CN108103197A - For detecting the primer sets of RPRM genes and PRDM5 gene methylations, reagent, kit and application - Google Patents
For detecting the primer sets of RPRM genes and PRDM5 gene methylations, reagent, kit and application Download PDFInfo
- Publication number
- CN108103197A CN108103197A CN201810127514.1A CN201810127514A CN108103197A CN 108103197 A CN108103197 A CN 108103197A CN 201810127514 A CN201810127514 A CN 201810127514A CN 108103197 A CN108103197 A CN 108103197A
- Authority
- CN
- China
- Prior art keywords
- genes
- prdm5
- rprm
- dna
- kit
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Hospice & Palliative Care (AREA)
- Biophysics (AREA)
- Oncology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a kind of for detecting primer sets, reagent, kit and the application of RPRM genes and PRDM5 gene methylations, it is related to biological technical field, provided by the present invention for detecting primer sets, reagent and the kit of RPRM genes and PRDM5 gene methylations simultaneously, including being used to detect the primer pair of RPRM genes and primer pair for detecting PRDM5 genes, high specificity, high sensitivity, can be easy, quick, accurate and detect the methylation state of RPRM genes and PRDM5 genes simultaneously.Method that is provided by the invention while detecting RPRM genes and PRDM5 gene methylations, it is easy to operate, be easy to grasp.The present invention also provides the application of above-mentioned primer sets, reagent or kit in preparing for the product of auxiliary diagnosis and/or early screening stomach cancer.
Description
Technical field
The present invention relates to biological technical fields, are used to detect RPRM genes and PRDM5 gene methyl more particularly, to one kind
Primer sets, reagent, kit and the application of change.
Background technology
Stomach cancer is one of most common tumour in worldwide, and incidence is in the 4th in all malignant tumours,
Its death rate is in the 2nd in malignant tumour, and has more than 70% gastric cancer cases and be present in developing country.China is annual
New hair gastric cancer cases about 400,000, account for the world always falls ill 40%.The early gastric caacer diagnosis ＜ 10% in China, one after diagnosing
Mostly middle and advanced stage, 5 annual survival rate ＜ 20%；At the same time, after developed country carries out whole people's examination, the early diagnostic rate of stomach cancer
Up to 50%, 5 annual survival rates are up to more than 60%.For this purpose, how to realize that the morning of stomach cancer is the discovery that the pass for reducing its death rate
Key.
Stomach cancer more than 90% is gland cancer, and nineteen sixty-five Lauren divides gland cancer according to the institutional framework and biological behaviour of stomach cancer
For visible peristalsis visible intestinal peristalsis and diffusion-type.The occurrence and development process of the two mainly includes following several stages：Normally-gastritis-atrophic stomach
Cancer-infiltrating cancer in inflammation-intestinal metaplasia-dysplasia-mucous membrane.1978, the World Health Organization was by atrophic gastritis
The precancerous condition of stomach cancer is classified as, before being referred to as precancerous lesions of chronic atrophic gastritis or stomach cancer with intestinal metaplasia, dysplasia
Phase lesion, the forme fruste intestinal metaplasia to occur together on the basis of withering slowly and (or) in, severe dysplasia be then considered as real
Precancerous lesion.The cure rate of preventing precancerous lesion of stomach cancer even early carcinoma of stomach is up to 90%, so early carcinoma of stomach, precancerous lesion are even
The discovery of disease is most important before cancer.
The examination main path of early gastric caacer includes general population generaI investigation, the examination of outpatient service opportunistic and people at highest risk's examination.
China is at present mainly using outpatient service opportunistic examination, mainly to participate in Physical Check-Ups person as examination object, by serum and stomach
Fluid markers checkout and diagnosis, electron microscopic diagnosis and imaging diagnosis section investigate stomach cancer, confirm the generation of stomach cancer.Serum PG
Level detection is to reflect the good index of stomach lining function and state, is had many advantages, such as noninvasive, easy, quick；Gastroscope detects quilt
It is considered " goldstandard " of stomach cancer detection, sensitivity and specificity are current stomach cancers most directly perceived, most effective more than 90%
Early detection means；Before scope use, canel barium meal contrast examination is diagnosing gastric cancer, the main means of examination, but since it cannot take
It obtains pathological diagnosis and is progressively detected and substitute by scope.When clinical routine diagnostics are applied to early gastric caacer diagnosis, gastric tissue has been sent out
Raw substantive lesion, cell have occurred canceration, really to have accomplished the early screening of stomach cancer, and gene diagnosis is sent to great expectations.
