Primer probe set for detecting human interleukin 4 receptor α (IL4RA) mrna, kit, and use thereof

Abstract

The present invention relates to the technical field of biological detection, and provides a primer probe set for detecting human interleukin 4 receptor α (IL4RA) mRNA, a kit, and a use thereof. The primer probe set of the present invention comprises IL4RA-F, IL4RA-R and a probe I4-Probe; the nucleotide sequence of IL4RA-F is shown in SEQ ID NO: 1, the nucleotide sequence of IL4RA-R is shown in SEQ ID NO: 2, and the nucleotide sequence of probe I4-Probe is shown in SEQ ID NO: 3. The present invention provides a kit comprising the primer probe set, which can detect the expression level of IL4RA mRNA by using an RNA one-step method, and provides a detection means having high accuracy, a broad detection range and high sensitivity for the detection of IL4RA protein.

Description

A primer and probe set, kit, and application for detecting human interleukin 4α receptor IL4RA mRNA. Technical Field

[0001] This invention belongs to the field of biological detection technology, and in particular relates to a primer and probe set, a kit for detecting human interleukin 4α receptor IL4RA mRNA, and their applications. Background Technology

[0002] The interleukin-4 receptor (IL-4R) complex is a heterodimeric structure composed of a common subunit, IL-4Rα, which pairs with different helper subunits to mediate the effects of IL-4 and IL-13 in various tissues. IL-4Rα pairs with the γc chain to form IL-4R type I, which is expressed on hematopoietic cells and specifically binds to IL-4. Furthermore, IL-4Rα binds to the low-affinity IL-13 binding receptor IL-13Rα1, forming a high-affinity IL-13 and IL-4 binding type II heterodimeric complex, which is expressed on both hematopoietic and non-hematopoietic cells (such as airway epithelium). Once IL-4 or IL-13 binds to its receptor, it triggers transphosphorylation and activation of receptor subunit-associated Janus family protein kinases (JAKs), including JAK1, JAK3, and JAK2, which are associated with the IL-4Rα, γc, and IL-13Rα1 chains, respectively. JAK activation initiates a phosphorylation cascade of specific tyrosine residues in the IL-4Rα cytoplasmic domain. These three tyrosine residues (human IL-4Rα Y575 / Y603 / Y633) act as a cassette (referring to DNA sequence components), and their phosphorylation allows transcription factor signaling and activator of transcription 6 (STAT6) to be recruited through the latter's SH2 domain, thereby activating it and initiating the transcriptional program it regulates. While the role of IL-4R signaling in promoting allergic effector pathways has been emphasized, recent studies have found that this pathway plays a fundamental role in regulating immune tolerance by disrupting allergen-specific regulatory T (Treg) cell responses. This disruption is crucial for chronic allergic inflammatory diseases such as asthma. A unique mechanism involving Treg cells in allergic diseases involves a variant of the IL-4Rα chain. In this variant, the glutamine residue at position 576 in the cytoplasmic domain is replaced with arginine (R576), which is closely associated with severe asthma and increased susceptibility to asthma. Once the latter is phosphorylated by JAK, R576 substitution allows the adaptor growth factor receptor-binding protein 2 (GRB2) to bind to the adjacent Y575, thereby leading to mitogen-activated kinase-like protein (MAPK) activation and IL6 gene induction.

[0003] Dupilumab is an IgG4 human monoclonal antibody (mAb) that binds to IL-4RA. Dupilumab inhibits IL-4R signaling induced by IL-4 and IL-13 and downregulates TH2 inflammation in a variety of allergic diseases, including atopic dermatitis, asthma, and possibly other allergic conditions. Developing a product capable of detecting human interleukin 4α receptor IL4RA is therefore essential. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a primer and probe set, a kit, and their applications for detecting human interleukin 4α receptor IL4RA mRNA, which can quantitatively detect the expression level of IL4RA mRNA in one step, providing a detection method with high accuracy, wide detection range, and high sensitivity for the detection of this protein.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a primer and probe set for detecting human interleukin 4α receptor IL4RA mRNA, the primer and probe set including IL4RA-F, IL4RA-R and probe I4-Probe;

[0007] The nucleotide sequence of IL4RA-F is shown in SEQ ID NO.1, the nucleotide sequence of IL4RA-R is shown in SEQ ID NO.2, and the nucleotide sequence of probe I4-Probe is shown in SEQ ID NO.3.

