WO2023010326A1 - PRIMER-PROBE SET AND KIT FOR HUMAN β-TRYPTASE MRNA RT-PCR DETECTION - Google Patents

Abstract

The present invention relates to a primer-probe set and a kit for human β-tryptase mRNA RT-PCR detection, which belongs to the technical field of biological detection. The primer-probe set of the present invention comprises a primer TPSB-F, a primer TPSB-R and a probe T-Probe, wherein the nucleotide sequence of the primer TPSB-F is as shown in SEQ ID NO: 1; the nucleotide sequence of the primer TPSB-R is as shown in SEQ ID NO: 2; and the nucleotide sequence of the probe T-Probe is as shown in SEQ ID NO: 3. By means of the present invention, a one-step TaqMan real-time fluorescent quantitative RT-PCR detection primer-probe set is established for human TPSB, and provides a detection means with high accuracy, a wide detection range and high sensitivity for detection of the protein.

Description

A kind of primer probe set and kit for human β-tryptase mRNA RT-PCR detection technical field

The invention relates to the technical field of biological detection, in particular to a primer probe set and a kit for human β-tryptase mRNA RT-PCR detection.

Background technique

Mast cells are activated during anaphylaxis, releasing inflammatory mediators including tryptase, a process that contributes to the signs and symptoms of anaphylaxis. Tryptase has the functions of promoting the repair of airway, regulating the tension and reactivity of airway smooth muscle cells, and stimulating the activation of mast cells. At present, tryptase is divided into three categories at the cDNA and protein levels: α, β, γ, among which β has the highest content. After an allergic reaction, a transient increase in the level of β-tryptase (TPSB) in the blood circulation helps to identify and assess the degree of the allergic reaction. 15 minutes to 3 hours of collection. Transiently elevated levels of beta-tryptase in the blood suggest an allergic reaction to a drug, insect venom, or food, and long-term, persistently elevated levels suggest mastocytosis or a hematological neoplasm. Elevated levels (or concentrations) of β-tryptase in nasal secretions suggest active allergic rhinitis or patients with allergic rhinitis are undergoing allergen challenge testing.

At present, the detection of TPSB 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 detecting TPSB mRNA. The ELISA method has the problems of small detection range and low sensitivity in the detection process, and its accuracy also has problems.

Contents of the invention

The object of the present invention is to provide a kind of primer probe group and kit for human β-tryptase 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 TPSB, 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 β-tryptase mRNA RT-PCR detection, the primer probe set includes primer TPSB-F, primer TPSB-R and probe T-Probe, the primer TPSB The nucleotide sequence of -F is as shown in SEQ ID NO.1, the nucleotide sequence of described primer TPSB-R is as shown in SEQ ID NO.2, and the nucleotide sequence of described probe T-Probe is as shown in SEQ ID NO.2 ID NO.3 is shown.

Preferably, the 5' end of the probe T-Probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group.

Preferably, primer GAPDH-F, primer GAPDH-R and probe G-Probe of the internal reference gene are also included, the nucleotide sequence of the primer GAPDH-F is shown in SEQ ID NO.4, and the primer GAPDH- The nucleotide sequence of R is shown in SEQ ID NO.5, and the nucleotide sequence of the probe G-Probe is shown in SEQ ID NO.6.

Preferably, 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 T-Probe Reporting groups are different.

Preferably, the fluorescent reporter group includes FAM or JOE, and the quencher group includes BHQ1.

The present invention also provides a kit for human β-tryptase mRNA RT-PCR detection, said kit comprising the primer probe set described in the technical scheme, PCR reaction solution, enzyme mixture, β-tryptase Standards, ROX reference dye, and nuclease-free water.

Preferably, the PCR reaction solution includes dNTP mix, MgCl ₂ and buffer.

Preferably, 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.

