WO2023010328A1 - 一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组和试剂盒 - Google Patents

一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组和试剂盒 Download PDF

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WO2023010328A1
WO2023010328A1 PCT/CN2021/110524 CN2021110524W WO2023010328A1 WO 2023010328 A1 WO2023010328 A1 WO 2023010328A1 CN 2021110524 W CN2021110524 W CN 2021110524W WO 2023010328 A1 WO2023010328 A1 WO 2023010328A1
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probe
primer
cysltr1
labeled
kit
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French (fr)
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刘奕
吴周杰
吴善东
蒋学翰
王教峰
雷薇
钱磊
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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/CN2021/110524 priority Critical patent/WO2023010328A1/zh
Priority to EP21952230.7A priority patent/EP4289968A4/en
Publication of WO2023010328A1 publication Critical patent/WO2023010328A1/zh

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    • 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
    • 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
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/10—Nucleotidyl transfering
    • C12Q2521/107—RNA dependent DNA polymerase,(i.e. reverse transcriptase)

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  • the invention relates to the technical field of biological detection, in particular to a primer probe set and a kit for RT-PCR detection of human leukotriene receptor CysLTR1 mRNA.
  • Leukotrienes are eicosaunsaturated acids with a conjugated triene structure isolated from the metabolites of arachidonic acid in leukocytes. It can be produced from arachidonic acid catalyzed by lipoxygenase (lipoxygenase). Although the content in the body is very low, it has high physiological activity and is a chemical mediator that triggers certain allergic reactions, inflammation, and cardiovascular diseases. Leukotrienes play an important role in inflammation of the upper and lower airways. Leukotrienes are more than 1000 times more potent than histamines in inducing nasal allergic reactions. Leukotrienes are significantly increased in both the immediate and delayed phases of allergen-induced nasal hypersensitivity reactions.
  • Cysteinyl leukotrienes are inflammatory mediators and regulators in the pathophysiology of asthma and allergic rhinitis (AR), and are key therapeutic targets. CysLTs can regulate hematopoietic progenitor cell production, eosinophilic Granulocyte recruitment and survival in inflamed tissues, cytokine and chemokine activity, amount of exhaled NO, contraction of smooth muscle and proliferation of fibroblasts.
  • CysLTs The biological effects of CysLTs depend on the expression of leukotriene receptors on the cell surface.
  • CysLTR1 There are two types of CysLTs receptors, CysLTR1 and CysLTR2, among which CysLTR1 is a G protein-coupled receptor, mainly expressed in spleen, lung, smooth muscle, etc.
  • CysLTR1 is a G protein-coupled receptor, mainly expressed in spleen, lung, smooth muscle, etc.
  • CysLTR1 is a G protein-coupled receptor, mainly expressed in spleen, lung, smooth muscle, etc.
  • CysLTR1 After CysLTR1 is activated by leukotrienes, it mediates continuous smooth muscle cell contraction and proliferation, mucosal edema, eosinophil aggregation, and increased mucus secretion, which directly leads to the occurrence and development of asthmatic airway inflammation, and plays a major role in the pathogenesis of asthma. effect.
  • CysLT1 receptor antagonists such as montelukast, zafirlukast, and promilukast
  • CysLTR1 receptor antagonists act on CysLTR1 and are used to block the asthmatic effect of CysLT1.
  • LTRA leukotriene receptor antagonists
  • LTRA can competitively inhibit the binding of leukotrienes to their receptors in vivo, block the activity of CysLTs, thereby inhibiting inflammatory and allergic reactions, but its curative effect has There are obvious individual differences, and its curative effect has a clear positive correlation with the expression level of leukotriene receptor gene mRNA.
  • CysLTR1 in the market still uses enzyme-linked immunosorbent assay (enzyme linked immunosorbent assay, ELISA) kit to detect its content in body fluids, and there is no commercial kit for detection of CysLTR1 mRNA.
  • ELISA enzyme linked immunosorbent assay
  • the object of the present invention is to provide a primer probe set and kit for human leukotriene receptor CysLTR1 mRNA RT-PCR detection.
  • the invention aims at the TaqMan real-time fluorescence quantitative one-step RT-PCR detection primer probe set established for human CysLTR1, and provides a detection means with high accuracy, wide detection range and high sensitivity for the detection of the protein.
  • the invention provides a primer probe set for human leukotriene receptor CysLTR1 mRNA RT-PCR detection, said primer probe set includes primer CysLTR1-F, primer CysLTR1-R and probe C1-Probe, said primer
  • the nucleotide sequence of CysLTR1-F is shown in SEQ ID NO.1
  • the nucleotide sequence of said primer CysLTR1-R is shown in SEQ ID NO.2
  • the nucleotide sequence of said probe C1-Probe is shown in Shown in SEQ ID NO.3.
  • the 5' end of the probe C1-Probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group.