[0008] Preferably, the primer and probe set further includes primers and probes for the internal reference gene GAPDH, wherein the primers for GAPDH include GAPDH-F and GAPDH-R, and the probe is a G-Probe.

[0009] The nucleotide sequence of GAPDH-F is shown in SEQ ID NO.4, the nucleotide sequence of GAPDH-R is shown in SEQ ID NO.5, and the nucleotide sequence of G-Probe is shown in SEQ ID NO.6.

[0010] Preferably, different fluorescent labeling groups are labeled at the 5′ ends of the probes I4-Probe and G-Probe, and the same or different quenching groups are labeled at the 3′ ends.

[0011] Preferably, the fluorescent labeling group includes FAM or JOE, and the quenching group includes BHQ1.

[0012] This invention provides a kit for detecting the expression level of human interleukin 4α receptor IL4RA mRNA. The kit includes a mixture of primer and probe sets, PCR reaction solution, enzyme mixture, human interleukin 4α receptor IL4RA standard, ROX reference dye, and nuclease-free water.

[0013] Preferably, the concentrations of IL4RA-F, IL4RA-R, I4-Probe, GAPDH-F, GAPDH-R, and G-Probe in the primer-probe mixture are 0.5–1.5 μM, 0.5–1.5 μM, 1–2 μM, 1–3 μM, 1–3 μM, and 2–4 μM, respectively.

[0014] Preferably, the PCR reaction solution includes dNTP mix, MgCl2, and buffer.

[0015] The enzyme mixture includes a hot-start Taq enzyme and a reverse transcriptase; the activity of the hot-start Taq enzyme is 1–5 U / μL, and the activity of the reverse transcriptase is 1–3 U / μL.

[0016] This invention also provides the application of the aforementioned primer-probe set in the preparation of products for detecting the expression level of human interleukin 4α receptor IL4RA mRNA.

[0017] Preferably, the reaction system is prepared using the primer and probe set described above, and a qRT-PCR reaction is performed.

[0018] The reaction system, in 20 μL, includes: 2.4 μL of nuclease-free water, 10 μL of PCR reaction solution, 0.5 μL of enzyme mixture, 0.1 μL of ROX reference dye, 2 μL of primer and probe mixture, and 5 μL of standard.

[0019] Preferably, the qRT-PCR reaction program includes: 42℃ for 30 min; 95℃ for 1 min; 95℃ for 5 s; 60℃ for 31 s; 40 cycles.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] (1) This invention provides a primer and probe set for detecting human interleukin 4α receptor IL4RA mRNA, and a kit containing the primer and probe set, which can detect the expression level of human interleukin 4α receptor (IL4RA) mRNA in one step using RNA. On the one hand, it can determine whether the patient's allergic symptoms are caused by the interleukin 4 (IL-4) pathway and guide the dosage of medication (high IL-4 receptor expression requires higher doses of anti-IL-4 drugs). On the other hand, it can dynamically monitor the effect of anti-IL-4 drug treatment, providing a detection method with high accuracy, wide detection range and high sensitivity for the detection of IL-4R protein.

[0022] (2) The primer probe set or kit described in this invention does not require separate reverse transcription, which greatly reduces the risk of aerosol contamination. Compared with immunological detection methods, this invention has high detection sensitivity, can detect low concentrations of clinical samples, can sensitively detect changes in IL4RA content, and can span at least 5 orders of magnitude, increasing the accuracy of detection results. It can complete the detection of at least 80 samples within 1 hour, thereby enabling earlier, more accurate, and faster dynamic monitoring and efficacy evaluation of treatment effects. Attached Figure Description

[0023] Figure 1 shows the plasmid map of the pGM-T vector;

[0024] Figure 2 shows the standard curve of IL4RAmRNA TaqMan real-time quantitative RT-PCR.

