WO2023040997A1 - Procédé de test monogénique et son application - Google Patents

Procédé de test monogénique et son application Download PDF

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WO2023040997A1
WO2023040997A1 PCT/CN2022/119180 CN2022119180W WO2023040997A1 WO 2023040997 A1 WO2023040997 A1 WO 2023040997A1 CN 2022119180 W CN2022119180 W CN 2022119180W WO 2023040997 A1 WO2023040997 A1 WO 2023040997A1
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region
sample
nucleic acid
clinical test
tested
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PCT/CN2022/119180
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Chinese (zh)
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林坚
陈航宇
许诺
陈龙
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北京大学
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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
    • 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
    • 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
    • C12Q1/6869—Methods for sequencing
    • 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
    • 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

  • This application relates to the field of biomedicine, in particular to a single gene detection method and its application.
  • a commonly used method for screening the beneficiaries of targeted drug therapy is to use the tumor tissue samples of tumor patients punctured or operated on to perform immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) tests of relevant target genes, so as to determine whether the tumor patients are Suitable for targeted drug therapy.
  • IHC immunohistochemistry
  • FISH fluorescence in situ hybridization
  • the present application provides an analysis method, comprising (S1) determining the area to be tested based on the quantity and/or presence of hydroxymethylcytosine in the clinical test-positive samples and the clinical test-negative samples of the target gene, (S2) Detecting the quantity and/or presence of said hydroxymethylcytosine in said test region of the test sample.
  • the present application provides a method for confirming the existence of a disease, assessing the formation or risk of developing the disease, assessing the progression and/or prognosis of the disease, and/or screening the population corresponding to the treatment, comprising (S1) based on the target gene The amount and/or presence of hydroxymethylcytosine in the clinical test positive sample and the clinical test negative sample, determine the area to be tested, (S2) detect the hydroxymethylcytosine in the test area of the test sample quantity and/or presence.
  • the present application provides an analysis method, comprising detecting the quantity and/or presence of hydroxymethylcytosine in the region to be tested of the single target gene of the sample to be tested by digital PCR based on a single target gene .
  • the present application provides a method for confirming the presence of a disease, assessing the development or risk of developing a disease, assessing the progression and/or prognosis of a disease, and/or screening a population responding to treatment, comprising based on a single target gene, by
  • the digital PCR method detects the quantity and/or presence of hydroxymethylcytosine in the test area of the test sample.
  • the present application provides a nucleic acid, which comprises a sequence capable of binding to the region to be tested determined by the method described in the present application, or its complementary region, or the above-mentioned fragments.
  • the present application provides a method for preparing a nucleic acid, comprising the sequence of the region to be tested, or its complementary region, or the above-mentioned fragment determined according to the method described in the application, designed to be able to bind to the region to be tested, or a complementary region thereof, or a nucleic acid of a fragment thereof.
  • the present application provides a kit comprising the nucleic acid described in the present application.
  • the present application provides the application of the nucleic acid described in the present application, and/or the kit described in the present application, in the preparation of disease detection products.
  • the present application provides the nucleic acid described in the present application, and/or the kit described in the present application, which can be used to confirm the existence of the disease, assess the formation or risk of the disease, assess the progress and/or prognosis of the disease and/or the nucleic acid described in the application. Or screen for applications in products that have appropriate populations for treatment.
  • the present application provides a database comprising the sequence of the region to be tested determined by the method described in the present application, or its complementary region, or the above-mentioned fragments.
  • the present application provides a storage medium, which records a program capable of running the method described in the present application.
  • the present application provides a device comprising the storage medium described in the present application.
  • the present application provides the device described in the present application, further comprising a processor coupled to the storage medium, and the processor is configured to execute based on a program stored in the storage medium to implement the present application the method described.
  • Figure 1 shows the copy number (copies of ng) of the PD-L1 hydroxymethylation site (CD274) single gene detection in the plasma cell-free DNA 5hmC library of 49 melanoma patients; among them, the PD-L1 positive group (PD -L1+) was significantly different from the PD-L1 negative group (PD-L1-) (p ⁇ 0.01).
  • Figure 2 shows the copy number (copies of ng) of EGFR hydroxymethylation site single gene detection in the plasma cell-free DNA 5hmC library of 56 patients with esophageal squamous cell carcinoma; among them, the EGFR positive group (EGFR+) and the EGFR negative group ( EGFR-) compared with significant difference (p ⁇ 0.01).
