NL2030975B1

NL2030975B1 - Primer probe set and kit for rt-pcr detection of human leukotriene receptor cysltr2 mrna

Info

Publication number: NL2030975B1
Application number: NL2030975A
Authority: NL
Inventors: Wu Zhoujie; Yang Xukai; Wu Shandong; Li Shidan; Liu Yi; Qian Lei; Jiang Xuehan
Original assignee: Hangzhou Zheda Dixun Biological Gene Eng Co Ltd
Priority date: 2021-08-04
Filing date: 2022-02-16
Publication date: 2023-02-21
Also published as: ZA202200491B; AU2021106588A4; CN113388679A

Abstract

L The present disclosure relates to a primer probe set and a kit for reverse transcription—polymerase chain reaction (RT—PCR) detection of a human leukotriene receptor CYSLTR2 mRNA, belonging to the technical field of biological detection. In the present disclosure, the primer probe set includes a primer CysLTRZ—F, a primer CysLTRZ—R and a probe CZ—Probe; where the primer CysLTRZ—F has a nucleotide sequence shown in SEQ ID NO. l, the primer CysLTRZ—R has a nucleotide sequence shown in SEQ ID NO.2, and the probe CZ—Probe has a nucleotide sequence shown in SEQ ID No.3. The primer probe set of TagMan real—time fluorescence quantitative one—step RT—PCR detection is established for a human CysLTRZ, and the primer probe set provides a detection method with high accuracy, wide detection range and high sensitivity for the CysLTRZ protein.

Description

PRIMER PROBE SET AND KIT FOR RT-PCR DETECTION OF HUMAN LEUKOTRIENE

RECEPTOR CYSLTRZ MRNA

TECHNICAL FIELD

The present disclosure belongs to the technical field of bio- logical detection, and specifically relates to a primer probe set and a kit for reverse transcription-polymerase chain reaction (RT-

PCR) detection of a human leukotriene receptor CYSLTR2 mRNA.

BACKGROUND ART

Leukotriene is an unsaturated eicosanoic acid with a conju- gated triene structure isolated from the metabolites of an arachi- donic acid in leukocytes. The leukotriene can be prepared by the arachidonic acid through catalyzation of a lipoxygenase. Although there is a low content of leukotriene in human body, the leukotri- ene show very high physiological activity and serves as a chemical mediator that triggers certain allergic reactions, inflammations and cardiovascular diseases. The leukotriene is important in in- flammations of the upper and lower respiratory tract. The leuko- triene is over 1,000 times stronger than histamine in inducing na- sal allergic reactions. The amount of leukotrienes increases sig- nificantly in both an immediate reaction stage and a late reaction stage in allergen-induced nasal allergic reactions. Cysteinyl leu- kotrienes (CysLTs) are inflammatory mediators and regulators in the pathophysiology of asthma and allergic rhinitis (AR), and are key therapeutic targets. The CysLTs can regulate the production of hematopoietic progenitor cells, the recruitment and survival of eosinophils in inflammatory tissues, the activity of cytokines and chemokines, the amount of exhaled NO, the contraction of smooth muscle and the proliferation of fibroblasts.

The biological role of CysLTs depends on the expression of leukotriene receptors on the cell surface. A CysLTs receptor in- cludes a CysLTR1 and a CysLTR2, where the CysLTR2 is a G protein- coupled receptor that is mainly expressed in peripheral tissue cells such as the heart, pulmonary veins, adrenal medulla, and in-

testinal myometrial neurons. In human brain, the CysLTR2 is ex- pressed on vascular smooth muscle cells and infiltrating granulo- cytes. After being activated by leukotrienes, the CysLTR2 will cause inflammation, increased vascular permeability and tissue fi- brosis, and will increase the risk of allergic asthma; and the

CysLTR2 is also closely related to cerebral ischemic injury.

During treatment, leukotriene receptor antagonists (LTRA) can competitively inhibit the binding of leukotrienes to their recep- tors in the body, and block the activity of CysLTs, thereby inhib- iting inflammatory and allergic reactions. However, the curative effect of the LTRA has obvious individual differences, and has a clear positive correlation with the expression level of a leuko- triene receptor gene mRNA. CysLT2 receptor antagonists that have been reported include an HAMI 3379 and a Bay U9773. The HAMI 3379 has a protective effect on acute and sub-acute ischemic brain in- juries and alleviates the microglia-related inflammations. The Bay

U9773 has the same affinity for CysLTl and CysLT2, and can bind to these two receptors to inhibit bronchial and venous muscle con- traction. By detecting the expression level of CYSLTR2 mRNA, whether the patient is attacked by diseases caused by the CysLTR2 pathway and whether the CysLT2 receptor antagonist is effective can be determined, and the dynamic monitoring of the therapeutic effect can be achieved.