In view of this, it is special to propose the present invention.
The content of the invention
First of the present invention is designed to provide one kind and is used for while detects RPRM genes and PRDM5 gene methylations
Primer sets, with alleviate it is in the prior art lack high specificity, high sensitivity and can detect simultaneously RPRM genes and
The technical issues of primer of PRDM5 gene methylations.
Second object of the present invention is that providing one kind is used for while detects RPRM genes and PRDM5 gene methylations
Reagent, with alleviate it is in the prior art lack high specificity, high sensitivity and can detect simultaneously RPRM genes and
The technical issues of reagent of PRDM5 gene methylations.
Third object of the present invention is that providing one kind is used for while detects RPRM genes and PRDM5 gene methylations
Kit, with alleviate it is in the prior art lack high specificity, high sensitivity and can detect simultaneously RPRM genes and
The technical issues of kit of PRDM5 gene methylations.
Fourth object of the present invention is that providing above-mentioned primer sets, reagent or kit is preparing for auxiliary diagnosis
And/or the application in the product of early screening stomach cancer, to alleviate in the prior art lack to stomach cancer progress early screening
The technical issues of effective means of diagnosis.
The 5th of the present invention is designed to provide a kind of method for detecting stomach cancer, to alleviate in the prior art lack
The technical issues of method of few accurate and effective early gastric caacer detection.
It is described to draw the present invention provides a kind of for detecting the primer sets of RPRM genes and PRDM5 gene methylations simultaneously
Object group includes detecting the primer pair of RPRM genes and the primer pair for detecting PRDM5 genes；
Draw the upstream for including the RPRM genes as shown in SEQ ID NO.1 for detecting the primer pair of RPRM genes
The anti-sense primer of object and the RPRM genes as shown in SEQ ID NO.2；
It is described to include the upstream of the PRDM5 genes as shown in SEQ ID NO.3 for detecting the primer pair of PRDM5 genes
The anti-sense primer of primer and the PRDM5 genes as shown in SEQ ID NO.4.
The present invention also provides a kind of for detecting the reagent of RPRM genes and PRDM5 gene methylations, the examination simultaneously
Agent includes above-mentioned primer sets.
The present invention also provides a kind of for detecting the kit of RPRM genes and PRDM5 gene methylations simultaneously, including
Above-mentioned primer sets or reagent.
Further, the kit includes PCR Mix and Taq enzyme Mix；
Reagents and dNTP of the PCR Mix described in including primer sets described in claim 1 or claim 2；
The Taq enzyme Mix includes Taq enzyme and probe.
Further, the kit further includes negative controls and positive reference substance；
Preferably, the negative controls include TE buffer solutions and RPRM genes and the non-base to methylate of PRDM5 genes
Because of a group DNA；
The positive control includes the genomic DNA that TE buffer solutions methylate with RPRM genes and PRDM5 genes.
Further, the kit further includes DNA processing articles for use；
Preferably, the DNA processing articles for use include the articles for use of extraction DNA and carry out sulphite conversion to the DNA
Articles for use；
Preferably, the articles for use of the extraction DNA include lysate, magnetic bead and extraction rinsing liquid, and sulfurous is carried out to the DNA
The articles for use of hydrochlorate conversion include sulfiding reagent, with reference to liquid, purifying rinsing liquid and eluent.
The present invention also provides mentioned reagent box in preparing for the product of auxiliary diagnosis and/or early screening stomach cancer
Using.
In addition, the present invention also provides the above-mentioned primer sets of application or reagent or the method for kit detection stomach cancer, the sides
Method includes：
It will be described in the plasma sample DNA after sulphite converts and primer sets described in claim 1 or claim 2
Reagent carry out quantitative fluorescent PCR reaction, judge whether to suffer from according to the Cp values of the plasma sample DNA after sulphite converts
Stomach cancer；
When the Cp values≤44.76 of the RPRM genes, then the risk height that gets a cancer of the stomach is judged；And/or
When the Cp values≤38.85 of the PRDM5 genes, then the risk height that gets a cancer of the stomach is judged.
