WO2023040910A1 - Hepatitis c and hepatic cancer detection reagent, and application thereof in hepatitis c and hepatic cancer detection - Google Patents
Hepatitis c and hepatic cancer detection reagent, and application thereof in hepatitis c and hepatic cancer detection Download PDFInfo
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- 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
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2440/00—Post-translational modifications [PTMs] in chemical analysis of biological material
- G01N2440/38—Post-translational modifications [PTMs] in chemical analysis of biological material addition of carbohydrates, e.g. glycosylation, glycation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
- G01N2800/085—Liver diseases, e.g. portal hypertension, fibrosis, cirrhosis, bilirubin
Definitions
- the invention belongs to the technical field of biomedicine and relates to a detection method of hepatitis C liver cancer, in particular to a detection method of hepatitis C liver cancer based on the specific fingerprint of serum glycoprotein oligosaccharide chain detection (G-Test).
- G-Test serum glycoprotein oligosaccharide chain detection
- Hepatitis C virus referred to as hepatitis C for short, is a kind of viral hepatitis caused by hepatitis C virus (HCV) infection, which is mainly transmitted through blood transfusion, acupuncture, drug abuse and other ways.
- HCV hepatitis C virus
- the pathological changes of hepatitis C are very similar to those of hepatitis B, with liver cell necrosis and lymphocyte infiltration being the main ones.
- Fibrous tissue proliferation in the portal area can occur in chronic hepatitis, leading to chronic inflammation, necrosis and fibrosis of the liver, and some patients may develop liver cirrhosis or even hepatocellular carcinoma (HCC).
- Hepatitis C is closely related to the occurrence of liver cancer, and the transition between the two is a relatively long process, which is divided into four stages: acute infection - chronic infection - liver cirrhosis - liver cancer.
- the initial stage of HCV infection (2 to 12 weeks) is the acute stage, and the infected person may have no obvious symptoms. 1 to 3 weeks after infection with HCV, HCV RNA can be detected in peripheral blood, and only a few people can clear the virus by themselves and recover.
- hepatitis C antibody is the main indicator for the diagnosis of hepatitis C virus.
- the anti-HCV antibody appears slowly, usually 2 to 6 months after the onset, or even 1 year before turning positive, so it cannot be used as an early diagnosis method.
- ALT single alanine aminotransferase
- Liver function including serum ALT, aspartate aminotransferase (AST), total bilirubin, direct bilirubin, indirect bilirubin, albumin, globulin, cholinesterase , alkaline phosphatase, transpeptidase, etc.
- AST aspartate aminotransferase
- Hepatitis C virus antibody against HCV 3.
- liver imaging Abdominal ultrasonography of liver, gallbladder and spleen to check whether there is chronic damage to the liver. If necessary, perform abdominal enhanced computerized tomography (CT) or magnetic resonance imaging (MRI) examination to understand the degree of damage. 5.
- CT computerized tomography
- MRI magnetic resonance imaging
- Liver transient elastic wave scanning is a non-invasive examination that can be used to evaluate the degree of liver fibrosis in patients with chronic hepatitis C. Assessment of liver fibrosis in patients with hepatitis C is important for determining treatment options. 6.
- Liver biopsy is the gold standard for assessing liver inflammation grade and fibrosis stage.
- HCV-RNA in blood can be directly detected by polymerase chain reaction (PCR), which can be used for early diagnosis of HCV infection.
- HCV-RNA and anti-HCV antibody are positive or HCV-RNA positive alone can be diagnosed as hepatitis C virus.
- patients with chronic hepatitis C belong to the high-risk group of liver cancer.
- the high-risk group of liver cancer refers to those who are over 35 years old, have serological evidence of HBV or HCV infection, or have a history of chronic hepatitis.
- Alpha-fetoprotein (AFP) detection + liver ultrasound examination is used for early detection of liver cancer.
- AFP detection and liver ultrasonography it is easy to cause false negatives and delay the diagnosis. Therefore, there is an urgent need for methods with high sensitivity and high specificity for the early detection of hepatitis C liver cancer.
- Protein glycosylation is the most common post-translational modification of proteins. It is a process in which sugars are transferred to proteins and special amino acid residues on proteins to form glycosidic bonds under the action of glycosyltransferases. Most glycoproteins are secreted proteins, widely present in cell membranes, interstitial cells, plasma, and mucus. Some enzymes and hormones are glycoproteins. Glycoproteins have a variety of biological functions. Some glycoproteins such as trocollagen are structural proteins.
- glycoproteins are glycoproteins
- fiber Proproteins are glycoproteins
- Lectins have the ability to aggregate cells, and sugar chains can also stabilize peptide chains. Another important function of glycoprotein is to directly or indirectly participate in various recognition phenomena on the cell surface.
- sugar chains Due to the importance of sugar chains in glycoproteins for maintaining biological functions of the body, changes in sugar chains help to elucidate the molecular mechanisms of abnormal biobehaviors such as inflammation, tumor cell invasion and metastasis of surrounding tissues. At present, changes in N-glycan chains have been found in various tumors.
- Sugar chains are important bioinformatics molecules that play unique roles in many physiological and pathological processes.
- the sugar chain structure is very complex and has microscopic heterogeneity. Its analysis and structural elucidation have always been the bottleneck of glycobiology research.
- the analysis methods of sugar chain structure are developing rapidly, mainly including: (1) High performance liquid chromatography (HPLC): high resolution, fast detection speed, high repeatability, high performance liquid chromatography column can be used repeatedly, but column efficiency will decrease It becomes lower with the increase of the number of uses, and the mobile phase is toxic, and the operation of the equipment requires strictly trained professionals, and the equipment is relatively expensive, and the solvent needs to be strictly purified; (2) mass spectrometry (MS): mass spectrometry has high sensitivity, It can obtain a variety of structural information and is suitable for the analysis of mixtures.
