WO2024002166A1 - Procédé de détection de canaux de fluorescence combinés multi-gènes - Google Patents

Procédé de détection de canaux de fluorescence combinés multi-gènes Download PDF

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WO2024002166A1
WO2024002166A1 PCT/CN2023/103215 CN2023103215W WO2024002166A1 WO 2024002166 A1 WO2024002166 A1 WO 2024002166A1 CN 2023103215 W CN2023103215 W CN 2023103215W WO 2024002166 A1 WO2024002166 A1 WO 2024002166A1
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gene
seq
dna
detection
fluorescence
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Chinese (zh)
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李彬
阮微媚
王洪
陈志伟
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广州市基准医疗有限责任公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the invention belongs to the field of gene detection, and specifically relates to a method for multi-gene combined fluorescence channel detection.
  • Circulating free DNA Circulating free DNA
  • cell free DNA Cell free DNA
  • cfDNA detection as a common form of liquid biopsy, has been used in clinical prenatal diagnosis of pregnant women, early diagnosis of tumors, rejection and infection detection after organ transplantation, and other fields.
  • cfDNA detection has great potential, it also faces a series of difficulties in technology development and clinical application.
  • the content of cfDNA in blood is generally low, and the fragments are small and difficult to extract.
  • the target cfDNA present in plasma is very small, improper specimen collection, anticoagulant use, and storage will affect the quality of the test, resulting in false positive or false negative results.
  • the randomness of the breaks may make the target region detected incomplete, further affecting the detection of target genes.
  • NGS Next Generation Sequencing
  • methylation sequencing methods which are divided into the following three categories according to their principles: 1. bisulfite sequencing; 2. sequencing based on restriction enzymes; 3. targeted enrichment of methylation Locus sequencing.
  • bisulfite sequencing data has good reproducibility, but its sensitivity is low and the cost is too high. Even with the continuous reduction of sequencing costs, it still makes scientific research more difficult. It’s hard for workers to accept.
  • the huge data generated in sequencing using this principle makes the analysis task arduous. Sequencing based on restriction endonucleases can determine methylation sites, reduce the amount of data, and have higher throughput.
  • the low efficiency of enzyme digestion may leave some methylated sites uncut, resulting in incomplete results.
  • Targeted enrichment of methylation site sequencing is more targeted, the sequencing cost is lower, and it is relatively easy to analyze the data.
  • the biggest drawback of this method is the uncertainty of the enrichment results, which often makes the results unverifiable.
  • even the low-cost enzyme digestion method i.e., the methylation sequencing method based on restriction enzymes mentioned above
  • Multi-gene combined testing can effectively solve the problem of low sensitivity and improve the accuracy of early screening and diagnosis of tumors (Olkhov-Mitsel, E., et al., (2014). "Novel multiplex MethyLight protocol for detection of DNA methylation in patient tissues and bodily fluids”, Scientific Reports 4:4432.).
  • Multi-gene joint testing is more suitable for precious clinical samples such as cfDNA. It only requires a small amount of templates to detect and analyze multiple genes.
  • the multi-gene combined testing solution is consistent with health economics and uses smaller amounts of reagents and testing materials. Moreover, detection time is saved and detection throughput and efficiency are improved.
  • the multi-gene joint detection in the existing technology also has some limitations.
  • the ABI 7500 real-time fluorescence quantitative PCR instrument supports up to five fluorescent dyes (including Green I, and ) (where ROX is used as a reference fluorescence), other platforms such as the Quantstudio TM 12K Flex real-time fluorescence quantitative PCR system supports the use of up to six fluorescent probes.
  • MethyLight is a method that uses real-time quantitative PCR to detect methylation. It detects DNA after bisulfite conversion. Bisulfite converts unmethylated cytosine (C) in DNA into uracil (U), while methylated cytosine is not changed. This process makes DNA The complexity is significantly reduced, making it more challenging to design highly specific primer probes. Special attention needs to be paid to the CG base content and oligonucleotide length of the target sequence and primer probe, the annealing temperature of the primer probe and the target sequence, and the multiplexing The possibility of secondary structure and dimer formation of primers and probes in the system.
  • the present invention provides a method for multi-gene combined fluorescence channel detection. This method can be used to realize detection of trace or trace amounts with a limited number of fluorescence channels. Simultaneous detection of as many targets as possible in quantitative DNA, with low cost, convenient operation and short cycle time.
