NL2030977B1 - Reagent for detecting expression level of human histamine receptor hrh4 mrna, kit and use - Google Patents
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Abstract
The present disclosure provides a reagent for detecting an expression level of a human histamine receptor HRH4 mRNA, a kit and use. In the present disclosure, the reagent includes a specific primer and a probe for a human histamine receptor HRH4, the specific primer includes an HRH4—E‘ and, an HRH4—R, and the probe includes an H4—Probe; and the HRH4—F has a nucleotide sequence shown in SEQ ID NO. l, the HRH4—R has a nucleotide sequence shown in SEQ ID NO. 2 and the H4—Probe has a nucleotide sequence shown in SEQ ID NO. 3. In the present disclosure, a kit for one—step detection based on the reagent is prepared, and the expression level of the HRH4 mRNA can be one—step quantitatively detected with simple operation and short detection time. The present disclosure provides a kit product that can guide the medication and accurately quantify the efficacy for H4 antihistamines.
Description
REAGENT FOR DETECTING EXPRESSION LEVEL OF HUMAN HISTAMINE RECEPTOR
HRH4 MRNA, KIT AND USE
The present disclosure belongs to the technical field of bio- logical detection, and specifically relates to a reagent for de- tecting an expression level of a human histamine receptor HRH4 mRNA, a kit and use.
Histamine is an active amine compound widely present in ani- mals and plants, is formed by the decarboxylation of a histidine, and is usually stored in the tissues. The histamine is an im- portant chemical conductive substance in the body, can affect many cell reactions, including allergies, inflammatory reactions, and gastric acid secretion, etc. When the body is stimulated to trig- ger an antigen-antibody reaction, the cell membrane permeability of mast cells changes to release histamine, and the histamine in- teracts with a histamine receptor to produce pathophysiological effects. The synthesis of a histamine mainly occurs in mast cells, basophils, lungs, skin and gastrointestinal mucosa, and is con- sistent with the tissues that store the histamine. The histamine, like other transmitters, binds to specific receptors on target cells, thereby changing the biological activity of cells and ex- erting wide physiological or pathological effects. The inflammato- ry effect of histamine, namely the effect of histamine on the im- mune homeostasis in the body depends on the expression and activi- ty of currently-known 4 histamine receptors. The 4 histamine re- ceptors are named in the order of discovery: a histamine 1 (H1) receptor, a histamine 2 (H2) receptor, a histamine 3 (H3) recep- tor, and a histamine 4 (H4) receptor; where, the H4 receptor is predominantly distributed in the immune system or hematopoietic- related tissues or cells, and is highly expressed in bone marrow, peripheral hematopoietic cells and other sites related to inflam- mation. Therefore, the H4 receptor has become a new therapeutic target in immune diseases such as allergic reactions and asthma.
The H4 receptor is involved in the chemotaxis of mast cells, eo- sinophils, and dendritic cells, as well as the production of cyto- kines in T cells and dendritic cells, and is involved in inflamma- tory reactions.
Most patients with allergic diseases use symptomatic treat- ment based on the experience of doctors and use antihistamines to relieve allergic symptoms. The antihistamines that target the H1 receptor are effective for some allergic patients, but still have limitations. H4 receptor antagonists can have a desirable thera- peutic effect on patients that have ineffective treatment effect with Hl receptor antagonists. However, the treatment with antihis- tamines is not effective for all patients, and cannot quantify the therapeutic effect; and long-term medication will also produce a series of side effects. There are no relevant studies and records on this at present.
In view of this, the present disclosure provides a reagent for detecting an expression level of a human histamine receptor
HRH4 mRNA, a kit and use. The expression level of the HRH4 mRNA can be detected using an RNA one-step method. The present disclo- sure provides a detection method with high accuracy, wide detec- tion range and high sensitivity for the detection of HRH4 pro- teins.
