WO2021243782A1 - 用于诊断肝癌和预测肝癌转移的血清miRNA标志物及其检测试剂盒 - Google Patents

用于诊断肝癌和预测肝癌转移的血清miRNA标志物及其检测试剂盒 Download PDF

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WO2021243782A1
WO2021243782A1 PCT/CN2020/099588 CN2020099588W WO2021243782A1 WO 2021243782 A1 WO2021243782 A1 WO 2021243782A1 CN 2020099588 W CN2020099588 W CN 2020099588W WO 2021243782 A1 WO2021243782 A1 WO 2021243782A1
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liver cancer
mir
kit
serum
reaction
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郑敏
厉双双
楼国华
周林福
邵佳佳
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浙江大学
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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
    • C12Q1/68—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
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

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  • the present invention belongs to the field of genetic engineering and medical diagnosis, and specifically relates to the application of serum miRNAs marker kits for diagnosing liver cancer and predicting liver cancer metastasis, and more specifically to the research and development of kits, analysis performance evaluation, and for early diagnosis of liver cancer and Transfer risk assessment.
  • MiRNA is a type of small non-coding RNA with a length of more than twenty bases. In recent years, more and more studies have found that they play an important role in the occurrence and development of various diseases, including liver cancer. Studies have shown that miRNAs play a huge role in cell proliferation, differentiation, apoptosis, migration and other abilities. miRNA generally inhibits the translation of the target gene or can cause the degradation of the target gene by binding to a specific site in the 3'-UTR region of the target gene's mRNA. Abnormal miRNA expression levels can cause abnormal expression of target genes to promote changes in cell physiological functions, promote the occurrence of tumor microenvironment, and ultimately lead to the occurrence of various diseases including tumors. miRNA exists stably in various human body fluids, so the detection of miRNA in serum can make it a potential non-invasive tumor biomarker.
  • liver cancer is the seventh-most malignant tumor in the world and the third-leading cause of cancer deaths in the world.
  • the abnormal expression of miRNAs has played a significant role in the diagnosis of liver cancer.
  • the present invention develops a three-index combined diagnostic kit for abnormally expressed miR-122-5p, miR-144-3p and miR-451a, which is more conducive to the actual detection and application of clinical samples, and can be used for early diagnosis of liver cancer and risk of metastasis evaluate.
  • the invention discloses the development and application of a kit containing three blood miRNAs markers related to human liver cancer.
  • the specific markers are serum-derived miR-122-5p, miR-144-3p and miR-451a.
  • One of the objectives of the present invention is to provide reagents for detecting serum miRNA markers.
  • the specific markers are serum-derived miR-122-5p, miR-144-3p and miR-451a.
  • the new reverse transcription primers and probe primers for serum miRNAs are:
  • the amplification method of the amplification primer is TaqMan probe method.
  • the invention also includes the application of the reagent in diagnosing liver cancer and predicting liver cancer metastasis.
  • the present invention also provides a kit for diagnosing human liver cancer and predicting liver cancer metastasis, which contains serum miRNA markers for detecting serum-derived miR-122-5p, miR-144-3p and miR-451a Reagents.
  • the reagents include reverse transcription primers and amplification primers used in QPCR experiments; the amplification method uses TaqMan probe method.
  • the invention also includes the application of the kit in diagnosing liver cancer and predicting liver cancer metastasis.
  • the present invention also provides the development and optimization of the above-mentioned serum miRNAs marker diagnostic kit.
  • the technical solutions for solving the technical problems of the present invention include:
  • This kit uses a two-step reverse transcription polymerase chain reaction method to detect miR-122-5p, miR-144-3p, and miR-451a in human serum. First, the miRNAs are reverse transcribed using long primers with a stem-loop structure, and then the cDNA obtained by reverse transcription is used as a template for PCR amplification.
  • Table 1 The main components of the three microribonucleic acid (miRNAs) detection kits.
  • This kit is suitable for ABI7500 real-time fluorescent quantitative PCR instrument.
  • a disposable syringe to collect 2 mL of peripheral blood (the blood collection time is generally morning or morning), and quickly transfer it to an EDTA anticoagulation tube or a biochemical tube, and mix. Centrifuge for 10 minutes at 3000 rpm, 4°C for 1 hour (at room temperature) or 2 hours (at 4°C), take the supernatant, continue with 16000g, 4°C, centrifuge for 10 minutes, and transfer the supernatant to a new centrifuge tube In the medium, store at -80°C for later use.
  • the separated serum samples in order to prevent the evaporation of water in the serum samples, they should be transferred to a test tube with a stopper. If it cannot be submitted for inspection in time, it should be stored in the refrigerator.
  • Clinical samples clinical samples to be tested
  • Positive quality control products positive quality control products in the kit.
  • reaction system is prepared according to the following ratio:
  • each reagent used that is, the number of people that should be satisfied by the total amount of each reverse transcription reaction solution, including: the number of clinical samples, 1 tube of positive quality control material and 1 PCR negative control; each reverse transcription reaction solution
  • the total amount number of people * reverse transcription reaction system
  • nucleic acid prepared in the "1 amplification reagent preparation (pre-PCR preparation period)" step to the reaction well containing the reverse transcription reaction solution.
