阅读说明：本技术 一种人白三烯受体CysLTR1 mRNA RT-PCR检测用引物探针组和试剂盒 (Primer probe set and kit for RT-PCR detection of human leukotriene receptor CysLTR1mRNA ) 是由 刘奕 吴周杰 吴善东 蒋学翰 王教峰 雷薇 钱磊 于 2021-08-04 设计创作，主要内容包括：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。(The invention relates to a primer probe set and a kit for RT-PCR detection of human leukotriene receptor CysLTR1mRNA, belonging to the technical field of biological detection. The primer Probe set comprises a primer CysLTR1-F, a primer CysLTR1-R and a Probe C1-Probe, wherein the nucleotide sequence of the primer CysLTR1-F is shown as SEQ ID NO.1, the nucleotide sequence of the primer CysLTR1-R is shown as SEQ ID NO.2, and the nucleotide sequence of the Probe C1-Probe is shown as SEQ ID NO. 3. The invention aims at the TaqMan real-time fluorescent quantitative one-step RT-PCR detection primer probe group established by human CysLTR1, and provides a detection means with high accuracy, wide detection range and high sensitivity for detecting the protein.)

1. A primer Probe set for RT-PCR detection of human leukotriene receptor CysLTR1mRNA is characterized by comprising a primer CysLTR1-F, a primer CysLTR1-R and a Probe C1-Probe, wherein the nucleotide sequence of the primer CysLTR1-F is shown as SEQ ID NO.1, the nucleotide sequence of the primer CysLTR1-R is shown as SEQ ID NO.2, and the nucleotide sequence of the Probe C1-Probe is shown as SEQ ID NO. 3.

2. The primer Probe set of claim 1, wherein the Probe C1-Probe is labeled with a fluorescent reporter group at the 5 'end and a quencher group at the 3' end.

3. The primer Probe set of claim 1, further comprising a primer GAPDH-F of an internal reference gene, a primer GAPDH-R and a Probe G-Probe, wherein the nucleotide sequence of the primer GAPDH-F is shown as SEQ ID NO.4, the nucleotide sequence of the primer GAPDH-R is shown as SEQ ID NO.5, and the nucleotide sequence of the Probe G-Probe is shown as SEQ ID NO. 6.

4. The primer Probe set of claim 3, wherein the Probe G-Probe is labeled with a fluorescent reporter group at the 5 'end and a quencher group at the 3' end; the fluorescent reporter group labeled with Probe G-Probe is different from the fluorescent reporter group labeled with Probe C1-Probe.

5. The primer probe set of claim 2 or 4, wherein the fluorescent reporter comprises FAM or JOE and the quencher comprises BHQ 1.

6. A kit for RT-PCR detection of human leukotriene receptor CysLTR1mRNA, which is characterized in that the kit comprises the primer probe set, PCR reaction solution, enzyme mixed solution, CysLTR1 standard substance, ROX reference dye and ribozyme-free water according to any one of claims 1 to 5.

7. The kit of claim 6, wherein the PCR reaction solution comprises dNTPmix, MgCl₂And a buffer.

8. The kit of claim 6, wherein the enzyme cocktail comprises Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody.

9. A method of using the kit of claim 6, 7 or 8, comprising the steps of: and mixing the primer probe group, the PCR reaction solution, the enzyme mixed solution, the standard substance or the sample to be detected, the ROX reference dye and the ribozyme-free water, and performing fluorescent quantitative amplification.

10. The use method according to claim 9, wherein the reaction system of the kit comprises, in 20 μ L: 10 mu L of primer probe group 2 mu L, PCR reaction solution, 0.5 mu L, ROX enzyme mixed solution 0.1 mu L of reference dye, 5 mu L of standard substance or sample to be detected and 2.4 mu L of ribozyme-free water; the conditions of the fluorescence quantitative amplification are as follows: 30min at 42 ℃; 1min at 95 ℃; amplification was carried out for 40 cycles at 95 ℃ for 5s and 60 ℃ for 31 s.