  • a method for multi-gene combined fluorescence channel detection includes the following steps:
  • step b) perform bisulfite conversion on the genomic DNA and/or cell-free DNA described in step a);
  • step b) Perform multi-gene combined fluorescence channel detection on the genomic DNA and/or cell-free DNA converted by bisulfite in step b);
  • step c) includes the following steps:
  • the detection target which is the DNA methylation region and/or mutation site related to the tumor in the biological sample to be tested, and use the DNA methylation region and/or mutation site as a marker , wherein the number of said markers is more than 1;
  • step ii Design amplification primers and probes for the markers described in step i); preferably, design amplification primers and probes for the internal reference gene at the same time;
  • step iii) Use the amplification primers and probes in step ii) to amplify the genomic DNA and/or cell-free DNA in step a) and/or the genomic DNA and/or cell-free DNA that have been subjected to bisulfite conversion treatment in step b).
  • DNA is subjected to fluorescent quantitative PCR, and the number of fluorescent quantitative channels in the fluorescent quantitative PCR is less than the number of markers in step i).
  • the marker is any one or more genes of SLC9A3, CPXM1, HOXA7, NEU1, SEMA6D and APC, and the internal reference gene is the ACTB gene.
  • the probe in step ii) is labeled with a fluorescent reporter group at the 5' end, and the fluorescent reporter group is selected from the group consisting of FAM, VIC, NED, CY5, TET, FITC, JOE, HEX, Any one from the group consisting of TAMRA, CY3, CY5.5, ROX and Texas Red; in some preferred embodiments, the fluorescent reporter group is FAM, VIC, NED and CY5;
  • the probe in step ii) is labeled with a fluorescence quenching group at the 3' end, and the fluorescence quenching group is any one selected from the group consisting of BHQ1, BHQ2, Dabcyl and TAMRA; in some preferred implementations In the scheme, the fluorescence quenching groups are BHQ1 and BHQ2.
  • any two genes of SLC9A3, CPXM1, HOXA7, NEU1, SEMA6D and APC are detected in one fluorescence quantitative channel;
  • the SLC9A3 and CPXM1 genes are detected in a fluorescence quantification channel
  • the HOXA7 and NEU1 genes are detected in a fluorescence quantification channel
  • the SEMA6D and APC genes are detected in a fluorescence quantification channel. Detection is performed, and the ACTB gene is detected in a fluorescence quantitative channel;
  • the SLC9A3 and CPXM1 genes are detected in the FAM fluorescence quantification channel
  • the HOXA7 and NEU1 genes are detected in the VIC fluorescence quantification channel
  • the SEMA6D and APC genes are detected in the NED fluorescence quantification channel. Detection was performed in , and the ACTB gene was detected in the CY5 fluorescence quantitative channel.
  • sequences of the amplification primers and probes of the SLC9A3 gene are respectively as shown in SEQ ID NO: 1-3; the sequences of the amplification primers and probes of the CPXM1 gene are as shown in SEQ.
  • sequences of the amplification primer and probe of the HOXA7 gene are shown in SEQ ID NO: 7-9; the sequences of the amplification primer and probe of the NEU1 gene are shown in SEQ ID NO: 10-12; the sequences of the amplification primers and probes of the SEMA6D gene are shown in SEQ ID NO: 13-15; the sequences of the amplification primers and probes of the APC gene are shown in SEQ ID NOs: 16-18; the sequences of the amplification primers and probes of the ACTB gene are shown in SEQ ID NOs: 19-21, respectively.
  • the amplification primers described in step ii) are designed to be mutated so that they can maintain the detection specificity in single-channel detection when combined with fluorescence channel detection;
  • amplification primers designed with the above variations can effectively improve the specificity of methylation detection of DNA converted by bisulfite, and is especially suitable for the reduction of specificity that may be encountered in combined fluorescence channel detection of multiple genes. Condition.
  • the method for variant design of the amplification primer is: according to the unmethylated conversion sequence, the methylated conversion sequence and primer design shown in Table 9 in Example 7 of the present invention. Improve the sequence, and carry out mutation design on the base adjacent to the CpG site closest to the 3' end of the amplification primer.
  • the amplification primers after mutation design include: the amplification primer of the NEU1 gene SEQ ID NO: 22, the amplification primer of the SEMA6D gene SEQ ID NO: 23 and the amplification primer of the APC gene SEQ ID NO: twenty four.
  • sequences of the amplification primer and probe of the SLC9A3 gene are respectively as shown in SEQ ID NO: 1-3; the sequences of the amplification primer and probe of the CPXM1 gene are as shown in SEQ ID NO: 4-6; the sequences of the amplification primers and probes of the HOXA7 gene are shown in SEQ ID NO: 7-9 respectively; the sequences of the amplification primers and probes of the NEU1 gene are shown in SEQ ID NO: 22.
  • SEQ ID NO: 11 and SEQ ID NO: 12; the sequences of the amplification primer and probe of the SEMA6D gene are shown in SEQ ID NO: 23, SEQ ID NO: 14 and SEQ ID NO: 15 respectively.