  • the addition amount of each nucleic acid is 5.0 ⁇ L/well, and no addition to the PCR negative control well Any sample or nucleic acid.
  • reaction system is prepared according to the following ratio:
  • step 2 Add the reverse transcription products 1, 2, 3, and 4 in step 2 to the reaction wells containing qPCR reagents 1, 2, 3, and 4, respectively, and the addition amount is 5.0 ⁇ L/well.
  • the collection of fluorescence signals was set as FAM, HEX and CY5, and the collection of data was set at 57°C.
  • FAM fluorescence signal
  • the instrument After the reaction is over, the instrument automatically saves the results, and after analyzing the image, adjust the Start value, End value and Threshold value of Baseline (self-adjustable, Start value can be between 3-15, End value between 5-20), adjust PCR Amplification curve of the negative control.
  • the Ct value of the PCR negative control should be: make the HEX(122)>33.73 of qPCR reaction 1; make the FAM(144)>36.50 of qPCR reaction 2; make the HEX(451)>34.10 of qPCR reaction 3;
  • the fluorescence Ct values of FAM and HEX are both ⁇ 31.00.
  • the analysis performance evaluation of the detection kit is mainly from the three aspects of sensitivity, specificity and precision.
  • This kit is used for the qualitative detection of three kinds of miRNAs (miR-122-5p, miR-144-3p, miR-451a) in human serum samples.
  • the experimental protocol and results are as follows.
  • RNA fragments for simulation test take synthetic miR-122-5p, miR-144-3p, miR-451a 3 kinds of RNA dry powder (2.5nmol/tube), respectively dissolve them with 150 ⁇ L sterile RNase-free water to make 1E13copies / ⁇ L of nucleic acid solution, use this as the stock solution for subsequent dilutions, and select a total of 3 concentrations for sensitivity testing.
  • the specific concentrations are shown in Table 7.
  • the detection of each miRNAs was repeated 20 times, and the concentration level with a positive detection rate of more than 90% was regarded as the lowest detection concentration.
  • the instrument used for detection was ABI7500 real-time fluorescent quantitative PCR instrument.
  • the test results are shown in Table 8 and Table 9. The results show that: the positive detection rate of the first two concentrations of the three miRNAs is greater than 90%, and the positive detection rate of the third concentration gradient is less than 20%, so this reagent
  • the lowest detectable concentration of the box for miR-122-5p is 5 ⁇ 10 3 copies/ ⁇ L
  • the lowest detectable concentration for miR-144-3p is 5 ⁇ 10 4 copies/ ⁇ L
  • the lowest detectable concentration for miR-451a It is 5 ⁇ 10 3 copies/ ⁇ L.
  • the instrument used in the experiment is ABI7500 real-time fluorescent quantitative PCR instrument.
  • 5 synthetic miRNAs (miR-122b, miR-122b-5p, miR-122-3p, miR-514a and miR- 677a)
  • ABI7500 real-time fluorescent quantitative PCR instrument for detection.
  • the test results are shown in Table 10. The results show that the test results of miR-122b, miR-122b-5p, miR-122-3p, miR-514a and miR-677a, which have similar sequences to the three miRNAs detected by this kit All are negative, so there is no cross-reaction in this kit.
  • Potential interfering substances mainly include free hemoglobin, bilirubin, triglycerides and total G-type immunoglobulin (IgG).
  • the verification experiment should be carried out under the potentially worst conditions, that is, samples with a cutoff concentration level should be selected (267 And 216), and add the potential maximum concentration of interfering substances related to the medical level.
  • test results are shown in Table 12.
  • the results show that after adding 4 different interfering substances, the target fluorescence Ct value of the sample with the threshold concentration level is not significantly different from the sample without interfering substance (control group) (single Factor analysis of variance and paired t test (p>0.05), that is, the test sample contains the following concentrations of interfering substances, which will not affect the determination of the results of the sample test by this kit:
  • Precision refers to the consistency between independent test results under specified conditions.
  • RNA solutions of miR-122-5p, miR-144-3p, and miR-451a were selected at two concentration levels of miRNAs, and a total of 6 RNA solutions with different concentrations were tested. Repeat the test for each RNA solution 20 times, and calculate the coefficient of variation (CV, %) of the Ct value of the test result. The CV value should be less than 5%.
  • the instrument used for detection is ABI7500 real-time fluorescent quantitative PCR instrument.
  • the test results are shown in Table 13. The results show that the 6 RNA solutions of different concentrations were repeatedly tested for 20 times, and the coefficient of variation of the Ct value was less than 5%.
  • this kit meets the standards in terms of sensitivity, specificity, and precision.
  • EV-RT P-HEX ddH2O; 1 ⁇ TE; Mg2+ (25mM); dNTP (25mM).
  • the miRNA in the sample was extracted according to the instructions of the mirVana TM PARIS TM RNA Isolation Kit (Applied Biosystem p/n AM1556).
