CN111154876B - Primer probe composition, kit and detection method for detecting methylation of human ADD3 and CDH23 genes - Google Patents
Primer probe composition, kit and detection method for detecting methylation of human ADD3 and CDH23 genes Download PDFInfo
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Abstract
The invention provides a primer probe composition, a kit and a detection method for detecting methylation of human ADD3 and CDH23 genes, wherein the primer probe composition comprises the following components: a first specific primer and a first specific probe for detecting the methylation of the human ADD3 gene, a second specific primer and a second specific probe for detecting the methylation of the human CDH23 gene, and a third specific primer and a third specific probe for detecting the beta-actin gene of the reference gene. By the primer probe composition, the kit and the detection method disclosed by the invention, the specificity detection on the methylation of the human ADD3 and CDH23 genes is realized, the accuracy of early screening and diagnosis on the human prostatic cancer is realized, and the primer probe composition, the kit and the detection method have the technical advantages of high sensitivity, high specificity, low cost, high flux, simplicity and convenience in operation, short experimental period and lower gene detection cost.
Description
Technical Field
The invention relates to the technical field of gene detection, in particular to a primer probe composition, a kit and a detection method for detecting methylation of human ADD3 and CDH23 genes.
Background
Prostate cancer is a high-grade malignancy in men. In the united states, the incidence of prostate cancer is first in the male malignancy, and its mortality rate is second only to that of lung cancer. With the change of factors such as life, eating habits, environment and the like, the incidence rate of the prostatic cancer in China is rapidly increased in recent years, and the prostatic cancer becomes the first major malignant tumor of the male urinary system in developed cities such as Shanghai and Beijing.
At present, early detection means of the prostate cancer mainly comprise detection of the concentration of prostate cancer antigen PSA in serum and digital rectal examination. Patients who are detected to be abnormal need further aspiration biopsy of the prostate to finally confirm whether they have prostate cancer. However, both of these early screening methods have certain shortcomings that may lead to unnecessary biopsy punctures and over-screening, over-treatment, etc. of patients who are not clinically obvious. PSA is widely accepted and applied as an early diagnosis marker of prostate cancer, so that the mortality of prostate cancer is greatly reduced, and meanwhile, clinical over-diagnosis and over-treatment are brought. In order to reduce over-diagnosis and over-treatment of prostate cancer patients, a new detection method with higher sensitivity, specificity and positive predictive value needs to be provided as an effective supplement for PSA early screening.
DNA methylation is an epigenetic modification that refers to the addition of a methyl group at the 5C position of cytosine, occurring primarily at CpG sites. DNA methylation plays a very important role in many biological functions, and abnormal methylation level can cause disorder of biological functions and cause certain diseases. The research finds that the DNA methylation abnormality is closely related to the occurrence and the development of tumors, and the DNA methylation abnormality of certain genes can be detected in the early stage of the occurrence of many cancers. In addition, since DNA methylation is a relatively stable epigenetic modification, more and more studies are being conducted to use DNA methylation as a marker for early diagnosis of cancer, and the DNA methylation marker is considered to be a powerful tool with great potential in the future clinical field.
The applicant finds that Chinese patent with publication number CN105074010B discloses a detection method of methylated DNA after careful search. The prior art relies on the exchange chromatography column to achieve peak separation of methylated DNA detection signals, which is not unlike the increase of detection cost of methylated DNA detection. And the technical scheme disclosed by the prior art is not suitable for rapidly screening and diagnosing whether the patient suffers from the prostate cancer in a hospital.
In view of the above, there is a need for an improved prostate cancer screening method in the prior art to solve the above problems.
Disclosure of Invention
The invention aims to disclose a primer probe composition, a kit and a detection method for detecting methylation of human ADD3 and CDH23 genes, and early screening of prostate cancer is realized on the basis of methylation indexes of the human ADD3 and CDH23 genes, so that early screening and diagnosis of the prostate cancer are realized.
