WO2023010327A1 - 一种人白三烯受体CysLTR2 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CysLTR2 mRNA RT-PCR检测用引物探针组和试剂盒 Download PDF

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WO2023010327A1
WO2023010327A1 PCT/CN2021/110522 CN2021110522W WO2023010327A1 WO 2023010327 A1 WO2023010327 A1 WO 2023010327A1 CN 2021110522 W CN2021110522 W CN 2021110522W WO 2023010327 A1 WO2023010327 A1 WO 2023010327A1
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probe
primer
cysltr2
labeled
seq
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吴周杰
吴善东
刘奕
蒋学翰
杨旭凯
李世旦
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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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
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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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  • the invention relates to the technical field of biological detection, in particular to a primer probe set and a kit for human leukotriene receptor CysLTR2 mRNA RT-PCR detection.
  • 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 and CysLTR2 are two types of CysLTs receptors, CysLTR1 and CysLTR2, among which CysLTR2 is a G protein-coupled receptor, which is mainly expressed in peripheral tissue cells such as the heart, pulmonary vein, adrenal medulla, and muscularis neurons.
  • CysLTR2 is expressed in On vascular smooth muscle cells and infiltrating granulocytes.
  • CysLTR2 When CysLTR2 is activated by leukotrienes, it will lead to inflammation, increased vascular permeability and tissue fibrosis, which will increase the risk of allergic asthma; it is also closely related to cerebral ischemic injury.
  • leukotriene receptor antagonists 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.
  • the reported CysLT2 receptor antagonists include HAMI 3379 and Bay U9773.
  • HAMI 3379 protects against acute and subacute ischemic brain injury and attenuates microglia-associated inflammation.
  • Bay U9773 has equal affinity to CysLT1 and CysLT2, can bind to these two receptors, and inhibit bronchial and venous muscle contraction.
  • CysLTR2 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 CysLTR2 mRNA.
  • ELISA enzyme linked immunosorbent assay
  • the object of the present invention is to provide a kind of primer probe group and kit for human leukotriene receptor CysLTR2 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 CysLTR2, 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 CysLTR2 mRNA RT-PCR detection, the primer probe set includes primer CysLTR2-F, primer CysLTR2-R and probe C2-Probe, the primer The nucleotide sequence of CysLTR2-F is shown in SEQ ID NO.1, the nucleotide sequence of said primer CysLTR2-R is shown in SEQ ID NO.2, and the nucleotide sequence of said probe C2-Probe is shown in Shown in SEQ ID NO.3.
  • the 5' end of the probe C2-Probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a 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 , the nucleotide sequence of the primer GAPDH-R is shown in SEQ ID NO.5, and 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 C2-Probe Reporting groups are different.
  • the fluorescent reporter group includes FAM or JOE, and the quencher group includes BHQ1.
  • the present invention also provides a kit for human leukotriene receptor CysLTR2 mRNA RT-PCR detection, said kit includes primer probe set, PCR reaction solution, enzyme mixture, CysLTR2 standard product, ROX reference dye and nuclease-free water.
  • the PCR reaction solution includes dNTP mix, 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 of 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 CysLTR2 mRNA.
  • the primer probe set of the present invention when used for detection, it has high sensitivity, can detect low concentration (10copies/ ⁇ L) clinical samples, and can more sensitively detect changes in the content of CysLTR2. It can span at least 6 orders of magnitude, increases the accuracy of the test results, and can complete at least 80 tests within one hour, so that the dynamic monitoring and curative effect evaluation of the treatment effect can be performed earlier, more accurately and faster.
  • Fig. 1 is the CysLTR2 mRNA TaqMan real-time fluorescent quantitative RT-PCR standard curve provided by the present invention
  • Figure 2 is a graph of the precision test results provided by the present invention, in which 1: 1.0 ⁇ 10 7 copies/ ⁇ L, 2: 1.0 ⁇ 10 4 copies/ ⁇ L;
  • Fig. 3 is the accuracy detection result figure that the present invention provides
  • Fig. 4 is the sensitivity detection result figure provided by the present invention.