Further, the DNA in the plasma sample after lysate cracks is extracted using magnetic bead absorption method, and by described in
DNA carries out sulphite conversion, then is purified using paramagnetic particle method, obtains the plasma sample after sulphite converts
DNA；
Preferably, the mixed liquor of application fetches rinsing liquid and absolute ethyl alcohol washs the DNA in the plasma sample
Afterwards, the DNA is subjected to sulphite conversion.
Further, by the combination liquid for being mixed with magnetic bead and the plasma sample DNA mixings after sulphite converts into
After row absorption, magnetic bead is washed using the mixed liquor of purifying rinsing liquid and absolute ethyl alcohol, then using elution institute
State the plasma sample DNA after sulphite converts.
Provided by the present invention for detecting the primer sets of RPRM genes and PRDM5 gene methylations simultaneously, including being used to examine
The primer pair of RPRM genes and the primer pair for detecting PRDM5 genes are surveyed, which can
It is easy, quick, accurate and detect the methylation level of RPRM genes and PRDM5 genes simultaneously.Provided by the present invention for simultaneously
The reagent of RPRM genes and PRDM5 gene methylations is detected, including above-mentioned primer sets, reagent high specificity, the high sensitivity, energy
It reaches easy, quick, accurate and detects the methylation level of RPRM genes and PRDM5 genes simultaneously.Provided by the present invention for same
When detect the kit of RPRM genes and PRDM5 gene methylations, including above-mentioned primer sets or reagent, can it is easy, quick,
Detect accurately and simultaneously the methylation state of RPRM genes and PRDM5 genes.The method of detection stomach cancer provided by the invention, will
Plasma sample DNA after sulphite converts carries out quantitative fluorescent PCR reaction with above-mentioned primer group reagent, according to through Asia
The Cp values of plasma sample DNA after sulfate conversion judge the risk that gets a cancer of the stomach, and this method applies primer sets provided by the invention
Or reagent or kit, it is easy to operate, be easy to grasp, it can accurately and efficiently judge the situation of getting a cancer of the stomach.The present invention also provides
The application of above-mentioned primer sets, reagent or kit in preparing for the product of auxiliary diagnosis and/or early screening stomach cancer, application
Primer sets provided by the invention, reagent or kit carry out early screening diagnosis to stomach cancer, have highly sensitive and high accuracy
The advantages of, and screening method is simple, is easy to grasp, and stomach cancer is detected exactly in preventing precancerous lesion of stomach cancer or early carcinoma of stomach period
Occur, the cure rate of stomach cancer can be greatly improved, there is great promotional value.
Specific embodiment
Technical scheme is clearly and completely described below in conjunction with embodiment, it is clear that described reality
It is part of the embodiment of the present invention to apply example, instead of all the embodiments.Based on the embodiments of the present invention, the common skill in this field
Art personnel all other embodiments obtained without making creative work belong to the model that the present invention protects
It encloses.
The present invention provides a kind of for detecting the primer sets of RPRM genes and PRDM5 gene methylations simultaneously, including with
Primer pair in detection RPRM genes and primer pair for detecting PRDM5 genes；
Primer pair for detecting RPRM genes includes：
The sense primer of RPRM genes：5’-GCGAGTGAGCGTTTAGTTC-3’(SEQ ID NO.1)；
The anti-sense primer of RPRM genes：5’-ACCCGCCACGTCCGTC-3’(SEQ ID NO.2).
Primer pair for detecting PRDM5 genes includes：
The sense primer of PRDM5 genes：5’-AGTTTTGTTTCGGGTTTCGC-3’(SEQ ID NO.3)；
The anti-sense primer of PRDM5 genes：5’-CATTCCTACTACGAAAACGC-3’(SEQ ID NO.4).
Provided by the present invention for detecting the primer sets of RPRM genes and PRDM5 gene methylations, high specificity, spirit simultaneously
Sensitivity is high, can be easy, quick, accurate and detects the methylation level of RPRM genes and PRDM5 genes simultaneously.
The present invention also provides a kind of for detecting the reagent of RPRM genes and PRDM5 gene methylations simultaneously, including upper
The primer sets stated.