Technical Field

The invention relates to the technical field of biological detection, and particularly relates to a primer probe set and a kit for RT-PCR detection of human leukotriene receptor CysLTR1 mRNA.

Background

Leukotrienes are eicosanoid unsaturated acids with conjugated triene structures isolated from arachidonic acid metabolites in leukocytes. Can be prepared from arachidonic acid catalyzed by lipoxygenase (lipoxygenase). The product has high physiological activity although its content is low, and is a chemical mediator for some allergic reaction, inflammation and cardiovascular diseases. Leukotrienes play an important role in inflammation of the upper and lower respiratory tract. Leukotrienes act more than 1000 times stronger than histamine in inducing nasal hypersensitivity. In the allergen-induced nasal allergy, the amount of leukotrienes increases significantly, whether in the immediate or delayed phase of the reaction. Cysteinyl leukotrienes (CysLTs), which are key therapeutic targets for the modulation of hematopoietic progenitor formation, eosinophil recruitment and survival in inflammatory tissues, cytokine and chemokine activities, the number of exhaled NO, smooth muscle contraction and fibroblast proliferation, are inflammatory mediators and regulators in the pathophysiology of asthma and Allergic Rhinitis (AR).

The biological effects of CysLTs depend on the expression of leukotriene receptors on the cell surface. CysLTs receptors include CysLTR1 and CysLTR2, wherein CysLTR1 is G protein coupled receptor, mainly expressed in spleen, lung, smooth muscle, etc. After being activated by leukotriene, CysLTR1 mediates the continuous contraction and proliferation of smooth muscle cells, mucosal edema, eosinophil aggregation and mucus secretion increase, thereby directly causing the generation and development of asthma airway inflammation and playing a main role in the pathogenesis of asthma. Approved CysLT1 receptor antagonists (e.g., montelukast, zafirlukast, and pruilast) act on CysLTR1 to block the asthmatic effect of CysLT 1. In the treatment process, the leukotriene receptor antagonist (LTRA) can competitively inhibit the combination of leukotriene and its receptor in vivo and block the activity of CysLTs, thereby inhibiting inflammatory and allergic reaction, but the curative effect has obvious individual difference and has definite positive correlation with the expression level of leukotriene receptor gene mRNA. By detecting the expression level of CysLTR1mRNA, whether the LTRA achieves the effect of blocking the activity of CysLTR1 can be judged, and the method has certain therapeutic significance for effectively treating anaphylactic reaction and inflammatory reaction caused by CysLTR1 pathway through medicaments.

Enzyme linked immunosorbent assay (ELISA) kit is still used for detecting CysLTR1 in the market to detect the content of CysLTR in body fluid, and no commercial kit for detecting CysLTR1mRNA is found. The ELISA method has the problems of small detection range and low sensitivity in the detection process, and the accuracy of the ELISA method also has a problem.

Disclosure of Invention

The invention aims to provide a primer probe set and a kit for RT-PCR detection of human leukotriene receptor CysLTR1 mRNA. The invention aims at the TaqMan real-time fluorescent quantitative one-step RT-PCR detection primer probe group established by human CysLTR1, and provides a detection means with high accuracy, wide detection range and high sensitivity for detecting the protein.

The invention provides a primer Probe set for RT-PCR detection of human leukotriene receptor CysLTR1mRNA, which comprises a primer CysLTR1-F, a primer CysLTR1-R and a Probe C1-Probe, wherein the nucleotide sequence of the primer CysLTR1-F is shown as SEQ ID NO.1, the nucleotide sequence of the primer CysLTR1-R is shown as SEQ ID NO.2, and the nucleotide sequence of the Probe C1-Probe is shown as SEQ ID NO. 3.

Preferably, the Probe C1-Probe is labeled with a fluorescent reporter group at the 5 'end and a quencher group at the 3' end.