At present, the detection of the content of CysLTR2 in the body fluids is still detected using an enzyme-linked immunosorbent assay (ELISA) kit. There is no commercial kit for detecting the

CYSLTR2 mRNA. The ELISA method has the problems of small detection range, low sensitivity and unsatisfactory accuracy during the de- tection.

SUMMARY

The purpose of the present disclosure is to provide a primer probe set and a kit for RT-PCR detection of a human leukotriene receptor CYSLTR2 mRNA. The primer probe set of TagMan real-time fluorescence quantitative one-step RT-PCR detection is established for a human CysLTR2, and the primer probe set provides a detection method with high accuracy, wide detection range and high sensitiv-

ity for the CysLTR2 protein.

The present disclosure provides a primer probe set for RT-PCR detection of a human leukotriene receptor CYSLTR2 mRNA, including a primer CysLTR2-F, a primer CysLTR2-R and a probe C2-Probe, where the primer CysLTR2-F has a nucleotide sequence shown in SEQ ID NO. 1, the primer CysLTR2-R has a nucleotide sequence shown in SEQ ID

NO.2, and the probe C2-Probe has a nucleotide sequence shown in

SEQ ID NO.3.

Preferably, a 5'-end of the probe C2-Probe may be labeled with a fluorescent reporter group, and a 3'-end of the probe C2-

Probe may be labeled with a guenching group.

Preferably, the primer probe set may further include a primer

GAPDH-F, a primer GAPDH-R and a probe G-Probe of a reference gene, where the primer GAPDH-F may have a nucleotide sequence shown in

SEQ ID NO.4, the primer GAPDH-R may have a nucleotide sequence shown in SEQ ID NO.5, and the probe G-Probe may have a nucleotide sequence shown in SEQ ID NO.6.

Preferably, a 5'-end of the probe G-Probe may be labeled with a fluorescent reporter group, and a 3'-end of the probe G-Probe may be labeled with a quenching group; and the fluorescent report- er group labeled on the probe G-Probe may be different from the fluorescent reporter group labeled on the probe C2-Probe.

Preferably, the fluorescent reporter group may include a 6- carboxyfluorescein (FAM) or a 2,7-dimethyl-4,5-dichloro-6- carboxyfluorescein (JOE), and the quenching group may include a

Black Hole Quencher-1 (BHQ1).

The present disclosure further provides a kit for RT-PCR de- tection of a human leukotriene receptor CYSLTRZ mRNA, including the primer probe set, a PCR reaction solution, an enzyme mixed so- lution, a CysLTR2 standard, a carboxy-X-rhodamine (ROX) reference dye and nuclease-free water.

Preferably, the PCR reaction solution may include a deoxy- ribonucleoside triphosphate (dNTP) mix, MgCl, and a buffer.

Preferably, the enzyme mixed solution may include a thermus aquaticus (Taq) enzyme, a reverse transcriptase, a ribonuclease (RNase) inhibitor and a Taq enzyme antibody.

The present disclosure further provides a method for using the kit, including the following steps: mixing the primer probe set, the PCR reaction solution, the enzyme mixed solution, the

CysLTR2 standard or a CysLTR2 sample to be tested, the ROX refer- ence dye and the nuclease-free water, and conducting fluorescence quantitative amplification.

Preferably, based on 20 pL, a reaction system of the kit may include: 2 pL of the primer probe set, 10 pL of the PCR reaction solution, 0.5 pL of the enzyme mixed solution, 0.1 pL of the ROX reference dye, 5 HL of the CysLTR2 standard or the CysLTR2 sample to be tested, and 2.4 pL of the nuclease-free water; and the fluo- rescence quantitative amplification may be conducted by: 42°C for 30 min; 95°C for 1 min; 95°C for 5 s, and 60°C for 31 s, for 40 cycles.