In order to realize the above objective, the present disclo- sure provides the following technical solutions:
The present disclosure provides a reagent for detecting an expression level of a human histamine receptor HRH4 mRNA, includ- ing a specific primer and a probe for a human histamine receptor
HRH4, where the specific primer includes an HRH4-F and an HRH4-R, and the probe includes an H4-Probe; and the HRH4-F has a nucleotide sequence shown in SEQ ID NO. 1, the HRH4-R has a nucleotide sequence shown in SEQ ID NO. 2 and the
H4-Probe has a nucleotide sequence shown in SEQ ID NO. 3.
Preferably, the reagent may further include a specific primer and a probe of a reference gene GAPDH, where the specific primer of the GAPDH may include a GAPDH-F and a GAPDH-R, and the probe of the GAPDH may include a G-Probe; and the GAPDH-F may have a nucleotide sequence shown in SEQ ID
NO. 4, the GAPDH-R may have a nucleotide sequence shown in SEQ ID
NO. 5 and the G-Probe may have a nucleotide sequence shown in SEQ
ID NO. ID NO. 6.
Preferably, 5'-ends of the H4-Probe and G-Probe may be sepa- rately labeled with different fluorescent reporter groups, and 3'- ends of the H4-Probe and G-Probe may be labeled with a same quenching group or different quenching groups.
Preferably, the fluorescent reporter group may include a 6- carboxyfluorescein (FAM) and a 2,7-dimethyl-4,5-dichloro-6- carboxyfluorescein (JOE), and the guenching group may include a
Black Hole Quencher-1 (BHQ1).
Preferably, the HRH4-F, the HRH4-R, the H4-Probe, the GAPDH-
F, the GAPDH-R and the G-Probe in the reagent may have a concen- tration of 2.25 nM, 1.5 nM, 1.5 nM, 1 nM, 1 nM and 1.5 nM, respec- tively.
The present disclosure further provides a kit for one-step detection of an expression level of a human histamine receptor
HRH4 mRNA, including the reagent, a PCR reaction solution, an en- zyme mixed solution, 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; and the enzyme mixed solution may include a thermus aquaticus (Taq) enzyme, a reverse transcriptase, a ribonuclease (RNase) in- hibitor and a Tag enzyme antibody with a mass ratio of 15:5:4:1.
Preferably, the kit may further include an RNA standard of the human histamine receptor HRH4.
The present disclosure further provides use of the reagent or the kit as a basis for the preparation of a medicine for treating immune diseases or as a tool for dynamically monitoring therapeu- tic effects.
Preferably, a reaction system may be prepared using the rea- gent or the kit to conduct a quantitative real-time polymerase chain reaction (qRT-PCR);
the reaction system, calculated in 20 pL, may include: 2.4 pL of the nuclease-free water, 10 uL of the PCR reaction solution, 0.5 pL of the enzyme mixed solution, 0.5 pL of the ROX reference dye, 2 pL of the reagent and 5 pL of the RNA standard or a to-be- tested RNA sample; and a qRT-PCR program may include: 42°C for 30 min; 95°C for 1 min; 95°C for 5 s, and 60°C for 31 s, 40 cycles.
The present disclosure provides a reagent for detecting an expression level of a human histamine receptor HRH4 mRNA, and a kit for one-step detection of the expression level of the HRH4 mRNA is prepared based on the reagent. The expression level of the
HRH4 mRNA in human blood, nasal secretions, bronchial irrigating fluid, saliva, and tear samples can be one-step quantitatively de- tected using the reagent or the kit with simple operation and short detection time. The present disclosure provides a kit prod- uct that can guide the medication and accurately quantify the ef- ficacy for H4 antihistamines.
In the present disclosure, the one-step detection is conduct- ed based on the reagent and the kit without separate reverse tran- scription, which greatly reduces the risk of causing aerosol pol- lution. Compared with immunological detection methods, the detec- tion method of the present disclosure has high sensitivity, can detect low-concentration clinical samples, can sensitively detect changes in HRH4 content, and has a detection range spanning at least 6 orders of magnitude. Accordingly, the accuracy of the de- tection results is increased, and at least 80 people can be de- tected within 1 hour, such that the treatment effect can be dynam- ically monitored and evaluated in an earlier, more accurate, and faster manner.