  • Figure 3 shows the copy number (copies of ng) of the HER2 hydroxymethylation site (ERBB2) single gene detection in the plasma cell-free DNA 5hmC library of 36 breast cancer patients; among them, the HER2 positive group (HER2+) and the HER2 negative group group (HER2-) was significantly different (p ⁇ 0.01).
  • sample generally refers to a material or mixture of materials, usually in liquid or other form, which may contain one or more analytes of interest.
  • the terms “determine”, “measure”, “evaluate”, “evaluate”, “determine” and “analyze” are used interchangeably herein and generally refer to any form of measurement, including determining the presence or absence of an element. These terms can include both quantitative and/or qualitative aspects. Evaluations can be relative or absolute. “Assessing the presence of” can include determining the amount of something present, as well as determining its presence or absence.
  • the simple steps of clinical immunohistochemical detection may include freezing or paraffin sections of clinical tissues, incubating and staining with antibodies after blocking, taking pictures and grading according to the intensity of the signal.
  • Test-positive samples; and samples with weaker signals and lower grades are clinical test-negative samples.
  • the simple steps of clinical FISH detection may include FISH sample preparation, probe preparation, probe labeling, hybridization, chromosome banding, fluorescence microscope detection, and result analysis.
  • FISH sample preparation may include FISH sample preparation, probe preparation, probe labeling, hybridization, chromosome banding, fluorescence microscope detection, and result analysis.
  • Slice the clinical tissue then use the probe to mark and hybridize, detect the fluorescent signal and grade it according to the signal intensity, among which the signal is stronger and the grade is higher as the positive sample of the clinical test; while the signal is weaker and the grade is lower. The low one is the negative sample of clinical test.
  • the simple steps of gene mutation detection may include taking patient tumor tissue samples, extracting DNA, and testing with kits and other methods to determine whether the patient has a mutation; those with mutations are regarded as positive samples in clinical tests; those without mutations are used as clinical tests. Negative samples.
  • methods for genetic detection may include: polymerase chain reaction-restriction fragment length polymorphism analysis technology, pyrosequencing method, single-strand conformational polymorphism analysis technology, probe amplification block mutation system, high-performance liquid Phase chromatography, micro-digital polymerase chain reaction, and high-resolution melting curve analysis techniques, etc.
  • the detection of gene expression can include selecting kits and other methods for detection according to the conditions of genes and tumors to determine whether the patient has gene expression variation; those with gene expression variation are regarded as positive samples in clinical tests; those without gene expression variation as clinically negative samples.
  • the genes for predicting the occurrence of diseases and related symptoms can include the genes related to diseases and related symptoms obtained through the existing sequencing data, or sequencing of clinical patient samples (including DNA, RNA and other sequencing methods), and data analysis. Genes, and genes that can predict the occurrence of diseases and their related symptoms through their expression levels in the absence of occurrence. Samples that are predicted to be highly correlated with diseases and their related symptoms can be regarded as positive samples of clinical tests, and samples that are predicted to be highly correlated with diseases and their related symptoms can be regarded as negative samples of clinical tests.
  • the blood test for tumor markers may include blood collection to check the levels of tumor markers in the blood.
  • the pleural effusion or ascites can be drained and sent for examination of tumor markers.
  • the changes in the content of tumor markers in the pleural effusion and ascites can be compared with the changes in the tumor markers in the blood of the patient, thereby strengthening the monitoring and control of the disease. diagnosis.
  • Samples that are predicted to be highly correlated with diseases and their related symptoms can be regarded as positive samples of clinical tests, and samples that are predicted to be highly correlated with diseases and their related symptoms can be regarded as negative samples of clinical tests.
  • sequencing generally refers to a method by which the identity of at least 10 contiguous nucleotides (e.g., at least 20, at least 50, at least 100, or at least 200 nucleotides) of a polynucleotide can be obtained. identities of one or more consecutive nucleotides).
  • UDP glucose modified with chemoselective groups generally refers to UDP glucose that has been functionalized, possibly at the 6-hydroxyl position, to include the ability to participate in 1,3- Groups for cycloaddition (or "click") reactions.
  • groups may include azido and alkynyl (eg cyclooctyne).