Preferably, in terms of 20 μL, the reaction system of the kit includes: 2 μL of primer probe set, 10 μL of PCR reaction solution, 0.5 μL of enzyme mixture, 0.1 μL of ROX reference dye, 5 μL of standard or test sample and no 2.4 μL 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 human β-tryptase mRNA RT-PCR detection. Compared with the immunological detection method, when the primer probe set of the present invention is used for detection, it has high sensitivity, can detect low concentration (10copies/μL) clinical samples, and can more sensitively detect changes in the content of TPSB. It can span at least 6 orders of magnitude, increasing the accuracy of the detection results, so that the dynamic monitoring and curative effect evaluation of the treatment effect can be performed earlier, more accurately and faster.

Instructions attached

Fig. 1 is the dilution operation process diagram that the present invention provides;

Fig. 2 is the TPSB mRNA TaqMan real-time fluorescence 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, in which 1: 1.0×10 ⁶ copies/μL, 2: 1.0×10 ³ copies/μL;

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;

Figure 6 is a diagram of the detection results of clinical samples provided by the present invention; wherein 1: positive sample 5GAPDH mRNA; 2: healthy control 3 GAPDH mRNA; 3: positive sample 5 TPSB mRNA; 4: healthy control 3 TPSB mRNA; 5: blank control NTC -GAPDH mRNA; 6: blank control NTC-TPSB mRNA;

Fig. 7 is provided by the present invention under the situation that primer, probe are not optimally designed, low value precision amplification curve;

Fig. 8 shows the amplification results of the non-optimal ratio enzyme mixture solution and the optimal ratio enzyme mixture solution provided by the present invention.

Detailed ways

The invention provides a primer probe set for human β-tryptase mRNA RT-PCR detection, the primer probe set includes primer TPSB-F, primer TPSB-R and probe T-Probe, the primer TPSB The nucleotide sequence of -F is as shown in SEQ ID NO.1: 5'-CAGCGAGTGGGCATCGTT-3', and the nucleotide sequence of the primer TPSB-R is as shown in SEQ ID NO.2: 5'-ATCCTTGACGTCCGGTCCC-3 ', the nucleotide sequence of the probe T-Probe is shown in SEQ ID NO.3: 5'-AGCCTGAGAGTCCGCGACCGAT-3'.

In the present invention, the 5' end of the probe T-Probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quenching group. In the present invention, the fluorescent reporter group preferably includes FAM or JOE, and the quencher group preferably includes BHQ1. In the embodiment of the present invention, the 5' end of the probe T-Probe is labeled with a FAM fluorescent reporter group, and the 3' end is labeled with a BHQ1 quencher group.

In the present invention, 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'. In the present invention, 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 T-Probe labeled The fluorescent reporter groups are preferably different. In the present invention, the fluorescent reporter group preferably includes FAM or JOE, and the quencher group preferably includes BHQ1. In the embodiment of the present invention, 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 β-tryptase mRNA RT-PCR detection, said kit comprising the primer probe set described in the technical scheme, PCR reaction solution, enzyme mixture, β-tryptase Standards, ROX reference dye, and nuclease-free water.

In the present invention, 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 0.1-1 mM. In the present invention, the use concentration of _MgCl2 is preferably 5～20mM; 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.

In the present invention, the enzyme mixture includes Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody. In the present invention, in the enzyme mixture, the volume ratio of Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody is preferably 14:5:5:1, and this ratio can obtain the best amplification effect . In the present invention, 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 with Taq enzyme, and can effectively inhibit the non-specific annealing of primers and primer dimer at low temperature. 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.

In the present invention, the β-tryptase standard product is preferably a β-tryptase mRNA standard product, which is used to prepare a quantitative 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. In the present invention, the kit of the present invention adopts a one-step RT-PCR quantitative detection method, which can detect the expression level of TPSB mRNA in human blood, nasal secretion, bronchial flushing fluid, saliva, and tear fluid samples.