  • primer GAPDH-F primer GAPDH-R of internal reference gene
  • probe G-Probe the nucleotide sequence of described primer GAPDH-F is as shown in SEQ ID NO.4, described primer GAPDH
  • SEQ ID NO.5 the nucleotide sequence of the probe G-Probe is shown in SEQ ID NO.6.
  • the 5' end of the probe G-Probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group; the fluorescent reporter group labeled with the probe G-Probe and the fluorescent reporter group labeled with the probe C1-Probe Reporting groups are different.
  • the fluorescent reporter group includes FAM and JOE, and the quencher group includes BHQ1.
  • the present invention also provides a kit for RT-PCR detection of human leukotriene receptor CysLTR1 mRNA, the kit includes the primer probe set described in the above technical scheme, PCR reaction solution, enzyme mixture, CysLTR1 standard product, ROX reference dye and nuclease-free water.
  • the PCR reaction solution includes dNTPmix, MgCl 2 and buffer.
  • the enzyme mixture comprises Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody.
  • the present invention also provides a method for using the kit described in the above technical scheme, comprising the steps of: mixing primer probe set, PCR reaction solution, enzyme mixture, standard or sample to be tested, ROX reference dye and nuclease-free water After mixing, fluorescent quantitative amplification is performed.
  • the reaction system of the kit includes: 2 ⁇ L of primer probe set, 10 ⁇ L of PCR reaction solution, 0.5 ⁇ L of enzyme mixture, 0.1 ⁇ L of ROX reference dye, 5 ⁇ L of standard or test sample and no 2.4 ⁇ L of ribozyme water; the conditions for the fluorescent quantitative amplification are: 42°C for 30 min; 95°C for 1 min; 95°C for 5 s, 60°C for 31 s, and 40 cycles of amplification.
  • the invention provides a primer probe set for RT-PCR detection of human leukotriene receptor CysLTR1 mRNA.
  • a one-step method is used for detection, without the need for a separate reverse transcription operation, which greatly reduces the risk of aerosol pollution; compared with the immunological detection method, using the primers of the present invention to detect
  • the detection method of the needle group has high detection sensitivity, can detect low concentration (10copies/ ⁇ L) clinical samples, and can more sensitively detect changes in the content of CysLTR1, and the detection range can span at least 5 orders of magnitude, increasing the accuracy of the detection results , can conduct dynamic monitoring and curative effect evaluation on the treatment effect earlier, more accurately and more quickly.
  • Fig. 1 is the dilution operation process diagram that the present invention provides
  • Fig. 2 is the CysLTR1 mRNA TaqMan real-time fluorescent quantitative RT-PCR standard curve provided by the present invention
  • Figure 3 is a graph of the precision test results provided by the present invention. where 1: 1.0 ⁇ 10 7 copies/ ⁇ L, 2: 1.0 ⁇ 10 4 copies/ ⁇ L;
  • Fig. 4 is the accuracy detection result figure that the present invention provides
  • Fig. 5 is the sensitivity detection result figure provided by the present invention.
  • Fig. 6 is the clinical sample detection result figure provided by the present invention.
  • 1 patient GAPDH mRNA
  • 2 healthy control GAPDH mRNA
  • 3 patient CysLTR1 mRNA
  • 4 healthy control CysLTR1 mRNA
  • Fig. 7 is the low-value precision amplification curve under the condition of unreasonable primer design provided by the present invention.
  • Fig. 8 shows the amplification results of the non-optimal ratio enzyme mixture solution (A) and the optimal ratio enzyme mixture solution (B) provided by the present invention.
  • the invention provides a primer probe set for human leukotriene receptor CysLTR1 mRNA RT-PCR detection, said primer probe set includes primer CysLTR1-F, primer CysLTR1-R and probe C1-Probe, said primer
  • the nucleotide sequence of CysLTR1-F is shown in SEQ ID NO.1: 5'-AACCTATCACAAGAAGTCAGC-3', and the nucleotide sequence of the primer CysLTR1-R is shown in SEQ ID NO.2: 5'-CCAAAGAGCCAAATGCCTTT- 3', the nucleotide sequence of the probe C1-Probe is shown in SEQ ID NO.3: 5'-CACTGCCTCTCCGTGTGGTC-3'.
  • the 5' end of the probe C1-Probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quenching group.
  • the fluorescent reporter group preferably includes FAM or JOE, and the quencher group preferably includes BHQ1.
  • the 5' end of the probe C1-Probe is labeled with a FAM fluorescent reporter group, and the 3' end is labeled with a BHQ1 quencher group.
  • the primer probe set also includes primer GAPDH-F, primer GAPDH-R and probe G-Probe of the internal reference gene, and the nucleotide sequence of the primer GAPDH-F is as shown in SEQ ID NO.4 Shown: 5'-GACAACAGCCTCAAGATCATC-3', the nucleotide sequence of the primer GAPDH-R is shown in SEQ ID NO.5: 5'-CGCCACAGTTTCCCGGAG-3', the nucleotide sequence of the probe G-Probe As shown in SEQ ID NO.6: 5'-ACTCATGACCACAGTCCATGCCAT-3'.
