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:48), reporter- Q has the nucleotide sequence shown in SEQ ID NO:49 (or the nucleotide sequence of reporter-Q consists of the nucleotide sequence shown in SEQ ID NO:49).
  • reporter-F has the nucleotide sequence shown in SEQ ID NO:48 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:48)
  • reporter- Q has the nucleotide sequence shown in SEQ ID NO:49 (or the nucleotide sequence of reporter
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group Fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group.
  • the above-mentioned reporter-F is 5'-FAM-TGTAC GGC CAA ACA GCT GG G TGC G-3'; reporter-Q is 5'-CCA GCT GTT TGG CC GTACA-Iowa Black FQ- 3'.
  • the present invention also provides a probe for detection of cancer-related gene mutation KRAS-G12A (for example, from the sequence shown in SEQ ID NO:50 to the sequence shown in SEQ ID NO:51), comprising: reporter-F and reporter -Q, wherein reporter-F has the nucleotide sequence shown in SEQ ID NO:54 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:54), reporter- Q has the nucleotide sequence shown in SEQ ID NO:55 (or the nucleotide sequence of reporter-Q consists of the nucleotide sequence shown in SEQ ID NO:55).
  • reporter-F has the nucleotide sequence shown in SEQ ID NO:54 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:54)
  • reporter- Q has the nucleotide sequence shown in SEQ ID NO:55 (or the nucleotide sequence of reporter-Q
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group A fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group.
  • the above-mentioned reporter-F is 5'-FAM-TGTAC TTG CCT ACG CCA GC A GCT C-3'; reporter-Q is 5'-GCT GGC GTA GGC AA GTACA-Iowa Black FQ- 3'.
  • the present invention also provides a probe for detection of cancer-related gene mutation KRAS-G13V (for example, from the sequence shown in SEQ ID NO: 56 to the sequence shown in SEQ ID NO: 57), comprising: reporter-F and reporter-F -Q, wherein reporter-F has the nucleotide sequence shown in SEQ ID NO:60 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:60), reporter- Q has the nucleotide sequence shown in SEQ ID NO:61 (or the nucleotide sequence of reporter-Q consists of the nucleotide sequence shown in SEQ ID NO:61).
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group A fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group.
  • the above-mentioned reporter-F is 5'-FAM-TGTAC TTG CCT ACG ACA CC A GCT C-3'; reporter-Q is 5'-GGT GTC GTA GGC AA GTACA-Iowa Black FQ- 3'.
  • the present invention also provides a probe for detection of cancer-related gene mutation PIK3CA-H1047R (for example, from the sequence shown in SEQ ID NO:62 to the sequence shown in SEQ ID NO:63), comprising: reporter-F and reporter-F -Q, wherein reporter-F has the nucleotide sequence shown in SEQ ID NO:66 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:66), reporter- Q has the nucleotide sequence shown in SEQ ID NO:67 (or the nucleotide sequence of reporter-Q consists of the nucleotide sequence shown in SEQ ID NO:67).
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group A fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group.
  • the above-mentioned reporter-F is 5'-CA GCC A CC ATG ACG TGC ATC CATGT-FAM-3'; reporter-Q is 5'-Iowa Black FQ-ACATG GAT GCA CGT CAT GG- 3'.
  • the present invention also provides a probe for the detection of cancer-related gene mutation STK11-F354L (for example, from the sequence shown in SEQ ID NO:68 to the sequence shown in SEQ ID NO:69), comprising: reporter-F and reporter-F -Q, wherein reporter-F has the nucleotide sequence shown in SEQ ID NO:72 (or the nucleotide sequence of reporter-F consists of the nucleotide sequence shown in SEQ ID NO:72), reporter- Q has the nucleotide sequence shown in SEQ ID NO:73 (or the nucleotide sequence of reporter-Q consists of the nucleotide sequence shown in SEQ ID NO:73).
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group A fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group.
  • the above-mentioned reporter-F is 5'-TTG GAC A TC GAG GAT GAC ATC CATGT-FAM-3'; reporter-Q is 5'-Iowa Black FQ-GTACA GAT GTC ATC CTC GA- 3'.
  • the present invention also provides a kit for detecting single nucleotide variation in Trichuris trichiura, which comprises the above-mentioned probe for detecting single nucleotide variation in Trichuris trichiura of the present invention.
  • the above-mentioned kit also includes DEP-1 and DEP-2, wherein the nucleotide sequences of DEP-1 and DEP-2 are the same as the partial sequence of the single-stranded DNA to be tested, the sequences of the two do not overlap, and DEP- 1.
  • the sequence combination of DEP-2 is the complete sequence or partial sequence of the single-stranded DNA to be tested (ie DEP-1+DEP-2 ⁇ single-stranded DNA to be tested).
  • DEP-1 has the nucleotide sequence shown in SEQ ID NO:3 (or DEP-1 is composed of the nucleotide sequence shown in SEQ ID NO:3)
  • DEP-2 has the nucleotide sequence shown in SEQ ID NO:3 : the nucleotide sequence shown in SEQ ID NO: 4 (or DEP-2 consists of the nucleotide sequence shown in SEQ ID NO: 4).
  • the above-mentioned kit also includes a standard having (or consisting of) the nucleotide sequence shown in SEQ ID NO: 4.
  • the present invention also provides a kit for detection of HBV single nucleotide variation, which comprises the above-mentioned probe for HBV single nucleotide variation detection of the present invention.
  • the above-mentioned kit also includes DEP-1 and DEP-2, wherein the nucleotide sequences of DEP-1 and DEP-2 are the same as the partial sequence of the single-stranded DNA to be tested, the sequences of the two do not overlap, and DEP- 1.
  • the sequence combination of DEP-2 is the complete sequence or partial sequence of the single-stranded DNA to be tested (ie DEP-1+DEP-2 ⁇ single-stranded DNA to be tested).
  • DEP-1 has (or consists of) a nucleotide sequence as shown in SEQ ID NO: 8
  • DEP-2 has (or consists of) a nucleotide sequence as shown in SEQ ID NO: 9 ).
  • the above-mentioned kit further comprises a standard having (or consisting of) the nucleotide sequence shown in SEQ ID NO:7.
  • the present invention also provides a kit for detecting single nucleotide variation of Trichuris trichiura, comprising: reporter-F1, reporter-Q1, reporter-F2, and reporter-Q2, wherein, reporter- F1 has the nucleotide sequence shown in SEQ ID NO: 16, reporter-Q1 has the nucleotide sequence shown in SEQ ID NO: 17, reporter-F2 has the nucleotide sequence shown in SEQ ID NO: 18 sequence, reporter-Q2 has the nucleotide sequence shown in SEQ ID NO:19.
  • the above-mentioned kit also includes DEP-1 and DEP-2, wherein the nucleotide sequences of DEP-1 and DEP-2 are the same as the internal partial sequence (not the edge) of the single-stranded DNA to be tested, and the sequences of the two are different. overlap, and the sequence combination of DEP-1 and DEP-2 is the internal partial sequence of the single-stranded DNA to be tested (ie DEP-1+DEP-2 ⁇ single-stranded DNA to be tested).
  • DEP-1 has (or consists of) the nucleotide sequence shown in SEQ ID NO:3
  • DEP-2 has (or consists of) the nucleotide sequence shown in SEQ ID NO:4 ).
  • the above kit also includes a pair of primers (DEP-3 and DEP-4), wherein the nucleotide sequence of the forward primer (DEP-3) is the same as the sequence of the edge portion of the single-stranded DNA to be tested, and does not It overlaps with DEP-1 and DEP-2, and the sequence combination of DEP-1, DEP-2 and DEP-3 is the partial sequence of the single-stranded DNA to be tested, and DEP-4 is the complement of the rest of the sequence of the single-stranded DNA to be tested. sequence.
  • DEP-3 has (or consists of) a nucleotide sequence as shown in SEQ ID NO: 14
  • DEP-4 has (or consists of) a nucleotide sequence as shown in SEQ ID NO: 15 ).
  • the above-mentioned reporter-F is labeled with a fluorescent reporter group, and the reporter-Q is labeled with a fluorescent quenching group; for example, the 5' end of reporter-F is labeled with a fluorescent reporter group, and the 3' end of reporter-Q is labeled with a fluorescent reporter group Fluorescence quenching group; or the 3' end of reporter-F is labeled with a fluorescent reporter group, and the 5' end of reporter-Q is labeled with a fluorescence quenching group; and the fluorescent reporter group labeled on reporter-F1 and reporter-F2 Groups are different.
  • the above-mentioned reporter-F1 is 5'-AT GAA GC G CTT TAC GAT ATT TGT TTC CGA-Cy5-3'; reporter-Q is 5'-Iowa Black RQ-TCG GAA ACA AAT ATC GTA AAG C–3'.
  • the above-mentioned reporter-F2 is 5'-G GAC GAA ACA TAC TGC ATA GA CATGT-FAM-3'; reporter-Q is 5'-Iowa Black FQ-ACATG TC TAT GCA GTA TGT- 3'.
  • the above-mentioned kit also includes a standard having (or consisting of) the nucleotide sequence shown in SEQ ID NO: 12.
  • the reporter in the above detection system covers mutation sites and non-mutation sites, which can be used to detect C. trichomoniasis infection, and screen or identify drug resistance of C. trichomes.
  • the present invention also provides a kit for detection of cancer-related gene mutation BRAF-D594G (for example, mutated from the sequence shown in SEQ ID NO: 20 to the sequence shown in SEQ ID NO: 21), which comprises the above-mentioned use in cancer of the present invention Probe for detection of related gene mutation BRAF-D594G.
  • the above-mentioned kit also includes DEP-1 and DEP-2, wherein the nucleotide sequences of DEP-1 and DEP-2 are the same as the partial sequence of the single-stranded DNA to be tested, the sequences of the two do not overlap, and DEP- 1.
  • the sequence combination of DEP-2 is the complete sequence or partial sequence of the single-stranded DNA to be tested (ie DEP-1+DEP-2 ⁇ single-stranded DNA to be tested).
  • DEP-1 has (or consists of) a nucleotide sequence as shown in SEQ ID NO: 22
  • DEP-2 has (or consists of) a nucleotide sequence as shown in SEQ ID NO: 23 ).
  • the above-mentioned kit further comprises a standard having (or consisting of) the nucleotide sequence shown in SEQ ID NO: 20.
  • the present invention also provides a kit for detection of cancer-related gene mutation BRAF-V600E (for example, mutation from the sequence shown in SEQ ID NO: 26 to the sequence shown in SEQ ID NO: 27), which comprises the above-mentioned use in cancer of the present invention.
  • Probe for detection of related gene mutation BRAF-V600E for example, mutation from the sequence shown in SEQ ID NO: 26 to the sequence shown in SEQ ID NO: 27.
  • the above-mentioned kit also includes DEP-1 and DEP-2, wherein the nucleotide sequences of DEP-1 and DEP-2 are the same as the partial sequences of the above-mentioned single-stranded DNA to be tested, and the sequences of the two do not overlap, and DEP -1.
  • the sequence combination of DEP-2 is the complete sequence or partial sequence of the single-stranded DNA to be tested (ie DEP-1+DEP-2 ⁇ single-stranded DNA to be tested).
  • DEP-1 has (or consists of) a nucleotide sequence as shown in SEQ ID NO: 28, and DEP-2 has (or consists of) a nucleotide sequence as shown in SEQ ID NO: 29 ).
  • the above-mentioned kit also includes a standard having (or consisting of) the nucleotide sequence shown in SEQ ID NO: 26.
  • the present invention also provides a kit for detection of cancer-related gene mutation EGFR-G719A (for example, from the sequence shown in SEQ ID NO: 32 to the sequence shown in SEQ ID NO: 33), which comprises the above-mentioned use in cancer of the present invention.
  • Probe for detection of related gene mutation EGFR-G719A for example, from the sequence shown in SEQ ID NO: 32 to the sequence shown in SEQ ID NO: 33.
  • the above-mentioned kit also includes DEP-1 and DEP-2, wherein the nucleotide sequences of DEP-1 and DEP-2 are the same as the partial sequence of the single-stranded DNA to be tested, the sequences of the two do not overlap, and DEP- 1.
  • the sequence combination of DEP-2 is the complete sequence or partial sequence of the single-stranded DNA to be tested (ie DEP-1+DEP-2 ⁇ single-stranded DNA to be tested).
  • DEP-1 has (or consists of) the nucleotide sequence shown in SEQ ID NO: 34
  • DEP-2 has (or consists of) the nucleotide sequence shown in SEQ ID NO: 35 ).
  • the above-mentioned kit also includes a standard having (or consisting of) the nucleotide sequence shown in SEQ ID NO:32.
  • the present invention also provides a kit for detection of cancer-related gene mutation EGFR-L858R (for example, from the sequence shown in SEQ ID NO: 38 to the sequence shown in SEQ ID NO: 39), which comprises the above-mentioned use in cancer of the present invention.
  • Probe for detection of related gene mutation EGFR-L858R for example, from the sequence shown in SEQ ID NO: 38 to the sequence shown in SEQ ID NO: 39.
  • the above-mentioned kit also includes DEP-1 and DEP-2, wherein the nucleotide sequences of DEP-1 and DEP-2 are the same as the partial sequences of the above-mentioned single-stranded DNA to be tested, and the sequences of the two do not overlap, and DEP -1.
  • the sequence combination of DEP-2 is the complete sequence or partial sequence of the single-stranded DNA to be tested (ie DEP-1+DEP-2 ⁇ single-stranded DNA to be tested).
  • DEP-1 has (or consists of) the nucleotide sequence shown in SEQ ID NO:40
  • DEP-2 has (or consists of) the nucleotide sequence shown in SEQ ID NO:41 ).
  • the above-mentioned kit also includes a standard having (or consisting of) the nucleotide sequence shown in SEQ ID NO:38.
  • the present invention also provides a kit for the detection of cancer-related gene mutation EGFR-L861Q (for example, from the sequence shown in SEQ ID NO: 44 to the sequence shown in SEQ ID NO: 45), which comprises the above-mentioned use in cancer of the present invention.
  • Probe for detection of related gene mutation EGFR-L861Q for example, from the sequence shown in SEQ ID NO: 44 to the sequence shown in SEQ ID NO: 45.
  • the above-mentioned kit also includes DEP-1 and DEP-2, wherein the nucleotide sequences of DEP-1 and DEP-2 are the same as the partial sequence of the single-stranded DNA to be tested, the sequences of the two do not overlap, and DEP- 1.
  • the sequence combination of DEP-2 is the complete sequence or partial sequence of the single-stranded DNA to be tested (ie DEP-1+DEP-2 ⁇ single-stranded DNA to be tested).
  • DEP-1 has (or consists of) the nucleotide sequence shown in SEQ ID NO: 46
  • DEP-2 has (or consists of) the nucleotide sequence shown in SEQ ID NO: 47 ).
  • the above-mentioned kit further comprises a standard having (or consisting of) the nucleotide sequence shown in SEQ ID NO:44.
  • the present invention also provides a kit for the detection of cancer-related gene mutation KRAS-G12A (for example, from the sequence shown in SEQ ID NO:50 to the sequence shown in SEQ ID NO:51), which comprises the above-mentioned use in cancer of the present invention.
  • Probe for detection of related gene mutation KRAS-G12A for example, from the sequence shown in SEQ ID NO:50 to the sequence shown in SEQ ID NO:51.
  • the above-mentioned kit also includes DEP-1 and DEP-2, wherein the nucleotide sequences of DEP-1 and DEP-2 are the same as the partial sequence of the single-stranded DNA to be tested, the sequences of the two do not overlap, and DEP- 1.
  • the sequence combination of DEP-2 is the complete sequence or partial sequence of the single-stranded DNA to be tested (ie DEP-1+DEP-2 ⁇ single-stranded DNA to be tested).
  • DEP-1 has (or consists of) the nucleotide sequence shown in SEQ ID NO: 52
  • DEP-2 has (or consists of) the nucleotide sequence shown in SEQ ID NO: 53 ).
  • the above-mentioned kit further comprises a standard having (or consisting of) the nucleotide sequence shown in SEQ ID NO:50.
  • the present invention also provides a kit for detection of cancer-related gene mutation KRAS-G13V (for example, from the sequence shown in SEQ ID NO: 56 to the sequence shown in SEQ ID NO: 57), which comprises the above-mentioned use in cancer of the present invention.
  • Probes for detection of related gene mutation KRAS-G13V are provided.
  • the above-mentioned kit also includes DEP-1 and DEP-2, wherein the nucleotide sequences of DEP-1 and DEP-2 are the same as the partial sequence of the single-stranded DNA to be tested, the sequences of the two do not overlap, and DEP- 1.
  • the sequence combination of DEP-2 is the complete sequence or partial sequence of the single-stranded DNA to be tested (ie DEP-1+DEP-2 ⁇ single-stranded DNA to be tested).
  • DEP-1 has (or consists of) the nucleotide sequence shown in SEQ ID NO: 58
  • DEP-2 has (or consists of) the nucleotide sequence shown in SEQ ID NO: 59 ).
  • the above-mentioned kit further comprises a standard having (or consisting of) the nucleotide sequence shown in SEQ ID NO:56.
  • the present invention also provides a kit for detection of cancer-related gene mutation PIK3CA-H1047R (for example, from the sequence shown in SEQ ID NO: 62 to the sequence shown in SEQ ID NO: 63), which comprises the above-mentioned use for cancer of the present invention Probe for detection of related gene mutation PIK3CA-H1047R.
  • the above-mentioned kit also includes DEP-1 and DEP-2, wherein the nucleotide sequences of DEP-1 and DEP-2 are the same as the partial sequence of the single-stranded DNA to be tested, the sequences of the two do not overlap, and DEP- 1.
  • the sequence combination of DEP-2 is the complete sequence or partial sequence of the single-stranded DNA to be tested (ie DEP-1+DEP-2 ⁇ single-stranded DNA to be tested).
  • DEP-1 has (or consists of) the nucleotide sequence shown in SEQ ID NO:64
  • DEP-2 has (or consists of) the nucleotide sequence shown in SEQ ID NO:65 ).
  • the above-mentioned kit also includes a standard having (or consisting of) the nucleotide sequence shown in SEQ ID NO:62.
  • the present invention also provides a kit for detection of cancer-related gene mutation STK11-F354L (for example, from the sequence shown in SEQ ID NO: 68 to the sequence shown in SEQ ID NO: 69), which comprises the above-mentioned use for cancer of the present invention Probe for detection of related gene mutation STK11-F354L.
  • the above-mentioned kit also includes DEP-1 and DEP-2, wherein the nucleotide sequences of DEP-1 and DEP-2 are the same as the partial sequence of the single-stranded DNA to be tested, the sequences of the two do not overlap, and DEP- 1.
  • the sequence combination of DEP-2 is the complete sequence or partial sequence of the single-stranded DNA to be tested (ie DEP-1+DEP-2 ⁇ single-stranded DNA to be tested).
  • DEP-1 has (or consists of) a nucleotide sequence as shown in SEQ ID NO:70
  • DEP-2 has (or consists of) a nucleotide sequence as shown in SEQ ID NO:71 ).
  • the above-mentioned kit also includes a standard having (or consisting of) the nucleotide sequence shown in SEQ ID NO:68.
  • the above-mentioned kit of the present invention may further comprise Mg 2+ , especially 0-10 mM (eg 0, 1.25, 2.5, 5, 10 mM) of Mg 2+ .
  • the above-mentioned kit of the present invention also includes a nucleotide protective agent, such as Tween 20, for preventing the potential loss of DNA oligonucleotides during dilution and pipetting; wherein, the content of the nucleotide protective agent can be 0.1 %-0.5% (v/v).
  • a nucleotide protective agent such as Tween 20
  • the content of the nucleotide protective agent can be 0.1 %-0.5% (v/v).
  • the above-mentioned kit of the present invention further comprises a buffer system, such as Tris buffer.
  • the above kit comprises: 1 mM Mg 2+ , 0.1% Tween 20 (v/v), 1 ⁇ Tris buffer, and corresponding probes and DEP.
  • the above-mentioned kit of the present invention may further comprise a negative control substance, which is a system without DNA to be tested, such as H 2 O (such as sterile double-distilled water, sterile deionized water, etc.).
  • a negative control substance which is a system without DNA to be tested, such as H 2 O (such as sterile double-distilled water, sterile deionized water, etc.).
  • the above-mentioned kit of the present invention may further comprise DNA extraction reagents and materials for extracting the DNA of the sample to be tested, and any suitable reagents and materials for DNA extraction known in the prior art may be used.
  • the above-mentioned kit may further include a pretreatment reagent for the sample to be tested, and the pretreatment reagent may be a reagent known in the prior art for pretreatment of samples for DNA extraction, for example, physiological saline and the like.
  • the present invention also provides the application of the above detection method, probe and kit in the detection of DNA single nucleotide variation.
  • the present invention also provides the above detection method, probe and kit, which are used for DNA single nucleotide variation.