  • miRNA-122 reaction system Per test/ ⁇ L 10 ⁇ buffer 2.5 HS Taq enzyme 0.4
  • miRNA-144 reaction system Per test/ ⁇ L 10 ⁇ buffer 2.5 HS Taq enzyme 0.4 Mg 2+ (25mM) 2 dNTP (25mM) 0.2 144RT-loop-F2 0.4 144RT-loop-R10 0.4 144-RT P-FAM 0.2 ddH 2 O 16.9 RT reaction product (template) 2 Total 25
  • miRNA-451 reaction system Per test/ ⁇ L 10 ⁇ buffer 2.5 HS Taq enzyme 0.4 Mg 2+ (25mM) 2 dNTP (25mM) 0.2 122RT-loop-F2* 0.4 451RT-loop-R6 0.4 122-RT P-HEX* 0.2 ddH 2 O 16.9 RT reaction product (template) 2 Total 25
  • 122RT-loop-F2 is the upstream primer for miRNA-122-5p
  • c 122-RT P-HEX is for miRNA-122 -5p probe
  • the suffix F represents the upstream primer
  • the suffix R represents the downstream primer
  • the suffix P represents the probe
  • the letter after P represents the modified fluorophore
  • 122-RT-loop2 is the reverse transcription primer for miRNA-122-5p
  • the downstream primer of 5p, d: 122-RT P-HEX is a probe for detecting miRNA-122-5p
  • the suffix F represents the upstream primer
  • the suffix R represents the downstream primer
  • the suffix P represents the probe
  • the letter after P represents the modified fluorescent group Group, the rest can be deduced by analogy.
  • miR-122-5p and miR-451a share upstream primers and probes
  • # internal standard is an external internal standard, artificially designed sequence, named EV
  • the invention discloses the development and application of a kit containing three blood miRNAs markers related to human liver cancer.
  • the specific markers are serum-derived miR-122-5p, miR-144-3p and miR-451a.
  • the present invention uses differential miRNAs as the core component of the detection kit, combined with corresponding internal references to assemble the detection kit, and through the actual detection application in clinical samples, optimizes the reaction conditions and reagent ratio of the kit, and adjusts its detection parameters. Preliminarily establish a standardized process for clinical detection and analysis, and use it for early diagnosis and metastasis risk assessment of liver cancer.
  • MiRNAs in serum are a new type of biomarker, which has the advantages of stability, minimally invasiveness, and easy detection, which can greatly improve the sensitivity and specificity of disease diagnosis.
  • the research and successful development of such small molecule biomarkers It is helpful for the auxiliary diagnosis of liver cancer and also provides reference for the research of other diseases.
  • the kit for diagnosing the occurrence of human liver cancer by detecting the expression of specific miRNAs is a systematic and comprehensive diagnostic kit that can be used for the auxiliary diagnosis of liver cancer patients and helps predict the metastasis of liver cancer patients. Clinically, it helps doctors better grasp the patient's condition, have a better grasp of the development trend of the disease, and can take more targeted and precise personalized treatment in a timely manner.
  • the TaqMan probe method for detecting miRNAs in serum samples has higher specificity, better repeatability, and more reliable results.
  • a diagnostic kit with multiple miRNAs indicators can improve the sensitivity and specificity of the results of the kit, and the detection of multi-index miRNAs can reduce the experimental results caused by the individual expression of a single indicator in the sample. Errors make the results more accurate and credible.
  • Figure 1 shows the use of the kit to detect the expression of miR-144-3P in serum in the three groups of normal people, liver cancer patients without metastasis, and liver cancer patients with metastasis;
  • Figure 2 shows the use of the kit to detect the expression of miR-451a in serum in the three groups of normal people, liver cancer patients without metastasis, and liver cancer patients with metastasis;
  • Figure 3 shows the use of the kit to detect the expression of miR-122-5p in serum in the three groups of normal people, liver cancer patients without metastasis, and liver cancer patients with metastasis;
  • Figure 4 shows the sensitivity and specificity of the ROC curve analysis of miR-144-3P, miR-451a and miR-122-5p when distinguishing between the liver cancer group and the normal group.
  • Example 1 Collection of samples and sorting of sample data
  • the samples were provided by the First affiliated Hospital of Zhejiang University School of Medicine (the samples were all processed by the same method within 2 hours of collection, the serum was obtained, divided into packaging, and the preservation conditions were uniform). After sorting out the sample data, the inventor selected the 42 cases of primary liver cancer patients (that is, no treatment for liver cancer, chemotherapy, drug treatment, etc. were performed before collection) and 39 cases of peripheral blood from normal people collected at the same period as the experimental group.
  • mirVana TM PARIS TM Kit (Applied Biosystem p/n AM1556) was used to extract the total RNA of the sample.
  • Example 3 the kit detects the target miRNAs of the sample
  • Reverse transcription reaction Add 4uL RNase Free dH2O, 2uL 5 ⁇ buffer (PrimeScriptBuffer2), 0.5uL specific reverse transcription primer, 0.5uL reverse transcriptase (PrimeScriptRT Enzyme Mix I), 3ul sample template, final volume into the reaction tube 10uL, incubate at 42°C for 15min, 85°C for 5s, 4°C ⁇ . The obtained cDNA template is stored at -20°C for later use.