  • UDP-6-N3-Glu can be UDP glucose modified with chemoselective groups.
  • biotin moiety generally refers to biotin or moieties such as desthiobiotin, oxidized biotin, 2-iminobiotin, diaminobiotin, biotin sulfoxide, biotin Affinity tags for biotin analogs such as Cytin.
  • the biotin moiety can bind streptavidin.
  • cycloaddition reaction and “click reaction” are generally interchangeably described terms, generally referring to the 1,3-cycloaddition between an azide and an alkynyl to form a five-membered heterocycle.
  • the alkynyl group can be strained (eg, in a ring such as cyclooctyne), and the cycloaddition reaction can be performed under copper-free conditions.
  • Dibenzocyclooctyne (DBCO) and difluorooctyne (DIFO) may be examples of alkynes capable of participating in copper-free cycloaddition reactions.
  • biotin-bound carrier generally refers to a carrier (eg a bead, which may be magnetic) attached to streptavidin or avidin or a functional equivalent thereof.
  • amplification generally refers to the use of a target nucleic acid as a template to produce one or more copies of the target nucleic acid.
  • copy of a fragment generally refers to an amplified product, wherein the copy of a fragment may be the reverse complement of the strand of the fragment, or may have the same sequence as the strand of the fragment.
  • enrichment generally refers to the separation of analytes with a certain characteristic (such as nucleic acids containing hydroxymethylcytosine) from analytes without that characteristic (such as nucleic acids containing hydroxymethylcytosine). partially purified. For example, enrichment typically increases the concentration of an analyte having the characteristic (eg, a nucleic acid comprising hydroxymethylcytosine) by at least 2-fold, at least 5-fold, or at least 10-fold relative to an analyte without the characteristic.
  • at least 10%, at least 20%, at least 50%, at least 80%, or at least 90% of the analytes in the sample may have the characteristic used for the enrichment.
  • at least 10%, at least 20%, at least 50%, at least 80%, or at least 90% of the nucleic acid molecules in the enriched composition may comprise chain of methylcytosine.
  • the present application provides an analysis method, comprising (S1) determining the area to be tested based on the quantity and/or presence of hydroxymethylcytosine in the clinical test-positive samples and clinical test-negative samples of the target gene, and (S2) detecting The quantity and/or presence of the hydroxymethylcytosine in the region to be tested of the sample to be tested.
  • the region to be tested may comprise a subregion of the region where a single target gene is located.
  • the present application provides a method of confirming the presence of a disease, assessing the development or risk of developing the disease, assessing the progression and/or prognosis of the disease and/or screening a population responding to treatment, comprising (S1) target-based The number and/or presence of hydroxymethylcytosine in the clinical test positive sample and the clinical test negative sample of the gene, determine the area to be tested, (S2) detect the hydroxymethylcytosine in the test area of the test sample the number and/or existence of
  • the region to be tested may comprise a subregion of the region where a single target gene is located.
  • the region to be tested can be determined based on the amount and/or presence of the hydroxymethylcytosine in the clinical test positive samples and the clinical test negative samples of a single target gene.
  • the step (S1) of the method of the present application may comprise a step (S1-1): determining the positive sample of the clinical test and the negative sample of the clinical test of the target gene.
  • the step (S1) of the method of the present application may include the step (S1-2): determining that the clinical test-positive sample and the clinical test-negative sample contain hydroxymethyl cells in the same region by a hydroxymethylation sequencing method. The number of nucleic acid fragments with pyrimidines.
  • the step (S1) of the method of the present application may include a step (S1-3): determining the region containing differential hydroxymethylcytosine in the clinical test positive sample and the clinical test negative sample, as the area to be tested.
  • the step (S2) of the method of the present application may include detecting the quantity and/or presence of the hydroxymethylcytosine in the region to be tested of the sample to be tested by sequencing.
  • the present application provides an analytical method or a method for confirming the presence of a disease, assessing the formation or risk of developing a disease, assessing the progression and/or prognosis of a disease and/or screening a population corresponding to treatment, comprising steps (S1- 1): determining the clinically positive samples and the clinically negative samples of the target gene; step (S1-2): determining the clinically positive samples and the clinically negative samples by hydroxymethylation sequencing The number of nucleic acid fragments containing hydroxymethylcytosine in the same region of the sample; step (S1-3): determining the region containing differential hydroxymethylcytosine in the clinical test positive sample and the clinical test negative sample, as The region to be tested; step (S2) may include detecting the quantity and/or presence of hydroxymethylcytosine in the region to be tested by sequencing.