In the present invention, the reaction system of the kit, in terms of 20 μL, preferably includes: 2 μL of primer probe set, 10 μL of PCR reaction solution, 0.5 μL of enzyme mixture, 0.1 μL of ROX reference dye, standard or test sample 5 μL and 2.4 μL of nuclease-free water. In the present invention, 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 β-tryptase mRNA RT-PCR detection primer probe set and kit described in the present invention are described in further detail below in conjunction with specific examples. The technical solutions of the present invention include but are not limited to the following examples.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents, etc. used in the following examples, if no special instructions, can be obtained from commercial sources

Example 1

1. The reagents and equipment involved are as follows:

1.1 Reagents

1.1.1 Whole blood total RNA kit (Hangzhou Xinjing Biological Reagent Development Co., Ltd., catalog number: 5201050)

1.1.2 HiScribe T7 High Yield RNA Synthesis Kit (New England Biolabs, Cat. No.: E2050S)

1.2 Main instruments

1.2.1 Applied Biosystems ^TM 7300 fluorescence quantitative PCR instrument: ThermoFisher, USA

1.2.2 -80℃ low temperature refrigerator: ThermoFisher, USA

1.2.3 High-speed and low-temperature desktop centrifuge: Eppendorf, Germany

1.2.4 Qubit 3 Fluorometer: ThermoFisher, USA

2. Method

2.1 Primer and probe design

According to the sequence of TPSB and GAPDH, use Primer 6.0 software to design fluorescent quantitative primers and probes. After a series of effect verification, the primer pairs TPSB-F, TPSB-R, GAPDH-F, GAPDH-R and probes of TPSB and GAPDH were obtained. T-Probe, G-Probe (see Table 1). Primers and probes were synthesized by Shanghai Sunny Biotechnology Co., Ltd.

Table 1 TaqMan real-time fluorescent quantitative PCR primer probe

2.2 Standard preparation

In vitro transcription. Using pGM-T ligation kit [Tiangen Biochemical Technology (Beijing) Co., Ltd., article number: VT202-01], using pGM-T as the carrier to construct TPSB plasmid DNA (entrusted to Nanjing GenScript Biotechnology Co., Ltd. to construct and synthesize), TPSB plasmid DNA was transcribed into mRNA in vitro with HiScribe T7 High Yield RNA Synthesis Kit (produced by NEW ENGLAND BioLabs, Cat. No.: E2040S).

According to the copy number calculation formula: copy number=[6.02×10 ²³ ×RNA concentration (ng/μL)×10 ^-9 ]/[RNA length (bp)×340], calculate the initial RNA copy number. Dilute with nuclease-free water to 1.0×10 ⁹ copies/μL, which is the TPSB standard.

2.3 Whole blood RNA extraction and dilution: 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/μL with nuclease-free water.

2.4 TaqMan real-time fluorescence quantitative PCR

Using the standard or whole blood RNA as a template, prepare a 20 μL system as shown in Table 2:

Table 2 20μL reaction system

The amplification reaction program is shown in Table 3:

Table 3 Amplification reaction program

2.5 Generation of standard curve

The TPSB standard was diluted in a 10-fold gradient, and 1.0×10 ⁷ to 1.0×10 ² copies/μL was selected as a template, and each dilution was replicated twice for TaqMan real-time fluorescent quantitative RT-PCR detection to generate a standard curve. The dilution operation process is shown in Figure 1, taking 50 μL/tube as an example, during each dilution process, take 5 μL of the sample before dilution and add it to a new tube containing 45 μL of water.

2.6 Precision testing

Select 1.0×10 ⁶ copies/μL, 1.0×10 ³ copies/μL standard as template, 10 replicates for each concentration, and perform 10 TaqMan real-time fluorescence quantitative RT-PCR detection, and calculate the logarithmic value of each concentration The coefficient of variation was statistically analyzed to analyze the precision of the detection method.

2.7 Accuracy detection

Select 1.0×10 ⁵ copies/μL standard as a template, and perform 3 times of TaqMan real-time fluorescence quantitative RT-PCR detection with 3 repetitions, calculate the absolute deviation of the logarithmic value of each concentration, and analyze the accuracy of the detection method.

2.8 Sensitivity detection

Choose 10.0copies/μL standard as the template, 25 repetitions, and perform 25 TaqMan real-time fluorescent quantitative RT-PCR detections to check whether there is any amplification and analyze the sensitivity of the detection method.