  • Example 4 Analysis of the predictive diagnosis of the occurrence and metastasis of liver cancer by miRNAs
  • metastasis patients with distant metastasis or intrahepatic metastasis or portal vein tumor thrombus on imaging
  • non-metastasis group The experimental results showed that there was metastasis group.
  • miR-122-5p has an AUC value of 0.783 when distinguishing liver cancer group from normal group
  • miR-144-3p has an AUC value of 0.808 when distinguishing liver cancer group from normal group
  • miR-451a is in When distinguishing the liver cancer group from the normal group, the AUC value is 0.858, and when the three indicators are combined, the AUC value is 0.938.
  • Experimental results show that serum miR-122-5p, miR-144-3P and miR-451a, as liver cancer biomarkers, have better sensitivity and specificity, and the combined diagnosis of the three indicators has better results.

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Abstract

本发明提供用于诊断肝癌和预测肝癌转移的血清miRNA标志物及其检测试剂盒，所述标志物为血清来源的miR-122-5p,miR-144-3p和miR-451a。

Description

用于诊断肝癌和预测肝癌转移的血清miRNA标志物及其检测试剂盒 技术领域
本发明属于基因工程和医学诊断领域，具体涉及用于诊断肝癌和预测肝癌转移的血清miRNAs标志物试剂盒的应用，更具体的涉及试剂盒的研发、分析性能评估，并用于肝癌的早期诊断和转移风险评估。
背景技术
MiRNA是一类小的非编码RNA，长度一般是二十几个碱基，近年来越来越多的研究发现它们在各种疾病包括肝癌的发生发展中发挥了重要的作用。研究表明miRNA在细胞的增殖，分化，凋亡，迁移等能力中承担着巨大的作用。miRNA一般通过结合靶基因mRNA的3’-UTR区的特定位点来抑制靶基因的翻译或者可以导致靶基因的降解。异常的miRNA表达水平，可以导致靶基因的表达异常从而促进细胞的生理功能改变，促进肿瘤微环境的发生，最终导致各种疾病包括肿瘤的发生。miRNA稳定存在于人类的各种体液中，所以血清中的miRNA检测可以使之成为有潜力的无创肿瘤生物标志物。
据2018年最新统计，肝癌是世界上第七大恶性肿瘤，也是世界上第三大癌症致死疾病。据已有研究，miRNAs异常表达在肝癌的诊断中发挥了不小的作用。本发明针对异常表达的miR-122-5p,miR-144-3p和miR-451a，研发出三指标联合诊断试剂盒，更有利于临床样本的实际检测应用，可用于肝癌的早期诊断和转移风险评估。
发明内容
本发明公开了包含三种与人类肝癌相关的血液miRNAs标志物的试剂盒的研发及其应用。具体的标志物为血清来源的miR-122-5p,miR-144-3p和miR-451a。
本发明的目的之一在于提供用于检测血清miRNA标志物的试剂，具体的标志物为血清来源的miR-122-5p,miR-144-3p和miR-451a。血清miRNAs的新的逆转录引物、探针引物为：
Figure PCTCN2020099588-appb-000001
Figure PCTCN2020099588-appb-000002
优选的，其中扩增引物的扩增方法为TaqMan探针法。
本发明还包括试剂在诊断肝癌和预测肝癌转移中的应用。
另外，本发明还提供了一种用于诊断人类肝癌和预测肝癌转移的试剂盒，包含用于检测血清来源的miR-122-5p,miR-144-3p和miR-451a的血清miRNA标志物的试剂。所述试剂包括QPCR实验中使用的反转录引物以及扩增引物；所述的扩增方法用的是TaqMan探针法。
本发明还包括试剂盒在诊断肝癌和预测肝癌转移中的应用。
本发明还提供上述血清miRNAs标志物的诊断试剂盒中的研发、优化。
具体来说，本发明解决技术问题的技术方案包括：
1.研究试剂盒。
2.试剂盒分析性能评估。
3.用试剂盒检测样本人群中的目的miRNAs。
1.研究试剂盒。
具体实行方案如下：
【检验原理】本试剂盒采用两步法逆转录聚合酶链式反应检测人血清中的miR-122-5p,miR-144-3p,miR-451a。首先，采用带茎环结构的长引物逆转录miRNAs，再以逆转录得到的cDNA为模板进行PCR扩增。
