WO2022193097A1 - 用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法 - Google Patents

用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法 Download PDF

Info

Publication number
WO2022193097A1
WO2022193097A1 PCT/CN2021/080864 CN2021080864W WO2022193097A1 WO 2022193097 A1 WO2022193097 A1 WO 2022193097A1 CN 2021080864 W CN2021080864 W CN 2021080864W WO 2022193097 A1 WO2022193097 A1 WO 2022193097A1
Authority
WO
WIPO (PCT)
Prior art keywords
methylation
nucleic acid
detection
gene
seq
Prior art date
Application number
PCT/CN2021/080864
Other languages
English (en)
French (fr)
Inventor
李德强
张茜
黄龙妹
刘刚
吕宁
陈一友
Original Assignee
杭州诺辉健康科技有限公司
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 杭州诺辉健康科技有限公司 filed Critical 杭州诺辉健康科技有限公司
Priority to PCT/CN2021/080864 priority Critical patent/WO2022193097A1/zh
Priority to PCT/CN2022/080695 priority patent/WO2022194101A1/zh
Publication of WO2022193097A1 publication Critical patent/WO2022193097A1/zh

Links

Images

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
    • C12Q1/6883—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
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the invention relates to a nucleic acid and protein detection target combination and a combined detection method for early screening of liver cancer, and a corresponding kit for liver cancer detection.
  • hepatocellular carcinoma hepatocellular carcinoma
  • Malignancy ranks second in death.
  • liver cancer patients the treatment and prognosis of liver cancer patients detected at an early stage are much better than those of advanced liver cancer patients.
  • the five-year survival rate of liver cancer patients in my country is only 14%, but the survival rate of early-stage liver cancer patients exceeds 50%. Therefore, early screening of high-risk groups of liver cancer can effectively reduce the mortality of liver cancer and reduce medical costs.
  • liquid biopsy technology based on the detection of circulating tumor DNA (ctDNA) mutations and methylation has been gradually applied to auxiliary diagnosis and early screening of cancer.
  • the combined detection of nucleic acid markers and protein markers can not only effectively improve the sensitivity and specificity of cancer detection, but also has the potential to detect multiple cancer species at one time.
  • the content of cfDNA derived from liver tissue accounts for about 1% of the total content, which is higher than that of cfDNA derived from other digestive organs. The proportion of cfDNA from liver tissue was higher. Therefore, blood samples are suitable for liver cancer diagnosis and screening based on liquid biopsy technology.
  • Driver gene mutations can promote the occurrence and progression of liver cancer by affecting key signaling pathways.
  • Some HBV-infected liver cancer patients can detect driver gene (such as TP53) point mutations in the liver cancer tissue DNA and blood ctDNA.
  • driver gene such as TP53
  • DNA methylation can activate or silence genes by regulating gene expression levels.
  • some tumor suppressor genes are hypermethylated, suggesting that changes in methylation levels can be detected markers of carcinogenesis.
  • the detection of liver cancer methylation markers in plasma cfDNA has also become an emerging non-invasive method for liver cancer screening, diagnosis and prognostic detection.
  • Hepatocellular carcinoma is heterogeneous, and there are differences in DNA level (point mutation, abnormal methylation) and protein level between different liver cancer patients and even several liver tumors in the same patient. Therefore, it is necessary to develop a detection method that can further improve the positive detection rate, sensitivity, and/or specificity of liver cancer.
  • Figure 1 shows a test flow for plasma samples.
  • Figure 3 is an analysis of the sensitivity and specificity of the predictive model for detecting the relevant population.
  • Normal represents healthy people
  • CHB represents hepatitis B patients
  • CIR represents liver cirrhosis patients
  • HCC liver cancer patients.
  • the present invention provides a liver cancer diagnosis method based on the combined detection of nucleic acid and protein multi-target points.
  • the combined detection of one or more of different types of markers such as point mutation, methylation, and protein can effectively improve the positive detection rate, sensitivity, and/or specificity of liver cancer.
  • the nucleic acid marker detected is a DNA marker.
  • the nucleic acid marker detected is an RNA marker.
  • the diagnostic markers employed in the present invention comprise protein markers, and two or more of gene mutation, DNA methylation, chromosomal copy number variation (CNV), cfDNA fragmentation patterns.
  • CNV chromosomal copy number variation
  • the multiple markers of the present invention are combined with detection methods and corresponding kits to detect point mutations, abnormal methylation and AFP protein levels in plasma free nucleic acid (such as cell-free DNA), so as to achieve a high-risk group from liver cancer. (Hepatitis B, liver cirrhosis patients, etc.) for the purpose of liver cancer screening.
  • advantages of the present invention include, but are not limited to:
  • the present invention has higher sensitivity and specificity, and can detect early-stage liver cancer individuals with AFP ⁇ 20ng/ml, thereby achieving the goal of "early diagnosis and early treatment”.
  • the present invention provides a kit for liver cancer detection.
  • the kit comprises one or more reagents selected from the group consisting of plasma free nucleic acid point mutation detection reagents, plasma free nucleic acid methylation detection reagents, and/or protein detection reagents.
  • the plasma free nucleic acid is plasma free DNA (cfDNA). In certain embodiments, the plasma free nucleic acid is plasma free RNA.
  • the kit comprises a reagent selected from the group consisting of plasma free nucleic acid point mutation detection reagents and plasma free nucleic acid methylation detection reagents. In certain embodiments, the kit comprises a reagent selected from the group consisting of plasma free nucleic acid point mutation detection reagents and protein detection reagents. In certain embodiments, the kit comprises a reagent selected from the group consisting of plasma free nucleic acid methylation detection reagents and protein detection reagents. In certain embodiments, the kit comprises a reagent selected from the group consisting of plasma free nucleic acid point mutation detection reagents, plasma free nucleic acid methylation detection reagents, and protein detection reagents.
  • kits comprise plasma free nucleic acid point mutation detection reagents.
  • the plasma free nucleic acid point mutation detection reagent is a TP53 gene mutation detection reagent.
  • the mutated site of the TP53 gene comprises a C-A mutation corresponding to nucleotide 88 of SEQ ID NO:43.
  • the TP53 gene mutation detection reagent comprises a forward primer selected from SEQ ID NO: 1-3, a reverse primer selected from SEQ ID NO: 4-6, and/or selected from SEQ ID NO: 4-6 Probes for NO: 7-9.
  • the plasma free nucleic acid point mutation detection reagent further comprises an internal reference gene.
  • the internal reference gene is the EIF2C gene.
  • the plasma free nucleic acid point mutation detection reagent is a plasma free DNA (cfDNA) point mutation detection reagent.
  • kits comprise plasma free nucleic acid methylation detection reagents.
  • the plasma free nucleic acid methylation detection reagent is a plasma free DNA methylation detection reagent.
  • the methylated genes detected by the plasma-free nucleic acid methylation detection reagent comprise F12 (Coagulation factor XII), CDKL2 (Cyclin dependent kinase like 2) and SCARF2 (Scavenger receptor class-F) one or more genes of member 2).
  • the methylated gene comprises F12.
  • the methylated gene comprises CDKL2.
  • the methylated gene comprises SCARF2.
  • the methylated genes comprise F12 and CDKL2. In certain embodiments, the methylated genes comprise F12 and SCARF2. In certain embodiments, the methylated genes comprise CDKL2 and SCARF2. In certain embodiments, the methylated genes comprise F12, CDKL2 and SCARF2.
  • plasma free nucleic acid is subjected to sulfite conversion prior to detection of methylated marker genes.
  • the plasma free nucleic acid methylation detection reagent comprises an internal reference gene.
  • the internal reference gene is a B2M gene, and a B2M fragment without a GpG site is selected as the target sequence.
  • the plasma free nucleic acid methylation detection reagent is a plasma free DNA methylation detection reagent.
  • the methylated sequence segment detected by the F12 gene methylation detection reagent comprises SEQ ID NO:44.
  • the F12 gene methylation detection reagent comprises forward primers selected from SEQ ID NOs: 13-15, reverse primers selected from SEQ ID NOs: 16-18, and/or selected from Probes of SEQ ID NOs: 31-33.
  • the methylated sequence segment detected by the CDKL2 gene methylation detection reagent comprises SEQ ID NO:45.
  • the CDKL2 gene methylation detection reagent comprises forward primers selected from SEQ ID NOs: 19-21, reverse primers selected from SEQ ID NOs: 22-24, and/or selected from Probes of SEQ ID NOs: 34-36.
  • the methylated sequence segment detected by the SCARF2 gene methylation detection reagent comprises SEQ ID NO:46.
  • the SCARF2 gene methylation detection reagent comprises forward primers selected from SEQ ID NOs: 25-27, reverse primers selected from SEQ ID NOs: 28-30, and/or selected from Probes of SEQ ID NOs: 37-39.
  • the kit comprises AFP protein detection reagents.
  • the liver cancer comprises early stage liver cancer.
  • liver cancer is staged using the Barcelona Clinic Liver Cancer (BCLC) system, see Table A below.
  • the early stage liver cancer comprises Barcelona Stage A.
  • the early stage liver cancer includes stages 0 and A of the Barcelona stage.
  • the liver cancer staging scheme adopts China liver cancer staging (CNLC), including: CNLC stage Ia, Ib, IIa, IIb, IIIa, IIIb, IV, as follows :
  • CNLC stage Ia Physical activity status (PS) score of 0-2, Child-Pugh A/B liver function, single tumor, diameter ⁇ 5cm, no vascular invasion and extrahepatic metastasis.
  • PS Physical activity status
  • CNLC stage Ib PS 0-2 points, Child-Pugh A/B liver function, single tumor, diameter > 5 cm, or 2 to 3 tumors, maximum diameter ⁇ 3 cm, no vascular invasion and extrahepatic metastasis.
  • CNLC stage IIa PS 0-2 points, Child-Pugh A/B liver function, 2-3 tumors, maximum diameter >3cm, no vascular invasion and extrahepatic metastasis.
  • CNLC stage IIb PS 0-2, Child-Pugh A/B liver function, ⁇ 4 tumors, regardless of tumor diameter, no vascular invasion or extrahepatic metastasis.
  • CNLC stage IIIa PS 0-2 points, Child-Pugh A/B liver function, regardless of tumor status, vascular invasion without extrahepatic metastasis.
  • CNLC stage IIIb PS 0-2 points, Child-Pugh A/B liver function, regardless of tumor status, vascular invasion, or extrahepatic metastasis.
  • CNLC stage IV PS 3-4, or Child-Pugh C grade of liver function, regardless of tumor status, vascular invasion, or extrahepatic metastasis.