  • sequences of the amplification primers and probes of the APC gene are shown in SEQ ID NO: 16, SEQ ID NO: 24 and SEQ ID NO: 18 respectively; the sequences of the amplification primers and probes of the ACTB gene are respectively As shown in SEQ ID NO: 19-21.
  • the multi-gene combined fluorescence channel detection method provided by the first aspect of the present invention can detect multiple methylation markers and/or point mutations in trace or trace amounts of genomic DNA and/or cell-free DNA. Markers are detected simultaneously.
  • a limited number of fluorescence channels can be used to simultaneously detect as many sites as possible in trace amounts of DNA.
  • the method uses four-channel combined detection for trace amounts of DNA and seven target sites including internal controls. This method is not only suitable for merging the fluorescence channels of more than two methylation regions, but also for simultaneous detection of methylation regions and point mutation detection in the same fluorescence channel. It is also suitable for the detection of multiple point mutations in the same fluorescence channel. Simultaneous detection in the channel.
  • the trace or trace amounts of genomic DNA and/or cell-free DNA are derived from single cell DNA or fragmented DNA; in some preferred embodiments, the fragmented DNA is free in plasma. DNA. In some embodiments, the minimum detectable amount of methylation in the micro or trace amount of DNA is 50 pg or less.
  • kits for tumor diagnosis includes a method for detecting methylation markers and/or point mutation markers in a biological sample to be tested.
  • Methylation level and/or mutation level reagents are provided, wherein the kit includes a method for detecting methylation markers and/or point mutation markers in a biological sample to be tested.
  • the tumor is gastric cancer
  • the methylation marker and/or point mutation marker is any one selected from the group consisting of SLC9A3, CPXM1, HOXA7, NEU1, SEMA6D and APC genes species or any combination thereof.
  • the reagents in the kit include amplification primers and probes without variation design and variation design as involved in the first aspect of the present invention.
  • the biological sample to be tested is selected from any one of the group consisting of cells, tissue samples, body fluid samples and excreta, or any combination of the above biological samples;
  • the body fluid sample is selected from any one or any combination thereof from the group consisting of plasma, saliva, and serum, and the excretion is selected from the group consisting of urine, feces, and colonic effluent. any one or any combination thereof;
  • the biological sample to be tested is selected from plasma.
  • the present invention realizes the simultaneous detection of as many markers as possible of trace or trace amounts of DNA using a limited number of fluorescence channels, and can detect a large number of non-methylated backgrounds. DNA and/or detecting trace amounts of methylated target sequences and/or mutated target sequences from a large amount of unmutated wild-type DNA, thereby improving detection efficiency and throughput, and improving detection sensitivity.
  • the present invention breaks through the limitation that the ABI 7500 detection platform can detect up to four gene sites to be detected in up to four fluorescence channels except ROX reference fluorescence, by calculating and analyzing the annealing between primers and probes in the combined detection system Temperature difference, the ability to form secondary structures and dimers, etc., enable this system to detect as many markers as possible with a limited number of fluorescence channels, further improving detection efficiency and throughput.
  • the present invention solves the problem of specificity deterioration that may occur in combined channel detection by adjusting the sequence design of primers and probes.
  • On the basis of maintaining the same specificity as single-channel detection it is possible to detect a large number of non-A methylated background DNA and/or detects trace amounts of methylated target sequences and/or mutated target sequences from a large amount of unmutated wild-type DNA, thereby improving detection sensitivity.
  • Figure 1A shows the heat map of methylation differences in fresh frozen tissue samples from 23 cases of gastric cancer and 165 cases of non-gastric cancer.
  • Figure 1B shows a heat map of methylation differences in paraffin-embedded tissue samples from 395 gastric cancer cases and 559 non-gastric cancer cases.
  • Figure 2 shows the ROC curves of different detection methods described in Example 8.
  • sample template refers to the nucleic acid derived from the sample used to analyze the presence of the "target".
  • background template is used to refer to nucleic acids other than the sample template, which may or may not be present in the sample. Background templates are often unintentional. This may be a legacy result, or it may This may be due to the presence of nucleic acid contaminants that are being attempted to be purified from the sample. For example, nucleic acids from an organism other than the nucleic acid to be detected may be present as background in the test sample.
  • primer refers to an oligonucleotide that occurs naturally or synthetically in a purified restriction digest when exposed to conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand (e.g., in It can serve as a starting point for synthesis in the presence of nucleotides and an inducer such as DNA polymerase and at the appropriate temperature and pH).
  • Primers are preferably single-stranded for maximum efficiency of amplification, but may be double-stranded. If double-stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primers are oligodeoxyribonucleotides. The primer must be long enough to initiate the synthesis of the extension product in the presence of the inducer. The exact length of the primer will depend on many factors, including temperature, source of primer, and method used.