  • the present application provides an analytical method or a method for confirming the presence of a disease, assessing the formation or risk of developing a disease, assessing the progression and/or prognosis of a disease and/or screening a population corresponding to treatment, comprising steps (S1- 1): determining the clinically positive samples and the clinically negative samples of the target gene; step (S1-2): determining the clinically positive samples and the clinically negative samples by hydroxymethylation sequencing The number of nucleic acid fragments containing hydroxymethylcytosine in the same region of the sample; step (S1-3): when the clinical test positive sample contains nucleic acid of hydroxymethylcytosine in the same region compared to the clinical test negative sample When the absolute value of the ratio log2 of the number of fragments after processing is 0.5 or more, it is determined that the region is the region to be tested; step (S2) may include detecting all parts of the region to be tested in the sample to be tested by sequencing. The amount and/or presence of the above-mentioned hydroxymethylcyto
  • the present application provides an analytical method or a method for confirming the presence of a disease, assessing the formation or risk of developing a disease, assessing the progression and/or prognosis of a disease and/or screening a population corresponding to treatment, comprising steps (S1- 1): determining the clinically positive samples and the clinically negative samples of the target gene; step (S1-2): determining the clinically positive samples and the clinically negative samples by hydroxymethylation sequencing The number of nucleic acid fragments containing hydroxymethylcytosine in the same region of the sample; step (S1-3): when the clinical test positive sample contains nucleic acid of hydroxymethylcytosine in the same region compared to the clinical test negative sample The absolute value of the ratio log2 of the number of fragments after processing is 0.5 or more, and when the difference between the clinical test positive sample and the clinical test negative sample in the same area is less than 0.05, it is determined that the area is The area to be tested; step (S2) may include detecting the quantity and/or presence of the hydroxy
  • the clinical test-positive samples and the clinical test-negative samples of the present application can be determined by the following clinical test methods: immunohistochemical detection, clinical FISH detection, gene mutation detection, gene expression detection, genes that predict the occurrence of diseases and related symptoms , and blood tests for tumor markers.
  • the clinical test-positive samples and the clinical test-negative samples of the present application can be determined by immunohistochemistry and/or fluorescence in situ hybridization.
  • methods for determining whether a clinical test sample is positive or negative can be based on diagnostic guidelines consensus in the art.
  • a sample with an immunohistochemical staining score of 1 or higher is judged to be a clinically positive sample according to the antibody staining signal standard of the immunohistochemical method.
  • the staining of SP142 can show brown dot or linear staining, distributed in the tumor or in the peritumoral stroma.
  • the judgment standard of immunohistochemical method can be as follows: adopt stepwise scoring method, divide into 4 grades according to the percentage of stained cells, namely: 0- ⁇ 1%; 1%- ⁇ 5%; 5%- ⁇ 10%; ⁇ 10%. Among them, ⁇ 1% were positive.
  • a sample with a fluorescence in situ hybridization ratio of 2.0 or higher is judged to be a clinically positive sample according to the fluorescent in situ hybridization fluorescent probe signal standard.
  • fluorescent in situ hybridization method in vitro fluorescently labeled DNA probes can be used, using the principle of complementary base pairing between the probe and the target gene to be detected. Fluorescent signals are used to obtain results, allowing the detection of chromosomal or genetic abnormalities in cells and tissues.
  • the judging criteria for fluorescence in situ hybridization can be: (1) red fluorescent signals are connected into clusters; (2) double microsome amplification occurs in red fluorescent signals; (3) red-green fluorescent signal ratio > 1.5.
  • the hydroxymethylation sequencing method of the present application may include the following steps: (S1-2a) extracting nucleic acid fragments in the sample, (S1-2b) labeling nucleic acid fragments containing hydroxymethylcytosine in the nucleic acid fragments , (S1-2c) enriching the nucleic acid fragments containing hydroxymethylcytosine, (S1-2d) sequencing the enriched nucleic acid fragments containing hydroxymethylcytosine.
  • the labeling described herein comprises contacting the nucleic acid fragment with DNA ⁇ -glucosyltransferase and UDP glucose modified with a chemoselective group.