  • the early stage liver cancer comprises stage Ia of the Chinese Liver Cancer Stage (CNLC). In certain embodiments, the early stage liver cancer comprises stage Ib of the Chinese Liver Cancer Stage (CNLC). In certain embodiments, the early-stage liver cancer includes stages Ia and Ib of the Chinese Liver Cancer Stage (CNLC).
  • the kit comprises free nucleic acid extraction reagents.
  • the free nucleic acid extraction reagent extracts and purifies plasma free nucleic acid by magnetic bead method.
  • the plasma-free nucleic acid is plasma-free DNA.
  • the kits comprise DNA sulfite conversion reagents.
  • the DNA sulfite conversion reagent is used for sulfite conversion of plasma cfDNA for subsequent methylation marker gene detection.
  • the kit can be used for the detection of liver cancer point mutations, methylation markers and AFP protein markers.
  • the present invention provides a method for detecting liver cancer, characterized in that the method comprises one or more of the following a)-c) three types of marker detection:
  • the methods provided by the present invention comprise plasma free nucleic acid point mutation marker detection and plasma free nucleic acid methylation marker detection. In certain embodiments, the methods provided by the present invention comprise plasma free nucleic acid point mutation marker detection and protein marker detection. In certain embodiments, the methods provided by the present invention comprise plasma free nucleic acid methylation marker detection and protein marker detection. In certain embodiments, the methods provided by the present invention comprise plasma free nucleic acid point mutation marker detection, plasma free nucleic acid methylation marker detection, and protein marker detection.
  • the plasma free nucleic acid is plasma free DNA (cfDNA). In certain embodiments, the plasma free nucleic acid is plasma free RNA.
  • the liver cancer is early stage liver cancer.
  • the plasma free nucleic acid point mutation marker detection comprises TP53 gene point mutation detection.
  • the TP53 gene point mutation marker comprises a C-A mutation corresponding to nucleotide position 88 of SEQ ID NO:43.
  • the plasma free nucleic acid point mutation marker detection is a plasma free DNA (cfDNA) point mutation marker detection.
  • the gene methylation signature detected by the plasma free nucleic acid methylation signature comprises the methylation signature of one or more genes selected from the group consisting of F12, CDKL2, and SCARF2. In certain embodiments, the gene methylation signature detected by the plasma free nucleic acid methylation signature comprises the methylation signature of the F12 gene. In certain embodiments, the gene methylation signature detected by the plasma free nucleic acid methylation signature comprises the methylation signature gene of CDKL2. In certain embodiments, the gene methylation signature detected by the plasma free nucleic acid methylation signature comprises the methylation signature of the SCARF2 gene.
  • the gene methylation signature detected by the plasma free nucleic acid methylation signature comprises the methylation signatures of two genes, F12 and CDKL2. In certain embodiments, the gene methylation signature detected by the plasma free nucleic acid methylation signature comprises the methylation signatures of two genes, F12 and SCARF2. In certain embodiments, the gene methylation signature detected by the plasma free nucleic acid methylation signature comprises the methylation signatures of both CDKL2 and SCARF2 genes. In certain embodiments, the gene methylation signatures detected by plasma free nucleic acid methylation signatures comprise methylation signatures of three genes, F12, CDKL2 and SCARF2. In certain embodiments, the plasma free nucleic acid methylation marker is a plasma free DNA (cfDNA) methylation marker.
  • cfDNA plasma free DNA
  • the method provided by the present invention comprises the following steps:
  • Part of the plasma free nucleic acid sample is used for detection of plasma free nucleic acid mutation marker detection.
  • the point mutation marker in step b) of the method is a TP53 gene mutation marker.
  • step b) of the method comprises using quantitative PCR method to obtain the Ct value corresponding to the gene mutation marker.
  • the gene methylation markers in step c) of the method comprise one or more selected from the group consisting of F12, CDKL2 and SCARF2 gene methylation markers.
  • step c) of the method comprises using quantitative PCR method to detect and obtain the Ct value corresponding to the gene methylation marker.
  • step e) of the method comprises multiplying the Ct value of the gene mutation marker detection result, the Ct value of the gene methylation marker detection result and the AFP protein marker detection result by their respective coefficients, and using logistic regression formula to determine the result.
  • a, b, c, d, f, and M are constants, and the above constants are determined by the distribution of clinical test data.
  • the determination threshold is determined according to the distribution of clinical sample test data (P value), the test result is determined to be positive when P ⁇ the threshold, and the test result is determined to be negative when P ⁇ the threshold.
  • the threshold for P is set to 0.20-0.30. In some embodiments, the threshold for P is set to 0.30-0.40. In some embodiments, the threshold for P is set to 0.40-0.50. In some embodiments, the threshold for P is set to 0.50-0.60. In some embodiments, the threshold for P is set to 0.60-0.70. In some embodiments, the threshold for P is set to 0.70-0.80. In some embodiments, the threshold for P is set to 0.80-0.90. In some embodiments, the threshold for P is set to 0.90-1.0. In some embodiments, the threshold for P is set to 0.42-0.50. In some embodiments, the threshold for P is set to 0.44-0.48.
  • the threshold for P is set to 0.44-0.45. In some embodiments, the threshold for P is set to 0.45-0.46. In some embodiments, the threshold for P is set to 0.46-0.47. In some embodiments, the threshold for P is set to 0.47-0.48.
  • the method sets a single marker positive threshold for the Ct TP53 and Ct F12 and AFP triple markers by comparing the data distribution of each of the markers in true negative and false positive samples.
  • a Ct TP53 ⁇ 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 is judged positive. In certain embodiments, it is determined to be positive when Ct TP53 ⁇ 26. In certain embodiments, a positive is determined when Ct F12 ⁇ 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35. In certain embodiments, it is determined to be positive when Ct F12 ⁇ 32.
  • AFP when AFP>50ng/ml, 100ng/ml, 150ng/ml, 200ng/ml, 250ng/ml, 300ng/ml, 350ng/ml, 400ng/ml, 450ng/ml, 500ng/ml, 550ng/ml, 600ng/ml, 650ng/ml, or 700ng/ml, it was judged as positive. In certain embodiments, AFP is judged positive when >400ng/ml. In certain embodiments, it is determined to be positive when Ct TP53 ⁇ 26 or Ct F12 ⁇ 32 or AFP > 400 ng/ml.
  • the present invention adopts the following technical solutions:
  • sequence comprising the mutation site of interest in the TP53 gene is as follows:
  • C indicates that the nucleotide corresponding to this site has CA mutation in the DNA of some liver cancer populations.
  • the methylation sequence contained in the F12 gene is as follows:
  • mCG indicates that the C of the CpG island can or has undergone methylation modification.
  • the CDKL2 gene comprises the following methylation sequences:
  • mCG indicates that the C of the CpG island can or has undergone methylation modification.
  • the SCARF2 gene comprises the following methylation sequences:
  • mCG indicates that the C of the CpG island can or has undergone methylation modification.
  • the reagent for detecting TP53 gene mutation comprises 1-3 mM MgCl 2 , 0.1-0.5 mM dNTP, 1-3 U/reaction Taq enzyme, 10x Taq Buffer, 0.1-0.6 ⁇ M point mutation Detection primer, 0.05-0.3 ⁇ M point mutation detection probe, 0.1-0.6 ⁇ M point mutation internal reference detection primer, 0.05-0.3 ⁇ M point mutation internal reference detection probe.
  • reagents for detecting methylation of F12, CDKL2, SCARF2 genes comprise 1-3 mM MgCl 2 , 0.1-0.5 mM dNTPs, 1-3 U/reaction Taq enzyme, 10x Taq Buffer, 0.1 -0.6 ⁇ M methylation detection primer, 0.05-0.3 ⁇ M methylation detection probe, 0.1-0.6 ⁇ M methylation internal reference detection primer, 0.05-0.3 ⁇ M methylation internal reference detection probe.
  • the above-mentioned primers for detecting point mutation markers include TP53 forward primer and/or TP53 reverse primer.
  • the TP53 forward primer comprises one or more of the following sequences:
  • SEQ ID NO. 2 TTCCTGCATGGGCGGCATG
  • the TP53 reverse primer comprises one or more of the following sequences:
  • SEQ ID NO. 5 ACCTGGAGTTCTTCCAGTGTG
  • SEQ ID NO. 6 AGTGTGATGATGGTGAGGA
  • the above-mentioned probe for detecting point mutation markers is a TP53 probe, comprising one or more of the following sequences:
  • SEQ ID NO. 7 AACCGGAGTCCCATCCTC
  • SEQ ID NO. 8 CCGGAGGGCCCATCCTCAC
  • the above-mentioned primers for point mutation detection are EIF2C forward primers, and/or EIF2C reverse primers.
  • the EIF2C forward primer comprises one or more of the following sequences:
  • SEQ ID NO. 10 GTTCGGCTTTCACCAGTCTG
  • the EIF2C reverse primer comprises one or more of the following sequences:
  • the probe used for point mutation detection is an EIF2C probe, comprising one or more of the following sequences:
  • SEQ ID NO. 12 CGCCCTGCCATGTGGAAGAT
  • the above-mentioned primers for detecting methylation markers include: F12 forward primer, F12 reverse primer, CDKL2 forward primer, CDKL2 reverse primer, SCARF2 forward primer, and/or SCARF2 reverse primer primers.
  • the F12 forward primer comprises one or more of the following sequences:
  • SEQ ID NO. 13 GATAGGAGCGCGTAGTTGTC
  • SEQ ID NO. 14 TATCGGTTGAACGTAAGGCGA
  • SEQ ID NO. 15 CGTTTGGTAGGTATATCGGTTG
  • the F12 reverse primer comprises one or more of the following sequences:
  • SEQ ID NO. 16 TCCTCTCCGCCCGAATTAACT
  • SEQ ID NO. 17 GAATTAACTCTATTACGCCTTCAAA
  • SEQ ID NO. 18 CGCCTTCAAAAAAATACGAACGAC
  • the CDKL2 forward primer comprises one or more of the following sequences:
  • SEQ ID NO. 21 GTTTCGGTTCGTCGTAAGTTTTATA
  • the CDKL2 reverse primer comprises one or more of the following sequences:
  • SEQ ID NO. 22 CATACGACCCGTAATCCTCCTAA
  • SEQ ID NO. 23 GACAACATACGACCCGTAATCC
  • SEQ ID NO. 24 ACAAAATAAAATCGTTACCTTAACG
  • the SCARF2 forward primer comprises one or more of the following sequences:
  • SEQ ID NO. 25 GCGTTTCGACGCGATTTAGTT
  • SEQ ID NO. 26 TTCGACGCGATTTAGTTCGGA
  • SEQ ID NO. 27 TGGATTCGGTTTGTTTCGGGTT
  • the SCARF2 reverse primer comprises one or more of the following sequences:
  • SEQ ID NO. 28 CGAATTAAAAAACGCCGCAC
  • SEQ ID NO. 29 AAACGCCGCACGAACTAACAA
  • SEQ ID NO. 30 AAAACGACCGCCGAACTCC
  • the above probes for detecting methylation markers are F12 probes, CDKL2 probes and/or SCARF2 probes, respectively.
  • the F12 probe comprises one or more of the following sequences:
  • SEQ ID NO. 31 ATTACGCCTTCAAAAAAATACGAAC
  • SEQ ID NO. 32 ACGAACGACAACTACGCGCTCCT
  • SEQ ID NO. 33 TACGCGCTCCTATCGCCTTACGTT
  • the CDKL2 probe comprises one or more of the following sequences:
  • SEQ ID NO. 34 AACTTACGACGAACCGAAACCC