  • Fig. 5 is the clinical sample detection result diagram provided by the present invention, wherein 1: patient GAPDH mRNA; 2: healthy control GAPDH mRNA; 3: patient CysLTR2 mRNA; 4: healthy control CysLTR2 mRNA;
  • Fig. 6 is provided by the present invention under the situation of non-optimally designed primers and probes, low value precision amplification curve
  • Fig. 7 The amplification results of the non-optimum 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 CysLTR2 mRNA RT-PCR detection
  • the primer probe set includes primer CysLTR2-F, primer CysLTR2-R and probe C2-Probe, the primer
  • the nucleotide sequence of CysLTR2-F is shown in SEQ ID NO.1: 5'-TCTGCTGATCATTCGGGTT-3'
  • the nucleotide sequence of the primer CysLTR2-R is shown in SEQ ID NO.2: 5'-TGATAGGGCAGGAAACACAA- 3'
  • the nucleotide sequence of the probe C2-Probe is shown in SEQ ID NO.3: 5'-TCACAGGAAGGCACTGACCAC-3'.
  • the 5' end of the probe C2-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 C2-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, and the 3' end is labeled with a quencher group; the fluorescent reporter group labeled with the probe G-Probe and the probe C2-Probe labeled
  • the fluorescent reporter groups are preferably different.
  • the fluorescent reporter group preferably includes FAM or JOE, and the quencher group preferably includes BHQ1.
  • the 5' end of the probe G-Probe is labeled with the JOE fluorescent reporter group, and the 3' end is labeled with the BHQ1 quencher group.
  • the present invention also provides a kit for human leukotriene receptor CysLTR2 mRNA RT-PCR detection, said kit includes primer probe set, PCR reaction solution, enzyme mixture, CysLTR2 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 ⁇ 10mM;
  • the buffer solution is preferably a Tris-HCl buffer solution, more preferably 10 ⁇ 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 12:4:3:1, and this ratio can obtain the best amplification effect .
  • Taq enzyme is a heat-resistant Taq DNA polymerase, utilizes its 3' ⁇ 5' polymerase activity to use DNA as a template, and deoxygenated mononucleotides in dNTPs are added 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 non-specific amplification of 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 CysLTR2 standard product is preferably an RNA standard product of CysLTR2, which is used to prepare 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 CysLTR2 mRNA in human blood, nasal secretion, bronchial flushing fluid, saliva, and tear fluid samples.
  • the reaction system of the kit in terms of 20 ⁇ L, preferably 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, standard or test sample 5 ⁇ L and 2.4 ⁇ L of nuclease-free water.
  • the conditions for the fluorescent quantitative amplification are preferably: 42°C for 30 min (reverse transcription); 95°C for 1 min (pre-denaturation); 95°C for 5 s, 60°C for 31 s, and 40 cycles of amplification.
  • a kind of human leukotriene receptor CysLTR2 mRNA RT-PCR detection primer probe set and kit for detection of a kind of human leukotriene receptor CysLTR2 mRNA RT-PCR according to the present invention will be described in further detail below in conjunction with specific examples. .
  • CysLTR2 and GAPDH use Primer 6.0 software to design fluorescent quantitative primers and probes. After a series of effect verification, the primer pairs CysLTR2-F, CysLTR2-R, GAPDH-F, GAPDH-R and probes of CysLTR2 and GAPDH were obtained. E-Probe, G-Probe (see Table 1). Primer probes were synthesized by Shanghai Sunny Biotechnology Co., Ltd.
  • CysLTR2 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 takes 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.
  • CysLTR2 Dilute the CysLTR2 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, CysLTR2
  • the present invention uses whole blood RNA for detection, and a certain domestic brand cysteinyl leukotriene receptor 2 (CYSLTR2) ELISA kit uses serum for detection.
  • CYSLTR2 cysteinyl leukotriene receptor 2
  • primers and probes in the system used by the present invention in embodiment 6 are replaced with other non-optimal primers and probes.
  • the amplification system and procedure are the same as in Example 1.