[0025] Figure 3 shows the precision test results, where 1: 1.0 × 10⁻⁶ 5 copies / μL, 2: 1.0×10 3 copies / μL;

[0026] Figure 4 shows the accuracy test results;

[0027] Figure 5 shows the sensitivity detection results;

[0028] Figure 6 shows the results of clinical sample testing, where 1 is GAPDH mRNA before treatment in case 1, 2 is GAPDH mRNA after treatment in case 1, 3 is IL4RA mRNA before treatment in case 1, and 4 is IL4RA mRNA after treatment in case 1.

[0029] Figure 7 shows the amplification curve for low precision when primer and probe design is unreasonable. Detailed Implementation

[0030] This invention provides a primer and probe set for detecting human interleukin 4α receptor IL4RA mRNA, the primer and probe set including IL4RA-F, IL4RA-R and probe I4-Probe;

[0031] In this invention, the primers and probes were synthesized by Shanghai Sunny Biotechnology Co., Ltd.; the nucleotide sequence of the IL4RA-F is shown in SEQ ID NO.1, specifically: 5′-CTCAGTGCTATAACACCA-3′;

[0032] The nucleotide sequence of the IL4RA-R is shown in SEQ ID NO.2, specifically: 5′-ATCCCACCATTCTTTCTTA-3′;

[0033] A fluorescent labeling group, FAM, is labeled at the 5′ end of the probe I4-Probe, and a quenching group, BHQ1, is labeled at the 3′ end. The nucleotide sequence of the probe I4-Probe is shown in SEQ ID NO.3, specifically: 5′-(FAM)-ACATAGCACAACAGGCAGACG-(BHQ1)-3′.

[0034] In this invention, the primer and probe set further includes primers and probes for the internal reference gene GAPDH. The primers for GAPDH include GAPDH-F and GAPDH-R, and the probe is a G-Probe. A fluorescent labeling group, JOE, is labeled at the 5′ end of the G-Probe, and a quenching group, BHQ1, is labeled at the 3′ end. The nucleotide sequence of GAPDH-F is shown in SEQ ID NO.4, specifically: 5′-GACAACAGCCTCAAGATCATC-3′.

[0035] The nucleotide sequence of the GAPDH-R is shown in SEQ ID NO.5, specifically: 5′-CGCCACAGTTTCCCGGAG-3′;

[0036] The nucleotide sequence of the G-Probe is shown in SEQ ID NO.6, specifically: 5′-(JOE)-ACTCATGACCACAGTCCATGCCAT-(BHQ1)-3′.

[0037] This invention provides a kit for detecting the expression level of human interleukin 4α receptor IL4RA mRNA. The kit includes a mixture of primer and probe sets, PCR reaction solution, enzyme mixture, human interleukin 4α receptor IL4RA standard, ROX reference dye, and nuclease-free water.

[0038] In this invention, the primer-probe set is preferably used in the form of a mixture. The concentration of IL4RA-F in the mixture is 0.5–1.5 μM, preferably 0.7–1.2 μM, and more preferably 1 μM; the concentration of IL4RA-R is 0.5–1.5 μM, preferably 0.7–1.2 μM, and more preferably 1 μM; the concentration of probe I4-Probe is 1–2 μM, preferably 1.3–1.8 μM, and more preferably 1.5 μM; the concentration of GAPDH-F is 1–3 μM, preferably 1.5–2.5 μM, and more preferably 2 μM; the concentration of GAPDH-R is 1–3 μM, preferably 1.5–2.5 μM, and more preferably 2 μM; and the concentration of G-Probe is 2–4 μM, preferably 2.5–3.5 μM, and more preferably 3 μM.