- HPLC High performance liquid chromatography
- MS mass spectrometry
- capillary electrophoresis capillary electrophoresis is low in cost, high in column efficiency, high in sensitivity, fast in speed, and easy to inject. The amount is small and the operation is simple, but the repeatability is not high and the stability is not as good as HPLC.
- the G-Test detection method is based on the capillary microelectrophoresis technology (DSA-FACE) of the DNA analyzer. After the N-sugar chain of the glycoprotein in the prostatic fluid sample is fluorescently labeled, it is separated by capillary microelectrophoresis. The content of the N-oligosaccharide chain obtained by measuring the fluorescent signal is the fingerprint spectrum (G-Test spectrum for short).
- This detection technology has the advantages of high sensitivity, simple operation, trace volume (2 ⁇ L serum), high repeatability, good stability, high throughput (96-well plate) and other sugar chain analysis technologies, and is suitable for general laboratory departments. It is expected to be used in clinical promotion.
- the purpose of the present invention is to provide a detection reagent for hepatitis C liver cancer.
- the reagent measures the glycome profile in serum, quantifies the peak value, and performs statistical analysis, thereby providing a method for establishing a model of the serum glycome profile of hepatitis C liver cancer.
- a hepatitis C liver cancer detection reagent which is formed by mixing the following reagents:
- Reagent A prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;
- Reagent B It is prepared by mixing 0.01 ⁇ 10U/10 ⁇ L glucosamidase and 0.01 ⁇ 10U/10 ⁇ L sialidase, and the pH value of the mixed solution is 4 ⁇ 9;
- Reagent C Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM ⁇ 1M;
- Reagent D stop solution.
- the volume ratio of the reagent A, reagent B and reagent C is 2:2:1.
- a preparation method of hepatitis C liver cancer detection reagent comprising the following steps:
- Step 3 Separation and analysis of oligosaccharide chains
- the denaturation temperature in the preparation of the step 1 oligosaccharide is not lower than 75°C, and the incubation temperature is not lower than 25°C.
- the temperature of fluorescent labeling in the second step is 50-90°C.
- composition detects hepatitis C liver cancer through the ratio of (NGA2F+NA2F)/NA3.
- the invention provides a method for establishing a serum glycoprotein N-glycan group pattern model of hepatitis C liver cancer, and performs statistical analysis by measuring the specific fingerprint pattern of the serum glycoprotein oligosaccharide chain G-Test.
- Test samples Serum from patients with hepatitis C liver cancer caused by hepatitis C virus and normal controls.
- Reagent A prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;
- Reagent B It is prepared by mixing 0.01 ⁇ 10U/10 ⁇ L glucosamidase and 0.01 ⁇ 10U/10 ⁇ L sialidase, and the pH value of the mixed solution is 4 ⁇ 9;
- Reagent C Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM ⁇ 1M;
- Reagent D stop solution.
- Step 3 Separation and analysis of oligosaccharide chains
- the composition detects hepatitis C liver cancer through the ratio of (NGA2F+NA2F)/NA3.
- the method of the present invention adopts the G-Test detection method with high sensitivity, simple operation, only needs a small amount of sample, high repeatability, good stability and high throughput, and has established a significant difference between patients with hepatitis C liver cancer and normal controls.
- -Model of the glycome map In subsequent applications, the N-glycan profile of the serum to be tested is calculated using the profile model established by this method, which can detect whether the sample has hepatitis C liver cancer. Compared with the prior art, it has higher accuracy, and the AUC area of the ROC curve for the hepatitis C liver cancer detection model reaches 0.864.
- the N-glycan map model constructed based on the method of the present invention can allow many patients to receive routine and non-invasive testing, and help doctors and patients detect the occurrence and progression of liver cancer caused by hepatitis C virus in a timely manner, and it is expected to be widely used in clinical practice .
- Figure 1 is the serum glycoprotein N-glycan map of the normal control group and the hepatitis C liver cancer group; the abbreviations of the oligosaccharides in the map are respectively expressed as: NGA2F, galactose missing two antennae containing core fucose (Agalacto core- ⁇ -1 , 6-fucosylated biantennary); NA2F, Bigalacto core- ⁇ -1, 6-fucosylated biantennary); NA3, triantennary.
- Test samples Serum from patients with hepatitis C liver cancer caused by hepatitis C virus and normal controls.
- Reagent A prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;
- Reagent B It is prepared by mixing 0.01 ⁇ 10U/10 ⁇ L glucosamidase and 0.01 ⁇ 10U/10 ⁇ L sialidase, and the pH value of the mixed solution is 4 ⁇ 9;
- Reagent C Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM ⁇ 1M;
- Reagent D stop solution.
- Step 3 Separation and analysis of oligosaccharide chains
- G-Test detection technology was used to process the collected serum samples of 66 cases of hepatitis C liver cancer patients and normal control group, including 36 cases of serum samples of hepatitis C liver cancer patients and 30 cases of serum samples of normal control group. Statistical analysis was carried out on the N-glycan profile obtained from samples measured by G-Test detection technology.
- the serum N-glycan profile shows about 9 N-glycan peaks, and the sugar chains show different mobility due to different molecular sizes, that is, the Different peaks represent different oligosaccharide chains, and the measured peak heights represent the relative concentration of oligosaccharide chains.
- Figure 1A is the normal control group
- Figure 1B is the hepatitis C liver cancer group.
- the composition of the N-glycan profile detects hepatitis C liver cancer by the ratio of (NGA2F+NA2F)/NA3.