  • the present invention also provides the application of the above detection method, probe and kit in detecting pathogen infection.
  • the present invention also provides the above detection method, probe and kit, which are used for detecting pathogen infection.
  • the above-mentioned pathogens may be microorganisms, parasites or other agents.
  • the above-mentioned microorganisms can be selected from one or more of viruses, chlamydia, rickettsia, mycoplasma, bacteria, spirochetes, fungi and the like.
  • the above-mentioned pathogen is a virus, such as, but not limited to, Adenoviridae (such as adenovirus), Herpesviridae (such as HSV1 (oral herpes), HSV2 (external genital herpes), VZV (varicella pox) ), EBV (Epstein-Barr virus), CMV (cytomegalovirus)), Poxviridae (such as variola virus, vaccinia virus), Lapaviridae (such as papillomavirus), Parvoviridae (such as B19 virus), hepadnaviridae (such as hepatitis B virus), polyomaviridae (such as polyoma virus), reoviridae (such as reovirus, rotavirus), picornaviridae (such as Such as enteroviruses, foot-and-mouth disease virus), caliciviridae (such as Norwalk virus, he
  • the above-mentioned pathogen is a parasite, such as, but not limited to, roundworm, whipworm, pinworm, hookworm, tapeworm, Entamoeba histolytica, Trichomonas vaginalis, liver fluke, hydatid , Paragonimus weisseri, Cysticercus suis, Toxoplasma gondii, Schistosoma, Trichinella, Filariasis, Plasmodium, Leishmania, sucking nematodes, mites, lice, ticks, etc.
  • a parasite such as, but not limited to, roundworm, whipworm, pinworm, hookworm, tapeworm, Entamoeba histolytica, Trichomonas vaginalis, liver fluke, hydatid , Paragonimus weisseri, Cysticercus suis, Toxoplasma gondii, Schistosoma, Trichinella, Filariasis, Plasmodium, Leishmania,
  • the present invention also provides the application of the above detection method, probe and kit in screening or identifying drug resistance of parasites.
  • the present invention also provides the above-mentioned detection method, probe and kit, which are used for screening or identifying drug resistance of parasites.
  • the above-mentioned parasite is C. trichomes.
  • the present invention also provides applications of the above detection methods, probes, and kits in disease diagnosis and disease risk assessment.
  • the present invention also provides the above-mentioned detection method, probe and kit, which are used for disease diagnosis and disease risk assessment.
  • the present invention also provides the application of the above probes in the preparation of products (eg, kits) for disease diagnosis and disease risk assessment.
  • the above-mentioned disease is a malignant tumor, including, but not limited to, lymphoma, blastoma, medulloblastoma, retinoblastoma, sarcoma, liposarcoma, synovial cell sarcoma, neuroendocrine Tumor, carcinoid tumor, gastrinoma, islet cell carcinoma, mesothelioma, schwannoma, acoustic neuroma, meningioma, adenocarcinoma, melanoma, leukemia or lymphoid malignancy, squamous cell carcinoma, epithelial squamous cell carcinoma Squamous cell carcinoma, lung cancer, small cell lung cancer, non-small cell lung cancer, adenocarcinoma lung cancer, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric cancer, colon cancer, pancreatic cancer, glioblastoma
  • the present invention also provides a method for disease diagnosis and disease risk assessment, which includes the steps of using the above-mentioned detection method, probe, and kit of the present invention.
  • the present invention discloses a new method for simulating and guiding the design of nucleic acid hybridization probes - DNA Equalizer Gate (DEG), which greatly expands the A detection window for distinguishing single nucleotide variations in double-stranded DNA (dsDNA).
  • DEG DNA Equalizer Gate
  • the invention also discloses a thermodynamically driven theoretical model for quantitatively simulating and predicting the performance of DEG.
  • the effectiveness of DEG for extending the detection window and improving sequence selectivity is demonstrated through computer simulations and experimental verifications. Since DEG acts directly on dsDNA, it is readily applicable to nucleic acid amplification techniques such as polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the utility of DEG was demonstrated through infection detection and drug resistance screening of clinical parasite samples collected from rural areas of Honduras.
  • the detection method provided by the present invention does not require the use of high-cost reagents such as enzymes and relatively high requirements for reaction conditions. Therefore, the method is simple in operation, low in
  • Figure 1 shows a schematic diagram of the experimental steps and DNA reactions in DEG.
  • Figure 2 shows optimization of asymmetric PCR using different ratios between forward and reverse primer concentrations; kinetic curves show measurements of ssDNA output generated by asymmetric PCR using a reporter probe using toehold exchange operate.
  • Figure 3 shows a schematic representation of DEG.
  • a Overall workflow for quantification of dsDNA using DEG. Using autonomous molecular computing, a mixture of target dsDNA and DEP is heated and rapidly cooled in a test tube to generate a controlled amount of ssDNA output. A fluorescent signal is then generated by the reporter probe.
  • the dsDNA target (AB) is denatured into A and B during heating and rapid cooling. The competition between DEP (C and D) and A for hybridization with B then occurs during renaturation. The net amount of ssDNA output (A) was quantitatively determined by an autonomic computational process that compared the initial concentration between target and DEP.
  • c Overall workflow for quantification of dsDNA using DEG. Using autonomous molecular computing, a mixture of target dsDNA and DEP is heated and rapidly cooled in a test tube to generate a controlled amount of ssDNA output. A fluorescent signal is then generated by the reporter probe.
  • the dsDNA target (AB) is
  • Figure 4 shows the simulation results obtained by DEG for an enlarged detection window (top) and an increase in the energy barrier for activation of the toehold exchange probe (bottom).
  • An increase in the energy barrier can be achieved by extending the length of the reverse toehold by 2 bp.
  • Figure 5 shows the theoretical model of DEG.
  • a Schematic representation of all possible elemental reactions occurring in the DEG.
  • c In silico prediction of A and AB yields based on dsDNA target concentration without probabilistic correction.
  • d A schematic diagram illustrating the need for probability correction when [AB]>[DEP].
  • Figure 6 shows the simulation results of DEG.
  • reaction yields as a function of target concentration and ⁇ G 0 for typical toehold-exchange (a) and DEG at DEP concentrations of 50, 100, 200 and 500 nM.
  • the yield of false targets is significantly suppressed over a wide concentration range, which can help improve specificity and expand detection range.
  • the detection window for distinguishing SNVs can be adjusted by changing the concentration of DEP.
  • the use of DEG dramatically increases the RF value from finite to infinite.
  • Figure 7 shows the experimental validation of DEG.
  • c Robustness factor plotted against target concentration and compared to computer simulations.
  • d Schematic representation of target and DEP sequences. Single nucleotide mutations were made to the target at positions 1, 6, 14 and 17. e.
  • Figure 8 shows the theoretical concentration dependence of the discrimination factor (DF).
  • DF discrimination factor
  • Figure 9 shows the dependence of RF on reaction yield and target concentration.
  • a Theoretical prediction of RF value as a function of reaction yield ⁇ .
  • b Absolute concentration difference between false and true target as a function of yield.
  • c RF as a function of target concentration.
  • d Absolute concentration difference between false and true target as a function of target concentration at the same yield.
  • e RF corrected using LOD and LOL.
  • Figure 10 shows the detection window of nucleic acid hybridization probes.
  • df In silico analysis of yields, DFs and RFs of all possible mutations with ⁇ G0 of 0-5 kcal/mol.
  • Figure 11 shows theoretical predictions for the yield of ssDNA output A from dsDNA input AB using DEG.
  • a Output ssDNA (A) and input dsDNA (AB) concentrations as a function of initial input concentration. The probability function is the key to ensuring the accuracy of the model.
  • b Yield of ssDNA (A) as a function of initial input concentration.
  • Figure 13 shows the determination of theoretical and experimental RF.
  • the theoretical RF was determined by extracting the concentrations of a pair of true and false targets that produced the same yield by Matlab software.
  • the experimental RF was determined by first fitting the experimental data to a calibration curve using a 4-parameter nonlinear model, and then using Matlab to extract the concentrations of a pair of true and false targets. RF at each concentration was then calculated using Equation 6 and plotted as a function of target (true target) concentration.
  • Figure 14 shows the effect of characterizing DEP and thermal scheme on DEG performance.
  • a Real-time fluorescence monitoring of the kinetics of the reporter gate to measure the X + produced by DEG.
  • b. Yield of each reaction based on A measured by endpoint fluorescence at 20 minutes. Yields were calculated by setting the fluorescence of the positive control to 1.
  • c. Detailed reactants and experimental procedures in each sample or control. Each sample was incubated at 37°C with 10 nM AB, 20 nM reporter and 200 nM DEP in IX Tris buffer (1 mM Mg 2+ and 0.1% Tween 20 (v/v)). Each error bar represents one standard deviation from replicate analyses.
  • Figure 15 shows the effect of DEP on DEG performance.
  • a Real-time fluorescence monitoring of the kinetics of the reporter gate to measure DEG production of A.
  • b Yield of each reaction based on A measured by endpoint fluorescence at 20 minutes.
  • c Detailed reactants and experimental procedures in each sample or control. Each error bar represents one standard deviation of replicate analyses.
  • Figure 16 shows the optimization of the thermal scheme for the denaturation and renaturation process.
  • Maximum yields were established when all DNA material was premixed in the same tube and heated, followed by rapid cooling to 4°C a.
  • the addition of DEP before (pre) or after (post) the thermal protocol was found to significantly affect the yield of A Rate.
  • the rapid cooling step was also found to be critical to ensure high yields of A.
  • Maximum yield was obtained when rapidly cooled to 4°C as the final temperature. Increasing the final temperature to 25, 55 and 75°C was found to gradually decrease the yield of the reaction.
  • Figure 17 shows the effect of Mg 2+ on DEG performance. Each error bar represents one standard deviation of replicate analyses.
  • Figure 18 shows the stability of export ssDNA A produced by DEG. Each error bar represents one standard deviation of replicate analyses.
  • Figure 19 shows an estimate of signal leakage from DEP.
  • a Schematic illustration of signal leakage caused by the interaction between DEP and reporter molecule.
  • b Estimated leakage (blank) as a function of DEP concentration, also compared to target specific fluorescence (sample). The target concentration was fixed at 10 nM, and the concentration of DEP varied from 10 nM to 5 ⁇ M. No fluorescence leakage was observed even when 1 ⁇ M DEP was applied. This indicates that there is no cross-reaction between DEP and the probe, and thus no competition between DEP and probe prior to ssDNA export. Each error bar represents one standard deviation of replicate analyses.
  • Figure 20 shows the use of DEG to estimate the LOD used to detect AB.
  • the LOD was estimated to be 0.5 nM. Error bars represent one standard deviation of replicate analyses.
  • Figure 21 is a schematic diagram showing the analysis of single nucleotide mutations in the T. trichomes (TT) subgenome.
  • TT T. trichomes
  • Figure 22 shows the experimental validation of DEGs used to identify single nucleotide T>A, T>G and T>C mutations in the 42-bp dsDNA TT target. Each error bar represents one standard deviation of replicate analyses.
  • Figure 23 shows a comparison of experimentally determined yields to yields predicted by simulations using DEG to analyze T>G and T>C mutations of double-stranded TT targets.
  • Figure 24 shows a comparison of experimentally measured and simulated DF using DEG for T>G and T>C mutations in double-stranded TT targets.
  • Figure 25 shows a comparison of experimental measurements and simulated RF using DEG for T>G and T>C mutations in double-stranded TT targets.
  • Figure 26 shows experimental measurements of DF for the target TT-28 with different mutations.
  • Figure 27 shows experimentally measured RF for the target TT-28 with different mutations.
  • Figure 28 is a schematic diagram showing the analysis of single nucleotide mutations in the HBV S gene subgenome.
  • Figure 29 shows the detection of synthetic HBV targets with various mutations and indels using DEG.
  • the shaded area represents the detection window where DEG outperforms toehold exchange beacons in discriminating the most challenging SNV27G.
  • Figure 30 shows DF values experimentally measured using DEG for three single nucleotide mutations (SNV27C, SNV27T and SNV27G) in the 44 bp HBV target.
  • the shaded area represents the detection window where DEG outperforms toehold exchange beacons in discriminating the most challenging SNV27G.
  • Figure 31 shows RF values experimentally measured using DEG for single nucleotide mutations in the 44bp HBV target.
  • Figure 32 shows the designs and sequences for nine clinically important single nucleotide variants commonly found in cancer.
  • Figure 33 shows experimentally measured yields, DF and RF for analysis of BRAF-D594G, BRAF-V600E, EGFR-G7119A, EGFR-L858R and EGFR-L861Q.
  • concentration of DEG was fixed at 200 nM.
  • Figure 34 shows experimentally measured yields, DF and RF for analysis of KRAS-G12A, KRAS-G13V, PIK3CA-H1047R and STK11-F354L.
  • concentration of DEG was fixed at 200 nM.
  • Figure 35 shows that DEG can effectively detect as low as 0.5% mutated targets against a background of high concentrations of unmutated sequence.
  • Figure 36 shows the results of simultaneous manipulation of TT and HBV targets using two sets of DEP in the same tube.
  • Figure 37 shows the sequences of different TT targets and corresponding DEP lengths to verify the target/DEP length effect.
  • Figure 38 shows experimentally measured yields, DF and RF of dsDNA target lengths (from 87 bp to 32 bp) using the corresponding DEP at a concentration of 200 nM.
  • Figure 39 shows experimentally measured yield, DF and RF of the target TT-32 using DEP at concentrations of 50, 100 and 200 nM, respectively.
  • Figure 40 shows integration of DEG with PCR.
  • a Schematic representation of dsDNA analysis using the 4-DEP design.
  • b Experimental validation of the 4-DEP design to detect 87bp dsDNA as a mimic for PCR amplicons. The concentration of external DEP was fixed at 500 nM and the concentration of internal DEP was set to 200 nM.
  • c Schematic representation of DEG-PCR designed using 4-DEP. The outer two DEPs were designed to be identical to PCR primers.
  • d Real-time monitoring of DEG-PCR using toehold exchange reporter molecules. A wide detection window was achieved, clearly distinguishing true templates down to 10 aM from 1 pM false targets containing single nucleotide mutations.
  • e Real-time monitoring of DEG-PCR using toehold exchange reporter molecules. A wide detection window was achieved, clearly distinguishing true templates down to 10 aM from 1 pM false targets containing single nucleotide mutations.
  • Figure 41 shows a schematic diagram of the sequence design of a set of four DEPs. These DEPs target the 87bp amplicon (AB) to detect hotspots of resistance in TT worms.
  • AB 87bp amplicon
  • a. Scheme shows 4-DEP design of PCR amplicons.
  • b. Design two internal DEPs to expose ssDNA domains that can be detected by reporter probes.
  • ssDNA export A
  • 2 external DEPs were designed with the same sequence as a pair of forward and reverse PCR primers.
  • c Design of reporter probes operated by toehold exchange.
  • Figure 42 shows validation of DEG detection of ssDNA output A by 4-DEP design validation.
  • Figure 43 shows DEG analysis of PCR amplicons using internal DEP with DEP concentrations ranging from 50 nM to 200 nM. showed that DEG allows detection of double-stranded PCR amplicons while identifying single nucleotide mutations over a wide concentration range.
  • Figure 44 shows the application of DEG-PCR in the analysis of clinical parasite samples.
  • a Typical workflow for analysis of parasite (Trichuris trichiura, TT) samples collected from fecal samples of school-aged children in rural Honduras, followed by detection using DEG-PCR.
  • b Simultaneous detection of parasitic infection and drug resistance screening using a dual channel design (FAM-Reporter and Cy5-Reporter).
  • PCR primers were designed to amplify nucleotides 1246-1333 in the beta-tubulin gene containing the 200th codon. The single nucleotide A to T mutation of this codon is a hotspot for drug resistance screening.
  • a toehold exchange reporter labeled with FAM was used to distinguish the point mutation, while a strand displacement reporter without reverse toehold (Cy5-reporter) was used to detect the conserved region around codon 200.
  • D.R.+ and D.R.- were defined using an error eclipse with 99% confidence interval and 2 degrees of freedom (two fluorescence channels).
  • Eight clinical helminth specimens including 6 T. trichomes (TT-1 to TT-6) and 2 Ascaris (AL, as negative control)) were tested and plotted in d.
  • Figure 45 shows the sequence design of DEG-PCR for analysis of clinical parasite samples.
  • a pair of primers were designed to amplify the ⁇ -tubulin gene from 1246 bp to 1333 bp by the primer design software BLAST.
  • a FAM-reporter (green) was designed to detect specific A to T mutations at codon 200 at bp 1271 to 1299 of the ⁇ -tubulin gene.
  • Cys 5-reporter (red) was designed to analyze bp 1301-13320 of the ⁇ -tubulin gene.
  • b Representative fluorescence kinetic curves indicating positive infection (red) and positive resistance (green, D.R.+).
  • d Representative fluorescence kinetic curves showing no infection and no drug resistance.
  • Figure 46 shows a schematic diagram of the sequence design of DEP and two reporter molecules.
  • Figure 47 shows the validation of 4-DEP-dual reporter DEG for detection of double-stranded TT targets with A to T mutations at codon 200 of ⁇ -tubulin.
  • AB double-stranded TT targets
  • Figure 48 shows the detection limit of 4-DEP-dual reporter DEG for analysis of synthetic DNA targets (resistance-positive (D.R.+) resistance mutants or resistance-negative (D.R.-) wild-type).
  • a Normalized fluorescence in FAM and Cys5 channels as a function of target concentration for detection of resistance-positive mutants.
  • b Fluorescence ratio at FAM and Cys5 channels as a function of target concentration.
  • c Dual channel fluorescence profiles of targets (D.R.+ or D.R.-) at 0.16, 0.31, 0.62, 1.25, 2.5, 5, 10, 20, 40 and 80 nM.
  • the detection limit for drug-resistant positive targets was 0.62 nM
  • the detection limit for drug-resistant negative targets was 1.25 nM.
  • the gray shaded areas in the subplots indicate fluorescence distributions that cannot differentiate between D.R+ and D.R.- targets.
  • Figure 49 shows the detection limit of 4-DEP-dual reporter DEG for analysis of synthetic DNA targets (using 800 nM DEP).
  • a Normalized fluorescence in FAM and Cys5 channels as a function of target concentration for detection of resistance-positive mutants.
  • b Fluorescence ratio at FAM and Cys5 channels as a function of target concentration.
  • c Dual channel fluorescence profiles of targets (D.R.+ or D.R.-) at 0.16, 0.31, 0.62, 1.25, 2.5, 5, 10, 20, 40 and 80 nM.
  • the detection limit of drug resistance positive target and negative negative target were both 0.62nM.
  • the gray shaded areas in the subplots indicate fluorescence distributions that cannot differentiate between D.R+ and D.R.- targets.
  • Figure 50 shows the detection of drug-resistant mutants in the presence of various concentrations of wild-type.
  • a Normalized fluorescence intensity of FAM and Cys5 channels as a function of percentage of spiked mutants in wild-type controls. The total target concentration was fixed at 20 nM.
  • b Experimental and theoretical calibration curves using the FAM/Cy5 ratio as readout.
  • c Experimentally calibrated linear regression.
  • Figure 51 shows the analysis of clinical parasite samples using dual reporter DEG-PCR.
  • a Normalized fluorescence intensity of FAM and Cys5 channels as a function of the original concentration of synthesized DNA template before PCR amplification. This template has the same subgenomic sequence as the drug-resistant mutant.
  • b Normalized fluorescence intensities of FAM and Cys5 channels for clinical parasite samples, including 6 Trichosanthes (TT) samples and 2 Ascaris (AL) samples. All TT samples were positive for infection and negative for drug resistance; while two AL samples showed negative for TT infection. Negative fluorescence intensity indicates that the fluorescence signal of AL is lower than that of blank. Each error bar represents one standard deviation of replicate analyses.
  • Figure 52 shows the results of detection of clinical parasite samples using standard PCR followed by polyacrylamide gel electrophoresis (PAGE) analysis.
  • PAGE polyacrylamide gel electrophoresis
  • Figure 53 shows genome sequencing data for clinical parasites.
  • the first row shows the DNA sequence from codon 186 to codon 214 of the wild-type C. trichomes ⁇ -tubulin gene. Codons 198 and 200 are highlighted as resistance mutation hotspots.
  • the sequences of the six worm samples taken from the patients were consistent with wild-type, which is in good agreement with the diagnostic results measured using DEG-PCR.
  • DNA oligonucleotides used in the examples of the present invention were purchased from Integrated DNA Technologies (IDT, Coralville, IA). DNA oligonucleotides of fluorophores (FAM- and Cy5-) and quencher (Iowa Blank) were purified by high performance liquid chromatography (HPLC). Other DNA material was used without purification. The sequences and modifications of the oligonucleotides used are listed in the table below.
  • TE tris-EDTA
  • 1 ⁇ TE buffer containing 10 mM MgCl 2 and 0.5% (v/v) TWEEN 20 (Sigma) was used as molecular reporter buffer.
