WO2023010325A1 - 一组检测人嗜酸性粒细胞阳离子蛋白mRNA表达的引物组、试剂盒和检测方法 - Google Patents

一组检测人嗜酸性粒细胞阳离子蛋白mRNA表达的引物组、试剂盒和检测方法 Download PDF

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WO2023010325A1
WO2023010325A1 PCT/CN2021/110517 CN2021110517W WO2023010325A1 WO 2023010325 A1 WO2023010325 A1 WO 2023010325A1 CN 2021110517 W CN2021110517 W CN 2021110517W WO 2023010325 A1 WO2023010325 A1 WO 2023010325A1
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probe
ecp
kit
primer set
pcr reaction
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PCT/CN2021/110517
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French (fr)
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程雷
沈华浩
吴善东
刘奕
吴周杰
蒋学翰
王教峰
王溢飞
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杭州浙大迪迅生物基因工程有限公司
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Priority to PCT/CN2021/110517 priority Critical patent/WO2023010325A1/zh
Publication of WO2023010325A1 publication Critical patent/WO2023010325A1/zh

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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • 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

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  • the invention belongs to the technical field of biological detection, and in particular relates to a set of primers, a kit and a detection method for detecting the expression of human eosinophilic cationic protein mRNA.
  • Eosinophil cationic protein is a strongly basic granule protein released by eosinophils (eosinophil, EOS) after activation. It exists in the matrix part of EOS granules, accounting for 30% of the granules. ECP is a specific marker of EOS activation, which can reflect the degree of EOS activation, and it also has a strong cytotoxic effect. ECP can be found in most body fluids of the human body, such as serum, nasal secretions, bronchial flushing fluid, saliva, tears, etc.
  • EOS degranulation protein For inflammatory diseases associated with eosinophil infiltration, the amount of EOS degranulation protein is more important than the total amount of EOS infiltration.
  • EOS granule proteins mainly include: ECP, EOS major basic protein (MBP), EOS peroxidase (eosinophil peroxidase, EPO) and EOS neurotoxin (eosinophil-derived neurotoxin, EDN).
  • ECP EOS major basic protein
  • EOS peroxidase eosinophil peroxidase, EPO
  • EOS neurotoxin eosinophil-derived neurotoxin
  • ECP is a very important airway active substance. It has various airway effects such as contracting bronchial smooth muscle, destroying alveolar surfactant, lysing cell membrane, and activating inflammatory cells. It is involved in airway hyperresponsiveness and airway epithelial damage in asthma. and other pathophysiological processes. Eosinophilic inflammation in asthmatic patients leads to increased ECP levels in blood and other body fluids such as bronchoalveolar fluid and sputum. ECP levels objectively reflect the degree of eosinophilic verification in asthmatic patients, and high levels indicate the inflammatory state of asthmatic patients. ECP measurements can be used to monitor asthmatic inflammation, guide steroid therapy for asthma, and identify patients who are not adhering to treatment.
  • ECP can also be used as a biochemical marker of allergic reactions. After people with allergies contact or inhale or ingest allergens, it will cause allergic asthma, allergic dermatitis, etc., and also increase the ECP level of allergic patients. In both immunotherapy and avoidance studies, ECP is an effective marker of whether avoidance and treatment are successful or effective.
  • ECP enzyme-linked immunosorbent assay
  • the object of the present invention is to provide a set of primers and kits for detecting the expression of human eosinophil cationic protein mRNA, which can quantitatively detect the expression level of human eosinophil cationic protein (ECP) mRNA in one step, A detection method with high accuracy, wide detection range and high sensitivity is provided for the detection of the protein.
  • ECP human eosinophil cationic protein
  • the invention provides a set of primers for detecting the expression of human eosinophil cationic protein mRNA, the primer set includes ECP-F, ECP-R, GAPDH-F and GAPDH-R;
  • the nucleotide sequence of described ECP-F is shown in SEQ ID NO.1;
  • the nucleotide sequence of described ECP-R is shown in SEQ ID NO.2;
  • the nucleotide sequence of described GAPDH-F is shown in SEQ ID NO.2; Shown in ID NO.3;
  • the nucleotide sequence of described GAPDH-R is shown in SEQ ID NO.4.
  • the primer set also includes probe E-Probe and probe G-Probe;
  • the nucleotide sequence of the probe E-Probe is shown in SEQ ID NO.5; the nucleotide sequence of the probe G-Probe is shown in SEQ ID NO.6.
  • both the 5' ends of the probe E-Probe and the probe G-Probe are connected with a fluorescent group, and the 3' ends are connected with a quenching group.
  • the 5' ends of the probe E-Probe and the probe G-Probe are connected to different fluorescent groups, and the 3' ends are connected to the same quenching group.
  • the present invention also provides a kit for one-step detection of human eosinophil cationic protein mRNA expression, said kit including the above primer set.