FIG. 1 is a standard curve of TagMan real-time fluorescence quantitative RT-PCR for HRH4 mRNA.
FIG. 2 is a result of precision detection, where 1: 1.0x107 copies/uL, and 2: 1.0x10° copies/upL.
FIG. 3 is a result of accuracy detection.
FIG. 4 is a result of sensitivity detection.
FIG. 5 is a result of clinical sample detection, where 1:
Case 3 GAPDH mRNA before treatment; 2: Case 3 GAPDH mRNA after treatment; 3: Case 3 HRH4 mRNA before treatment; 4: Case 3 HRH4 mRNA after treatment. 5 FIG. 6 is a low-precision amplification curve in the case of non-optimal primer and probe designs.
FIG. 7 is an effect of the enzyme mixed solution on amplifi- cation.
FIG 8 is a plasmid structure map of pGM-T vector.
The present disclosure provides a reagent for detecting an expression level of a human histamine receptor HRH4 mRNA, includ- ing a specific primer and a probe for a human histamine receptor
HRH4, where the specific primer includes an HRH4-F and an HRH4-R, and the probe includes an H4-Probe; and the HRH4-F has a nucleotide sequence shown in SEQ ID NO. 1, the HRH4-R has a nucleotide sequence shown in SEQ ID NO. 2 and the
H4-Probe has a nucleotide sequence shown in SEQ ID NO. 3.
In the present disclosure, the reagent preferably further in- cludes a specific primer and a probe of a reference gene GAPDH; the specific primer of the reference gene GAPDH preferably in- cludes a GAPDH-F and a GAPDH-R, and the probe of the reference gene GAPDH preferably includes a G-Probe; the GAPDH-F preferably has a nucleotide sequence preferably shown in SEQ ID NO. 4, the
GAPDH-R preferably has a nucleotide sequence preferably shown in
SEQ ID NO. 5 and the G-Probe preferably has a nucleotide sequence preferably shown in SEQ ID NO. 6. 5'-ends of the H4-Probe and G-
Probe are separately labeled with different fluorescent reporter groups, and 3'-ends of the H4-Probe and G-Probe are labeled with a same quenching group or different quenching groups. In an example, the fluorescent reporter group preferably includes an FAM and a
JOE, and the quenching group includes a BHQ1l, and Shanghai Sunny
Biotechnology Co., Ltd. is entrusted to synthesize the specific primer and the probe (Table 1).
Table 1 TagMan real-time fluorescence quantitative PCR of primer probe
In the present disclosure, the HRH4-F, the HRH4-R, the H4-
Probe, the GAPDH-F, the GAPDH-R and the G-Probe in the reagent have a concentration or number of moles of 2.25 nM, 1.5 nM, 1.5 nM, 1 nM, 1 nM and 1.5 nM, or respectively.
The present disclosure further provides a kit for one-step detection of an expression level of a human histamine receptor
HRH4 mRNA, including the reagent, a PCR reaction solution, an en- zyme mixed solution, an ROX reference dye and nuclease-free water.
In the present disclosure, the PCR reaction solution prefera- bly includes a dNTP mix, MgCl, and a buffer; the dNTP mix is pref- erably a mixture of a dATP, a dTTP, a dCTP, and a dGTP, which is purchased from Thermo Fisher Scientific (product number: R0132), and has a working concentration of preferably 0.1-1 mM; the MgCl, has a concentration of preferably 5-20 mM; and the buffer is a 10- 50 mM Tris-HCl buffer (at pH 8.0).
The enzyme mixed solution includes a Tag enzyme, a reverse transcriptase, an RNase inhibitor and a Taq enzyme antibody with a mass ratio of 15:5:4:1 to obtain the best amplification effect.
In the present disclosure, the kit preferably further in- cludes an RNA standard of the human histamine receptor HRH4 to prepare a standard curve.