【主要组成成分】
表1：三种微小核糖核酸(miRNAs)检测试剂盒主要组成成分表
Figure PCTCN2020099588-appb-000003
Figure PCTCN2020099588-appb-000004
【适用仪器】
本试剂盒适用于ABI7500实时荧光定量PCR仪。
【样本要求】
用一次性针筒采集外周血2mL(采血时间一般为早晨或上午)，迅速转入EDTA抗凝管或生化管中，混匀。在1小时(室温条件下)或2小时(4℃条件下)内于3000rpm，4℃，离心10分钟，取上清，继续16000g,4℃,离心10分钟，吸取上清到新的离心管中，置于-80℃保存 备用。
注意：对于分离好的血清样本，为了防止血清样本中水分的挥发，应移入带塞的试管中。如不能及时送检，应置冰箱中冷藏保存。
【检验方法】
1扩增试剂准备(PCR前准备期)
根据用户选用的miRNAs核酸抽提试剂盒说明书提取人血清中的miRNAs。提取时，在裂解的步骤中加入内标，内标添加量为5uL/样本。需要处理的样本种类包括：
1.1临床样本：待检测的临床样本；
1.2阳性质控品：试剂盒内阳性质控品。
2逆转录
2.1扩增试剂准备(PCRⅠ室)
从试剂盒中取出检测缓冲液和引物，在冰上或2～8℃融化后，将所有组分轻微摇晃混匀并低速简短离心。每一反应按照以下比例配制反应体系：
表2：试剂盒逆转录反应体系表
Figure PCTCN2020099588-appb-000005
计算好各试剂使用量(即每一个逆转录反应液的总量应满足的人份数包括：临床样本的个数、1管阳性质控品和1个PCR阴性对照；每一个逆转录反应液的总量＝人份数*逆转录反应体系)，加入适当体积离心管中，充分混匀，简短离心。反应液配置完毕，进行分装，在每一个逆转录反应孔加入5μL反应液，分装完毕，转移至PCRⅡ室。
2.2加样(PCRⅡ室)
在装有逆转录反应液的反应孔中加入“1扩增试剂准备(PCR前准备期)”步骤制备的核酸，每一份核酸的添加量是5.0μL/孔，PCR阴性对照孔中不添加任何样本或核酸。
2.3逆转录反应(检测区)
将反应管放入荧光PCR检测仪中，循环参数设定如下：
表3：试剂盒逆转录反应步骤表
Figure PCTCN2020099588-appb-000006
3 PCR检测
3.1扩增试剂准备(PCRⅠ室)
从试剂盒中取出检测缓冲液和引物探针，在冰上或2～8℃融化后，将所有组分轻微摇晃混匀并低速简短离心。每一反应按照以下比例配制反应体系：
表4：试剂盒qPCR反应体系表
Figure PCTCN2020099588-appb-000007
计算好各试剂使用量(即每一个qPCR反应液的总量应满足的人份数包括：临床样本的个数、1管阳性质控品和1个PCR阴性对照，每一个qPCR反应液的总量＝人份数*qpcr反应体系)，加入适当体积离心管中，充分混匀，简短离心。反应液配置完毕，进行PCR反应液的分装，在每个聚合酶链式反应孔中加入20μL反应液，分装完毕，转移至PCRⅡ室。
3.2加样(PCRⅡ室)
将步骤2中的逆转录产物1、2、3、4分别加入装有qPCR反应试剂1、2、3、4的反应孔中，添加量是5.0μL/孔。
3.3 PCR反应(检测区)
将反应管放入荧光PCR检测仪中，循环参数设定如下：
表5：试剂盒PCR反应步骤表
Figure PCTCN2020099588-appb-000008
Figure PCTCN2020099588-appb-000009
荧光信号的收集定为FAM、HEX和CY5，数据的采集定在57℃。采用ABI7500仪器时，请将“Quencher”一栏设置为“none”，“passive reference”一栏选为“none”。
【检验结果的解释】
反应结束后，仪器自动保存结果，分析图像后调节Baseline的Start值、End值和Threshold值(可自行调节，Start值可以在3-15之间，End值位于5-20之间)，调整PCR阴性对照的扩增曲线。
PCR阴性对照的Ct值应为：使qPCR反应1的HEX(122)>33.73；使qPCR反应2的FAM(144)>36.50；使qPCR反应3的HEX(451)>34.10；阳性质控品的FAM、HEX的荧光Ct值均≤31.00。
以上条件需要在同一次试验中同时满足，否则认为PCR反应无效，需要重新进行检测。具体如下：
表6：试剂盒PCR反应结果判定表
Figure PCTCN2020099588-appb-000010
2.试剂盒分析性能评估
具体实行方案如下：
主要从灵敏度、特异性、精密度三个方面对检测试剂盒进行分析性能评估。
1.灵敏度
本试剂盒用于人血清样本中:3种miRNAs(miR-122-5p,miR-144-3p,miR-451a)的定性检测，针对其灵敏度的测试，实验方案与结果如下。
1.1实验方案
采用合成的RNA片段进行模拟试验，取合成的miR-122-5p,miR-144-3p,miR-451a的3种RNA干粉(2.5nmol/管)，分别用150μL无菌无RNase水溶解为1E13copies/μL的核酸溶液，以此为原液进行后续的稀释，共选取3个浓度进行灵敏度测试，具体的浓度如表7所示。每个miRNAs重复检测20次，将具有90％以上的阳性检出率的浓度水平作为最低检出浓度，检测所用的仪器为ABI7500实时荧光定量PCR仪。
表7：最低检测限实验所用的RNA信息表