  • TWEEN 20 is used to prevent potential loss of DNA oligonucleotides during dilution and pipetting.
  • SDR strand displacement
  • TER toehold exchange
  • the free energies of DNA strands and complexes are estimated by NUPACK.
  • the temperature was set to 4°C (in an ice-water bath), the Na + concentration was 0.1 M, and the Mg2+ concentration was 0.001 M; while the temperature of the DNA species in the toehold exchange reaction was set to 37°C.
  • Other parameters use default settings.
  • An ice-water cooling bath (4°C) was prepared in advance. Mix the double-stranded DNA target and the DNA probe at a user-defined concentration in a 0.2 mL PCR tube, adjusting the volume to 100 ⁇ L. The sample tubes were then placed in a thermal cycler (Bio-Rad T100TM) and heated to 95°C for 5 minutes (set to 10 minutes for the next step). While the samples were kept hot in the thermal cycler, the tubes were quickly transferred and immersed in an ice-water bath (4°C) for 2 minutes ( Figure 1). 90 ⁇ L of sample was transferred to a microplate (Corning) and heated in a microplate reader (Molecular Devices) set at 37°C for 5 minutes. Thereafter, 10 ⁇ L of 200 nM toehold exchange reporter was added to trigger the reaction.
  • PCR In a typical PCR protocol, mix 4 ⁇ L of DNA template, 20 ⁇ L of Taq 2 ⁇ Master Mix, and forward and reverse primers at appropriate concentrations (usually 500 nM) to 40 ⁇ L. PCR was started by incubating at 94°C for 3 minutes, followed by 35 cycles (denaturation at 94°C, annealing at 52°C and extension at 72°C for 30 seconds), and finally extension at 72°C for 30 minutes. 5 min in a Bio-Rad T100TM thermal cycler. The thermal protocol for asymmetric PCR remained unchanged, while primer concentrations were unbalanced (500 nM forward primer and 40 nM reverse primer, Figure 2).
  • the PCR amplicons were then mixed with 4 DEPs and the volume was adjusted to 90 ⁇ L.
  • Real-time fluorescence data was acquired using a SpectraMax i3 microplate reader (Molecular Devices). The temperature was set to 37°C and the fluorescence was monitored for 1 hour at a frequency of 1 data point per minute.
  • the excitation/emission wavelength of the FAM channel was set to 485nm/515nm, and the excitation/emission wavelength of the Cy5 channel was set to 640nm/675nm.
  • STH worm samples were recovered from eight school-aged children in the rural area of La Chicaca in northwestern Honduras. Ethical approval was obtained from the National Autonomous University of Honduras and Brock University. Eight participants received a regimen based on pyrantel pamoate and ocantenamate (Conmetel) on the first three days and albendazole on the fourth day. Adult worms excreted in feces were washed with saline solution and stored in 70% ethanol. After recovery of the samples, DNA was extracted using the Automate Express DNA extraction system (Thermo Fisher Scientific Inc.) and the commercial kit PrepFiler Express BTA according to the manufacturer's protocol. Thereafter, these clinical DNA samples were tested following a typical DEG-PCR procedure (250 nM per PCR primer; 200 nM per probe).
  • DNA templates were synthesized from two batches (two replicates of each batch) from 1 aM to 1 pM (containing D.R.(-) and D.R.(+)) by the same DEG-PCR protocol and clinical samples to establish fluorescence profiles. Furthermore, to simulate a heterozygous genotype with a D.R.(+) mutation in only one chromosome, WT and MT synthetic DNA templates were mixed equally at final concentrations of 1 aM to 1 pM. The complete fluorescence distribution map is shown in Figure 10. An error eclipse was used instead of a linear fit curve due to batch-to-batch PCR errors.
  • PCR amplicon solution 5 ⁇ L was mixed with loading buffer (Bio-Rad) and then loaded onto an 8% native PAGE gel to validate and evaluate the PCR procedure. A voltage of 110V was used to drive electrophoresis. Afterwards, the gel was stained with ethidium bromide and imaged using the Gel Doc XR+ imager system (Bio-Rad).
  • DEGs are designed to suppress the detection signal of false targets by inverting the quantitative relationship between detection signal and target concentration, thereby maximizing the detection window for distinguishing single nucleotide variants.
  • RF robustness factor
  • RF the larger the RF value, the wider the detection window.
  • DEG acts on dsDNA, detection of single-stranded DNA (ssDNA) is also possible. And when single-stranded DNA (ssDNA) is detected, the concentration of DNA equalizer probe (DEP) is close to infinity.
  • the design of the DEG is shown in Figure 3.
  • the double-stranded input AB was rapidly heated at 95°C and then rapidly cooled to 0°C in a splitter gate, yielding single-stranded target A and its complement B (Fig. 3c).
  • B is consumed by DEP, which has the same sequence as A, and DEP is divided into two or more parts in annihilator gate 1 ( ⁇ 1, Fig. 3d). Therefore, the yield ( ⁇ ) of A was quantitatively determined by the concentration of DEP.
  • the concentration of AB is less than that of DEP, A is the main product, although there is competition between A and DEP for hybridization with B.
  • thermodynamically driven model successfully predicted the distribution of A and AB over a concentration range where [AB] ⁇ [DEP] (Fig. 5c). However, it cannot simulate the thermodynamic behavior of DEG when [AB] ⁇ [DEP].
  • the inventors then corrected the model by introducing a probability function that considered the possible distribution of DEP on AB (Fig. 5d). Mathematically, the probability of successful formation of a DEP-B triplex (BCD) is [DEPs] 0 /[AB] 0 ) 2 (Fig. 5d).
  • the combination of the thermodynamically driven model and the probabilistic correction resulted in a characteristic asymmetric unimodal curve (Fig. 5e), which was also confirmed experimentally.
  • ⁇ , DF, and RF were first quantitatively analyzed in silico for three key factors in DEG, including target concentration, sequence design ( ⁇ G 0 ), and defined by DEP. detection window.
  • the detection of ssDNA can also be described in the model of the present invention by setting the concentration of DEP to infinity, where the yield of A is 100%.
  • frustrating probes Toeholdexchange or molecular beacons
  • Concentration is an important variable for evaluating the sensitivity and specificity of DNA hybridization probes, however the concentration dependence of yield, discrimination factor (DF) and robustness factor (RF) remains unexplored systematically.
  • the inventors first analyzed the concentration dependence of hybridization yield and sequence specificity over a wide range of concentrations. In the inventor's system, the toehold exchange probe was chosen as the test platform. To highlight the numerical relationship between the variables, the inventors applied a dimensionless transformation to all concentrations prior to derivation.
  • the toehold exchange reaction can be simplified to a bimolecular reversible reaction (Equation 1):
  • thermodynamics of toehold exchange probes can be tuned by varying the length of the forward and reverse toeholds or by controlling the stoichiometry between CP and P.
  • the free energy of each reactant and product can be calculated using NUPACK software.
  • the equilibrium constant can follow the law of conservation of mass from the reaction free energy (Equation 2) and the concentration of all nucleic acid species.
  • the inventors further performed dimensionless processing of the formula for converting the concentration into a numerical value, wherein the target concentration in the dimensionless form is expressed as ⁇ and the target concentration of [P] 0 is expressed as ⁇ (Equation 3).
  • Equation 4 The concentration dependence of ⁇ is solved by Equation 4:
  • DF discrimination factor
  • the DF values for a pair of true and false targets are a function of sequence design ( ⁇ G and Keq) and target concentration ⁇ (Equations 5, 6).
  • Figure 8 shows the mathematical prediction of the DF of the true target for the five false targets as a function of target concentration ( ⁇ ).
  • the DF values of all pseudotargets containing single nucleotide mutations decreased monotonically with increasing target concentration ⁇ .
  • the simulated DF value reaches a maximum in the low target concentration range when the yield of the false target is close to a minimum.
  • LOD limit of detection
  • the yield and DF values become meaningless.
  • the simulations show that the absolute difference in yield between true and false targets becomes so small in the concentration range ⁇ ⁇ 0.6 that it is difficult to resolve experimentally (Fig. 8b).
  • LOD can be arbitrarily defined as the minimum yield that allows an experiment to distinguish signal from background from true or false targets.
  • Figure 8c shows a simulation of DF using the modified model when false targets became undetectable (LOD set to 1% yield). Indeed, the inventors could also set the LOD to be the minimum detectable yield of the true target.
  • RF robustness factor
  • DEGs are designed to convert dsDNA targets to ssDNA output in a quantitative manner with a well-defined detection window.
  • the inventors assumed that all reactions are thermodynamically driven and that all DNA species are in their thermodynamically stable states. Under this assumption, a set of equilibrium equations can be used to predict the concentration distribution of the newly formed DNA species (the main content in Fig. 5a). However, only the independent equations need to be solved, otherwise meaningless answers will be produced.
  • RM numerical response matrix
  • RM The order of RM is 4 (verified by Matlab), which is less than the dimension of RM. Therefore, there are only 4 independent reaction formulas in this reaction system, and the inventors selected the first 4 reactions in the model. Use NUPACK to predict all value, the balance equation is as follows:
  • [A] 0 , [B] 0 , [C] 0 , and [D] 0 are initial concentrations
  • Figure 11 shows the mathematical prediction of the yield of each DNA species at different target concentrations. Without correction using the probability function, the yield of output DNA (A) decreased linearly as a function of input target concentration (dashed lines in Figures 11a and 11b). With the probability correction, a sharp transition occurs when [target] equals [DEP], which is also confirmed experimentally. This transition is determined solely by the concentration of DEP, thus allowing the detection window to be defined in Figure 11c and suppressing the signal of false targets as shown in Figure S5d.
  • the combination of the DEG model with the classical toehold exchange model allowed the inventors to accurately model the yield and discrimination factors for the true target and any given mutation.
  • the reaction yield was first converted to the ssDNA output concentration using the toehold exchange model, and then the ssDNA concentration was converted to the dsDNA target concentration using the DEG model.
  • the detection window that has been established for identifying single nucleotide mismatches can be expanded (Figure 12 to the left). As shown by the simulation results in FIG. 12 .
  • the inventors' DEG method performs better at scale (essentially to infinity) and is more sensitive at low concentration ranges.
  • the stability results of the output DNA A produced by DEG are shown in Figure 18. Once produced by DEG, B is blocked by DEP and thus cannot react with B by renaturation. For accurate quantification of X by DEG and reporter molecules, it is critical to ensure the stability of free A in the reaction mixture.
  • the inventors monitored the concentration in solution A after DEG reaction at room temperature for 5 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours and 24 hours, respectively. The results show that A is highly stable with no apparent loss during the first 2 h. In fact, the inventors analyzed A with a reporter probe within the first 30 minutes.
  • the LOD results for detecting X using DEG estimation are shown in Figure 20.
  • the results showed that the LOD was estimated to be 0.5 nM when 200 nM DEP was used to generate single-stranded output A.
  • the inventors used two sets of synthetic targets, namely the 42bp subgenome of the ⁇ -tubulin gene of the parasite Trichuris trichiura (TT) and the 44bp subgenome sequence of the hepatitis B virus (HBV) S gene, to test The analytical performance and versatility of DEGs to identify single nucleotide variants. Both diseases are major threats to human health worldwide. Different types of mutations and insertions/deletions are tested using the DEG detection platform of the present invention. The inventors also demonstrated the possibility of DEG multiplexing by mixing the two sets of targets and the corresponding DEPs into the same tube.
  • Figure 21 shows a schematic diagram for the analysis of single nucleotide mutations in the T. trichomes (TT) subgenome.
  • the purpose of designing a double-stranded synthetic HBV S gene target was to test the versatility of the DEG approach. As shown in Figure 28, a pair of DEP and reporter probes were designed for this synthetic target. Single-nucleotide mutations and base insertions/deletions were introduced in this system and tested using DEG. To validate the DEG method for identifying challenging single-nucleotide mutations, the inventors deliberately introduced the A to G mutation, a well-known challenging SNV because the formation of G-T wobble reduces true target interaction with Free energy difference between fake targets. The inventors found that the DEG approach effectively improved the specificity and concentration robustness of the analysis of this challenging SNV compared to direct analysis using toehold exchange beacons (highlighted in Figures 29-31).
  • Results evaluating DEGs for detection of rare mutations are shown in Figure 35.
  • the results show that using DEG can effectively detect as low as 0.5% of mutated targets in the background of high concentrations of unmutated sequences.
  • the DNA hybridization probe used in practice should be compatible with commonly used nucleic acid amplification techniques such as PCR. Because DEG acts directly on dsDNA, it is an ideal probe for analyzing dsDNA amplicons. Therefore, the inventors went on to demonstrate the adaptability of DEG to PCR. As a proof of principle, a set of four DEPs was designed for a representative 87 bp dsDNA amplicon (Figure 40a), which was shown to be fully compatible with DEGs. To avoid potential cross-reactivity, the two outer DEPs were purposely designed to be identical to PCR primers ( Figure 40c).
  • FIG. 41 A schematic diagram of the sequence design of a set of four DEPs is shown in Figure 41. These DEPs target the 87bp amplicon (T) to detect hotspots of resistance in TT worms.
  • T 87bp amplicon
  • PCR amplicons were analyzed using DEGs with DEP concentrations ranging from 50 nM to 500 nM as shown in Figure 43.
  • DEG allows detection of double-stranded PCR amplicons while identifying single-nucleotide mutations over a wide concentration range.
  • Kinetic curves show measurements of ssDNA output generated by asymmetric PCR using a reporter probe using a toehold exchange operation.
  • the first reporter (FAM-reporter) operating by the principle of toehold exchange was designed to target a specific A to T mutation at codon 200 of ⁇ -tubulin, a well-established TT-resistant benzimidazole (BZ , drugs) hot spots. Therefore, the fluorescence of this reporter molecule (FAM) is turned on only when resistance develops in TT infection (D.R.+TT infection).
  • a second reporter (Cys-reporter) operated by toehold-mediated strand displacement was designed to detect TT infection.
  • the reporter has a reverse toehold of 0 and is therefore insensitive to single-nucleotide mutations, which ensures that infection can be detected regardless of the presence of the SNP. Simultaneous detection of two fluorescence channels (FAM and Cys) allows detection of infection and screening for drug resistance in a single assay.
  • the inventors used DEG-PCR to diagnose soil-transmitted helminth (STH) infection using clinical samples collected from school-age children living in the highly endemic rural area of Honduras.
  • the inventors used DEG-PCR to detect STH infection while screening for resistance in the same assay ( Figure 44a).
  • Two fluorescent reporter molecules were designed to test codons 196 to 203 and codons 206 to 213 of the ⁇ -tubulin gene of C. trichomes ( Figure 44b).
  • the single nucleotide A to T mutation at codon 200 of beta tubulin is a well-established genetic variation for drug resistance screening (Figure 45).
  • the toehold-exchange reporter that tested this domain was designed to be highly sensitive to this SNV, whereas the reporter targeting codons 206 to 213 was not designed to reverse toehold.
  • the two reporter molecules were labeled with spectrally distinct fluorescent dyes (FAM and Cy5) and thus operated simultaneously in solution ( Figures 46 and 47).