  • the label of the present application may be that the chemoselective group is attached to a nucleic acid fragment containing hydroxymethylcytosine in the sample.
  • a chemoselective group described herein may comprise an azido group.
  • the chemoselective groups of the present application may comprise any chemical click group.
  • labeling as described herein may also comprise contacting said nucleic acid fragment bearing a chemoselective group with biotin comprising biotin capable of reacting with said chemoselective group.
  • biotin capable of reacting with the chemoselective group may comprise biotin containing a dibenzocyclooctyne modification.
  • the label may be that a first label is linked to a nucleic acid fragment containing hydroxymethylcytosine in the sample, and may include linking the nucleic acid fragment to which the first label is linked with a second label, the The second label is selectively reactive with said first label.
  • said enriching can comprise contacting said nucleic acid fragments bearing biotin with magnetic beads comprising streptavidin.
  • the labeled nucleic acid fragments containing hydroxymethylcytosine can be combined with magnetic beads containing streptavidin.
  • said enrichment may comprise separating magnetic beads bearing said hydroxymethylcytosine nucleic acid fragments by magnetic force.
  • the step (S1) may include a step (S1-3): determining a region containing differential hydroxymethylcytosine in the clinical test positive sample and the clinical test negative sample as the region to be tested.
  • the region of differentiated hydroxymethylcytosine may comprise, in the clinical test positive sample, compared to the clinical test negative sample, the number of nucleic acid fragments comprising hydroxymethylcytosine in the region is significantly increased and / or areas of significant reduction.
  • the area to be tested can be determined by determining the multiple of difference in the number of nucleic acid fragments containing hydroxymethylcytosine in the same area in the clinical test positive sample compared to the clinical test negative sample.
  • the multiple of difference may be related to the following statistical value: the difference in the number of nucleic acid fragments containing hydroxymethylcytosine in the same region in the clinical test positive sample compared to the clinical test negative sample, the clinical test The log2-processed ratio of the number of nucleic acid fragments containing hydroxymethylcytosine in the same region in the positive sample compared to the negative sample in the clinical test and/or in the positive sample compared to the negative sample in the clinical test Absolute value of the ratio of the number of nucleic acid fragments containing hydroxymethylcytosine in the same region after log2 treatment.
  • the ratio log2 of the number of nucleic acid fragments containing hydroxymethylcytosine in the same region in the clinical test positive sample compared to the clinical test negative sample has an absolute value of 0.5 or more after processing, it can be determined that The above-mentioned area is the area to be tested.
  • the area can be determined as the area to be tested.
  • the area can be determined as the area to be tested.
  • the step (S2) of the present application may include detecting the quantity and/or presence of the hydroxymethylcytosine in the region to be tested in the sample to be tested by sequencing.
  • the present application may sequence the sample to be tested by a method selected from the following group: digital PCR and high-throughput sequencing.
  • the sequencing method of the present application can be selected from any sequencing method known in the art.
  • the present application also provides a method for analyzing, confirming the existence of a disease, assessing the formation or risk of developing a disease, assessing the progress and/or prognosis of a disease and/or screening a population that is suitable for treatment, which may include
  • the target gene the quantity and/or presence of hydroxymethylcytosine in the region to be tested of the single target gene in the sample to be tested is detected by digital PCR.
  • the present application provides an analysis method, comprising detecting the hydroxymethylcytosine in the region to be tested of the single target gene in the sample to be tested by digital PCR based on the region to be tested obtained through preliminary screening in the single target gene the number and/or existence of
  • the step of primary screening may be the step (S1) of any analysis method in the present application.
  • the region to be measured in the present application is determined by step (S1-1), step (S1-2), and step (S1-3) of any analysis method in the present application.
  • the present application also provides a method for determining a region to be detected, and the region to be detected is used in a detection method based on the quantity and/or presence of hydroxymethylcytosine.
  • the method for determining the area to be tested in the present application includes step (S1-1), step (S1-2), and step (S1-3) of any analysis method in the present application.
  • the present application also provides a database, which may contain the sequence of the region to be tested determined by the method described in the present application, or its complementary region, or the above-mentioned fragments.
  • the region to be tested determined by PD-L1 may be chr9:5449218-5450100.
  • the region to be tested determined by PD-L1 may be chr9:5460114-5460435.