  • the 5' end of the probe G-Probe is labeled with a fluorescent reporter group
  • the 3' end is labeled with a quencher group
  • the fluorescent reporter group labeled with the probe G-Probe and the probe C1-Probe labeled The fluorescent reporter groups are preferably different.
  • the fluorescent reporter group preferably includes FAM or JOE
  • the quencher group preferably includes BHQ1.
  • the 5' end of the probe G-Probe is labeled with the JOE fluorescent reporter group
  • the 3' end is labeled with the BHQ1 quencher group.
  • the present invention also provides a kit for RT-PCR detection of human leukotriene receptor CysLTR1 mRNA, the kit includes the primer probe set described in the above technical scheme, PCR reaction solution, enzyme mixture, CysLTR1 standard product, ROX reference dye and nuclease-free water.
  • the PCR reaction solution includes dNTP mix, MgCl 2 and buffer; the dNTP mix is deoxyribonucleoside triphosphate, including dATP, dCTP, dGTP and dTTP, and the dNTP mix of the present invention is preferably purchased Available from ThermoFisher (Cat. No. R0192), the working concentration is preferably 0.3-0.8 mM.
  • the use concentration of MgCl2 is preferably 5 ⁇ 12mM;
  • the buffer solution is preferably a Tris-HCl buffer solution, more preferably 20 ⁇ 50mM Tris-HCl buffer solution, and the pH value of the Tris-HCl buffer solution is preferably 8.0.
  • the enzyme mixture includes Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody.
  • the volume ratio of Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody is preferably 15:5:3:1, and this ratio can obtain the best amplification effect .
  • the Taq enzyme is a heat-resistant Taq DNA polymerase, using its 3' ⁇ 5' polymerase activity to use DNA as a template to add deoxygenated mononucleotides in dNTPs to the 3-OH end one by one;
  • the 5' ⁇ 3' exonuclease activity can identify and eliminate mismatched primer ends, which is related to the correction function during replication, and can also hydrolyze nucleotides from the 5' end, and can also work through several nucleotides, Excision of 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 Exogenous RNase activity; Taq enzyme antibody is an anti-Taq antibody for hot-start PCR, which inhibits DNA polymerase activity after binding to Taq enzyme, and can effectively inhibit non-specific annealing of primers and primer dimers at low temperatures.
  • the Taq enzyme antibody is denatured in the initial DNA denaturation step of the PCR reaction, and the Taq enzyme restores its activity to achieve PCR amplification.
  • the CysLTR1 standard product is preferably an RNA standard product of CysLTR1 for preparing a quantification curve.
  • the present invention also provides a method for using the kit described in the above technical scheme, comprising the steps of: mixing primer probe set, PCR reaction solution, enzyme mixture, standard or sample to be tested, ROX reference dye and nuclease-free water After mixing, fluorescent quantitative amplification is performed.
  • the kit of the present invention adopts a one-step RT-PCR quantitative detection method, which can detect the expression level of CysLTR1 mRNA in human blood.
  • the reaction system of the kit in 20 ⁇ L, includes: 2 ⁇ L of primer probe set, 10 ⁇ L of PCR reaction solution, 0.5 ⁇ L of enzyme mixture, 0.1 ⁇ L of ROX reference dye, 5 ⁇ L of standard or test sample and 2.4 ⁇ L of nuclease-free water.
  • the conditions for the fluorescent quantitative amplification are preferably: 42°C for 30min (reverse transcription); 95°C for 1min (pre-denaturation); 95°C for 5s, 60°C for 31s, and 40 cycles of amplification.
  • the kit of the invention has a simple operation method and short detection time, and provides a kit product that can guide medication and accurately quantify curative effect for CysLTR1 receptor antagonists.
  • Cysteinyl leukotrienes (CysLTs), inflammatory mediators and regulators in the pathophysiology of asthma and allergic rhinitis (AR), are key therapeutic targets.
  • leukotriene receptor CysLTR1 antagonists can reduce allergic inflammation by blocking the activity of CysLTR1, resulting in a wide range of clinical effects.
  • leukotriene receptor CysLTR1 The mRNA expression of leukotriene receptor CysLTR1 was detected to be higher than the normal reference range, indicating that the treatment with leukotriene receptor CysLTR1 antagonists would be effective; the level of CysLTR1 in the blood was reduced after treatment, indicating that the treatment was effective . If there are obvious allergic symptoms, but the expression level of leukotriene receptor CysLTR1 is very low, it means that the allergic symptoms are not caused by the leukotriene pathway, and treatment with leukotriene receptor CysLTR1 antagonists is ineffective.
  • CysLTR1 and GAPDH use Primer 6.0 software to design fluorescent quantitative primers and probes. After a series of effect verification, the primer pairs CysLTR1-F, CysLTR1-R, GAPDH-F, GAPDH-R and probes of CysLTR1 and GAPDH were obtained. E-Probe, G-Probe (see Table 1). Primer probes were synthesized by Shanghai Sunny Biotechnology Co., Ltd.