  • probe refers to an oligonucleotide (e.g., a nucleotide sequence) that occurs naturally in a purified restriction digest or is synthesized, recombinant, or produced by PCR amplification, which is capable of interacting with another A sensing target oligonucleotide hybridizes. Probes can be single-stranded or double-stranded. Probes can be used for the detection, identification and isolation of specific genetic sequences (eg, "capture probes"). It is contemplated that in some embodiments, any probe used in the invention may be labeled with any "reporter” such that it is detectable in any detection system.
  • gastric cancer gastric cancer
  • gastric cancer gastric cancer
  • stomach cancer epithelial malignant tumors originating in the stomach.
  • gastric cancer is used in the broadest sense and refers to all cancers that begin in the stomach. It includes the following subtypes that begin in the stomach: adenocarcinoma, lymphoma, gastrointestinal stromal tumor (GIST), carcinoid tumors and squamous cell carcinoma, small cell carcinoma and leiomyosarcoma.
  • GIST gastrointestinal stromal tumor
  • Stage 0 (Tis, N0, M0), Stage IA (T1, N0, M0), Stage IB (T1, N1, M0; or T2, N0, M0), stage IIA (T1, N2, M0; T2, N1, M0; or T2, N0, M0), stage IIB (T1, N3, M0; T2, N2, M0; T3, N1, M0; or T4a, N0, M0), stage IIIA (T1, N2, M0; T2, N1, M0; or T2, N0, M0), stage IIIB (T3, N3, M0; T4a, N2, M0; or T4b, N0 or N1, M0), stage IIIC (T4a, N3, M0; or T4b, N2 or N3, M0) and stage IV (any Other T, N and M).
  • TNM Tumor Node Metastasis
  • stage I stage II, stage III, stage IV, etc.
  • the stages of gastric cancer include but are not strictly limited to the following stages within its definition:
  • Stage I In stage I, cancer has formed in the lining of the stomach wall's mucosa (the innermost layer). Stage I is divided into stages IA and IB based on where the cancer has spread.
  • Stage IA The cancer may have spread into the submucosa (the layer of tissue next to the mucosa) of the stomach wall.
  • Stage IB The cancer may have spread to the submucosa (the layer of tissue next to the mucosa) of the stomach wall and is found in 1 or 2 lymph nodes close to the tumor; or it may have spread to the muscle layer of the stomach wall.
  • submucosa the layer of tissue next to the mucosa
  • Stage II stomach cancer is divided into stages IIA and IIB based on where the cancer has spread.
  • Stage IIA The cancer has spread to the subserosal layer of the stomach wall (the layer of tissue next to the serosa); or it has spread to the muscular layer of the stomach wall and is found in 1 or 2 lymph nodes close to the tumor; or it may have spread to the submucosa (the layer of tissue next to the mucosa) of the stomach wall and is found in 3 to 6 lymph nodes close to the tumor.
  • Stage IIB The cancer has spread to the serosa (the outermost layer) of the stomach wall; or it has spread to the subserosal layer of the stomach wall (the layer of tissue next to the serosa) and is found in 1 or 2 lymph nodes close to the tumor ; or may have spread to the muscular layer of the stomach wall and be found in 3 to 6 lymph nodes close to the tumor; or may have spread to the submucosa (the layer of tissue next to the mucosa) of the stomach wall and be found in 7 or 7 lymph nodes close to the tumor More lymph nodes were found.
  • Stage III stomach cancer is divided into Stage IIIA, Stage IIIB, and Stage IIIC based on where the cancer has spread.
  • Stage IIIA The cancer has spread to the serosa (the outermost layer) of the stomach wall and is found in 1 or 2 lymph nodes close to the tumor; or it has spread to the subserosa (the layer of tissue next to the serosa) of the stomach wall ) and is found in 3 to 6 lymph nodes close to the tumor; or has spread to the muscle layer of the stomach wall and is found in 7 or more lymph nodes close to the tumor.
  • Stage IIIB The cancer has spread to nearby organs, such as the spleen, transverse colon, liver, diaphragm, pancreas, kidneys, adrenal glands, or small intestine, and can be found in 1 or 2 lymph nodes close to the tumor; or it has spread to the stomach wall Serosa (the outermost layer) and is found in 3 to 6 lymph nodes close to the tumor; or spreads to the subserosal layer (the tissue layer immediately next to the serosa) of the stomach wall and is found in 7 or more lymph nodes close to the tumor were found in lymph nodes.
  • organs such as the spleen, transverse colon, liver, diaphragm, pancreas, kidneys, adrenal glands, or small intestine
  • Stage IIIC The cancer has spread to nearby organs such as the spleen, transverse colon, liver, diaphragm, pancreas, kidneys, adrenal glands, or small intestine and can be found in 3 or more lymph nodes close to the tumor; or it has spread to the stomach wall of the serosa (outermost layer) and is found in 7 or more lymph nodes close to the tumor.