2.10 Clinical sample testing

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.

3. Experimental results

3.1 Standard curve

Dilute the TPSB standard product according to a 10-fold gradient, select 1.0×10 ⁷ ～1.0×10 ² copies/μL as the template, and perform 2 replicates for each dilution, perform TaqMan real-time fluorescent quantitative RT-PCR detection, and generate a standard curve, TPSB The mRNA TaqMan real-time fluorescent quantitative RT-PCR standard curve is shown in Figure 2. Taking the copy number logarithmic value as the abscissa and the Ct value as the ordinate, the regression equation is obtained: y=-3.46x+38.261 (R ² =0.996), the R ² of the regression equation is 0.996, and the linear range is 1.0×10 ² ～ 1.0×10 ⁷ copies/μL. It shows that the copy number logarithmic value of the standard equation has a very high correlation with the Ct value.

3.2 Precision testing

Select 1.0×10 ⁶ copies/μL, 1.0×10 ³ copies/μL standard as template, 10 replicates for each concentration, and perform 10 TaqMan real-time fluorescence quantitative RT-PCR detection, and calculate the logarithmic value of each concentration The coefficient of variation was used for statistical analysis. Precision detection result is as shown in table 4 and Fig. 3, and the result shows that the coefficient of variation of each concentration logarithmic value is respectively 0.765%, 2.547%, less than 5%, shows that the TaqMan real-time fluorescent quantitative RT-PCR detection method that the present invention establishes Has excellent precision.

Table 4 Precision test results

theoretical copy number copy number log mean SD C.V. 1.0×10 ⁶ 5.934 0.045 0.765% 1.0×10 ³ 2.916 0.074 2.547%

3.3 Accuracy detection

Select 1.0×10 ⁵ copies/μL standard as a template, and perform 3 repetitions of TaqMan real-time fluorescent quantitative RT-PCR detection, and calculate the absolute deviation of the logarithmic value of each concentration. The results are shown in Figure 4 and Table 5, and the results show that the absolute deviations of each concentration logarithmic value are respectively -0.115, -0.079, -0.103, within the scope of ± 0.5, showing that the TaqMan real-time fluorescent quantitative RT-PCR established by the present invention The detection method has excellent accuracy.

Table 5 Accuracy test results

3.4 Sensitivity detection

Select 10.0copies/μL standard as a template, 25 replicates, and perform 25 TaqMan real-time fluorescent quantitative RT-PCR tests to check whether there is any amplification. Sensitivity detection results are shown in Table 6 and Figure 5, the results show that a total of 25 detection results, up to 100%, show that the TaqMan real-time fluorescent quantitative RT-PCR detection method established by the present invention has very high sensitivity, the lowest detection copy Count <10copies/μL.

Table 6 Sensitivity detection Ct value results

33.112 33.655 34.996 33.374 33.072 35.019 33.432 34.864 35.830 34.680 33.495 37.118 33.542 33.172 34.855 33.883 34.704 34.883 34.509 33.579 37.455 33.027 34.572 32.982 33.334

3.5 Clinical sample testing

The results of the comparison between the present invention and the widely used ImmunCAP ^TM TPSB reagent abroad are shown in Table 7 and Figure 6:

Table 7 comparison results

The present invention uses whole blood RNA for detection, and the ImmunCAP ^TM TPSB reagent uses serum for detection.

The above results show that the TaqMan real-time fluorescent quantitative RT-PCR detection method established by the present invention has higher specificity and better sensitivity than imported fluorescent enzyme immunoassay reagents.

Comparative example 1

Amplification results using other non-optimal primers and probes

The primers and probes in the system used in the present invention 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 7, when non-optimal TPSB primers and probes are used, such as:

TPSB-F: TGCAGCGAGTGGGCATCGT (SEQ ID NO. 7);

TPSB-R: TCTGGGCGGTGTAGAACTGT (SEQ ID NO. 8);

T-Probe: (FAM)-CACTTCTGCGGGGGCTCCCTC (SEQ ID NO. 9)-(BHQ1).