Preferably, the kit further comprises a primer GAPDH-F of the reference gene, a primer GAPDH-R and a Probe G-Probe, wherein the nucleotide sequence of the primer GAPDH-F is shown as SEQ ID NO.4, the nucleotide sequence of the primer GAPDH-R is shown as SEQ ID NO.5, and the nucleotide sequence of the Probe G-Probe is shown as SEQ ID NO. 6.

Preferably, the 5 'end of the Probe G-Probe is labeled with a fluorescent reporter group, and the 3' end of the Probe G-Probe is labeled with a quenching group; the Probe G-Probe labeled fluorescent reporter is different from the Probe C1-Probe labeled fluorescent reporter.

Preferably, the fluorescent reporter group comprises FAM and JOE and the quencher group comprises BHQ 1.

The invention also provides a kit for RT-PCR detection of human leukotriene receptor CysLTR1mRNA, which comprises the primer probe group, PCR reaction solution, enzyme mixed solution, CysLTR1 standard substance, ROX reference dye and ribozyme-free water.

Preferably, the PCR reaction solution comprises dNTPmix and MgCl₂And a buffer.

Preferably, the enzyme mixture includes Taq enzyme, reverse transcriptase, rnase inhibitor and Taq enzyme antibody.

The invention also provides a using method of the kit in the technical scheme, which comprises the following steps: and mixing the primer probe group, the PCR reaction solution, the enzyme mixed solution, the standard substance or the sample to be detected, the ROX reference dye and the ribozyme-free water, and performing fluorescent quantitative amplification.

Preferably, the reaction system of the kit comprises, in 20 μ L: 10 mu L of primer probe group 2 mu L, PCR reaction solution, 0.5 mu L, ROX enzyme mixed solution 0.1 mu L of reference dye, 5 mu L of standard substance or sample to be detected and 2.4 mu L of ribozyme-free water;

the conditions of the fluorescence quantitative amplification are as follows: 30min at 42 ℃; 1min at 95 ℃; amplification was carried out for 40 cycles at 95 ℃ for 5s and 60 ℃ for 31 s.

The invention provides a primer probe set for RT-PCR detection of human leukotriene receptor CysLTR1 mRNA. When the primer probe group is used for detection, a one-step method is adopted for detection, reverse transcription operation is not required to be carried out independently, and the risk of aerosol pollution is greatly reduced; compared with an immunological detection method, the detection method utilizing the primer probe set has high detection sensitivity, can detect a clinical sample with low concentration (10 copies/mu L), can detect the content change of CysLTR1 more sensitively, has a detection range spanning at least 5 orders of magnitude, increases the accuracy of a detection result, and can perform dynamic monitoring and curative effect evaluation on a treatment effect more early, more accurately and more quickly.

Drawings

FIG. 1 is a diagram of the dilution operation provided by the present invention;

FIG. 2 is CysLTR1 mRNAtaqMan real-time fluorescent quantitative RT-PCR standard curve provided by the present invention;

FIG. 3 is a diagram showing the results of the precision measurement according to the present invention; wherein 1: 1.0X 10⁷copies/μL，2：1.0×10⁴copies/μL；

FIG. 4 is a graph of accuracy testing results provided by the present invention;

FIG. 5 is a graph of the sensitivity detection results provided by the present invention;

FIG. 6 is a graph of the results of clinical sample testing provided by the present invention; wherein 1: patient GAPDH mRNA; 2: healthy control GAPDH mRNA; 3: patient CysLTR1 mRNA; 4: healthy control CysLTR1 mRNA;

FIG. 7 is a low value precision amplification curve chart provided by the present invention under the condition of unreasonable primer design;

FIG. 8 shows the amplification results of the enzyme mixture (A) at a non-optimal ratio and the enzyme mixture (B) at an optimal ratio according to the present invention.

Detailed Description

The invention provides a primer Probe set for RT-PCR detection of human leukotriene receptor CysLTR1mRNA, which comprises a primer CysLTR1-F, a primer CysLTR1-R and a Probe C1-Probe, wherein the nucleotide sequence of the primer CysLTR1-F is shown as SEQ ID NO. 1: 5'-AACCTATCACAAGAAGTCAGC-3', the nucleotide sequence of the primer CysLTR1-R is shown as 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' are provided.