  • organs such as the spleen, transverse colon, liver, diaphragm, pancreas, kidneys, adrenal glands, or small intestine and can be found in 3 or more lymph nodes close to the tumor; or it has spread to the stomach wall of the serosa (outermost layer) and is found in 7 or more lymph nodes close to the tumor.
  • Stage IV In stage IV, the cancer has spread to distant parts of the body.
  • the term "marker” refers to measurable substances such as genes, proteins, metabolites, etc. quantity of molecules. Measurement of disease-related variables that can be used to diagnose a disease or indicate the severity of a disease. The presence or risk of a disease can be inferred from the biomarker parameter without measuring the disease itself.
  • the term "real-time fluorescence quantitative PCR” refers to a method of using fluorescent chemicals to measure the total amount of product after each polymerase chain reaction (PCR) cycle in a DNA amplification reaction.
  • PCR polymerase chain reaction
  • the cycle threshold means: the number of cycles experienced when the fluorescence signal in each reaction tube reaches the set threshold.
  • the setting method of fluorescence threshold is as follows: the fluorescence signal of the first 15 cycles of the PCR reaction is used as the fluorescence background signal.
  • the default (default) setting of the fluorescence threshold is the standard deviation of the fluorescence signal of 3 to 15 cycles. 10 times.
  • the term "amplification efficiency" is a stable and reliable method to evaluate PCR amplification efficiency.
  • a series of diluted samples are used, and a standard qPCR program is used to amplify to obtain the Ct value.
  • the term "cut off value” refers to a critical Ct value for judging whether a sample is negative or positive for a certain biomarker.
  • the "cut-off value” i.e., positive judgment value
  • the critical Ct value can be based on the required sensitivity or specificity. Sexual requirements vary.
  • sensitivity refers to the proportion of positive samples detected as positive.
  • Truste positives refer to the recognized golden The standard diagnosis is positive.
  • the term "subject” may be a mammal or a cell, tissue, organ or part of the mammal.
  • mammal refers to any kind of mammal, preferably human (including humans, human subjects or human patients).
  • Subjects and mammals include, but are not limited to, farm animals, sporting animals, pets, primates, horses, dogs, cats and rodents such as mice and rat.
  • diagnosis includes the detection or identification of a disease state or condition in a subject, the determination of the likelihood that a subject will develop a given disease or condition, or the determination of the likelihood that a subject suffering from a disease or condition will respond to treatment. properties, determining the prognosis of a subject suffering from a disease or disorder (or its likely progression or regression), and determining the effect of a treatment in a subject suffering from a disease or disorder.
  • methylation refers to the methylation of cytosine at the C5 or N4 position of cytosine (Cytosine, abbreviated as C), the N6 position of adenine (Adenine, abbreviated as A) or other types of nucleic acids. Methylation. In vitro amplified DNA is usually unmethylated because generally in vitro DNA amplification methods do not preserve the methylation pattern of the amplified template. However, "unmethylated DNA” or “methylated DNA” may also refer to amplified DNA that is unmethylated or methylated from the original template, respectively.
  • methylated nucleotide or “methylated nucleotide base” refers to the presence of a methyl moiety on a nucleotide base, wherein the methyl moiety is not present in the generally accepted typical in nucleotide bases.
  • cytosine does not contain a methyl moiety on its pyrimidine ring, but 5-methylcytosine contains a methyl moiety at position 5 of its pyrimidine ring. Therefore, cytosine is not a methylated nucleotide and 5-methylcytosine is.
  • Thymine (T) contains a methyl moiety at position 5 of its pyrimidine ring; however, for the purposes of this article, thymine is not considered a methylated nucleoside when present in DNA acid because thymine is the typical nucleotide base of DNA.
  • methylation status may optionally be represented or indicated by the term "methylation value” (eg, indicating methylation frequency, fraction, ratio, percentage, etc.).
  • the term "methylation value” can be used, for example, to quantify the amount of intact nucleic acid present after restriction digestion with a methylation-dependent restriction enzyme, or by comparing amplification profiles after a bisulfite reaction, or by comparing bisulfite Sequences of salt-treated and untreated nucleic acids were generated. Therefore, values such as methylation values represent methylation status and can therefore be used as quantitative indicators of methylation status in multiple copies of a locus.
  • the degree of co-methylation is expressed or indicated by the methylation status of more than one methylation site. Within a methylation region, when the methylation status of more than one methylation site is methylated is defined as comethylation.
  • methylation frequency or “methylation percent (%)” refers to the number of instances of a molecule or locus that is methylated relative to the number of instances of a molecule or locus that is unmethylated.