The coefficient of variation of the concentration logarithmic value of the low-value precision exceeds 5%, reaching 10.267%.

Table 8 The coefficient of variation results of the concentration logarithmic value of non-optimal primers and probes with low precision

theoretical copy number copy number log mean SD C.V.

1.0×10 ³ 2.504 0.257 10.267%

Comparative example 2

Enzyme mixture effect comparison

Amplify with a non-optimal ratio of enzyme mixture (the volume ratio of Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody is 11:4:3:1) and an optimal ratio of enzyme mixture For 3 whole blood RNA samples, primers and probes for amplification, amplification system, and procedures were the same as 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, and the amplification result using the best enzyme mixture ratio is shown in Figure 8 B Show. The concentration of the enzyme mixture in different proportions will affect the amplification effect of the sample, and the poor amplification effect will lead to deviation and inaccuracy in the measured final concentration results. Therefore, the optimal ratio of enzyme mixture should be used. Compared with the two (non-optimal ratio of enzyme mixture and optimal ratio of enzyme mixture), the Ct value of the amplification result of the non-optimal ratio of enzyme mixture is relatively delayed by 3 cycles The above, and the repeated results of the same sample under the optimal enzyme mixture ratio are more consistent and the difference is smaller. It can be seen that the amplification effect of the best enzyme mixture is better.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

Claims (13)

A kind of human β-tryptase mRNA RT-PCR detects primer probe group, it is characterized in that, described primer probe group comprises primer TPSB-F, primer TPSB-R and probe T-Probe, and described primer TPSB The nucleotide sequence of -F is as shown in SEQ ID NO.1, the nucleotide sequence of described primer TPSB-R is as shown in SEQ ID NO.2, and the nucleotide sequence of described probe T-Probe is as shown in SEQ ID NO.2 ID NO.3 is shown. The primer-probe set according to claim 1, wherein the 5' end of the probe T-Probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a quencher group. The primer probe group according to claim 1, is characterized in that, also comprises the primer GAPDH-F of internal reference gene, primer GAPDH-R and probe G-Probe, the nucleotide sequence of described primer GAPDH-F is as SEQ As shown in 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 primer probe set according to claim 3, wherein 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 fluorescence of the probe G-Probe is labeled The reporter group is different from the fluorescent reporter group labeled with the probe T-Probe. The primer-probe set according to claim 2 or 4, wherein the fluorescent reporter group includes FAM or JOE, and the quencher group includes BHQ1. The primer-probe set according to claim 5, wherein the 5' end of the probe T-Probe is labeled with a FAM fluorescent reporter group, and the 3' end is labeled with a BHQ1 quencher group. The primer-probe set according to claim 5, wherein the 5' end of the probe G-Probe is marked with a JOE fluorescent reporter group, and the 3' end is marked with a BHQ1 quencher group. A kit for human β-tryptase mRNA RT-PCR detection, characterized in that, the kit includes the primer probe set described in any one of claims 1 to 7, PCR reaction solution, enzyme mixture, β - Tryptase standard, ROX reference dye and nuclease-free water. The kit according to claim 8, wherein the PCR reaction solution comprises dNTP mix, MgCl ₂ and a buffer. The test kit according to claim 8, wherein the enzyme mixture comprises Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody. The kit according to claim 10, characterized in that, in the enzyme mixture, the volume ratio of Taq enzyme, reverse transcriptase, RNase inhibitor and Taq enzyme antibody is 14:5:5:1. The method for using the kit according to any one of claims 8 to 11, comprising the steps of: mixing the primer probe set, PCR reaction solution, enzyme mixture, standard product or sample to be tested, ROX reference dye and nuclease-free water After mixing, fluorescent quantitative amplification is performed. The method according to claim 12, characterized in that, in terms of 20 μL, the reaction system of the kit includes: 2 μL of primer probe set, 10 μL of PCR reaction solution, 0.5 μL of enzyme mixture, and 0.1 μL of ROX reference dye , 5 μL of standard or sample to be tested and 2.4 μL of nuclease-free water; The conditions for the fluorescence 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.

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