Figure PCTCN2020099588-appb-000011
1.2结果与分析
检测结果如表8和表9所示，结果表明，:3种miRNAs的前两个浓度的阳性检出率均大于90％，第三个浓度梯度的阳性检出率小于20％，因此本试剂盒对miR-122-5p的最低检出浓度为5×10 3copies/μL，对miR-144-3p的最低检出浓度为5×10 4copies/μL，对miR-451a的最低检出浓度为5×10 3copies/μL。实验所用仪器为ABI7500实时荧光定量PCR仪。
表8：miR-122-5p最低检出浓度的实验结果
Figure PCTCN2020099588-appb-000012
Figure PCTCN2020099588-appb-000013
表9：miR-144-3p和miR-451a最低检出浓度的实验结果
Figure PCTCN2020099588-appb-000014
Figure PCTCN2020099588-appb-000015
2.特异性
本试剂盒的特异性主要从交叉反应和干扰物质两个方面来验证，具体实验方案和结果如下。
2.1交叉反应
2.1.1实验方案
本实验选用合成的5个与miR-122-5p、miR-144-3p和miR-451a序列相近的miRNAs(miR-122b、miR-122b-5p、miR-122-3p、miR-514a和miR-677a)作为检测对象，用ABI7500实时荧光定量PCR仪进行检测。
2.1.2结果与分析
检测结果如表10所示，结果表明，与本试剂盒所检测的3种miRNAs序列相近的miR-122b、miR-122b-5p、miR-122-3p、miR-514a和miR-677a的检测结果均为阴性，因此本试剂盒无交叉反应。
表10：交叉反应实验结果
Figure PCTCN2020099588-appb-000016
2.2干扰物质
2.2.1实验方案
潜在的干扰物质主要包括游离血红蛋白、胆红素、甘油三酯和总G型免疫球蛋白(IgG)， 验证实验应在潜在的最差条件下进行，即应选择临界值浓度水平的样本(267和216)，并对其添加医学水平相关的潜在最大浓度的干扰物质。
2.2.2结果与分析
检测结果如表12所示，结果表明，临界值浓度水平的样本在加入4种不同的干扰物质之后，目标荧光Ct值与不加干扰物质的样本(对照组)相比没有显著性差异(单因素方差分析和配对t检验p＞0.05)，即检验样本中含有以下浓度的干扰物质，不会影响本试剂盒对样本检测而结果的判定：
表11：干扰物质及相应的浓度
干扰物质名称 胆红素 游离血红蛋白 甘油三脂 总G型免疫球蛋白(IgG)
浓度 20mg/L 2mg/mL 1mg/mL 25mg/L
表12:干扰物质实验结果
Figure PCTCN2020099588-appb-000017
结论：由2.1和2.2的实验结果可知，本试剂盒的特异性良好。
3.精密度
精密度是指在规定条件下，相互独立的检测结果间的一致性。
3.1实验方案
本实验分别选取了高低两个浓度水平的miRNAs的miR-122-5p、miR-144-3p和miR-451a的RNA溶液，共6个不同浓度的RNA溶液进行测试。每个RNA溶液重复检测20次，计算检测结果Ct值的变异系数(CV，％)，CV值应小于5％。检测所用仪器为ABI7500实时荧光定量PCR仪。
3.2结果与分析
检测结果如表13所示，结果表明，6个不同浓度的RNA溶液重复检测20次，Ct值的变异系数均小于5％。
表13：精密度实验结果
Figure PCTCN2020099588-appb-000018
Figure PCTCN2020099588-appb-000019
3.3结论
上述实验结果表明，本试剂盒精密度良好。
综上，本试剂盒在灵敏度、特异性、精密度三个方面都符合标准。
3.用试剂盒检测样本人群中的目的miRNAs。
具体实行方案如下：
【实验目的】
用TaqMan探针法检测血清样本里的miR-122-5p,miR-144-3p,miR-451a，看这3种miRNAs在各样本中的含量是否有差异。
【实验原料】
TAKARA TaqTM Hot Start Version；MaqMAXTM miVanaTM Total RNA Isolation Kit；PrimeScriptTMRT reagent Kit；122RT-loop2；144RT-loop2；451RT-loop2；EV-RT-loop2；122RT-loop-F2；122RT-loop-R7；122-RT P-HEX；144RT-loop-F2；144RT-loop-R10；144-RT P-FAM；451RT-loop-R6；EV-RT-loop-F；EV-RT-loop-R；
EV-RT P-HEX；ddH2O；1×TE；Mg2+(25mM)；dNTP(25mM)。
【实验步骤】
根据mirVana TM PARIS TM RNA Isolation Kit(Applied Biosystem p/n AM1556)的说明书提取样品中的miRNA。
用1×TE将特异的逆转录引物122RT-loop2、144RT-loop2、451RT-loop2和EV-RT-loop2稀释至2uM，再使用PrimeScriptTMRT reagent Kit中的成分按以下表格配制逆转录反应体系：
Figure PCTCN2020099588-appb-000020