The present disclosure provides a primer probe set for RT-PCR detection of a human leukotriene receptor CYSLTRZ mRNA. Compared with immunological detection methods, the prime probe set of the present disclosure has high sensitivity during detection, can de- tect low-concentration (10 copies/uL) clinical samples, can sensi- tively detect changes in CysLTR2 content, and has a detection range spanning at least 6 orders of magnitude. Accordingly, the accuracy of the detection results is increased, and at least 80 people can be detected within 1 hour, such that the treatment ef- fect can be dynamically monitored and evaluated in an earlier, more accurate, and faster manner.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a standard curve of a TaqMan real-time fluorescence quantitative RT-PCR for CYSLTR2 mRNA provided by the present dis- closure.

FIG. 2 is a result of a precision detection provided by the present disclosure. Herein, 1: 1.0x107 copies/pL, and 2: 1.0x10° copies/uL.

FIG. 3 is a result of an accuracy detection provided by the present disclosure.

FIG. 4 is a result of a sensitivity detection provided by the present disclosure.

FIG. 5 is a result of a clinical sample detection provided by the present disclosure. Herein, 1: patient GAPDH mRNA; 2: healthy control GAPDH mRNA; 3: patient CYSLTR2 mRNA; and 4: healthy con- trol CYSLTRZ mRNA.

FIG. 6 is a low-precision amplification curve in the case of 5 non-optimal primer and probe designs provided by the present dis- closure.

FIG. 7 is an amplification result of an enzyme mixed solution (A) with the non-optimal ratio and an enzyme mixed solution (B) with the optimal ratio provided by the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

The present disclosure provides a primer probe set for RT-PCR detection of a human leukotriene receptor CYSLTRZ mRNA, including a primer CysLTR2-F, a primer CysLTR2-R and a probe C2-Probe, where the primer CysLTR2-F has a nucleotide sequence shown in SEQ ID NO. 1: 5'- TCTGCTGATCATTCGGGTT-3', the primer CysLTR2-R has a nucleo- tide sequence shown in SEQ ID NO.2: 5'- TGATAGGGCAGGAAACACAA-3', and the probe C2-Probe has a nucleotide sequence shown in SEQ ID

NO.3: 5'-TCACAGGAAGGCACTGACCAC-3'.

In the present disclosure, a 5'-end of the probe C2-Probe is labeled with a fluorescent reporter group, and a 3'-end of the probe C2-Probe is labeled with a quenching group. The fluorescent reporter group preferably includes an FAM or a JOE, and the quenching group preferably includes a BHQ1. A 5'-end of the probe

C2-Probe is labeled with an FAM fluorescent reporter group, and a 3'-end of the probe C2-Probe is labeled with a BHQ1 quenching group.

In the present disclosure, the primer probe set further in- cludes a primer GAPDH-F, a primer GAPDH-R and a probe G-Probe of a reference gene, where the primer GAPDH-F may have a nucleotide se- quence shown in SEQ ID NO.4: 5'-GACAACAGCCTCAAGATCATC-3', the pri- mer GAPDH-R may have a nucleotide sequence shown in SEQ ID NO.5: 5'-CGCCACAGTTTCCCGGAG-3"', and the probe G-Probe may have a nucleo- tide sequence shown in SEQ ID NO.6: 5'-ACTCATGACCACAGTCCATGCCAT- 3'. A 5'-end of the probe G-Probe is labeled with a fluorescent reporter group, and a 3'-end of the probe G-Probe is labeled with a quenching group; and the fluorescent reporter group labeled on the probe G-Probe is preferably different from the fluorescent re- porter group labeled on the probe C2-Probe. The fluorescent re- porter group preferably includes the FAM or the JOE, and the quenching group preferably includes the BHQ1. In an example, a 5'- end of the probe G-Probe is labeled with a JOE fluorescent report- er group, and a 3'-end of the probe G-Probe is labeled with a BHQ1 quenching group.

The present disclosure further provides a kit for RT-PCR de- tection of a human leukotriene receptor CYSLTRZ mRNA, including the primer probe set, a PCR reaction solution, an enzyme mixed so- lution, a CysLTR2 standard, an ROX reference dye and nuclease-free water.

In the present disclosure, the PCR reaction solution includes a deoxy-ribonucleoside triphosphate (dNTP) mix, MgCl, and a buffer; the dNTP mix includes a dATP, a dCTP, a dGTP and a dTTP; and the dNTP mix is preferably purchased from Thermo Fisher Scientific (product number: R0192), and has a working concentration of pref- erably 0.3~0.8 mM. The MgCl; has a concentration of preferably 5-10 mM during using; and the buffer is preferably a Tris-HCl buffer, more preferably a 10-50 mM Tris-HCl buffer with a pH of preferably 8.0.