  • SEQ ID NO. 35 TATAAACTTACGACGAACCGAAACCCA
  • SEQ ID NO. 36 CAAAATAAAATCGTTACCTTAACG
  • the SCARF2 probe comprises one or more of the following sequences:
  • SEQ ID NO. 37 TAACAACCGCAAAAACGACCGC
  • SEQ ID NO. 38 CGCAAAAACGACCGCCGAACT
  • SEQ ID NO. 39 TAAATCGCGTCGAAACGCCTAAAC
  • the above-mentioned primers for methylation internal control detection are B2M forward primers and/or B2M reverse primers.
  • the B2M forward primer comprises one or more of the following sequences:
  • SEQ ID NO. 40 GTAGGTTTGGGTAATTTTAAATAGTGGA
  • the B2M reverse primer comprises one or more of the following sequences:
  • SEQ ID NO. 41 TTCTTTCAAAATATCATCCCCCAAT
  • the probe used for methylation internal reference detection is a B2M probe, comprising one or more of the following sequences:
  • SEQ ID NO. 42 TTCCTACAAATCTTCCCCCCAAACACC
  • the 5' end of any of the probes mentioned above carries a fluorophore, and the 3' end all carries a quenching group.
  • the fluorophore may be a FAM or JOE group.
  • the quenching group is a BHQ1 label.
  • the present invention also provides a method of using the above-mentioned early-stage liver cancer detection kit, which comprises the following steps:
  • the extracted cfDNA is divided into two parts, one is used to detect TP53 gene mutation, and the Ct value corresponding to the above markers is obtained by real-time fluorescence quantitative PCR method.
  • the other part was converted by sulfite conversion reagent, and then the methylation levels of F12, CDKL2, and SCARF2 genes were detected by real-time fluorescence quantitative PCR, and the Ct values corresponding to the above markers were obtained.
  • TP53 gene mutation detection results (Ct value), F12, CDKL2, SCARF2 gene methylation detection results (Ct value) and AFP protein detection results were multiplied by their respective coefficients, and the results were determined by logistic regression formula.
  • a, b, c, d, f, and M are constants, and the above constants are determined by the distribution of clinical test data.
  • the determination threshold is determined.
  • P ⁇ threshold the test result is judged as positive
  • P ⁇ threshold the test result is judged as negative
  • a single marker positive threshold was set for Ct TP53 and Ct F12 and AFP triple marker, that is, when Ct TP53 ⁇ 26 or Ct F12
  • ⁇ 32 or AFP>400ng/ml regardless of other marker detection data, the final result is judged to be positive.
  • Example 1 Obtaining markers for liver cancer detection (point mutation, methylation, protein)
  • the gene point mutation target used in the present invention is TP53 mutation.
  • the above mutation sites were obtained by the inventors in combination with the whole exome (WES) sequencing results of cancer tissue/peripheral blood samples from 20 liver cancer patients and database information.
  • WES whole exome
  • methylation markers the inventors combined various databases and the results of DNA methylation chips of liver cancer clinical samples to perform marker screening, and verified the methylation of candidate markers in liver cancer and adjacent tissues through methylation PCR and sequencing. level differences and identify methylation markers that will ultimately be used in liver cancer detection.
  • a total of 3 methylation marker genes were used for sample detection, namely F12 (Coagulation factor XII), CDKL2 (Cyclin dependent kinase like 2) and SCARF2 (Scavenger receptor class-F member 2).
  • the applicant has found through a large number of studies that point mutation markers and methylation markers are complementary to the marker of liver cancer, and the combined detection of the two markers can help to improve the detection sensitivity.
  • the present invention also includes a protein marker, alpha-fetoprotein (AFP).
  • AFP is an important indicator for the current clinical diagnosis of liver cancer, and about 60% of liver cancer patients will have elevated plasma/serum AFP levels.
  • China's "Guidelines for the Diagnosis and Treatment of Primary Liver Cancer" recommends that people at high risk of liver cancer should undergo AFP markers and liver ultrasound examinations every 6 months.
  • primers and probes were designed to detect the point mutation of TP53 gene and the methylation sequences of F12, CDKL2 and SCARF2.
  • the primer probe sequences corresponding to point mutation markers and methylation markers are shown in Table 1:
  • primer names with the last letter “F” indicate upstream primers
  • those with the last letter “R” indicate downstream primers
  • those with the last letter “P” indicate probes.
  • the fluorescent groups that label the above target probes are all FAM, and the quenching groups are all BHQ1.
  • the EIF2C gene was used as the internal reference, and the internal control primers and internal control probes were designed for the target sequence of EIF2C, and the corresponding performance verification was carried out, so that the non-template system (NTC) did not appear obvious amplification curve (no peak). , and the sample has an obvious amplification curve (peak).
  • NTC non-template system
  • EIF2C forward primer is SEQ ID NO.10: GTTCGGCTTCACCAGTCTG
  • the EIF2C reverse primer is SEQ ID NO. 11: CTCATAGCTCTCCCCACTC
  • the B2M gene was used as an internal reference, and fragments without GpG sites (all C converted to T) were selected as the target sequence.
  • NTC non-template system
  • B2M forward primer is SEQ ID NO.40: GTAGGTTTGGGTAATTTTAAATAGTGGA
  • B2M reverse primer is SEQ ID NO.41: TTCTTTCAAAATATCATCCCCCAAT
  • the fluorescent groups that label the above-mentioned internal reference probes are all JOE, and the quenching groups are all BHQ1.
  • the plasma samples of 251 subjects to be tested are used for the detection of liver cancer markers.
  • 30 samples from healthy individuals constituted the healthy group
  • 98 samples from patients with liver cancer constituted the liver cancer group.
  • the detection process of plasma samples is shown in Figure 1.
  • the joint inspection steps include: sample collection, cfDNA extraction and sulfite conversion, point mutation marker detection, methylation marker detection, AFP detection, logistic regression formula calculation and result judgment.
  • the elution volume of the last step was 50 ⁇ l, and 10 ⁇ l of the eluted product was transferred to a new tube for subsequent point mutation marker detection, and the remaining 40 ⁇ l was used for methylation marker detection.
  • the Ct value of TP53 mutant gene was determined by fluorescence quantitative PCR, and the detection target was the TP53R249S mutation site.
  • the internal reference gene is used for the quality control of DNA template and reaction system. If the internal reference EIF2C Ct value is greater than 18, the experimental result is invalid and should be re-tested.
  • the above three mutation sites and the internal reference gene were detected in the same reaction tube.
  • the reaction system is shown in Table 2, and the reaction program is shown in Table 3.
  • one group of primer-probe combinations was selected to detect plasma samples.
  • the primer-probe stock solution concentration was 50 ⁇ M.
  • the probe was diluted to 10 ⁇ M with TE buffer as the working solution; the upstream and downstream primers of EIF2C and TP53 genes were mixed in equal volumes, and the primer Mix was diluted with TE buffer to 10 ⁇ M, which was used as the working solution for PCR reaction. Finally Ct TP53 is obtained.
  • the methylation marker genes detected by the three reaction wells are:
  • Reaction well 1 F12
  • Reaction well 2 CDKL2
  • Reaction well 3 SCARF2.
  • each methylation marker gene among the three designed primer-probe combinations, one set of primer-probe combinations was selected to detect the primer-probes.
  • reaction well 1 the reaction system is shown in Table 4, and the reaction procedure is shown in Table 5.
  • the primer-probe stock solution concentration was 50 ⁇ M.
  • the probe is diluted to 10 ⁇ M with TE buffer to be the working solution; the upstream and downstream primers of the target gene F12 and the internal reference gene B2M are mixed in equal volumes, and then the primer Mix is diluted with TE buffer to 10 ⁇ M, which is used as the working solution for PCR detection.
  • the dilution mode and final concentration of primer probes corresponding to each marker gene are the same as those in reaction well 1, and other steps and PCR reaction conditions remain unchanged.
  • Ct F12 , Ct CDKL2 and Ct SCARF2 were obtained .
  • Plasma AFP concentration was quantitatively detected by Elisa method, and 50 ⁇ l of each plasma sample was taken for detection.
  • the minimum detection limit of plasma AFP in the present invention is 1 ng/ml.
  • a single-label positive threshold was set for the Ct TP53 and Ct F12 and AFP triple markers, that is, when Ct TP53 ⁇ 26 or Ct F12 ⁇ 32 or AFP > 400ng/ml, a single result can facilitate the detection Positive result.
  • N and Normal represent healthy people
  • CHB represents hepatitis B patients
  • CIR represents liver cirrhosis patients
  • HCC liver cancer patients.
  • ROC curve analysis was performed on the results of step 5 using SPSS 20.0 software.
  • the threshold corresponding to the point with the largest Youden index (sensitivity+specificity-1) on the ROC curve was 0.465.
  • the judgment threshold is determined according to the distribution of clinical sample test data (P value), and the test result is judged to be positive when P ⁇ threshold, and negative when P ⁇ threshold.
  • Sensitivity (true positive rate) true positive/(true positive+false negative)*100%. This refers to the percentage of patients with liver cancer who were correctly diagnosed with liver cancer by the test method.
  • the main detection population of the present invention that is, the domestic high-risk population of liver cancer mainly includes liver cirrhosis patients and hepatitis B patients
  • the formula algorithm and positive threshold were unchanged, and the sensitivity and specificity analysis were performed again.
  • FIG. 3 is a performance comparison of the model including the healthy human population and the model not including the healthy human population in this embodiment.
  • the results show that the specificity of the existing diagnostic model in the hepatitis B + cirrhosis population is about 90%, which is similar to the specificity of the control group including healthy people.
  • the detection kit and the corresponding combined detection method of DNA point mutation, methylation and protein in the present invention can achieve higher detection sensitivity than AFP single-label detection. , while ensuring that specificity is not reduced. In particular, it can detect more than half of the early-stage liver cancer patients with AFP ⁇ 20ng/ml, which provides the possibility for the screening of early-stage liver cancer patients and the timely prevention of postoperative liver cancer patients.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pathology (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • Hospice & Palliative Care (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Oncology (AREA)
  • Microbiology (AREA)
  • Genetics & Genomics (AREA)
  • General Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Food Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

