WO2022247523A1 - 一种用于结直肠癌早期诊断的生物标志物组合物及应用 - Google Patents

一种用于结直肠癌早期诊断的生物标志物组合物及应用 Download PDF

Info

Publication number
WO2022247523A1
WO2022247523A1 PCT/CN2022/087384 CN2022087384W WO2022247523A1 WO 2022247523 A1 WO2022247523 A1 WO 2022247523A1 CN 2022087384 W CN2022087384 W CN 2022087384W WO 2022247523 A1 WO2022247523 A1 WO 2022247523A1
Authority
WO
WIPO (PCT)
Prior art keywords
value
colorectal cancer
kit
occult blood
biomarker
Prior art date
Application number
PCT/CN2022/087384
Other languages
English (en)
French (fr)
Inventor
陈朋
周忠坤
李洋
马万通
刘宇恒
张仁涛
马云浩
杜康嘉
Original Assignee
杭州微度生物科技有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 杭州微度生物科技有限公司 filed Critical 杭州微度生物科技有限公司
Priority to EP22810256.2A priority Critical patent/EP4350008A1/en
Priority to US18/560,848 priority patent/US20240254569A1/en
Publication of WO2022247523A1 publication Critical patent/WO2022247523A1/zh

Links

Classifications

    • 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
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
    • 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
    • G—PHYSICS
    • G06—COMPUTING; CALCULATING OR COUNTING
    • G06F—ELECTRIC DIGITAL DATA PROCESSING
    • G06F17/00—Digital computing or data processing equipment or methods, specially adapted for specific functions
    • G06F17/10—Complex mathematical operations
    • G06F17/18—Complex mathematical operations for evaluating statistical data, e.g. average values, frequency distributions, probability functions, regression analysis
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention belongs to the field of colorectal cancer detection, and in particular relates to a biomarker composition for early diagnosis of colorectal cancer and its application.
  • Colorectal cancer is a common clinical tumor with high morbidity and mortality. Among new cancer cases in China, the incidence of colorectal cancer ranks fourth among men, third among women, and second globally . If colorectal cancer lesions can be detected in the early stage and surgically removed, the survival rate will exceed 90%, while in the late stage it will be reduced to 13%. Therefore, early screening is the most effective means to reduce the mortality rate of colorectal cancer. Developed countries have established effective early screening programs and diagnostic techniques, which have become an important part of the method of selecting treatment and predicting the outcome, resulting in great improvement in the risk of death and the quality of treatment, which provides us with guidance and learn from.
  • Non-invasive fecal occult blood tests include: based on guaiac-based chemical method (gFOBT); immunological method, etc., all of which have the problems of low sensitivity and susceptibility to diet and medication.
  • gFOBT guaiac-based chemical method
  • immunological method etc.
  • Stool genes are another reliable biomarker that can be used for early screening through DNA testing.
  • Multi-target gene detection has been recommended by the "Guidelines for Early Screening of Colorectal Cancer” for colorectal cancer screening every three years, but it is expensive and has a poor diagnostic effect on adenomas, and has not yet been validated in Asian populations.
  • Cologuard is an effective molecular (gene mutation, methylation and occult blood test) screening technology, which combines the detection of fecal occult blood with the detection of tumor-related DNA markers in blood to quantitatively detect DNA markers associated with colorectal tumors and occult hemoglobin in human feces enable early diagnosis of colorectal cancer, but they are expensive, require blood tests, and are less effective in diagnosing adenomas. Therefore, we need to develop sensitive, non-invasive and economical non-invasive screening technology to improve the status quo.
  • Chinese patent CN106574294A discloses a method, primers and kit for diagnosing colorectal cancer from human stool samples by quantitative PCR. and/or adenomatous polyps for early detection, risk screening, and monitoring, but this method has low accuracy and sensitivity for colorectal cancer detection.
  • Chinese patent CN110512015A discloses a colon cancer biomarker composition and its application.
  • the biomarker composition includes Clostridiaceae, Porphyromonas, Peptostreptococcus, Fusobacterium nucleatum, and Bacillus.
  • the inventors collected a large number of stool samples from patients with colorectal cancer and precancerous lesions (colorectal adenoma and colorectal neoplastic polyps) and healthy individuals, and used high-throughput sequencing, real-time fluorescent quantitative PCR, high-resolution melting curve method or biological Microarray and other methods, through comparative analysis and verification of the differences in the abundance and relative content of microorganisms in stool samples, microorganisms related to colorectal cancer and precancerous lesions are determined, and different combinations of the above microorganisms are used as biomarker compositions. By detecting the abundance of the biomarker composition in the sample, it is predicted whether there is colorectal cancer. However, this method has poor detection specificity.
  • the present invention screens specific intestinal microorganisms as a biomarker composition for early diagnosis of colorectal cancer
  • the biomarker composition includes Peptostreptococcus stomatis, Fusobacterium nucleatum, micromicrobial Parvimonas micra, Bifidobacterium Bifidobacterium; and through the combination of the quantitative level detection of the above-mentioned microbial composition and the fecal occult blood test, and through the regression equation, it is used for the diagnosis of colorectal cancer.
  • the present invention utilizes the results based on high-throughput sequencing Specificity, combined with early diagnosis of colorectal cancer, high sensitivity and specificity, can avoid the pain, discomfort and postoperative complications caused by colonoscopy invasive examination, and significantly reduce the false positive rate of early diagnosis of colorectal cancer , can be widely used in the early diagnosis of colorectal cancer.
  • the object of the present invention is to provide a colorectal cancer biomarker composition, said biomarker composition comprises Peptostreptococcus stomatis, Fusobacterium nucleatum, micromonocytes Bacteria Parvimonas micra, Bifidobacterium Bifidobacterium.
  • Another object of the present invention is to provide a kit for detecting colorectal cancer, said kit comprising quantification reagents for a biomarker composition, said biomarker composition comprising Peptostreptococcus stomatis, nucleated Fusobacterium nucleatum, Parvimonas micra, Bifidobacterium.
  • the quantification reagents are used to quantify each component of the biomarker composition.
  • the principle adopted by the quantification reagent is real-time quantitative PCR method.
  • the quantification reagent is a real-time quantitative PCR quantification reagent.
  • said quantification reagents comprise a primer set for a biomarker composition.
  • the primer set includes primer pairs for amplifying each component of the biomarker composition.
  • nucleotide sequence of the primer set is shown in SEQ ID NO.1-8.
  • the quantification reagent also includes a pair of primers for detecting total bacteria.
  • nucleotide sequence of the primer pair for detecting total bacteria is shown in SEQ ID NO.9-10.
  • the quantification reagents also include real-time quantitative PCR reaction reagents.
  • the kit further includes an occult blood detection reagent for detecting human hemoglobin in feces.
  • the occult blood detection reagent includes diluent and detection test paper.
  • Another object of the present invention is to provide an application of the above-mentioned kit in the early diagnosis of colorectal cancer, the step when the kit is used to detect colorectal cancer is: detecting the biomarker composition described in claim 1 The abundance of each component in the sample and the value of fecal occult blood, and then judge the test results through the regression equation. Using the subject's fecal genomic DNA as a template, the primer set in the kit was used to detect the abundance of biomarkers in the sample.
  • the kit when used to detect colorectal cancer, it includes the following steps:
  • X 0 is the result value of occult blood
  • X 1 is the Ct value of Peptostreptococcus stomatis
  • X 2 is the Ct value of Fusobacterium nucleatum
  • X 3 is the Ct value of Parvimonas micra
  • X 4 is the Ct value of Bifidobacterium
  • a and ⁇ 0 - ⁇ 4 are constants, and A and ⁇ 0 - ⁇ 4 are obtained from clinical experiment data.
  • the regression equation in the step (4) includes any one of formula (IV)-formula (VIII):
  • the regression equation in the step (4) is:
  • the kit also includes DNA extraction reagents.