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Abstract
Provided are a hepatitis C and hepatic cancer detection reagent and a preparation method therefor. The detection reagent is made from mixing the following reagents, reagent A, which is prepared by adding SDS having a mass concentration of 0.5-5% in an ammonium bicarbonate solution having a concentration of 10 mM; reagent B, which is prepared by mixing 0.01-10 U / 10 μL glycosaminoacylase with 0.01-10 U/ 10 μL sialidase, the mixed solution having a pH value of 4-9; reagent C, which is prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration being 0.01 mM to 1 M; and reagent D, which is a stop solution; a glucome graph of a serum is determined by means of the detection reagent, a peak is quantified and statistical analysis is performed, and a method for constructing a hepatitis C and hepatic cancer serum glucome graph model is provided to detect hepatitis C and hepatic cancer.
Description
本发明属于生物医药技术领域，涉及一种丙肝肝癌的检测方法，具体涉及一种基于血清糖蛋白寡糖链检测(G-Test)特异指纹图谱的丙肝肝癌检测方法。The invention belongs to the technical field of biomedicine and relates to a detection method of hepatitis C liver cancer, in particular to a detection method of hepatitis C liver cancer based on the specific fingerprint of serum glycoprotein oligosaccharide chain detection (G-Test).
慢性丙型病毒性肝炎(Chronic Hepatitis C Virus-HCV)是导致肝硬化与肝癌的主要原因之一。丙型病毒性肝炎，简称为丙型肝炎是一种由丙型肝炎病毒(HCV)感染引起的病毒性肝炎，主要经输血、针刺、吸毒等途径传播。丙肝的病理改变与乙肝极为相似，以肝细胞坏死和淋巴细胞浸润为主。慢性肝炎可出现汇管区纤维组织增生，导致肝脏慢性炎症坏死和纤维化，部分患者可发展为肝硬化甚至肝细胞癌(HCC)。丙肝肝炎与肝癌的发生密切相关，两者间的转变是一个较漫长的过程，分为四个阶段：急性感染—慢性感染—肝硬化—肝癌。HCV感染初期(2～12周)为急性期，感染者可无明显症状。感染HCV后1～3周，在外周血可检测到HCV RNA，只有少数人可自行清除病毒而痊愈。慢性期50％～85％的患者会进入慢性炎症阶段，发展为慢性丙肝。HCV慢性感染者具有较高发生肝癌的风险。HCV慢性感染者发生肝癌的风险是普通人群的15～20倍。目前全球有1.3亿～2.1亿的HCV慢性感染者。10％～40％的慢性丙肝患者将进展为肝硬化，其中，又有1％～5％最终进展为肝癌。目前丙肝抗体是诊断丙型病毒性肝炎的主要指标。但因感染HCV后，抗HCV抗体出现较慢，一般在发病后2～6个月，甚至1年才转阳，故不能作为早期诊断的方法。Chronic Hepatitis C Virus-HCV is one of the main causes of liver cirrhosis and liver cancer. Hepatitis C virus, referred to as hepatitis C for short, is a kind of viral hepatitis caused by hepatitis C virus (HCV) infection, which is mainly transmitted through blood transfusion, acupuncture, drug abuse and other ways. The pathological changes of hepatitis C are very similar to those of hepatitis B, with liver cell necrosis and lymphocyte infiltration being the main ones. Fibrous tissue proliferation in the portal area can occur in chronic hepatitis, leading to chronic inflammation, necrosis and fibrosis of the liver, and some patients may develop liver cirrhosis or even hepatocellular carcinoma (HCC). Hepatitis C is closely related to the occurrence of liver cancer, and the transition between the two is a relatively long process, which is divided into four stages: acute infection - chronic infection - liver cirrhosis - liver cancer. The initial stage of HCV infection (2 to 12 weeks) is the acute stage, and the infected person may have no obvious symptoms. 1 to 3 weeks after infection with HCV, HCV RNA can be detected in peripheral blood, and only a few people can clear the virus by themselves and recover. In the chronic phase, 50% to 85% of patients will enter the chronic inflammation phase and develop into chronic hepatitis C. People with chronic HCV infection have a higher risk of developing liver cancer. The risk of liver cancer in patients with chronic HCV infection is 15-20 times that of the general population. Currently, there are 130 million to 210 million HCV chronically infected patients worldwide. 10% to 40% of chronic hepatitis C patients will progress to liver cirrhosis, and 1% to 5% of them will eventually develop into liver cancer. At present, hepatitis C antibody is the main indicator for the diagnosis of hepatitis C virus. However, after HCV infection, the anti-HCV antibody appears slowly, usually 2 to 6 months after the onset, or even 1 year before turning positive, so it cannot be used as an early diagnosis method.
当各型病毒性肝炎特异性标志检测阴性，临床症状及单项谷丙转氨酶(ALT)升高，提示急性病毒性肝炎时，应考虑是否为丙型病毒性肝炎。鉴别诊断主要依据特异性血清学检查：1.肝功能：包括血清ALT、谷草转氨酶(AST)、总胆红素、直接胆红素、间接胆红素、白蛋白、球蛋白，胆碱酯酶、碱性磷酸酶、转肽酶等。2.丙肝病毒抗体抗HCV。3.丙肝病毒定量血清HCV RNA。4.影像学：腹部肝胆脾超声检查了解肝脏有无慢性损伤，必要时行腹部增强电子计算机断层扫描(CT)或核磁共振成像(MRI)检查，以了解病情损伤程度。5.肝脏瞬时弹 性波扫描，是一种无创检查可用于慢性丙型肝炎患者肝脏纤维化程度评估。丙型肝炎患者评估肝脏纤维化程度对于确定治疗方案非常重要。6.肝组织活检，是评估患者肝脏炎症分级与纤维化分期的金标准。When the detection of specific markers of various types of viral hepatitis is negative, and the clinical symptoms and single alanine aminotransferase (ALT) increase suggest acute viral hepatitis, it should be considered whether it is hepatitis C virus. Differential diagnosis is mainly based on specific serological tests: 1. Liver function: including serum ALT, aspartate aminotransferase (AST), total bilirubin, direct bilirubin, indirect bilirubin, albumin, globulin, cholinesterase , alkaline phosphatase, transpeptidase, etc. 2. Hepatitis C virus antibody against HCV. 3. Quantitative serum HCV RNA of hepatitis C virus. 4. Imaging: Abdominal ultrasonography of liver, gallbladder and spleen to check whether there is chronic damage to the liver. If necessary, perform abdominal enhanced computerized tomography (CT) or magnetic resonance imaging (MRI) examination to understand the degree of damage. 5. Liver transient elastic wave scanning is a non-invasive examination that can be used to evaluate the degree of liver fibrosis in patients with chronic hepatitis C. Assessment of liver fibrosis in patients with hepatitis C is important for determining treatment options. 6. Liver biopsy is the gold standard for assessing liver inflammation grade and fibrosis stage.