提供用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法，包含血浆游离核酸点突变检测、血浆游离核酸甲基化检测、和/或蛋白检测，以及相应的用于肝癌检测的试剂盒。

Description

用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法 技术领域
本发明涉及一种用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法，以及相应的用于肝癌检测的试剂盒。
背景技术
国家癌症中心的统计结果显示，2015年中国肝细胞癌(简称肝癌)发病数约37.0万，占全球50％以上，在所有癌种中排列第四位；而肝癌死亡数约32.6万，居我国恶性肿瘤死亡第二位。对于肝癌患者而言，早期被发现的肝癌患者，其治疗和预后效果远优于进展期肝癌患者。我国肝癌患者的五年生存率只有14％，但早期肝癌患者的生存率超过50％，故对肝癌高风险发病人群进行早期筛查，能够有效降低肝癌的死亡率并减少医疗成本。2018年欧洲肝脏研究学会(EASL)和美国肝病研究学会(AASLD)发表的指南中均指出，成年的肝硬化患者(HBV感染、HCV感染、NAFLD及其它病因造成)和未患肝硬化的乙肝病毒携带者均应接受肝癌监测，故上述人群是肝癌早期筛查的主要对象。
中国卫健委发布的《原发性肝癌诊疗规范(2019年版)》建议，肝癌高危人群应至少每隔6个月进行一次肝脏超声检查和血清甲胎蛋白(AFP)水平检测。尽管超声与AFP联合检测能在一定程度上提升患者整体生存时长，但超声影像检查对医生操作经验和结果判读的准确性依赖较高；而常规甲胎蛋白(AFP)检测肝癌(正常参考范围0-20ng/ml)的灵敏度只有约60％，特异性只有约80％，容易出现较高的假阳性结果。若AFP的阳性判断值提升到400ng/ml，则灵敏度只有35％甚至更低。所以开发一些用于肝癌早筛早诊的新型标记依然非常有必要。
近年来，基于循环肿瘤DNA(ctDNA)突变及甲基化检测的液体活检技术逐渐应用于癌症辅助诊断和早筛。核酸标记与蛋白标记的联合检测不仅能有效提高癌症检出的灵敏性与特异性，而且有希望一次性检测多个癌种。另外，健康人血浆游离DNA(cfDNA)中，来源于肝组织的cfDNA含量约占总含量的1％，高于其它 消化器官来源的cfDNA；而肝病患者(乙肝、肝硬化、肝癌)血浆中的肝组织来源cfDNA含量占比更高。故血液样本适用于基于液体活检技术的肝癌诊断和筛查。
驱动基因突变可通过影响关键信号通路促进肝癌发生和进展，一些被HBV感染过的肝癌患者，其肝癌组织DNA和血液ctDNA中可检出驱动基因(如TP53)点突变。DNA甲基化作为一种表观遗传调节因子，可通过调节基因表达水平进行基因激活或沉默。在肝癌早期发生的过程中，一些抑癌基因出现高甲基化，这表明甲基化水平改变能够成为被检测到的癌症发生标记。血浆cfDNA中肝癌甲基化标记检测也成为一种新兴的，用于肝癌筛查、诊断和预后检测的无创手段。肝癌具有异质性，不同肝癌患者，甚至同一患者的数个肝部肿瘤之间存在DNA水平(点突变、异常甲基化)和蛋白水平差异。所以需要开发能够进一步提高肝癌的阳性检出率，灵敏度，和/或特异性的检测方法。
附图说明
图1显示的是血浆样本的一种检测流程。
图2是对251例样本以相关公式计算的P值分布。Normal代表健康人，CHB代表乙肝患者，CIR代表肝硬化患者，HCC代表肝癌患者。“**”代表小于显著性水平α＝0.01。
图3是预测模型对相关人群进行检测的灵敏度(sensitivity)和特异性(specificity)分析。Normal代表健康人，CHB代表乙肝患者，CIR代表肝硬化患者，HCC代表肝癌患者。
具体实施方式
本发明立足于临床需求，提供一种基于核酸及蛋白多靶点联合检测的肝癌诊断方法。通过点突变、甲基化、蛋白等不同类型标记的一种或多种进行联合检测，能有效提高肝癌的阳性检出率，灵敏度，和/或特异性。在某些实施例中，所检测的核酸标记为DNA标记。在某些实施例中，所检测的核酸标记为RNA标记。
在某些实施例中，本发明采用的诊断标记包含蛋白标记，以及基因突变、DNA甲基化、染色体拷贝数变异(CNV)、cfDNA片段化模式中的两种或多种。通过采 集待测者的外周血样本，联合蛋白检测结果和血浆cfDNA检测结果，用于早期肝癌的筛查和辅助诊断。在某些实施例中，本发明的多种标记联合检测手段及对应的试剂盒，通过检测血浆游离核酸(如游离DNA)的点突变、异常甲基化以及AFP蛋白水平，达到从肝癌高危人群(乙肝、肝硬化患者等)中进行肝癌筛查的目的。
在某些实施例中，本发明的优点包括，但不限于：
1)实现了基于血液的检测，具有无创性和非侵入性，无需进行手术和穿刺这类有创的组织活检。
2)本发明相较于常规的AFP血清学检测，具有更高的灵敏度和特异性，并且能检出AFP<20ng/ml的早期肝癌个体，进而实现“早诊早治”的目标。
3)相比起二代测序技术，实时荧光定量PCR检测周期更短，操作步骤更简便，经济成本更低。
在某些实施例中，本发明提供了一种用于肝癌检测的试剂盒。在某些实施例中，所述试剂盒包含选自血浆游离核酸点突变检测试剂、血浆游离核酸甲基化检测试剂、和/或蛋白检测试剂的一种或多种试剂。
在某些实施例中，所述血浆游离核酸为血浆游离DNA(cfDNA)。在某些实施例中，所述血浆游离核酸为血浆游离RNA。
在某些实施例中，所述试剂盒包含选自血浆游离核酸点突变检测试剂和血浆游离核酸甲基化检测试剂。在某些实施例中，所述试剂盒包含选自血浆游离核酸点突变检测试剂和蛋白检测试剂。在某些实施例中，所述试剂盒包含选自血浆游离核酸甲基化检测试剂和蛋白检测试剂。在某些实施例中，所述试剂盒包含选自血浆游离核酸点突变检测试剂、血浆游离核酸甲基化检测试剂、和蛋白检测试剂。
在某些实施例中，所述试剂盒包含血浆游离核酸点突变检测试剂。在某些实施例中，所述血浆游离核酸点突变检测试剂为TP53基因突变检测试剂。在某些实施例中，所述TP53基因突变的位点包含相当于SEQ ID NO:43的第88位核苷酸的C-A突变。在某些实施例中，所述TP53基因突变检测试剂包含选自SEQ ID NO:1-3的正向引物，选自SEQ ID NO:4-6的反向引物，和/或选自SEQ ID NO:7-9的探针。在某些实施例中，所述血浆游离核酸点突变检测试剂还包含一个内参基因。在某些实施例中，所述内参基因为EIF2C基因。在某些实施例中，所述血浆游离核酸点突变检测试剂为血浆游离DNA(cfDNA)点突变检测试剂。
在某些实施例中，所述试剂盒包含血浆游离核酸甲基化检测试剂。在某些实施例中，所述血浆游离核酸甲基化检测试剂为血浆游离DNA甲基化检测试剂。在某些实施例中，所述包含血浆游离核酸甲基化检测试剂检测的甲基化基因包含选自F12(Coagulation factor XII)、CDKL2(Cyclin dependent kinase like 2)和SCARF2(Scavenger receptor class-F member 2)的一种或多种基因。在某些实施例中，所述甲基化基因包含F12。在某些实施例中，所述甲基化基因包含CDKL2。在某些实施例中，所述甲基化基因包含SCARF2。在某些实施例中，所述甲基化基因包含F12和CDKL2。在某些实施例中，所述甲基化基因包含F12和SCARF2。在某些实施例中，所述甲基化基因包含CDKL2和SCARF2。在某些实施例中，所述甲基化基因包含F12、CDKL2和SCARF2。
在某些实施例中，在对甲基化标记基因进行检测之前，将血浆游离核酸进行亚硫酸盐转化。在某些实施例中，所述血浆游离核酸甲基化检测试剂包含一个内参基因。在某些实施例中，内参基因为B2M基因，并选取不含GpG位点的B2M片段作为目的序列。在某些实施例中，所述血浆游离核酸甲基化检测试剂为血浆游离DNA甲基化检测试剂。
在某些实施例中，所述F12基因甲基化检测试剂所检测的甲基化序列区段包含SEQ ID NO：44。在某些实施例中，所述F12基因甲基化检测试剂包含选自SEQ ID NO:13-15的正向引物，选自SEQ ID NO:16-18的反向引物，和/或选自SEQ ID NO:31-33的探针。
在某些实施例中，所述CDKL2基因甲基化检测试剂所检测的甲基化序列区段包含SEQ ID NO：45。在某些实施例中，所述CDKL2基因甲基化检测试剂包含选自SEQ ID NO:19-21的正向引物，选自SEQ ID NO:22-24的反向引物，和/或选自SEQ ID NO:34-36的探针。
在某些实施例中，所述SCARF2基因甲基化检测试剂所检测的甲基化序列区段包含SEQ ID NO：46。在某些实施例中，所述SCARF2基因甲基化检测试剂包含选自SEQ ID NO:25-27的正向引物，选自SEQ ID NO:28-30的反向引物，和/或选自SEQ ID NO:37-39的探针。
在某些实施例中，所述试剂盒包含AFP蛋白检测试剂。
在某些实施例中，所述肝癌包括早期肝癌。
在某些实施例中，肝癌分期采用巴塞罗那肝癌临床分期系统(Barcelona Clinic Liver Cancer(BCLC)，见下方表A)。在某些实施例中，所述早期肝癌包括巴塞罗那分期的A期。在某些实施例中，所述早期肝癌包括巴塞罗那分期的0期和A期。
表A：巴塞罗那肝癌临床分期
Figure PCTCN2021080864-appb-000001
在某些实施例中，肝癌分期采用中国肝癌的分期方案(China liver cancer staging，CNLC)，包括：CNLC Ⅰa期、Ⅰb期、Ⅱa期、Ⅱb期、Ⅲa期、Ⅲb期、Ⅳ期，具体如下：
CNLC Ⅰa期：体力活动状态(performance status，PS)评分0～2分，肝功能Child-Pugh A/B级，单个肿瘤、直径≤5cm，无血管侵犯和肝外转移。
CNLC Ⅰb期：PS 0～2分，肝功能Child-Pugh A/B级，单个肿瘤、直径＞5cm，或2～3个肿瘤、最大直径≤3cm，无血管侵犯和肝外转移。
CNLC Ⅱa期：PS 0～2分，肝功能Child-Pugh A/B级，2～3个肿瘤、最大直径＞3cm，无血管侵犯和肝外转移。
CNLC Ⅱb期：PS 0～2分，肝功能Child-Pugh A/B级，肿瘤数目≥4个、肿瘤直径不论，无血管侵犯和肝外转移。
CNLC Ⅲa期：PS 0～2分，肝功能Child-Pugh A/B级，肿瘤情况不论、有血管侵犯而无肝外转移。
CNLC Ⅲb期：PS 0～2分，肝功能Child-Pugh A/B级，肿瘤情况不论、血管侵犯不论、有肝外转移。
CNLC Ⅳ期：PS 3～4，或肝功能Child-Pugh C级，肿瘤情况不论、血管侵犯不论、肝外转移不论。
在某些实施例中，所述早期肝癌包括中国肝癌分期(CNLC)的Ⅰa期。在某些实施例中，所述早期肝癌包括中国肝癌分期(CNLC)的Ⅰb期。在某些实施例中，所述早期肝癌包括中国肝癌分期(CNLC)的Ⅰa期和Ⅰb期。
在某些实施例中，所述试剂盒包含游离核酸提取试剂。在某些实施例中，所述游离核酸提取试剂通过磁珠法提取并纯化血浆游离核酸。
在某些实施例中，所述血浆游离核酸为血浆游离DNA。在某些实施例中，所述试剂盒包含DNA亚硫酸盐转化试剂。在某些实施例中，所述DNA亚硫酸盐转化试剂用于将血浆cfDNA进行亚硫酸盐转化，用于后续的甲基化标记基因检测。
在某些实施例中，所述试剂盒可用于肝癌点突变、甲基化标记和AFP蛋白标记的检测。
在某些实施例中，本发明提供了一种用于检测肝癌的方法，其特征在于，所述方法包含以下a)-c)三类标记检测的一种或多种：
a)血浆游离核酸点突变标记检测；
b)血浆游离核酸甲基化标记检测；
c)蛋白标记检测。
在某些实施例中，本发明提供的方法包含血浆游离核酸点突变标记检测和血浆游离核酸甲基化标记检测。在某些实施例中，本发明提供的方法包含血浆游离核酸点突变标记检测和蛋白标记检测。在某些实施例中，本发明提供的方法包含血浆游离核酸甲基化标记检测和蛋白标记检测。在某些实施例中，本发明提供的方法包含血浆游离核酸点突变标记检测，血浆游离核酸甲基化标记检测，和蛋白标记检测。
在某些实施例中，所述血浆游离核酸为血浆游离DNA(cfDNA)。在某些实施例中，所述血浆游离核酸为血浆游离RNA。
在某些实施例中，所述肝癌是早期肝癌。
在某些实施例中，血浆游离核酸点突变标记检测包含TP53基因点突变检测。在某些实施例中，TP53基因点突变标记包含相当于SEQ ID NO:43的第88位核苷酸的C-A突变。在某些实施例中，所述血浆游离核酸点突变标记检测为血浆游离DNA(cfDNA)点突变标记检测。