In the present disclosure, the TagMan real-time fluorescent quantitative PCR is directly conducted using the RNA as a template without a separate reverse transcription when detecting by the kit; the Taq enzyme is a heat-resistant Tag DNA polymerase, deox- ynucleotides in the dNTP are added to a 3-0H terminus one by one using the 3'—5' polymerase activity of the Taq enzyme and using
DNA as a template. Meanwhile, mismatched primer ends can be iden- tified and eliminated using the 5'3' 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 nu- cleotides. In this way, the chain replacement is realized during the chain extension, and the replaced probe is cut off. The re- verse transcriptase can reverse transcribe an mRNA into a cDNA for
PCR reaction. The RNase inhibitor is used to suppress the activity of an exogenous RNase. The Tag enzyme antibody is an anti-Taq an- tibody for hot-start PCR, inhibits 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 an- tibody is denatured during the initial DNA denaturation of the PCR reaction, and the Tag enzyme recovers the activity to realize PCR amplification.
The present disclosure further provides use of the reagent or the kit as a basis for the preparation of a medicine for treating immune diseases or as a tool for dynamically monitoring therapeu- tic effects.
In the present disclosure, the immune diseases preferably in- clude allergic diseases. The expression of the histamine H4 recep- tor (HRH4) mRNA is detected using the reagent or the kit of the present disclosure. On the one hand, whether the patient's aller- gic symptoms are caused by the histamine activation pathway of the
H4 receptor can be determined (allergy symptoms caused by a non- histamine pathway do not express histamine receptors) and the dos- age can be guided (a higher H4 receptor expression requires a higher dose of H4 receptor antagonists}. On the other hand, the treatment effect of H4 receptor antagonists can be dynamically monitored.
In the present disclosure, preferably a reaction system is prepared using the reagent or the kit to conduct a gRT-PCR reac- tion; the reaction system, calculated in 20 pL, preferably in- cludes: 2.4 nL of the nuclease-free water, 10 pL of the PCR reac- tion solution, 0.5 pL of the enzyme mixed solution, 0.5 4uL of the
ROX reference dye, 2 pL of the reagent and 5 pL of the standard or the RNA; a RT-PCR program includes: 42°C for 30 min; 95°C for 1 min; 95°C for 5 s, and 60°C for 31 s, 40 cycles; and an expression of HRH4 mRNA is calculated according to the standard curve pre- pared with the standard. The standard curve uses a copy number logarithm as an abscissa (x) and a Ct value as an ordinate (vy): y=-3.177x + 34.178 (R2=0.996). There is no special limitation on the source of the RNA, and the RNA can be extracted from human blood, nasal secretions, bronchial irrigating fluid, saliva or tear samples.
The reagent for detecting an expression level of a human his- tamine receptor HRH4 mRNA, the kit and the use provided by the present disclosure are described in detail below with reference to the examples, but these examples should not be understood as lim- iting the claimed protection scope of the present disclosure.
Unless otherwise specified, the reagents, kits and instru- ments used in the present disclosure are all commercially- available conventional in the art: a whole-blood total RNA kit (Hangzhou Simgen Biological Rea- gent Development Co., Ltd., product number: 5201050); a HiScribe T7 High Yield RNA Synthesis Kit (New England Bi- olabs, product number: E20405); an Applied Biosystems™ 7300 fluorescence quantitative PCR in- strument (Thermo Fisher Scientific, USA); a -80°C low-temperature refrigerator (Thermo Fisher Scien- tific, USA); a high-speed and low-temperature table centrifuge (Eppendorf,
Germany); and a Qubit 3 fluorometer (Thermo Fisher Scientific, USA).
Example 1 1. Shanghai Sunny Biotechnology Co., Ltd. was entrusted to synthesize the primers and probes shown in Table 1. 2. Preparation of a standard
In-vitro transcription: a pGM-T ligation kit [TIANGEN Biotech (Beijing) Co., Ltd., product number: VT202-01] was used, a HRH4 plasmid DNA (constructed and synthesized by entrusting Nanjing
GenScript Biotech Co., Ltd., FIG. 8) was constructed using a pGM-T as a vector, and the HRH4 plasmid DNA was transcribed into an mRNA in vitro using a HiScribe T7 High Yield RNA Synthesis Kit (NEW
ENGLAND Biolabs, product number: E20408).