3.完成逆转录反应后，用1×TE将122RT-loop-F2、122RT-loop-R7、122-RT P-HEX、144RT-loop-F2、144RT-loop-R10、144-RT P-FAM、451RT-loop-R6、EV-RT-loop-F；EV-RT-loop-R；和EV-RT P-HEX稀释至20μM，然后使用TAKARA Taq TM Hot Start Version中的成分按以下表格配制qPCR反应体系：
miRNA-122反应体系 每测试/μL
10×buffer 2.5
HS Taq酶 0.4
Mg 2+(25mM) 2
dNTP(25mM) 0.2
122RT-loop-F2 a 0.4
122RT-loop-R7 b 0.4
122-RT P-HEX c 0.2
ddH 2O 16.9
RT反应产物(模板) 2
Total 25
miRNA-144反应体系 每测试/μL
10×buffer 2.5
HS Taq酶 0.4
Mg 2+(25mM) 2
dNTP(25mM) 0.2
144RT-loop-F2 0.4
144RT-loop-R10 0.4
144-RT P-FAM 0.2
ddH 2O 16.9
RT反应产物(模板) 2
Total 25
miRNA-451反应体系 每测试/μL
10×buffer 2.5
HS Taq酶 0.4
Mg 2+(25mM) 2
dNTP(25mM) 0.2
122RT-loop-F2* 0.4
451RT-loop-R6 0.4
122-RT P-HEX* 0.2
ddH 2O 16.9
RT反应产物(模板) 2
Total 25
内标(EV)反应体系 # 每测试/μL
10×buffer 2.5
HS Taq酶 0.4
Mg 2+(25mM) 2
dNTP(25mM) 0.2
EV-RT-loop-F 0.4
EV-RT-loop-R 0.4
EV-RT P-HEX 0.2
ddH 2O 16.9
RT反应产物(模板) 2
Total 25
(a：122RT-loop-F2是检测miRNA-122-5p的上游引物，b：122RT-loop-R7是检测miRNA-122-5p的下游引物，c：122-RT P-HEX是检测miRNA-122-5p的探针，后缀F代表上游引物，后缀R代表下游引物后缀P代表探针，P后面的字母代表修饰的荧光基团，其余以此类推。*：miR-122-5p和miR-451a共用上游引物和探针；#:内标是外源内标，人为设计的序列，名称为EV)
反应程序为：
Figure PCTCN2020099588-appb-000021
具体引物探针序列
Figure PCTCN2020099588-appb-000022
(a：122-RT-loop2是miRNA-122-5p的逆转录引物，b：122RT-loop-F2是检测miRNA-122-5p的上游引物，c：122RT-loop-R7是检测miRNA-122-5p的下游引物，d：122-RT P-HEX是检测miRNA-122-5p的探针，后缀F代表上游引物，后缀R代表下游引物后缀P代表探针，P后面的字母代表修饰的荧光基团，其余以此类推。*：miR-122-5p和miR-451a共用上游引物和探针；#:内标是外源内标，人为设计的序列，名称为EV)
本发明公开了包含三种与人类肝癌相关的血液miRNAs标志物的试剂盒的研发及其应用。具体的标志物为血清来源的miR-122-5p,miR-144-3p和miR-451a。本发明以差异miRNAs作为检测试剂盒的核心组分，配合对应的内参组装成检测试剂盒，并通过在临床样本中的实际检测应用，优化试剂盒反应条件和试剂配比，调整其检测参数，初步建立临床检测及分析的标准化流程，并用于肝癌的早期诊断和转移风险评估。
本发明的优点和有益效果如下：
1.血清中miRNAs是一种新型生物标志物，拥有稳定，微创，易于检测等优点，可以大大提高疾病诊断的敏感性和特异性，对该类小分子生物标志物的研究和成功开发有助于肝癌的辅助诊断，也为其它疾病的研究提供借鉴。
2.通过检测特定的miRNAs的表达，来诊断人类肝癌是否发生的试剂盒是一种系统，全面的诊断试剂盒，可用于肝癌患者的辅助诊断，有助于预测肝癌病人的转移情况，可以在临床上帮助医生更好的掌握患者病情，对疾病发展趋势有更好的把握，可以及时采取更具有针对性，更加精准的个性化治疗。
3.用TaqMan探针法检测血清样本里的miRNAs相较于SYBR GREEN法，前者特异性更高，重复性更好，结果更可靠。
4.多个miRNAs指标的诊断试剂盒相较于单一miRNAs指标检测，可以提高试剂盒结果的灵敏度和特异性，且多指标miRNAs检测可以减少因单一指标在样本中的个体表达差异导致的实验结果误差，使结果更加精准、可信。
附图说明
图1显示利用试剂盒检测血清中miR-144-3P在正常人、肝癌病人无转移和肝癌病人伴转移三组中的表达情况；
图2显示利用试剂盒检测血清中miR-451a在正常人、肝癌病人无转移和肝癌病人伴转移三组中的表达情况；
图3显示利用试剂盒检测血清中miR-122-5p在正常人、肝癌病人无转移和肝癌病人伴转 移三组中的表达情况；
图4显示利用ROC曲线分析miR-144-3P、miR-451a和miR-122-5p各自区分以及联合检测区分肝癌组和正常组时的敏感性和特异性。
具体的实施方式
实施例1，样本的收集和样本资料的整理