  • the kit also includes PCR reaction solution, enzyme mixture, ROX reference dye and nuclease-free water;
  • the enzyme in described enzyme mixed solution comprises Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody;
  • the mass ratio of described Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody is 13:5: 3:1.
  • the PCR reaction solution includes dNTP mix, MgCl 2 and buffer.
  • the kit also includes human eosinophil cationic protein mRNA standard.
  • the present invention also provides a method for detecting the expression level of ECP mRNA in one step based on the above kit, comprising the following steps: using the ECP standard product as a template, preparing a qRT-PCR reaction system, performing a qRT-PCR reaction, and comparing The value is the abscissa, and the quantitative curve is set with the Ct value as the ordinate;
  • RNA extracted from the sample as a template, prepare a qRT-PCR reaction system, perform a qRT-PCR reaction, and use the quantitative curve to measure the expression level of ECP.
  • the qRT-PCR reaction system is calculated in 20 ⁇ l, including: 2.4 ⁇ l of nuclease-free water, 10 ⁇ l of PCR reaction solution, 0.5 ⁇ l of enzyme mixture, 0.1 ⁇ l of ROX reference dye, 2 ⁇ l of primer set and probe mixture, and Template 5 ⁇ l.
  • the qRT-PCR reaction program includes: 42°C for 30 min; 95°C for 1 min; 95°C for 5s, 60°C for 31s, 40 cycles.
  • the present invention Compared with the prior art, the present invention has the beneficial effects that: the present invention provides a set of primers for detecting the expression of human eosinophil cationic protein (ECP) mRNA, and utilizes the primer set to construct a one-step detection kit .
  • ECP eosinophil cationic protein
  • the detection sensitivity is higher, clinical samples with low concentration can be detected, and ECP can be detected more sensitively
  • the detection range can span at least 6 orders of magnitude, which increases the accuracy of the detection results and can complete the detection of at least 80 people within 1 hour, so that the treatment effect can be dynamically monitored earlier, more accurately and faster and efficacy evaluation.
  • Using the primer set or kit of the present invention to detect changes in the level of ECP mRNA in patients can be used to monitor allergic diseases such as asthma or eosinophil infiltration inflammation, guide glucocorticoid treatment programs, and find patients who do not comply.
  • Figure 1 is the ECP mRNA TaqMan real-time fluorescent quantitative RT-PCR standard curve
  • Figure 2 is the results of precision testing, where 1: 1.0 ⁇ 10 6 copies/ ⁇ l, 2: 1.0 ⁇ 10 3 copies/ ⁇ l;
  • Figure 3 is a graph of the accuracy test results
  • Fig. 4 is sensitivity detection result figure
  • Figure 5 is a diagram of the detection results of clinical samples, in which 1: positive sample 4 GAPDH mRNA; 2: healthy control 2 GAPDH mRNA; 3: positive sample 4 ECP mRNA; 4: healthy control 2 ECP mRNA; 5: blank control NTC-GAPDH mRNA 6: blank control NTC-ECP mRNA;
  • Fig. 6 is under the situation of non-optimal primer, probe design, low value precision amplification curve
  • Fig. 7 is the influence of enzyme mixture on the amplification curve
  • Fig. 8 is the plasmid structure of pGM-T vector.
  • the present invention provides a set of primers for detecting the expression of human eosinophil cationic protein mRNA, said primer set includes ECP-F, ECP-R, GAPDH-F and GAPDH-R; both sets of primers are necessary technical features?
  • the nucleotide sequence of described ECP-F is shown in SEQ ID NO.1;
  • the nucleotide sequence of described ECP-R is shown in SEQ ID NO.2;
  • the nucleotide sequence of described GAPDH-F is shown in SEQ ID NO.2; Shown in ID NO.3;
  • the nucleotide sequence of described GAPDH-R is shown in SEQ ID NO.4.
  • the primer set preferably also includes probe E-Probe and probe G-Probe; the nucleotide sequence of the probe E-Probe is preferably as shown in SEQ ID NO.5; the probe The nucleotide sequence of G-Probe is preferably as shown in SEQ ID NO.6.
  • the combinations of probes and primers in the present invention are preferably: ECP-F, ECP-R and E-probe, GAPDH-F, GAPDH-R and G-Probe.
  • the 5' ends of the probe E-Probe and the probe G-Probe of the present invention are preferably connected with a fluorescent group, and the 3' ends are connected with a quenching group, and the probe E-Probe and the probe G -The 5' ends of the Probe are connected with different fluorescent groups, and the 3' ends are connected with the same quenching group.
  • the 5' end of the nucleotide sequence of the probe E-Probe is labeled with a FAM fluorescent reporter group, and the 3' end is labeled with a BHQ1 quencher group; the nucleoside of the probe E-Probe The 5' end of the acid sequence is labeled with the JOE fluorescent reporter group, and the 3' end is labeled with the BHQ1 quencher group.