[0039] In this invention, the PCR reaction solution includes dNTP mix, MgCl2, and buffer; the dNTP mix is ​​preferably a mixture of dATP, dTTP, dCTP, and dGTP, purchased from Thermo Fisher Scientific (catalog number: R0192), with a working concentration preferably of 0.1–1 mM; the MgCl2 concentration is preferably 5–20 mM; the buffer is 10–50 mM Tris-HCl buffer (pH 8.0); the enzyme mixture includes hot-start Taq enzyme and reverse transcriptase; the activity of the hot-start Taq enzyme is 1–5 U / μL, preferably 2–4 U / μL, more preferably 3 U / μL, and the activity of the reverse transcriptase is 1–3 U / μL, preferably 1.5–2.5 U / μL, more preferably 2 U / μL; the mass ratio of the hot-start Taq enzyme to the reverse transcriptase is 5:2, under which optimal amplification efficiency can be achieved; the hot-start Taq enzyme is a heat-resistant hot-start Taq enzyme. DNA polymerase, utilizing its 3′→5′ polymerase activity, uses DNA as a template to add deoxymononucleotides from dNTPs one by one to the 3′-OH end; simultaneously, its 5′→3′ exonuclease activity can both recognize and eliminate mismatched primer ends, which is related to the proofreading function during replication, and can also hydrolyze nucleotides from the 5′ end, and can also act through several nucleotides to cleave mismatched nucleotides, thereby achieving strand substitution during strand elongation and cutting the replaced probe; reverse transcriptase can reverse transcribe mRNA into cDNA for PCR reaction; the interleukin 4α receptor IL4RA standard is preferably an RNA standard, used to prepare a standard curve.

[0040] This invention also provides the application of the aforementioned primer-probe set in the preparation of products for detecting the expression level of human interleukin 4α receptor IL4RA mRNA.

[0041] In this invention, the primer and probe set is used to prepare the reaction system and perform qRT-PCR. The reaction system, in 20 μL, includes: 2.4 μL of nuclease-free water, 10 μL of PCR reaction solution, 0.5 μL of enzyme mixture, 0.1 μL of ROX reference dye, 2 μL of primer and probe set mixture, and 5 μL of standard. The qRT-PCR reaction program includes: 42℃ for 30 min; 95℃ for 1 min; 95℃ for 5 s; 60℃ for 31 s, for 40 cycles.

[0042] In this invention, the expression level of human interleukin 4α receptor IL4RA mRNA is calculated based on a standard curve prepared from standard samples. The standard curve is plotted with the copy number logarithm as the abscissa (x) and the Ct value as the ordinate (y): y = -3.36419x + 36.59100 (|r| = 0.99984). This invention does not have any special limitation on the source of the RNA, which can be extracted from human blood, nasal secretions, bronchial lavage fluid, saliva, or tear samples.

[0043] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Unless otherwise specified, the reagents, kits, and instruments involved in the embodiments of this invention are all conventional commercially available products in the art.

[0045] The whole blood total RNA kit was purchased from Hangzhou Xinjing Bio-Reagent Development Co., Ltd., catalog number: 5201050; the HiScribe T7 High Yield RNA Synthesis Kit was purchased from New England Biolabs, catalog number: E2050S. Quantitative Real-Time PCR Instrument, Shanghai Hongshi Medical Technology Co., Ltd.; -80℃ Low-Temperature Freezer, ThermoFisher, USA; High-Speed ​​Low-Temperature Benchtop Centrifuge, Eppendorf, Germany; Qubit 3 Fluorometer, ThermoFisher, USA.

[0046] Example

[0047] 1. We commissioned Shanghai Sunny Biotechnology Co., Ltd. to synthesize primers and probes.

[0048] 2. Preparation of Standards

[0049] In vitro transcription: The pGM-T ligation kit (purchased from Tiangen Biotech (Beijing) Co., Ltd., catalog number: VT202-01) was used to construct IL4RA plasmid DNA using pGM-T as a vector (construction and synthesis were commissioned to Nanjing Genscript Biotech Co., Ltd., Figure 1). The IL4RA plasmid DNA was then transcribed into mRNA in vitro using the HiScribe T7 HighYield RNA Synthesis Kit (manufactured by NEW ENGLAND BioLabs, catalog number: E2040S).

[0050] According to the copy number calculation formula: Copy number = [6.02 × 10^6] 23 ×RNA concentration (ng / μL) × 10 -9 Calculate the initial RNA copy number by dividing the RNA length by 340 (bp). Dilute to 1.0 × 10⁻⁶ with nuclease-free water. 9 The copeies / μL is the IL4RA standard.