  • bisulfite reagent refers to a reagent that in some embodiments includes bisulfite (bisulfite), disulfite (disulfite), hydrogen sulfite (hydrogen sulfite), or a combination thereof,
  • bisulfite bisulfite
  • disulfite disulfite
  • hydrogen sulfite hydrogen sulfite
  • a combination thereof After DNA is treated with bisulfite reagent, its unmethylated cytosine nucleotides will be converted into uracil, while the methylated cytosine and other bases will remain unchanged, so it can distinguish, for example, CpG dinuclear Methylated and unmethylated cytidines in nucleotide sequences.
  • methylation assay refers to any assay used to determine the methylation status of one or more CpG dinucleotide sequences within a nucleic acid sequence.
  • the detection of the above-mentioned methylated regions of the present invention includes the following main steps:
  • DNA eg., plasma cell-free DNA
  • any standard means in the art including the use of commercially available kits.
  • the biological sample to be tested is a biopsy or autopsy sample.
  • the biological sample is a cell or tissue sample.
  • the biological sample is a body fluid sample, including any one of plasma, saliva, and serum.
  • the biological sample is excreta, including any of urine, feces, and colonic effluent.
  • AUC is an abbreviation for "area under the curve.” Specifically, it refers to the area under the receiver operating characteristic (ROC) curve.
  • ROC receiver operating characteristic
  • a ROC curve is a plot of the true positive rate versus the false positive rate for different possible block cut points of a diagnostic test. It illustrates the trade-off between sensitivity and specificity depending on the chosen cleavage point (any increase in sensitivity will be accompanied by a decrease in specificity).
  • the area under the ROC curve (AUC) is a measure of the accuracy of a diagnostic test (the larger the area, the better; optimal is 1; a random test will have an ROC curve on the diagonal with an area of 0.5; see: JPEgan. (1975) Signal Detection Theory and ROC Analysis, Academic Press, New York).
  • multi-gene joint testing can effectively solve the problem of low sensitivity and improve the accuracy of early screening and diagnosis of tumors.
  • the multi-gene combined testing solution is consistent with health economics and uses smaller amounts of reagents and testing materials.
  • the 450K chip methylation information of paraffin-embedded tissue samples from 395 cases of gastric cancer and 559 cases of non-gastric cancer was used to perform limma (moderated t-test) differential analysis. According to the results of differential analysis, it is required that FDR ⁇ 0.05 and beta value difference be greater than 0.15 to obtain candidate differential CpG sites. Then combined with the AUC, sensitivity (Sensitivity), specificity (Specificity) of the candidate CpG site and the co-methylation status of CpG at adjacent positions, etc., comprehensive screening to 6 co-methylated regions, that is, 6 methylation markers, as shown in Figure 1B.
  • methylation markers i.e., markers of gastric cancer
  • primer pairs including forward primers and reverse primers
  • probe combinations as shown in Table 1 were designed:
  • gene name-F in Table 1-2 indicates the forward primer of the gene detection fragment
  • gene name-R indicates the reverse primer of the gene detection fragment
  • gene name-P indicates the Probes for gene detection fragments (the corresponding modification groups are in parentheses).
  • the commercial fully methylated standard (used as a positive control in this example) and the non-methylated standard (used as a negative control in this example) were prepared with nuclease-free water to 5ng/ ⁇ L and diluted
  • the final commercial fully methylated standard was diluted with the diluted unmethylated standard, and the ratios were 1:0, 1:9, 1:19, 1:39, 1:99, and 1:199. , 0:1, corresponding methylation rate They are 100%, 10%, 5%, 2.5%, 1%, 0.5% and 0% respectively.
  • Fluorescent quantitative PCR reaction program 95°C for 5 minutes; 95°C for 15 seconds, 60°C for 40 seconds, 15 cycles; 95°C for 15 seconds, 62°C for 40 seconds (fluorescence collection), 45 cycles.
  • “Undetermined” described in Table 4 in this example means that the corresponding Ct value was not detected under the amplification conditions.
  • the “optimal linear range” mentioned in Table 4 means that the fully methylated standard corresponding to the percentage mass content shows a good linear relationship within this range when the completely unmethylated standard is used as the background.
  • the “linear correlation coefficient” described in Table 4 is used to reflect the relationship between methylation percentage and Ct value. The closer it is to 1, the stronger the correlation between methylation percentage and Ct value.
  • Example 4 Combined fluorescence channel method to detect standards and evaluate the performance of primers and probes
  • “Undetermined” described in Table 6 of this example means that the corresponding Ct value was not detected under this amplification condition.
  • the "optimal linear range” mentioned in Table 6 means that the fully methylated standard corresponding to the percentage mass content shows a good linear relationship within this range when the completely unmethylated standard is used as the background.
  • the "linear correlation coefficient” described in Table 6 is used to reflect the relationship between methylation percentage and Ct value. The closer it is to 1, the stronger the correlation between methylation percentage and Ct value.