在某些实施例中，血浆游离核酸甲基化标记检测的基因甲基化标记包含选自F12、CDKL2和SCARF2的一种或多种基因的甲基化标记。在某些实施例中，血浆游离核酸甲基化标记检测的基因甲基化标记包含F12基因的甲基化标记。在某些实施例中，血浆游离核酸甲基化标记检测的基因甲基化标记包含CDKL2的甲基化标记基因。在某些实施例中，血浆游离核酸甲基化标记检测的基因甲基化标记包含SCARF2基因的甲基化标记。在某些实施例中，血浆游离核酸甲基化标记检测的基因甲基化标记包含F12和CDKL2两种基因的甲基化标记。在某些实施例中，血浆游离核酸甲基化标记检测的基因甲基化标记包含F12和SCARF2两种基因的甲基化标记。在某些实施例中，血浆游离核酸甲基化标记检测的基因甲基化标记包含CDKL2和SCARF2两种基因的甲基化标记。在某些实施例中，血浆游离核酸甲基化标记检测的基因甲基化标记包含F12、CDKL2和SCARF2三种基因的甲基化标记。在某些实施例中，所述血浆游离核酸甲基化标记为血浆游离DNA(cfDNA)甲基化标记。
在某些实施例中，本发明提供的方法包含以下步骤：
a)提取并纯化所述血浆游离核酸的样本。
b)将部分所述血浆游离核酸的样本用于检测血浆游离核酸突变标记检测。
c)将部分所述血浆游离核酸的样本通过亚硫酸盐转化试剂进行转化，随后检测一个或多个基因标记的甲基化水平。
d)对样本中的AFP蛋白标记进行定量检测。
e)对血浆游离核酸突变标记，基因甲基化标记和AFP蛋白标记检测结果通过公式进行结果判定。
在某些实施例中，该方法步骤b)中的点突变标记为TP53基因突变标记。在某些实施例中，该方法步骤b)中包含利用定量PCR法获得基因突变标记对应的Ct值。在某些实施例中，该方法步骤c)中的基因甲基化标记包含选自F12、CDKL2和SCARF2基因甲基化标记的一个或多个。在某些实施例中，该方法步骤c)包含利用定量PCR法检测并获得基因甲基化标记对应的Ct值。在某些实施例中，该方法步骤e)中包含对基因突变标记检测结果的Ct值、基因甲基化标记检测结果的Ct值和AFP蛋白标记检测结果均乘以各自系数，并通过逻辑回归公式进行结果判定。
在某些实施例中，该方法所述逻辑回归计算公式为：P＝e K/(1+e K)，上式中，P为综合指数，其范围为0<P<1；e为自然常数；K＝a*Ct TP53+b*Ct F12+c*Ct CDKL2+d*Ct SCARF2+f*log 2(AFP)+M。其中a、b、c、d、f、M为常数，上述常数通过临床测试数据分布确定。根据临床样本检测数据(P值)的分布确定判定阈值，P≥所述阈值时检测结果判定为阳性，P<所述阈值时检测结果判定为阴性。
在某些实施例中，针对P的所述阈值设置为0.20-0.30。在某些实施例中，针对P的所述阈值设置为0.30-0.40。在某些实施例中，针对P的所述阈值设置为0.40-0.50。在某些实施例中，针对P的所述阈值设置为0.50-0.60。在某些实施例中，针对P的所述阈值设置为0.60-0.70。在某些实施例中，针对P的所述阈值设置为0.70-0.80。在某些实施例中，针对P的所述阈值设置为0.80-0.90。在某些实施例中，针对P的所述阈值设置为0.90-1.0。在某些实施例中，针对P的所述阈值设置为0.42-0.50。在某些实施例中，针对P的所述阈值设置为0.44-0.48。在某些实施例中，针对P的所述阈值设置为0.44-0.45。在某些实施例中，针对P的所述阈值设置为0.45-0.46。在某些实施例中，针对P的所述阈值设置 为0.46-0.47。在某些实施例中，针对P的所述阈值设置为0.47-0.48。
在某些实施例中，该方法通过比较各所述标记在真阴性与假阳性样本中的数据分布，对Ct TP53和Ct F12以及AFP三标记设置单标记阳性阈值。
在某些实施例中，当Ct TP53<22，23，24，25，26，27，28，29，30，31，32，33，34，或35时即判定为阳性。在某些实施例中，当Ct TP53<26时即判定为阳性。在某些实施例中，当Ct F12<22，23，24，25，26，27，28，29，30，31，32，33，34，或35时即判定为阳性。在某些实施例中，当Ct F12<32时即判定为阳性。在某些实施例中，当AFP>50ng/ml，100ng/ml，150ng/ml，200ng/ml，250ng/ml，300ng/ml，350ng/ml，400ng/ml，450ng/ml，500ng/ml，550ng/ml，600ng/ml，650ng/ml，或700ng/ml，时即判定为阳性。在某些实施例中，当AFP>400ng/ml时即判定为阳性。在某些实施例中，当Ct TP53<26或Ct F12<32或AFP>400ng/ml时，即判定为阳性。
在某些实施例中，本发明采用如下技术方案：
在某些实施例中，包含TP53基因目的突变位点的序列如下：
Figure PCTCN2021080864-appb-000002
C表示该位点对应的核苷酸在部分肝癌人群DNA中发生C-A突变。
在某些实施例中，F12基因包含的甲基化序列如下：
Figure PCTCN2021080864-appb-000003
mCG表示CpG岛的C可以发生或发生了甲基化修饰。
在某些实施例中，CDKL2基因包含的甲基化序列如下：
Figure PCTCN2021080864-appb-000004
Figure PCTCN2021080864-appb-000005
mCG表示CpG岛的C可以发生或发生了甲基化修饰。
在某些实施例中，SCARF2基因包含的甲基化序列如下：
Figure PCTCN2021080864-appb-000006
mCG表示CpG岛的C可以发生或发生了甲基化修饰。
在某些实施例中，用于检测TP53基因突变的试剂包含1-3mM的MgCl 2、0.1-0.5mM的dNTP、1-3U/反应的Taq酶、10x Taq Buffer、0.1-0.6μM的点突变检测引物、0.05-0.3μM的点突变检测探针、0.1-0.6μM的点突变内参检测引物、0.05-0.3μM的点突变内参检测探针。
在某些实施例中，用于检测F12、CDKL2、SCARF2基因甲基化的试剂包含1-3mM的MgCl 2、0.1-0.5mM的dNTP、1-3U/反应的Taq酶、10x Taq Buffer、0.1-0.6μM的甲基化检测引物、0.05-0.3μM的甲基化检测探针、0.1-0.6μM的甲基化内参检测引物、0.05-0.3μM的甲基化内参检测探针。
在某些实施例中，上述用于检测点突变标记的引物包括TP53正向引物和/或TP53反向引物。
在某些实施例中，TP53正向引物包含以下的一个或多个序列：
SEQ ID NO.1：GTGTAACAGTTCCTGCATGG
SEQ ID NO.2：TTCCTGCATGGGCGGCATG
SEQ ID NO.3：TAACAGTTCCTGCATGGGCG
在某些实施例中，TP53反向引物包含以下的一个或多个序列：
SEQ ID NO.4：CCAGTGTGATGATGGTGAG
SEQ ID NO.5：ACCTGGAGTCTTCCAGTGTG
SEQ ID NO.6：AGTGTGATGATGGTGAGGA
在某些实施例中，上述用于检测点突变标记的探针为TP53探针，包含以下的一个或多个序列：
SEQ ID NO.7：AACCGGAGTCCCATCCTC
SEQ ID NO.8：CCGGAGGCCCATCCTCAC
SEQ ID NO.9：TGAACCGGAGGCCCATCC
在某些实施例中，上述用于点突变内参检测的引物为EIF2C正向引物，和/或EIF2C反向引物。
在某些实施例中，EIF2C正向引物包含以下的一个或多个序列：
SEQ ID NO.10：GTTCGGCTTTCACCAGTCTG
在某些实施例中，EIF2C反向引物包含以下的一个或多个序列：
SEQ ID NO.11：CTCCATAGCTCTCCCCACTC
在某些实施例中，用于点突变内参检测的探针为EIF2C探针，包含以下的一个或多个序列：
SEQ ID NO.12：CGCCCTGCCATGTGGAAGAT
在某些实施例中，上述用于检测甲基化标记的引物包括：F12正向引物、F12反向引物、CDKL2正向引物、CDKL2反向引物、SCARF2正向引物，和/或SCARF2反向引物。
在某些实施例中，F12正向引物包含以下的一个或多个序列：
SEQ ID NO.13：GATAGGAGCGCGTAGTTGTC
SEQ ID NO.14：TATCGGTTGAACGTAAGGCGA
SEQ ID NO.15：CGTTTGGTAGGTATATCGGTTG
在某些实施例中，F12反向引物包含以下的一个或多个序列：
SEQ ID NO.16：TCCTCTCCGCCCGAATTAACT
SEQ ID NO.17：GAATTAACTCTATTACGCCTTCAAA
SEQ ID NO.18：CGCCTTCAAAAAAATACGAACGAC
在某些实施例中，CDKL2正向引物包含以下的一个或多个序列：
SEQ ID NO.19：GAAGTAGGTAGGGAGGTAGGT
SEQ ID NO.20：AGGGCGAAGTAGGTAGGGAG
SEQ ID NO.21：GTTTCGGTTCGTCGTAAGTTTATA
在某些实施例中，CDKL2反向引物包含以下的一个或多个序列：
SEQ ID NO.22：CATACGACCCGTAATCCTCCTAA
SEQ ID NO.23：GACAACATACGACCCGTAATCC
SEQ ID NO.24：ACAAAATAAAATCGTTACCTTAACG
在某些实施例中，SCARF2正向引物包含以下的一个或多个序列：
SEQ ID NO.25：GCGTTTCGACGCGATTTAGTT
SEQ ID NO.26：TTCGACGCGATTTAGTTCGGA
SEQ ID NO.27：TGGATTCGGTTTGTTTCGGGTT
在某些实施例中，SCARF2反向引物包含以下的一个或多个序列：
SEQ ID NO.28：CGAATTAAAAACGCCGCAC
SEQ ID NO.29：AAACGCCGCACGAACTAACAA
SEQ ID NO.30：AAAACGACCGCCGAACTCC
在某些实施例中，上述用于检测甲基化标记的探针分别为F12探针、CDKL2探针和/或SCARF2探针。
在某些实施例中，F12探针包含以下的一个或多个序列：
SEQ ID NO.31：ATTACGCCTTCAAAAAAATACGAAC
SEQ ID NO.32：ACGAACGACAACTACGCGCTCCT
SEQ ID NO.33：TACGCGCTCCTATCGCCTTACGTT
在某些实施例中，CDKL2探针包含以下的一个或多个序列：
SEQ ID NO.34：AACTTACGACGAACCGAAACCC
SEQ ID NO.35：TATAAACTTACGACGAACCGAAACCCA
SEQ ID NO.36：CAAAATAAAATCGTTACCTTAACG
在某些实施例中，SCARF2探针包含以下的一个或多个序列：
SEQ ID NO.37：TAACAACCGCAAAAACGACCGC
SEQ ID NO.38：CGCAAAAACGACCGCCGAACT
SEQ ID NO.39：TAAATCGCGTCGAAACGCCTAAAC
在某些实施例中，上述用于甲基化内参检测的引物为B2M正向引物和/或B2M反向引物。
在某些实施例中，B2M正向引物包含以下的一个或多个序列：
SEQ ID NO.40：GTAGGTTTGGGTAATTTTAAATAGTGGA
在某些实施例中，B2M反向引物包含以下的一个或多个序列：
SEQ ID NO.41：TTCTTTCAAAATATCATCCCCCAAT
在某些实施例中，用于甲基化内参检测的探针为B2M探针，包含以下的一个或多个序列：
SEQ ID NO.42：TTCCTACAAATCTTCCCCCAAACACC
在某些实施例中，上述任一所述探针5’端均携带荧光基团，3’端均携带淬灭基团。在某些实施例中，所述荧光基团可为FAM或JOE基团。在某些实施例中，所述淬灭基团为BHQ1标记。
在某些实施例中，本发明还提供一种上述早期肝癌检测试剂盒的使用方法，所述使用方法包括以下步骤：
1、采用游离核酸提取试剂，通过磁珠法提取并纯化血浆cfDNA。
2、提取后的cfDNA分为两份，一份用于检测TP53基因突变，利用实时荧光定量PCR法获得上述标记对应的Ct值。
3、另一份通过亚硫酸盐转化试剂进行转化，随后利用实时荧光定量PCR法检测F12、CDKL2、SCARF2基因甲基化水平，并获得上述标记对应的Ct值。
4、血浆AFP水平检测，利用Elisa方法对样本中的AFP蛋白进行定量检测。
5、TP53基因突变检测结果(Ct值)、F12、CDKL2、SCARF2基因甲基化检测结果(Ct值)和AFP蛋白检测结果均乘以各自系数，并通过逻辑回归公式进行结果判定。
所述逻辑回归计算公式为：P＝e K/(1+e K)。
上式中，P为综合指数，其范围为0<P<1；e为自然常数；K＝a*Ct TP53+b*Ct F12+c*Ct CDKL2+d*Ct SCARF2+f*log 2(AFP)+M。其中a、b、c、d、f、M为常数，上述常数通过临床测试数据分布确定。