In the present disclosure, the enzyme mixed solution includes a Taq enzyme, a reverse transcriptase, an RNase inhibitor and a

Tag enzyme antibody with a volume ratio of preferably 12:4:3:1 to obtain the optimal amplification effect. The Taq enzyme is a heat- resistant Tag DNA polymerase, deoxynucleotides in the dNTP are added to a 3-OH terminus one by one using the 3'35' polymerase ac- tivity of the Taq enzyme and using DNA as a template. Meanwhile, mismatched primer ends can be identified and eliminated using the 513" exonuclease activity of the Tag enzyme, which is related to the correction function during the replication, nucleotides can also be hydrolyzed from the 5'-end and mismatched nucleotides can also be excised through several nucleotides. In this way, the chain replacement is realized during the chain extension, and the replaced probe is cut off. The reverse transcriptase can reverse transcribe an mRNA into a cDNA for PCR reaction. The RNase inhibi- tor is used to suppress the activity of an exogenous RNase. The

Taq enzyme antibody is an anti-Taq antibody for hot-start PCR, in- hibits DNA polymerase activity after binding to the Tag enzyme, and can effectively suppress the non-specific annealing of primers and the non-specific amplification caused by primer dimers under low temperature. The Taq enzyme antibody is denatured during the initial DNA denaturation of the PCR reaction, and the Tag enzyme recovers the activity to realize PCR amplification. The CysLTR2 standard is preferably a RNA standard of the CysLTR2 for preparing a quantitative one curve.

CysLTR2 standard or a CysLTR2 sample to be tested, the ROX refer- ence dye and the nuclease-free water, and conducting fluorescence quantitative amplification. In the present disclosure, the kit adopts a quantitative detection method of one-step RT-PCR technol- ogy, which can detect an expression level of the CYSLTRZ mRNA in human blood, nasal secretions, bronchial irrigating fluid, saliva, and tear samples.

In the present disclosure, based on 20 pL, a reaction system of the kit preferably includes: 2 pL of the primer probe set, 10 pL of the PCR reaction solution, 0.5 pL of the enzyme mixed solu- tion, 0.1 pL of the ROX reference dye, 5 pL of the CysLTR2 stand- ard or the CysLTR2 sample to be tested, and 2.4 pL of the nucle- ase-free water. The fluorescence quantitative amplification is preferably conducted by: 42°C for 30 min (reverse transcription); 95°C for 1 min (pre-denaturation}; 95°C for 5 s, and 60°C for 31 s, for 40 cycles.

The primer probe set and the kit for RT-PCR detection of a human leukotriene receptor CYSLTR2 mRNA according to the present disclosure will be further described in detail below with refer- ence to specific examples. The technical solutions of the present disclosure include, but are not limited to, the following exam- ples.

Unless otherwise specified, the experimental methods de- scribed in the following examples are all conventional methods.

The methods shall be conducted in accordance with the techniques or conditions described in the literature in the art or in accord- ance with the product specification. The materials, and reagents and the like used in the following examples are all commercially available, unless otherwise specified.

Example 1 1. The reagents and equipment involved were as follows: 1.1 Reagents 1.1.1 A whole-blood total RNA kit (Hangzhou Simgen Biological

Reagent Development Co., Ltd., product number: 5201050). 1.1.2 A HiScribe T7 High Yield RNA Synthesis Kit (New England

Biolabs, product number: E20408S). 1.2 Main instruments 1.2.1 An Applied Biosystems™ 7300 fluorescence quantitative

PCR instrument (Thermo Fisher Scientific, USA). 1.2.2 A -80°C low-temperature refrigerator (Thermo Fisher

Scientific, USA). 1.2.3 A high-speed and low-temperature table centrifuge (Ep- pendorf, Germany). 1.2.4 A Qubit 3 fluorometer (Thermo Fisher Scientific, USA). 2. Method 2.1 Design of primers and probes

Fluorescent quantitative primers and probes were designed us- ing a Primer 6.0 software according to the sequence of a CysLTR2 and a GAPDH; after a series of effect verification, primer pairs

CysLTR2-F, CysLTR2-R, GAPDH-F, and GAPDH-R and probes E-Probe, and

G-Probe of the CysLTR2 and the GAPDH were obtained (Table 1). The primers and probes were synthesized by Shanghai Sunny Biotechnolo- gy Co., Ltd.