  • CysLTR1 plasmid DNA was transcribed into mRNA in vitro with HiScribe T7 High Yield RNA Synthesis Kit (produced by NEW ENGLAND BioLabs, Cat. No.: E2040S).
  • 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 10 copies/ ⁇ L, which is the CysLTR1 standard.
  • EDTA anticoagulated whole blood samples were extracted with a whole blood total RNA kit, quantified with a Qubit 3 fluorometer, and then diluted to 20 ng/ ⁇ L with nuclease-free water.
  • the CysLTR1 standard was diluted in a 10-fold gradient, and 1.0 ⁇ 10 8 to 1.0 ⁇ 10 3 copies/ ⁇ L was selected as a template, and each dilution was replicated three times for TaqMan real-time fluorescent quantitative RT-PCR detection to generate a standard curve.
  • the dilution operation process is shown in Figure 1, taking 50 ⁇ L/tube as an example, during each dilution process, take 5 ⁇ L of the sample before dilution and add it to a new tube containing 45 ⁇ L of water.
  • step 2.3 Take positive samples and healthy control whole blood samples for whole blood RNA extraction and dilution according to step 2.3, and perform TaqMan real-time fluorescent quantitative RT-PCR detection according to step 2.4.
  • CysLTR1 Dilute the CysLTR1 standard product according to a 10-fold gradient, select 1.0 ⁇ 10 8 ⁇ 1.0 ⁇ 10 3 copies/ ⁇ L as the template, and perform 3 replicates for each dilution, perform TaqMan real-time fluorescence quantitative RT-PCR detection, and generate a standard curve, CysLTR1
  • the present invention uses whole blood RNA for detection, and a domestic brand of cysteinyl leukotriene receptor 1 (CYSLTR1) ELISA kit uses serum for detection.
  • CYSLTR1 cysteinyl leukotriene receptor 1
  • CysLTR1-F GTATCTTCTGCCACATGCC (SEQ ID NO. 7);
  • CysLTR1-R TTGCCAAAGAAGCCTACAACA (SEQ ID NO.8);
  • Non-optimal ratio of enzyme mixture the volume ratio of Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody is 14:4:5:1
  • an optimal ratio of enzyme mixture to test the standard Products were amplified, and 4 gradients of 1.0 ⁇ 10 3 to 1.0 ⁇ 10 6 copies/ ⁇ L were obtained from the amplification on the standard curve.
  • the primers and probes, amplification system, and procedures for amplification were the same as those in Example 1.
  • the result is shown in Figure 8
  • the amplification result using the non-optimal enzyme mixture ratio is shown in A in Figure 8
  • the amplification result using the best enzyme mixture ratio is shown in Figure 8 B Show.
  • Both are compared, and the standard curve amplification results of the best enzyme mixture are more reproducible.
  • the Ct differences of the adjacent concentrations were 3.3, 3.3, 3.4, respectively, while the Ct differences of the adjacent concentrations of the standard curve amplification results of the non-optimal enzyme mixture were 3.8, 3.1, 3.6, indicating that the optimal enzyme mixture