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Abstract

一种用于DNA单核苷酸变异检测的方法、探针及试剂盒及其应用。方法通过用户可定义的检测信号和靶标浓度之间的定量关系转换，极大地扩展了用于区分双链DNA中单核苷酸变异的检测窗口。通过计算机模拟和实验验证，方法用于扩展检测窗和改进序列选择性的有效性得到证明。由于方法直接作用于dsDNA，因此容易适用于核酸扩增技术，如聚合酶链式反应(PCR)。通过对从Honduras农村地区收集的临床寄生虫样品进行感染检测和耐药性筛选，证实了其实用性。另外，方法中不需要使用酶等成本高、对反应条件要求比较高的试剂，因此，方法操作简单，成本较低，便于企业和实验室应用。

Description

一种用于DNA单核苷酸变异检测的方法、探针及试剂盒及其应用 技术领域
本发明涉及生物检测技术领域，具体涉及一种用于DNA单核苷酸变异检测的方法、探针及试剂盒及其应用。
背景技术
单核苷酸变异(single nucleotide variation，SNV)因其与疾病易感性、药物反应差异性、人类进化及种群多样性等相关而广受关注。SNV是指在基因组中单个核苷酸的变异，包括置换、颠换、缺失和插入。SNV如果位于编码区可能会影响所编码的氨基酸从而影响蛋白质功能。一般而言，人群中变异频率小于1％的SNV称为点突变，而变异频率不小于1％的SNV称为SNP，目前已在dbSNP存放的人基因组SNP位点数目已超过900万个。
SNV用作遗传标记具有以下优点：(1)SNV在人群中是二等位基因性的，在任何人群中其等位基因频率都可估计出来；(2)它在基因组中的分布广泛；(3)与串联重复的微卫星位点相比，SNV是高度稳定的，尤其是处于编码区的SNV，而前者的高突变率容易引起对人群的遗传分析出现困难；(4)部分位于基因内部的SNV可能会直接影响产物蛋白质的结构或基因表达水平，因此，它们本身可能就是疾病遗传机制的候选改变位点；(5)易于进行自动化分析，缩短了研究时间。由于SNV具有以上优点，其在分子诊断、临床检验、法医学、病原检测、药效评估、新药研发和群体进化等方面具有重要应用价值。
目前检测SNV的传统技术主要基于4种基本原理：内切酶酶切技术、等位基因特异性杂交、引物延伸和寡核苷酸连接技术。
基于酶切技术的检测方法：
包括限制性内切酶片段长度多态性和引物入侵分析技术等。限制性内切酶是一类识别DNA特异位点，并在特异位点进行切割的酶类。该技术的前提是待检测的SNP位点的两侧需含有限制性内切酶识别序列。此法简便、快速，可进行大量样本的分析，缺点是不能直接分析DNA的排列顺序，且有近半数的SNP位点并不导致酶切位点的改变。
基于等位基因特异性杂交的检测方法：
其反应原理是碱基的互补配对原则，在杂交法中，针对变异位点及侧翼序列设计探针，通常探针间只有一个碱基的差异，分别对应不同的等位基因。将所有序列特异的寡核苷酸探针经过处理后，点样在尼龙膜、玻片或硅片等固相或液相载体上，与待检测的DNA模板进行杂交，根据检测到的信号进行SNV的分型。包括DNA芯片检测技术、TaqMan探针技术、等位基因特异性寡核苷酸杂交和分子信标技术等。该方法具有很高的特异性和灵敏性，可用于有基因变异的遗传病的基因诊断和筛选，在有固定点突变的筛选中具有重要价值，如对p53基因、人乳腺癌基因1和2突变热点的筛选。但必须己知待检基因的缺陷，才能有针对性地合成变异的寡核苷酸探针，且分型费用相对较高。
基于引物延伸的检测方法：
该方法的反应原理是首先扩增出含有变异位点的一段DNA，然后一条寡核苷酸引物直接在待检测碱基的上游或下游发生退火，通过加入不同荧光标记的dNTP或ddNTP进行一个或几个碱基的延伸，根据延伸反应的信号来确定位点信息。主要有单碱基延伸技术和焦测序技术等。该方法的优点是检测的敏感度和分辨率均较高，但其缺点是必须使用多色荧光系统以及相应的检测系统，而且多种染料的激发光和发射荧光的光谱往往会有较大部分的重叠，从而干扰对荧光强度的测量精度。
基于寡核苷酸连接反应的检测方法：
两条相邻的寡核苷酸序列与模板退火，只有当其在接合处与模板完全匹配时才会在连接酶的作用下连接在一起，因而等位基因特异性寡核苷酸序列连接技术能探察单碱基变异位点的性质。寡核苷酸连接分析于1994年由Samiotaki等人建立，其原理是将PCR产物变性为单链，然后加入两条探针A和B(约20个核苷酸长)，A和B的序列分别与目标DNA中变异位点两旁的序列互补配对，A的5'端与B的3’端相邻。如果两条相邻探针与目标DNA单链完全互补配对结合时，在DNA连接酶的作用下，两条探针 的5'端磷酸基和3'羟基形成磷酸二酯键而连接起来。
如果B探针的3’端存在错配碱基的话，则连接反应不发生。连接产物变性后，可以作为引物的模板，重复上述变性-复性-连接反应循环后，根据特殊的测量手段测量信号，来判断是否发生连接反应，并以此作为模板DNA单链是否存在碱基变化的依据。寡核苷酸连接分析技术的优点是仅需使用1/10常用量的DNA样本，对DNA内部序列进行评价，且检测结果不受PCR扩增反应中非特异性产物的影响。此外，结果可直接传入计算机储存和统计分析，自动化检测，大大提高了检测效率。缺点是必须已知变异位点所在，才能有针对性地合成探针，而且存在多个位点时，需要合成多个探针进行检测。
上述内切酶酶切技术、等位基因特异性杂交、引物延伸和寡核苷酸连接技术存在如操作程序复杂，灵敏性不足，检测通量低或DNA样本需求量大等缺点。而理想的检测方法应该具有高准确度、操作简单、高通量和低成本的特点，因此探索和建立高效且检测成本低的SNV的检测方法仍然十分必要。
发明内容
为克服现有技术的不足，本发明提供一种用于DNA单核苷酸变异检测的方法，其包括：
将待测DNA与反应体系混合，加热解旋，然后快速冷却，检测。
其中，待测DNA的两条链分别记为A链、B链，待测链为A链。
具体地，上述加热解旋温度为80-99℃(具体如80、82、84、86、88、90、91、92、93、94、95、96、97、98、99℃)；在本发明的一个实施例中，该温度为95℃。
具体地，上述加热时间为1-10分钟(具体如1、2、3、4、5、6、7、8、9、10分钟)；在本发明的一个实施例中，该时间为5分钟。
具体地，上述快速冷却可以为在1-5分钟内(例如1、2、3、4或5分钟内)冷却至0-4℃(例如0、1、2、3、4℃)；在本发明的一个实施例中，上述快速冷却为在2分钟内冷却至4℃。
具体地，上述检测在快速冷却后2小时内(例如2小时内，1小时内，30分钟内，15分钟内，5分钟内)进行。
具体地，上述反应体系包含：n条DNA平衡探针(DNA equalizer probe，DEP)：
DEP-1，…，DEP-n；
其中，n为大于1的整数(例如2、3、4、5、6、7、8、9、10)；
DEP-1，…，DEP-n的核苷酸序列与待测单链DNA的部分序列相同，且均不重叠，且DEP-1，…，DEP-n的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+…+DEP-n≤待测单链DNA)。
在本发明的一个实施例中，n＝2。
在本发明的一个实施例中，DEP-1，…，DEP-n的序列组合为待测单链DNA的完整序列(即DEP-1+…+DEP-n＝待测单链DNA)。
在本发明的一个实施例中，n＝2，且DEP-1、DEP-2的核苷酸序列与待测单链DNA的部分序列相同，且DEP-1、DEP-2的序列组合为待测单链DNA的完整序列(即DEP-1+DEP-2＝待测单链DNA)。
具体地，DEP与待测DNA的摩尔比为1-500:1(例如1:1、2:1、5:1、10:1、20:1、30:1、40:1、50:1、60:1、70:1、80:1、90:1、100:1、200:1、300:1、400:1、500:1)。
具体地，上述反应体系还可包含Mg 2+，特别是0-10mM(例如0、1.25、2.5、5、10mM)的Mg 2+。
具体地，上述反应体系还包含核苷酸保护剂，如Tween 20，用于防止稀释和移液过程中DNA寡核苷酸的潜在损失；其中，核苷酸保护剂的含量可以为0.1％-0.5％(v/v)。
具体地，上述反应体系还包含缓冲体系，如Tris缓冲液。
具体地，上述检测包括如下步骤：将快速冷却的混合物在35-40℃(例如35、36、37、38、39、40℃)下加热1-10分钟(例如1、2、3、4、5、6、7、8、9、10分钟)，添加检测单链DNA的分子探针触发检测反应；在本发明的一个实施例中，上述检测包括：将快速冷却的混合物转移到微孔板中，并在设置为37℃的微孔板读数器中加热5分钟，添加检测单链DNA的分子探针。其中，该检测探针可以经过荧光标记，上述检测还包括收集检测反应达到平衡时的荧光数据的步骤。
具体地，上述检测探针包含：2m个报告探针(reporter)：
reporter-F 1，
reporter-Q 1，
……
reporter-F m，
reporter-Q m；
其中，m为大于等于1的整数(例如1、2、3、4、5、6、7、8、9、10)；
各reporter-F的核苷酸序列由序列F-S1和F-S2组成，其中，F-S1的核苷酸序列与待测单链DNA的部分序列互补，F-S2为与待测单链DNA无关的任意序列(其不与待测单链DNA互补，长度可以为1-10个核苷酸)；
各reporter-Q的核苷酸序列与相应的reporter-F的核苷酸序列互补，且reporter-Q的核苷酸序列长度小于相应的reporter-F的核苷酸序列长度；
且各F-S1序列不同且不重叠。
具体地，待测单链DNA中与各F-S1序列互补的序列间隔至少1个(例如1个，2个，3个，4个，5个)核苷酸。
具体地，上述各F-S1序列中至少一个与待测单链DNA中待检测位点(根据已公布的信息，可能发生单核苷酸变异而引起所要关注的问题(例如产生耐药性、引起疾病)的位点)互补。
具体地，各F-S1序列的长度分别独立地为15-30个核苷酸(例如15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30个核苷酸)。
具体地，各F-S2序列的长度分别独立地为1-10个核苷酸(例如1、2、3、4、5、6、7、8、9、10个核苷酸)，例如TGTAC，CGCTT。
具体地，各F-S2序列各自独立，可以相同或不同。
具体地，各reporter-Q的序列长度分别独立地为15-25个核苷酸(例如15、16、17、18、19、20、21、22、23、24、25个核苷酸)。
具体地，各个reporter-Q的序列长度与reporter-Q的序列长度相差5-10个核苷酸(例如5、6、7、8、9、10个核苷酸)。
具体地，上述各reporter通过荧光标记；其中，各reporter-F标记有荧光报告基团，各reporter-Q标记有荧光淬灭基团，例如，reporter-F的5’端标记有荧光报告基团，其相应的reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，其相应的reporter-Q的5’端标记有荧光淬灭基团。
具体地，上述荧光报告基团可以为，例如，FAM、Texas Red、ROX、TET、VIC、JOE、HEX、Cy3、Cy3.5、Cy5、Cy5.5、LC RED640、LC RED705等。
具体地，上述荧光淬灭基团可以为，例如，Iowa Black、TAMRA、DABCYL、ECLIPSE、BHQ1、BHQ2、BHQ3等。
具体地，上述各reporter-F上标记的荧光报告基团各不相同。
在本发明的一个实施例中，m＝1。
在本发明的另一个实施例中，m＝2。
具体地，上述各个reporter与待测DNA的摩尔比为1-500:1(例如1:1、2:1、5:1、10:1、20:1、30:1、40:1、50:1、60:1、70:1、80:1、90:1、100:1、200:1、300:1、400:1、500:1)。
在本发明的一个实施方式中，上述方法还可以包括将待测DNA通过核酸扩增技术(如聚合酶链式反应(PCR))进行扩增的步骤。
具体地，上述方法还包括对标准品(序列与无变异的待测DNA相同，可通过人工合成)进行检测的步骤(与待测DNA相同的检测步骤)。
具体地，上述检测可以为定性检测，也可以为定量检测。
在本发明的一个实施方式中，上述检测为定性检测，可通过将待测DNA样品与等量的标准品的检测信号(例如荧光信号)强弱对比进行。
具体地，上述方法包括建立标准曲线的步骤，例如，利用标准品的量与相应的标准品的检测信号(例如荧光信号)建立标准曲线。
在本发明的一个实施方式中，上述检测为定量检测，可以通过测得的待测DNA样品的检测信号(例如荧光信号)，利用标准曲线，计算出待测DNA样品中无变异DNA的量，进而得到该样品中变异DNA的量。
在本发明的一个实施方式中，上述检测为定量检测，检测信号为荧光信号，可以通过将荧光数据归一化并根据公式η＝((F-F b))/((F m-F b))转化成表观杂交产率，其中F为处于平衡状态的样品荧光读数，F m表示对于50倍过量的真ssDNA靶标至链置换信标观察到的最大荧光，F b为仅由受保护信标产生的背景荧光。
具体地，本方法适用于不同长度范围的靶标，上述待测DNA的长度可以为，例如，32-87bp。
具体地，上述单核苷酸变异可以为取代(取代为A、T、C、G中的一个)、插入(插入A、T、C、G中的一个)或缺失。
具体地，上述方法可同时检测同一待测DNA中一个或多个单核苷酸变异位点，也可以同时检测多个不同待测DNA。
具体地，上述方法检测的受试者可以为寄生虫(例如毛首鞭形线虫(Trichuris trichiura)、蛔虫(Ascaris lumbricoides))、病毒(例如HBV)或动物(如哺乳动物(例如人类))。
本发明还提供一种用于DNA单核苷酸变异检测的探针，其包含：2m个报告探针(reporter)：
reporter-F 1，
reporter-Q 1，
……
reporter-F m，
reporter-Q m；
其中，m为大于等于1的整数(例如1、2、3、4、5、6、7、8、9、10)；
各reporter-F的核苷酸序列由序列F-S1和F-S2组成，其中，F-S1的核苷酸序列与待测单链DNA的部分序列互补，F-S2为与待测单链DNA无关的任意序列(其不与待测单链DNA互补，长度可以为1-10个核苷酸)；
各reporter-Q的核苷酸序列与相应的reporter-F的核苷酸序列互补，且reporter-Q的核苷酸序列长度小于相应的reporter-F的核苷酸序列长度；
且各F-S1序列不同且不重叠。
具体地，上述报告探针具有本发明上述相应定义。
本发明还提供一种用于DNA单核苷酸变异检测的试剂盒，其包含本发明上述用于DNA单核苷酸变异检测的探针。
具体地，上述试剂盒还包含：n条DNA平衡探针(DNA equalizer probe，DEP)：
DEP-1，…，DEP-n；
其中，n为大于1的整数(例如2、3、4、5、6、7、8、9、10)；
DEP-1，…，DEP-n的核苷酸序列与待测单链DNA的部分序列相同，且均不重叠，且DEP-1，…，DEP-n的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+…+DEP-n≤待测单链DNA)。
具体地，上述DEP具有本发明上述相应定义。
具体地，上述试剂盒还可包含Mg 2+，特别是0-10mM(例如0、1.25、2.5、5、10mM)的Mg 2+。
具体地，上述试剂盒还包含核苷酸保护剂，如Tween 20，用于防止稀释和移液过程中DNA寡核苷酸的潜在损失；其中，核苷酸保护剂的含量可以为0.1％-0.5％(v/v)。
具体地，上述试剂盒还包含缓冲体系，如Tris缓冲液。
在本发明的一个实施例中，上述检测体系包含：1mM Mg 2+，0.1％Tween 20(v/v)，1×Tris缓冲液，DEP，报告探针。
具体地，上述试剂盒还包含标准品，其序列与无变异的待测DNA相同，可通过人工合成。
具体地，上述试剂盒还可包含阴性对照品，其为不含待测DNA的体系，例如H 2O(如无菌双蒸水、无菌去离子水等)。
具体地，上述试剂盒还可以包含DNA提取试剂和材料，用于提取待测样品DNA，可以采用现有技术已知任何合适的用于DNA提取的试剂和材料。
具体地，必要时，上述试剂盒还可以包含待测样品预处理试剂，该预处理试剂可以采用现有技术已知用于预处理样品以便于DNA提取的试剂，例如，生理盐水等。
本发明还提供一种用于毛首鞭形线虫(Trichuris trichiura)单核苷酸变异(例如从SEQ ID NO:1所示序列突变为SEQ ID NO:2所示序列)检测的探针，其包含：reporter-F和reporter-Q；其中，reporter-F具有如SEQ ID NO:5所示的核苷酸序列(或reporter-F的核苷酸序列由SEQ ID NO:5所示的核苷酸序列组成)，reporter-Q具有如SEQ ID NO:6所示的核苷酸序列(或reporter-Q的核苷酸序列由SEQ ID NO:6所示的核苷酸序列组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团。
在本发明的一个实施例中，上述reporter-F为5’–G GAC GAA ACA TAC TGC ATA GA CATGT–FAM–3’；reporter-Q为5’–Iowa Black FQ–ACATG TC TAT GCA GTA TGT–3’。
本发明还提供一种用于HBV单核苷酸变异检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:10所示的核苷酸序列(或reporter-F的核苷酸序列由SEQ ID NO:10 所示的核苷酸序列组成)，reporter-Q具有如SEQ ID NO:11所示的核苷酸序列(或reporter-Q的核苷酸序列由SEQ ID NO:11所示的核苷酸序列组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团。
在本发明的一个实施例中，上述reporter-F为5’–FAM–CGCTT AGG TTG GTG AGT GATT GG AGG TT–3’；reporter-Q为5’–A ATC ACT CAC CAA CCT AAGCG–Iowa Black FQ–3’。
本发明还提供一种用于毛首鞭形线虫(Trichuris trichiura)单核苷酸变异(例如从SEQ ID NO:12所示序列突变为SEQ ID NO:13所示序列)检测的探针，其包含：reporter-F1、reporter-Q1、reporter-F2、reporter-Q2，其中，reporter-F1具有如SEQ ID NO:16所示的核苷酸序列(或reporter-F1的核苷酸序列由SEQ ID NO:16所示的核苷酸序列组成)，reporter-Q1具有如SEQ ID NO:17所示的核苷酸序列(或reporter-Q1的核苷酸序列由SEQ ID NO:17所示的核苷酸序列组成)，reporter-F2具有如SEQ ID NO:18所示的核苷酸序列(或reporter-F2的核苷酸序列由SEQ ID NO:18所示的核苷酸序列组成)，reporter-Q2具有如SEQ ID NO:19所示的核苷酸序列(或reporter-Q2的核苷酸序列由SEQ ID NO:19所示的核苷酸序列组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团；且reporter-F1和reporter-F2上标记的荧光报告基团不同。
在本发明的一个实施例中，上述reporter-F1为5’–AT GAA GC G CTT TAC GAT ATT TGT TTC CGA-Cy5–3’；reporter-Q为5’–Iowa Black RQ–TCG GAA ACA AAT ATC GTA AAG C–3’。
本发明还提供一种用于癌症相关基因突变BRAF-D594G(例如从SEQ ID NO:20所示序列突变为SEQ ID NO:21所示序列)检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:24所示的核苷酸序列(或reporter-F的核苷酸序列由SEQ ID NO:24所示的核苷酸序列组成)，reporter-Q具有如SEQ ID NO:25所示的核苷酸序列(或reporter-Q的核苷酸序列由SEQ ID NO:25所示的核苷酸序列组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团。
在本发明的一个实施例中，上述reporter-F为5'–AGA CCA A AA CCA CCT ATT TTT CATGT-FAM–3'；reporter-Q为5'–Iowa Black FQ-ACATG AAA AAT AGG TGG TT–3'。
本发明还提供一种用于癌症相关基因突变BRAF-V600E(例如从SEQ ID NO:26所示序列突变为SEQ ID NO:27所示序列)检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:30所示的核苷酸序列(或reporter-F的核苷酸序列由SEQ ID NO:30所示的核苷酸序列组成)，reporter-Q具有如SEQ ID NO:31所示的核苷酸序列(或reporter-Q的核苷酸序列由SEQ ID NO:31所示的核苷酸序列组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团。
在本发明的一个实施例中，上述reporter-F为5'–ATC GAG A TT TCT CTG TAG CTA CATGT-FAM–3'；reporter-Q为5'–Iowa Black FQ-ACATG TAG CTA CAG AGA AA–3'。
本发明还提供一种用于癌症相关基因突变EGFR-G719A(例如从SEQ ID NO:32所示序列突变为SEQ ID NO:33所示序列)检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:36所示的核苷酸序列(或reporter-F的核苷酸序列由SEQ ID NO:36所示的核苷酸序列组成)，reporter-Q具有如SEQ ID NO:37所示的核苷酸序列(或reporter-Q的核苷酸序列由SEQ ID NO:37所示的核苷酸序列组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团。