  • the region to be tested determined by EGFR may be chr7:55127173-55127790.
  • the region to be tested determined by HER2 may be chr17:37852095-37852700.
  • the present application provides a nucleic acid, and the nucleic acid may comprise a region to be tested that can be determined in combination with the method described in the present application, or a complementary region thereof, or a sequence of a fragment thereof.
  • the nucleic acid can be a probe.
  • the present application provides a method for preparing a nucleic acid, which may include the sequence of the region to be tested determined according to the method described in the application, or its complementary region, or the above-mentioned fragments, designed to be able to bind to the region to be tested region, or a complementary region thereof, or a nucleic acid of a fragment thereof.
  • the nucleic acid of the present application can be CTGTATTGCCACATAATGTCTATA as shown in SEQ ID NO:1.
  • the nucleic acid of the present application can be ATTGAGAAATTGGACTCTTCGTTG as shown in SEQ ID NO:2.
  • the nucleic acid of the present application can be TTGCATGAATGCAGGAAAAA as shown in SEQ ID NO:3.
  • the nucleic acid of the present application can be AGTTGGCACTGGGTCTCTGT as shown in SEQ ID NO:4.
  • the sequences shown in SEQ ID NO:1 and/or SEQ ID NO:2 can bind chr9:5449218-5450100.
  • sequences shown in SEQ ID NO:3 and/or SEQ ID NO:4 can bind chr7:55127173-55127790.
  • sequences shown in SEQ ID NO:5 and/or SEQ ID NO:6 can bind chr17:37852095-37852700.
  • the present application provides a kit that can comprise the nucleic acid described in the present application.
  • the kits of the present application may also contain other materials required for the kits.
  • the present application provides the application of the nucleic acid of the present application, and/or the kit of the present application, in the preparation of disease detection products.
  • the present application provides the nucleic acid of the present application, and/or the kit of the present application, which can be used for disease detection.
  • the present application provides a method for disease detection, which may include providing the nucleic acid of the present application, and/or the kit of the present application.
  • the application provides the nucleic acid of the application, and/or the kit of the application, which can be prepared to confirm the existence of the disease, assess the formation of the disease or the risk of formation, assess the progress and/or prognosis of the disease and/or screen for Therapy has an application in the product for the corresponding population.
  • the present application provides the nucleic acid of the present application, and/or the kit of the present application, which can be used to confirm the existence of a disease, assess the formation of a disease or the risk of formation, assess the progression and/or prognosis of a disease and/or screen There is a corresponding population for treatment.
  • the present application provides a method for confirming the presence of a disease, assessing the formation or risk of developing a disease, assessing the progression and/or prognosis of a disease and/or screening a population responding to treatment, which may comprise providing the nucleic acid of the present application, and /or the test kit of the present application.
  • a disease in this application may comprise a tumor.
  • diseases in this application may include solid tumors.
  • diseases in the present application may comprise melanoma, esophageal tumor, and/or breast tumor.
  • a disease in this application may comprise melanoma.
  • the diseases in the present application may include squamous cell carcinoma of the esophagus.
  • a disease in the present application may comprise breast cancer.
  • any one or more of the methods of the present application may be for non-diagnostic purposes.
  • any one or more of the methods of the present application may be for diagnostic purposes.
  • the present application also provides a storage medium, which can record a program capable of running the method described in the present application.
  • the present application also provides a device comprising the storage medium described in the present application.
  • the non-transitory computer readable storage medium may include a floppy disk, a flexible disk, a hard disk, a solid state storage (SSS) (such as a solid state drive (SSD)), a solid state card (SSC), a solid state module (SSM)), an enterprise high-grade flash drives, tape, or any other non-transitory magnetic media, etc.
  • SSD solid state drive
  • SSC solid state card
  • SSM solid state module
  • Non-transitory computer readable storage media may also include punched cards, paper tape, cursor sheets (or any other physical media having a pattern of holes or other optically identifiable markings), compact disc read only memory (CD-ROM) , Rewritable Disc (CD-RW), Digital Versatile Disc (DVD), Blu-ray Disc (BD) and/or any other non-transitory optical media.
  • CD-ROM compact disc read only memory
  • CD-RW Rewritable Disc
  • DVD Digital Versatile Disc
  • BD Blu-ray Disc
  • the device as described in the present application further includes a processor coupled to the storage medium, and the processor is configured to execute based on the program stored in the storage medium to implement the method described in the present application.