  • the optimal linear range of the SLC9A3 gene and CPXM1 gene i.e., the lowest methylation rate in the detection linear range
  • the results corresponding to the SLC9A3 gene and CPXM1 gene were reduced to 0.5% (for details, please refer to the results corresponding to the SLC9A3 & CPXM1 genes in the second column of Table 6), effectively achieving an improvement in detection sensitivity.
  • the optimal linear range of the SEMA6D gene and the APC gene i.e., the lowest methylation rate for detecting the linear range
  • the optimal linear range of the SEMA6D gene and the APC gene also ranged from 1% in a single channel (see the results corresponding to the SEMA6D gene and the APC gene in column 2 of Table 4 for details) It was reduced to 0.5% (for details, please refer to the results corresponding to SEMA6D&APC genes in the second column of Table 6), which effectively improved the detection sensitivity.
  • HOXA7 and NEU1 genes and SEMA6D and APC genes were combined and non-specifically detected in the 0% methylation standard in this example (see the last column of Table 6 for details, HOXA7&NEU1 genes and SEMA6D&APC genes Ct value detection results).
  • Example 5 Improved detection method and primer design to improve detection specificity
  • Example 3 Regarding the problem of non-specific detection of the 0% methylation standard described in Example 3 (for details, see the Ct value of the APC gene in Table 4, which is 31.52) and the 0% methylation standard described in Example 4 (for details, see the Ct value of the HOXA7&NEU1 gene in Table 6 The value is 29.00, and the Ct value of the SEMA6D&APC gene in Table 6 is 31.97).
  • the primer design a mismatch design for one base of the methylated sequence after sulfite conversion was introduced.
  • the base mismatch greatly increases the specificity for non-methylated sequences, but has no significant effect on the amplification of methylated sequences, so the specificity of DNA methylation detection can be improved.
  • a mismatch base was introduced at the second or third base from the penultimate 3' end of the forward or reverse primers of NEU1, SEMA6D and APC respectively.
  • the sequences of the redesigned primer pairs (including forward primer and reverse primer) and probe sequences are shown in Table 7:
  • gene name-F in Table 7 indicates the forward primer of the gene detection fragment
  • gene name-R indicates the reverse primer of the gene detection fragment
  • gene name-P indicates the gene detection fragment.
  • the specific operation of detecting a single methylated region is the same as that in Example 3.
  • the specific operation of methylation detection by combining fluorescence channels is the same as that in Example 4.
  • the target sequence of the target region is amplified, and the detection result is evaluated through the amplification cycle threshold Ct.
  • the specific operations are as follows:
  • “Undetermined” in Table 8 in this example means that the corresponding Ct value was not detected under the amplification conditions.
  • the "optimal linear range” mentioned in Table 8 means that the fully methylated standard with the corresponding percentage mass content shows a good linear relationship within this range when the completely unmethylated standard is used as the background.
  • the "linear correlation coefficient” described in Table 8 is used to reflect the relationship between the methylation percentage and the Ct value. The closer it is to 1, the stronger the correlation between the methylation percentage and the Ct value.
  • Example 7 Method for designing methylation primers to improve detection specificity
  • the primer design principles for methylation PCR detection require that the last three bases at the 3' end of the primer should contain at least 1 CpG site to enhance the specificity of the primer, combined with the principle of base mismatch PCR amplification (Li , B., et al. (2004). "Genotyping with TaqMAMA.” Genomics 83(2):311-320.), the bases adjacent to the CpG site closest to the 3' end of the primer are designated as specific according to the following table Improved design.
  • the unconverted sequence of SEQ ID NO: 10 is The sequence of the corresponding unmethylated cytosine after bisulfite conversion is SEQ ID NO: 25: (That is, the second C at the 3' end in SEQ ID NO: 10 is converted to T), and the sequence of the methylated cytosine after bisulfite conversion is still SEQ ID NO: 10: As shown in Table 9 below, the corresponding modified primer sequences are: (i.e. SEQ ID NO: 22).
  • the improved primers designed as described above may have performance differences in actual methylation detection.
  • appropriate primers will be selected based on the detection results of specific methylated regions.
  • preferred primer sequences are listed.
  • this includes, but is not limited to, the preferred primer sequences described above.