  • the Ct difference of the standard curve amplification results is more uniform. It can be seen that the amplification effect of the best enzyme mixture is better.

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Abstract

本发明涉及一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组和试剂盒，属于生物检测技术领域。本发明所述引物探针组包括引物CysLTR1-F、引物CysLTR1-R和探针C1-Probe，所述引物CysLTR1-F的核苷酸序列如SEQ ID NO.1所示，所述引物CysLTR1-R的核苷酸序列如SEQ ID NO.2所示，所述探针C1-Probe的核苷酸序列如SEQ ID NO.3所示。本发明针对人CysLTR1建立了TaqMan实时荧光定量一步法RT-PCR检测方法。

Description

一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组和试剂盒 技术领域
本发明涉及生物检测技术领域，具体涉及一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组和试剂盒。
背景技术
白三烯是从花生四烯酸在白细胞中代谢产物分离得到的具有共轭三烯结构的二十碳不饱和酸。可由花生四烯酸经脂(肪)氧合酶(lipoxygenase)催化而制得。在体内含量虽然很低，但却具有很高的生理活性，是引发某些过敏反应、炎症以及心血管等疾病的化学介质。白三烯在上下呼吸道的炎症中起重要作用。在诱导鼻过敏反应方面，白三烯的作用比组织胺强1000多倍。在变应原诱导的鼻过敏反应中，无论是在速发反应还是迟发反应阶段，白三烯的数量都显着增加。半胱氨酰白三烯(Cysteonyl leukotrienes，CysLTs)是哮喘和过敏性鼻炎(AR)的病理生理中的炎症介质和调节因子，是关键的治疗靶点，CysLTs可以调节造血祖细胞生成、嗜酸性粒细胞在炎症组织募集和存活、细胞因子和趋化因子的活性、呼出气NO的数量、平滑肌的收缩和成纤维细胞的增殖。
CysLTs的生物学作用取决于细胞表面白三烯受体的表达。CysLTs受体有CysLTR1和CysLTR2两种，其中CysLTR1为G蛋白偶联受体，主要表达于脾、肺、平滑肌等。CysLTR1被白三烯激活后，介导持续的平滑肌细胞收缩及增生、黏膜水肿、嗜酸性细胞聚集、黏液分泌增多，从而直接导致哮喘气道炎症的发生与发展，在哮喘的发病机制中起主要作用。已被批准的CysLT1受体拮抗剂(如孟鲁司特、扎鲁司特和普鲁司特)作用于CysLTR1，用于阻断CysLT1的致哮喘作用。在治疗的过程中，白三烯受体拮抗剂(LTRA)可以在体内竞争性抑制白三烯与其受体的结合，阻断CysLTs的活性，从而抑制炎性、过敏性反应，但其疗效具有明显的个体差异，并且其疗效与白三烯受体基因mRNA表达水平有明确的正相 关。通过对CysLTR1 mRNA表达水平的检测，可判断出LTRA是否达到阻断CysLTR1的活性的效果，这对药物治疗CysLTR1通路引起的过敏反应及炎症反应是否有效具有一定的治疗意义。
现在市场上对于CysLTR1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CysLTR1 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。
发明内容
本发明的目的在于提供一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组和试剂盒。本发明针对人CysLTR1建立的TaqMan实时荧光定量一步法RT-PCR检测引物探针组，为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。
本发明提供了一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组，所述引物探针组包括引物CysLTR1-F、引物CysLTR1-R和探针C1-Probe，所述引物CysLTR1-F的核苷酸序列如SEQ ID NO.1所示，所述引物CysLTR1-R的核苷酸序列如SEQ ID NO.2所示，所述探针C1-Probe的核苷酸序列如SEQ ID NO.3所示。
优选的是，所述探针C1-Probe的5'端标记荧光报告基团，3'端标记淬灭基团。
优选的是，还包括内参基因的引物GAPDH-F、引物GAPDH-R，和探针G-Probe，所述引物GAPDH-F的核苷酸序列如SEQ ID NO.4所示，所述引物GAPDH-R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。
优选的是，所述探针G-Probe的5'端标记荧光报告基团，3'端标记淬灭基团；探针G-Probe标记的荧光报告基团与探针C1-Probe标记的荧光报告基团不同。
优选的是，所述荧光报告基团包括FAM和JOE，所述淬灭基团包括BHQ1。
本发明还提供了一种人白三烯受体CysLTR1 mRNA RT-PCR检测用 试剂盒，所述试剂盒包括上述技术方案所述引物探针组、PCR反应液、酶混合液、CysLTR1标准品、ROX参比染料和无核酶水。
优选的是，所述PCR反应液包括dNTPmix、MgCl 2和缓冲液。
优选的是，所述酶混合液包括Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体。
本发明还提供了上述技术方案所述试剂盒的使用方法，包括以下步骤：将引物探针组、PCR反应液、酶混合液、标准品或待测样品、ROX参比染料和无核酶水混合后，进行荧光定量扩增。
优选的是，以20μL计，所述试剂盒的反应体系包括：引物探针组2μL、PCR反应液10μL、酶混合液0.5μL、ROX参比染料0.1μL、标准品或待测样品5μL和无核酶水2.4μL；所述荧光定量扩增的条件为：42℃ 30min；95℃ 1min；95℃ 5s，60℃ 31s，扩增40个循环。