Table 1 TagMan real-time fluorescence quantitative PCR of primers and probes

Name of Amplified primers and Use Primer sequence {5'>3'} (SEQ ID NO.} fragment probes size titative amplifica- | TGATAGGGCAGGAAACACAA (2) 129 bp

Je Joa titative amplifica- | CGCCACAGTTTCCCGGAG (5) {JOE)-ACTCATGACCACAGTCCATGCCAT {6}-

G-Probe gene GAPDH frag- {BHQ1) ment 2.2. Preparation of a standard

A pGM-T ligation kit [TIANGEN Biotech (Beijing) Co., Ltd., product number: VT202-01] was used, a CysLTR2 plasmid DNA (con- structed and synthesized by entrusting Nanjing GenScript Biotech

Co., Ltd.) was constructed using a pGM-T as a vector, and the Cys-

LTR2 plasmid DNA was transcribed into an mRNA in vitro using a

HiScribe T7 High Yield RNA Synthesis Kit (NEW ENGLAND BioLabs, product number: E20405).

An initial copy number of RNA was calculated according to a copy number calculation formula: copy number=[6.02x10°°xRNA concen- traticn (ng/uL) x10] / [RNA length (bp) *x340]. The CYSLTR2 mRNA was diluted with nuclease-free water to 1.0x10'° copies/pL to obtain a

CYSLTR2 mRNA standard. 2.3. Extraction and dilution of whole-blood RNA: whole-blood total RNA was extracted from ethylenediaminetetraacetic acid (EDTA) anticoagulated whole-blood samples with the whole-blood to- tal RNA kit, quantificated with the Qubit 3 fluorometer and dilut- ed with the nuclease-free water to 20 ng/uL. 2.4 TagMan real-time fluorescent quantitative PCR

A 20 pL system was prepared using the CYSLTR2 mRNA standard or a whole blood RNA as a template, the system was shown in Table 2:

Table 2 Reaction system

TT oysttR2

PCR re- Enzyme ROXref- Primer-probe

Nuclease- mRNA Stand- Total action mixed erence mixed solu- free water ard or whole- volume solution solution dye tion blood RNA 24pl 10u O5 Oul 2ul spb 20m

An amplification reaction program was shown in Table 3:

Table 3 Reaction program

Stage | | Time Number of cycles | Other parameter settings ture

Detection fluorescein: FAM, JOE {42°C 30min | 1 1 | Reference fluorescence: ROX

Ce {95°C ilmin 11 2 i Fluorescence signal collection: Stage 3 ‘Stage | 95°C Ssec 60°C for 31 sec ee ee 40 3 | 60°C | 31sec

TEE Generation of a standard curve

The CYSLTR2 mRNA standard was diluted in a 10-fold gradient using 1.0x10%°-1.0x10° copies/puL as a template, 3 replicates were conducted for each dilution, and TagMan real-time fluorescence quantitative RT-PCR detection was conducted to generate a standard curve. The dilution operation was shown in FIG. 1. A 50 pL/tube was taken as an example: for each dilution, 5 uL of a sample be- fore dilution was added to a new tube containing 45 pL of water. 2.6 Precision detection 1.0x107 copies/pL and 1.0x10% copies/uL of CYSLTRZ mRNA stand- ards were taken as a template, 10 replicates were conducted for each concentration; 10 times of TagMan real-time fluorescent quan- titative RT-PCR detections were conducted, the coefficient of var- iation of the logarithm of each concentration was calculated, re- spectively; and statistical analysis was conducted to analyze the precision of the detection method. 2.7 Accuracy detection

A 1.0x10° copies/pL of CYSLTR2 mRNA standard was subjected to 30-fold dilution (2 pL 1.0x10° copies/uL of a CYSLTR2 mRNA standard + 58 pL of nuclease-free water) as a template, for 3 replicates; 3 times of TagMan real-time fluorescence quantitative RT-PCR detec- tions were conducted, and the accuracy of the detection method was analyzed. 2.8 Sensitivity detection

A 10.0 copiles/pL of CYSLTR2 mRNA standard was taken as a tem- plate, for 25 replicates, 25 times of TagMan real-time fluores- cence quantitative RT-PCR detection were conducted to check wheth- er there were amplifications, and the sensitivity of the detection method was analyzed. 2.9 Clinical sample detection