  • the beneficial effects of the present invention are: 1 the present invention provides a biomarker composition for early diagnosis of colorectal cancer, and primer pairs and kits for quantitative detection of the biomarker composition; 2 the present invention Using the specificity of high-throughput sequencing results combined with fecal occult blood test for early diagnosis of colorectal cancer has high sensitivity and specificity, and can avoid the pain, discomfort and postoperative complications caused by invasive colonoscopy. Significantly reduce the false positive rate of early diagnosis of colorectal cancer, realize the non-invasive diagnosis of colorectal cancer, and have a wide range of application value.
  • biomarker refers to a disease marker that is a readily measurable substance commonly present in a body sample, the amount measured can be correlated to the underlying disease pathophysiology, eg the presence or absence of CRC.
  • Quantification refers to the ability to quantify the amount of a specific nucleic acid sequence in a sample.
  • Molecular biology methods for determining the amount of a target nucleic acid sequence include, but are not limited to, endpoint PCR, competitive PCR, reverse transcriptase PCR (PT-PCR) , quantitative PCR (qPCR), PCR-ELISA, DNA microarray, etc.
  • the term "quantification level" may be concentration (DNA amount per unit volume), DNA amount per cell number, cycle threshold (Ct value).
  • concentration DNA amount per unit volume
  • Ct value cycle threshold
  • the quantification of the bacterial sequence is performed by qPCR; in a more preferred embodiment below, the quantification of the bacterial sequence is performed by qPCR, and the quantification level is the Ct value.
  • the Ct value refers to the number of qPCR cycles experienced when the fluorescent signal in each reaction tube reaches the set threshold value.
  • the Ct level is inversely proportional to the quantification of the target nucleic acid in the sample, ie, the lower the Ct level, the greater the amount of target nucleic acid in the sample.
  • Quantitation of the abundance of a target nucleic acid sequence in a sample can be absolute or relative. Relative quantification is based on one or more internal reference genes, i.e., the 16S rRNA gene from a reference strain, such as the use of universal primers to determine the total bacterial count and express the abundance of this target nucleic acid sequence as a percentage of bacteria; absolute quantification is achieved by comparison with a DNA standard Get the exact number of target molecules.
  • primer refers to an oligonucleotide that can be used in an amplification method such as the polymerase chain reaction (PCR) to amplify a nucleotide sequence. Primers are polynucleotide sequences based on specific target sequences.
  • PCR polymerase chain reaction
  • sensitivity, specificity, accuracy and other combinations are used to describe the goodness and reliability of the detection method of the present invention.
  • sensitivity, specificity, accuracy include: True Positive (TP), True Negative (TN), False Positive (FP), False Negative (FN); If the given screening test also shows the presence of the disease, the test result is considered a true positive; if it proves that the patient does not have the disease, and the given screening test also shows the absence of the disease, the test result is considered positive. is a true negative; a test result is a false positive if the results of a screening experiment indicate that a patient who actually does not have the disease has the disease; , the test result is false negative.
  • TN/(TN+FP) number of true negative assessments/number of all negative assessments
  • the primer pairs were designed for Peptostreptococcus stomatis, Fusobacterium nucleatum, Parvimonas micra, Bifidobacterium and total bacteria as follows (1)-(5):
  • Primer pair (1) upstream primer 5-AAGTGTTAGCGGTATAGGATG-3; downstream primer 5-CGTGTCTCAGTTCCAATGT-3;
  • Primer pair (2) upstream primer 5-GGATTTATTGGGCGTAAAGC-3; downstream primer 5-GGCATTCCTACAAATATCTACGAA-3;
  • Primer pair (3) upstream primer 5-GCGTAGATATTAGGAGGAATAC-3; downstream primer 5-GCGGAATGCTTAATGTGTT-3;
  • Primer pair (4) upstream primer 5-CATCGCTTAACGGTGGAT-3-3; downstream primer 5-TTCGCCATTGGTGTTCTT-3;
  • Primer pair (5) upstream primer 5-GCAGGCCTAACACATGCAAGTC-3; downstream primer 5-CTGCTGCCTCCCGTAGGAGT-3.
  • a kit for detecting colorectal cancer comprising biomarker compositions Peptostreptococcus stomatis, Fusobacterium nucleatum, Parvimonas micra, and quantification of Bifidobacterium Reagent, reaction buffer system, Mix, ROX and RNase-free ddH 2 O, the quantitative reagent includes a primer set for detecting the biomarker composition, the primer set is as the primer pair in Example 1 (1 )-(4), and a pair of primers for detecting Total bacteria, said primer pair is shown in the primer pair (5) in Example 1.
  • Method of use extract the stool genomic DNA from the subject, and use the above primer pair (1)-(5) to detect the abundance of the biomarker in the sample as the template of the stool genomic DNA; according to the abundance of the biomarker in the sample Determine whether you have colorectal cancer.
  • a kit for detecting colorectal cancer comprising quantification of biomarker compositions Peptostreptococcus stomatis, Fusobacterium nucleatum, Parvimonas micra, Bifidobacterium Reagent and occult blood detection reagent;
  • the quantitative reagent includes primer set, reaction buffer system, Mix, ROX and RNase-free ddH 2 O, and the primer set is as shown in the primer pair (1)-(4) in Example 1 , and a primer pair for detecting Total bacteria, said primer pair is shown in the primer pair (5) in Example 1;
  • said occult blood detection reagent includes diluent and detection test paper.
  • X 0 is the result value of occult blood
  • X 1 is the digestive chain of stomatitis
  • X 2 is the Ct value of Fusobacterium nucleatum
  • X 3 is the Ct value of Parvimonas micra
  • X 4 is the Ct value of Bifidobacterium
  • a and ⁇ 0 - ⁇ 4 is constant and obtained by clinical experiment data analysis
  • described regression equation can be any one in following formula (IV)-formula (VIII):
  • a kit for detecting colorectal cancer comprising quantification of biomarker compositions Peptostreptococcus stomatis, Fusobacterium nucleatum, Parvimonas micra, Bifidobacterium Reagent, occult blood detection reagent and DNA extraction reagent;
  • Said quantitative reagent comprises primer set, reaction buffer system, Mix, ROX and RNase-free ddH 2 O, said primer set is the primer pair (1)-( 4), and a primer pair for detecting total bacteria, said primer pair is shown in primer pair (5) in Example 1;
  • said kit also includes; said occult blood detection reagent includes diluent and detection test strips.
  • X 0 is the result value of occult blood
  • X 1 is the digestive chain of stomatitis
  • X 2 is the Ct value of Fusobacterium nucleatum
  • X 3 is the Ct value of Parvimonas micra
  • X 4 is the Ct value of Bifidobacterium
  • a and ⁇ 0 - ⁇ 4 is constant and obtained by clinical experiment data analysis
  • described regression equation can be any one in following formula (IV)-formula (VIII):
  • kits provided by the invention to screen the above cases, and according to the test results, judge that the kit provided by the invention is used for colorectal cancer Sensitivity, specificity and accuracy of detection.
  • each 10 ⁇ L reaction system contains 5 ⁇ L, 2 ⁇ Mix; 0.3 ⁇ L, upstream primer; 0.3 ⁇ L, downstream primer; 0.2 ⁇ L, 50 ⁇ ROX; 3.2 ⁇ L, RNase-free ddH 2 O; 20ng, genomic DNA (40, 20, 10, 5ng genomic DNA were added when constructing the standard song);
  • reaction conditions are: 95°C, 15min; 45 cycles (95°C, 10s; 58/60°C, 60s or 95°C, 10s; 58/60°C 20s, 72°C 30s) ; 1.6°C/s (95°C, 15s; 60°C, 60s).
  • the primer pair (1) has an annealing temperature of 56° C., a two-step method
  • the primer pair (2) has an annealing temperature of 58° C., a two-step method
  • the primer pair (3) has an annealing temperature of 56° C., a three-step method
  • the primer pair (4) has an annealing temperature of 60°C, a three-step method
  • the primer pair (5) has an annealing temperature of 60°C, a two-step method.
  • the P value was obtained by the following formula. When P>0.5, the result was judged as positive; when P ⁇ 0.5, the result was judged as negative.
  • X 0 is the result value of occult blood
  • X 1 is the Ct value of Peptostreptococcus stomatis
  • X 2 is Ct value of Fusobacterium nucleatum
  • X 3 is the Ct value of Parvimonas micra
  • X 4 is the Ct value of Bifidobacterium.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • Data Mining & Analysis (AREA)
  • General Physics & Mathematics (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • Pure & Applied Mathematics (AREA)
  • Theoretical Computer Science (AREA)
  • Computational Mathematics (AREA)
  • Mathematical Analysis (AREA)
  • Mathematical Optimization (AREA)
  • Mathematical Physics (AREA)
  • Evolutionary Biology (AREA)
  • Probability & Statistics with Applications (AREA)
  • Algebra (AREA)
  • Operations Research (AREA)
  • Databases & Information Systems (AREA)
  • Software Systems (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明属于结直肠癌检测领域，具体涉及一种用于结直肠癌早期诊断的生物标志物组合物及应用。本发明具体公开了生物标志物在结直肠癌早期诊断中的应用，通过测定该生物标志物的含量和粪便隐血实验结合用于结直肠癌的早期诊断。