本发明提供了一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组。本发明所述引物探针组用于检测时，采用一步法检测，无需单独进行反转录操作，极大降低了造成气溶胶污染的风险；与免疫学检测方法相比，利用本发明引物探针组的检测方法检测的灵敏度高，可以检测低浓度(10copies/μL)的临床样本，能够更灵敏地探测到CysLTR1的含量变化，检测范围可跨越至少5个数量级，增加了检测结果的准确性，能更早期、更准确、更快速地对治疗效果进行动态监测和疗效评估。
说明书附图
图1为本发明提供的稀释操作过程图；
图2为本发明提供的CysLTR1 mRNA TaqMan实时荧光定量RT-PCR标准曲线；
图3为本发明提供的精密度检测结果图；其中1：1.0×10 7copies/μL，2：1.0×10 4copies/μL；
图4为本发明提供的准确度检测结果图；
图5为本发明提供的灵敏度检测结果图；
图6为本发明提供的临床样本检测结果图；其中1：患者GAPDH mRNA；2：健康对照GAPDH mRNA；3：患者CysLTR1 mRNA；4：健 康对照CysLTR1 mRNA；
图7为本发明提供的在引物设计不合理的情况下，低值精密度扩增曲线图；
图8为本发明提供的采用非最佳配比的酶混合液(A)和最佳配比的酶混合液(B)扩增结果。
具体实施方式
本发明提供了一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组，所述引物探针组包括引物CysLTR1-F、引物CysLTR1-R和探针C1-Probe，所述引物CysLTR1-F的核苷酸序列如SEQ ID NO.1所示：5'-AACCTATCACAAGAAGTCAGC-3'，所述引物CysLTR1-R的核苷酸序列如SEQ ID NO.2所示：5'-CCAAAGAGCCAAATGCCTTT-3'，所述探针C1-Probe的核苷酸序列如SEQ ID NO.3所示：5'-CACTGCCTCTCCGTGTGGTC-3'。
在本发明中，所述探针C1-Probe的5'端标记荧光报告基团，3'端标记淬灭基团。在本发明中，所述荧光报告基团优选包括FAM或JOE，所述淬灭基团优选包括BHQ1。在本发明实施例中，所述探针C1-Probe的5'端标记FAM荧光报告基团，3'端标记BHQ1淬灭基团。
在本发明中，所述引物探针组还包括内参基因的引物GAPDH-F、引物GAPDH-R和探针G-Probe，所述引物GAPDH-F的核苷酸序列如SEQ ID NO.4所示：5'-GACAACAGCCTCAAGATCATC-3'，所述引物GAPDH-R的核苷酸序列如SEQ ID NO.5所示：5'-CGCCACAGTTTCCCGGAG-3'，所述探针G-Probe的核苷酸序列如SEQ ID NO.6所示：5'-ACTCATGACCACAGTCCATGCCAT-3'。在本发明中，所述探针G-Probe的5'端标记荧光报告基团，3'端标记淬灭基团，探针G-Probe标记的荧光报告基团与探针C1-Probe标记的荧光报告基团优选不同。在本发明中，所述荧光报告基团优选包括FAM或JOE，所述淬灭基团优选包括BHQ1。在本发明实施例中，所述探针G-Probe的5'端标记JOE荧光报告基团，3'端标记BHQ1淬灭基团。
本发明还提供了一种人白三烯受体CysLTR1 mRNA RT-PCR检测用试剂盒，所述试剂盒包括上述技术方案所述引物探针组、PCR反应液、酶混合液、CysLTR1标准品、ROX参比染料和无核酶水。
在本发明中，所述PCR反应液包括dNTP mix、MgCl 2和缓冲液；所述dNTP mix为脱氧核糖核苷三磷酸，包括dATP，dCTP，dGTP和dTTP，本发明所述dNTP mix优选购自ThermoFisher公司(货号：R0192)，工作浓度优选为0.3～0.8mM。在本发明中，MgCl 2的使用浓度优选为5～12mM；缓冲液优选为Tris-HCl缓冲液，更优选为20～50mM Tris-HCl缓冲液，所述Tris-HCl缓冲液的pH值优选为8.0。
在本发明中，所述酶混合液包括Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体。在本发明中，所述酶混合液中，Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体的体积比优选为15:5:3:1，此比例能够获得最佳的扩增效果。在本发明中，Taq酶为耐热的TaqDNA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。在本发明中，所述CysLTR1标准品优选为CysLTR1的RNA标准品，用于配制定量曲线。
本发明还提供了上述技术方案所述试剂盒的使用方法，包括以下步骤：将引物探针组、PCR反应液、酶混合液、标准品或待测样品、ROX参比染料和无核酶水混合后，进行荧光定量扩增。在本发明中，本发明所述试剂盒采用一步法RT-PCR技术定量检测方法，能够检测人血液中 CysLTR1 mRNA的表达水平。
在本发明中，所述试剂盒的反应体系，以20μL计，包括：引物探针组2μL、PCR反应液10μL、酶混合液0.5μL、ROX参比染料0.1μL、标准品或待测样品5μL和无核酶水2.4μL。在本发明中，所述荧光定量扩增的条件优选为：42℃ 30min(逆转录)；95℃ 1min(预变性)；95℃ 5s，60℃ 31s，扩增40个循环。
本发明所述试剂盒操作方法简单，检测时间较短，为CysLTR1受体拮抗剂提供了可指导用药及准确量化疗效的试剂盒产品。半胱氨酰白三烯(CysLTs)是哮喘和过敏性鼻炎(AR)的病理生理中的炎症介质和调节因子，是关键的治疗靶点。在治疗的过程中，白三烯受体CysLTR1拮抗剂可以通过阻断CysLTR1的活性减少过敏性炎症，产生广泛的临床效应。检测到白三烯受体CysLTR1的mRNA表达量高于正常参考范围，表明患者使用白三烯受体CysLTR1拮抗剂进行治疗会有疗效；治疗后监测到血液中的CysLTR1含量降低，说明治疗有效果。如果表现出明显的过敏症状，但白三烯受体CysLTR1表达量很低，说明该过敏症状不是通过白三烯途径引起的，用白三烯受体CysLTR1拮抗剂治疗无效。
下面结合具体实施例对本发明所述的一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组和试剂盒做进一步详细的介绍，本发明的技术方案包括但不限于以下实施例。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到