目前用聚合酶链式反应(PCR)方法可以直接检测血中的HCV-RNA，可用于HCV感染的早期诊断。HCV-RNA及抗HCV抗体均阳性或HCV-RNA单独阳性即可确诊为丙型病毒性肝炎。和乙肝一样，慢性丙肝患者属于肝癌高危人群。肝癌高危人群是指年龄在35岁以上，有HBV或HCV感染的血清学证据，或有慢性肝炎病史者，甲胎蛋白(AFP)检测+肝脏超声检查，用于肝癌早发现。但是由于AFP检测和肝脏超声检查的灵敏度不高，容易造成假阴性，延误诊断。因此急需高灵敏度和高特异度的方法用于丙肝肝癌的早期检测。At present, HCV-RNA in blood can be directly detected by polymerase chain reaction (PCR), which can be used for early diagnosis of HCV infection. HCV-RNA and anti-HCV antibody are positive or HCV-RNA positive alone can be diagnosed as hepatitis C virus. Like hepatitis B, patients with chronic hepatitis C belong to the high-risk group of liver cancer. The high-risk group of liver cancer refers to those who are over 35 years old, have serological evidence of HBV or HCV infection, or have a history of chronic hepatitis. Alpha-fetoprotein (AFP) detection + liver ultrasound examination is used for early detection of liver cancer. However, due to the low sensitivity of AFP detection and liver ultrasonography, it is easy to cause false negatives and delay the diagnosis. Therefore, there is an urgent need for methods with high sensitivity and high specificity for the early detection of hepatitis C liver cancer.
蛋白质的糖基化(Glycosylation)是一种最常见的蛋白翻译后修饰，是在糖基转移酶作用下将糖类转移至蛋白质和蛋白质上特殊的氨基酸残基形成糖苷键的过程。大多数的糖蛋白都是分泌蛋白，广泛存在于细胞膜、细胞间质、血浆以及粘液中。有些酶和激素是糖蛋白。糖蛋白具有多种生物功能。有些糖蛋白如原胶原是结构蛋白质。许多酶和激素(如***、促甲状腺激素等)有糖蛋白结构，血液中的许多糖蛋白担负无机离子(Fe2+、Ca2+、Cu2+等)和激素等生物活性物质的运输，血液凝固(纤维蛋白原是糖蛋白)和抗体活性等生物功能。凝集素有凝集细胞的能力，糖链还可起稳定肽链的作用。糖蛋白的另一重要功能是直接或间接地参与细胞表面的种种识别现象。由于糖蛋白中糖链对于维持机体生物学功能的重要性，糖链的改变有助于阐明炎症、肿瘤细胞对周围组织侵袭及转移等异常生物行为学的分子机理。目前，己经在多种肿瘤中发现了N-糖链的改变。Protein glycosylation (Glycosylation) is the most common post-translational modification of proteins. It is a process in which sugars are transferred to proteins and special amino acid residues on proteins to form glycosidic bonds under the action of glycosyltransferases. Most glycoproteins are secreted proteins, widely present in cell membranes, interstitial cells, plasma, and mucus. Some enzymes and hormones are glycoproteins. Glycoproteins have a variety of biological functions. Some glycoproteins such as trocollagen are structural proteins. Many enzymes and hormones (such as luteinizing hormone, thyroid-stimulating hormone, etc.) have glycoprotein structures, and many glycoproteins in the blood are responsible for the transport of biologically active substances such as inorganic ions (Fe2+, Ca2+, Cu2+, etc.) and hormones, blood coagulation (fiber Proproteins are glycoproteins) and have biological functions such as antibody activity. Lectins have the ability to aggregate cells, and sugar chains can also stabilize peptide chains. Another important function of glycoprotein is to directly or indirectly participate in various recognition phenomena on the cell surface. Due to the importance of sugar chains in glycoproteins for maintaining biological functions of the body, changes in sugar chains help to elucidate the molecular mechanisms of abnormal biobehaviors such as inflammation, tumor cell invasion and metastasis of surrounding tissues. At present, changes in N-glycan chains have been found in various tumors.