It is high specificity, sensitive provided by the present invention for detecting the reagent of RPRM genes and PRDM5 gene methylations simultaneously
Degree is high, can be easy, quick, accurate and detects the methylation level of RPRM genes and PRDM5 genes simultaneously.
The present invention also provides a kind of for detecting the kit of RPRM genes and PRDM5 gene methylations simultaneously, including
Above-mentioned primer sets or reagent.
It, can be easy, fast provided by the present invention for detecting the kit of RPRM genes and PRDM5 gene methylations simultaneously
It is speed, accurate and detect the methylation level of RPRM genes and PRDM5 genes simultaneously.
In one preferred embodiment, kit includes PCR Mix and Taq enzyme Mix.
PCR Mix include above-mentioned primer sets or reagent and dNTP；Taq enzyme Mix includes Taq enzyme and probe.
Wherein, the probe sequence of RPRM is：FAM-CGTCGTTTTTTTATTTTTCG-MGB (SEQ ID NO.5)；
The probe sequence of PRDM5 is：FAM-AGCGTTTAGGTTCGCGTTTT-MGB (SEQ ID NO.6)；
The probe sequence of ACTB is：VIC-AGTAAGTTTTTTGGATTGTG-MGB(SEQ ID NO.7).
What is applied during conventional progress DNA methylation assay is dye method, and application is MGB in a preferred embodiment of the invention
Sonde method, sensitivity higher, specificity are stronger.
By the component comprising primer and dNTP composition PCR Mix premixed liquids, while Taq enzyme and probe are formed into Taq enzyme Mix
Premixed liquid on the basis of can not failing before use ensureing the reagent for detection, makes the kit only need when in use
2 kinds of premixed liquids are mixed, is prepared into PCR pre-reaction liquid, then dispenses into PCR reaction vessels, add in isometric treat
Sample template, sealing.It is easy to operate, it is easy to grasp.
In one preferred embodiment, PCR Mix and Taq enzyme Mix respectively further comprise solvent.It is wrapped in PCR Mix
The solvent contained for example can be, but the one or more being not limited in sterilizing purified water, water for injection or distilled water；Taq enzyme Mix
Included in solvent for example can be but one kind being not limited in sterilizing purified water, water for injection, distilled water or TE buffer solutions
It is or a variety of.
In one preferred embodiment, kit further includes negative controls and positive reference substance.
Preferably, negative controls include TE buffer solutions and RPRM genes and the non-genome to methylate of PRDM5 genes
DNA；Positive control includes the genomic DNA that TE buffer solutions methylate with RPRM genes and PRDM5 genes.
Directly it can ensure to apply reagent provided by the invention comprising negative controls and positive reference substance in kit
When box is detected, there is the control of standard to the testing result of sample to be tested.Pass through the inspection of negative controls and positive reference substance
It surveys as a result, it is possible to effectively judge the testing result of sample to be tested, while ensure the accuracy and validity of experiment.
In one preferred embodiment, kit further includes DNA processing articles for use.
Preferably, DNA handles articles for use of the articles for use including extraction DNA and the DNA to extracting carries out sulphite conversion
Articles for use.
Articles for use directly are handled comprising DNA in kit, only need a set of kit provided by the invention, it can either be to be measured
Sample is detected, and meets the pretreatments such as the extraction before detection and sulphite conversion, can be reached a box and is mostly used, uses
Convenient purpose.
Preferably, extracting the articles for use of DNA includes lysate, magnetic bead and extraction rinsing liquid, and sulfurous is carried out to the DNA extracted
The articles for use of hydrochlorate conversion include sulfiding reagent, with reference to liquid, purifying rinsing liquid and eluent.
Preferably, lysate includes guanidinium isothiocyanate；Magnetic bead includes silicon substrate magnetic bead；Extracting rinsing liquid includes isothiocyanic acid
Guanidine；Sulfiding reagent includes sulfite solution, is preferably sodium hydrogensulfite；Include guanidine hydrochloride with reference to liquid；Purifying rinsing liquid includes
One or more in Tris-HCl solution, sodium hydroxide solution or acetic acid solution.
The present invention also provides above-mentioned primer sets or reagent or kit to prepare for auxiliary diagnosis and/or early stage
Application in the product of examination stomach cancer.