An initial copy number of the RNA was calculated according to a copy number calculation formula: copy number=[6.02x10%xRNA con- centration (ng/pL)x10°]/[RNA length (bp)=x340]. The HRH4 mRNA was diluted with nuclease-free water to 1.0x10'° copies/pL to obtain a
HRH4 mRNA standard. 3. Extraction and dilution of whole-blood RNA
Whole-blood total RNA was extracted from ethylenediaminetet- raacetic acid (EDTA) anticoagulated whole-blood samples with the whole-blood total RNA kit, quantificated with the Qubit 3 fluorom- eter, and diluted with the nuclease-free water to 20 ng/uL. 4. TagMan real-time fluorescent quantitative PCR
A 20 uL system was prepared using the standard/whole-blood
RNA as a template with: 2.4 uL of the nuclease-free water, 10 uL of the PCR reaction solution, 0.5 uL of the enzyme mixed solution, 0.5 pL of the ROX reference dye, 2 pL of a mixed solution of the primer and the probe, and 5 pL of the standard or the RNA.
A gRT-PCR program was as follow: 42°C for 30 min; 95°C for 1 min; 95°C for 5 s, and 60°C for 31 =, 40 cycles.
A detection fluorescein was set up: FAM, JOE; a reference fluorescence was: ROX; the reaction system was: 20 pL; and a fluo- rescence signal collection was: 60°C for 31 sec. 5. Generation of a standard curve
The HRH4 standard was diluted in a 10-fold gradient using 1.0x10%-1.0%x10° copies/pl as a template, 3 replicates were conduct- ed for each dilution, and TagMan real-time fluorescence quantita- tive RT-PCR detection was conducted to generate the standard curve.
The result is shown in FIG. 1. A copy number logarithm is taken as an abscissa and a Ct value is taken as an ordinate, and a regression equation is obtained: y=-3.177x+34.178 (R*=0.996), indi- cating the copy number logarithm of a standard equation has very high correlation with the Ct value. 6. Precision detection 1.0x107 copies/pL and 1.0x10°% copies/pL of standards were tak- en as templates, 10 replicates were conducted for each concentra- tion; 10 times of TagMan real-time fluorescent quantitative RT-PCR detections were conducted, the coefficient of variation of the logarithm of each concentration was calculated, respectively; and statistical analysis was conducted to analyze the precision of the detection method.
The results are shown in Table 2 and FIG. 2. The coefficient of variation of the logarithm of each concentration is 0.443% and 0.430% separately, 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 2 Precision detection result
Theoretical copy Mean logarithm of copy er
SD C.V number number 7. Accuracy detection
A 1.0x10° copies/pL of standard was subjected to 30-fold di- lution (2 uL 1.0x10° copies/pL of a standard + 58 pL of nuclease- free water) as a template, for 3 replicates; 3 times of TagMan re- al-time fluorescence quantitative RT-PCR detections were conduct- ed, and the absolute deviation of the logarithm of each concentra- tion was calculated. The results are shown in FIG. 3. The absolute deviation of the logarithm of each concentration is 0.126, 0.137, and 0.131, respectively, within the range of 0.5, indicating that the TagMan real-time fluorescent guantitative RT-PCR detection method established by the present disclosure has excellent accura- cy.
Table 3 Accuracy detection data
TT Copy num- | Theoretical | Theoretical
Results Absolute © {copies/nL) Copy number | copy number deviation rithm (copies/uL) | logarithm 4.570x10° 4.660 3.333x10° 4.523 0.137 15.353 [4505.00 | 4.654 0.131 8. Sensitivity detection
A 10.0 copies/pL of standard was taken as a template, for 25 replicates, 25 times of TagMan real-time fluorescence quantitative
RT-PCR detection were conducted to check whether there were ampli- fications, and the sensitivity of the detection method was ana-
lyzed.