To achieve the first object, the present invention provides a primer probe composition for detecting methylation of human ADD3 and CDH23 genes, comprising: a first specific primer and a first specific probe for detecting methylation of a human ADD3 gene, a second specific primer and a second specific probe for detecting methylation of a human CDH23 gene, and a third specific primer and a third specific probe for detecting beta-actin of an internal reference gene;
the forward primer of the first specific primer has a nucleotide sequence shown as SEQ NO.1, the reverse primer of the first specific primer has a nucleotide sequence shown as SEQ NO.2, and the first specific probe has a nucleotide sequence shown as SEQ NO. 3;
the forward primer of the second specific primer has a nucleotide sequence shown as SEQ NO.4, the reverse primer of the second specific primer has a nucleotide sequence shown as SEQ NO.5, and the second specific probe has a nucleotide sequence shown as SEQ NO. 6;
the forward primer of the third specific primer has a nucleotide sequence shown as SEQ NO.7, the reverse primer of the third specific primer has a nucleotide sequence shown as SEQ NO.8, and the third specific probe has a nucleotide sequence shown as SEQ NO. 9.
As a further improvement of the invention, the 5 'ends of the first specific probe and the second specific probe are marked with a fluorescent reporter group, and the 3' ends are marked with a fluorescent quenching group; the fluorescence reporter group is selected from any one of FAM, HEX, TET, JOE, NED, VIC, CY3, CY5, ROX or TAMRA, and the fluorescence quencher group is selected from any one of MGB, BHQ or thiophosphate.
In order to achieve the second object of the present application, the present invention also discloses a kit for detecting methylation of human ADD3 and CDH23 genes, comprising a primer probe premix solution, an amplification premix solution, a negative quality control product and a positive quality control product, wherein the primer probe premix solution is composed of the primer probe composition disclosed by any one of the inventions of the present invention;
the amplification premixed solution consists of PCR buffer solution, PCR protective agent, taq enzyme, dNTPs and MgCl 2 Forming;
the positive quality control product is bisulfite modified fully methylated human genome DNA;
the negative quality control product is non-methylated human genome DNA modified by bisulfite.
Finally, the present application also discloses a detection method for detecting methylation of human ADD3 and CDH23 genes, comprising the steps of:
extracting a biological sample;
extracting sample DNA from a biological sample, and adjusting the sample DNA to standard purity and standard concentration;
step (3) adding bisulfite into the sample DNA for conversion;
step (4) adding the transformed sample DNA, the positive quality control product and the negative quality control product into the kit according to claim 3 respectively, and performing PCR amplification reaction;
and (5) analyzing the detection result.
As a further improvement of the invention, the biological sample in step (1) is cells in the first section of urine after the massage of the prostate;
the step (2) of extracting sample DNA from the biological sample material is specifically as follows: extracting by using a cell lysate or a purification column kit containing a nonionic detergent, converting by using a sulfite conversion kit, and measuring the purity of the converted sample DNA by using an ultraviolet spectrophotometer.
As a further improvement of the invention, in the step (2), the standard purity of the sample DNA is OD260/OD280 of 1.8-2.0, and the standard concentration is 0.2-2 mug/ml;
if the concentration of the sample DNA is less than 0.2. Mu.g/ml, the sample DNA is concentrated by ethanol precipitation, and if the concentration of the sample DNA is more than 2. Mu.g/ml, TE buffer is added to dilute the concentration of the sample DNA to a standard concentration.
As a further improvement of the invention, saidThe reaction system of the amplification reaction in the step (4) is 50 μ l:10 XPCR buffer, mgCl 2 50mM, dNTPs 250. Mu.M, 10 XPCR protectant, forward primer 100. Mu.M of the first specific primer, reverse primer 100. Mu.M of the first specific primer, first specific probe 100. Mu.M, forward primer 100. Mu.M of the second specific primer, reverse primer 100. Mu.M of the second specific primer, second specific probe 100. Mu.M, sterilized ultrapure water to 50. Mu.l;
the concentration of the positive quality control substance contained in the reaction system of the amplification reaction is 10-2000 copies/mu l, and the concentration of the negative quality control substance is 10-2000 copies/mu l.