  • the database system may implement various mechanisms to ensure that the methods described herein performed on the database system produce correct results.
  • the database system may use disks as permanent data storage.
  • the database system can provide database storage and processing services for multiple database clients.
  • the database client may store database data across multiple shared storage devices, and/or may utilize one or more execution platforms with multiple execution nodes.
  • the database system can be organized such that storage and computing resources can be effectively scaled indefinitely.
  • the method of the present application can accurately determine the hydroxymethylation site of the target gene, and realize the detection of a single gene site based on 5-hydroxymethylcytosine (5hmC), which can be used for disease detection, screening of people benefiting from standard disease treatment programs, Targeted drug benefit population screening.
  • the detection method based on 5-hydroxymethylcytosine (5hmC) of the present application can be convenient, fast and accurate compared with existing target gene detection methods (such as IHC, FISH or multi-site detection).
  • centrifuge For the first centrifugation, centrifuge at 1350g at 4°C for 12 minutes at low temperature, take out the light yellow supernatant and transfer it to a 2mL DNase free sterile centrifuge tube.
  • the amplified product was purified with AmpureXP beads (KAPA, KK8001), and finally eluted with 20uL eluent to obtain the final 5hmC library.
  • Library concentration determination can be performed with Qubit 3.0.
  • Preparation of samples prepare five 1.5mL EP tubes, and mix every 4 samples to be sequenced into one EP tube. There are 20 samples in 5 tubes (index cannot be repeated); each 5hmC library sample absorbs 5ng, and each EP The total volume of the tube is 20uL, and the final concentration is 1ng/uL;
  • sample reaction system (20uL) is as follows: draw 4uL of the library in each EP tube for qPCR quantification.
  • Result analysis analyze according to the qPCR operating software to determine whether the 5hmC library is degraded and whether it meets the sequencing requirements;
  • the library (16uL) that passed the quality inspection was sequenced with Illumina NextSeq500, the sequencing kit used was High Output Kit v2 (75cycles), the sequencing throughput of each sample was 1.5Gb, and the sequencing band size was 75bp.
  • Each original sequencing FASTQ data is first trimmed with low-quality data using Trimmomatic software, and then compared to the human genome hg19 using Bowtie2 software;
  • the corresponding site of the following PD-L1 primer can be chr9:5449218-5450100, for example, the following EGFR
  • the site corresponding to the primer may be chr7:55127173-55127790, for example, the site corresponding to the following HER2 primer may be chr17:37852095-37852700.
  • the amount of 5hmC library added to the sample is 10ng, the standard primer concentration is 10uM, and the dye method master mix (Yongnuo, S0200020301);
  • Blood samples were collected from 49 melanoma patients, including 14 PD-L1 positive patients and 35 negative patients. Take the peripheral blood samples (8-10mL) of the above 49 melanoma patients, and separate the plasma (4-5mL), extract cfDNA from the plasma for 5hmC-Seal high-throughput sequencing, and the sequencing throughput of each sample is 1.5Gb , the size of the sequencing band is 75bp;
  • the clinical samples were divided into two groups: PD-L1 positive and negative; through the comparative analysis between the positive group and the negative group, according to
  • > 0.5, pvalue ⁇
  • the 0.01 screening condition screens 5hmC differential markers to find the hydroxymethylation site (CD274) corresponding to the PD-L1 gene;
  • CD274 (PD-L1) hydroxymethylation site primers (Qingke Biotechnology Co., Ltd.), for example, the site corresponding to the following PD-L1 primers can be chr9:5449218-5450100.
  • the amount of 5hmC library input for each melanoma patient was 10ng, and four groups of experimental cohorts were designed: blank control group (Blank control), negative control group (Negative control), PD-L1 positive group (PD-L1+) and PD-L1 Negative group (PD-L1-); Among them, the blank control group is RNase-Free water, and the sample volume is 9.2uL.
  • the negative control group was a melanoma cfDNA sample (without 5hmC enrichment), and the loading amount was 10 ng.
  • Figure 1 shows the copy number (copies of ng) of PD-L1 hydroxymethylation site (CD274) single gene detection in the plasma cell-free DNA 5hmC library of 49 melanoma patients; among them, PD-L1 There was a significant difference (p ⁇ 0.01) between the positive group (PD-L1+) and the PD-L1 negative group (PD-L1-).