The results are shown in Table 4 and FIG. 4. A total of 25 detection results are obtained, reaching 100%. This indicates that the TagMan real-time fluorescent guantitative RT-PCR detection method established by the present disclosure has very high sensi- tivity, and the minimum number of detected copies is less than 10 copies/pL.
Table 4 Ct value results of sensitivity detection 32.533 | 32.716 | 33.514 | 32.321 | 31.944 32.110 | 31.967 | 33.336 | 33.215 | 31.506 31.673 | 33.513 | 32.747 | 31.747 | 31.961 31.605 | 32.968 | 33.157 | 31.965 | 32.189 32.769 | 32.178 | 32.692 | 32.465 | 31.669 9. Clinical sample detection
Whole-blood samples of patients and normal persons were taken to extract and dilute whole-blood RNA according to the above steps, and TagMan real-time fluorescent quantitative RT-PCR detec- tion was conducted according to the above steps.
Compared with a domestic brand of histamine H4 receptor (HRH4) kit of enzyme-linked immunosorbent assay (ELISA) , the re- sults are shown in Table 5 and FIG. 5. The TagMan real-time fluo- rescent 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 effect.
Table 5 Comparison of clinical sample detection effect
Histamine H4 receptor (HRH4) kit of
Product of the present disclosure
ELISA
SN | Sample type positive/
Results {copies/uL) Result (ng/ml) | positive/ negative negative
Case 1 before 1 1154.669 + 678.3 + treatment
Case 1 after 960.673 + 153.2 + treatment
Case 2 before 3 911.955 + 487.3 + treatment
Case 2 after 496.380 37.6 - treatment
Case 3 before 1620.624 + 781.4 + treatment
Case 3 after 32.378 - 147.6 + treatment
Healthy control 756.543 356.4 + 1
Healthy control 30.457 19.2 2
Healthy control 10 61.125 - 28.7 3
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 5 were replaced with other non-optimal primers and probes. The results are shown in FIG. 6. When using non- optimal HRH4 primers and probes, the amplification results of the standard curve are poor, with almost no amplification.
Non-optimal HRH4 primers and probes were as follows:
HRH4-F (SEQ ID NO.7): GCCAGATACTAATAGCACAAT;
HRH4-R (SEQ ID NO.8): CCACAAAAGCTAAAATGACCAA; and
H4-Probe (SEQ ID NO.9): (FAM) -AAGCACTCGTGTTACTTTAGCA- (BHQ1}).
Comparative Example 2 Comparison of the effect of enzyme mixed solution
An amplification was conducted using a non-optimal ratio of enzyme mixed solution (the Tag enzyme, reverse transcriptase,
RNase inhibitor and Tag enzyme antibody had a mass ratio of 16:3:5:1) and a best ratio of enzyme mixed solution on 4 gradients 1.0x10°%-1.0x10% copies/ul on a calibration curve. An amplification result using the non-optimal ratio of enzyme mixed solution is shown in FIG. 7A, and an amplification result using the best ratio of enzyme mixed solution is shown in FIG. 7B. It can be seen that the best enzyme mixed solution has better amplification effect.
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.