As a further improvement of the invention, the reaction procedure of the amplification reaction in the step (4) is as follows:
(1) Pre-denaturation at 95 ℃ for 5min, with the cycle number of 1;
(2) Denaturation at 95 ℃ for 10sec, cycle number 45;
(3) 60 ℃ annealing, extension and fluorescence detection for 34sec with cycle number of 45, the fluorescence detection channels are FAM signal channel and VIC signal channel.
Compared with the prior art, the invention has the beneficial effects that:
by the primer probe composition, the kit and the detection method disclosed by the invention, the specificity detection on the methylation of the human ADD3 and CDH23 genes is realized, the accuracy of early screening and diagnosis on the human prostatic cancer is realized, and the primer probe composition, the kit and the detection method have the technical advantages of high sensitivity, high specificity, low cost, high flux, simplicity and convenience in operation, short experimental period and lower gene detection cost.
Drawings
FIG. 1 is an amplification curve of a positive quality control for detecting methylation of the human ADD3 gene;
FIG. 2 is an amplification curve of a negative quality control for detecting methylation of the human ADD3 gene;
FIG. 3 is an amplification curve of a positive sample for detecting methylation of the human ADD3 gene by the detection method for detecting methylation of the human ADD3 and CDH23 genes disclosed herein;
FIG. 4 is an amplification curve of a negative sample for detecting methylation of the human ADD3 gene by the detection method for detecting methylation of the human ADD3 and CDH23 genes disclosed by the invention;
FIG. 5 is an amplification curve of a positive quality control for detecting methylation of the human CDH23 gene;
FIG. 6 is an amplification curve of a negative quality control for detecting methylation of the human CDH23 gene;
FIG. 7 is an amplification curve of a positive sample for detecting methylation of the human CDH23 gene by the detection method for detecting methylation of the human ADD3 and CDH23 genes disclosed in the present invention;
FIG. 8 is an amplification curve of a negative sample for detecting methylation of the human CDH23 gene by the detection method for detecting methylation of the human ADD3 and CDH23 genes disclosed in the present invention;
FIG. 9 shows the amplification curve of the reference gene β -actin.
Detailed Description
The present invention is described in detail with reference to the embodiments shown in the drawings, but it should be understood that these embodiments are not intended to limit the present invention, and those skilled in the art should understand that functional, methodological, or structural equivalents or substitutions made by these embodiments are within the scope of the present invention.
Briefly, the primer probe composition, the kit and the detection method for detecting the methylation of the human ADD3 and CDH23 genes disclosed by the application can be used for screening the prostate cancer of human ADD3 and CDH23 gene methylated DNA methylation markers, avoiding various problems and disadvantages existing in vivo puncture and prostate cancer screening depending on PSA indexes, and can be used for early-stage auxiliary screening of the prostate cancer, so that an accurate basis is provided for targeted drug therapy or surgical therapy. Meanwhile, the term "sec" referred to in the present embodiment is a unit of time: second, the term "rpm" is the unit of rotation: rotating per minute; the term "min" is a unit of time: and (3) minutes.
First, the present application discloses a primer probe composition (hereinafter referred to as "primer probe composition") for detecting methylation of human ADD3 and CDH23 genes, wherein whether methylation occurs in human ADD3 and CDH23 site genes is used as a medical basis for judging whether a patient suffers from prostate cancer or not by qPCR amplification reaction based on the primer probe composition, and prostate biopsy is avoided. Specifically, the primer probe composition comprises: a first specific primer and a first specific probe for detecting the methylation of the human ADD3 gene, a second specific primer and a second specific probe for detecting the methylation of the human CDH23 gene, and a third specific primer and a third specific probe for detecting the beta-actin gene of the reference gene.