实施例1
1.所涉及试剂及设备如下：
1.1 试剂
1.1.1 全血总RNA试剂盒(杭州新景生物试剂开发有限公司，货号：5201050)
1.1.2 HiScribe T7 High Yield RNA Synthesis Kit(New England Biolabs，货号：E2050S)
1.2 主要仪器
1.2.1 Applied Biosystems TM 7300荧光定量PCR仪：ThermoFisher，美国
1.2.2 -80℃低温冰箱：ThermoFisher，美国
1.2.3 高速低温台式离心机：Eppendorf，德国
1.2.4 Qubit 3荧光计：ThermoFisher，美国
2.方法
2.1 引物和探针设计
根据CysLTR1和GAPDH序列，利用Primer 6.0软件，设计荧光定量引物和探针，经过系列效果验证，获得了CysLTR1和GAPDH的引物对CysLTR1-F、CysLTR1-R、GAPDH-F、GAPDH-R和探针E-Probe、G-Probe(见表1)。引物探针由上海桑尼生物科技有限公司合成。
表1 TaqMan实时荧光定量PCR引物探针
Figure PCTCN2021110524-appb-000001
2.2 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CysLTR1质粒DNA(委托南京金斯瑞生物科技有限公司构建及合成)，将CysLTR1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 10copies/μL，即为CysLTR1标准品。
2.3 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。
2.4 TaqMan实时荧光定量PCR
以标准品或全血RNA为模板，配制20μL体系如表2所示：
表2 反应体系
Figure PCTCN2021110524-appb-000002
扩增反应程序如表3所示：
表3 反应程序
Figure PCTCN2021110524-appb-000003
2.5 标准曲线的生成
将CysLTR1标准品按10倍梯度进行稀释，选择1.0×10 8～1.0×10 3copies/μL作为模板，每个稀释度3个重复，进行TaqMan实时荧光定量RT-PCR检测，生成标准曲线。稀释操作过程如图1所示，以50μL/管为例，每次稀释的过程，取5μL稀释前样品，加入到含有45μL水的新管中。
2.6 精密度检测
选择1.0×10 7copies/μL、1.0×10 4copies/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。
2.7 准确度检测
选择1.0×10 6copies/μL标准品进行30倍稀释(2μL 1.0×10 6copies/μL标准品+58μL无核酶水)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，分析该检测方法的准确度。
2.8 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。
2.9 临床样本检测
取阳性样本和健康对照全血样本按照2.3步骤进行全血RNA提取和稀释，按照2.4步骤进行TaqMan实时荧光定量RT-PCR检测。
3.实验结果
3.1 标准曲线
将CysLTR1标准品按10倍梯度进行稀释，选择1.0×10 8～1.0×10 3copies/μL作为模板，每个稀释度3个重复，进行TaqMan实时荧光定量RT-PCR检测，生成标准曲线，CysLTR1 mRNA TaqMan实时荧光定量RT-PCR标准曲线如图2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＝-3.398x+35.344(R 2＝1.000)，该回归方程的R 2＝1.000，线性范围为1.0×10 3～1.0×10 8copies/μL。说明标准方程的拷贝数对数值与Ct值具有极高的相关性。
3.2 精密度检测
选择1.0×10 7copies/μL、1.0×10 4copies/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。精密度检测结果如图3和表4所示，结果显示，每个浓度对数值的变异系数分别为0.320％、0.444％，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4 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 7 7.042 0.023 0.320％
1.0×10 4 3.996 0.018 0.444％
3.3 准确度检测
选择1.0×10 6copies/μL标准品进行30倍稀释(2μL 1.0×10 6copies/μL标准品+58μL无核酶水)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。结果如图4和表5所示，结果显示，每个浓度对数值的绝对偏差分别为0.002、0.011、0.008，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5 准确度检测结果
Figure PCTCN2021110524-appb-000004
3.4 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。灵敏度检测结果如图5和表6所示，结果显示，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6 灵敏度检测Ct值结果
33.271 33.123 33.228 32.975 33.800
33.150 33.284 32.547 33.408 33.416
33.270 33.216 34.086 32.939 32.709
33.312 33.788 33.070 33.653 32.923
32.789 33.700 33.624 33.562 32.810
3.5 临床样本检测
本发明和国内某品牌半胱氨酰白三烯受体1(CYSLTR1)ELISA试剂盒比对结果如图6和表7所示：