DNA methylation is closely related with tumour, and abnormal methylation state is the key factor of carcinogenesis.RPRM is located at
On chromosome 2q23 sites, its abnormal hyper-methylation and the occurrence and development of stomach cancer are closely related；PRDM5, positioned at chromosome
On 4q25-26 sites, it is hypermethylated with early carcinoma of stomach in dysplasia, and two kinds of combination can be used as stomach cancer auxiliary to examine
The disconnected important symbol object with early screening.Early stage sieve is carried out to stomach cancer using primer sets provided by the invention, reagent or kit
Look into diagnosis, there is highly sensitive and high accuracy, and screening method is simple, is easy to grasp, in preventing precancerous lesion of stomach cancer or
Early carcinoma of stomach period detects the generation of stomach cancer exactly, can greatly improve the cure rate of stomach cancer, has great promotion price
Value.
In addition, the present invention also provides a kind of method for detecting stomach cancer using above-mentioned primer sets or reagent or kit, bags
It includes：
Plasma sample DNA after sulphite converts and above-mentioned primer sets or reagent progress quantitative fluorescent PCR is anti-
Should, judged whether according to the Cp values of the plasma sample DNA after sulphite converts with stomach cancer；
When the Cp values≤44.76 of RPRM genes, then the risk height that gets a cancer of the stomach is judged；And/or the Cp values when PRDM5 genes
When≤38.85, then the risk height that gets a cancer of the stomach is judged.
The method of detection stomach cancer provided by the invention, it is easy to operate, be easy to grasp, it can quickly and accurately detect and suffer from cancer
Situation.
In one preferred embodiment, quantitative fluorescent PCR reaction is carried out to include the following steps：
A), by PCR Mix and Taq enzyme Mix mixings, PCR pre-reaction liquid is obtained；
B), PCR pre-reaction liquid is dispensed into PCR reaction vessels, adds in isometric sample to be tested template, is sealed；
PCR programs when c), for detecting are as follows：95 DEG C first are denatured 5 minutes, and then 95 DEG C of denaturation are moved back for 20 seconds, 61 DEG C
Fire 40 seconds, and 50 Xun Huans are carried out, during PCR cycle, instrument warming and cooling rate is set as 1.3 DEG C/S, last 40 DEG C of maintenances
30 seconds.
In one preferred embodiment, using ACTB as the internal reference of PCR amplification.
Wherein, the sense primer of internal reference ACTB is：5’-TGGGGTGGTGATGGAGGAGG-3’ (SEQ ID NO.8)；
The anti-sense primer of internal reference ACTB is：5’-CCAACACACAATAACAAACA-3’(SEQ ID NO.9).
In one preferred embodiment, extracted using magnetic bead absorption method in the plasma sample after lysate cracks
DNA, and DNA is subjected to sulphite conversion, then purified using paramagnetic particle method, obtain the blood plasma sample after sulphite converts
This DNA.
Preferably, the mixed liquor of application fetches rinsing liquid and absolute ethyl alcohol washs the DNA in the plasma sample
Afterwards, DNA is subjected to sulphite conversion.
Specifically comprise the following steps：
A) lysate of 1.0-4.0mL, is added in the plasma sample of 0.5-2.0mL, is vibrated under conditions of 10-30 DEG C
It is incubated 10-30min；
B), using the dissociative DNA in magnetic bead absorption method extraction step a)；
C), the mixed liquor of application fetches rinsing liquid and absolute ethyl alcohol washs the DNA obtained in step b)；
D) DNA after washing in step c), is subjected to sulphite conversion, and is purified using paramagnetic particle method, is obtained
BisDNA (DNA after sulphite vulcanization).
In one preferred embodiment, after the combination liquid for being mixed with magnetic bead and above-mentioned bisDNA mixings being adsorbed,
Magnetic bead is washed using the mixed liquor of purifying rinsing liquid and absolute ethyl alcohol, then using the above-mentioned bisDNA of elution.
Specifically comprise the following steps：
A), above-mentioned bisDNA is adsorbed onto with combining liquid, magnetic bead mixing, bisDNA on magnetic bead；
B), purifying rinsing liquid mixed with absolute ethyl alcohol, for step A) in be adsorbed with bisDNA magnetic bead washing, then
It is eluted using eluent, obtains bisDNA.
In order to contribute to it is clearer understand present disclosure, be described in detail as follows in conjunction with specific embodiment.