糖链是重要的生物信息分子，在许多生理和病理过程中都发挥着独特作用。糖链结构非常复杂，具有微观不均一性，其分析和结构解析一直是糖生物学研究的瓶颈。目前糖链结构的分析方法发展迅速，主要包括：(1)高效液相色谱法(HPLC)：分辨率高，检测速度快，重复性高，高效液相色谱柱可以反复使用，但是柱效会随着使用次数的增加而变低，且流动相有毒，设备操作需要受过严格培训的专业人才进行，且设备相对昂贵，溶剂需要严格纯化；(2)质谱法(MS)：质谱具有灵敏度高、可获得多种结构信息和适于分析混合物等优点，是糖链定性定量分析的一种理想手段。但是质谱仪器精密，设备操作复杂，且质谱仪价格昂 贵，不适合临床上普及推广使用；(3)毛细管电泳法(CE)：毛细管电泳成本低、柱效高、灵敏度高、速度快、进样量少、操作简单，但是重复性不高，稳定性不如HPLC。Sugar chains are important bioinformatics molecules that play unique roles in many physiological and pathological processes. The sugar chain structure is very complex and has microscopic heterogeneity. Its analysis and structural elucidation have always been the bottleneck of glycobiology research. At present, the analysis methods of sugar chain structure are developing rapidly, mainly including: (1) High performance liquid chromatography (HPLC): high resolution, fast detection speed, high repeatability, high performance liquid chromatography column can be used repeatedly, but column efficiency will decrease It becomes lower with the increase of the number of uses, and the mobile phase is toxic, and the operation of the equipment requires strictly trained professionals, and the equipment is relatively expensive, and the solvent needs to be strictly purified; (2) mass spectrometry (MS): mass spectrometry has high sensitivity, It can obtain a variety of structural information and is suitable for the analysis of mixtures. It is an ideal method for qualitative and quantitative analysis of sugar chains. However, the mass spectrometry instrument is precise, the operation of the equipment is complicated, and the mass spectrometer is expensive, so it is not suitable for popularization and use in clinical practice; (3) capillary electrophoresis (CE): capillary electrophoresis is low in cost, high in column efficiency, high in sensitivity, fast in speed, and easy to inject. The amount is small and the operation is simple, but the repeatability is not high and the stability is not as good as HPLC.
G-Test检测法(Glycan-Test)是基于DNA分析仪的毛细管微电泳技术(DSA-FACE)，将***液样本中糖蛋白的N-糖链进行荧光标记后，用毛细管微电泳进行分离，通过测量荧光信号得到的N-寡糖链的含量即指纹图谱(简称G-Test图谱)。该检测技术具有灵敏度高、操作简单、微量(2μL血清)、重复性高、稳定性好、高通量(96-孔板)等其他糖链分析技术无法比拟的优点，适用于一般检验科室，可望用于临床推广使用。The G-Test detection method (Glycan-Test) is based on the capillary microelectrophoresis technology (DSA-FACE) of the DNA analyzer. After the N-sugar chain of the glycoprotein in the prostatic fluid sample is fluorescently labeled, it is separated by capillary microelectrophoresis. The content of the N-oligosaccharide chain obtained by measuring the fluorescent signal is the fingerprint spectrum (G-Test spectrum for short). This detection technology has the advantages of high sensitivity, simple operation, trace volume (2μL serum), high repeatability, good stability, high throughput (96-well plate) and other sugar chain analysis technologies, and is suitable for general laboratory departments. It is expected to be used in clinical promotion.
发明内容Contents of the invention
本发明目的在于提供一种丙肝肝癌检测试剂，通过试剂测定血清中糖组图谱，将峰值量化，进行统计学分析，从而提供一种丙肝肝癌血清糖组图谱的模型的建立方法。The purpose of the present invention is to provide a detection reagent for hepatitis C liver cancer. The reagent measures the glycome profile in serum, quantifies the peak value, and performs statistical analysis, thereby providing a method for establishing a model of the serum glycome profile of hepatitis C liver cancer.
本发明采用的技术方案如下：The technical scheme that the present invention adopts is as follows:
一种丙肝肝癌检测试剂，由以下试剂混合而成：A hepatitis C liver cancer detection reagent, which is formed by mixing the following reagents:
试剂A：浓度为10mM的碳酸氢铵溶液中加入质量浓度0.5～5％的SDS配制而成；Reagent A: prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;
试剂B：由0.01～10U/10μL糖胺酰酶和0.01～10U/10μL唾液酸酶混合配制而成，混合溶液pH值为4～9；Reagent B: It is prepared by mixing 0.01~10U/10μL glucosamidase and 0.01~10U/10μL sialidase, and the pH value of the mixed solution is 4~9;
试剂C：由8-氨基芘-1,3,6-三磺酸溶于DMSO中配制而成，浓度为0.01mM～1M；Reagent C: Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM～1M;
试剂D：终止液。Reagent D: stop solution.
优选地，所述试剂A、试剂B与试剂C的体积比为2：2：1。Preferably, the volume ratio of the reagent A, reagent B and reagent C is 2:2:1.
一种丙肝肝癌检测试剂的制备方法，包括以下步骤：A preparation method of hepatitis C liver cancer detection reagent, comprising the following steps:
步骤一 寡糖链的制备Step 1 Preparation of oligosaccharide chains
在经过灭活处理的2μL血清样品中加入4μL试剂A，进行变性，降温到室温后，加入4μL试剂B，孵育1～6h；Add 4 μL of reagent A to 2 μL of inactivated serum samples to denature, after cooling down to room temperature, add 4 μL of reagent B, and incubate for 1-6 hours;
步骤二 寡糖链的标记Step 2 Labeling of oligosaccharide chains
在步骤一得到的液体中加入2μL试剂C，进行荧光标记，然后加入150μL试剂D终止标记反应；Add 2 μL of reagent C to the liquid obtained in step 1 for fluorescent labeling, then add 150 μL of reagent D to terminate the labeling reaction;
步骤三 寡糖链分离分析Step 3: Separation and analysis of oligosaccharide chains
取10μL步骤二处理后的液体，用分析仪进行糖链分离，得到图谱。Take 10 μL of the liquid treated in step 2, and use an analyzer to separate sugar chains to obtain a spectrum.
优选地，所述步骤一寡糖的制备中变性温度为不低于75℃加热，孵育温度为不低于25℃。Preferably, the denaturation temperature in the preparation of the step 1 oligosaccharide is not lower than 75°C, and the incubation temperature is not lower than 25°C.