Whole-blood samples of positive samples and healthy control were taken to extract and dilute whole-blood RNA according to the steps of 2.3, and TagMan real-time fluorescent quantitative RT-PCR detection was conducted according to the steps of 2.4. 3. Experimental results 3.1 Standard curve

The CYSLTR2 mRNA standard was diluted in a 10-fold gradient using 1.0x10°-1.0x10° copies/uL as a template, 3 replicates were conducted for each dilution, and TagMan real-time fluorescence quantitative RT-PCR detection was conducted to generate a standard curve. The standard curve of the TagMan real-time fluorescence quantitative RT-PCR of the CYSLTR2 mRNA is shown in FIG. 1. A copy number logarithm was taken as an abscissa and a Ct value is taken as an ordinate, and a regression equation was obtained: y=- 3.321x+35.602 (R*=0.997), where the regression equation has

R’=0.997, and a linear range of 1.0x10°-1.0x10° copies/pL. It indi- cates that the copy number logarithm of the standard equation has a very high correlation with the Ct value. 3.2 Precision detection 1.0x107 copies/pL and 1.0x10° copies/nL of CYSLTR2 mRNA stand- ards were taken as templates, 10 replicates were conducted for each concentration; 10 times of TagMan real-time fluorescent quan- titative RT-PCR detections were conducted, the coefficient of var- iation of the logarithm of each concentration was calculated, re- spectively; and statistical analysis was conducted. The results are shown in FIG. 2 and Table 4. The coefficient of variation of the logarithm of each concentration is 0.418% and 0.382% separate- ly, which are less than 5%, indicating that the TagMan real-time fluorescent quantitative RT-PCR detection method established by the present disclosure has excellent precision.

Table 4 Precision detection result oo

Mean of copy number logarithm SD CV ber

3.3 Accuracy detection

A 1.0x10° copies/pL of CYSLTR2 mRNA standard was subjected to 30-time dilution (2 nL 1.0x10° copies/pL of a CYSLTR2 mRNA standard + 58 pL of nuclease-free water) as a template, for 3 replicates; 3 times of TagMan real-time fluorescence quantitative RT-PCR detec- tions were conducted, and the absolute deviation of the logarithm of each concentration was calculated. The results are shown in

FIG. 3 and Table 5. The absolute deviation of the logarithm of each concentration is 0.142, 0.099, and 0.114, respectively, with- in the range of 20.5, indicating that the TagMan real-time fluo- rescent quantitative RT-PCR detection method established by the present disclosure has excellent accuracy.

Table 5 Accuracy detection result

Results Copy num- Theoretical copy Theoretical Absolute

Cr (cop- ber loga- number (cop- copy number devia- ies/tuL) rithm ies/ tl) logarithm tion 3.4 Sensitivity detection

A 10.0 copies/pL of CYSLTR2 mRNA standard was taken as a tem- plate, for 25 replicates, 25 times of TagMan real-time fluores- cence quantitative RT-PCR detection were conducted to check wheth- er there were amplifications. The results are shown in FIG. 4 and

Table 6. A total of 25 detection results are obtained, reaching 100%. This indicates that the TagMan real-time fluorescent quanti- tative RT-PCR detection method established by the present disclo- sure has very high sensitivity, and the minimum of detected copy number is less than 10 copies/uL.

Table 6 Ct value result of sensitivity detection

3.5 Clinical sample detection

A comparison result of the present disclosure and a certain domestic brand of cysteinyl leukotriene receptor 2 (CYSLTRZ) kit of enzyme-linked immunosorbent assay (ELISA) are shown in FIG. 5 and Table 7.

Table 7 Comparison result

Product of the present disclosure CYSLTR2 kit of ELISA

SN | Sample type Positive/

Results (copies/ul) | Positive/negative | Result {ng/mL} negative

Sample 1 before 1 1941.692 + 632.6 + treatment

Sample 1 after 2 674.853 + 276.8 + treatment

Sample 2 before 3 605.634 + 157.9 treatment

Sample 2 after 163.405 35.2 treatment

Sample 3 before 5 1135.179 + 483.9 + treatment

Sample 3 after 101.044 60.4 treatment 7 | Positive sample 4 854.882 257.6 + [Foams 7 5 ee Jon | Healthy control 5 2.972 - -

In the present disclosure, the detection was conducted using the whole-blood RNA; and the certain domestic brand of CYSLTR2 kit of ELISA was detected using a serum. 10 Comparative Example 1

Results of amplification using other non-optimal primers and probes

The primers and probes in the system used in the present dis- closure in Example 6 were replaced with other non-optimal primers and probes. An amplification system and a program were the same as those in Example 1. The results are shown in FIG. 6 and Table 8.