表7 比对结果
Figure PCTCN2021110524-appb-000005
Figure PCTCN2021110524-appb-000006
本发明采用全血RNA进行检测，国内某品牌半胱氨酰白三烯受体1(CYSLTR1)ELISA试剂盒采用血清进行检测。
对比例1
采用其它非最佳引物、探针进行扩增的结果
将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。扩增体系、程序与实施例1相同。结果如图7和表8，在采用非最佳的CysLTR2引物、探针，如：
CysLTR1-F：GTATCTTCTGCCACATGCC(SEQ ID NO.7)；
CysLTR1-R：TTGCCAAAGAAGCCTACAACA(SEQ ID NO.8)；
C1-Probe：(FAM)-CCGCAATCAAGTGTATTCCACC(SEQ ID NO.9)-(BHQ1)。
低值精密度的浓度对数值的变异系数结果超出5％，达8.813％。
表8 非最佳引物、探针进行扩增的结果
理论拷贝数 拷贝数对数值均值 SD C.V
1.0×10 4 3.637 0.321 8.813％
对比例2
非最佳酶混合液的扩增结果
用非最佳配比的酶混合液(Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体的体积比为14:4:5:1)和最佳配比的酶混合液分别对标准品进行扩增，扩增得到标准曲线上1.0×10 3～1.0×10 6copies/μL 4个梯度，扩增用引 物和探针、扩增体系、程序与实施例1相同。结果如图8所示，采用非最佳的酶混合液配比的扩增结果如图8中的A所示，采用最佳的酶混合液配比的扩增结果如图8中的B所示。两者(最佳酶混合液的标准曲线扩增结果和非最佳酶混合液的标准曲线扩增结果)相比之下，最佳酶混合液的标准曲线扩增结果重复性更好，相邻浓度的Ct差值分别为3.3、3.3、3.4，而非最佳酶混合液的标准曲线扩增结果的相邻浓度的Ct差值分别为3.8、3.1、3.6，说明最佳酶混合液的标准曲线扩增结果的Ct差值更均一。可见最佳酶混合液的扩增效果较好。
以上结果表明本发明建立的TaqMan实时荧光定量RT-PCR检测方法具有比对试剂更好的灵敏度和特异性，同时可以有效监测治疗效果。
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. 一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组，其特征在于，所述引物探针组包括引物CysLTR1-F、引物CysLTR1-R和探针C1-Probe，所述引物CysLTR1-F的核苷酸序列如SEQ ID NO.1所示，所述引物CysLTR1-R的核苷酸序列如SEQ ID NO.2所示，所述探针C1-Probe的核苷酸序列如SEQ ID NO.3所示。
  2. 根据权利要求1所述的引物探针组，其特征在于，所述探针C1-Probe的5'端标记荧光报告基团，3'端标记淬灭基团。
  3. 根据权利要求1所述的引物探针组，其特征在于，还包括内参基因的引物GAPDH-F、引物GAPDH-R和探针G-Probe，所述引物GAPDH-F的核苷酸序列如SEQ ID NO.4所示，所述引物GAPDH-R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。
  4. 根据权利要求3所述的引物探针组，其特征在于，所述探针G-Probe的5'端标记荧光报告基团，3'端标记淬灭基团；探针G-Probe标记的荧光报告基团与探针C1-Probe不同标记的荧光报告基团。
  5. 根据权利要求2或4所述的引物探针组，其特征在于，所述荧光报告基团包括FAM或JOE，所述淬灭基团包括BHQ1。
  6. 根据权利要求5所述的引物探针组，其特征在于，所述探针C1-Probe的5'端标记FAM荧光报告基团，3'端标记BHQ1淬灭基团。
  7. 根据权利要求5所述的引物探针组，其特征在于，所述探针G-Probe的5'端标记JOE荧光报告基团，3'端标记BHQ1淬灭基团。
  8. 一种人白三烯受体CysLTR1 mRNA RT-PCR检测用试剂盒，其特征在于，所述试剂盒包括权利要求1～7任一项所述引物探针组、PCR反应液、酶混合液、CysLTR1标准品、ROX参比染料和无核酶水。
  9. 根据权利要求8所述的试剂盒，其特征在于，所述PCR反应液包括dNTP mix、MgCl 2和缓冲液。
  10. 根据权利要求8所述的试剂盒，其特征在于，所述酶混合液包括 Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体。
  11. 根据权利要求10所述的试剂盒，其特征在于，所述酶混合液中，Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体的体积比为15:5:3:1。
  12. 权利要求8～11任一项所述试剂盒的使用方法，包括以下步骤：将引物探针组、PCR反应液、酶混合液、标准品或待测样品、ROX参比染料和无核酶水混合后，进行荧光定量扩增。
  13. 根据权利要求12所述的使用方法，其特征在于，以20μL计，所述试剂盒的反应体系包括：引物探针组2μL、PCR反应液10μL、酶混合液0.5μL、ROX参比染料0.1μL、标准品或待测样品5μL和无核酶水2.4μL；
    所述荧光定量扩增的条件为：42℃ 30min；95℃ 1min；95℃ 5s，60℃ 31s，扩增40个循环。
PCT/CN2021/110524 2021-08-04 2021-08-04 一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组和试剂盒 WO2023010328A1 (zh)

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EP21952230.7A EP4289968A4 (en) 2021-08-04 2021-08-04 PRIMER-PROBE ASSEMBLY AND KIT FOR RT-PCR DETECTION OF HUMAN LEUKOTRIENE RECEPTOR CYSLTR1 MRNA

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