The processing of the composition and sample to be tested of 1 kit of embodiment
1st, provided by the present invention for detecting the kit of RPRM genes and PRDM5 gene methylations simultaneously, at DNA
Manage kit and methylated genes detection kit.Wherein, the composition of DNA treatment kits is as shown in table 1, methylated genes inspection
The composition of test agent box is as shown in table 2.
1 DNA treatment kits of table
2 methylated genes detection kit of table
| Reagent | Main component |
| PCR Mix | DNTP, primer, buffer salt |
| Taq enzyme Mix | Taq enzyme, probe, buffer salt |
| Negative controls | TE buffer solutions, unmethylated human gene group DNA |
| Positive reference substance | TE buffer solutions, the human gene group DNA to methylate |
2nd, instrument and equipment：Instrument and equipment is as follows needed for detection：
Whirlpool concussion instrument, rotation vortex mixer, 15mL magnetic frames, 2.0mL magnetic frames, metal bath, PCR instrument, palm centrifugation
Machine, micropipettor, superclean bench, Lightcycler 480 etc..
3rd, the extraction of plasma DNA and vulcanization purifying
Specific method is as follows：
1. reagent prepares
1) rinsing liquid is extracted：Isometric absolute ethyl alcohol is added in using preceding for the first time, vibrates mixing.
2) rinsing liquid is purified：For the first time using 4 times of volume absolute ethyl alcohols of preceding addition, mixing is vibrated.
2. operating procedure
1) a 15mL centrifuge tube (providing for oneself) is taken, adds in 2mL plasma samples, positive reference substance, negative controls respectively take 1
Thaw at RT adds in 4mL lysates, vortex mixing, shaken at room temperature 10-30min.
2) magnetic bead of 100 μ L, vortex mixing, shaken at room temperature 10-30min are added in.
3) centrifuge tube is placed on magnetic frame, after magnetic bead absorption completely, supernatant discarding.
4) 1mL extraction rinsing liquids are added in, magnetic bead mixing is transferred in 1.5-2mL centrifuge tubes, centrifuge tube is placed on magnetic
On power frame, after magnetic bead absorption completely, supernatant discarding.
5) it is repeated 1 times step 4).
6) 80% ethyl alcohol of 1mL is added in, centrifuge tube is placed on magnetic frame by vortex mixing, after magnetic bead absorption completely, is abandoned
Remove supernatant.
7) it is repeated 1 times step 6).
8) wink from, centrifuge tube is placed on magnetic frame, magnetic bead completely absorption after, carefully remove residual liquid, room with rifle
Temperature places 5-10min.
9) 100 μ L purified water vortex mixings are added in into centrifuge tube, 56 DEG C of incubation 5-10min, wink is from centrifuge tube is placed
On magnetic frame, after magnetic bead absorption completely, draw DNA solution and preserve.
10) 1 600 μ L centrifuge tube is taken, adds in 100 μ L DNA solutions, adds sulfiding reagent, the DNA of 30 μ L of 190 μ L
Protect liquid, vortex mixing, wink from.
10) by centrifuge tube 98 DEG C of incubations 10min, 80 DEG C of incubation 1h.
11) be cooled to room temperature, wink from.1 2mL centrifuge tube is taken, the combination liquid of 1.2mL is added in, is transferred to above-mentioned sulfur product,
Add 50 μ L magnetic beads, vortex mixing, shaken at room temperature 10-30min, centrifuge tube from being placed on magnetic frame, magnetic bead is complete by wink
After absorption, supernatant is abandoned.
12) the purifying rinsing liquid 1 of 500 μ L is added in, vortex mixing, centrifuge tube from being placed on magnetic frame, magnetic bead is complete by wink
After full absorption, supernatant is abandoned.
13) the purifying rinsing liquid 2 of 500 μ L is added in, vortex mixing, shaken at room temperature 10-30min, wink is from centrifuge tube is placed
On magnetic frame, after magnetic bead absorption completely, supernatant is abandoned.
14) the purifying rinsing liquid 3 of 500 μ L is added in, vortex mixing, centrifuge tube from being placed on magnetic frame, magnetic bead is complete by wink
After full absorption, supernatant is abandoned.