优选地，所述步骤二中荧光标记的温度为50～90℃。Preferably, the temperature of fluorescent labeling in the second step is 50-90°C.
一种组合物在制备丙肝肝癌检测试剂中的应用，所述组合物通过(NGA2F+NA2F)/NA3的比值来检测丙肝肝癌。An application of a composition in the preparation of a detection reagent for hepatitis C liver cancer, the composition detects hepatitis C liver cancer through the ratio of (NGA2F+NA2F)/NA3.
本发明提供一种丙肝肝癌的血清糖蛋白N-糖组图谱模型的建立方法，通过测定血清糖蛋白寡糖链G-Test特异指纹图谱，进行统计学分析。The invention provides a method for establishing a serum glycoprotein N-glycan group pattern model of hepatitis C liver cancer, and performs statistical analysis by measuring the specific fingerprint pattern of the serum glycoprotein oligosaccharide chain G-Test.
材料和方法：Materials and methods:
一、检测样本：丙肝病毒引起的丙肝肝癌患者与正常对照人的血清。1. Test samples: Serum from patients with hepatitis C liver cancer caused by hepatitis C virus and normal controls.
二、实验设备：糖组分析仪，PCR，离心机。2. Experimental equipment: sugar group analyzer, PCR, centrifuge.
三、试剂制备：3. Reagent preparation:
试剂A：浓度为10mM的碳酸氢铵溶液中加入质量浓度0.5～5％的SDS配制而成；Reagent A: prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;
试剂B：由0.01～10U/10μL糖胺酰酶和0.01～10U/10μL唾液酸酶混合配制而成，混合溶液pH值为4～9；Reagent B: It is prepared by mixing 0.01~10U/10μL glucosamidase and 0.01~10U/10μL sialidase, and the pH value of the mixed solution is 4~9;
试剂C：由8-氨基芘-1,3,6-三磺酸溶于DMSO中配制而成，浓度为0.01mM～1M；Reagent C: Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM～1M;
试剂D：终止液。Reagent D: stop solution.
四、糖测序检测步骤：4. Glycosequencing detection steps:
步骤一 寡糖链的制备Step 1 Preparation of oligosaccharide chains
在经过灭活处理的2μL血清样品中加入4μL试剂A，进行变性，降温到室温后，加入4μL试剂B，孵育1～6h；Add 4 μL of reagent A to 2 μL of inactivated serum samples to denature, after cooling down to room temperature, add 4 μL of reagent B, and incubate for 1-6 hours;
步骤二 寡糖链的标记Step 2 Labeling of oligosaccharide chains
在步骤一得到的液体中加入2μL试剂C，进行荧光标记，然后加入150μL试剂D终止标记反应；Add 2 μL of reagent C to the liquid obtained in step 1 for fluorescent labeling, then add 150 μL of reagent D to terminate the labeling reaction;
步骤三 寡糖链分离分析Step 3: Separation and analysis of oligosaccharide chains
取10μL步骤二处理后的液体，用分析仪进行糖链分离，得到图谱。Take 10 μL of the liquid treated in step 2, and use an analyzer to separate sugar chains to obtain a spectrum.
五、监测对比分析5. Monitoring comparative analysis
将每个峰峰高值除以所有峰高度的总和，定量计算得到每个峰的相对含量，即N-糖组图谱的峰值量化，然后对量化后的丙肝肝癌组和正常对照组N-糖组图谱中9个寡糖峰进行比对统计分析。所述组合物通过(NGA2F+NA2F)/NA3的比值来检测丙肝肝癌。Divide the peak height of each peak by the sum of all peak heights, and quantitatively calculate the relative content of each peak, that is, the peak quantification of the N-glycan group profile, and then quantify the N- The 9 oligosaccharide peaks in the glycome map were compared and analyzed statistically. The composition detects hepatitis C liver cancer through the ratio of (NGA2F+NA2F)/NA3.
与现有技术相比，本发明的有益效果：Compared with prior art, the beneficial effect of the present invention:
(1)本发明方法采用灵敏度高、操作简单、仅需微量样品、重复性高、稳定性好和高通量的G-Test检测方法，建立了丙肝肝癌患者和正常对照人具有显著差异的N-糖组图谱模型。在后续应用中，待检测血清的N-糖组图谱用本方法建立的图谱模型来计算，能够检测样本是否具有丙肝肝癌。与现有技术相比，具有更高的准确度，对丙肝肝癌检测模型所做ROC曲线的AUC面积达到0.864。(1) The method of the present invention adopts the G-Test detection method with high sensitivity, simple operation, only needs a small amount of sample, high repeatability, good stability and high throughput, and has established a significant difference between patients with hepatitis C liver cancer and normal controls. -Model of the glycome map. In subsequent applications, the N-glycan profile of the serum to be tested is calculated using the profile model established by this method, which can detect whether the sample has hepatitis C liver cancer. Compared with the prior art, it has higher accuracy, and the AUC area of the ROC curve for the hepatitis C liver cancer detection model reaches 0.864.
(2)基于本发明方法构建的N-糖组图谱模型，能够让众多患者接受常规、无创检测，帮助医生及患者及时检测丙肝病毒引起的肝癌的发生和病情进展，有望在临床中推广使用。(2) The N-glycan map model constructed based on the method of the present invention can allow many patients to receive routine and non-invasive testing, and help doctors and patients detect the occurrence and progression of liver cancer caused by hepatitis C virus in a timely manner, and it is expected to be widely used in clinical practice .