The forward primer (namely ADD3 forward primer) of the first specific primer has a nucleotide sequence shown as SEQ NO.1, the reverse primer (namely ADD3 reverse primer) of the first specific primer has a nucleotide sequence shown as SEQ NO.2, and the first specific probe (namely ADD3 specific probe) has a nucleotide sequence shown as SEQ NO. 3.
The forward primer (i.e., CDH23 forward primer) of the second specific primer has the nucleotide sequence shown in SEQ NO.4, the reverse primer (i.e., CDH23 forward primer) of the second specific primer has the nucleotide sequence shown in SEQ NO.5, and the second specific probe (i.e., CDH23 specific probe) has the nucleotide sequence shown in SEQ NO. 6.
The forward primer (namely beta-actin forward primer) of the third specific primer has a nucleotide sequence shown as SEQ NO.7, the reverse primer (namely beta-actin reverse primer) of the third specific primer has a nucleotide sequence shown as SEQ NO.8, and the third specific probe (namely beta-actin specific probe) has a nucleotide sequence shown as SEQ NO. 9.
The forward primer of the first specific primer, the reverse primer of the first specific primer and the first specific probe are used for carrying out gene detection on the methylation of the ADD3 gene in the human Y chromosome together, and the forward primer of the second specific primer, the reverse primer of the second specific primer and the second specific probe are used for carrying out gene detection on the methylation of the CDH23 gene in the human Y chromosome together. The reference Gene β -actin, as a housekeeping Gene (Home-eating Gene, HKG), for mammalian cell expression generally refers to the protein expressed by the housekeeping Gene code. Their expression in various tissues and cells is relatively constant, and it is commonly used as a reference substance when detecting the change of the expression level of protein, thereby providing an accurate reference basis for gene detection of human ADD3 and CDH23 gene methylation.
The 5 'ends of the first specific probe and the second specific probe are marked with fluorescent reporter groups, and the 3' ends are marked with fluorescent quenching groups. Specifically, the fluorescence reporter group is selected from any one of FAM, HEX, TET, JOE, NED, VIC, CY3, CY5, ROX or TAMRA, and the fluorescence quencher group is selected from any one of MGB, BHQ or thiophanate.
For example, in this example, the sequence of the first specific probe is as follows:
FAM-AGGTAGTCGTTTCGAATTAGGT-MGB。
the sequence of the second specific probe is shown below:
FAM-TCGGGCGTTTAGGG-MGB。
the primer probe compositions comprising the primers and probes disclosed in this example are designed as shown in Table I below.
Watch 1
The primer probe composition disclosed by the embodiment realizes efficient and specific detection on the methylation of the gene loci of ADD3 and CDH23 in human Y chromosome, has the advantages of high sensitivity and strong specificity, improves the detection accuracy of positive samples of prostate cancer, and improves unnecessary live body puncture and repeated puncture biopsy caused by poor PSA specificity, so that a kit prepared based on the primer probe composition does not cause wound to a patient when used for detection, and accurate clinical medical detection data is provided for early screening of the prostate cancer.
Meanwhile, based on the primer probe composition for detecting methylation of human ADD3 and CDH23 genes disclosed above, this example also discloses a kit for detecting methylation of human ADD3 and CDH23 genes (hereinafter referred to as kit). The kit comprises a primer probe premix (B-PPmix), an amplification premix (B-APmix), a negative quality control product and a positive quality control product, wherein the primer probe premix (B-PPmix) consists of the primer probe composition.