根据临床样本检测数据(P值)的分布确定判定阈值，P≥阈值时检测结果判定为阳性，P<阈值时检测结果判定为阴性。另外，为提高公式的检测灵敏度，通过比较各标记在真阴性与假阳性样本中的数据分布，对Ct TP53和Ct F12以及AFP三标记设置单标记阳性阈值，即当Ct TP53<26或Ct F12<32或AFP>400ng/ml 时，无论其它标记检测数据如何，最终结果均判定为阳性。
具体实施例
以下结合附图和具体实施例对本发明作具体的介绍，所描述的实施例便于更好地理解本发明，但并不限定本发明。
实施例1-获得用于肝癌检测的标记(点突变、甲基化、蛋白)
本发明中使用的基因点突变靶标为TP53突变。上述突变位点由发明人结合20例肝癌患者癌组织/外周血样本全外显子(WES)测序结果，以及数据库信息而获得。对于甲基化标记，发明人结合多种数据库以及肝癌临床样本DNA甲基化芯片结果，进行标记初筛，并通过甲基化PCR和测序，验证候选标记在肝癌和癌旁组织中甲基化水平的差异，并确定最终用于肝癌检测的甲基化标记。用于样本检测的甲基化标记基因共3个，分别为F12(Coagulation factor XII)、CDKL2(Cyclin dependent kinase like 2)和SCARF2(Scavenger receptor class-F member 2)。
申请人通过大量研究发现，点突变标记和甲基化标记对肝癌的标示具有互补性，两种标记联合检测可有助于提升检测灵敏度。本发明还包含一种蛋白标记，即甲胎蛋白(AFP)。AFP是当前临床诊断肝癌的重要指标，约60％的肝癌患者会出现血浆/血清AFP含量上升。2020年中国《原发性肝癌诊疗指南》推荐肝癌高危人群每6个月进行一次AFP标志物和肝脏超声检查。
实施例2-DNA标记对应的引物探针
根据前期测序结果，设计检测TP53基因点突变以及F12、CDKL2、SCARF2甲基化序列的引物和探针。点突变标记和甲基化标记对应的引物探针序列如表1所示：
表1
Figure PCTCN2021080864-appb-000007
Figure PCTCN2021080864-appb-000008
Figure PCTCN2021080864-appb-000009
表1中，引物名称最后一个字母为“F”的表示上游引物，最后一个字母为“R”的表示下游引物，最后一个字母为“P”的表示探针。标记上述靶标探针的荧光基团均为FAM，淬灭基团均为BHQ1。
对于点突变标记检测，用EIF2C基因作为内参，针对EIF2C目的序列设计内控引物和内控探针，并进行相应的性能验证，使非模板体系(NTC)不出现明显的扩增曲线(不起峰)，且样本出现明显扩增曲线(起峰)。
EIF2C正向引物为SEQ ID NO.10：GTTCGGCTTTCACCAGTCTG
EIF2C反向引物为SEQ ID NO.11：CTCCATAGCTCTCCCCACTC
EIF2C探针核苷酸序列SEQ ID NO.12：CGCCCTGCCATGTGGAAGAT
对于甲基化标记检测，用B2M基因作为内参，并选取不含GpG位点的片段(C全部转换成T)作为目的序列。针对B2M目的序列设计内控引物和内控探针，并进行相应的性能验证，使非模板体系(NTC)不出现明显的扩增曲线(不起峰)，且样本出现明显扩增曲线(起峰)。
B2M正向引物为SEQ ID NO.40：GTAGGTTTGGGTAATTTTAAATAGTGGA
B2M反向引物为SEQ ID NO.41：TTCTTTCAAAATATCATCCCCCAAT
B2M探针核苷酸序列SEQ ID NO.42：TTCCTACAAATCTTCCCCCAAACACC
标记上述内参探针的荧光基团均为JOE，淬灭基团均为BHQ1。
实施例3-采用DNA标记和蛋白标记进行肝癌联检
本发明的一个实施例中，251例待测者的血浆样本用于肝癌标记检测。其中30例健康人样本组成健康组，62例乙肝患者和61例肝硬化患者样本组成高危组，98例肝癌患者样本组成肝癌组。
血浆样本的检测流程如图1所示。其联检步骤包括：样本收集、cfDNA的提 取与亚硫酸盐转化、点突变标记的检测、甲基化标记的检测、AFP检测、逻辑回归公式计算及结果判定。
下面结合实施例对本发明做进一步说明。
1、血浆cfDNA提取、分装和转化
取2ml待测者血浆，利用游离DNA提取试剂盒分离并纯化出血浆样本的cfDNA。最后一步的洗脱体积为50μl，取10μl洗脱产物至新管，用于之后的点突变标记检测，其余40μl用于甲基化标记检测。
2、点突变标记的PCR检测
利用荧光定量PCR法测定TP53突变基因Ct值，检测靶点为TP53R249S突变位点。而内参基因用于DNA模板及反应体系的质量控制，若内参EIF2C Ct值大于18，则实验结果无效，应重新检测。
上述3个突变位点连同内参基因在同一个反应管中检测，反应体系如表2所示，反应程序如表3所示。在设计的3组TP53引物探针组合中，选取其中1组引物探针组合对血浆样本进行检测。引物探针母液浓度均为50μM。探针用TE缓冲液稀释至10μM后即为工作液；EIF2C、TP53基因各自的上下游引物等体积混合，引物Mix用TE缓冲液稀释至10μM后，即作为工作液用于PCR反应。最终得到Ct TP53。
表2
试剂与模板 体积(μl)
10x Taq Buffer 2.5
MgCl 2(50mM) 2
dNTP mix(10mM) 0.5
EIF2C引物Mix(10μM) 0.5
EIF2C探针(10μM) 0.25
TP53引物Mix(10μM) 0.75
TP53探针(10μM) 0.25
Taq酶 0.12
超纯水 13.13
cfDNA模板 5
表3
Figure PCTCN2021080864-appb-000010
3、甲基化标记的PCR检测
40μl cfDNA经亚硫酸盐转化和纯化后，以荧光定量PCR法对3个甲基化标记进行检测。转化后的cfDNA中，未甲基化的胞嘧啶(C)被转化成尿嘧啶(U)，而甲基化的胞嘧啶仍旧不变。3个甲基化标记分3个反应孔进行PCR反应，每个反应孔检测一个靶标基因和一个内参基因，这三个基因被不同荧光基团区分。最终获得各个甲基化标记基因的Ct值，而内参基因B2M用于DNA模板和反应体系的质量控制，若内参B2M Ct值大于35，则实验结果无效，应重新检测。
3个反应孔检测的甲基化标记基因分别是：
反应孔1——F12；反应孔2——CDKL2；反应孔3——SCARF2。
对于每个甲基化标记基因，在设计好的3组引物探针组合中，选取其中1组引物探针组合对引物探针进行检测。
以反应孔1为例，反应体系如表4所示，反应程序如表5所示。引物探针母液浓度均为50μM。探针用TE缓冲液稀释至10μM后即为工作液；靶标基因F12和内参基因B2M各自的上下游引物等体积混合，随后用TE缓冲液将引物Mix稀释至10μM，即作为工作液用于PCR检测。其余两个反应孔中，各标记基因对应的引物探针稀释方式和终浓度与反应孔1一致，其它步骤和PCR反应条件均不变。最终得到Ct F12、Ct CDKL2和Ct SCARF2。
表4
试剂与模板 体积(μl)
10x Taq Buffer 3
MgCl 2(50mM) 2.4
dNTP mix(10mM) 0.75
B2M引物Mix(10μM) 0.6
B2M探针(10μM) 0.3
F12/CDKL2/SCARF2引物Mix(10μM) 0.9
F12/CDKL2/SCARF2探针(10μM) 0.3
Taq酶 0.15
超纯水 11.6
转化后的cfDNA模板 10
表5
Figure PCTCN2021080864-appb-000011
4、蛋白标记的PCR检测
利用Elisa法对血浆AFP浓度进行定量检测，每例血浆样本取50μl进行检测。
本发明中的血浆AFP最低检测限为1ng/ml。
5、逻辑回归公式建立及P值计算
数据分析采用了R软件和SPSS 20.0软件(IBM)。在建立公式环节，4个核酸标记检测结果(Ct值)、AFP蛋白标记检测结果(转换为对数)与临床诊断结果(肝癌记为1，健康、乙肝、肝硬化均记为0)进行逻辑回归分析，并获得每个标记在公式中对应的系数。最终算出的综合指数(P值)综合了各项标记数据经回归系数相乘后的得分，并由逻辑回归公式而得出。另外，为提高公式的检测 灵敏度，对Ct TP53和Ct F12以及AFP三标记设置单标记阳性阈值，即当Ct TP53<26或Ct F12<32或AFP>400ng/ml时，单项结果即可促成检测阳性结果。
所述逻辑回归计算公式为：P＝e K/(1+e K)。
上式中，P为综合指数，其范围为0<P<1；e为自然常数；K＝a*Ct TP53+b*Ct F12+c*Ct CDKL2+d*Ct SCARF2+f*log 2(AFP)+M+X；a、b、c、d、f、M为临床常数；X为附加分数，若Ct TP53<26或Ct F12<32和AFP>400ng/ml三项条件中任何一项达成，X＝10，否则X＝0。
利用R软件计算并输出每例样本对应的P值，251例待测值的P值如表6和图2所示。
表6
Figure PCTCN2021080864-appb-000012
Figure PCTCN2021080864-appb-000013
Figure PCTCN2021080864-appb-000014
其中，N和Normal代表健康人，CHB代表乙肝患者，CIR代表肝硬化患者，HCC代表肝癌患者。
6、阈值的获得
利用SPSS 20.0软件进行对步骤5的结果进行ROC曲线分析。ROC曲线上Youden指数(灵敏度+特异性-1)最大的点对应的阈值为0.465。根据临床样本检测数据(P值)的分布确定判定阈值，P≥阈值时检测结果判定为阳性，P<阈值时检测结果判定为阴性。
7、临床试验结果对比
我们利用本发明的核酸及蛋白标记联合检测方法，和建立的逻辑回归算式及判定方法，对251例肝癌及非肝癌样本的阴阳性判定结果，与医院的临床诊断结果(影像学或病理报告)进行比较，得出训练集和验证集样本的灵敏度和特异性。结果如表7所示。
表7
Figure PCTCN2021080864-appb-000015
注：灵敏度(真阳性率)＝真阳性/(真阳性+假阴性)*100％。此处指肝癌患者被检测方法正确诊断出肝癌的百分比。
特异性(真阴性率)＝真阴性/(真阴性+假阳性)*100％。此处指非肝癌对照人群(肝硬化、乙肝患者和健康人)被检测方法正确诊断为未患肝癌的百分比。
为了验证本发明的有益效果，本实施例还进行了性能对比，以检测灵敏度和特异性为指标，以免疫化学发光法AFP单标记检测结果(Cutoff＝20ng/ml)为对照。对比结果如表8所示。
表8
  灵敏度 特异性
本发明 73.5％ 92.2％
AFP单标记 49.0％ 88.9％
我们将检测的肝癌患者样本数据根据AFP水平分成3组，并分别统计本发明在3组人群中的检测灵敏度和特异性，结果如表9所示。
表9
AFP水平(ng/ml) 灵敏度 特异性
<20 52.0％(26/50) 95.6％(130/136)
20-400 93.9％(31/33) 64.7％(11/17)
>400 100％(15/15)  
另外，由于本发明的主要检测人群，即国内肝癌高危人群主要包含肝硬化患者和乙肝患者，我们将251例样本中的乙肝患者和肝硬化患者样本设为对照组(剔除健康人检测结果)，公式算法和阳性阈值不变，重新进行灵敏度(sensitivity)和特异性分析(specificity)。图3是本实施例中包含健康人人群与不包含健康人人群的模型性能比较。结果表明，现有诊断模型在乙肝+肝硬化人群的特异性约90％，与包含健康人人群对照组的特异性相近。
显而易见的，本发明中检测试剂盒及对应的DNA点突变、甲基化和蛋白三种标记联合检测方法，作为一种无创液体活检方式，可以实现相较于AFP单标记检测更高的检测灵敏度，而保证特异性不降低。特别是能够检出过半数AFP<20ng/ml的早期肝癌患者，从而对早期肝癌患者的筛查和术后肝癌患者的及时预防提供了可能。
尽管在本申请中已经将权利要求阐述为特征的特定组合，但应该理解的是，本公开的范围还包括本文明确或隐含地公开的任何新颖特征或任何新颖特征组合或其任何概括，无论它是否涉及与任何权利要求中目前要求保护的相同的发明，并且无论它是否减轻了与本发明相同的技术问题中的任何一个或全部问题。申请人在此提供通知，在本申请或由此衍生的任何进一步申请的审查期间，新的权利要求可以被制定为这样的特征和/或特征的组合。
尽管已经示出和描述了一些实施例，但是本领域技术人员应该理解，在不脱离本发明的原理的情况下可以对这些实施例进行改变，本发明的范围在权利要求中限定。
本领域技术人员会理解，在不脱离本发明的全部范围和精神的情况下，可对本申请描述的部件、方法、步骤、结构、运动、配合进行修改(添加和/或去除)，本发明的范围和精神涵盖这样的修改以及其任何和全部等同物。