<110> Hangzhou Zheda Dixun Biological Gene Engineering Co., Ltd. <120> REAGENT FOR DETECTING EXPRESSION LEVEL OF HUMAN HISTAMINE <130> HKJP2021121072 <150> 202110890787.3 <151> 2021-08-04 <160> 9 <170> Patentln version 3.5 <210> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer HRH4-F <400> 1 ctecatatte tctgttcaca at 22 <210> 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer HRH4-R
<400> 2 tggatacaaa agaggattga ca 22 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of H4-Probe <400> 3 aacaggtcct aaatcagttt ggta 24
<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 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 G-Probe <400> 6 actcatgacc acagtccatg ccat 24 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Non-optimal primer HRH4-F
<400> 7 gccagatact aatagcacaa t 21 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220>
<223> Non-optimal primer HRH4-R <400> 8 ccacaaaagc taaaatgacc aa 22
<210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Non-optimal H4-Probe <400> 9 aagcactcgt gttactttag ca 22
<110> Hangzhou Zheda Dixun Biological Gene Engineering Co., Ltd. <120> REAGENT FOR DETECTING EXPRESSION LEVEL OF HUMAN HISTAMINE <130> HKJIP2021121072 <150> 202110890787.3 <151> 2021-08-04 <160> 9 <170> PatentIn version 3.5 <21e> 1 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer HRH4-F <400> 1 ctccatattc tctgttcaca at 22 <2105 2 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer HRH4-R <400> 2 tggatacaaa agaggattga ca 22 <2105 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of H4-Probe <400> 3 aacaggtcct aaatcagttt ggta 24 <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 G-Probe
<400> 6 actcatgacc acagtccatg ccat 24 <210> 7
<211> 21
<212> DNA
<213> Artificial Sequence
<220>
<223> Non-optimal primer HRH4-F
<400> 7 gccagatact aatagcacaa t 21 <2105 8
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Non-optimal primer HRH4-R
<400> 8 ccacaaaagc taaaatgacc aa 22 <210> 9
<211> 22
<212> DNA
<213> Artificial Sequence
<220>
<223> Non-optimal H4-Probe
<400> 9 aagcactcgt gttactttag ca 22
Claims (10)
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| CN202110890787.3A CN113584154A (en) | 2021-08-04 | 2021-08-04 | Reagent and kit for detecting human histamine receptor HRH4 mRNA expression level and application |
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| WO2015010442A1 (en) * | 2013-07-24 | 2015-01-29 | The Chinese University Of Hong Kong | Biomarkers for premature birth |
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| AU4171301A (en) * | 2000-05-31 | 2001-12-11 | Ortho Mcneil Pharm Inc | Dnas encoding mammalian histamine receptor of the h4 subtype |
| CN104195255B (en) * | 2014-09-09 | 2016-08-17 | 广州蓝吉生物技术有限公司 | The test kit of a kind of detection fusion Gene A ML1-ETO mrna expression and detection method thereof |
| CN105017385B (en) * | 2015-07-02 | 2018-08-03 | 吉林大学 | Based on simulation people's histamine receptor 4（HR4）The vaccine and its construction method of epitope |
| CN110592290B (en) * | 2019-10-31 | 2022-10-04 | 上海市动物疫病预防控制中心 | Kit and method for detecting feline calicivirus |
| CN112877469A (en) * | 2019-11-29 | 2021-06-01 | 中国疾病预防控制中心病毒病预防控制所 | Real-time PCR detection method of human H group rotavirus |
| WO2023010329A1 (en) * | 2021-08-04 | 2023-02-09 | 杭州浙大迪迅生物基因工程有限公司 | Reagent for detecting expression level of human histamine receptor hrh4 mrna, reagent kit, and detection method |
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Non-Patent Citations (2)
| Title |
|---|
| CHEN B ET AL: "Association between copy-number variations of the human histamine H4 receptor gene and atopic dermatitis in a Chinese population", CLINICAL AND EXPERIMENTAL DERMATOLOGY, BLACKWELL SCIENTIFIC PUBLICATIONS, GB, vol. 38, no. 3, 21 March 2013 (2013-03-21), pages 295 - 301, XP071608886, ISSN: 0307-6938, DOI: 10.1111/CED.12117 * |
| YUAN ZHU ET AL: "CLONING, EXPRESSION, AND PHARMACOLOGICAL CHARACTERIZATION OF A NOVEL HUMAN HISTAMINE RECEPTOR", MOLECULAR PHARMACOLOGY, AMERICAN SOCIETY FOR PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS, US, vol. 59, no. 3, 1 January 2001 (2001-01-01), pages 434 - 441, XP002941038, ISSN: 0026-895X * |
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