图1是正常对照组和丙肝肝癌组的血清糖蛋白N-糖组图谱；图谱中的寡糖缩写分别表示为：NGA2F，半乳糖缺失含核心岩藻糖两天线(Agalacto core-α-1，6-fucosylated biantennary)；NA2F，核心岩藻糖两天线(Bigalacto core-α-1，6-fucosylated biantennary)；NA3，三天线(Triantennary)。Figure 1 is the serum glycoprotein N-glycan map of the normal control group and the hepatitis C liver cancer group; the abbreviations of the oligosaccharides in the map are respectively expressed as: NGA2F, galactose missing two antennae containing core fucose (Agalacto core-α-1 , 6-fucosylated biantennary); NA2F, Bigalacto core-α-1, 6-fucosylated biantennary); NA3, triantennary.
图2是模型建立后的ROC曲线图；检测样本通过函数(NGA2F+NA2F)/NA3用于鉴别丙肝肝癌的ROC曲线；检测样本总数为66例，其中丙肝肝癌患者血清36例，正常人对照组血清30例，得到曲线下面积AUC＝0.864。Figure 2 is the ROC curve after the model is established; the detection sample is used to identify the ROC curve of hepatitis C liver cancer through the function (NGA2F+NA2F)/NA3; the total number of detection samples is 66 cases, including 36 cases of serum from patients with hepatitis C liver cancer, and the normal control group Sera from 30 cases obtained the area under the curve AUC=0.864.
下面结合实施例和附图对本发明作进一步详述。需要说明的是，下列实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件试验，或按照制造厂商建议的条件。The present invention will be described in further detail below in conjunction with the embodiments and accompanying drawings. It should be noted that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods that do not specify specific conditions in the following examples, they are usually tested according to conventional conditions, or according to the conditions suggested by the manufacturer.
实施例1 检测丙肝肝癌Example 1 Detection of hepatitis C liver cancer
通过测定血清糖蛋白寡糖链G-Test特异指纹图谱，进行统计学分析，采用的材料和方法：Statistical analysis was carried out by measuring the G-Test specific fingerprint of serum glycoprotein oligosaccharide chains, and the materials and methods used:
一、检测样本：丙肝病毒引起的丙肝肝癌患者与正常对照人的血清。1. Test samples: Serum from patients with hepatitis C liver cancer caused by hepatitis C virus and normal controls.
二、实验设备：糖组分析仪，PCR，离心机。2. Experimental equipment: sugar group analyzer, PCR, centrifuge.
三、试剂制备：3. Reagent preparation:
试剂A：浓度为10mM的碳酸氢铵溶液中加入质量浓度0.5～5％的SDS配制而成；Reagent A: prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;
试剂B：由0.01～10U/10μL糖胺酰酶和0.01～10U/10μL唾液酸酶混合配制而成，混合溶液pH值为4～9；Reagent B: It is prepared by mixing 0.01~10U/10μL glucosamidase and 0.01~10U/10μL sialidase, and the pH value of the mixed solution is 4~9;
试剂C：由8-氨基芘-1,3,6-三磺酸溶于DMSO中配制而成，浓度为0.01mM～1M；Reagent C: Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM～1M;
试剂D：终止液。Reagent D: stop solution.
四、糖测序检测步骤：4. Glycosequencing detection steps:
步骤一 寡糖链的制备Step 1 Preparation of oligosaccharide chains
在经过灭活处理的2μL血清样品中加入4μL试剂A，进行变性，降温到室温后，加入4μL试剂B，孵育1～6h；Add 4 μL of reagent A to 2 μL of inactivated serum samples to denature, after cooling down to room temperature, add 4 μL of reagent B, and incubate for 1-6 hours;
步骤二 寡糖链的标记Step 2 Labeling of oligosaccharide chains
在步骤一得到的液体中加入2μL试剂C，进行荧光标记，然后加入150μL试剂D终止标记反应；Add 2 μL of reagent C to the liquid obtained in step 1 for fluorescent labeling, then add 150 μL of reagent D to terminate the labeling reaction;
步骤三 寡糖链分离分析Step 3: Separation and analysis of oligosaccharide chains
取10μL步骤二处理后的液体，用分析仪进行糖链分离，得到图谱。Take 10 μL of the liquid treated in step 2, and use an analyzer to separate sugar chains to obtain a spectrum.
五、检测对比分析5. Detection and comparative analysis
利用G-Test检测技术对收集的66例丙肝肝癌患者和正常人对照组的血清样本进行处理，其中丙肝肝癌患者血清36例，正常人对照组血清30例。对G-Test检测技术测定样本得到的N-糖组图谱进行统计学分析。G-Test detection technology was used to process the collected serum samples of 66 cases of hepatitis C liver cancer patients and normal control group, including 36 cases of serum samples of hepatitis C liver cancer patients and 30 cases of serum samples of normal control group. Statistical analysis was carried out on the N-glycan profile obtained from samples measured by G-Test detection technology.
将每个峰峰高值除以所有峰高度的总和，定量计算得到每个峰的相对含量，即N-糖组图谱峰值量化，然后对量化后的丙肝肝癌组和正常对照组N-糖组图谱中9个糖峰进行比对统计分析。如图1所示，血清N-糖组图谱大概显示出9个N-糖链峰，糖链因分子大小的不同而表现出不同的迁移率，即表现在N-糖组图谱上的不同的峰则代表了不同的寡糖链，所测出的峰高代表了寡糖链的相对浓度含量，图1A为正常对照组，图1B为丙肝肝癌组。N-糖组图谱的组合物通过(NGA2F+NA2F)/NA3的比值来检测丙肝肝癌。Divide the peak height of each peak by the sum of all peak heights, and quantitatively calculate the relative content of each peak, that is, the peak quantification of the N-glycan group map, and then quantify the N-glycan of the hepatitis C liver cancer group and the normal control group The 9 sugar peaks in the group map were compared and analyzed statistically. As shown in Figure 1, the serum N-glycan profile shows about 9 N-glycan peaks, and the sugar chains show different mobility due to different molecular sizes, that is, the Different peaks represent different oligosaccharide chains, and the measured peak heights represent the relative concentration of oligosaccharide chains. Figure 1A is the normal control group, and Figure 1B is the hepatitis C liver cancer group. The composition of the N-glycan profile detects hepatitis C liver cancer by the ratio of (NGA2F+NA2F)/NA3.