In the present invention, the Probe C1-Probe is labeled with a fluorescent reporter at the 5 'end and a quencher at the 3' end. In the present invention, the fluorescent reporter group preferably comprises FAM or JOE, and the quencher group preferably comprises BHQ 1. In the embodiment of the invention, 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 quenching group.

In the present invention, the primer Probe set further comprises a primer GAPDH-F of an internal reference gene, a primer GAPDH-R and a Probe G-Probe, wherein the nucleotide sequence of the primer GAPDH-F is shown in SEQ ID NO. 4: 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 is shown in SEQ ID NO. 6: 5'-ACTCATGACCACAGTCCATGCCAT-3' are provided. In the present invention, the Probe G-Probe is preferably different from the Probe C1-Probe-labeled fluorescent reporter group in that the Probe G-Probe is labeled with a fluorescent reporter group at its 5 'end and a quencher group at its 3' end. In the present invention, the fluorescent reporter group preferably comprises FAM or JOE, and the quencher group preferably comprises BHQ 1. In the embodiment of the invention, the 5 'end of the Probe G-Probe is marked with a JOE fluorescent reporter group, and the 3' end of the Probe G-Probe is marked with a BHQ1 quenching group.

The invention also provides a kit for RT-PCR detection of human leukotriene receptor CysLTR1mRNA, which comprises the primer probe group, PCR reaction solution, enzyme mixed solution, CysLTR1 standard substance, ROX reference dye and ribozyme-free water.

In the invention, the PCR reaction solution comprises dNTPmix and MgCl₂And a buffer; the dNTPmix is deoxyribonucleoside triphosphate and comprises dATP, dCTP, dGTP and dTTP, is preferably purchased from ThermoFisher company (product number: R0192), and has the working concentration of preferably 0.3-0.8 mM. In the present invention, MgCl₂The use concentration of (b) is preferably 5-12 mM; the buffer solution is preferably Tris-HCl buffer solution, more preferably 20-50 mM Tris-HCl buffer solution, and the pH value of the Tris-HCl buffer solution is preferably 8.0.

In the invention, the enzyme mixed solution comprises Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody. In the present invention, the volume ratio of Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody in the enzyme mixture is preferably 15:5:3:1, and the optimal amplification effect can be obtained. In the present invention, Taq enzyme is thermostable Taq DNA polymerase, and deoxymononucleotides in dNTPs are added one by one to the 3-OH terminus using the 3'→ 5' polymerase activity thereof and DNA as a template; meanwhile, the activity of 5' → 3' exonuclease of the primer can be utilized to recognize and eliminate mismatched primer ends, is related to a correction function in a copying process, can hydrolyze nucleotides from the 5' end, can act through a plurality of nucleotides, and can cut off mismatched nucleotides, so that strand replacement is realized in a strand extension process, and a replaced probe is cut off; reverse transcriptase can reverse transcribe mRNA into cDNA for PCR reactions; the RNase inhibitor is used for inhibiting the activity of exogenous RNase; the Taq enzyme antibody is an anti-Taq antibody for hot start PCR, inhibits DNA polymerase activity after being combined with Taq enzyme, can effectively inhibit non-specific annealing of a primer and non-specific amplification caused by a primer dimer under a low temperature condition, is denatured in an initial DNA denaturation step of PCR reaction, recovers activity of the Taq enzyme, and realizes PCR amplification. In the present invention, the CysLTR1 standard is preferably an RNA standard of CysLTR1, which is used for preparing a quantitative curve.

The invention also provides a using method of the kit in the technical scheme, which comprises the following steps: and mixing the primer probe group, the PCR reaction solution, the enzyme mixed solution, the standard substance or the sample to be detected, the ROX reference dye and the ribozyme-free water, and performing fluorescent quantitative amplification. In the invention, the kit adopts a quantitative detection method of a one-step RT-PCR technology, and can detect the expression level of CysLTR1mRNA in human blood.