Description

一种用于结直肠癌早期诊断的生物标志物组合物及应用 技术领域
本发明属于结直肠癌检测领域，具体涉及一种用于结直肠癌早期诊断的生物标志物组合物及应用。
背景技术
结直肠癌是临床常见肿瘤，具有高发病率及高死亡率，在中国新增癌症病例中结直肠癌发病率在男性中排第四，在女性中排第三，在全球范围排第二位。如果能在早期检测到结直肠癌病变并通过手术切除，则存活率超过90％，而晚期则降低至13％，因此，早筛是降低结直肠癌死亡率最有效的手段。发达国家已经建立了有效的早期筛查计划和诊断技术，成为选择治疗方法并预测结果的方法的重要组成部分，使得死亡风险和治疗质量都获得了很大的改善，这为我们提供了指导与借鉴。
目前对于筛查方法，结肠镜检查仍然是明确诊断的金标准。虽然它有效，但它也可能引起恐惧，尴尬，可能会导致严重的并发症，参与率低，不适合早期筛查。其它的侵入式筛查如气钡灌肠、核磁共振成像等，有技术难度大，费用高等缺点。而非侵入式的粪便隐血检查有：基于愈创木脂化学法(gFOBT)；免疫法等，均存在灵敏度低、易受饮食用药影响的问题。《结直肠癌早期筛查指南》推荐：高敏gFOBT每年检查一次，高敏免疫化学法每年检查一次，但这种方法具有较高的假阳性率，对早期息肉敏感性低。
粪便基因是另一种可靠的生物标志物，可以通过DNA检测来进行早期筛查。多靶点基因检测已被《结直肠癌早期筛查指南》推荐用于结直肠癌筛查，每三年一次，但是价格昂贵，对腺瘤诊断效果差，尚未在亚洲人群进行验证。FDA批准的Cologuard(大肠卫士)是一种有效的分子(基因突变，甲基化和潜血实验)筛查技术，是将粪便潜血检测与血液中肿瘤相关DNA标志物检测相结合，定量检测血液中与结直肠肿瘤相关的DNA标志物，及人类粪便中存在的潜血红蛋白，实现结直肠癌的早期诊断，但是其价格昂贵，且需要抽血检查，并且对腺瘤的诊断效果较差。因此，需要我们开发灵敏、无创、经济的无创筛查技术来改善现状。
研究表明肠道生物群在人类健康和许多疾病的过程中发挥着重要作用，可用 作诊断，预后和分层等生物标志物，并且可以通过患者粪便进行检测。例如，中国专利CN106574294A，公开了一种通过定量PCR从人类粪便样本诊断结肠直肠癌的方法、引物及试剂盒，通过检测粪便中一种或多种16SrDNA细菌序列的量化水平，对人类对象的CRC和/或腺瘤性息肉进行早期检测、风险筛查和监控的方法，但是这种方法对结直肠癌的检测准确率和灵敏度较低。中国专利CN110512015A，公开了一种肠癌生物标志物组合物及其应用，所述生物标志物组合物包括梭菌科、卟啉单胞菌属、消化链球菌属、具核梭杆菌和微单胞菌属。发明人采集大量结直肠癌和癌前病变(结直肠腺瘤和结直肠肿瘤性息肉)患者和健康个体的粪便样本，利用高通量测序、实时荧光定量PCR、高分辨率熔解曲线法或生物芯片等方法，通过对粪便样本中微生物的丰度和相对含量差异进行对比分析和验证，确定了与结直肠癌和癌前病变相关的微生物，采用上述微生物的不同组合作为生物标志物组合物，通过检测生物标志物组合物在样本中的丰度，预测是否有结直肠癌发生。但是该方法检测特异性差。
本发明筛选了特定的肠道微生物作为用于结直肠癌早期诊断的生物标志物组合物，所述生物标志物组合物包括口炎消化链球菌Peptostreptococcus stomatis,具核梭状杆菌Fusobacterium nucleatum,微小微单胞菌Parvimonas micra,双歧杆菌Bifidobacterium；并通过上述微生物组合物的定量水平检测和粪便潜血实验结合，通过回归方程，用于结直肠癌的诊断结果，本发明利用基于高通量测序结果的特异性，并结合对结直肠癌进行早期诊断，灵敏度高和特异性强，能够避免肠镜侵入式检查导致的痛苦、不适及并术后发症，显著降低结直肠癌早期诊断的假阳性率，可以广泛用于结直肠癌的早期诊断。
发明内容
针对上述技术问题，本发明的目的在于提供一种结直肠癌生物标志物组合物，所述生物标志物组合物包括口炎消化链球菌Peptostreptococcus stomatis，具核梭状杆菌Fusobacterium nucleatum，微小微单胞菌Parvimonas micra,双歧杆菌Bifidobacterium。
本发明的另一目的在于提供一种检测结直肠癌的试剂盒，所述试剂盒包括生物标志物组合物的量化试剂，所述生物标志物组合物包括口炎消化链球菌 Peptostreptococcus stomatis,具核梭状杆菌Fusobacterium nucleatum,微小微单胞菌Parvimonas micra,双歧杆菌Bifidobacterium。
优选地，所述量化试剂用于量化生物标志物组合物中的每一组分。
优选地，所述量化试剂采用的原理为实时定量PCR法。
优选地，所述量化试剂为实时定量PCR量化试剂。
优选地，所述量化试剂包括用于生物标志物组合物的引物组。
优选地，所述引物组包括用于扩增生物标志物组合物每一组分的引物对。
优选地，所述引物组的核苷酸序列如SEQ ID NO.1-8所示。
优选地，所述量化试剂还包括用于检测总菌的引物对。
优选地，所述用于检测总菌的引物对的核苷酸序列如SEQ ID NO.9-10所示。
优选地，所述量化试剂还包括实时定量PCR反应试剂。
优选地，所述试剂盒还包括用于检测粪便中人血红蛋白的隐血检测试剂。
优选地，所述隐血检测试剂包括稀释液和检测试纸。
本发明的另一目的在于提供一种上述试剂盒在结直肠癌早期诊断中的应用，所述试剂盒用于检测结直肠癌时的步骤为：检测权利要求1所述的生物标志物组合物中每一组分在样本中的丰度以及粪便隐血值，再通过回归方程判断检测结果。以受试者粪便基因组DNA为模板，采用试剂盒中的引物组检测生物标志物在样本中的丰度。
优选地，所述试剂盒用于检测结直肠癌时，包括以下步骤：
(1)粪便隐血值测定：采用粪便隐血检测试剂盒测定受试者粪便样品的隐血结果值X 0，其中隐血结果为阳性时X 0＝1，隐血结果为阳性时X 0＝0；