[0051] 3. Whole blood RNA extraction and dilution: Total RNA was extracted from EDTA-anticoagulated whole blood samples using a whole blood total RNA kit. After quantification using a Qubit 3 fluorometer, the RNA was diluted to 20 ng / μL with nuclease-free water.

[0052] 4. TaqMan Real-Time Quantitative PCR

[0053] Using standard / whole blood RNA as a template, prepare a 20 μL system: 2.4 μL nuclease-free water, 10 μL PCR reaction solution, 0.5 μL enzyme mixture, 0.1 μL ROX reference dye, 2 μL primer and probe mixture, and 5 μL standard or RNA.

[0054] qRT-PCR reaction program: 42℃ for 30 min; 95℃ for 1 min; 95℃ for 5 s; 60℃ for 31 s, 40 cycles. Detection fluorescent dyes: FAM, JOE; reference fluorescent dye: ROX; reaction volume: 20 μL; fluorescence signal collection: 60℃ for 31 s.

[0055] 5. Generation of the standard curve

[0056] The IL4RA standard was diluted in 10-fold serial dilutions, selecting 1.0 × 10⁻⁶. 7 ~1.0×10 1 Using copes / μL as a template, two replicates were performed for each dilution, and TaqMan real-time quantitative RT-PCR was used to generate a standard curve.

[0057] The results are shown in Figure 2. With the copy number logarithm as the x-axis and the Ct value as the y-axis, the regression equation is obtained as: y = -3.36419x + 36.59100 (|r| = 0.99984). This shows that the copy number logarithm of the standard equation has a very high correlation with the Ct value.

[0058] 6. Precision testing

[0059] Select 1.0×10 6 copies / μL, 1.0×10 3 Using a standard of copies / μL as a template, 10 replicates were performed for each concentration, and 10 TaqMan real-time quantitative RT-PCR assays were conducted. The coefficient of variation of the logarithmic values ​​for each concentration was calculated and statistically analyzed to determine the precision of the assay.

[0060] The results are shown in Table 1 and Figure 3. The coefficients of variation for each concentration logarithm were 0.250% and 0.886%, respectively, which are less than 5%, indicating that the TaqMan real-time fluorescence quantitative RT-PCR detection method established in this invention has excellent precision.

[0061] Table 1 Precision test results

[0062] 7. Accuracy Testing

[0063] Select 1.0×10 5 Using copies / μL of standard as templates, three replicates were performed for three TaqMan real-time quantitative RT-PCR assays, and the absolute deviation of the logarithmic values ​​for each concentration was calculated. The results are shown in Table 2 and Figure 4. The absolute deviations of the logarithmic values ​​for each concentration were -0.062, -0.071, and -0.060, respectively, within ±0.5, indicating that the TaqMan real-time quantitative RT-PCR detection method established in this invention has excellent accuracy.

[0064] Table 2 Accuracy Test Data

[0065] 8. Sensitivity Testing

[0066] Using 10.0 copies / μL of standard as a template, 25 replicates were performed for 25 TaqMan real-time quantitative RT-PCR assays to check for amplification spikes and analyze the sensitivity of the assay.

[0067] The results are shown in Table 3 and Figure 5. A total of 25 detections were successful, achieving a 100% success rate. This indicates that the TaqMan real-time quantitative RT-PCR detection method established in this invention has high sensitivity, with a minimum detectable copy number of <10 copies / μL.

[0068] Table 3. Ct values ​​from 25 sensitivity tests.

[0069] 9. Clinical sample testing

[0070] Whole blood samples from patients and healthy individuals were collected, and whole blood RNA was extracted and diluted according to the steps described above. TaqMan real-time quantitative RT-PCR detection was then performed according to the steps described above.

[0071] Compared with a domestic brand of human interleukin 4 receptor (IL4R) detection kit (enzyme-linked immunosorbent assay), the results are shown in Table 4 and Figure 6. The results show that the TaqMan real-time fluorescence quantitative RT-PCR detection method established in this invention has better sensitivity and specificity than existing detection kits, and can effectively monitor the treatment effect.