在本发明的一个实施例中，上述reporter-F为5'–FAM-TGTAC CGC ACC GGA GGC CA G CAC TTT–3'；reporter-Q为5'–T GGC CTC CGG TGC G GTACA-Iowa Black FQ–3'。
本发明还提供一种用于癌症相关基因突变EGFR-L858R(例如从SEQ ID NO:38所示序列突变为SEQ ID NO:39所示序列)检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:42所示的核苷酸序列(或reporter-F的核苷酸序列由SEQ ID NO:42所示的核苷酸序列组成)，reporter-Q具有如SEQ ID NO:43所示的核苷酸序列(或reporter-Q的核苷酸序列由SEQ ID NO:43所示的核苷酸序列组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团。
在本发明的一个实施例中，上述reporter-F为5'–ACA GAT T TT GGG CGG GCC AAA CATGT A-FAM–3'；reporter-Q为5'–Iowa Black FQ-T ACATG T TTG GCC CGC CCA A–3'。
本发明还提供一种用于癌症相关基因突变EGFR-L861Q(例如从SEQ ID NO:44所示序列突变为SEQ ID NO:45所示序列)检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:48所示的核苷酸序列(或reporter-F的核苷酸序列由SEQ ID NO:48所示的核苷酸序列组成)，reporter-Q具有如SEQ ID NO:49所示的核苷酸序列(或reporter-Q的核苷酸序列由SEQ ID NO:49所示的核苷酸序列组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团。
在本发明的一个实施例中，上述reporter-F为5'–FAM-TGTAC GGC CAA ACA GCT GG G TGC G–3'；reporter-Q为5'–CCA GCT GTT TGG CC GTACA-Iowa Black FQ–3'。
本发明还提供一种用于癌症相关基因突变KRAS-G12A(例如从SEQ ID NO:50所示序列突变为SEQ ID NO:51所示序列)检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:54所示的核苷酸序列(或reporter-F的核苷酸序列由SEQ ID NO:54所示的核苷酸序列组成)，reporter-Q具有如SEQ ID NO:55所示的核苷酸序列(或reporter-Q的核苷酸序列由SEQ ID NO:55所示的核苷酸序列组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团。
在本发明的一个实施例中，上述reporter-F为5'–FAM-TGTAC TTG CCT ACG CCA GC A GCT C–3'；reporter-Q为5'–GCT GGC GTA GGC AA GTACA-Iowa Black FQ–3'。
本发明还提供一种用于癌症相关基因突变KRAS-G13V(例如从SEQ ID NO:56所示序列突变为SEQ ID NO:57所示序列)检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:60所示的核苷酸序列(或reporter-F的核苷酸序列由SEQ ID NO:60所示的核苷酸序列组成)，reporter-Q具有如SEQ ID NO:61所示的核苷酸序列(或reporter-Q的核苷酸序列由SEQ ID NO:61所示的核苷酸序列组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团。
在本发明的一个实施例中，上述reporter-F为5'–FAM-TGTAC TTG CCT ACG ACA CC A GCT C–3'；reporter-Q为5'–GGT GTC GTA GGC AA GTACA-Iowa Black FQ–3'。
本发明还提供一种用于癌症相关基因突变PIK3CA-H1047R(例如从SEQ ID NO:62所示序列突变为SEQ ID NO:63所示序列)检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:66所示的核苷酸序列(或reporter-F的核苷酸序列由SEQ ID NO:66所示的核苷酸序列组成)，reporter-Q具有如SEQ ID NO:67所示的核苷酸序列(或reporter-Q的核苷酸序列由SEQ ID NO:67所示的核苷酸序列组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团。
在本发明的一个实施例中，上述reporter-F为5'–CA GCC A CC ATG ACG TGC ATC CATGT-FAM–3'；reporter-Q为5'–Iowa Black FQ-ACATG GAT GCA CGT CAT GG–3'。
本发明还提供一种用于癌症相关基因突变STK11-F354L(例如从SEQ ID NO:68所示序列突变 为SEQ ID NO:69所示序列)检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:72所示的核苷酸序列(或reporter-F的核苷酸序列由SEQ ID NO:72所示的核苷酸序列组成)，reporter-Q具有如SEQ ID NO:73所示的核苷酸序列(或reporter-Q的核苷酸序列由SEQ ID NO:73所示的核苷酸序列组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团。
在本发明的一个实施例中，上述reporter-F为5'–TTG GAC A TC GAG GAT GAC ATC CATGT-FAM–3'；reporter-Q为5'–Iowa Black FQ-GTACA GAT GTC ATC CTC GA–3'。
本发明还提供一种用于毛首鞭形线虫(Trichuris trichiura)单核苷酸变异检测的试剂盒，其包含本发明上述用于毛首鞭形线虫单核苷酸变异检测的探针。
具体地，上述试剂盒还包含DEP-1、DEP-2，其中，DEP-1、DEP-2的核苷酸序列与待测单链DNA的部分序列相同，二者序列不重叠，且DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+DEP-2≤待测单链DNA)。
具体地，上述DEP-1具有如SEQ ID NO:3所示的核苷酸序列(或DEP-1由SEQ ID NO:3所示的核苷酸序列组成)，DEP-2具有如SEQ ID NO:4所示的核苷酸序列(或DEP-2由SEQ ID NO:4所示的核苷酸序列组成)。
具体地，上述试剂盒还包含标准品，其具有如SEQ ID NO:4所示的核苷酸序列(或由其组成)。
具体地，由于毛首鞭形线虫的β-微管蛋白第200位密码子的特定A到T突变(如SEQ ID NO:1到SEQ ID NO:2的突变)是一个公认的TT耐苯并咪唑(BZ，药物)的热点，上述试剂盒可用于筛选或鉴定毛首鞭形线虫的耐药性。
本发明还提供一种用于HBV单核苷酸变异检测的试剂盒，其包含本发明上述用于HBV单核苷酸变异检测的探针。
具体地，上述试剂盒还包含DEP-1、DEP-2，其中，DEP-1、DEP-2的核苷酸序列与待测单链DNA的部分序列相同，二者序列不重叠，且DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+DEP-2≤待测单链DNA)。
具体地，上述DEP-1具有如SEQ ID NO:8所示的核苷酸序列(或由其组成)，DEP-2具有如SEQ ID NO:9所示的核苷酸序列(或由其组成)。
具体地，上述试剂盒还包含标准品，其具有如SEQ ID NO:7所示的核苷酸序列(或由其组成)。
本发明还提供一种用于毛首鞭形线虫(Trichuris trichiura)单核苷酸变异检测的试剂盒，其包含：reporter-F1、reporter-Q1、reporter-F2、reporter-Q2，其中，reporter-F1具有如SEQ ID NO:16所示的核苷酸序列，reporter-Q1具有如SEQ ID NO:17所示的核苷酸序列，reporter-F2具有如SEQ ID NO:18所示的核苷酸序列，reporter-Q2具有如SEQ ID NO:19所示的核苷酸序列。
具体地，上述试剂盒还包含DEP-1、DEP-2，其中，DEP-1、DEP-2的核苷酸序列与待测单链DNA的内部部分序列相同(非边缘)，二者序列不重叠，且DEP-1、DEP-2的序列组合为待测单链DNA的内部部分序列(即DEP-1+DEP-2≤待测单链DNA)。
具体地，上述DEP-1具有如SEQ ID NO:3所示的核苷酸序列(或由其组成)，DEP-2具有如SEQ ID NO:4所示的核苷酸序列(或由其组成)。
具体地，上述试剂盒还包含一对引物(DEP-3和DEP-4)，其中，正向引物(DEP-3)的核苷酸序列与待测单链DNA的边缘部分序列相同，且不与DEP-1、DEP-2重叠，且DEP-1、DEP-2、DEP-3的序列组合为待测单链DNA的部分序列，DEP-4为待测单链DNA的其余部分序列的互补序列。
具体地，上述DEP-3具有如SEQ ID NO:14所示的核苷酸序列(或由其组成)，DEP-4具有如SEQ ID NO:15所示的核苷酸序列(或由其组成)。
具体地，上述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团；例如，reporter-F的5’端标记有荧光报告基团，reporter-Q的3’端标记有荧光淬灭基团；或reporter-F的3’端标记有荧光报告基团，reporter-Q的5’端标记有荧光淬灭基团；且reporter-F1和reporter-F2上标记的荧光报告基团不同。
在本发明的一个实施例中，上述reporter-F1为5’–AT GAA GC G CTT TAC GAT ATT TGT TTC CGA-Cy5–3’；reporter-Q为5’–Iowa Black RQ–TCG GAA ACA AAT ATC GTA AAG C–3’。
在本发明的一个实施例中，上述reporter-F2为5’–G GAC GAA ACA TAC TGC ATA GA CATGT–FAM–3’；reporter-Q为5’–Iowa Black FQ–ACATG TC TAT GCA GTA TGT–3’。
具体地，上述试剂盒还包含标准品，其具有如SEQ ID NO:12所示的核苷酸序列(或由其组成)。
具体地，由于毛首鞭形线虫的β-微管蛋白第200位密码子的特定A到T突变(如SEQ ID NO:12到SEQ ID NO:13的突变)是一个公认的TT耐苯并咪唑(BZ，药物)的热点，上述检测体系中reporter涵盖突变位点和非突变位点，可用于检测毛首鞭形线虫感染，并筛选或鉴定毛首鞭形线虫的耐药性。
本发明还提供一种用于癌症相关基因突变BRAF-D594G(例如从SEQ ID NO:20所示序列突变为SEQ ID NO:21所示序列)检测的试剂盒，其包含本发明上述用于癌症相关基因突变BRAF-D594G检测的探针。
具体地，上述试剂盒还包含DEP-1、DEP-2，其中，DEP-1、DEP-2的核苷酸序列与待测单链DNA的部分序列相同，二者序列不重叠，且DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+DEP-2≤待测单链DNA)。
具体地，上述DEP-1具有如SEQ ID NO:22所示的核苷酸序列(或由其组成)，DEP-2具有如SEQ ID NO:23所示的核苷酸序列(或由其组成)。
具体地，上述试剂盒还包含标准品，其具有如SEQ ID NO:20所示的核苷酸序列(或由其组成)。
本发明还提供一种用于癌症相关基因突变BRAF-V600E(例如从SEQ ID NO:26所示序列突变为SEQ ID NO:27所示序列)检测的试剂盒，其包含本发明上述用于癌症相关基因突变BRAF-V600E检测的探针。
具体地，上述试剂盒还包含DEP-1、DEP-2，其中，DEP-1、DEP-2的核苷酸序列与上述待测单链DNA的部分序列相同，二者序列不重叠，且DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+DEP-2≤待测单链DNA)。
具体地，上述DEP-1具有如SEQ ID NO:28所示的核苷酸序列(或由其组成)，DEP-2具有如SEQ ID NO:29所示的核苷酸序列(或由其组成)。
具体地，上述试剂盒还包含标准品，其具有如SEQ ID NO:26所示的核苷酸序列(或由其组成)。
本发明还提供一种用于癌症相关基因突变EGFR-G719A(例如从SEQ ID NO:32所示序列突变为SEQ ID NO:33所示序列)检测的试剂盒，其包含本发明上述用于癌症相关基因突变EGFR-G719A检测的探针。
具体地，上述试剂盒还包含DEP-1、DEP-2，其中，DEP-1、DEP-2的核苷酸序列与待测单链DNA的部分序列相同，二者序列不重叠，且DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+DEP-2≤待测单链DNA)。
具体地，上述DEP-1具有如SEQ ID NO:34所示的核苷酸序列(或由其组成)，DEP-2具有如SEQ ID NO:35所示的核苷酸序列(或由其组成)。
具体地，上述试剂盒还包含标准品，其具有如SEQ ID NO:32所示的核苷酸序列(或由其组成)。
本发明还提供一种用于癌症相关基因突变EGFR-L858R(例如从SEQ ID NO:38所示序列突变为SEQ ID NO:39所示序列)检测的试剂盒，其包含本发明上述用于癌症相关基因突变EGFR-L858R检测的探针。
具体地，上述试剂盒还包含DEP-1、DEP-2，其中，DEP-1、DEP-2的核苷酸序列与上述待测单链DNA的部分序列相同，二者序列不重叠，且DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+DEP-2≤待测单链DNA)。
具体地，上述DEP-1具有如SEQ ID NO:40所示的核苷酸序列(或由其组成)，DEP-2具有如SEQ ID NO:41所示的核苷酸序列(或由其组成)。
具体地，上述试剂盒还包含标准品，其具有如SEQ ID NO:38所示的核苷酸序列(或由其组成)。
本发明还提供一种用于癌症相关基因突变EGFR-L861Q(例如从SEQ ID NO:44所示序列突变为SEQ ID NO:45所示序列)检测的试剂盒，其包含本发明上述用于癌症相关基因突变EGFR-L861Q检测的探针。
具体地，上述试剂盒还包含DEP-1、DEP-2，其中，DEP-1、DEP-2的核苷酸序列与待测单链DNA的部分序列相同，二者序列不重叠，且DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+DEP-2≤待测单链DNA)。
具体地，上述DEP-1具有如SEQ ID NO:46所示的核苷酸序列(或由其组成)，DEP-2具有如SEQ ID NO:47所示的核苷酸序列(或由其组成)。
具体地，上述试剂盒还包含标准品，其具有如SEQ ID NO:44所示的核苷酸序列(或由其组成)。
本发明还提供一种用于癌症相关基因突变KRAS-G12A(例如从SEQ ID NO:50所示序列突变为SEQ ID NO:51所示序列)检测的试剂盒，其包含本发明上述用于癌症相关基因突变KRAS-G12A检 测的探针。
具体地，上述试剂盒还包含DEP-1、DEP-2，其中，DEP-1、DEP-2的核苷酸序列与待测单链DNA的部分序列相同，二者序列不重叠，且DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+DEP-2≤待测单链DNA)。
具体地，上述DEP-1具有如SEQ ID NO:52所示的核苷酸序列(或由其组成)，DEP-2具有如SEQ ID NO:53所示的核苷酸序列(或由其组成)。
具体地，上述试剂盒还包含标准品，其具有如SEQ ID NO:50所示的核苷酸序列(或由其组成)。
本发明还提供一种用于癌症相关基因突变KRAS-G13V(例如从SEQ ID NO:56所示序列突变为SEQ ID NO:57所示序列)检测的试剂盒，其包含本发明上述用于癌症相关基因突变KRAS-G13V检测的探针。
具体地，上述试剂盒还包含DEP-1、DEP-2，其中，DEP-1、DEP-2的核苷酸序列与待测单链DNA的部分序列相同，二者序列不重叠，且DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+DEP-2≤待测单链DNA)。
具体地，上述DEP-1具有如SEQ ID NO:58所示的核苷酸序列(或由其组成)，DEP-2具有如SEQ ID NO:59所示的核苷酸序列(或由其组成)。
具体地，上述试剂盒还包含标准品，其具有如SEQ ID NO:56所示的核苷酸序列(或由其组成)。
本发明还提供一种用于癌症相关基因突变PIK3CA-H1047R(例如从SEQ ID NO:62所示序列突变为SEQ ID NO:63所示序列)检测的试剂盒，其包含本发明上述用于癌症相关基因突变PIK3CA-H1047R检测的探针。
具体地，上述试剂盒还包含DEP-1、DEP-2，其中，DEP-1、DEP-2的核苷酸序列与待测单链DNA的部分序列相同，二者序列不重叠，且DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+DEP-2≤待测单链DNA)。
具体地，上述DEP-1具有如SEQ ID NO:64所示的核苷酸序列(或由其组成)，DEP-2具有如SEQ ID NO:65所示的核苷酸序列(或由其组成)。
具体地，上述试剂盒还包含标准品，其具有如SEQ ID NO:62所示的核苷酸序列(或由其组成)。
本发明还提供一种用于癌症相关基因突变STK11-F354L(例如从SEQ ID NO:68所示序列突变为SEQ ID NO:69所示序列)检测的试剂盒，其包含本发明上述用于癌症相关基因突变STK11-F354L检测的探针。
具体地，上述试剂盒还包含DEP-1、DEP-2，其中，DEP-1、DEP-2的核苷酸序列与待测单链DNA的部分序列相同，二者序列不重叠，且DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列(即DEP-1+DEP-2≤待测单链DNA)。
具体地，上述DEP-1具有如SEQ ID NO:70所示的核苷酸序列(或由其组成)，DEP-2具有如SEQ ID NO:71所示的核苷酸序列(或由其组成)。
具体地，上述试剂盒还包含标准品，其具有如SEQ ID NO:68所示的核苷酸序列(或由其组成)。
具体地，本发明上述试剂盒还可包含Mg 2+，特别是0-10mM(例如0、1.25、2.5、5、10mM)的Mg 2+。
具体地，本发明上述试剂盒还包含核苷酸保护剂，如Tween 20，用于防止稀释和移液过程中DNA寡核苷酸的潜在损失；其中，核苷酸保护剂的含量可以为0.1％-0.5％(v/v)。
具体地，本发明上述试剂盒还包含缓冲体系，如Tris缓冲液。
在本发明的一个实施例中，上述试剂盒包含：1mM Mg 2+，0.1％Tween 20(v/v)，1×Tris缓冲液，以及相应的探针和DEP。
具体地，本发明上述试剂盒还可包含阴性对照品，其为不含待测DNA的体系，例如H 2O(如无菌双蒸水、无菌去离子水等)。
具体地，本发明上述试剂盒还可以包含DNA提取试剂和材料，用于提取待测样品DNA，可以采用现有技术已知任何合适的用于DNA提取的试剂和材料。
具体地，必要时，上述试剂盒还可以包含待测样品预处理试剂，该预处理试剂可以采用现有技术已知用于预处理样品以便于DNA提取的试剂，例如，生理盐水等。
本发明还提供上述检测方法、探针、试剂盒在DNA单核苷酸变异的检测中的应用。
本发明还提供上述检测方法、探针、试剂盒，其用于DNA单核苷酸变异。
本发明还提供上述检测方法、探针、试剂盒在检测病原体感染中的应用。
本发明还提供上述检测方法、探针、试剂盒，其用于检测病原体感染。
具体地，上述病原体可以为微生物、寄生虫或其他媒介。具体地，上述微生物可选自：病毒、衣原体、立克次体、支原体、细菌、螺旋体、真菌等中的一种或多种。
在本发明的一个实施方式中，上述病原体为病毒，例如，但不限于，腺病毒科(如腺病毒)、疱疹病毒科(如HSV1(口腔疱疹)、HSV2(外生殖器疱疹)、VZV(水痘)、EBV(埃-巴二氏病毒)、CMV(巨细胞病毒))、痘病毒科(如天花病毒、牛痘病毒)、乳多泡病毒科(如乳头瘤病毒)、细小病毒科(如B19病毒)、嗜肝DNA病毒科(如乙型肝炎病毒)、多瘤病毒科(如多瘤病毒)、呼肠孤病毒科(如呼肠弧病毒、轮状病毒)、小核糖核酸病毒科(如肠道病毒、口蹄疫病毒)、嵌杯样病毒科(如诺沃克病毒、戊型肝炎病毒)、披膜病毒科(如风疹病毒)、沙粒病毒科(如淋巴细胞性脉络丛脑膜炎病毒)、逆转录病毒科(HIV-1、HIV-2、HTLV-1)、黄病毒科(如登革热病毒、寨卡病毒、乙型脑炎病毒、基孔肯亚病毒、黄热病病毒、丙型肝炎病毒、西尼罗病毒等)、正粘病毒科(如流感病毒(如甲型流感病毒、乙型流感病毒、丙型流感病毒等))、副粘病毒科(如1型人副流感病毒(HPV)、2型HPV、3型HPV、4型HPV、仙台病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞病毒、新城疫病毒等)、布尼亚病毒科(如加利福尼亚脑炎病毒、汉坦病毒)、弹状病毒科(如狂犬病毒)、丝状病毒科(如埃博拉病毒、马尔堡病毒)、冠状病毒科(如HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV、SARS-CoV-2等)、星状病毒科(如星状病毒)、博尔纳病毒科(如博尔纳病毒)。
在本发明的一个实施方式中，上述病原体为寄生虫，例如，但不限于，蛔虫、鞭虫、蛲虫、钩虫、绦虫、溶组织内阿米巴、阴道毛滴虫、肝吸虫、包虫、卫氏并殖吸虫、猪囊虫、弓形虫、血吸虫、旋毛虫、丝虫、疟原虫、利士曼原虫、吸吮线虫、螨虫、虱、蜱等。
本发明还提供上述检测方法、探针、试剂盒在筛选或鉴定寄生虫耐药性中的应用。
本发明还提供上述检测方法、探针、试剂盒，其用于筛选或鉴定寄生虫耐药性。