The amplification premixed solution (B-APmix) consists of PCR buffer solution, PCR protective agent, taq enzyme, dNTPs and MgCl 2 And (4) forming. Positive quality control is bisulfite (e.g., sodium bisulfite or potassium bisulfite) modified, fully methylated human genomic DNA (Qiagen, 59655); the negative quality control was bisulfite (e.g., sodium bisulfite or potassium bisulfite) modified non-methylated human genomic DNA (Qiagen, 59665). The target site of the ADD3 gene is at least one CpG site contained in the promoter region of the ADD3 gene; the target site of the CDH23 gene is at least one CpG site contained in the promoter region of the CDH23 gene.
Finally, the present application also discloses a detection method for detecting methylation of human ADD3 and CDH23 genes, which is characterized by comprising the following steps:
first, step (1) is performed to extract a biological sample. The biological sample in the step (1) is cells in the initial urine after the prostate massage, and the initial urine is about 40-50 ml. The urine of the first section contains a large amount of shed renal epithelial cells, and the renal epithelial cells are used as biological detection materials to carry out gene detection on ADD3 and CDH23 gene methylation indexes in human Y chromosome. Preferably, in this embodiment, the step (1) further comprises centrifuging the initial urine (10min, 4 ℃), taking the supernatant, resuspending the supernatant with 1ml of precooled PBS, transferring the supernatant into a new 1.5ml EP tube, centrifuging again (10min, 4 ℃) to collect the precipitate, and extracting the sample DNA.
Then, step (2) is performed to extract sample DNA from the biological sample material, and the sample DNA is adjusted to standard purity and standard concentration.
The step (2) of extracting the sample DNA from the biological sample material specifically comprises the following steps: extracting by using a cell lysate or a purification column kit containing a nonionic detergent, converting by using a sulfite conversion kit, and measuring the purity of the converted sample DNA by using an ultraviolet spectrophotometer. The standard purity of the sample DNA in the step (2) is OD260/OD280 of 1.8-2.0, and the standard concentration is 0.2-2 mug/ml; if the concentration of the sample DNA is less than 0.2. Mu.g/ml, the sample DNA is concentrated by ethanol precipitation, and if the concentration of the sample DNA is more than 2. Mu.g/ml, TE buffer is added to dilute the concentration of the sample DNA to a standard concentration.
Then, step (3) is carried out to add bisulfite (200-600 ng) to the sample DNA for conversion.
Then, step (4) is performed to add the transformed sample DNA, positive quality control (PC) and negative quality control (NC) to the kits disclosed in the above examples, respectively, and perform PCR amplification reaction.
And (4) adding 5 mu l of sample DNA prepared in the step (3) into a reaction hole of a PCR instrument (model ABI 7500), sealing a fluorescent quantitative PCR-grade sealing film or an upper tube cover, putting the sample DNA into a centrifuge, centrifuging for 30sec at the rotating speed of 2000rpm to ensure that the sample DNA solution and the reagent in the kit fully react and completely sink to the bottom of a PCR tube.
Specifically, the reaction system of the amplification reaction in step (4) is 50 μ l:10 XPCR buffer, mgCl 2 50mM, dNTPs 250. Mu.M, 10 XPCR protectant, forward primer 100. Mu.M of the first specific primer, reverse primer 100. Mu.M of the first specific primer, first specific probe 100. Mu.M, forward primer 100. Mu.M of the second specific primer, reverse primer 100. Mu.M of the second specific primer, second specific probe 100. Mu.M, sterilized ultrapure water to 50. Mu.l. The reaction system of the amplification reaction contains the positive quality control substance with the concentration of 10-2000 copies/mu l and the negative quality control substance with the concentration of 10-2000 copies/mu l.
The kit comprises a primer probe premix (namely B-PPmix), an amplification premix (namely B-APmix), a negative quality control product (NC) and a positive quality control Product (PC). The system of the kit is shown in table two below.