  • the preferred highly specific primer sequences can be used in many other different methods to detect co-methylation in DNA methylation regions, such as: methylation-specific PCR (MSP), DNA methylation chip, Targeted DNA methylation sequencing, digital PCR quantitative and fluorescence quantitative PCR, methylation-sensitive restriction endonuclease (MS-RE)-PCR/Southern method, direct sequencing method, methylation-sensitive single nucleotide Primer extension (Ms-SnuPE), combined bisulfite restriction enzyme method (COBRA), methylation-sensitive single-strand conformation analysis (MS-SSCA), methylation-sensitive denaturing gradient gel electrophoresis ( MS DGGE), methylation-specific denaturing high-performance liquid chromatography (MS-DHPLC), methylation-specific Heterogeneous microarray (MSO), methylation-sensitive melting curve analysis (MS-MCA), methylation-sensitive spot analysis (MS-DBA), methylation-specific multiligation-dependent probe a
  • Example 8 Comparison of clinical diagnostic performance between single channel multi-gene methylation detection and multi-gene combined channel detection
  • Example 10 On the clinical plasma samples of 58 cases of gastric cancer and 59 cases of non-gastric cancer confirmed by endoscopic pathology or clinical diagnosis, the methylation of multiple methylation regions was detected according to the detection technology in Example 3 and Example 4 of the present invention. .
  • the clinical information statistics of the test samples are shown in Table 10 below:
  • Plasma separation method centrifuge at 1,600g, 4°C for 10 minutes, carefully transfer the upper plasma to a 2mL centrifuge tube; plasma 16,000g, 4°C, centrifuge a second time for 10 minutes, carefully transfer the upper plasma to a new 2mL centrifuge tube; The separated plasma should be extracted immediately or frozen below -80°C for extraction within one year.
  • Plasma free DNA was extracted using LIFE's commercial kit MagMAX TM Cell-Free DNA Isolation Kit in accordance with the instructions. The extracted DNA was used after passing the quality inspection of Qubit 4.0 and Agilent 2100 bioanalyzer.
  • the DNA bisulfite conversion kit was purchased from ZYMO RESEARCH Company and was performed according to the kit instructions.
  • Example 5 of the present invention Single-plex fluorescence quantitative PCR detection and multi-gene combined channel PCR detection were performed according to the methods of Example 3 and Example 4 of the present invention, respectively.
  • each The distribution of ⁇ Ct values of each fluorescence channel (combined or single SEQ ID) in the gastric cancer group and non-gastric cancer group create a ROC curve, and calculate the detection sensitivity and specificity under its AUC value and Youden index, and compare using single-plex fluorescence quantitative PCR
  • the AUC of detecting a single methylated region, the sensitivity and specificity under Youden index cut-off, and the AUC, sensitivity and specificity of the combined interpretation of two methylated regions that is, the combined interpretation of the two-gene single-plex detection described in Table 11) sex, and the comparative results of the AUC, sensitivity and specificity of the two methylated regions in the multi-gene combined channel PCR detection (i.e. the two gene combined channel detection interpretation described in Table 11) are shown in Table 11 below, and their ROC curves
  • Table 11 the comparative results of the AUC, sensitivity and specificity of the two methylated regions in the multi-gene combined channel PCR detection

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Abstract

La présente invention concerne un procédé de détection de canaux de fluorescence combinés multi-gènes. Le procédé comprend les étapes suivantes : a) extraction de l'ADN génomique et/ou de l'ADN acellulaire d'un échantillon biologique à détecter ; b) conversion au bisulfite de l'ADN génomique et/ou de l'ADN acellulaire à l'étape a) ; et c) détection de canaux de fluorescence combinés multi-gènes sur l'ADN génomique et/ou l'ADN acellulaire soumis à la conversion au bisulfite à l'étape b). Le procédé de détection selon l'invention peut mettre en œuvre une détection synchrone cible autant que possible pour une petite quantité ou une petite quantité d'ADN au moyen d'un nombre limité de canaux de fluorescence, et mettre en œuvre la détection d'une quantité de trace de séquences cibles méthylées à partir d'une grande quantité d'ADN non méthylé, ce qui permet d'améliorer la sensibilité de détection. De plus, le procédé peut mettre en œuvre la détection autant de sites à détecter que possible au moyen d'un nombre limité de canaux de fluorescence, ce qui permet d'améliorer l'efficacité et le débit de détection.
PCT/CN2023/103215 2022-06-29 2023-06-28 Procédé de détection de canaux de fluorescence combinés multi-gènes WO2024002166A1 (fr)

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WO2012055409A1 (fr) * 2010-10-27 2012-05-03 Quantibact A/S Capture de séquences d'arn et/ou d'adn méthylé(s) par des sondes spécifiques
US20160083792A1 (en) * 2013-01-25 2016-03-24 Murdoch Childrens Research Institute An assay for quantitating the extent of methylation of a target site
CN106399577A (zh) * 2016-12-15 2017-02-15 湖南圣湘生物科技有限公司 一种用于单通道双靶点核酸检测的实时荧光pcr检测方法
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CN112280839A (zh) * 2020-10-16 2021-01-29 李凯 检测靶点数多于荧光通道数的实时pcr技术及其应用
CN114277135A (zh) * 2021-10-25 2022-04-05 广州市基准医疗有限责任公司 胃癌淋巴结转移相关的甲基化生物标记物及其组合和检测试剂盒
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