  • the present invention lists the information of the above primer sets in Table 1.
  • the present invention also provides a kit for one-step detection of human eosinophil cationic protein mRNA expression, said kit including the primer set described in the above technical scheme.
  • PCR reaction solution Preferably also include PCR reaction solution, enzyme mixed solution, ROX reference dye and nuclease-free water in the test kit of the present invention
  • the enzyme in described enzyme mixed solution comprises Taq enzyme, reverse transcriptase, RNase inhibitor and Taq Enzyme antibody; the mass ratio of the Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody is preferably 13:5:3:1. Use this ratio for optimal amplification.
  • the PCR reaction solution of the present invention preferably includes dNTP mix, MgCl 2 and buffer.
  • the source of the buffer is not particularly limited in the present invention.
  • the kit of the present invention further includes a human eosinophil cationic protein mRNA standard.
  • the preparation method of the human eosinophilic cationic protein (ECP) mRNA standard product is not particularly limited in the present invention, preferably entrusted to Nanjing GenScript Biotechnology Co., Ltd. to construct and synthesize, and dilute the RNA nuclease to 1.0 ⁇ 10 9 copeies/ ⁇ L is the ECP standard.
  • the standard product described in the present invention is preferably an ECP mRNA standard product, which is used to prepare a quantitative curve.
  • the upstream and downstream primers and probes it is preferable to prepare the upstream and downstream primers and probes to the working concentration before use.
  • the working concentration of the upstream and downstream primers is preferably 0.5-1 ⁇ M; the working concentration of the probe is preferably 1.5 ⁇ 2 ⁇ M.
  • Taq enzyme is a heat-resistant Taq DNA polymerase, utilizes its 3' ⁇ 5' polymerase activity to use DNA as a template, and adds deoxygenated mononucleotides in dNTPs to the 3-OH end one by one; Its 5′ ⁇ 3′exonuclease activity can recognize and eliminate mismatched primer ends, which is related to the correction function during the replication process, and can also hydrolyze nucleotides from the 5’ end, and can also work through several nucleotides , to excise mismatched nucleotides, thereby realizing strand replacement during chain elongation, and cutting off the replaced probe; reverse transcriptase can reverse transcribe mRNA into cDNA for PCR reaction; RNase inhibitor is used to Inhibit the activity of exogenous RNase; Taq enzyme antibody is an anti-Taq antibody for hot-start PCR, which inhibits the activity of DNA polymerase after binding to Taq enzyme, and can effectively inhibit the non-specific anne
  • the present invention also provides a method for detecting the expression level of ECP mRNA in one step based on the above kit, comprising the following steps: using the ECP standard product as a template, preparing a qRT-PCR reaction system, performing a qRT-PCR reaction, and comparing The value is the abscissa, and the quantitative curve is set with the Ct value as the ordinate;
  • RNA extracted from the sample as a template, prepare a qRT-PCR reaction system, perform a qRT-PCR reaction, and use the quantitative curve to measure the expression level of ECP.
  • the present invention does not specifically limit the extraction source of the RNA, which preferably includes whole blood, serum, nasal secretion, bronchial flushing fluid, saliva or tears. In the embodiment of the present invention, it is preferred to extract RNA from whole blood for the above detection.
  • the qRT-PCR reaction system of the present invention is calculated in 20 ⁇ l, and preferably includes: 2.4 ⁇ l of nuclease-free water, 10 ⁇ l of PCR reaction solution, 0.5 ⁇ l of enzyme mixture, 0.1 ⁇ l of ROX reference dye, 2 ⁇ l of primer set and probe mixture, and Template 5 ⁇ l.
  • the qRT-PCR reaction procedure of the present invention includes: 42°C for 30 minutes; 95°C for 1 minute; 95°C for 5s, 60°C for 31s, 40 cycles.
  • a set of primers, kits and detection methods for detecting human eosinophil cationic protein mRNA expression provided by the present invention are described in detail below in conjunction with the examples, but they cannot be interpreted as limiting the protection scope of the present invention.
  • test materials involved in the present invention are conventional purchases:
  • copy number [6.02 ⁇ 10 23 ⁇ RNA concentration (ng/ ⁇ l) ⁇ 10 -9 ]/[RNA length (bp) ⁇ 340], calculate the initial RNA copy number. Dilute with nuclease-free water to 1.0 ⁇ 10 9 copeies/ ⁇ l, which is the ECP standard.
  • EDTA anticoagulated whole blood samples were extracted with a whole blood total RNA kit, quantified with a Qubit 3 fluorometer, and diluted to 20 ng/ ⁇ l with nuclease-free water.