Watch two
In order to ensure the success of the test operation and the subsequent analysis of the test result, a PC control group (namely a positive quality control material) and an NC control group (namely a negative quality control material) are required to be arranged in each process of detecting the methylation of the ADD3 and CDH23 genes of the human. And (3) sequentially putting the PCR tube or the PCR96 pore plate into a PCR instrument, performing real-time fluorescent quantitative PCR amplification according to the amplification program in the step (4), and adopting a fluorescent probe method to utilize a fluorescent reporter group (such as FAM or VIC) marked at the 5 'end and a fluorescent quencher group (such as MGB or BHQ) marked at the 3' end. The BHQ as the fluorescence quenching group may be BHQ-1, BHQ-2 or BHQ-3.
The reaction procedure of the amplification reaction in step (4) is as follows:
(1) Pre-denaturation at 95 ℃ for 5min, with the cycle number of 1;
(2) Denaturation at 95 ℃ for 10sec, and cycle number of 45;
(3) 60 ℃ annealing, extension and fluorescence detection for 34sec, cycle number 45, the fluorescence detection channels are FAM signal channel and VIC signal channel.
And finally, performing detection result analysis in the step (5).
(1) Quality control standard
(1) The positive control has positive determination result and Ct value less than or equal to 36.
(2) The negative control was negative and the Ct value was not interpreted (determined).
(3) The amplification curve should have a distinct exponential phase, and the baseline and exponential phase should have distinct inflection points.
If the three quality control standards are met, the test is determined to be effective; otherwise, the test is deemed invalid.
(2) Interpretation of results
(1) The PC has typical amplification curves under both an FAM signal channel and a VIC signal channel, and the Ct value of the VIC signal channel is less than or equal to 32;
(2) the NC has no typical amplification curve or Ct value in the FAM signal channel, and the Ct value of the VIC signal channel is less than or equal to 32;
if the two requirements are met, the experiment is successful, the detection result of the sample can be analyzed, and the judgment of the detection result is shown in table 3.
Table 3: determination of detection result
Referring to fig. 1 and 2, and fig. 5 and 6, the positive quality control substances have typical amplification curves in the FAM signal channel, the negative quality control substances have no typical amplification curve in the FAM signal channel, and the Ct value is less than or equal to 32, which meets the judgment standard of the positive quality control substances as the positive control group, and indicates that the detection result is reliable. Referring to fig. 3 and 4, the beta-actin gene has a typical amplification curve under the VIC signal channel, and the Ct value is less than 32, which indicates that the sample DNA is qualified and the detection result is credible. Referring to FIG. 3, the sample DNA has a typical amplification curve in the FAM signal channel, and the Δ Ct is less than 12, which indicates that the sample is a positive sample; in FIG. 4, the sample DNA has no typical amplification curve in the FAM channel, indicating that the test sample is a negative sample. Referring to fig. 7 and 8, the beta-actin gene has a typical amplification curve under the VIC signal channel, and the Ct value is less than 32, which indicates that the sample DNA is qualified and the detection result is credible. In FIG. 7, the sample DNA has a typical amplification curve in the FAM signal channel, and the Ct value is < 42, indicating that the sample is a positive sample; in FIG. 8, the sample DNA has no typical amplification curve in the FAM channel, indicating that the test sample is a negative sample. Referring to FIG. 9, the beta-actin gene has a typical amplification curve in the VIC channel, which proves that the sample DNA to be detected can be used for detecting whether methylation occurs in human ADD3 gene locus and CDH23 gene locus or not by the detection method disclosed by the application, and the sample DNA is further divided into a positive sample or a negative sample.