Claims (34)

  1. 一种用于肝癌检测的试剂盒，其特征在于，所述试剂盒包含选自血浆游离核酸点突变检测试剂、血浆游离核酸甲基化检测试剂、和/或蛋白检测试剂的一种或多种试剂。
  2. 根据权利要求1所述的试剂盒，其特征在于，所述试剂盒包含血浆游离核酸点突变检测试剂。
  3. 根据权利要求2所述的试剂盒，其特征在于，所述血浆游离核酸点突变检测试剂为TP53基因突变检测试剂。
  4. 根据权利要求3所述的试剂盒，其特征在于，所述TP53基因突变的位点包含相当于SEQ ID NO:43的第88位核苷酸的C-A突变。
  5. 根据权利要求3或4所述的试剂盒，其特征在于，所述TP53基因突变检测试剂包含选自SEQ ID NO:1-3的正向引物，选自SEQ ID NO:4-6的反向引物，和/或选自SEQ ID NO:7-9的探针。
  6. 根据权利要求2-5任一项所述的试剂盒，其特征在于，用EIF2C基因作为所述血浆游离核酸点突变检测试剂的内参。
  7. 根据权利要求1-6任一项所述的试剂盒，其特征在于，所述游离核酸点突变检测试剂为游离DNA(cfDNA)点突变检测试剂。
  8. 根据权利要求1-7任一项所述的试剂盒，其特征在于，包含血浆游离核酸甲基化检测试剂。
  9. 根据权利要求8所述的试剂盒，其特征在于，所述血浆游离核酸甲基化检测试剂检测的甲基化基因包含选自F12(Coagulation factor XII)、CDKL2(Cyclin dependent kinase like 2)和SCARF2(Scavenger receptor class-F member 2)的一种或多种基因。
  10. 根据权利要求9所述的试剂盒，其特征在于，所述甲基化基因包含F12、CDKL2和SCARF2。
  11. 根据权利要求9或10所述的试剂盒，其特征在于，在对甲基化标记基因进行检测之前，将血浆游离核酸进行亚硫酸盐转化。
  12. 根据权利要求9-11任一项所述的试剂盒，其特征在于，用B2M基因作为内 参，并选取不含GpG位点的B2M片段作为目的序列。
  13. 根据权利要求9-12任一项所述的试剂盒，其特征在于，所述F12基因甲基化检测试剂所检测的甲基化序列区段为SEQ ID NO：44。
  14. 根据权利要求9-13任一项所述的试剂盒，其特征在于，所述F12基因甲基化检测试剂包含选自SEQ ID NO:13-15的正向引物，选自SEQ ID NO:16-18的反向引物，和/或选自SEQ ID NO:31-33的探针。
  15. 根据权利要求9-14任一项所述的试剂盒，其特征在于，所述CDKL2基因甲基化检测试剂所检测的甲基化序列区段为SEQ ID NO：45。
  16. 根据权利要求9-15任一项所述的试剂盒，其特征在于，所述CDKL2基因甲基化检测试剂包含选自SEQ ID NO:19-21的正向引物，选自SEQ ID NO:22-24的反向引物，和/或选自SEQ ID NO:34-36的探针。
  17. 根据权利要求9-16任一项所述的试剂盒，其特征在于，所述SCARF2基因甲基化检测试剂所检测的甲基化序列区段为SEQ ID NO：46。
  18. 根据权利要求9-17任一项所述的试剂盒，其特征在于，所述SCARF2基因甲基化检测试剂包含选自SEQ ID NO:25-27的正向引物，选自SEQ ID NO:28-30的反向引物，和/或选自SEQ ID NO:37-39的探针。
  19. 根据权利要求8-18任一项所述的试剂盒，其特征在于，所述血浆游离核酸甲基化检测试剂为血浆游离DNA甲基化检测试剂。
  20. 根据权利要求1-19任一项所述的试剂盒，其特征在于，所述试剂盒包含AFP蛋白检测试剂。
  21. 根据权利要求1-20任一项所述的试剂盒，其特征在于，所述肝癌包括早期肝癌。
  22. 根据权利要求1-21任一项所述的试剂盒，其特征在于，还包含游离核酸提取试剂，所述游离核酸提取试剂通过磁珠法提取并纯化血浆游离核酸。
  23. 根据权利要求22所述的试剂盒，其特征在于，所述血浆游离核酸为血浆游离DNA。
  24. 一种检测肝癌的方法，其特征在于，所述方法包含以下a)-c)三类标记检测的一种或多种：
    a)血浆游离核酸点突变标记检测；b)血浆游离核酸甲基化标记检测；
    c)蛋白标记检测。
  25. 如权利要求24所述的方法，其特征在于，所述血浆游离核酸点突变标记检测包含TP53基因点突变检测。
  26. 如权利要求25所述的方法，其特征在于，所述TP53基因点突变标记包含相当于SEQ ID NO:43的第88位核苷酸的C-A突变。
  27. 如权利要求24-26任一项所述的方法，其特征在于，所述血浆游离核酸甲基化标记检测的基因甲基化标记包含选自F12、CDKL2和SCARF2的一种或多种基因的甲基化标记。
  28. 如权利要求27所述的方法，其特征在于，所述基因甲基化标记包含F12、CDKL2和SCARF2。
  29. 如权利要求24-28任一项所述的方法，其特征在于，所述血浆游离核酸为血浆游离DNA。
  30. 如权利要求24-29任一项所述的方法，其特征在于，所述肝癌是早期肝癌。
  31. 如权利要求24-30任一项所述的方法，其特征在于，所述方法包含以下步骤：
    a)提取并纯化所述血浆游离核酸的样本。
    b)将部分所述血浆游离核酸的样本用于检测TP53基因突变标记，利用定量PCR法获得上述标记对应的Ct值。
    c)将部分所述血浆游离核酸的样本通过亚硫酸盐转化试剂进行转化，随后利用定量PCR法检测F12、CDKL2和SCARF2基因标记的甲基化水平，并获得上述标记对应的Ct值。
    d)对样本中的AFP蛋白标记进行定量检测。
    e)对P53基因突变标记检测结果(Ct值)、F12、CDKL2、SCARF2基因甲基化标记检测结果(Ct值)和AFP蛋白标记检测结果均乘以各自系数，并通过逻辑回归公式进行结果判定。
  32. 如权力要求31所述的方法，其特征在于，所述逻辑回归计算公式为：P＝e K/(1+e K)，
    上式中，P为综合指数，其范围为0<P<1；e为自然常数；K＝a*Ct TP53+b*Ct F12+c*Ct CDKL2+d*Ct SCARF2+f*log 2(AFP)+M。其中a、b、c、d、f、M为常数， 上述常数通过临床测试数据分布确定。根据临床样本检测数据(P值)的分布确定判定阈值，P≥所述阈值时检测结果判定为阳性，P<所述阈值时检测结果判定为阴性。
  33. 如权力要求32所述的方法，其特征在于，通过比较各所述标记在真阴性与假阳性样本中的数据分布，对Ct TP53和Ct F12以及AFP三标记设置单标记阳性阈值，当Ct TP53<26或Ct F12<32或AFP>400ng/ml时，即判定为阳性。
  34. 如权力要求32或33所述的方法，其特征在于，所述阈值设置为0.4-0.55，优选的，所述阈值设置为0.42-0.5，更优选的，所述阈值设置为0.44-0.48。
PCT/CN2021/080864 2021-03-15 2021-03-15 用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法 WO2022193097A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2021/080864 WO2022193097A1 (zh) 2021-03-15 2021-03-15 用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法
PCT/CN2022/080695 WO2022194101A1 (zh) 2021-03-15 2022-03-14 用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2021/080864 WO2022193097A1 (zh) 2021-03-15 2021-03-15 用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法