在本发明的一个实施例中，上述寄生虫为毛首鞭形线虫。
本发明还提供上述检测方法、探针、试剂盒在疾病的诊断和患病风险的评估中的应用。
本发明还提供上述检测方法、探针、试剂盒，其用于疾病的诊断和患病风险评估。
本发明还提供上述探针在制备用于疾病的诊断和患病风险的评估的产品(如试剂盒)中的应用。
在本发明的一个实施例中，上述疾病为恶性肿瘤，包括，但不限于，淋巴瘤、母细胞瘤、髓母细胞瘤、视网膜母细胞瘤、肉瘤、脂肪肉瘤、滑膜细胞肉瘤、神经内分泌肿瘤、类癌肿瘤、胃泌素瘤、胰岛细胞癌、间皮瘤、神经鞘瘤、听神经瘤、脑膜瘤、腺癌、黑素瘤、白血病或淋巴样恶性肿瘤、鳞状细胞癌、上皮鳞状细胞癌、肺癌、小细胞肺癌、非小细胞肺癌、腺癌肺癌、肺鳞癌、腹膜癌、肝细胞癌、胃癌、肠癌、胰腺癌、成胶质细胞瘤、子宫颈癌、卵巢癌、肝癌、膀胱癌、肝癌、乳腺癌、转移性乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫癌、唾液腺癌、肾癌、前列腺癌、外阴癌、甲状腺癌、肝癌、肛门癌、阴茎癌、梅克尔细胞癌、食管癌、胆道肿瘤、头颈部癌和血液恶性肿瘤。
本发明还提供一种用于疾病的诊断和患病风险评估的方法，其包括使用本发明上述检测方法、探针、试剂盒的步骤。
本发明公开了一种新的模拟并引导设计核酸杂交探针的方法-DNA均衡器门(DEG)，其通过用户可定义的检测信号和靶标浓度之间的定量关系的转换，极大地扩展了用于区分双链DNA(dsDNA)中单核苷酸变异的检测窗口。本发明还公开了一种热力学驱动的理论模型，定量模拟和预测DEG的性能。通过计算机模拟和实验验证，DEG用于扩展检测窗和改进序列选择性的有效性得到证明。由于DEG直接作用于dsDNA，因此其容易适用于核酸扩增技术，如聚合酶链式反应(PCR)。通过对从Honduras农村地区收集的临床寄生虫样品进行感染检测和耐药性筛选，证实了DEG的实用性。本发明所提供的检测方法中不需要使用酶等成本高、对反应条件要求比较高的试剂，因此，该方法操作简单，成本较低，便于企业和实验室应用。
附图说明
图1所示为DEG中实验步骤和DNA反应的示意图。
图2所示为使用正向和反向引物浓度之间的不同比例优化不对称PCR；动力学曲线显示，使用报告探针通过不对称PCR产生的ssDNA输出的测量，该报告探针使用toehold交换操作。
图3所示为DEG的示意图。a.使用DEG定量dsDNA的总体工作流程。使用自主分子计算，在试管中，将靶标dsDNA和DEP的混合物加热并快速冷却，以产生数量可控的ssDNA输出。然后通 过报告探针产生荧光信号。b.从机制上讲，在加热和快速冷却过程中，dsDNA靶标(AB)被变性为A和B。然后在复性过程中发生DEP(C和D)与A之间的竞争，以便与B杂交。ssDNA输出(A)的净含量由自主计算过程定量确定，该过程将目标和DEP之间的初始浓度进行比较。c.当[AB]≤[DEP]时，B和DEP之间的反应(即BCD的形成)在热力学上是有利的，从而使A的产量最大化。d.当[AB]>[DEP]时，BC和BD作为中间体生成，然后通过链置换消耗A。e.通过此计算过程，DEG将检测信号与目标浓度之间的定量关系从典型的S型函数转换为单峰函数。这样，可以有效地抑制虚假目标的检测信号，从而可以大大扩大检测窗口并提高鉴别系数(DF)。
图4所示为通过DEG获得扩大的检测窗口(顶部)和用于激活toehold交换探针的能量势垒的增加(底部)的模拟结果。通过将反向toehold的长度延长2bp，可以实现能量势垒的增加。
图5所示为DEG的理论模型。a.DEG中发生的所有可能的元件反应的示意图。b.将DEG中的复杂反应网络线性化为0＝RM·反应物以提取独立方程式，其中RM是化学计量矩阵，反应物代表DNA物质。RM的秩确定为4，表明需要求解四个独立的平衡方程。这样，选择反应[i-iv]建立数学模型。c.在不进行概率校正的情况下，根据dsDNA靶标浓度对A和AB的产量进行计算机预测。d.说明当[AB]>[DEP]时需要进行概率校正的示意图。
图6所示为DEG的模拟结果。在计算机模拟中，对于典型的toehold-exchange(a)以及DEP浓度在50、100、200和500nM下的DEG，反应产率随靶标浓度和ΔΔG 0的变化。经典的toehold-exchange可以视为DEG的一种特殊情况，其中[DEPs]＝∞。DEG存在最大产率，其中[AB]＝[DEP]。在很宽的浓度范围内，假靶标的产率得到显著抑制，这可以帮助提高特异性并扩大检测范围。在计算机上预测经典toehold-exchange(c)和DEG(d)的鉴别因子。区分SNV的检测窗口可通过改变DEP的浓度进行调整。在计算机上预测经典toehold-exchange(e)和DEG(f)的鲁棒性因子。DEG的使用将RF值从有限值急剧增加到无限。
图7所示为DEG的实验验证。a.将不同DEP浓度的DEG的靶标浓度绘制成实验测定的产率(Exp)，并与模拟(Sim)进行比较。经典的toehold-exchange可以视为DEG的一种特殊情况，其中[DEPs]＝∞。b.针对靶标浓度绘制的使用一对合成的真靶标和假靶标(ΔΔG 0＝2.29kcal/mol)的实验测定的鉴别因子，并与计算机模拟的靶标浓度进行比较。c.针对靶标浓度绘制的稳健性因子，并与计算机模拟进行比较。d.靶标和DEP序列的示意图。在位置1、6、14和17对靶标进行了单核苷酸突变。e.针对真靶标和假靶标的靶标浓度的实验测定的产率，靶标在四个指定位置携带突变。所有实验均在含有1mM Mg2+和20nM toehold交换信标的1×PBS缓冲液中于37℃下进行。每个误差条代表重复分析的一个标准偏差。
图8所示为鉴别因子(DF)的理论浓度依赖性。a.相对于由其热力学参数定义的五个理论假靶标，作为真靶标的靶标浓度的函数的理论DF。假靶标的反应自由能在方框中示出，其颜色与DF曲线相同。b.作为靶标浓度的函数的一对真靶标和假靶标之间的产率差异。c.通过包括使用LOD的校正来模拟DF。
图9所示为RF对反应产率和目标浓度的依赖性。a.作为反应产率η的函数的RF值的理论预测。b.假靶标和真靶标之间的绝对浓度差作为产率的函数。c.RF作为目标浓度的函数。d.在相同产率下，假靶标和真靶标的绝对浓度差作为靶标浓度的函数。e.使用LOD和LOL修正的RF。
图10所示为核酸杂交探针的检测窗口。a-c.通过模拟计算，理论预测了ΔΔG 0＝2.30kcal/mol的一对真靶标和假靶标的反应产率、DF、RF。d-f.计算机分析ΔΔG 0为0-5kcal/mol的所有可能突变的产率、DF和RF。
图11所示为用于使用DEG从dsDNA输入AB产生ssDNA输出A的产率的理论预测。a.作为初始输入浓度的函数的输出ssDNA(A)和输入dsDNA(AB)浓度。概率函数是保证模型精确度的关键。b.ssDNA(A)的产率作为初始输入浓度的函数。c.DEP对DEG产生ssDNA输出的影响。对于每个滴定曲线存在最大产率，其中[输入]＝[DEP]。d.用于分析针对三个单核苷酸突变的正确dsDNA靶标的滴定曲线。[DEP]＝500nM。
图12所示为
Figure PCTCN2020119612-appb-000001
的修正。通过使用toehold交换报告分子测量真靶标和三个单核苷酸突变来进行修正。产率可以使用每种DNA物质的预测ΔG来预测(左侧曲线)。为了用实验结果拟合理论曲线，需要-1.575kcal/mol的修正。这一修正应用于整个工作中的所有模拟。[AB＝10nM，[报告分子]＝20nM，使用NUPACK软件预测所有DNA物质的理论ΔG。
图13所示为理论和实验RF的确定。通过Matlab软件提取产生相同产率的一对真靶标和假靶 标的浓度来确定理论RF。首先使用4参数非线性模型将实验数据拟合到校准曲线中，然后使用Matlab提取一对真靶标和假靶标的浓度，从而确定实验RF。然后使用式6计算各浓度下的RF，并绘制为靶标(真靶标)浓度的函数。
图14所示为表征DEP和热方案对DEG性能的影响。a.实时荧光监测报告分子门(reporter gate)的动力学，以测量DEG产生的X +。b.根据在20分钟时通过终点荧光测量的A，每个反应的产率。通过将阳性对照的荧光设定为1来计算产率。c.每个样品或对照中的详细反应物和实验步骤。将每个样品在37℃下孵育，其中样品在1×Tris缓冲液(1mM Mg 2+和0.1％Tween 20(v/v))中含有10nM AB、20nM报告分子和200nM DEP。每个误差条代表来自重复分析的一个标准偏差。
图15所示为DEP对DEG性能的影响。a.实时荧光监测报告分子门(reporter gate)的动力学，以测量DEG产生的A。b.根据在20分钟时通过终点荧光测量的A，每个反应的产率。c.每个样品或对照中的详细反应物和实验步骤。每个误差条代表重复分析的一个标准偏差。
图16所示为变性和复性过程的热方案的优化。当所有DNA物质在同一试管中预混合并进行加热时，然后快速冷却至4℃时，可建立最大产率a.发现在热方案之前(pre)或之后(post)加入DEP显著影响A的产率。b.还发现快速冷却步骤对于确保的高产率的A至关重要。c.当快速冷却至4℃作为最终温度时，获得最大产率。发现将最终温度增加至25、55和75℃会逐渐降低反应的产率。
图17所示为Mg 2+对DEG性能的影响。每个误差条代表重复分析的一个标准偏差。
图18所示为由DEG产生的输出ssDNA A的稳定性。每个误差条代表重复分析的一个标准偏差。
图19所示为来自DEP的信号泄漏的预估。a.由DEP和报告分子之间的相互作用引起的信号泄漏的示意图。b.作为DEP浓度的函数的预估泄漏(空白)，也将其与靶标特异性荧光(样品)进行比较。靶标浓度固定在10nM，DEP的浓度从10nM到5μM不等。即使当施加1μM DEP时也未观察到荧光泄漏。这表明DEP与探针之间无交叉反应，因此DEP与探针的ssDNA输出之前无竞争。每个误差条代表重复分析的一个标准偏差。
图20所示为使用DEG估计用于检测AB的LOD。当使用200nM DEP产生单链输出A时，LOD估计为0.5nM。误差条表示重复分析的一个标准偏差。
图21所示为分析毛首鞭形线虫(TT)亚基因组单核苷酸突变的示意图。a.TT靶标的序列和点突变。b.为TT靶标设计的一对DEP的序列。c.通过toehold交换操作的报告探针的序列设计。
图22所示为用于鉴别42-bp dsDNA TT靶标中的单核苷酸T>A、T>G和T>C突变的DEG的实验验证。每个误差条代表重复分析的一个标准偏差。
图23所示为实验测定的产率与通过使用DEG分析双链TT靶标的T>G和T>C突变的模拟预测的产率的比较。
图24所示为使用DEG对双链TT靶标中的T>G和T>C突变进行实验测量和模拟DF的比较。
图25所示为使用DEG对双链TT靶标中的T>G和T>C突变进行实验测量和模拟RF的比较。
图26所示为对具有不同突变的靶标TT-28的实验测量DF。
图27所示为对具有不同突变的靶标TT-28的实验测量RF。
图28所示为分析HBV S基因亚基因组的单核苷酸突变的示意图。a.靶序列和点突变。b.针对HBV靶标设计的一对DEP的序列。c.通过toehold交换操作的报告探针的设计.
图29所示为使用DEG检测具有不同突变和插入/缺失的合成HBV靶标。阴影区域表示检测窗口，其中DEG在鉴别最具挑战性的SNV27G方面胜过toehold交换信标。
图30所示为使用DEG对44bp HBV靶标中的三个单核苷酸突变(SNV27C、SNV27T和SNV27G)进行实验测量的DF值。阴影区域表示检测窗口，其中DEG在鉴别最具挑战性的SNV27G方面胜过toehold交换信标。
图31所示为使用DEG对44bp HBV靶标中的单核苷酸突变进行实验测量的RF值。
图32所示为对于9种在癌症中常见的临床上重要的单核苷酸变体的设计和序列。
图33所示为用于分析BRAF-D594G、BRAF-V600E、EGFR-G7119A、EGFR-L858R和EGFR-L861Q的实验测量的产率、DF和RF。DEG的浓度固定为200nM。
图34所示为用于分析KRAS-G12A、KRAS-G13V、PIK3CA-H1047R和STK11-F354L的实验测量的产率、DF和RF。DEG的浓度固定为200nM。
图35所示为使用DEG可以有效检测出高浓度的未突变序列背景下低至0.5％的突变靶标。
图36所示为在同一试管中使用两组DEP同时操作TT和HBV靶标的结果。
图37所示为不同TT靶标和相应DEP长度的序列，以验证靶/DEP的长度效应。
图38所示为使用200nM浓度的相应DEP，通过实验测量的dsDNA靶标长度(从87bp到32bp)的产率、DF和RF。
图39所示为使用浓度分别为50、100和200nM的DEP，实验测量的靶标TT-32的产率、DF和RF。
图40所示为DEG与PCR的整合。a.使用4-DEP设计分析dsDNA的示意图。b.4-DEP设计的实验验证，用于检测87bp dsDNA作为PCR扩增子的模拟物。外部DEP的浓度固定为500nM，内部DEP的浓度设置为200nM。c.使用4-DEP设计的DEG-PCR的示意图。外部两个DEP设计为与PCR引物相同。d.使用toehold交换报告分子实时监测DEG-PCR。实现了一个宽的检测窗口，可将低至10aM的真模板与包含单个核苷酸突变的1pM假靶标清晰地区分开。e.不对称PCR的示意图，然后使用toehold交换报告分子进行检测。f.实时监测非对称PCR扩增子的检测，发现检测窗口比DEG-PCR窄得多，在DEG-PCR中，只有在1fM以上才能进行正确的区分。
图41所示为一组四个DEP的序列设计的示意图。这些DEP靶向87bp扩增子(AB)以检测TT蠕虫中耐药性的热点。a.方案显示了PCR扩增子的4-DEP设计。b.设计两个内部DEP，以暴露可被报告探针检测的ssDNA结构域。为促进ssDNA输出(A)的产生，设计了2个外部DEP，其序列与一对正向和反向PCR引物相同。c.通过toehold交换操作的报告探针的设计。
图42所示为通过4-DEP设计验证DEG对ssDNA输出A的检测的验证。
图43所示为使用DEP浓度从50nM至200nM不等的内部DEP的DEG分析PCR扩增子。表明DEG允许检测双链PCR扩增子，同时通过宽浓度范围识别单核苷酸突变。
图44所示为DEG-PCR在分析临床寄生虫样品中的应用。a.用于分析从洪都拉斯农村地区学龄儿童粪便样本中收集的寄生虫(Trichuris trichiura，TT)样本的典型工作流程，然后使用DEG-PCR进行检测。b.使用双通道设计(FAM-Reporter和Cy5-Reporter)同时检测寄生虫感染和进行耐药性筛查。设计PCR引物以扩增包含第200个密码子的β-微管蛋白基因中的核苷酸1246-1333。该密码子的单核苷酸A至T突变是耐药性筛选的热点。使用标记有FAM的toehold交换报告分子(FAM-reporter)来区分该点突变，而使用无反向toehold的链置换报告分子(Cy5-reporter)检测200密码子附近的保守区域。使用合成DNA标准品(点)和13个(D.R.-)临床样品(圆圈)作为训练集(c)和8个未知临床寄生虫样品的双通道DEG-PCR的实验测试。d.测试结果分为三个区域，分别定义为感染阳性和耐药性(D.R.+)、感染阳性和无耐药性(D.R.-)和感染阴性(N.C.)。使用具有99％置信区间和2个自由度(两个荧光通道)的误差eclipse来定义D.R.+和D.R.-。测试了8个临床蠕虫标本(包括6个毛首鞭形线虫(TT-1至TT-6)和2个蛔虫(AL，作为阴性对照))，并在d中作图。
图45所示为用于分析临床寄生虫样品的DEG-PCR的序列设计。a.通过引物设计软件BLAST设计了一对将β-微管蛋白基因从1246bp扩增到1333bp的引物。设计FAM-报告分子(绿色)以检测β-微管蛋白基因第1271至1299bp密码子200处的特异A至T突变。设计Cys 5-报告分子(红色)以分析β-微管蛋白基因的第1301-13320bp。b.指示阳性感染(红色)和阳性耐药性(绿色，D.R.+)的代表性荧光动力学曲线。c.指示阳性感染(红色)但阴性耐药性(绿色，D.R.-)的代表性荧光动力学曲线。d.显示无感染和无耐药性的代表性荧光动力学曲线。
图46所示为DEP和两个报告分子的序列设计的示意图。
图47所示为4-DEP-双报告分子DEG用于检测在β-微管蛋白密码子200处具有A至T突变的双链TT靶标的验证。实时荧光监测FAM-和Cys5-报告分子的动力学，以在两个不同位点测定双链TT靶标(AB)，如图45所示。当使用一组4个DEP(每个200nM)检测20nM A时，在两个通道中观察到荧光的快速增加。
图48所示为4-DEP-双报告分子DEG用于分析合成DNA靶标(耐药阳性(D.R.+)的耐药突变体或耐药阴性(D.R.-)的野生型)的检出限。a.FAM和Cys5通道中的归一化荧光随检测耐药性阳性突变体的靶标浓度的变化。b.FAM和Cys5通道处的荧光比随靶标浓度的变化。c.0.16、0.31、0.62、1.25、2.5、5、10、20、40和80nM的靶标(D.R.+或D.R.-)的双通道荧光分布图。耐药阳性靶标的检出限为0.62nM，耐药阴性靶标的检出限为1.25nM。子图中的灰色阴影区域指示无法区分D.R+和D.R.-靶标的荧光分布。
图49所示为4-DEP-双报告分子DEG用于分析合成DNA靶标的检出限(使用800nM DEP)。a.FAM和Cys5通道中的归一化荧光随检测耐药性阳性突变体的靶标浓度的变化。b.FAM和Cys5通道处的荧光比随靶标浓度的变化。c.0.16、0.31、0.62、1.25、2.5、5、10、20、40和80nM的靶标(D.R.+或D.R.-)的双通道荧光分布图。耐药阳性靶标和阴性阴性靶标的检出限均为0.62nM。 子图中的灰色阴影区域指示无法区分D.R+和D.R.-靶标的荧光分布。
图50所示为在不同浓度的野生型存在下检测耐药突变体。a.FAM和Cys5通道的归一化荧光强度随野生型对照中加标突变体的百分比的变化。总靶标浓度固定在20nM。b.使用FAM/Cy5比作为读出值的实验和理论校准曲线。c.实验校准的线性回归。
图51所示为应用双报告分子DEG-PCR分析临床寄生虫样品。a.FAM和Cys5通道的归一化荧光强度随PCR扩增前合成DNA模板的原始浓度的变化。该模板与耐药突变株具有相同的亚基因组序列。b.用于临床寄生虫样品(包括6个毛首鞭形线虫(TT)样品和2个蛔虫(AL)样品)的FAM和Cys5通道的归一化荧光强度。所有TT样品均呈阳性感染，阴性耐药；而两个AL样品显示TT感染阴性。负荧光强度表明AL的荧光信号低于空白的荧光信号。每个误差条代表重复分析的一个标准偏差。
图52所示为使用标准PCR和随后的聚丙烯酰胺凝胶电泳(PAGE)分析检测临床寄生虫样品的结果。a.1μM至1pM范围内的标准合成DNA模板的PCR扩增子的PAGE分析。b.8个临床寄生虫样品的PCR扩增子的PAGE分析。
图53所示为临床寄生虫的基因组测序数据。第一行显示了野生型毛首鞭形线虫β-微管蛋白基因的密码子186至密码子214的DNA序列。密码子198和200被突出显示为耐药性突变热点。从患者身上提取的6份蠕虫样本的序列与野生型一致，这与使用DEG-PCR测量的诊断结果非常一致。
具体实施方式
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
本文中出现的部分缩写及相应含义如下：
DEG       DNA均衡器门(DNA equalizer gate)
DEP       DNA平衡探针(DNA equalizer probe)
dsDNA     双链DNA
ssDNA     单链DNA
SNV       单核苷酸变异
本文所引用的各种出版物、专利和公开的专利说明书，其公开内容通过引用整体并入本文。
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例中所采用的材料和方法如下：
DNA寡核苷酸
本发明实施例中使用的DNA寡核苷酸购自Integrated DNA Technologies(IDT，Coralville，IA)。荧光团(FAM-和Cy5-)和淬灭剂(Iowa Blank)的DNA寡核苷酸通过高效液相色谱(HPLC)纯化。其他DNA物质未经纯化即使用。所用寡核苷酸的序列和修饰列于下表。
表1 DNA序列信息
Figure PCTCN2020119612-appb-000002
Figure PCTCN2020119612-appb-000003
Figure PCTCN2020119612-appb-000004
Figure PCTCN2020119612-appb-000005
Figure PCTCN2020119612-appb-000006
Figure PCTCN2020119612-appb-000007
缓冲条件
通过使用1×tris-EDTA(TE)缓冲液(10mM Tris-HCl，pH＝8.0，1M EDTA，购自Sigma)溶解寡核苷酸来重悬DNA寡核苷酸，然后在-20℃下保存。将包含10mM MgCl 2和0.5％(v/v)TWEEN 20(Sigma)的1×TE缓冲液用作分子报告缓冲液。将含有1mM MgCl 2和0.5％(v/v)TWEEN 20的1×PBS(pH＝7.4，购自Sigma)用作反应缓冲液。TWEEN 20用于防止稀释和移液过程中DNA寡核苷酸的潜在损失。
荧光报告分子(reporter)的制备
使用BioRad T100热循环仪在分子报告缓冲液中对所有链置换(SDR)和toehold交换(TER)报告分子进行退火。将样品(通常终浓度为5μM)95℃加热5分钟，然后在40分钟的时间内以恒定速率逐渐冷却到室温。用于SDR的猝灭剂与荧光团的浓度比为1.5，而用于TER的猝灭剂与荧光团的浓度比为3。将制备的报告分子溶液保存在4℃下待用。
数学模型建立
DNA链和复合物的自由能通过NUPACK估算。对于DEG的热力学参数设置，温度设置为4℃(在冰水浴中)，Na +浓度为0.1M，Mg 2+浓度为0.001M；而toehold交换反应中DNA物质的温度设置为37℃。其他参数使用默认设置。
在MATLAB(2019a，MathWorks)中通过符号方法计算η、DF和RF的浓度依赖性方程的解析解。在同一平台上进行矩阵(RM)分析和求解平衡方程组。特别是在方程系统中，由于变量之间的强耦合(三阶)，数值计算方法是必要的。边界条件仅限于真实值和合理解答(例如，产率必须大于0但小于1)。为了计算理论RF值，采用两个反向函数：第一个是将产率(归一化)转化为ssDNA靶标的浓度；第二个是考虑概率函数将ssDNA靶标反向至相应的dsDNA浓度。通过拟合非线性曲线函数计算实验RF值。在Graphpad Prism 8中绘制二维曲线，并在MATLAB中绘制三维热图。