  • the ECP standard was diluted in a 10-fold gradient, and 1.0 ⁇ 10 7 to 1.0 ⁇ 10 2 copeies/ ⁇ l was selected as a template, and each dilution was replicated twice for TaqMan real-time fluorescent quantitative RT-PCR detection to generate a standard curve.
  • the TaqMan real-time fluorescence quantification established by the present invention is compared with a certain domestic brand human ribonuclease A3 (RNASE3/ECP) enzyme-linked immunosorbent assay kit and the widely used ImmunCAPTM ECP reagent abroad.
  • RNASE3/ECP human ribonuclease A3
  • the RT-PCR detection method has better sensitivity than imported fluorescent enzyme immunoassay reagents and better specificity than domestic enzyme immunoassay reagents.
  • ECP-F (SEQ ID NO.7): TGAACCCCAGAACAACCAG;
  • ECP-R (SEQ ID NO. 8): CAGTTTATTGCAGGGTTCACA;
  • Amplify with a non-optimal ratio of enzyme mixture (the mass ratio of Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody is 11:6:4:1) and an optimal ratio of enzyme mixture
  • amplification results using the non-optimal enzyme mixture ratio are shown in A in Figure 7, and the amplification results using the optimal enzyme mixture ratio are shown in Figure 7B. It can be seen that the amplification effect of the best enzyme mixture is better.

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Abstract

本发明提供了一组检测人嗜酸性粒细胞阳离子蛋白mRNA表达的引物组、试剂盒和检测方法，属于生物检测技术领域。本发明所述引物组包括ECP-F、ECP-R、GAPDH-F和GAPDH-R：所述ECP-F的核苷酸序列如SEQ ID NO．1所示；所述ECP-R的核苷酸序列如SEQ ID NO．2所示；所述GAPDH-F的核苷酸序列如SEQ ID NO．3所示；所述GAPDH-R的核苷酸序列如SEQ ID NO．4所示。本发明还提供了一种包含上述引物组的试剂盒，可一步法定量检测人ECPmRNA的表达水平。

Description

一组检测人嗜酸性粒细胞阳离子蛋白mRNA表达的引物组、试剂盒和检测方法 技术领域
本发明属于生物检测技术领域，具体涉及一组检测人嗜酸性粒细胞阳离子蛋白mRNA表达的引物组、试剂盒和检测方法。
背景技术
嗜酸性粒细胞阳离子蛋白(eosinophil cationic protein，ECP)是嗜酸性粒细胞(eosinophil，EOS)活化后释放出的强碱性颗粒蛋白，存在于EOS颗粒的基质部分，占颗粒的30％。ECP是EOS激活后的特异性标志物，可以反映EOS的活化程度，其本身亦具有极强的细胞毒性作用。ECP可在人体大多数体液中发现，如血清、鼻腔分泌物、支气管冲洗液、唾液、泪液等。
对于嗜酸性粒细胞浸润相关的炎症疾病，EOS脱颗粒蛋白的含量多少比EOS浸润总数更重要。EOS颗粒蛋白主要有：ECP、EOS主要碱性蛋白(major basic protein，MBP)、EOS过氧化物酶(eosinophil peroxidase，EPO)及EOS神经毒素(eosinophil-derived neurotoxin，EDN)，其中ECP的毒性作用尤其显著。在炎症过程中，EOS被活化，发生脱颗粒，导致粘膜损伤，进而增加组织的过敏性，引起慢性炎症，释放ECP，同时引起外周血及膜粘液中ECP水平上升。