In the present invention, the reaction system of the kit, in 20 μ L, comprises: 10 mu L of primer probe group 2 mu L, PCR reaction solution, 0.5 mu L, ROX enzyme mixture solution 0.1 mu L of reference dye, 5 mu L of standard substance or sample to be detected and 2.4 mu L of ribozyme-free water. In the present invention, the conditions for the fluorescent quantitative amplification are preferably: 30min at 42 ℃ (reverse transcription); 1min at 95 ℃ (pre-denaturation); amplification was carried out for 40 cycles at 95 ℃ for 5s and 60 ℃ for 31 s.

The kit has simple operation method and short detection time, and provides a kit product capable of guiding medication and accurately quantifying the curative effect for CysLTR1 receptor antagonist. Cysteinyl leukotrienes (CysLTs) are mediators of inflammation and regulators in the pathophysiology of asthma and Allergic Rhinitis (AR), and are key therapeutic targets. During the course of treatment, leukotriene receptor CysLTR1 antagonists can reduce allergic inflammation by blocking the activity of CysLTR1, producing a wide range of clinical effects. The mRNA expression of the leukotriene receptor CysLTR1 is detected to be higher than the normal reference range, which indicates that the patient has curative effect when the patient is treated by using the leukotriene receptor CysLTR1 antagonist; a decrease in CysLTR1 levels in the blood was monitored after treatment, indicating that the treatment was effective. If there is a clear allergic symptom but the leukotriene receptor CysLTR1 is expressed in a very low amount, it indicates that the allergic symptom is not caused by the leukotriene pathway, and the treatment with leukotriene receptor CysLTR1 antagonist is not effective.

The primer probe set and the kit for RT-PCR detection of human leukotriene receptor CysLTR1mRNA according to the present invention will be described in further detail with reference to the following embodiments, but the technical scheme of the present invention includes but is not limited to the following embodiments.

The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified

Example 1

1. The reagents and equipment involved are as follows:

1.1 reagents

1.1.1 Whole blood Total RNA kit (Hangzhou Xinjing Bioreagent development Co., Ltd., cat # 5201050)

1.1.2 HiScribe T7High Yield RNA Synthesis Kit (New England Biolabs, cat # E2050S)

1.2 Main instruments

1.2.1 Applied Biosystems^TM7300 fluorescent quantitative PCR instrument: ThermoFisher, USA

1.2.2-80 ℃ low-temperature refrigerator: ThermoFisher, USA

1.2.3 high speed cryogenic table centrifuge: eppendorf, Germany

1.2.4 Qubit 3 fluorometer: ThermoFisher, USA

2. Method of producing a composite material

2.1 primer and Probe design

Fluorescent quantitative primers and probes were designed based on CysLTR1 and GAPDH sequences by using Primer 6.0 software, and the Primer pair CysLTR1-F, CysLTR1-R, GAPDH-F, GAPDH-R and probes E-Probe and G-Probe (see Table 1) of CysLTR1 and GAPDH were obtained after series of effect verification. The primer probe was synthesized by Shanghai Sangni Biotech Co., Ltd.

TABLE 1 TaqMan real-time fluorescent quantitative PCR primer probes

2.2 preparation of standards

In vitro transcription. pGM-T ligation kit (Tiangen Biochemical technology (Beijing) Co., Ltd., Cat. No.: VT202-01], CysLTR1 plasmid DNA was constructed using pGM-T as a vector (constructed and synthesized by Nanjing King Shirui Biotech Co., Ltd.), and CysLTR1 plasmid DNA was transcribed into mRNA in vitro using the HiScribe T7High Yield RNA Synthesis Kit (NEW ENGLAND Biolabs, cat # E2040S).

According to a copy number calculation formula: copy number ═ 6.02X 10²³X RNA concentration (ng/. mu.L). times.10^-9]/[ RNA Length (bp) × 340]And calculating the initial copy number of the RNA. Diluting with ribozyme-free water to 1.0X 10¹⁰copies/mu L, namely CysLTR1 standard substance.