样本由浙江大学医学院附属第一医院提供(样本在收集的2h内全都用相同的方法进行处理，获取血清，分装，保存条件均一)，通过对样本资料的整理，发明人从中选择了符合条件的原发性肝癌病人42例(即收集前未进行任何针对肝癌的手术，化疗，药物治疗等处理)和39例与实验组同一时期收集的正常人的外周血。
实施例2，样本miRNA的提取
在本发明的具体操作中，用mirVana TM PARIS TM Kit(Applied Biosystem p/n AM1556)提取样本Total RNA。
2.1在2×Denaturing Solution溶液中添加375μLβ-巯基乙醇，混匀后放置备用；在miRNA Wash Solution 1中添加21mL无水乙醇，混匀后放置备用；在Wash Solution 2/3中添加40mL无水乙醇，混匀后放置备用。
2.2取300μL血清，加入等量的2×Denaturing Solution，涡旋混匀，冰上放置5分钟，每个样加5ul外源内标。
2.3添加与总体积等量的酚/氯仿，涡旋混匀30-60秒，室温，16000g离心10min。
2.4小心吸取上清到新的1.5mL离心管中，添加1.25倍体积的无水乙醇，涡旋混匀。
2.5将洁净的离心柱放置到洁净的收集管中，吸取上一步的液体到柱子中，10000g，室温离心30秒，弃流过液，将离心柱重新放置到收集管中。重复该步骤，直到所有的液体过柱。
2.6吸取700uL miRNA Wash Solution 1到离心柱中，10000g，室温离心15秒，弃流过液，将离心柱重新放置到收集管中
2.7吸取500uL Wash Solution 2/3过柱两次，空柱离心1分钟。将离心柱放置到新的收集管中，柱中心加入50uL 95℃预热的nuclease-free水，室温最高转速离心30秒，收集管中的液体即为提取的Total RNA，可放置在-70℃保存。
实施例3，试剂盒检测样本目的miRNAs
对实施例1中收集的样本进行miRNAs的QPCR检测
3.1反转录反应：在反应管加入4uL RNase Free dH2O、2uL 5×缓冲液 (PrimeScriptBuffer2)、0.5uL特异地逆转录引物、0.5uL逆转录酶(PrimeScriptRT Enzyme Mix I)，3ul样本模板，最后体积10uL，42℃孵育15min，85℃5s，4℃∞。所获得的cDNA模板保存在-20℃备用。
3.3上机跑QPCR：采用25uL反应体系，配置反应体系如表格所示。配置和加样步骤均于冰上进行。本反应适用于ABI7500实时荧光定量PCR仪。
实施例4分析各miRNAs对肝癌发生和转移的预测诊断
以miR-122-5p,miR-144-3p,miR-451a为研究对象，用试剂盒对42例肝癌血清样本，39例正常人血清样本进行检测，根据所得ct值结果分析各miRNAs对肝癌发生和转移的预测诊断，并对正常对照组和肝癌病人组的敏感性和特异性作比较。
具体结果：实验证明肝癌组血清中的miR-144-3p,miR-451a明显低于正常组。对肝癌病人组的数据进行进一步的分析，将肝癌病人分为有转移(影像学显示有远处转移或者肝内转移或者门静脉癌栓的病人)和无转移组，实验结果表明，有转移组的肝癌病人的miR-144-3p，miR-451a表达水平明显低于无转移组,表明这两个指标与肝癌恶性程度相关，而肝癌组血清中的miR-122-5p明显高于正常组，但在肝癌有无转移组中无明显差异。
利用ROC曲线分析显示，miR-122-5p在区分肝癌组和正常组时，其AUC值为0.783，miR-144-3p在区分肝癌组和正常组时，其AUC值为0.808，miR-451a在区分肝癌组和正常组时，其AUC值为0.858，而三个指标联合时，AUC值为0.938。实验结果显示血清miR-122-5p、miR-144-3P和miR-451a作为肝癌生物标志物，具有较好的灵敏性和特异性，同时三个指标联合诊断具有更好的效果。
曲线下的面积
Figure PCTCN2020099588-appb-000023
Figure PCTCN2020099588-appb-000024
a.在非参数假设下
b.零假设：实面积＝0.5。

Claims (7)

  1. 用于诊断肝癌和预测肝癌转移的血清miRNA标志物，所述的标志物为血清来源的miR-122-5p,miR-144-3p和miR-451a。
  2. 用于检测权利要求1所述血清miRNA标志物的试剂，包括QPCR实验中使用的反转录引物以及扩增引物
    Figure PCTCN2020099588-appb-100001
  3. 根据权利要求2所述的试剂，其特征在于：其中扩增引物的扩增方法为TaqMan探针法。
  4. 权利要求2或3所述试剂在诊断肝癌和预测肝癌转移中的应用。
  5. 用于诊断人类肝癌和预测肝癌转移的试剂盒，包含用于检测权利要求1所述血清miRNA标志物的试剂。
  6. 根据权利要求5所述的试剂盒，其特征在于：所述试剂包括QPCR实验中使用的反转录引物以及扩增引物；所述的扩增方法用的是TaqMan探针法。
  7. 权利要求5或6所述试剂盒在诊断肝癌和预测肝癌转移中的应用。
PCT/CN2020/099588 2020-05-31 2020-06-30 用于诊断肝癌和预测肝癌转移的血清miRNA标志物及其检测试剂盒 WO2021243782A1 (zh)

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