(2)以受试者粪便基因组DNA为模板，采用试剂盒中的引物组分别检测每个生物标志物的扩增值Ct 标志物，和总菌的扩增值Ct 总菌；
(3)根据回归方程计出算Y值，并将Y值带入公式(Ⅱ)计算P值，其中，e为自然常数，
Figure PCTCN2022087384-appb-000001
(5)结果判定：当P>0.5，结直肠癌诊断结果为阳性；当P≤0.5，结直肠癌诊断结果为阴性。
优选地，所述步骤(4)中的回归方程为式(Ⅲ)所示，
Y＝A+β 0X 0+β 1X 1+β 2X 2+β 3X 3+β 4X 4      (Ⅲ)；
其中，X 0为隐血结果值，X 1为口炎消化链球菌Peptostreptococcus stomatis的Ct值，X 2为具核梭状杆菌Fusobacterium nucleatum的Ct值，X 3为微小微单胞菌Parvimonas micra的Ct值，X 4为双歧杆菌Bifidobacterium的Ct值；A和β 0-β 4均为常数，A和β 0-β 4由临床实验数据获得。
优选地，所述步骤(4)中的回归方程包括式(Ⅳ)-式(Ⅷ)中的任一种：
Y＝-3.9583+3.1015X 0-0.6478X 1-0.3980X 2+0.2912X 3-0.5130X 4      (Ⅳ)；
Y＝-2.965+2.7005X 0-1.2309X 1-0.6054X 2+0.3328X 3-0.2359X 4       (Ⅴ)；
Y＝-3.099+1.9907X 0-2.0011X 1-0.3290X 2+0.5476X 3-0.6620X 4       (Ⅵ)；
Y＝-3.771+4.0089X 0-0.9983X 1-0.4482X 2+0.1288X 3-0.4917X 4        (Ⅶ)；
Y＝-2.9043+2.8920X 0-0.5471X 1-0.1049X 2+0.2281X 3-0.6194X 4       (Ⅷ)。
优选地，所述步骤(4)中的回归方程为：
Y＝-2.965+2.7005X 0-1.2309X 1-0.6054X 2+0.3328X 3-0.2359X 4。
优选地，所述试剂盒还包括DNA提取试剂。
本发明的有益效果是：①本发明提供了一种用于结直肠癌早期诊断的生物标志物组合物，及用于定量检测所述生物标志物组合物的引物对和试剂盒；②本发明利用基于高通量测序结果的特异性，并结合粪便潜血实验对结直肠癌进行早期诊断，灵敏度高和特异性强，能够避免肠镜侵入式检查导致的痛苦、不适及并术后发症，显著降低结直肠癌早期诊断的假阳性率，实现了结直肠癌的无创诊断，还具有广泛的应用价值。
具体实施方式
以下的实施例便于更好地理解本发明，但并不限定本发明。下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为市售。
术语“生物标志物”是指作为通常存在于身体样本中且可容易测定的物质的疾病标记物，所测量的量可以关联到潜在的疾病病理生理学，例如CRC的存在或不存在。
术语“量化”是指量化样本中的特定核酸序列的量的能力，用于测定目标核酸序列的量的分子生物学方法包括但不限于终点PCR、竞争PCR、逆转录酶PCR(PT-PCR)、定量PCR(qPCR)、PCR-ELISA、DNA微阵列等。
术语“量化水平”可以是浓度(每单位体积的DNA量)，每细胞数量的DNA量，循环阈值(Ct值)。在以下一个优选的实施例中，所述细菌序列的量化通过qPCR进行；在以下一个更优选的实施例中，所述细菌序列的定量是通过qPCR进行，且量化等级是Ct值。Ct值是指每个反应管内的荧光信号到达设定的域值时所经历的qPCR循环数。Ct水平与样本中的目标核酸的量化成反比，即，Ct水平越低，样本中目标核酸的量越大。
样本中目标核酸序列丰度的定量可以是绝对或相对的。相对定量是基于一个或多个内部参照基因，即来自参照菌株的16S rRNA基因，如使用通用引物测定总细菌数并将该目标核酸序列的丰度表达为细菌百分比；绝对定量通过与DNA标准比较得到目标分子的确切数目。
术语“引物”是指可在扩增方法例如聚合酶链式反应(PCR)中使用以扩增核苷酸序列的寡核苷酸。引物是基于特定目标序列的多核苷酸序列。
在一个具体实施例中，使用灵敏度、特异性、准确性及其他组合用来描述本发明所述检测方法的良好性和可靠性。与灵敏度、特异性、准确性的描述一起使用的若干术语包括：真阳性(TP)、真阴性(TN)、假阳性(FP)、假阴性(FN)；其中，如果证明患者患有疾病，且给定的筛查实验也表明该疾病的存在，则测试结果被认为是真阳性；如果证明患者未患有疾病，且给定的筛查实验也表明该疾病不存在，则测试结果被认为是真阴性；如果筛查实验的结果表明实际上未患有疾病的患者患有该疾病，则测试结果为假阳性；如果筛查实验的结果表明实际上患有疾病的患者未患有该疾病，则测试结果为假阴性。
灵敏度＝TP/(TP+FN)＝真阳性评估的数量/所有阳性评估的数量；
特异性＝TN/(TN+FP)＝真阴性评估的数量/所有阴性评估的数量；
准确度＝(TN+TP)/(TN+TP+FN+FP)＝正确评估数/所有评估的数量。
实施例1引物组的设计
分别针对口炎消化链球菌Peptostreptococcus stomatis、具核梭状杆菌Fusobacterium nucleatum、微小微单胞菌Parvimonas micra、双歧杆菌Bifidobacterium以及总菌设计引物对如下(1)-(5)所示：
引物对(1)：上游引物5-AAGTGTTAGCGGTATAGGATG-3；下游引物5-CGTGTCTCAGTTCCAATGT-3；
引物对(2)：上游引物5-GGATTTATTGGGCGTAAAGC-3；下游引物5-GGCATTCCTACAAATATCTACGAA-3；