15) the purifying rinsing liquid 1 of 500 μ L is added in, vortex mixing, centrifuge tube from being placed on magnetic frame, magnetic bead is complete by wink
After full absorption, supernatant is abandoned.
16) it is repeated 1 times step 15).
17) centrifuge tube is removed, centrifuge tube from being placed on magnetic frame, after magnetic bead absorption completely, carefully removed with rifle by wink
Residual liquid is placed at room temperature for 5-10min.
18) 56 DEG C of aqua sterilisa is preheating to magnetic bead addition 30-60 μ L, is closed the lid, vortex mixing, 56 DEG C of incubation 5-
10min, intermediate mixing again is once.
19) centrifuge tube from being placed on magnetic frame, treat that magnetic bead adsorbs completely, draw supernatant bisDNA to new centrifugation by wink
It is preserved in pipe.
The detection of 2 RPRM and PRDM5 methylated genes of embodiment
1) according to 3 reaction system of table, prepare PCR pre-reaction liquid, after mixing packing to PCR pipe, sample to be tested, reference substance and
Purified water is added in as template, and each reaction volume is 20 μ L；
3 PCR reaction systems of table
2) quantitative fluorescent PCR reaction is carried out according to the reaction condition of table 4.
4 reaction condition of table
3) on sample after machine, Experiment name, operation PCR reactions is preserved, and sample-adding typesetting is write according to experimental layout in time
And sample ID.
Embodiment 3PCR interpretations of result
1." Analysis " is selected in 480 basic software modules columns.
2. select " Abs Quant/Fit Points " analytical model.
3. it selects " Filter Comb 465-510 ".
4. selecting " Cycle Range " window, " First Cycle " is set as " 1 "；" Last Cycle " is " 50 ".
It is " 5-22 " by background set 5. in " Cycle Range " window, " Background " for clicking on blueness is pressed
" Min Offset " is set as " 4 ", " Max Offset " is set as " 21 " by button.
6. selecting " Noise Band " window, " Noise Band " is set as " Noise Band (Fluoresc) ", and
The value of " Noise Band " is manually changed to " 2.0 ".
Note：If background value is higher than 2.0, the value of baseline, i.e. " Noise Band " can be adjusted according to actual conditions, until not having
The amplification curve for having apparent Exponential growth stage should be feminine gender.
7. select " Analysis " window, " Threshold (Auto) " will " Threshold " value be automatically adjusted to and
" Noise Band " value is consistent, and " Fit Points " is set as " 2 ".
8. clicking on " CalcuLate ", software provides the Cp values of target gene RPRM and PRDM5 automatically.
9. it selects " Filter Comb 533-580 ".
10. clicking on " CalcuLate ", software provides the Cp values of reference gene ACTB automatically.
The specific interpretation of 11.PCR results is as shown in table 5：
If sentence read result is feminine gender, the risk that gets a cancer of the stomach is relatively low；If sentence read result is the positive, the risk that gets a cancer of the stomach is higher, builds
Microscopy is discussed to confirm.
The interpretation of 5 PCR results of table
The detection of experimental example clinical sample
It collects altogether and detects 56 clinical samples using provided by the present invention for detecting RPRM genes and PRDM5 genes simultaneously
The kit to methylate is detected, and testing result is as shown in the table.Wherein Healthy People 9, non-atrophic gastritis 17,
Atrophic gastritis, intestinal metaplasia and dysplasia amount to 14, stomach cancer 16 (2 I phases, 3 III phases, 1 IV phase and
10 unknown samples by stages), clinical sample the results show stomach cancer detection sensitivity 94%, gastric precancerous lesion detection sensitivity
86%, overall sensitivity=A/ (A+C) * 100%=90%, specificity=D/ (B+D) * 100%=73%, positive predictive value=
A/ (A+B) * 100%=79%, negative predictive value=D/ (C+D) * 100%=86%.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe is described in detail the present invention with reference to foregoing embodiments, it will be understood by those of ordinary skill in the art that：Its according to
Can so modify to the technical solution recorded in foregoing embodiments either to which part or all technical characteristic into
Row equivalent substitution；And these modifications or replacement, the essence of appropriate technical solution is not made to depart from various embodiments of the present invention technology
The scope of scheme.