ECP是一种非常重要的气道活性物质，具有收缩支气管平滑肌、破坏肺泡表面活性物质、裂解细胞膜、活化炎性细胞等多种气道效应，参与哮喘的气道高反应性、气道上皮损伤等病理生理过程。哮喘患者嗜酸细胞性炎症导致血液和其它体液如支气管肺泡液和痰液的ECP水平升高，ECP水平客观反映了哮喘患者的嗜酸细胞验证程度，高水平提示哮喘患者的炎症状态。ECP测定可用于监测哮喘炎症，指导哮喘的激素治疗，以及发 现对治疗不依从的患者。同时ECP也可用作过敏反应的生化标志物，过敏人群接触或吸入或摄入过敏原后，会引发过敏性哮喘、过敏性皮炎等，也使过敏患者的ECP水平升高。在免疫治疗和回避研究中，ECP都是回避和治疗是否成功或有效的一个有效标志物。
现在市场上对于ECP的检测仍使用酶联免疫吸附法(enzyme linked immunosorbent assay，ELISA)试剂盒检测其在体液中的含量，尚未见检测ECP mRNA的商业化试剂盒。ELISA方法在检测过程中存在着检测范围小和灵敏度低的问题，且其准确性也存在问题，所以建立一种准确度高、检测范围广及灵敏度高的方法来检测ECP mRNA表达量是非常必要的。
发明内容
有鉴于此，本发明的目的在于提供提供一组检测人嗜酸性粒细胞阳离子蛋白mRNA表达的引物组和试剂盒，可一步法定量检测人嗜酸性粒细胞阳离子蛋白(ECP)mRNA的表达水平，为该该蛋白的检出提供准确度高、检测范围广及灵敏度高的检测手段。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一组检测人嗜酸性粒细胞阳离子蛋白mRNA表达的引物组，所述引物组包括ECP-F、ECP-R、GAPDH-F和GAPDH-R；
所述ECP-F的核苷酸序列如SEQ ID NO.1所示；所述ECP-R的核苷酸序列如SEQ ID NO.2所示；所述GAPDH-F的核苷酸序列如SEQ ID NO.3所示；所述GAPDH-R的核苷酸序列如SEQ ID NO.4所示。
优选的，所述引物组还包括探针E-Probe和探针G-Probe；
所述探针E-Probe的核苷酸序列如SEQ ID NO.5所示；所述探针G-Probe的核苷酸序列如SEQ ID NO.6所示。
优选的，所述探针E-Probe和探针G-Probe的5’端均连接有荧光基团，3’端均连接有淬灭基团。
优选的，所述探针E-Probe和探针G-Probe的5’端连接不同的荧光基团，3’端连接相同的淬灭基团。
本发明还提供了一种一步法检测人嗜酸性粒细胞阳离子蛋白mRNA 表达的试剂盒，所述试剂盒包括上述引物组。
优选的，所述试剂盒中还包括PCR反应液、酶混合液、ROX参比染料和无核酶水；
所述酶混合液中的酶包括Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体；所述Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体的质量比为13:5:3:1。
优选的，所述PCR反应液包括dNTP mix、MgCl 2和缓冲液。
优选的，所述试剂盒中还包括人嗜酸性粒细胞阳离子蛋白mRNA标准品。
本发明还提供了一种基于上述试剂盒的一步法检测ECP mRNA表达水平的方法，包括以下步骤：以ECP标准品为模板，配制qRT-PCR反应体系，进行qRT-PCR反应，以拷贝数对数值为横坐标，以Ct值为纵坐标设置定量曲线；
以样本提取得到的RNA为模板，配制qRT-PCR反应体系，进行qRT-PCR反应，利用所述定量曲线，测算ECP的表达水平。
优选的，所述qRT-PCR反应体系以20μl计，包括：无核酶水2.4μl、PCR反应液10μl、酶混合液0.5μl、ROX参比染料0.1μl、引物组和探针混合液2μl和模板5μl。
优选的，所述qRT-PCR反应的程序包括：42℃30min；95℃1min；95℃5s，60℃31s，40循环。
本发明与现有技术相比具有的有益效果是：本发明提供了一组检测人嗜酸性粒细胞阳离子蛋白(ECP)mRNA表达的引物组，并利用所述引物组构建了一步法检测试剂盒。利用所述引物组或所述试剂盒进行一步法检测ECP mRNA的表达水平时，与免疫学检测方法相比，检测的灵敏度更高，可以检测低浓度的临床样本，能够更灵敏地探测到ECP的含量变化，检测范围可跨越至少6个数量级，增加了检测结果的准确性，能在1小时之内完成至少80人份检测，从而更早期、更准确、更快速地对治疗效果进行动态监测和疗效评估。利用本发明所述引物组或试剂盒，检测患者ECP mRNA水平变化，可以用于监测哮喘等过敏性疾病或嗜酸性粒细胞 浸润类炎症，指导糖皮质激素治疗方案，发现不依从的患者。
附图说明
图1为ECP mRNA TaqMan实时荧光定量RT-PCR标准曲线；
图2为精密度检测结果图，其中1：1.0×10 6copies/μl，2：1.0×10 3copies/μl；
图3为准确度检测结果图；
图4为灵敏度检测结果图；
图5为临床样本检测结果图，其中1：阳性样本4 GAPDH mRNA；2：健康对照2 GAPDH mRNA；3：阳性样本4 ECP mRNA；4：健康对照2 ECP mRNA；5：空白对照NTC-GAPDH mRNA；6：空白对照NTC-ECP mRNA；