使用DEG的方法
预先准备冰水冷却浴(4℃)。将双链DNA靶标和用户自定义浓度的DNA探针在0.2mL PCR管中混合，将体积调节至100μL。然后将样品管放入热循环仪(Bio-Rad T100TM)中，并加热到95℃持续5分钟(设置为10分钟，用于下一步)。当样品在热循环仪中保持高温时，将试管快速转移并浸入冰水浴(4℃)中2分钟(图1)。将90μL样品转移到微孔板(Corning)中，并在设置为37℃的微孔板读数器(Molecular Devices)中加热5分钟。此后，添加10μL的200nM toehold交换报告分子以触发反应。
DEG-PCR
在典型的PCR方案中，将4μL DNA模板，20μL Taq 2×Master Mix以及适当浓度(通常为500nM)的正向和反向引物混合至40μL。通过在94℃孵育3分钟开始PCR，然后进行35个循环(在94℃变性，在52℃退火和在72℃延伸30秒)，最后在72℃延伸30分钟。在Bio-Rad T100TM热循环仪中5分钟。不对称PCR的热方案保持不变，而引物浓度不平衡(500nM正向引物和40nM反向引物，图2)。然后将PCR扩增子与4个DEP混合，并将体积调节至90μL。为避免潜在的副反应，将外部DEP(与引物相同)设置为500nM，内部两个DEP为200nM，添加双重报告分子(分开的FAM和Cy5荧光通道)以开始反应。
基于时间的荧光研究
使用SpectraMax i3酶标仪(Molecular Devices)来获取实时荧光数据。将温度设置为37℃，以每分钟1个数据点的频率监控荧光1小时。FAM通道的激发/发射波长设置为485nm/515nm，Cy5通道的激发/发射波长设置为640nm/675nm。将荧光数据归一化并根据公式η＝((F-F b))/((F m-F b))转化成表观杂交产率，其中F为处于平衡状态的样品荧光读数，F m表示对于50倍过量的真ssDNA靶标至链置换信标观察到的最大荧光，F b为仅由受保护信标产生的背景荧光。出于实际目的，当反应大致达到平衡时，在约20-30分钟时收集平衡荧光数据。
使用DEG-PCR分析STH临床样品
STH蠕虫样本是从洪都拉斯西北部拉希卡卡农村地区的八个学龄儿童中回收的。洪都拉斯National Autonomous University和Brock University均获得了伦理批准。八名参与者在前三天接受了基于双羟萘酸噻嘧啶和奥坎特氨酸盐(Conmetel)的治疗方案，并在第四天接受了阿苯达唑治疗方案。用盐溶液清洗在粪便中排出的成虫，并将其储存在70％的乙醇中。回收样本后，根据制造商的规程，使用Automate Express DNA提取系统(Thermo Fisher Scientific Inc.)和商业试剂盒PrepFiler Express BTA提取DNA。此后，遵循典型的DEG-PCR程序(每个PCR引物250nM；每个探针200nM)检测这些临床DNA样品。
通过相同的DEG-PCR方案和临床样品，从1aM到1pM(包含D.R.(-)和D.R.(+))的两批(每批重复两次)合成DNA模板，以建立荧光分布图。此外，为了模拟仅一个染色体具有D.R.(+)突变的杂合基因型，将WT和MT合成DNA模板以1aM至1pM的终浓度均等混合。完整的荧光分布图如图10所示。由于批次间的PCR错误，因此采用误差eclipse而不是线性拟合曲线。
聚丙烯酰胺凝胶电泳
将5μL PCR扩增子溶液与上样缓冲液(Bio-Rad)混合，然后上样到8％天然PAGE凝胶上，以验证和评估PCR程序。使用110V的电压来驱动电泳。之后，将凝胶用溴化乙锭染色，并使用Gel Doc XR+成像仪系统(Bio-Rad)成像。
实施例1：DNA均衡器门(DNA equalizer gate，DEG)设计原理
设计DEG的目的是通过检测信号和靶标浓度之间的定量关系的转化来抑制假靶的检测信号，从而最大化用于区分单核苷酸变体的检测窗口。为了定量地描述检测窗口，发明人引入了鲁棒性因子(RF)，其被定义为产生相同水平的检测信号的乱真目标和正确目标之间的浓度比RF＝[T] spurious/[T] correct(图2b)。这样，RF值越大，检测窗口越宽。尽管DEG作用于dsDNA，但是单链DNA(ssDNA)的检测也可以。并且当对单链DNA(ssDNA)进行检测时，其中DNA平衡探针(DNA equalizer probe，DEP)的浓度接近无穷大。
DEG的设计在图3中示出。双链输入物AB通过在95℃快速加热，然后在分离器闸(splitter gate)中快速冷却至0℃，产生单链靶标A及其互补序列B(图3c)。然后，B被DEP消耗，DEP与A具有相同的序列，DEP在湮没器门(annihilator gate)1中被分成两个或更多部分(Σ1，图3d)。因此，A的产率(η)通过DEP的浓度定量测定。当AB的浓度小于DEP的浓度时，A是主要产物，尽管在A和DEP之间存在与B杂交的竞争。当AB的浓度大于DEP的浓度时，未消耗的B将与湮没器门2中的A再杂交(∑2，图3e)。因此，当AB的浓度等于DEP的浓度时，存在A的最大产率。最后，使用设计成对SNV敏感的toehold交换报告分子定量剩余的A(图3f)。由于每个DEP被设计成仅含有报告分子的toehold结构域或分支迁移结构域的序列，因此在不存在靶标的情况下不能产生荧光信号。通过DEG，杂交探针的常规S形检测曲线被转化为不对称的单峰曲线(图3b)。
检测信号与目标浓度之间的定量关系从S形函数到不对称的单峰函数的转换提供了三个明显的优点。首先，该变换仅抑制较高浓度端处的检测信号。这样，它允许检测窗口的显著扩展而不损害在较低浓度端的灵敏度(图6)。第二，检测窗口的操作是用户可定义的，并且可以在任何目标浓度下实现。原则上，真靶标和假靶标(correct and spurious targets)同时实现了以DEG为单位的最大产量，这两者都由DEP浓度决定。这样，通过简单地改变DEP的浓度，即可确定和调整检测窗口。此外，在整个浓度范围内，真靶标的检测信号保持比假靶标的检测信号高得多(RF＝∞，图3b右侧)，而常规探针的检测窗口窄得多(图3b左侧，图4)。第三，由于假靶标的检测信号被显著抑制，鉴别因子(DF)在宽的浓度范围内被显著增强(图2b右侧)。在分子水平上，B作为竞争性消耗A的分子汇(molecular sink)，而与突变的同一性或位置无关，这与利用为已知突变特别设计的分子汇或储库的现有策略显著不同。为了定量模拟和预测DEG扩展检测窗口和提高序列选择性的有效性，建立了理论模型并在下一部分中进行了详细介绍。
实施例2：DEG的理论模型
引入数学模型以通过考虑所有可能的反应来定量分析DEG(图5a)。为了得出该反应网络中每种DNA物质的产率与序列设计((ΔΔG 0)和均衡器探针浓度的函数关系，需要求解一组八个平衡方程。但是，发明人发现这些方程是相互耦合的，这在数学上很难解决。因此，引入了化学计量矩阵RM来帮助简化计算(图5a)，其中前四行被列为必不可少的(具体如实施例4第4.4所述)。然后，通过数值方法求解该基本的平衡方程组，其中求解A和AB的分布作为目标浓度的函数，并在图5c中进行绘制。
热力学驱动模型成功地预测了浓度范围内A和AB的分布，其中[AB]≥[DEP](图5c)。然而，当[AB]<[DEP]时，它不能模拟DEG的热力学行为。然后，发明人通过引入考虑DEP在AB上的可能分布的概率函数来校正该模型(图5d)。在数学上，DEP-B三链体(BCD)成功形成的概率是[DEPs] 0/[AB] 0) 2(图5d)。热力学驱动模型与概率校正的组合导致特征性不对称单峰曲线(图5e)，其也在实验上得到证实。
实施例3：计算机预测和实验验证
使用本发明的理论模型，首先在计算机中针对DEG中的三个关键因素对η、DF和RF进行了定量分析，这三个关键因素包括靶标浓度、序列设计(ΔΔG 0)和由DEP定义的检测窗口。ssDNA的检测也可以在本发明的模型中通过将DEP的浓度设定为无穷大来描述，其中产生A的产率是100％。图6中的模拟结果描述了在50、100、200和500nM的不同DEP浓度下，从ssDNA([DEP]＝∞)检测到dsDNA检测的理论转变。与η饱和超过某一靶标浓度的常规抑制探针(frustrating probe)(Toeholdexchange或分子信标)不同(图6a)，在单一靶标浓度下，DEG中存在最大η，其仅由DEP([T] max＝[DEP])定义并且与序列无关(图6b)。模拟结果还揭示了检测窗口的显著扩展，其中可以实现单核苷酸突变的高度特异性识别(图6d)。改善的DF水平也可以由DEP的浓度来确定(图6d)。由于高浓度SNVs的η被完全抑制，观察到RF从有限值(图6e)到无穷(图6f)的显著转变。
在图7中绘制了在不同浓度的合成dsDNA靶标下实验测量的η、DF和RF，以与计算机预测的值进行比较。实验验证和优化如下实施例5中进行详细说明。发现
Figure PCTCN2020119612-appb-000008
校正+1.58kcal/mol显著改善了实验观察和计算机预测之间的一致性。使用来自报告分子的荧光读数直接计算特定靶标浓度下的η和DF。与计算机预测一致，对于真靶标和假靶标，均观察到最大η，其由DEP的浓度严格定义(图7a)。如理论上所预测的，假靶标的η被DEG显著抑制了，结果，也观察到了改进的DF，这也与模拟很好地一致(图7b)。通过首先使用非线性模型拟合校准曲线，然后根据定义计算，间接测量RF，然后根据定义进行计算(具体可见实施例4第4.2部分，式8)。再次，在宽浓度范围内确定了无限RF(图7c)。DEG的有效性和灵活性通过不同的单核苷酸突变的类型和位置、不同长度的dsDNA靶标以及9组临床上重要的SNV进行了实验验证(具体可见实施例6-9)。除在dsDNA的最边缘发生突变时，DEG对所有靶标均有效。
实施例4：DEG的理论框架和数学模拟
浓度是评价DNA杂交探针的灵敏度和特异性的重要变量，然而产率、鉴别因子(DF)和鲁棒性因子(RF)的浓度依赖性仍然未被系统探索。发明人首先分析了杂交产率和序列特异性在宽浓度范围内的浓度依赖性。在发明人的系统中，选择toehold交换探针作为测试平台。为了突出变量之间的数值关系，发明人在推导之前对所有浓度应用了无量纲变换。
4.1浓度依赖性和鲁棒性
toehold交换反应可以简化成双分子可逆反应(式1)：
Figure PCTCN2020119612-appb-000009
其中T为靶标，C为T的部分互补链，P为C的保护链。toehold交换探针的热力学可通过改变正向和反向toehold的长度或通过控制CP和P之间的化学计量来调节。可以使用NUPACK软件计算各反应物和产物的自由能。
Zhang和同事先前已经将反应的产率定义为：
Figure PCTCN2020119612-appb-000010
其中[T] 0和[CP] 0分别是T和CP的初始浓度。发明人认为该定义可用于指导Toehold交换探针的序列设计，但不适用于预测探针在指定实验条件下的分析行为，因为初始目标浓度通常是系统中未知的变量。实际上，CP的浓度是固定的，并且T是变量，因此发明人将反应产率定义为：
Figure PCTCN2020119612-appb-000011
发明人还选择[CP] 0作为无量纲变换的特征浓度。
对于典型的可逆反应，平衡常数可以由反应自由能(式2)和所有核酸物质的浓度遵循质量守恒定律。
Figure PCTCN2020119612-appb-000012
发明人进一步将浓度转化成数值的公式进行了无量纲化处理，其中无量纲形式的目标浓度分别表示为τ和[P] 0的目标浓度表示为γ(式3)。
Figure PCTCN2020119612-appb-000013
where γ：＝[P] 0/[CP] 0and τ：＝[T] 0/[CP] 0respectively.
η的浓度依赖性通过式4求解：
Figure PCTCN2020119612-appb-000014
当K eq＝1,
Figure PCTCN2020119612-appb-000015
为了定量描述和比较序列特异性，通常使用鉴别因子(DF)，其中DF＝η 真/η 假。在一般情况下，真靶标(K eq，c)和假靶标(K eq，s)的平衡常数都不为1，鉴别因子(DF)表示为：
Figure PCTCN2020119612-appb-000016
对于(K eq，c)调整为1的设计良好的探针，DF公式可以简化为：
Figure PCTCN2020119612-appb-000017
实际上，一对真靶标和假靶标的DF值是序列设计(ΔG和Keq)和靶标浓度τ的函数(式5、6)。
图8示出了作为靶标浓度(τ)的函数的针对五个假靶标的真靶标的DF的数学预测。所有含有单核苷酸突变的假靶标的DF值随着靶标浓度τ的增加而单调降低。当假靶标的产率接近极小值时，模拟的DF值在低靶标浓度范围内达到最大。然而，当靶标浓度接近或低于特定分析技术的检测极限(LOD)时，产率和DF的数值变得无意义。尽管DF高，模拟显示，在浓度范围τ<0.6时，真靶标和假靶标之间的产率的绝对差异变得很小，因此难以用实验方法解决(图8b)。因此，发明人通过将分析方法的LOD引入到模拟中来修正数学模型。LOD可以被任意定义为允许实验区分由真靶标或假靶标产生的信号与背景的最小产量。图8c显示了当假靶标变得不可检测时(LOD设置为1％产率)，使用修正的模型对DF进行的模拟。实际上，发明人还可以将LOD设置为真靶标的最小可检测产率。
4.2鲁棒性因素
为了定量描述鉴别SNV的检测窗口，发明人在数学上定义了一个鲁棒性因子(RF)，其为一对假靶标和真靶标的产率相同时的浓度比。为此，发明人首先导出目标浓度τ作为产率和平衡常数的函数(式7)。然后可以使用式8从数学上推导出RF。
Figure PCTCN2020119612-appb-000018
Figure PCTCN2020119612-appb-000019
在灵敏度和特异性之间的最佳权衡下，其中K eq，correct＝1，产率为50％，实际有用的RF可以简化为：
Figure PCTCN2020119612-appb-000020
理论RF值作为η的函数线性增加(图9a)。然而，这明显偏离了实验观察结果，因为当杂交方法的 产率接近0或100％时，假靶标和真靶标之间的绝对浓度差(τS-τC)变得不太显著(图9b)。为了更好地反映分析性能，发明人考虑LOD和线性极限(LOL)来修正模型。图9e显示了通过将LOD设定为1％产率并将LOL设定为95％产率，修正的RF模拟。为了理解RF的浓度依赖性，发明人进一步将x轴从产率η转化成靶标浓度τ_correct(图9c-e)。
4.3 toehold交换探针的检测窗口
为了证明toehold交换探针的检测窗口的浓度依赖性。发明人接下来通过MATLAB使用数值方法模拟η、DF和RF。使用42nt合成DNA(参见实施例6第6.1部分)作为模型靶标，并引入单个T至A突变以产生假靶标。可以使用NUPACK软件计算每种DNA物质的标准吉布斯自由能(ΔG 0)，并且因此可以如下计算对于每种toehold交换反应的
Figure PCTCN2020119612-appb-000021
Figure PCTCN2020119612-appb-000022
一对真靶标和假靶标之间的热力学差异可以用ΔΔG 0定量，其中
Figure PCTCN2020119612-appb-000023
在发明人的模型系统中，ΔΔG 0确定为2.30kcal/mol。然后可以通过计算机预测这对合成序列的产率(图10a)、序列选择性(图10b)和浓度鲁棒性(图10c)。正如所预期的那样，所有三个参数都强烈地依赖于浓度(图10a-10c)，这表明，良好设计和优化的toehold交换探针可能仅在一定浓度范围内表现出色。
通过在模型中进一步包括ΔΔG 0作为变量，发明人能够模拟toehold交换探针对所有可能突变的浓度依赖性，这些突变在输血上反应为不同的ΔΔG 0值(图10d-10f)。发明人的模拟结果定量地反映了检测窗口与鉴别某个突变的难度成反比：ΔΔG 0值越小，允许有效鉴别的浓度鲁棒性范围越窄(图10e)。
4.4 DNA均衡器门(DEG)
DEG被设计为以具有明确的检测窗口的定量方式将dsDNA靶转化为ssDNA输出。为了模拟这个过程，发明人假设所有的反应都是热力学驱动的，并且所有的DNA物质都处于其热力学稳定状态。在这种假设下，可以用一组平衡方程来预测新形成的DNA物质的浓度分布(图5a中的主要内容)。然而，仅需要求解独立方程，否则将产生无意义的答案。为了帮助确定独立的平衡方程，发明人从反应体系中提取数值反应矩阵(RM)：
Figure PCTCN2020119612-appb-000024
RM的阶数为4(通过Matlab验证)，小于RM的维数。因此，在该反应体系中只存在4个独立的反应式，发明人在模型中选择了前4个反应。使用NUPACK预测所有
Figure PCTCN2020119612-appb-000025
值，平衡方程如下所示：
Figure PCTCN2020119612-appb-000026
Figure PCTCN2020119612-appb-000027
Figure PCTCN2020119612-appb-000028
Figure PCTCN2020119612-appb-000029
where[A] 0＝[A] eq+[AB] eq；[B] 0＝[B] eq+[BCD] eq+[BC] eq+[BD] eq；
[C] 0＝[C] eq+[BC] eq；[D] 0＝[D] eq+[BD] eq
[A] 0，[B] 0，[C] 0，and[D] 0为初始浓度
根据DEG的实验条件，在4℃下计算标准反应自由能。当[Target]＞[DEP]时，在模型中引入概率函数以定量描述DEP和互补链之间发生的概率结合。
图11表示在不同靶标浓度下每种DNA物质的产率的数学预测。在没有使用概率函数修正的情况下，输出DNA(A)的产量作为输入靶标浓度的函数线性降低(图11a和11b中的虚线)。通过概率修正，当[靶标]等于[DEP]时发生急剧转变，这也通过实验证实。这种转变仅仅由DEP的浓度决定，因此允许在图11c中限定检测窗口，并如图S5d中所示抑制假靶标的信号。
DEG模型与经典toehold交换模型的结合，使发明人能够精确地模拟针对真靶标和任何给定突变的产量和鉴别因子。为了模拟DEG体系中的RF，利用Matlab内置的数学反函数，首先利用toehold交换模型将反应产率转化为ssDNA输出浓度，然后利用DEG模型将ssDNA浓度转化为dsDNA靶标浓度。
4.5 DEG和用于扩大检测窗口的能量势垒的增加之间的比较
通过增加用于激活探针的能量势垒(图4的右下方)或使用DEG方法(图6的右上方)，可以扩大已经建立的用于鉴别单核苷酸错配的检测窗口(图12的左侧)。如通过图12中的模拟结果所展示的。发明人的DEG方法在扩展程度(基本上到无限)上表现更好，并在低浓度范围下更灵敏。
4.6参数修正与拟合
4.6.1
Figure PCTCN2020119612-appb-000030
的修正
Zhang及其同事先前发现，对使用NUPACK软件预测的
Figure PCTCN2020119612-appb-000031
值进行修正是必要的，以改进理论预测和实验观测之间的一致性。在发明人的研究中还进行了类似的修正，以提高数学预测的准确性(图12)。通过比较在不同的
Figure PCTCN2020119612-appb-000032
下的理论预测和实验测定的产率，确定了1.575kcal/mol的修正值，并应用于在整个研究中。
4.6.2通过拟合确定实验RF
由于真靶标和假靶标的两个校准曲线均是使用散射数据点建立的，因此无法直接确定实验RF。因此，发明人结合实验拟合和数学转换来解决这个问题(图13)。首先采用4参数非线性拟合对实验结果进行拟合。将通过拟合确定一组四个参数，包括M、L、s和E(式12)。M和L表示曲线中的最高和最低信号；E表示介于最大和最小极限之间的靶标浓度；s表示拟合曲线的陡度。一旦通过拟合建立了该数学模型，发明人就能够将toehold交换反应的任何产率转化成真靶标或假靶标的相应浓度。然后可以确定实验RF。对于DEG系统，首先需要将校准曲线分为两个部分：[靶标]＝<[DEP]和[靶标]>[DEP](图13)。
非线性模型：
Figure PCTCN2020119612-appb-000033
其中M、L、s和E是要拟合的参数。
实施例5：DEG的实验验证和优化
DEG中实验步骤和DNA反应的示意图如图1所示。
表征DEP和热方案对DEG性能的影响如图14所示，表明DEP和热方案对于确保用于产生单链输出的DEG的高产率至关重要。将在含有1mM Mg 2+和0.1％Tween 20(v/v)的1×Tris缓冲液中含有10nM X、20nM报告分子和200nM DEP的每个样品在37℃下孵育。
DEP对DEG性能的影响如图15所示。发现每个DEP探针部分湮没B并因此促进X向A的转化。然而，只有当两种DEP存在于反应中时才能实现最大产率。
用于分离器和湮灭器门的热方案的优化如图16所示。当所有DNA物质在同一试管中预混合并加热，然后快速冷却至4℃时，确定最大产率。其中，a.发现在热方案之前(pre)或之后(post)加入DEP显著影响的产率。选择DEP和靶标的预混合作为最佳步骤，因为其既提高了反应产率又简化了操作。b.还发现快速冷却步骤对于确保的高产率的A至关重要。c.当快速冷却至4℃作为最终温度时，获得最大产率。发现将最终温度增加至25、55和75℃会逐渐降低反应的产率。
Mg 2+对DEG性能的影响如图17所示。发现DEG在Mg 2+在0-10mM范围内是稳定的。当增加Mg 2+浓度至20mM时发现反应产率略有下降，因为高浓度的Mg 2+可能通过加速复性而促进X的形成。
由DEG产生的输出DNA A的稳定性结果如图18所示。一旦由DEG产生，B被DEP阻断，并因此不能通过复性与B反应。为了通过DEG和报告分子准确定量X，确保反应混合物中游离A的稳定性至关重要。发明人在室温下分别在5分钟，15分钟，30分钟，1小时，2小时，3小时，6小时，12小时和24小时监测DEG反应后溶液A中的浓度。结果表明，A是高度稳定的，在前2小时内没有明显的损失。实际上，发明人在前30分钟内使用报告探针对A进行分析。
对来自DEP的信号泄漏的预估结果如图19所示。荧光背景的可能来源是由DEP和报告探针的相互作用引起的信号。因此，发明人预估了在不同DEP浓度下的信号泄漏。靶标浓度固定在10nM，DEP的浓度从20nM到5μM不等。即使当施加1μM DEP时也未观察到荧光泄漏。当使用5μM DEP时，观察到小于5％的泄漏。
使用DEG估计用于检测X的LOD结果如图20所示。结果表明，当使用200nM DEP产生单链输出A时，LOD估计为0.5nM。