引物对(3)：上游引物5-GCGTAGATATTAGGAGGAATAC-3；下游引物5-GCGGAATGCTTAATGTGTT-3；
引物对(4)：上游引物5-CATCGCTTAACGGTGGAT-3-3；下游引物5-TTCGCCATTGGTGTTCTT-3；
引物对(5)：上游引物5-GCAGGCCTAACACATGCAAGTC-3；下游引物5-CTGCTGCCTCCCGTAGGAGT-3。
实施例2试剂盒的制备和使用方法
2.1试剂盒1
一种检测结直肠癌的试剂盒，所述试剂盒包括生物标志物组合物口炎消化链球菌Peptostreptococcus stomatis,具核梭状杆菌Fusobacterium nucleatum,微小微单胞菌Parvimonas micra,双歧杆菌Bifidobacterium的量化试剂、反应缓冲体系、Mix、ROX和RNase-free ddH 2O，所述量化试剂包括用于检测所述生物标志物组合物的引物组，所述引物组如实施例1中的引物对(1)-(4)所示，以及用于检测总菌Total bacterica的引物对，所述引物对如实施例1中的引物对(5)所示。
使用方法：提取受试者粪便基因组DNA，并以为粪便基因组DNA模板，采用上述引物对(1)-(5)检测生物标志物在样本中的丰度；根据生物标志物在样本中的丰度判断是否患有结直肠癌。
2.2试剂盒2
一种检测结直肠癌的试剂盒，所述试剂盒包括生物标志物组合物口炎消化链球菌Peptostreptococcus stomatis，具核梭状杆菌Fusobacterium nucleatum，微小微单胞菌Parvimonas micra，双歧杆菌Bifidobacterium的量化试剂和隐血检测试剂；所述量化试剂包括引物组、反应缓冲体系、Mix、ROX和RNase-free ddH 2O，所述引物组如实施例1中的引物对(1)-(4)所示，以及用于检测总菌Total bacterica的引物对，所述引物对如实施例1中的引物对(5)所示；所述隐血检测试剂包括稀释液和检测试纸。
使用方法：
(1)粪便隐血值测定：采用粪便隐血检测试剂盒测定受试者粪便样品的隐血结果值X 0，其中隐血结果为阳性时X 0＝1，隐血结果为阳性时X 0＝0；
(2)以受试者粪便基因组DNA为模板，采用试剂盒中的引物组分别检测每个生物标志物的扩增值Ct 标志物，和总菌的扩增值Ct 总菌；
(3)根据回归方程Y＝A+β 0X 0+β 1X 1+β 2X 2+β 3X 3+β 4X 4(Ⅲ)，计出算Y值，并将Y值带入公式(Ⅱ)计算P值，其中，e为自然常数，
Figure PCTCN2022087384-appb-000002
(4)结果判定：当P>0.5，结直肠癌诊断结果为阳性；当P≤0.5，结直肠癌诊断结果为阴性。
其中回归方程Y＝A+β 0X 0+β 1X 1+β 2X 2+β 3X 3+β 4X 4(Ⅲ)中，X 0为隐血结果值，X 1为口炎消化链球菌Peptostreptococcus stomatis的Ct值，X 2为具核梭状杆菌Fusobacterium nucleatum的Ct值，X 3为微小微单胞菌Parvimonas micra的Ct值，X 4为双歧杆菌Bifidobacterium的Ct值；A和β 0-β 4均为常数并且由临床实验数据分析获得；
根据临床实验分析，所述回归方程可以为下式(Ⅳ)-式(Ⅷ)中的任一种：
Y＝-3.9583+3.1015X 0-0.6478X 1-0.3980X 2+0.2912X 3-0.5130X 4    (Ⅳ)；
Y＝-2.965+2.7005X 0-1.2309X 1-0.6054X 2+0.3328X 3-0.2359X 4    (Ⅴ)；
Y＝-3.099+1.9907X 0-2.0011X 1-0.3290X 2+0.5476X 3-0.6620X 4    (Ⅵ)；
Y＝-3.771+4.0089X 0-0.9983X 1-0.4482X 2+0.1288X 3-0.4917X 4    (Ⅶ)；
Y＝-2.9043+2.8920X 0-0.5471X 1-0.1049X 2+0.2281X 3-0.6194X 4    (Ⅷ)。
2.3试剂盒3
一种检测结直肠癌的试剂盒，所述试剂盒包括生物标志物组合物口炎消化链球菌Peptostreptococcus stomatis，具核梭状杆菌Fusobacterium nucleatum，微小微单胞菌Parvimonas micra，双歧杆菌Bifidobacterium的量化试剂、隐血检测试剂和DNA提取试剂；所述量化试剂包括引物组、反应缓冲体系、Mix、ROX和RNase-free ddH 2O，所述引物组如实施例1中的引物对(1)-(4)所示，以及用于检测总菌的引物对，所述引物对如实施例1中的引物对(5)所示；所述试剂盒还包括；所述隐血检测试剂包括稀释液和检测试纸。
使用方法：
(1)粪便隐血值测定：采用粪便隐血检测试剂盒测定受试者粪便样品的隐血结果值X 0，其中隐血结果为阳性时X 0＝1，隐血结果为阳性时X 0＝0；
(2)以受试者粪便基因组DNA为模板，采用试剂盒中的引物组分别检测每个生物标志物的扩增值Ct 标志物，和总菌的扩增值Ct 总菌；
(3)根据回归方程Y＝A+β 0X 0+β 1X 1+β 2X 2+β 3X 3+β 4X 4(Ⅲ)，计出算Y值，并将Y值带入公式(Ⅱ)计算P值，其中，e为自然常数，
Figure PCTCN2022087384-appb-000003
(4)结果判定：当P>0.5，结直肠癌诊断结果为阳性；当P≤0.5，结直肠癌诊断结果为阴性。
其中回归方程Y＝A+β 0X 0+β 1X 1+β 2X 2+β 3X 3+β 4X 4(Ⅲ)中，X 0为隐血结果值，X 1为口炎消化链球菌Peptostreptococcus stomatis的Ct值，X 2为具核梭状杆菌Fusobacterium nucleatum的Ct值，X 3为微小微单胞菌Parvimonas micra的Ct值，X 4为双歧杆菌Bifidobacterium的Ct值；A和β 0-β 4均为常数并且由临床 实验数据分析获得；