图6为在非最佳引物、探针设计的情况下，低值精密度扩增曲线图；
图7为酶混合液对扩增曲线的影响；
图8为pGM-T载体的质粒结构。
下面结合实施例和附图对本发明进一步说明。
具体实施方式
本发明提供了一组检测人嗜酸性粒细胞阳离子蛋白mRNA表达的引物组，所述引物组包括ECP-F、ECP-R、GAPDH-F和GAPDH-R；两组引物都是必要技术特征？
所述ECP-F的核苷酸序列如SEQ ID NO.1所示；所述ECP-R的核苷酸序列如SEQ ID NO.2所示；所述GAPDH-F的核苷酸序列如SEQ ID NO.3所示；所述GAPDH-R的核苷酸序列如SEQ ID NO.4所示。
在本发明中，所述引物组优选还包括探针E-Probe和探针G-Probe；所述探针E-Probe的核苷酸序列优选如SEQ ID NO.5所示；所述探针G-Probe的核苷酸序列优选如SEQ ID NO.6所示。本发明所述探针和引物的组合方式优选为：ECP-F、ECP-R和E-probe，GAPDH-F、GAPDH-R和G-Probe。
本发明所述探针E-Probe和探针G-Probe的5’端优选均连接有荧光基团，3’端均连接有淬灭基团，且所述探针E-Probe和探针G-Probe的5’端分别连接不同的荧光基团，3’端连接相同的淬灭基团。在本发明实施例中， 所述探针E-Probe的核苷酸序列的5’端标记FAM荧光报告基团，3’端标记BHQ1淬灭基团；所述探针E-Probe的核苷酸序列的5’端标记JOE荧光报告基团，3’端标记BHQ1淬灭基团。
本发明将上述引物组的信息列于表1。
表1 TaqMan实时荧光定量PCR引物组信息
Figure PCTCN2021110517-appb-000001
本发明还提供了一种一步法检测人嗜酸性粒细胞阳离子蛋白mRNA表达的试剂盒，所述试剂盒包括上述技术方案所述引物组。
本发明所述试剂盒中优选还包括PCR反应液、酶混合液、ROX参比染料和无核酶水；所述酶混合液中的酶包括Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体；所述Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体的质量比优选为13:5:3:1。使用该比例可以得到最佳的扩增效果。
本发明所述PCR反应液优选包括dNTP mix、MgCl 2和缓冲液。本发明对所述缓冲液的来源并没有特殊限定。
本发明所述试剂盒中优选还包括人嗜酸性粒细胞阳离子蛋白mRNA标准品。本发明对所述人嗜酸性粒细胞阳离子蛋白(ECP)mRNA标准品的制备方法并没有特殊限定，优选委托南京金斯瑞生物科技有限公司构建及合成，并将RNA无核酶水稀释至1.0×10 9copeies/μL，即为ECP标准品。本发明所述标准品优选为ECP mRNA标准品，用于配制定量曲线。
本发明所述试剂盒中，优选将上、下游引物和探针配制到工作浓度后，再行使用，所述上、下游引物的工作浓度优选为0.5～1μM；所述探针的工作浓度优选为1.5～2μM。
在本发明中，Taq酶为耐热的Taq DNA聚合酶，利用其3′→5′聚合 酶活性以DNA为模板，将dNTP中的脱氧单核苷酸逐个加到3-OH末端；同时利用其5′→3′外切酶活性即能识别和消除错配的引物末端，与复制过程中校正功能有关，又可以从5’端水解核苷酸，还能经过几个核苷酸起作用，切除错配的核苷酸，由此在链延伸过程中实现链替换，并将被替换的探针切断；逆转录酶可将mRNA逆转录成cDNA以进行PCR反应；RNA酶抑制剂用来抑制外源性RNase的活性；Taq酶抗体是热启动PCR用抗Taq抗体，其与Taq酶结合后抑制DNA聚合酶活性，能够在低温条件下有效抑制引物的非特异性退火及引物二聚体引起的非特异性扩增，Taq酶抗体在PCR反应最初的DNA变性步骤中变性，Taq酶恢复活性，实现PCR扩增。
本发明还提供了一种基于上述试剂盒的一步法检测ECP mRNA表达水平的方法，包括以下步骤：以ECP标准品为模板，配制qRT-PCR反应体系，进行qRT-PCR反应，以拷贝数对数值为横坐标，以Ct值为纵坐标设置定量曲线；
以样本提取得到的RNA为模板，配制qRT-PCR反应体系，进行qRT-PCR反应，利用所述定量曲线，测算ECP的表达水平。
本发明对所述RNA的提取来源并没有特殊限定，优选包括全血、血清、鼻腔分泌物、支气管冲洗液、唾液或泪液，本发明实施例中，优选提取全血的RNA进行上述检测。
本发明所述qRT-PCR反应体系以20μl计，优选包括：无核酶水2.4μl、PCR反应液10μl、酶混合液0.5μl、ROX参比染料0.1μl、引物组和探针混合液2μl和模板5μl。
本发明所述qRT-PCR反应的程序包括：42℃30min；95℃1min；95℃5s，60℃31s，40循环。