对G-Test指纹图谱的各个峰值进行量化，然后对丙肝肝癌组(36例)和正常对照组(30例)进行统计学分析，通过九个峰建立模型来预测丙肝肝癌，发 现两组的区分上具有统计学意义(p＜0.05)。ROC曲线分析显示，模型在检测丙肝肝癌患者时具有显著的临床意义，即AUC可达0.864(图2)。由此说明血清中九个寡糖峰可以作为丙肝肝癌的标志物。Quantify each peak of the G-Test fingerprint, and then conduct statistical analysis on the hepatitis C liver cancer group (36 cases) and the normal control group (30 cases), build a model through nine peaks to predict hepatitis C liver cancer, and find the difference between the two groups It was statistically significant (p<0.05). ROC curve analysis showed that the model had significant clinical significance in detecting patients with hepatitis C liver cancer, that is, the AUC could reach 0.864 (Figure 2). This shows that the nine oligosaccharide peaks in serum can be used as markers of hepatitis C liver cancer.
以上所述的具体实施例，结合附图对本发明的目的、技术方案和有益效果进行了进一步详细说明，所应理解的是，以上所述仅为本发明的具体实施例并不用于限制本发明，凡在本发明的精神和原则之内，本领域技术人员所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The specific embodiments described above, in conjunction with the accompanying drawings, further describe the purpose, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above descriptions are only specific embodiments of the present invention and are not intended to limit the present invention. , within the spirit and principles of the present invention, any modifications, equivalent replacements, improvements, etc. made by those skilled in the art shall be included within the protection scope of the present invention.
Claims (6)
- 一种丙肝肝癌检测试剂，其特征在于，由以下试剂混合而成：A hepatitis C liver cancer detection reagent is characterized in that it is formed by mixing the following reagents:试剂A：浓度为10mM的碳酸氢铵溶液中加入质量浓度0.5～5％的SDS配制而成；Reagent A: prepared by adding SDS with a mass concentration of 0.5 to 5% in ammonium bicarbonate solution with a concentration of 10 mM;试剂B：由0.01～10U/10μL糖胺酰酶和0.01～10U/10μL唾液酸酶混合配制而成，混合溶液pH值为4～9；Reagent B: It is prepared by mixing 0.01~10U/10μL glucosamidase and 0.01~10U/10μL sialidase, and the pH value of the mixed solution is 4~9;试剂C：由8-氨基芘-1,3,6-三磺酸溶于DMSO中配制而成，浓度为0.01mM～1M；Reagent C: Prepared by dissolving 8-aminopyrene-1,3,6-trisulfonic acid in DMSO, the concentration is 0.01mM～1M;试剂D：终止液。Reagent D: stop solution.
- 根据权利要求1所述的丙肝肝癌检测试剂，其特征在于，所述试剂A、试剂B与试剂C的体积比是2：2：1。The hepatitis C liver cancer detection reagent according to claim 1, wherein the volume ratio of the reagent A, reagent B and reagent C is 2:2:1.
- 根据权利要求1所述的丙肝肝癌检测试剂的制备方法，其特征在于，包括以下步骤：The preparation method of hepatitis C liver cancer detection reagent according to claim 1, is characterized in that, comprises the following steps:步骤一 寡糖链的制备Step 1 Preparation of oligosaccharide chains在经过灭活处理的2μL血清样品中加入4μL试剂A，进行变性，降温到室温后，加入4μL试剂B，孵育1～6h；Add 4 μL of reagent A to 2 μL of inactivated serum samples to denature, after cooling down to room temperature, add 4 μL of reagent B, and incubate for 1-6 hours;步骤二 寡糖链的标记Step 2 Labeling of oligosaccharide chains在步骤一得到的液体中加入2μL试剂C，进行荧光标记，然后加入150μL试剂D终止标记反应；Add 2 μL of reagent C to the liquid obtained in step 1 for fluorescent labeling, then add 150 μL of reagent D to terminate the labeling reaction;步骤三 寡糖链分离分析Step 3: Separation and analysis of oligosaccharide chains取10μL步骤二处理后的液体，用分析仪进行糖链分离，得到图谱。Take 10 μL of the liquid treated in step 2, and use an analyzer to separate sugar chains to obtain a spectrum.
- 根据权利要求3所述的丙肝肝癌检测试剂的制备方法，其特征在于，所述步骤一寡糖的制备中变性温度为不低于75℃加热，孵育温度为不低于25℃。The preparation method of hepatitis C liver cancer detection reagent according to claim 3, characterized in that, in the preparation of the step 1 oligosaccharide, the denaturation temperature is not lower than 75°C heating, and the incubation temperature is not lower than 25°C.
- 根据权利要求3所述的丙肝肝癌检测试剂的制备方法，其特征在于，所述步骤二中荧光标记的温度为50～90℃。The preparation method of hepatitis C liver cancer detection reagent according to claim 3, characterized in that the temperature of fluorescent labeling in the second step is 50-90°C.
- 一种组合物在制备丙肝肝癌检测试剂中的应用，其特征在于，所述组合物通过(NGA2F+NA2F)/NA3的比值来检测丙肝肝癌。An application of a composition in the preparation of a hepatitis C liver cancer detection reagent is characterized in that the composition detects the hepatitis C liver cancer by the ratio of (NGA2F+NA2F)/NA3.
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