实施例6：使用DEG检测不同的单核苷酸突变
发明人用两组合成靶标，即寄生虫毛首鞭形线虫(Trichuris trichiura，TT)的β-微管蛋白基因的42bp亚基因组和乙型肝炎病毒(HBV)S基因的44bp亚基因组序列，检验了DEG鉴别单核苷酸变体的分析性能和多功能性。这两种疾病都是全世界人类健康的主要威胁。使用本发明的DEG检测平台测试不同类型的突变和插入/缺失。发明人还通过将两组靶标和相应的DEP混合到同一试管中证明了DEG多重化的可能性。
6.1.毛首鞭形线虫中单核苷酸突变的检测
分析毛首鞭形线虫(TT)亚基因组单核苷酸突变的示意图如图21所示。
用于鉴别42-bp dsDNA TT靶标中的单核苷酸T>A、T>G和T>C突变的DEG的实验验证如图22所示。在DEP浓度为100nM、200nM和500nM时检测正确的TT靶标和三个单核苷酸突变的原始荧光信号。还使用报告探针直接分析单链TT靶标，其相当于具有无限DEP的DEG系统。
实验测定的产率与通过使用DEG分析双链TT靶标的T>G和T>C突变的模拟预测的产率的比较如图23所示。
使用DEG对双链TT靶标中的T>G和T>C突变进行实验测量和模拟DF的比较如图24所示。
使用DEG对双链TT靶标中的T>G和T>C突变进行实验测量和模拟RF的比较如图25所示。
对具有不同突变的靶标TT-28的实验测量DF如图26所示。
对具有不同突变的靶标TT-28的实验测量RF如图27所示。
6.2.HBV单核苷酸变异的检测
设计双链合成HBV S基因靶标的目的是测试DEG方法的多功能性。如图28中所示，为该合成靶设计了一对DEP和报告探针。在该系统中引入了单核苷酸突变和碱基插入/缺失，并使用DEG进行了测试。为了验证用于鉴别具有挑战性的单核苷酸突变的DEG方法，发明人特意引入了A至G突变，这是一种众所周知的具有挑战性的SNV，因为G-T摆动的形成减少了真靶标与假靶标之间的自由能差异。发明人发现，与使用toehold交换信标的直接分析相比，DEG方法有效地提高了分析这种具有挑战性的SNV的特异性和浓度鲁棒性(在图29-31中突出显示)。
6.3检测癌症中临床上重要的单核苷酸变异
为了进一步证明DEG方法的多功能性和鲁棒性，发明人针对癌症中经常检测到的临床上重要的单核苷酸变体设计了9组DEG和toehold交换探针。序列和设计如图32所示，DEG用于分析9组靶标的性能如图33和34所示。
实施例7：评估DEG对罕见突变的检测
评估DEG对罕见突变的检测的结果如图35所示。结果表明，使用DEG可以有效检测出高浓度的未突变序列背景下低至0.5％的突变靶标。
实施例8：DEG的多重性
在同一试管中使用两组DEP同时操作TT和HBV靶标。观察各靶标的特征检测曲线，检测窗口由其对应的DEP(对于TT，[DEP]＝50nM，对于HBV，[DEP]＝100nM)控制。结果如图36所示。该实验表明，可在同一试管中进行多重DEG以独立控制多个链置换反应。
实施例9：靶标和DEP的长度效应
如图37所示，利用不同TT靶标和相应DEP长度的序列，验证靶标/DEP的长度效应。使用200nM浓度的相应DEP，通过实验测量的dsDNA靶标长度(从87bp到32bp)的产率、DF和RF，结果如图38所示。这些结果表明，DEG方法适用于不同长度范围的靶标，对分析性能的影响最小。使用浓度分别为50、100和200nM的DEP，实验测量了靶标TT-32的产率、DF和RF，结果如图39所示。
实施例10：DEG与PCR的整合
实际应用的DNA杂交探针应与常用的核酸扩增技术如PCR相容。由于DEG直接作用于dsDNA，因此它是用于分析dsDNA扩增子的理想探针。因此，发明人接着证实了DEG对PCR的适应性。作为原理的证明，设计了一组四个DEP用于代表性的87bp dsDNA扩增子(图40a)，其显示与DEG完全相容。为了避免潜在的交叉反应，特意设计两个外部DEP与PCR引物相同(图40c)。
图41d中的结果证明DEG-PCR既高度灵敏，又具有特异性。可以检测到低至1aM的合成DNA模板。更重要的是，使用DEG，含有单核苷酸突变的1pM假模板的荧光信号被显著抑制，这比10aM真模板的荧光信号低得多(图40d)。相反，当使用不对称PCR产生可检测的ssDNA扩增子，然后使用相同的Toehold-交换报告分子读出时，观察到的检测窗口窄得多(高于1fM)(图40e、40f)。
实施例11：DEG PCR
一组四个DEP的序列设计的示意图如图41所示。这些DEP靶向87bp扩增子(T)以检测TT蠕虫中耐药性的热点。
通过4-DEP设计验证DEG对ssDNA输出A的检测如图42所示。实时荧光监测报告分子的动力学，以用于测量4-DEP均衡器门产生的A。将所有DEP的浓度固定在200nM，以检测20nM具有耐药性突变的靶标A(突变体)，产生80％的荧光产率。DEG方法允许鉴别具有高序列特异性的单核苷酸突变。
使用DEP浓度从50nM至500nM不等的DEG分析PCR扩增子如图43所示。DEG允许检测双链PCR扩增子，同时通过宽浓度范围识别单核苷酸突变。
使用正向和反向引物浓度之间的不同比例优化不对称PCR如图2所示。动力学曲线显示，使用报告探针通过不对称PCR产生的ssDNA输出的测量，该报告探针使用toehold交换操作。
实施例12：DEG-PCR的临床验证
由病毒、细菌和寄生虫引起的感染是全世界人类的主要威胁。抗生素用于治疗各种感染性疾病(通常由于诊断不足)的广泛使用也导致耐药性问题。因此，诊断传染病的理想试验不仅要准确检测特定病原体，还要筛选或鉴定耐药性，从而指导治疗。为此，发明人通过引入双报告系统设计了DEG-PCR，该系统允许同时检测由毛首鞭形线虫(TT)引起的感染并筛选耐药性。第一个通过toehold交换原理操作的报道分子(FAM-报道分子)被设计为针对β-微管蛋白第200位密码子的特定A到T突变，这是一个公认的TT耐苯并咪唑(BZ，药物)的热点。因此，只有当在TT感染中出现耐药性(D.R.+TT感染)时，该报告分子(FAM)的荧光才会开启。通过toehold介导的链置换操作的第二个报告分子(Cys-报告分子)被设计用于检测TT感染。报告分子的反向toehold为0，因此对单核苷酸突变不敏感，这确保了无论SNP的存在都能检测到感染。同时检测两个荧光通道(FAM和Cys)允许在单个试验中检测感染并筛选耐药性。
发明人使用从在Honduras的高度流行性农村地区生活的学龄儿童收集的临床样品，使用DEG-PCR来诊断土壤传播的蠕虫(STH)感染。发明人使用DEG-PCR检测STH感染，同时在同一试验中筛选耐药性(图44a)。设计两个荧光报告分子以测试毛首鞭形线虫的β-微管蛋白基因的密码子196至203和密码子206至213(图44b)。β微管蛋白第200位密码子处的单核苷酸A至T突变是用于药物抗性筛选的成熟遗传变异(图45)。通过包括5-nt反向toehold，将测试该结构域(196至203位密码子)的toehold-exchange报告分子设计为对该SNV高度敏感，而靶向密码子206至213的报告分子没有设计反向toehold。用光谱上不同的荧光染料(FAM和Cy5)标记这两种报告分子，因此在溶液中同时操作(图46和47)。首先使用双通道DEG-PCR测试不同浓度的合成DNA标准品和13种耐药阴性的临床TT样品(图48-51)并绘制在图45c中，其中可定义三个区域(具有99％置信度的错误重叠)，表示阳性感染和阳性耐药性(D.R.+)、阳性感染但阴性耐药性(D.R.-)以及无可检测感染(N.C.)。对接受了阿苯达唑治疗的Hondrams患者排出的六份临床寄生虫样品进行了测试，发现其为TT阳性，但无耐药性(图45d)。还测试了两个用作阴性对照的临床蛔虫样品，发现其均为TT阴性。所有结果与使用显微镜(Kato-Katz)、PCR后凝胶分析(图52)和DNA测序(图53)的诊断测试一致。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。
本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。

Claims (61)

  1. 一种用于DNA单核苷酸变异检测的方法，其包括：
    将待测DNA与反应体系混合，加热解旋，然后快速冷却，检测。
  2. 如权利要求1所述的方法，其特征在于，所述加热解旋温度为80-99℃，时间为1-10分钟。
  3. 如权利要求1所述的方法，其特征在于，所述快速冷却为在1-5分钟内冷却至0-4℃。
  4. 如权利要求1所述的方法，其特征在于，所述反应体系包含：n条DNA平衡探针：
    DEP-1，…，DEP-n；
    其中，n为大于1的整数；
    DEP-1，…，DEP-n的核苷酸序列与待测单链DNA的部分序列相同，且均不重叠，且DEP-1，…，DEP-n的序列组合为待测单链DNA的完整序列或部分序列。
  5. 如权利要求4所述的方法，其特征在于，所述n＝2，DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列。
  6. 如权利要求4所述的方法，其特征在于，所述DEP与待测DNA的摩尔比为1-500:1。
  7. 如权利要求1所述的方法，其特征在于，所述检测包括如下步骤：将快速冷却的混合物在35-40℃下加热1-10分钟，添加检测单链DNA的分子探针触发检测反应。
  8. 如权利要求7所述的方法，其特征在于，所述检测探针包含：2m个报告探针：
    reporter-F 1，
    reporter-Q 1，
    ……
    reporter-F m，
    reporter-Q m；
    其中，m为大于等于1的整数；
    各reporter-F的核苷酸序列由序列F-S1和F-S2组成，其中，F-S1的核苷酸序列与待测单链DNA的部分序列互补，F-S2为与待测单链DNA无关的任意序列；
    各reporter-Q的核苷酸序列与相应的reporter-F的核苷酸序列互补，且reporter-Q的核苷酸序列长度小于相应的reporter-F的核苷酸序列长度；
    且各F-S1序列不同且不重叠。
  9. 如权利要求8所述的方法，其特征在于，各F-S1序列中至少一个与待测单链DNA中待检测位点互补。
  10. 如权利要求8所述的方法，其特征在于，各F-S1序列的长度分别独立地为15-30个核苷酸；
    各F-S2序列的长度分别独立地为1-10个核苷酸；
    各reporter-Q的序列长度分别独立地为15-25个核苷酸；
    各reporter-Q的序列长度与reporter-Q的序列长度相差5-10个核苷酸
  11. 如权利要求8所述的方法，其特征在于，各reporter-F标记有荧光报告基团，各reporter-Q标记有荧光淬灭基团，且各reporter-F上标记的荧光报告基团各不相同。
  12. 如权利要求1所述的方法，其特征在于，所述单核苷酸变异为取代、插入或缺失。
  13. 一种用于DNA单核苷酸变异检测的探针，其包含：2m个报告探针：
    reporter-F 1，
    reporter-Q 1，
    ……
    reporter-F m，
    reporter-Q m；
    其中，m为大于等于1的整数；
    各reporter-F的核苷酸序列由序列F-S1和F-S2组成，其中，F-S1的核苷酸序列与待测单链DNA的部分序列互补，F-S2为与待测单链DNA无关的任意序列；
    各reporter-Q的核苷酸序列与相应的reporter-F的核苷酸序列互补，且reporter-Q的核苷酸序列长度小于相应的reporter-F的核苷酸序列长度；
    且各F-S1序列不同且不重叠。
  14. 如权利要求13所述的探针，其特征在于，各F-S1序列中至少一个与待测单链DNA中待检测位点互补。
  15. 如权利要求13所述的探针，各F-S1序列的长度分别独立地为15-30个核苷酸；
    各F-S2序列的长度分别独立地为1-10个核苷酸；
    各reporter-Q的序列长度分别独立地为15-25个核苷酸；
    各reporter-Q的序列长度与reporter-Q的序列长度相差5-10个核苷酸
  16. 如权利要求13所述的探针，其特征在于，各reporter-F标记有荧光报告基团，各reporter-Q标记有荧光淬灭基团，且各reporter-F上标记的荧光报告基团各不相同。
  17. 一种用于DNA单核苷酸变异检测的试剂盒，其包含权利要求13-16任一项所述的探针。
  18. 如权利要求17所述的试剂盒，其还包含：n条DNA平衡探针：
    DEP-1，…，DEP-n；
    其中，n为大于1的整数；
    DEP-1，…，DEP-n的核苷酸序列与待测单链DNA的部分序列相同，且均不重叠，且DEP-1，…，DEP-n的序列组合为待测单链DNA的完整序列或部分序列。
  19. 如权利要求17所述的试剂盒，其特征在于，所述n＝2，DEP-1、DEP-2的序列组合为待测单链DNA的完整序列或部分序列。
  20. 如权利要求17所述的试剂盒，其特征在于，所述试剂盒还可包含选自：Mg 2+、核苷酸保护剂、缓冲体系中的一种或多种组分。
  21. 一种用于毛首鞭形线虫单核苷酸变异检测的探针，其包含：reporter-F和reporter-Q；其中，reporter-F具有如SEQ ID NO:5所示的核苷酸序列，reporter-Q具有如SEQ ID NO:6所示的核苷酸序列。
  22. 一种用于HBV单核苷酸变异检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:10所示的核苷酸序列，reporter-Q具有如SEQ ID NO:11所示的核苷酸序列。
  23. 一种用于毛首鞭形线虫单核苷酸变异检测的探针，其包含：reporter-F1、reporter-Q1、reporter-F2、reporter-Q2，其中，reporter-F1具有如SEQ ID NO:16所示的核苷酸序列，reporter-Q1具有如SEQ ID NO:17所示的核苷酸序列，reporter-F2具有如SEQ ID NO:18所示的核苷酸序列，reporter-Q2具有如SEQ ID NO:19所示的核苷酸序列。
  24. 一种用于癌症相关基因突变BRAF-D594G检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:24所示的核苷酸序列，reporter-Q具有如SEQ ID NO:25所示的核苷酸序列。
  25. 一种用于癌症相关基因突变BRAF-V600E检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:30所示的核苷酸序列，reporter-Q具有如SEQ ID NO:31所示的核苷酸序列。
  26. 一种用于癌症相关基因突变EGFR-G719A检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:36所示的核苷酸序列，reporter-Q具有如SEQ ID NO:37所示的核苷酸序列。
  27. 一种用于癌症相关基因突变EGFR-L858R检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:42所示的核苷酸序列，reporter-Q具有如SEQ ID NO:43所示的核苷酸序列。
  28. 一种用于癌症相关基因突变EGFR-L861Q检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:48所示的核苷酸序列，reporter-Q具有如SEQ ID NO:49所示的核苷酸序列。
  29. 一种用于癌症相关基因突变KRAS-G12A检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:54所示的核苷酸序列，reporter-Q具有如SEQ ID NO:55所示的核苷酸序列。
  30. 一种用于癌症相关基因突变KRAS-G13V检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:60所示的核苷酸序列，reporter-Q具有如SEQ ID NO:61所示的核苷酸序列。
  31. 一种用于癌症相关基因突变PIK3CA-H1047R检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:66所示的核苷酸序列，reporter-Q具有如SEQ ID NO:67所示的核苷酸序列。
  32. 一种用于癌症相关基因突变STK11-F354L检测的探针，其包含：reporter-F和reporter-Q，其中，reporter-F具有如SEQ ID NO:72所示的核苷酸序列，reporter-Q具有如SEQ ID NO:73所示的核苷酸序列。
  33. 如权利要求21-32任一项所述的探针，其特征在于，所述reporter-F标记有荧光报告基团，reporter-Q标记有荧光淬灭基团。
  34. 一种用于毛首鞭形线虫单核苷酸变异检测的试剂盒，其包含权利要求21或33所述的探针。
  35. 如权利要求34所述的试剂盒，其还包含DNA平衡探针：DEP-1、DEP-2，其中，所述DEP-1具有如SEQ ID NO:3所示的核苷酸序列，DEP-2具有如SEQ ID NO:4所示的核苷酸序列。
  36. 一种用于HBV单核苷酸变异检测的试剂盒，其包含权利要求22或33所述的探针。
  37. 如权利要求36所述的试剂盒，其还包含DNA平衡探针：DEP-1、DEP-2，其中，所述DEP-1具有如SEQ ID NO:8所示的核苷酸序列，DEP-2具有如SEQ ID NO:9所示的核苷酸序列。
  38. 一种用于毛首鞭形线虫单核苷酸变异检测的试剂盒，其包含权利要求23或33所述的探针。
  39. 如权利要求38所述的试剂盒，其还包含DNA平衡探针：DEP-1、DEP-2、DEP-3和DEP-4，其中，所述DEP-1具有如SEQ ID NO:3所示的核苷酸序列，DEP-2具有如SEQ ID NO:4所示的核苷酸序列，DEP-3具有如SEQ ID NO:14所示的核苷酸序列，DEP-4具有如SEQ ID NO:15所示的核苷酸序列。
  40. 一种用于癌症相关基因突变BRAF-D594G检测的试剂盒，其包含权利要求24或33所述的探针。
  41. 如权利要求40所述的试剂盒，其还包含DNA平衡探针：DEP-1、DEP-2，其中，所述DEP-1具有如SEQ ID NO:22所示的核苷酸序列，DEP-2具有如SEQ ID NO:23所示的核苷酸序列。
  42. 一种用于癌症相关基因突变BRAF-V600E检测的试剂盒，其包含权利要求25或33所述的探针。
  43. 如权利要求42所述的试剂盒，其还包含DNA平衡探针：DEP-1、DEP-2，其中，所述DEP-1具有如SEQ ID NO:28所示的核苷酸序列，DEP-2具有如SEQ ID NO:29所示的核苷酸序列。
  44. 一种用于癌症相关基因突变EGFR-G719A检测的试剂盒，其包含权利要求26或33所述的探针。
  45. 如权利要求44所述的试剂盒，其还包含DNA平衡探针：DEP-1、DEP-2，其中，所述DEP-1具有如SEQ ID NO:34所示的核苷酸序列，DEP-2具有如SEQ ID NO:35所示的核苷酸序列。
  46. 一种用于癌症相关基因突变EGFR-L858R检测的试剂盒，其包含权利要求27或33所述的探针。
  47. 如权利要求46所述的试剂盒，其还包含DNA平衡探针：DEP-1、DEP-2，其中，所述DEP-1具有如SEQ ID NO:40所示的核苷酸序列，DEP-2具有如SEQ ID NO:41所示的核苷酸序列。
  48. 一种用于癌症相关基因突变EGFR-L861Q检测的试剂盒，其包含权利要求28或33所述的探针。
  49. 如权利要求48所述的试剂盒，其还包含DNA平衡探针：DEP-1、DEP-2，其中，所述DEP-1具有如SEQ ID NO:46所示的核苷酸序列，DEP-2具有如SEQ ID NO:47所示的核苷酸序列。
  50. 一种用于癌症相关基因突变KRAS-G12A检测的试剂盒，其包含权利要求29或33所述的探针。
  51. 如权利要求50所述的试剂盒，其还包含DNA平衡探针：DEP-1、DEP-2，其中，所述DEP-1具有如SEQ ID NO:52所示的核苷酸序列，DEP-2具有如SEQ ID NO:53所示的核苷酸序列。
  52. 一种用于癌症相关基因突变KRAS-G13V检测的试剂盒，其包含权利要求30或33所述的探针。
  53. 如权利要求52所述的试剂盒，其还包含DNA平衡探针：DEP-1、DEP-2，其中，所述DEP-1具有如SEQ ID NO:58所示的核苷酸序列，DEP-2具有如SEQ ID NO:59所示的核苷酸序列。
  54. 一种用于癌症相关基因突变PIK3CA-H1047R检测的试剂盒，其包含权利要求31或33所述的探针。
  55. 如权利要求54所述的试剂盒，其还包含DNA平衡探针：DEP-1、DEP-2，其中，所述DEP-1具有如SEQ ID NO:64所示的核苷酸序列，DEP-2具有如SEQ ID NO:65所示的核苷酸序列。
  56. 一种用于癌症相关基因突变STK11-F354L检测的试剂盒，其包含权利要求32或33所述的探针。
  57. 如权利要求56所述的试剂盒，其还包含DNA平衡探针：DEP-1、DEP-2，其中，所述DEP-1具有如SEQ ID NO:70所示的核苷酸序列，DEP-2具有如SEQ ID NO:71所示的核苷酸序列。
  58. 如权利要求34-57任一项所述的试剂盒，其还包含选自：Mg 2+、核苷酸保护剂、缓冲体系中的一种或多种组分。
  59. 如权利要求1-12任一项所述的方法、如权利要求13-16、21-33任一项所述的探针、如权利要求17-20、34-58任一项所述的试剂盒在DNA单核苷酸变异的检测中的应用。
  60. 如权利要求13-16、24-32任一项所述的探针或如权利要求17-20、40-57任一项所述的试剂盒在制备用于疾病的诊断和患病风险的评估或检测病原体感染的产品中的应用。
  61. 如权利要求13-16、21、23任一项所述的探针或如权利要求17-20、34-35、38-39任一项所述的试剂盒在制备用于检测病原体感染的产品中的应用。
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