在本发明中，所述定量曲线，以拷贝数对数值为横坐标(x)，Ct值为纵坐标(y)：y＝-3.27x+35.533(R 2＝0.999)；线性范围为1.0×10 2-1.0×10 7copies/μL。利用本发明所述方法对ECP mRNA进行检测，具有比进口荧光酶免法试剂更好的灵敏度和比国产酶免试剂更好的特异性。
下面结合实施例对本发明提供的一组检测人嗜酸性粒细胞阳离子蛋 白mRNA表达的引物组、试剂盒和检测方法进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
本发明中所涉及的试材如无特殊说明，均为常规购买得到：
全血总RNA试剂盒(杭州新景生物试剂开发有限公司，货号：5201050)；
HiScribe T7 High Yield RNA Synthesis Kit(New England Biolabs，货号：E2050S)；
Applied Biosystems TM7300荧光定量PCR仪(ThermoFisher，美国)；
-80℃低温冰箱(ThermoFisher，美国)；
高速低温台式离心机(Eppendorf，德国)；
Qubit 3荧光计(ThermoFisher，美国)。
实施例1
1、委托上海桑尼生物科技有限公司合成表1所示的引物和探针。
2、标准品制备
体外转录。采用pGM-T连接试剂盒[天根生化科技(北京)有限公司，货号：VT202-01]，以pGM-T为载体构建ECP质粒DNA(委托南京金斯瑞生物科技有限公司构建及合成，图8)，将ECP质粒DNA用HiScribe T7 High Yield RNA Synthesis Kit(NEW ENGLAND BioLabs公司生产，货号：E2040S)体外转录成mRNA。
根据拷贝数计算公式：拷贝数＝[6.02×10 23×RNA浓度(ng/μl)×10 -9]/[RNA长度(bp)×340]，计算RNA初始拷贝数。用无核酶水稀释至1.0×10 9copeies/μl，即为ECP标准品。
3、全血RNA提取及稀释
EDTA抗凝全血样本用全血总RNA试剂盒提取全血总RNA，采用Qubit 3荧光计定量后，用无核酶水稀释至20ng/μl。
4、TaqMan实时荧光定量PCR
以标准品/全血RNA为模板，配制20μL体系：无核酶水2.4μl、PCR反应液10μl、酶混合液0.5μl、ROX参比染料0.1μl、引物组和探针混合液2μl和标准品/全血RNA 5μl。
qRT-PCR反应的程序：42℃30min；95℃1min；95℃5s，60℃31s，40循环，并设置检测荧光素：FAM、JOE，参比荧光：ROX，反应体系：20μl，荧光信号收集：60℃31sec。
5、标准曲线的生成
将ECP标准品按10倍梯度进行稀释，选择1.0×10 7～1.0×10 2copeies/μl作为模板，每个稀释度2个重复，进行TaqMan实时荧光定量RT-PCR检测，生成标准曲线。
标准曲线如图1所示，以拷贝数对数值为横坐标，Ct值为纵坐标，得到回归方程式：y＝-3.27x+35.533(R 2＝0.999)，说明标准方程的拷贝数对数值与Ct值具有极高的相关性。
6、精密度检测
选择1.0×10 6copies/μl、1.0×10 3copies/μl的标准品作为模板，每个浓度10个重复量，进行10次TaqMan实时荧光定量RT-PCR检测，分别计算每个浓度对数值的变异系数进行统计学分析，分析该检测方法的精密度。
结果如表2和图2所示，每个浓度对数值的变异系数分别为0.460％、1.575％，小于5％，表明本发明建立的TaqMan实时荧光定量RT-PCR检测方法具有极好的精密度。
表2 精密度检测结果
理论拷贝数 拷贝数对数值均值 SD C.V
1.0×10 6 5.950 0.027 0.460％
1.0×10 3 2.952 0.046 1.575％
7、准确度检测
选择1.0×10 5copies/μL标准品作为模板，3个重复量，进行3次TaqMan实时荧光定量RT-PCR检测，计算每个浓度对数值的绝对偏差。结果如表3所示，每个浓度对数值的绝对偏差分别为-0.130、-0.135、-0.143，在±0.5范围内，表明本发明建立的TaqMan实时荧光定量RT-PCR检测方法具有 极好的准确度。
表3 准确度检测
Figure PCTCN2021110517-appb-000002
8、灵敏度检测
选择10.0copies/μl标准品作为模板，25个重复量，进行25次TaqMan实时荧光定量RT-PCR检测，查看是否有扩增抬头，分析该检测方法的灵敏度。
结果如表4和图4所示，共计25次检测出结果，达100％，表明本发明建立的TaqMan实时荧光定量RT-PCR检测方法具有很高的灵敏度，最低检出拷贝数＜10copies/μl。
表4 灵敏度检测Ct值结果
32.951 32.621 32.816 32.644 33.448
32.658 32.922 32.140 32.984 33.033
32.850 32.916 33.579 32.549 32.359
33.004 33.427 32.696 33.106 32.580
32.437 33.301 33.245 33.015 32.425