  • Blood samples were collected from 56 patients with esophageal squamous cell carcinoma, including 34 EGFR-positive patients and 22 EGFR-negative patients.
  • Peripheral blood samples (8-10mL) were collected from the above 56 patients with esophageal squamous cell carcinoma, and plasma (4-5mL) was separated, and cfDNA was extracted from the plasma for 5hmC-Seal high-throughput sequencing.
  • the sequencing throughput of each sample was 1.5 Gb, the size of the sequencing band is 75bp;
  • the clinical samples were divided into EGFR positive and negative groups; through the comparative analysis of the positive group and the negative group, filter according to
  • > 0.5, pvalue ⁇ 0.01 5hmC differential markers, find the hydroxymethylation site corresponding to the EGFR gene;
  • Synthesize EGFR hydroxymethylation site primers (Qingke Biotechnology Co., Ltd.), for example, the site corresponding to the following EGFR primers can be chr7:55127173-55127790.
  • the amount of 5hmC library input for each patient with esophageal squamous cell carcinoma was 10 ng, and four groups of experimental cohorts were designed: blank control group (Blank control), negative control group (Negative control), EGFR positive group (EGFR+) and EGFR negative group (EGFR- ); wherein, the blank control group was RNase-Free water, and the loading volume was 9.2uL.
  • the negative control group was esophageal squamous cell carcinoma cfDNA samples (without 5hmC enrichment), and the loading amount was 10 ng.
  • Figure 2 shows the copy number (copies of ng) of EGFR hydroxymethylation site single gene detection in the plasma cell-free DNA 5hmC library of 56 patients with esophageal squamous cell carcinoma; among them, the EGFR positive group (EGFR+) and Compared with EGFR-negative group (EGFR-), there was a significant difference (p ⁇ 0.01).
  • Blood samples were collected from 36 breast cancer patients, including 16 HER2 (ERBB2) positive patients and 20 negative patients. Take the peripheral blood samples (8-10mL) of the above 36 breast cancer patients, and separate the plasma (4-5mL), extract cfDNA from the plasma for 5hmC-Seal high-throughput sequencing, and the sequencing throughput of each sample is 1.5Gb , using a paired-end 75bp sequencing strategy;
  • HER2 detection by clinical immunohistochemistry the clinical samples were divided into HER2 positive and negative groups; through the comparative analysis of the positive group and the negative group, filter according to
  • > 0.5, pvalue ⁇ 0.01 5hmC differential markers, find the hydroxymethylation site corresponding to HER2 (ERBB2);
  • ERBB2 hydroxymethylation site primers (Qingke Biotechnology Co., Ltd.), for example, the site corresponding to the following ERBB2 primers can be chr17:37852095-37852700.
  • the 5hmC library input amount for each breast cancer patient was 10ng, and four experimental cohorts were designed: blank control group (Blank control), negative control group (Negative control), HER2 positive group (HER2+) and HER2 negative group (HER2-) ; Among them, the blank control group was RNase-Free water, and the loading volume was 9.2uL.
  • the negative control group was breast cancer cfDNA samples (without 5hmC enrichment), and the loading amount was 10ng.
  • FIG. 3 shows the copy number (copies of ng) of the HER2 hydroxymethylation site (ERBB2) single gene detection in the plasma cell-free DNA 5hmC library of 36 breast cancer patients; among them, the HER2 positive group (HER2+ ) compared with the HER2-negative group (HER2-) had a significant difference (p ⁇ 0.01).

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Abstract

L'invention concerne un procédé de test monogénique et une application de celui-ci. Plus précisément, l'invention concerne un procédé de test monogénique, comprenant l'étape (S1) consistant, sur la base de la quantité et/ou de la présence d'hydroxyméthylcytosine dans un échantillon positif d'examen clinique et un échantillon négatif d'examen clinique d'un gène cible, à déterminer une région à tester, et l'étape (S2) consistant à détecter la quantité et/ou la présence d'hydroxyméthylcytosine dans ladite région d'un échantillon à tester.
PCT/CN2022/119180 2021-09-17 2022-09-16 Procédé de test monogénique et son application WO2023040997A1 (fr)

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