Publications (1)

Publication Number Publication Date
WO2022193097A1 true WO2022193097A1 (zh) 2022-09-22

Family

ID=83321589

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/CN2021/080864 WO2022193097A1 (zh) 2021-03-15 2021-03-15 用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法
PCT/CN2022/080695 WO2022194101A1 (zh) 2021-03-15 2022-03-14 用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法

Family Applications After (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/080695 WO2022194101A1 (zh) 2021-03-15 2022-03-14 用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法

Country Status (1)

Country Link
WO (2) WO2022193097A1 (zh)

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130337449A1 (en) * 2010-12-13 2013-12-19 Samsung Life Public Welfare Foundation Marker for predicting gastric cancer prognosis and method for predicting gastric cancer prognosis using the same
CN106222267A (zh) * 2016-08-01 2016-12-14 博尔诚（北京）科技有限公司 检测肝癌的组合物及其用途
KR20170071724A (ko) * 2015-12-16 2017-06-26 연세대학교 산학협력단 간암 발생 특이적 유전자 발현에 관여하는 유전자 구조 내 cpg 섬의 dna 메틸화 변이를 이용한 간암의 예측 또는 진단 방법
CN110117652A (zh) * 2018-02-05 2019-08-13 箐健科技(天津)有限公司 肝癌早期诊断方法
CN110904231A (zh) * 2019-12-05 2020-03-24 圣湘生物科技股份有限公司 一种用于辅助诊断肝癌的试剂及其在制备试剂盒中的用途
CN110904225A (zh) * 2019-11-19 2020-03-24 中国医学科学院肿瘤医院 用于肝癌检测的组合标志物及其应用
WO2020140927A1 (en) * 2019-01-02 2020-07-09 Crownmab Biotech Inc. Cancer treatment using multi-targeted kinase inhibitor in combination of protein kinase biomarkers
CN111690740A (zh) * 2019-03-11 2020-09-22 中国医学科学院肿瘤医院 肝细胞癌早筛试剂盒及其制备方法和用途

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130337449A1 (en) * 2010-12-13 2013-12-19 Samsung Life Public Welfare Foundation Marker for predicting gastric cancer prognosis and method for predicting gastric cancer prognosis using the same
KR20170071724A (ko) * 2015-12-16 2017-06-26 연세대학교 산학협력단 간암 발생 특이적 유전자 발현에 관여하는 유전자 구조 내 cpg 섬의 dna 메틸화 변이를 이용한 간암의 예측 또는 진단 방법
CN106222267A (zh) * 2016-08-01 2016-12-14 博尔诚（北京）科技有限公司 检测肝癌的组合物及其用途
CN110117652A (zh) * 2018-02-05 2019-08-13 箐健科技(天津)有限公司 肝癌早期诊断方法
WO2020140927A1 (en) * 2019-01-02 2020-07-09 Crownmab Biotech Inc. Cancer treatment using multi-targeted kinase inhibitor in combination of protein kinase biomarkers
CN111690740A (zh) * 2019-03-11 2020-09-22 中国医学科学院肿瘤医院 肝细胞癌早筛试剂盒及其制备方法和用途
CN110904225A (zh) * 2019-11-19 2020-03-24 中国医学科学院肿瘤医院 用于肝癌检测的组合标志物及其应用
CN110904231A (zh) * 2019-12-05 2020-03-24 圣湘生物科技股份有限公司 一种用于辅助诊断肝癌的试剂及其在制备试剂盒中的用途

Also Published As

Publication number Publication date
WO2022194101A1 (zh) 2022-09-22

Similar Documents

Publication Publication Date Title
CN110904225B (zh) 用于肝癌检测的组合标志物及其应用
CN113604563B (zh) 一种肝癌诊断或辅助诊断的核酸组合、检测试剂盒及其应用
CN109929919B (zh) Dna甲基化检测方法及相关应用
CN111676287B (zh) 一种基因标志物组合及其应用
TWI730429B (zh) Hoxa7甲基化檢測試劑
CN110229908A (zh) 用于早期检测肺癌基因甲基化的引物、探针及试剂盒
CN114672568B (zh) 一种用于检测宫颈细胞基因甲基化的试剂盒
CN113846167B (zh) 一种原发性肝癌的分子标记物检测试剂盒、核酸组合及应用
CN111705130B (zh) 一种基因标志物组合及其应用
TW202028463A (zh) Hoxa7和hoxa9甲基化檢測試劑在製備肺癌診斷試劑中的用途
TWI815044B (zh) 肺癌檢測試劑及試劑盒
CN113215260A (zh) 一种gstp1，apc和rassf1在制备前列腺癌标志物中的应用及其试剂盒
TWI774992B (zh) Hoxa7甲基化檢測試劑在製備肺癌診斷試劑中的用途
CN117089614A (zh) 一种宫颈癌相关基因dna甲基化pcr-荧光探针法检测试剂盒
WO2022193097A1 (zh) 用于肝癌早筛的核酸及蛋白检测靶标组合及其联合检测方法
CN113337608B (zh) 用于肝癌早期诊断的组合标志物及其应用
CN113322318B (zh) Linc00485作为分子标志物在制备用于诊断和/或预后肝细胞癌的产品中的应用
WO2019095541A1 (zh) 一种诊断预示乳腺癌骨转移的组合物及诊断预示方法
CN115851959B (zh) 一种用于食管鳞状细胞癌及癌前病变的诊断或辅助诊断的试剂及检测试剂盒
CN114150065B (zh) 一种结直肠癌或癌前病变的标记物及其应用
CN116004821A (zh) 用于检测肠癌的标记物及其应用
WO2022253247A1 (zh) 一种肝癌诊断或辅助诊断的核酸组合、检测试剂盒及其应用
CN116179693A (zh) 检测目标区域的甲基化水平的试剂在制备妇科恶性肿瘤诊断产品中的应用
CN115725733A (zh) 一种znf135基因甲基化检测试剂及其应用
CN116516005A (zh) 一种用于检测头颈鳞癌的核酸产品、试剂盒及应用

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: 21930693

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21930693

Country of ref document: EP

Kind code of ref document: A1