根据临床实验分析，所述回归方程可以为下式(Ⅳ)-式(Ⅷ)中的任一种：
Y＝-3.9583+3.1015X 0-0.6478X 1-0.3980X 2+0.2912X 3-0.5130X 4    (Ⅳ)；
Y＝-2.965+2.7005X 0-1.2309X 1-0.6054X 2+0.3328X 3-0.2359X 4    (Ⅴ)；
Y＝-3.099+1.9907X 0-2.0011X 1-0.3290X 2+0.5476X 3-0.6620X 4    (Ⅵ)；
Y＝-3.771+4.0089X 0-0.9983X 1-0.4482X 2+0.1288X 3-0.4917X 4    (Ⅶ)；
Y＝-2.9043+2.8920X 0-0.5471X 1-0.1049X 2+0.2281X 3-0.6194X 4    (Ⅷ)。
实施例3结直肠癌诊断
选取患有结直肠癌和腺瘤息肉患者40例和健康人18例，使用本发明提供的试剂盒对上述病例进行筛查，并根据检测结果，判断本发明提供的试剂盒用于结直肠癌检测的灵敏度、特异性和准确度。
1.粪便样品采集
准备两支采样管，标记为a管与b管，使用采集管采集受试患者的粪便1平勺，约0.25ml，2g；
2.粪便隐血检测
取上述a管，依照粪便隐血检测试(胶体金法)剂盒说明，在自动粪便处理分析系统上进行粪便隐血检测，其中质控线条带与检测线条带均为阳性，则为阳性结果，记为X 0＝1；质控线条带阳性，检测线条带阴性，则为阴性结果，记为X 0＝0。
3.粪便生物标志物检测
3.1粪便基因组DNA提取
(1)称取上述b管中的粪便样本180-220mg至2ml离心管中，根据基因组提取试剂及说明提取基因组DNA：
(2)称取粪便样本180-220mg(如果是液态样本则转移200μl至离心管中)至2ml离心管中，并将管子置于冰上；
(3)向样本中加入500μl葡萄糖及EDTA缓冲液，100μl裂解液，15μlProteinase K，0.25g的研磨珠，间歇振荡1min至样本充分混匀；95℃孵育15min，孵育期间震荡2-3次；
(4)涡旋15s，12,000rpm离心3min，转移上清液至新的离心管中，加入10μl的RNase A，震荡混匀后室温放置5min；
(5)加入200μlNaOH-SDS缓冲液，震荡混匀，置冰上5min；12,000rpm离心3min，将所得上清液转移至新的1.5ml离心管，加入等体积醋酸钠溶液，并加入到一个吸附柱CR2中(吸附柱放入收集管中)，12,000rpm离心30s，倒掉废液，将吸附柱CR2放入收集管中；
(6)向吸附柱CR2中加入500μl无水乙醇，12,000rpm离心30s，倒掉废液，将吸附柱CR2放入收集管中；向吸附柱CR2中加入700μl漂洗液，12,000rpm离心30s，倒掉废液，吸附柱CR2放入收集管中，重复操作；
(7)将吸附柱CR2放回收集管中，12,000rpm离心2min，倒掉废液，将吸附柱CR2置于室温放置数分钟，以彻底晾干吸附材料中残余的漂洗液；
(8)用紫外分光光度计检测浓度与纯度，其中OD260/OD280比值应为1.7-1.9，确保提取的DNA无污染，浓度≥50ng/μL为最佳，即获得粪便基因组DNA。
3.2实时qPCR检测
(1)Real Time PCR反应液的配制：冰上进行反应液的配制，每10μL的反应体系含5μL，2×Mix；0.3μL，上游引物；0.3μL，下游引物；0.2μL，50×ROX；3.2μL，RNase-free ddH 2O；20ng，基因组DNA(构建标曲时分别添加40、20、10、5ng基因组DNA)；
(2)在qPCR仪上进行实验，反应条件为：95℃，15min；45个循环(95℃，10s；58/60℃，60s或者95℃，10s；58/60℃20s，72℃30s)；1.6℃/s(95℃，15s；60℃，60s)。其中，所述引物对(1)56℃退火温度，两步法；所述引物对(2)58℃退火温度，两步法；所述引物对(3)56℃退火温度，三步法；所述引物对(4)60℃退火温度，三步法；所述引物对(5)60℃退火温度，两步法。
(3)绘制标准曲线，按效率＝10(-1/斜率)-1计算扩增效率，90％～110％合格；
(4)根据上述步骤进行样本实验，记录上述生物标志物相应的引物扩增值Ct 标志物，及总菌的引物扩增值Ct 总菌，计算每个生物标志物的循环值Ct，其中所述Ct＝lg(2^(Ct 标志物-Ct 总菌))。
4.诊断结果判定
通过下述公式得到P值，当P>0.5，判定结果为阳性；当P≤0.5，判定结果为阴性。
Figure PCTCN2022087384-appb-000004
其中Y＝-3.9583+3.1015X 0-0.6478X 1-0.3980X 2+0.2912X 3-0.5130X 4，X 0为隐血结果值，X 1为口炎消化链球菌Peptostreptococcus stomatis的Ct值，X 2为具核梭状杆菌Fusobacterium nucleatum的Ct值，X 3为微小微单胞菌Parvimonas micra的Ct值，X 4为双歧杆菌Bifidobacterium的Ct值。
当P>0.5，判定结果为阳性，即确定患有结直肠癌；当P≤0.5，判定结果为阴性，即确定不患有结直肠癌。
5结果分析
实验结果表明，以发明所述的试剂盒用于结直肠癌诊断，具有较高的准确率、特异性和灵敏度。
上述说明是针对本发明较佳可行实施例的详细说明，但实施例并非用以限定本发明的专利申请范围，凡本发明所提示的技术精神下所完成的同等变化或修饰变更，均应属于本发明所涵盖专利范围。