When non-optimal CysLTR2 primers and probes are used, such as:

CysLTR2-F: GCACCTTCAGCAATAACAACA (SEQ ID NO.7};

CysLTR2-R: GCTGCAGGAAAACATATATGG (SEQ ID NO. 8); and

C2-Probe: (FAM)-caacccatttcccaagactccece (SEQ ID NO.9)-(BH9Q1).

The coefficient of variation of the logarithm of the low- precision concentration exceeds 5%, reaching 7.815%.

Table 8 Results of amplification with non-optimal primers and probes maen mem je Je

SD C.V copy number arithm

Comparative Example 2

Amplification result of non-optimal enzyme mixed solution

An amplification was conducted using a non-optimal ratio of enzyme mixed solution (the Taq enzyme, reverse transcriptase,

RNase inhibitor and Tag enzyme antibody had a mass ratio of 10:5:4:1) on the CYSLTR2 mRNA standard with the primers and probes, the amplification system, and the program the same as those in Example 1. 4 gradients 1.0x10°-1.0x10°% copies/pL of a standard curve were obtained. An amplification result using the non-optimal enzyme mixed solution is shown in FIG. 7A. Compared with the standard curve of the amplification result using the op- timal enzyme mixed solution (FIG. 7B), the Ct of the corresponding concentration of the standard curve of the amplification result using the non-optimal enzyme mixed solution is delayed by more than 10 cycles. In addition, the reproducibility of CYSLTR2 mRNA standards at low concentrations (such as 1.0x10° copies/ulL) varies greatly, and has poor overall linearity effect, and an unqualified standard curve. It can be seen that the amplification result using the non-optimal enzyme mixed solution has poor amplification ef- fect.

The above results show that the TagMan real-time fluorescent quantitative RT-PCR detection method established in the present disclosure has better sensitivity and specificity than that of a counterpart reagent, and can effectively monitor the treatment ef- fect.

The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of or- dinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present dis- closure.

SEQUENCE LISTING

<110> Hangzhou Zheda Dixun Biological Gene Engineering Co., Ltd. <120> PRIMER PROBE SET AND KIT FOR RT-PCR DETECTION OF HUMAN <130> HKJP2021121070 <150> 202110891433.0 <151> 2021-08-04 <160> 9 <170> Patentin version 3.5 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer CysLTR2-F <400> 1 tctgctgatc attcgggtt 19 <210> 2 <211> 20 <212> DNA <213> Artificial Seguence <220> <223> Primer CysLTR2-R

<400> 2 tgalagggca ggaaacacaa 20

<210> 3 <211> 21 <212> DNA <213> Artificial Sequence

<220> <223> DNA sequence of probe C2-Probe <400> 3 tcacaggaag gcactgacca C 21 <210> 4 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Primer GAPDH-F

<400> 4 gacaacagcc tcaagatcat C 21 <210> 5 <211> 18 <212> DNA <213> Artificial Seguence

<220> <223> Primer GAPDH-R <400> 5 cgccacagtt tcccggag 18 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DNA seguence of probe G-Probe

<400> 6 actcatgacc acagtccatg ccat 24 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Non-optimal primer CysLTR2-F <400> 7 gcaccttcag caataacaac a 21

<210> 8 <211> 21 <212> DNA

<213> Artificial Sequence <220> <223> Non-optimal primer CysLTR2-R

<400> 8 gctgcaggaa aacatatatg g 21 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of non-optimal CysLTR2 probe C2-Probe <400> 9 caacccattt cccaagacte cc 22

SEQUENCE LISTING

<110> Hangzhou Zheda Dixun Biological Gene Engineering Co., Ltd. <120> PRIMER PROBE SET AND KIT FOR RT-PCR DETECTION OF HUMAN <130> HKJP2021121070 <150> 202110891433.0 <151> 2021-08-04 <160> 9 <170> PatentIn version 3.5 <21e> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Primer CysLTR2-F <400> 1 tctgctgatc attcgggtt 19 <2105 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Primer CysLTR2-R <400> 2 tgatagggca ggaaacacaa 20 <2105 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of probe C2-Probe <400> 3 tcacaggaag gcactgacca c 21 <2105 4 <211> 21 <212> DNA