  • the results are shown in Figure 6 and Table 8, when using non-optimal CysLTR2 primers and probes, such as:
  • CysLTR2-F GCACCTTCAGCAATAACAACA (SEQ ID NO. 7)
  • CysLTR2-R GCTGCAGGAAAACATATATGG (SEQ ID NO.8)
  • the coefficient of variation of the concentration logarithmic value of the low-value precision exceeds 5%, reaching 7.815%.
  • Amplify the standard with an enzyme mixture with a non-optimal ratio (the volume ratio of Taq enzyme, reverse transcriptase, RNase inhibitor, and Taq enzyme antibody is 10:5:4:1), and amplify to obtain a standard curve 1.0 ⁇ 10 3 to 1.0 ⁇ 10 6 copies/ ⁇ L in 4 gradients, primers and probes for amplification, amplification system, and procedure are the same as in Example 1.
  • Amplification results using a non-optimal enzyme mix are shown in A in Figure 7. Compared with the standard curve amplification result (B in Fig.

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Abstract

本发明涉及一种人白三烯受体CysLTR2 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CysLTR2-F、引物CysLTR2-R和探针C2-Probe，所述引物CysLTR2-F的核苷酸序列如SEQ ID NO.1所示，所述引物CysLTR2-R的核苷酸序列如SEQ ID NO.2所示，所述探针C2-Probe的核苷酸序列如SEQ ID NO.3所示。

Description

一种人白三烯受体CysLTR2 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CysLTR2 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CysLTR2为G蛋白偶联受体，主要表达于心脏、肺静脉、肾上腺髓质、肠肌层神经元等外周组织细胞中，在人脑中，CysLTR2表达在血管平滑肌细胞及浸润的粒细胞上。CysLTR2被白三烯激活后，将导致炎症、血管通透性增加和组织的纤维化，会增加过敏性哮喘的风险；与脑缺血损伤也密切相关。
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。现在已有报道的CysLT2受体拮抗剂包 含HAMI 3379和Bay U9773。HAMI 3379对急性和亚急性缺血性脑损伤具有保护作用，并减轻小胶质细胞相关的炎症。Bay U9773对CysLT1和CysLT2有同等的亲和力，可与这两种受体结合，抑制支气管和静脉肌肉收缩。通过对CysLTR2 mRNA表达水平的检测，可判断出患者是否因CysLTR2途径导致的病症，采用CysLT2受体拮抗剂是否有效果及实现治疗效果的动态监测。
现在市场上对于CysLTR2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CysLTR2 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CysLTR2 mRNA RT-PCR检测用引物探针组和试剂盒。本发明针对人CysLTR2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CysLTR2 mRNA RT-PCR检测用引物探针组，所述引物探针组包括引物CysLTR2-F、引物CysLTR2-R和探针C2-Probe，所述引物CysLTR2-F的核苷酸序列如SEQ ID NO.1所示，所述引物CysLTR2-R的核苷酸序列如SEQ ID NO.2所示，所述探针C2-Probe的核苷酸序列如SEQ ID NO.3所示。
优选的是，所述探针C2-Probe的5'端标记荧光报告基团，3'端标记淬灭基团。
优选的是，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，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C2-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或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CysLTR2 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、CysLTR2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dNTP mix、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CysLTR2 mRNA RT-PCR检测用引物探针组。与免疫学检测方法相比，本发明所述引物探针组用于检测时，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CysLTR2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。
说明书附图
图1为本发明提供的CysLTR2 mRNA TaqMan实时荧光定量RT-PCR标准曲线；
图2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3为本发明提供的准确度检测结果图；
图4为本发明提供的灵敏度检测结果图；
图5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CysLTR2 mRNA；4：健康对照CysLTR2 mRNA；
图6为本发明提供的在非最佳设计引物、探针的情况下，低值精密度扩增曲线图；
图7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CysLTR2 mRNA RT-PCR检测用引物探针组，所述引物探针组包括引物CysLTR2-F、引物CysLTR2-R和探针C2-Probe，所述引物CysLTR2-F的核苷酸序列如SEQ ID NO.1所示：5'-TCTGCTGATCATTCGGGTT-3'，所述引物CysLTR2-R的核苷酸序列如SEQ ID NO.2所示：5'-TGATAGGGCAGGAAACACAA-3'，所述探针C2-Probe的核苷酸序列如SEQ ID NO.3所示：5'-TCACAGGAAGGCACTGACCAC-3'。