Claims (13)

  1. 一种结直肠癌生物标志物组合物，其特征在于，所述生物标志物组合物包括口炎消化链球菌Peptostreptococcus stomatis，具核梭状杆菌Fusobacterium nucleatum，微小微单胞菌Parvimonas micra，双歧杆菌Bifidobacterium。
  2. 一种检测结直肠癌的试剂盒，其特征在于，所述试剂盒包括权利要求1所述生物标志物组合物的量化试剂，所述量化试剂用于量化权利要求1所述的生物标志物组合物中的每一组分。
  3. 如权利要求2所述的试剂盒，其特征在于，所述量化试剂采用的原理为实时定量PCR法。
  4. 如权利要求3所述的试剂盒，其特征在于，所述量化试剂包括用于扩增权利要求1所述生物标志物组合物的引物组，所述引物组包括用于扩增权利要求1所述生物标志物组合物每一组分的引物对。
  5. 如权利要求4所述的试剂盒，其特征在于，所述引物组的核苷酸序列如SEQ ID NO.1-8所示。
  6. 如权利要求5所述的试剂盒，其特征在于，所述量化试剂还包括用于扩增总菌的引物对。
  7. 如权利要求6所述的试剂盒，其特征在于，所述用于扩增总菌的引物对的核苷酸序列如SEQ ID NO.9-10所示。
  8. 如权利要求7所述的试剂盒，其特征在于，所述量化试剂还包括实时定量PCR反应试剂。
  9. 如权利要求2-8任一所述的试剂盒，其特征在于，所述试剂盒还包括用于检测粪便中人血红蛋白的隐血检测试剂。
  10. 如权利要求9所述的试剂盒在结直肠癌早期诊断中的应用，其特征在于，所述试剂盒用于检测结直肠癌时的步骤为：检测权利要求1所述的生物标志物组合物中每一组分在样本中的丰度以及粪便隐血值，再通过回归方程判断检测结果。
  11. 如权利要求10所述的试剂盒在结直肠癌早期诊断中的应用，其特征在于，所述试剂盒用于检测结直肠癌时，包括以下步骤：
    (1)粪便隐血值测定：采用粪便隐血检测试剂盒测定受试者粪便样品的隐血结果值X 0，其中隐血结果为阳性时X 0＝1，隐血结果为阳性时X 0＝0；
    (2)以受试者粪便基因组DNA为模板，采用试剂盒中的量化试剂分别检测每个生物标志物的扩增值Ct 标志物，和总菌的扩增值Ct 总菌；
    (3)根据公式(Ⅰ)分别确定样本中生物标志物组合物中每一组分在总菌中的相对含量Ct值，
    Ct＝lg(2^(Ct 标志物-Ct 总菌))
    (Ⅰ)；
    (4)根据回归方程计出算Y值，并将Y值带入公式(Ⅱ)计算P值，其中，e为自然常数，
    Figure PCTCN2022087384-appb-100001
    (5)结果判定：当P>0.5，结直肠癌诊断结果为阳性；当P≤0.5，结直肠癌诊断结果为阴性。
  12. 如权利要求11所述的应用，其特征在于，所述步骤(4)中的回归方程为式(Ⅲ)所示，
    Y＝A+β 0X 0+β 1X 1+β 2X 2+β 3X 3+β 4X 4
    (Ⅲ)；
    其中，X 0为隐血结果值，X 1为口炎消化链球菌Peptostreptococcus stomatis的Ct值，X 2为具核梭状杆菌Fusobacterium nucleatum的Ct值，X 3为微小微单胞菌Parvimonas micra的Ct值，X 4为双歧杆菌Bifidobacterium的Ct值；A和β 0-β 4均为常数，A和β 0-β 4由临床实验数据获得。
  13. 如权利要求12所述的应用，其特征在于，所述步骤(4)中的回归方程包括式(Ⅳ)-式(Ⅷ)中的任一种：
    Y＝-3.9583+3.1015X 0-0.6478X 1-0.3980X 2+0.2912X 3-0.5130X 4
    (Ⅳ)；
    Y＝-2.965+2.7005X 0-1.2309X 1-0.6054X 2+0.3328X 3-0.2359X 4
    (Ⅴ)；
    Y＝-3.099+1.9907X 0-2.0011X 1-0.3290X 2+0.5476X 3-0.6620X 4
    (Ⅵ)；
    Y＝-3.771+4.0089X 0-0.9983X 1-0.4482X 2+0.1288X 3-0.4917X 4
    (Ⅶ)；
    Y＝-2.9043+2.8920X 0-0.5471X 1-0.1049X 2+0.2281X 3-0.6194X 4
    (Ⅷ)。
PCT/CN2022/087384 2020-05-27 2022-04-18 一种用于结直肠癌早期诊断的生物标志物组合物及应用 WO2022247523A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
EP22810256.2A EP4350008A1 (en) 2020-05-27 2022-04-18 Biomarker composition for early diagnosis of colorectal cancer and application
US18/560,848 US20240254569A1 (en) 2020-05-27 2022-04-18 Biomarker combination for colorectal cancer early diagnosis and use thereof

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
CN202010458730 2020-05-27
CN202110562717.5A CN113106163B (zh) 2020-05-27 2021-05-24 一种用于结直肠癌早期诊断的生物标志物组合物及应用
CN202110562717.5 2021-05-24

Publications (1)

Publication Number Publication Date
WO2022247523A1 true WO2022247523A1 (zh) 2022-12-01

Family

ID=76722936

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/087384 WO2022247523A1 (zh) 2020-05-27 2022-04-18 一种用于结直肠癌早期诊断的生物标志物组合物及应用

Country Status (4)