The above-listed detailed description is merely a detailed description of possible embodiments of the present invention, and it is not intended to limit the scope of the invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention are intended to be included within the scope of the present invention.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
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Claims (3)
1. A primer probe composition for detecting methylation of human ADD3 and CDH23 genes, comprising: a first specific primer and a first specific probe for detecting methylation of a human ADD3 gene, a second specific primer and a second specific probe for detecting methylation of a human CDH23 gene, and a third specific primer and a third specific probe for detecting beta-actin of an internal reference gene;
the forward primer of the first specific primer is shown as a nucleotide sequence shown in SEQ NO.1, the reverse primer of the first specific primer is shown as a nucleotide sequence shown in SEQ NO.2, and the first specific probe is shown as a nucleotide sequence shown in SEQ NO. 3;
the forward primer of the second specific primer is shown as a nucleotide sequence shown in SEQ NO.4, the reverse primer of the second specific primer is shown as a nucleotide sequence shown in SEQ NO.5, and the second specific probe is shown as a nucleotide sequence shown in SEQ NO. 6;
the forward primer of the third specific primer is shown as a nucleotide sequence shown in SEQ NO.7, the reverse primer of the third specific primer is shown as a nucleotide sequence shown in SEQ NO.8, and the third specific probe is shown as a nucleotide sequence shown in SEQ NO. 9.
2. The primer-probe composition of claim 1, wherein the first specific probe and the second specific probe are labeled with a fluorescent reporter at their 5 'end and a fluorescent quencher at their 3' end; the fluorescence reporter group is selected from any one of FAM, HEX, TET, JOE, NED, VIC, CY3, CY5, ROX or TAMRA, and the fluorescence quencher group is selected from any one of MGB, BHQ or thiophosphate.
3. A kit for detecting methylation of human ADD3 and CDH23 genes, which is characterized by comprising a primer probe premix solution, an amplification premix solution, a negative quality control product and a positive quality control product, wherein the primer probe premix solution is composed of the primer probe composition as claimed in claim 1 or 2;
the amplification premix consists of PCR buffer solution, PCR protective agent and Taq enzyme、dNTPs、MgCl 2 Forming;
the positive quality control product is bisulfite modified fully methylated human genome DNA;
the negative quality control product is non-methylated human genome DNA modified by bisulfite.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010053489.4A CN111154876B (en) | 2020-01-17 | 2020-01-17 | Primer probe composition, kit and detection method for detecting methylation of human ADD3 and CDH23 genes |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010053489.4A CN111154876B (en) | 2020-01-17 | 2020-01-17 | Primer probe composition, kit and detection method for detecting methylation of human ADD3 and CDH23 genes |
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| Publication Number | Publication Date |
|---|---|
| CN111154876A CN111154876A (en) | 2020-05-15 |
| CN111154876B true CN111154876B (en) | 2023-01-20 |
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| CN108396063A (en) * | 2017-02-07 | 2018-08-14 | 复旦大学附属华山医院 | Application of the CDH23 gene mutations in the diagnosis of pituitary adenoma molecule |
| CN110484625A (en) * | 2019-08-29 | 2019-11-22 | 无锡市申瑞生物制品有限公司 | For detecting primer combination of probe object, kit and the detection method of PRKY gene methylation |
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| GB201402771D0 (en) * | 2014-02-17 | 2014-04-02 | Isis Innovation | Biomarker & therapeutic target for sarcoma |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108396063A (en) * | 2017-02-07 | 2018-08-14 | 复旦大学附属华山医院 | Application of the CDH23 gene mutations in the diagnosis of pituitary adenoma molecule |
| CN110484625A (en) * | 2019-08-29 | 2019-11-22 | 无锡市申瑞生物制品有限公司 | For detecting primer combination of probe object, kit and the detection method of PRKY gene methylation |
Non-Patent Citations (1)
| Title |
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| CDH23 Methylation Status and Presbycusis Risk in Elderly Women;Amal Bouzid，等;《frontiers in aging neuroscience》;20180831(第10期);第1-7页 * |
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Denomination of invention: Primer probe composition, kit, and detection method for detecting methylation of human ADD3 and CDH23 genes Effective date of registration: 20230925 Granted publication date: 20230120 Pledgee: Agricultural Bank of China Limited by Share Ltd. Wuxi Huishan branch Pledgor: WUXI SHENRUI BIO-PHARMACEUTICALS Co.,Ltd. Registration number: Y2023980058229 |