[0072] Table 4 Comparison of Clinical Sample Detection Experiments

[0073] Note: + indicates positive, - indicates negative.

[0074] 10. Results of amplification using other suboptimal primers and probes

[0075] The primers and probes used in the system of this invention were replaced with other non-optimal primers and probes. The results are shown in Table 5 and Figure 7.

[0076] Non-optimal primers and probes are:

[0077] F:5'-CCCAGCGAGCATGTGAAAC-3' (SEQ ID NO.7);

[0078] R: 5′-GATACGGGTTGCTCCAGGTC-3′ (SEQ ID NO. 8);

[0079] Probe: 5′-(FAM)-ACACCAATGTCTCCGACACTCTGCTGC-(BHQ1)-3′ (SEQ ID NO. 9).

[0080] Table 5. Amplification using suboptimal primers and probes.

[0081] The results showed that when primers and probes were not designed properly, the coefficient of variation of the concentration logarithm of low precision exceeded 5%, reaching 6.132%. This indicates that using different primers and probes for amplification will result in poor precision and thus have an adverse effect on the detection results.

[0082] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A primer and probe set for detecting human interleukin 4α receptor IL4RA mRNA, characterized in that, The primer-probe set includes IL4RA-F, IL4RA-R and probe I4-Probe; The nucleotide sequence of IL4RA-F is shown in SEQ ID NO.1, the nucleotide sequence of IL4RA-R is shown in SEQ ID NO.2, and the nucleotide sequence of probe I4-Probe is shown in SEQ ID NO.

3.

2. The primer-probe set according to claim 1, characterized in that, The primer and probe set also includes primers and probes for the internal reference gene GAPDH, wherein the primers for GAPDH include GAPDH-F and GAPDH-R, and the probe is a G-Probe. The nucleotide sequence of GAPDH-F is shown in SEQ ID NO.4, the nucleotide sequence of GAPDH-R is shown in SEQ ID NO.5, and the nucleotide sequence of G-Probe is shown in SEQ ID NO.

6.

3. The primer-probe set according to claim 2, characterized in that, Different fluorescent labeling groups are marked at the 5′ ends of the probes I4-Probe and G-Probe, and the same or different quenching groups are marked at the 3′ ends.

4. The primer-probe set according to claim 3, characterized in that, The fluorescent labeling group includes FAM or JOE, and the quenching group includes BHQ1.

5. A kit for detecting the expression level of human interleukin 4α receptor IL4RA mRNA, characterized in that, The kit comprises a mixture of primer and probe sets as described in any one of claims 1 to 4, a PCR reaction solution, an enzyme mixture, a human interleukin 4α receptor IL4RA standard, a ROX reference dye, and nuclease-free water.

6. The reagent kit according to claim 5, characterized in that, The concentrations of IL4RA-F, IL4RA-R, I4-Probe, GAPDH-F, GAPDH-R, and G-Probe in the primer-probe mixture were 0.5–1.5 μM, 0.5–1.5 μM, 1–2 μM, 1–3 μM, 1–3 μM, and 2–4 μM, respectively.

7. The reagent kit according to claim 5, characterized in that, The PCR reaction solution includes dNTP mix, MgCl2, and buffer solution; The enzyme mixture includes a hot-start Taq enzyme and a reverse transcriptase; the activity of the hot-start Taq enzyme is 1–5 U / μL, and the activity of the reverse transcriptase is 1–3 U / μL.

8. The use of the primer and probe set according to any one of claims 1 to 4 in the preparation of a product for detecting the expression level of human interleukin 4α receptor IL4RA mRNA.

9. The application according to claim 8, characterized in that, The reaction system was prepared using the primer and probe set described above, and qRT-PCR reaction was performed. The reaction system, in 20 μL, includes: 2.4 μL of nuclease-free water, 10 μL of PCR reaction solution, 0.5 μL of enzyme mixture, 0.1 μL of ROX reference dye, 2 μL of primer and probe mixture, and 5 μL of standard.

10. The application according to claim 8, characterized in that, The qRT-PCR reaction program includes: 42℃ for 30 min; 95℃ for 1 min; 95℃ for 5 s; 60℃ for 31 s; 40 cycles.

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