在本发明中，所述探针C2-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C2-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C2-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CysLTR2 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、CysLTR2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5～10mM；缓冲液优选为Tris-HCl缓冲液，更优选为10～50mM 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12:4:3:1，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。在本发明中，所述CysLTR2标准品优选为CysLTR2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、鼻腔分泌物、支气管冲洗液、唾液、泪液样本中CysLTR2 mRNA的表达水平。
在本发明中，所述试剂盒的反应体系，以20μL计，优选包括：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。
下面结合具体实施例对本发明所述的一种人白三烯受体CysLTR2 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 TM7300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CysLTR2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CysLTR2和GAPDH的引物对CysLTR2-F、CysLTR2-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 PCTCN2021110522-appb-000001
2.2标准品制备
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CysLTR2质粒DNA(委托南京金斯瑞生物科技有限公司构建及合成)，将CysLTR2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 PCTCN2021110522-appb-000002
扩增反应程序如表3所示：
表3 反应程序
Figure PCTCN2021110522-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CysLTR2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，CysLTR2 mRNA TaqMan实时荧光定量RT-PCR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＝-3.321x+35.602(R 2＝0.997)，该回归方程的R 2＝0.997，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2和表4所示，结果显示，每个浓度对数值的变异系数分别为0.418％、0.382％，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 6.945 0.029 0.418％
1.0×10 4 3.973 0.015 0.382％
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3和表5显示，每个浓度对数值的绝对偏差分别为0.142、0.099、0.144，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 PCTCN2021110522-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4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
33.025 32.678 33.317 32.759 32.854
32.096 32.901 32.974 32.421 32.840
33.098 32.625 32.872 33.481 32.313
32.652 32.445 32.729 32.055 32.980
33.432 32.717 32.271 34.375 32.580
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2(CYSLTR2)ELISA试剂盒比对结果如表7和图5所示：
表7 比对结果
Figure PCTCN2021110522-appb-000005
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2(CYSLTR2)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
将实施例6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。扩增体系、程序与实施例1相同。结果如图6和表8，在采用非最佳的CysLTR2引物、探针，如：
CysLTR2-F：GCACCTTCAGCAATAACAACA(SEQ ID NO.7)
CysLTR2-R：GCTGCAGGAAAACATATATGG(SEQ ID NO.8)
C2-Probe：(FAM)-CAACCCATTTCCCAAGACTCCC(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7.815％。
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 3.381 0.264 7.815％
对比例2
非最佳酶混合液的扩增结果
用非最佳配比(Taq酶、逆转录酶、RNA酶抑制剂和Taq酶抗体的体积比为10:5:4:1)的酶混合液对标准品进行扩增，扩增得到标准曲线1.0×10 3～1.0×10 6copies/μL4个梯度，扩增用引物和探针、扩增体系、程序与实施例1相同。采用非最佳酶混合液扩增结果如图7中的A所示。相比采用最佳酶混合液的标准曲线扩增结果(图7中的B)，非最佳酶混合液的标准曲线扩增结果的相应浓度的Ct往后推延10个循环数以上，且低浓度下的标准品(如1.0×10 3copies/μL)重复性相差较大，整体线性效果差，标准曲线不合格。可见非最佳酶混合液扩增效果不好。
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CysLTR2 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CysLTR2-F、引物CysLTR2-R和探针C2-Probe，所述引物CysLTR2-F的核苷酸序列如SEQ ID NO.1所示，所述引物CysLTR2-R的核苷酸序列如SEQ ID NO.2所示，所述探针C2-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C2-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C2-Probe标记的荧光报告基团不同。
  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C2-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CysLTR2 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、CysLTR2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10:5:4:1。
  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。
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