Country Link
US (1) US20240254569A1 (zh)
EP (1) EP4350008A1 (zh)
CN (2) CN117305487A (zh)
WO (1) WO2022247523A1 (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110903997B (zh) * 2019-10-23 2021-11-23 上海市第十人民医院 一株从大肠癌肿瘤组织中获取的具核梭杆菌及其应用
CN117305487A (zh) * 2020-05-27 2023-12-29 微度(苏州)生物科技有限公司 一种用于结直肠癌早期诊断的生物标志物组合物及应用
CN114574604A (zh) * 2022-03-31 2022-06-03 聚点(重庆)生物医学研究有限公司 一种辅助诊断结直肠癌高危人群的生物标志物及方法

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106399570A (zh) * 2016-11-30 2017-02-15 杭州诺辉健康科技有限公司 用于早期结直肠癌辅助诊断的试剂盒及其使用方法和检测系统
CN106574294A (zh) 2014-03-03 2017-04-19 何塞普特鲁塔博士赫罗纳生物医学研究所 用于通过定量pcr从人粪便样本诊断结肠直肠癌的方法、引物及试剂盒
US20180122511A1 (en) * 2014-10-21 2018-05-03 uBiome, Inc. Method and system for characterizing microorganism-related conditions
CN110512015A (zh) 2019-09-11 2019-11-29 苏州普瑞森基因科技有限公司 一种肠癌生物标志物组合物及其应用
US20210000885A1 (en) * 2015-11-30 2021-01-07 Joseph E. Kovarik Method for Reducing the Likelihood of Developing Bladder or Colorectal Cancer in an Individual Human Being
CN113106163A (zh) * 2020-05-27 2021-07-13 兰州大学 一种用于结直肠癌早期诊断的生物标志物组合物及应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2927327A1 (en) * 2014-04-02 2015-10-07 EntreChem, S.L. Non-invasive in vitro method for diagnosis and prognosis of colorectal cancer
EP3518946A4 (en) * 2016-09-27 2020-09-09 Board of Regents, The University of Texas System METHODS TO IMPROVE IMMUNE CHECKPOINT BLOCKING TREATMENT BY MODULATING THE MICROBIOME
AR110378A1 (es) * 2016-12-15 2019-03-20 Univ College Cork National Univ Of Ireland Cork Métodos para determinar el estado del cáncer colorrectal en una persona
US20200011873A1 (en) * 2017-03-17 2020-01-09 Second Genome, Inc. Leveraging sequence-based fecal microbial community survey data to identify a composite biomarker for colorectal cancer

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106574294A (zh) 2014-03-03 2017-04-19 何塞普特鲁塔博士赫罗纳生物医学研究所 用于通过定量pcr从人粪便样本诊断结肠直肠癌的方法、引物及试剂盒
US20180122511A1 (en) * 2014-10-21 2018-05-03 uBiome, Inc. Method and system for characterizing microorganism-related conditions
US20210000885A1 (en) * 2015-11-30 2021-01-07 Joseph E. Kovarik Method for Reducing the Likelihood of Developing Bladder or Colorectal Cancer in an Individual Human Being
CN106399570A (zh) * 2016-11-30 2017-02-15 杭州诺辉健康科技有限公司 用于早期结直肠癌辅助诊断的试剂盒及其使用方法和检测系统
CN110512015A (zh) 2019-09-11 2019-11-29 苏州普瑞森基因科技有限公司 一种肠癌生物标志物组合物及其应用
CN113106163A (zh) * 2020-05-27 2021-07-13 兰州大学 一种用于结直肠癌早期诊断的生物标志物组合物及应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG HE;SHI XINLONG;LI JINGJING;ZHU CHENGZHANG;LU JIYONG;WANG SHUAI;WANG TAO;DU BINBIN;ZHANG WEISHENG;YANG XIONGFE: "Research Progress in Relationship between Gut Microbiota and Colorectal Cancer", CHINESE JOURNAL OF GENERAL SURGERY, vol. 29, no. 10, 25 October 2020 (2020-10-25), pages 1261 - 1269, XP093008534, ISSN: 1005-6947, DOI: 10.7659/j.issn.1005-6947.2020.10.013 *

Also Published As

Publication number Publication date
CN113106163A (zh) 2021-07-13
US20240254569A1 (en) 2024-08-01
CN113106163B (zh) 2024-06-04
EP4350008A1 (en) 2024-04-10
CN117305487A (zh) 2023-12-29

Similar Documents

Publication Publication Date Title
WO2022247523A1 (zh) 一种用于结直肠癌早期诊断的生物标志物组合物及应用
CN110387421A (zh) 用于肺癌检测的DNA甲基化qPCR试剂盒及使用方法
US20220259672A1 (en) Gene marker combination and use thereof
CN109371133B (zh) 一组与胰腺癌相关的LncRNA分子标记物及其应用
CN113186309B (zh) 泌尿系统细菌感染检测体系及其试剂盒和应用
CN106755464A (zh) 用于筛选肠癌和/或胃癌的基因标志物的方法、用该方法筛选的基因标志物及其用途
CN101855348A (zh) 肝癌相关基因、以及肝癌风险的判定方法
CN109055555B (zh) 一种肺癌早期转移诊断标志物及其试剂盒和应用
Akutsu et al. Development of a multiplex RT-PCR assay and statistical evaluation of its use in forensic identification of vaginal fluid
CN107630093B (zh) 用于诊断肝癌的试剂、试剂盒、检测方法及用途
WO2018062743A1 (ko) 질 내 미생물을 포함하는 조성물
CN112501278A (zh) Smim26作为结核病诊断分子标识的用途
WO2017026691A1 (ko) 비만의 진단용 조성물 및 이의 용도
AU2015246009B2 (en) Methods and kits for identifying pre-cancerous colorectal polyps and colorectal cancer
CN111154880B (zh) 一种膀胱癌体液活检生物标志物及其应用
CN114107498A (zh) 结直肠癌血液检测标记物及其应用
CN116068193B (zh) 结核病分子标志物组合及其用途
WO2024031860A1 (zh) 一种用于结直肠癌诊断的多基因dna甲基化联合检测试剂盒及应用
WO2024036785A1 (zh) 胃癌早期筛查的dna甲基化标志物组合及试剂盒
CN111575377B (zh) 用于line-1的检测引物组及其应用
RU2794198C1 (ru) Способ диагностики нарушения функционального состояния щитовидной железы у детей 4-10 лет, проживающих в условиях Крайнего Севера
CN112522404B (zh) 一种用于检测前列腺癌的多重荧光pcr试剂盒
CN107130028A (zh) Syce3在结直肠癌诊断和治疗效果评价中的应用
CN117604101A (zh) 一种基因标志物组合、试剂盒及检测方法
CN116083583A (zh) miRNA-483-5p在制备胰腺癌肝转移诊断试剂盒中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22810256

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 18560848

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 11202308748U

Country of ref document: SG

WWE Wipo information: entry into national phase

Ref document number: 2022810256

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2022810256

Country of ref document: EP

Effective date: 20240102