<213> Artificial Sequence

<220>

<223> Primer GAPDH-F

<400> 4 gacaacagcc tcaagatcat c 21 <210>5 5

<211> 18

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer GAPDH-R

<400> 5 cgccacagtt tcccggag 18 <210> 6

<211> 24

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of probe G-Probe

<400> 6 actcatgacc acagtccatg ccat 24 <210> 7

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Non-optimal primer CysLTR2-F

<400> 7 gcaccttcag caataacaac a 21 <2105 8

<211> 21

<212> DNA

<213> Artificial Sequence

<220>

<223> Non-optimal primer CysLTR2-R

<400> 8 gctgcaggaa aacatatatg g 21 <210> 9

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> DNA sequence of non-optimal CysLTR2 probe C2-Probe

<400> 9 caacccattt cccaagactc cc 22

Claims

CONCLUSIONS

A primer probe set for reverse transcription polymerase chain reaction (RT-PCR) detection of a human leukotriene receptor CYSLTR2 mRNA, comprising a primer CysLTR2-F, a primer Cys-LTR2-R and a probe C2-Probe, wherein the primer CysLTR2-F has a nucleotide sequence shown in SEQ ID NO. 1, the primer CysLTR2-R has a nucleotide sequence shown in SEQ ID NO.2 and the probe C2-Probe has a nucleotide sequence shown in SEQ ID NO.3.

A primer probe set according to claim 1, wherein a 5' end of the probe C2-Probe is labeled with a fluorescent reporter group, and a 3' end of the probe C2-Probe is labeled with a quench group.

The primer probe set of claim 1, further comprising a primer GAPDH-F, a primer GAPDH-R and a probe G-Probe of a reference gene, wherein the primer GAPDH-F has a nucleotide sequence shown in SEQ ID NO. 4, the primer GAPDH-R has a nucleotide sequence shown in SEQ ID NO. 5 and the probe G-Probe has a nucleotide sequence shown in SEQ ID NO. 6.

A primer probe set according to claim 3, wherein a 5' end of the probe G-Probe is labeled with a fluorescent reporter group, and a 3' end of the probe G-Probe is labeled with a quench group; and the fluorescent reporter group labeled on the probe G-Probe differs from the fluorescent reporter group labeled on the probe C2-Probe.

A primer probe set according to claim 2 or 4, wherein the fluorescent reporter group comprises a 6-carboxyfluorescein (FAM) or a 2,7-dimethyl-4,5-dichloro-6-carboxyfluorescein (JOE), and the duench group comprises a Black Hole Quencher-1 (BHQ1).

A kit for RT-PCR detection of a human leukotriene receptor CYSLTR2 mRNA, comprising the primer probe set according to any one of claims 1 to 5, a PCR reaction solution, a mixed enzyme solution, a CysLTR2 standard, a carboxy- X-rhodamine (ROX) reference dye and nuclease free water.

The kit of claim 6, wherein the PCR reaction solution is a mixture of deoxy ribonucleoside triphosphate (dNTP), MgCl; and includes a buffer.

The kit of claim 6, wherein the mixed enzyme solution comprises a thermus aquaticus (Taq) enzyme, a reverse transcriptase, a ribonuclease (RNase) inhibitor and a Taq enzyme antibody.

A method of using the kit according to claim 6, 7 or 8, comprising the steps of: mixing the primer probe set, the PCR reaction solution, the mixed enzyme solution, the CysLTR2 standard or a CysLTR2 sample to be tested, the ROX - reference dye and the nuclease-free water, and performing quantitative fluorescence amplification.

The method of claim 9, wherein on a 20 µL basis, a reaction system of the kit comprises: 2 µL of the primer probe set, 10 µL of the PCR reaction solution, 0.5 µL of the mixed enzyme solution, 0.1 pL of the ROX reference dye, 5 pL of the CysLTR2 standard or CysLTR2 sample to be tested, and 2.4 HL of the nuclease-free water; and wherein the quantitative fluorescence amplification is performed at: 42°C for 30 minutes; 95°C for 1 minute; 95 °C for 5 s and 60 °C for 31 s, for 40 cycles.