9、临床样本检测
取阳性样本和健康对照全血样本按照3进行全血RNA提取和稀释，按照4进行TaqMan实时荧光定量RT-PCR检测。
和国内某品牌人核糖核酸酶A3(RNASE3/ECP)酶联免疫吸附测定试剂盒及国外应用较广的ImmunCAPTM ECP试剂比对结果如表5和图5所示，本发明建立的TaqMan实时荧光定量RT-PCR检测方法具有比进口荧光酶免法试剂更好的灵敏度和比国产酶免试剂更好的特异性。
表5 临床样本检测对比结果
Figure PCTCN2021110517-appb-000003
Figure PCTCN2021110517-appb-000004
比较例1、采用其它非最佳引物、探针进行扩增的结果
将实施例5中本发明所用体系中的引物、探针替换成其它非最佳引物、探针。结果如图6所示，标准品曲线扩增结果差，且扩增效率仅有68.547％。
采用的非最佳ECP引物、探针：
ECP-F(SEQ ID NO.7)：TGAACCCCAGAACAACCAG；
ECP-R(SEQ ID NO.8)：CAGTTTATTGCAGGGTTCACA；
E-Probe(SEQ ID NO.9)：(FAM)-CCAAAATCAAGTGGGGCGAT-(BHQ1)。
比较例2、酶混合液效果比较
用非最佳配比的酶混合液(Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体的质量比为11:6:4:1)和最佳配比的酶混合液分别扩增2例全血RNA样本，采用非最佳的酶混合液配比的扩增结果如图7中A所示，采用最佳的酶混合液配比的扩增结果如图7中B所示。可见最佳酶混合液的扩增效果较好。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。

Claims (13)

  1. 一组检测人嗜酸性粒细胞阳离子蛋白mRNA表达的引物组，其特征在于，所述引物组包括ECP-F、ECP-R、GAPDH-F和GAPDH-R；
    所述ECP-F的核苷酸序列如SEQ ID NO.1所示；所述ECP-R的核苷酸序列如SEQ ID NO.2所示；所述GAPDH-F的核苷酸序列如SEQ ID NO.3所示；所述GAPDH-R的核苷酸序列如SEQ ID NO.4所示。
  2. 根据权利要求1所述引物组，其特征在于，所述引物组还包括探针E-Probe和探针G-Probe；
    所述探针E-Probe的核苷酸序列如SEQ ID NO.5所示；所述探针G-Probe的核苷酸序列如SEQ ID NO.6所示。
  3. 根据权利要求2所述引物组，其特征在于，所述探针E-Probe和探针G-Probe的5’端均连接有荧光基团，3’端均连接有淬灭基团。
  4. 根据权利要求2或3所述引物组，其特征在于，所述探针E-Probe和探针G-Probe的5’端连接不同的荧光基团，3’端连接相同的淬灭基团。
  5. 根据权利要求4所述引物组，其特征在于，所述荧光基团包括FAM或JOE，所述淬灭基团包括BHQ1。
  6. 一种一步法检测人嗜酸性粒细胞阳离子蛋白mRNA表达的试剂盒，其特征在于，所述试剂盒包括权利要求1～5任一项所述引物组。
  7. 根据权利要求6所述试剂盒，其特征在于，所述试剂盒中还包括PCR反应液、酶混合液、ROX参比染料和无核酶水；
    所述酶混合液中的酶包括Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体；所述Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体的质量比为13:5:3:1。
  8. 根据权利要求7所述试剂盒，其特征在于，所述PCR反应液包括dNTP mix、MgCl 2和缓冲液。
  9. 根据权利要求6或7所述试剂盒，其特征在于，所述试剂盒中还包括人嗜酸性粒细胞阳离子蛋白mRNA标准品。
  10. 一种基于权利要求6～9任一项所述试剂盒的一步法检测ECP mRNA表达水平的方法，其特征在于，包括以下步骤：以样本提取得到的RNA为模板，配制qRT-PCR反应体系，进行qRT-PCR反应，利用ECP获得的定量曲线，测算ECP的表达水平。
  11. 根据权利要求10所述方法，其特征在于，所述定量曲线的获得，包括：以ECP标准品为模板，配制qRT-PCR反应体系，进行qRT-PCR反应，以拷贝数对数值为横坐标，以Ct值为纵坐标设置定量曲线。
  12. 根据权利要求10或权利要求11所述方法，其特征在于，所述qRT-PCR反应体系以20μl计，包括：无核酶水2.4μl、PCR反应液10μl、酶混合液0.5μl、ROX参比染料0.1μl、引物组的水溶液2μl和模板5μl。
  13. 根据权利要求10或权利要求11所述方法，其特征在于，所述qRT-PCR反应的程序包括：42℃ 30min；95℃ 1min；95℃ 5s，60℃ 31s，40循环。
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