2.3 Whole blood RNA extraction and dilution: EDTA anticoagulated whole blood sample whole blood total RNA is extracted by a whole blood total RNA kit, and after quantification by a Qubit 3 fluorometer, the whole blood total RNA is diluted to 20 ng/. mu.L by ribozyme-free water.

2.4TaqMan real-time fluorescent quantitative PCR

Using standard or whole blood RNA as template, 20. mu.L system was prepared as shown in Table 2:

TABLE 2 reaction System

The amplification reaction procedure is shown in table 3:

TABLE 3 reaction procedure

2.5 Generation of Standard Curve

CysLTR1 standard was diluted 10-fold and selected at 1.0X 10⁸～1.0×10³Take copies/mu L as template, repeat 3 times for each dilution, carry on TaqMan real-time fluorescent quantitative RT-PCR detection, produce the standard curve. The procedure of the dilution is shown in FIG. 1, and in each dilution procedure, 5. mu.L of the sample before dilution is added to a new tube containing 45. mu.L of water, taking 50. mu.L/tube as an example.

2.6 detection of precision

Selection of 1.0X 10⁷copies/μL、1.0×10⁴Taking a copies/mu L standard substance as a template, carrying out 10 times of TaqMan real-time fluorescence quantitative RT-PCR detection on each concentration by 10 times of repeated weight, respectively calculating the variation coefficient of each concentration logarithmic value, carrying out statistical analysis, and analyzing the precision of the detection method.

2.7 accuracy testing

Selection of 1.0X 10⁶30-fold dilution of copies/. mu.L standard (2. mu.L of 1.0X 10)⁶copies/muL standard +58 muL ribozyme-free water) as a template, 3 times of TaqMan real-time fluorescent quantitative RT-PCR detection is carried out for 3 times, the absolute deviation of each concentration logarithmic value is calculated, and the accuracy of the detection method is analyzed.

2.8 sensitivity detection

Selecting 10.0 copies/. mu.L standard substance as a template, carrying out 25 times of TaqMan real-time fluorescent quantitative RT-PCR detection by 25 repeated amounts, checking whether amplification is carried out, and analyzing the sensitivity of the detection method.

2.9 clinical sample testing

Taking the positive sample and the healthy control whole blood sample, extracting and diluting the whole blood RNA according to 2.3 steps, and carrying out TaqMan real-time fluorescence quantitative RT-PCR detection according to 2.4 steps.

3. Results of the experiment

3.1 Standard Curve

CysLTR1 standard was diluted 10-fold and selected at 1.0X 10⁸～1.0×10³copies/. mu.L as template, 3 replicates per dilution, TaqMan real-time fluorescenceQuantitative RT-PCR detection, generating a standard curve, wherein CysLTR1 mNATaqMan real-time fluorescence quantitative RT-PCR standard curve is shown in figure 2. Taking the logarithm of the copy number as an abscissa and the Ct value as an ordinate, obtaining a regression equation: -3.398x +35.344 (R)²1.000), R of the regression equation²1.000, linear range 1.0 × 10³～1.0×10⁸copies/. mu.L. The log copy number value of the standard equation is shown to have a very high correlation with the Ct value.

3.2 precision determination

Selection of 1.0X 10⁷copies/μL、1.0×10⁴Taking a copies/mu L standard substance as a template, carrying out 10 times of TaqMan real-time fluorescence quantitative RT-PCR detection on each concentration by 10 repeated weight, and respectively calculating the variation coefficient of each concentration logarithmic value for statistical analysis. The results of the precision detection are shown in fig. 3 and table 4, and the results show that the coefficient of variation of each log concentration value is 0.320%, 0.444% and less than 5%, respectively, indicating that the TaqMan real-time fluorescence quantitative RT-PCR detection method established by the invention has excellent precision.

TABLE 4 results of precision measurements

Theoretical number of copies	Logarithmic mean of copy number	SD	C.V
				1.0×107	7.042	0.023	0.320％
1.0×104	3.996	0.018	0.444％

3.3 accuracy detection

Selection of 1.0X 10⁶30-fold dilution of copies/. mu.L standard (2. mu.L of 1.0X 10)⁶copies/muL standard +58 muL ribozyme-free water) as template, 3 replicates, 3 TaqMan real-time fluorescent quantitative RT-PCR assays were performed, and the absolute deviation of each concentration log value was calculated. The results are shown in FIG. 4 and Table 5, and the results show that the absolute deviation of each log-concentration value is 0.002, 0.011 and 0.008 within the range of +/-0.5, which indicates that the TaqMan real-time fluorescence quantitative RT-PCR detection method established by the invention has excellent accuracy.

TABLE 5 accuracy test results

3.4 sensitivity detection

Selecting 10.0 copies/. mu.L standard substance as a template, carrying out 25 times of TaqMan real-time fluorescent quantitative RT-PCR detection by 25 repeated amounts, and checking whether amplification is new. The sensitivity detection results are shown in FIG. 5 and Table 6, and the results show that the total detection results of 25 times reach 100%, which indicates that the TaqMan real-time fluorescence quantitative RT-PCR detection method established by the invention has very high sensitivity, and the lowest detected copy number is less than 10 copies/mu L.

TABLE 6 Ct value results of sensitivity detection

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 clinical sample testing

The comparison result of the invention and domestic ELISA kit of a certain brand of cysteinyl leukotriene receptor 1(CYSLTR1) is shown in FIG. 6 and Table 7:

TABLE 7 alignment results

The invention adopts whole blood RNA for detection, and domestic cysteine acyl leukotriene receptor 1(CYSLTR1) ELISA kit of a certain brand adopts serum for detection.

Comparative example 1

Results of amplification with other non-optimal primers, probes

The primers and probes in the system used by the invention are replaced by other non-optimal primers and probes. The amplification system and procedure were the same as in example 1. The results are shown in fig. 7 and table 8, with non-optimal CysLTR2 primers, probes, such as:

CysLTR1-F：GTATCTTCTGCCACATGCC(SEQ ID NO.7)；

CysLTR1-R：TTGCCAAAGAAGCCTACAACA(SEQ ID NO.8)；

C1-Probe：(FAM)-CCGCAATCAAGTGTATTCCACC(SEQ ID NO.9)-(BHQ1)。

the result of the coefficient of variation of the log values of low value precision was more than 5%, which reached 8.813%.

TABLE 8 results of amplification with non-optimal primers and probes

Theoretical number of copies	Logarithmic mean of copy number	SD	C.V
				1.0×104	3.637	0.321	8.813％

Comparative example 2

Amplification results of non-optimal enzyme mixtures

Respectively amplifying the standard substance with enzyme mixture (the volume ratio of Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody is 14:4:5:1) and enzyme mixture with optimal ratio, and amplifying to obtain 1.0 × 10 on the standard curve³～1.0×10⁶The primers and probes for amplification, amplification system, and procedure were the same as in example 1. As a result, FIG. 8 shows the results of amplification using the non-optimal enzyme mixture ratio, A in FIG. 8, and B in FIG. 8, using the optimal enzyme mixture ratio. Compared with the standard curve amplification result of the optimal enzyme mixed liquor and the standard curve amplification result of the non-optimal enzyme mixed liquor, the standard curve amplification result of the optimal enzyme mixed liquor has better repeatability, the Ct difference values of adjacent concentrations are respectively 3.3, 3.3 and 3.4, and the Ct difference values of adjacent concentrations of the standard curve amplification result of the non-optimal enzyme mixed liquor are respectively 3.8, 3.1 and 3.6, which indicates that the Ct difference values of the standard curve amplification result of the optimal enzyme mixed liquor are more uniform. Therefore, the best enzyme mixed solution has better amplification effect.

The results show that the TaqMan real-time fluorescent quantitative RT-PCR detection method established by the invention has better sensitivity and specificity compared with a reagent, and can effectively monitor the treatment effect.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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