SEQUENCE LISTING
<110>Jiangsu is true biological medicine technology limited company
<120>For detecting the primer sets of RPRM genes and PRDM5 gene methylations, reagent, kit and application
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
gcgagtgagc gtttagttc 19
<210> 2
<211> 16
<212> DNA
<213>Artificial sequence
<400> 2
acccgccacg tccgtc 16
<210> 3
<211> 20
<212> DNA
<213>Artificial sequence
<400> 3
agttttgttt cgggtttcgc 20
<210> 4
<211> 20
<212> DNA
<213>Artificial sequence
<400> 4
cattcctact acgaaaacgc 20
<210> 5
<211> 20
<212> DNA
<213>Artificial sequence
<400> 5
cgtcgttttt ttatttttcg 20
<210> 6
<211> 20
<212> DNA
<213>Artificial sequence
<400> 6
agcgtttagg ttcgcgtttt 20
<210> 7
<211> 20
<212> DNA
<213>Artificial sequence
<400> 7
agtaagtttt ttggattgtg 20
<210> 8
<211> 20
<212> DNA
<213>Artificial sequence
<400> 8
tggggtggtg atggaggagg 20
<210> 9
<211> 20
<212> DNA
<213>Artificial sequence
<400> 9
ccaacacaca ataacaaaca 20
Claims (10)
- A kind of 1. primer sets for being used to detect RPRM genes and PRDM5 gene methylations simultaneously, which is characterized in that the primer sets Including being used to detect the primer pair of RPRM genes and primer pair for detecting PRDM5 genes；It is described to include the sense primer and such as of RPRM genes as shown in SEQ ID NO.1 for detecting the primer pair of RPRM genes The anti-sense primer of RPRM genes shown in SEQ ID NO.2；It is described for detect the primer pair of PRDM5 genes include the sense primers of PRDM5 genes as shown in SEQ ID NO.3 with The anti-sense primer of PRDM5 genes as shown in SEQ ID NO.4.
- 2. a kind of reagent for being used to detect RPRM genes and PRDM5 gene methylations simultaneously, which is characterized in that the reagent includes Primer sets described in claim 1.
- 3. a kind of kit for being used to detect RPRM genes and PRDM5 gene methylations simultaneously, which is characterized in that will including right Seek the reagent described in primer sets or the claim 2 described in 1.
- 4. the kit according to claim 3 for being used to detect RPRM genes and PRDM5 gene methylations simultaneously, feature It is, the kit includes PCR Mix and Taq enzyme Mix；Reagents and dNTP of the PCR Mix described in including primer sets described in claim 1 or claim 2；The Taq enzyme Mix includes Taq enzyme and probe.
- 5. the kit according to claim 3 for being used to detect RPRM genes and PRDM5 gene methylations simultaneously, feature It is, the kit further includes negative controls and positive reference substance；Preferably, the negative controls include TE buffer solutions and RPRM genes and the non-genome to methylate of PRDM5 genes DNA；The positive control includes the genomic DNA that TE buffer solutions methylate with RPRM genes and PRDM5 genes.
- 6. the kit according to claim 3 for being used to detect RPRM genes and PRDM5 gene methylations simultaneously, feature It is, the kit further includes DNA processing articles for use；Preferably, the DNA processing articles for use include the articles for use of extraction DNA and sulphite conversion purifying are carried out to the DNA Articles for use；Preferably, the articles for use of the extraction DNA include lysate, magnetic bead and extraction rinsing liquid, and sulphite is carried out to the DNA The articles for use of conversion purifying include sulfiding reagent, DNA protections liquid, with reference to liquid, purifying rinsing liquid and eluent.
- 7. such as application of the claim 3-6 any one of them kit in gastric cancer auxiliary diagnosis and/or early screening.
- 8. reagent or claim 3-6 any one of them described in application primer sets described in claim 1 or claim 2 The method that kit detects stomach cancer, which is characterized in that the described method includes：By the examination described in the plasma sample DNA after sulphite converts and primer sets described in claim 1 or claim 2 Agent carries out quantitative fluorescent PCR reaction, is judged whether according to the Cp values of the plasma sample DNA after sulphite converts with stomach Cancer；When the Cp values≤44.76 of the RPRM genes, then the risk height that gets a cancer of the stomach is judged；And/orWhen the Cp values≤38.85 of the PRDM5 genes, then the risk height that gets a cancer of the stomach is judged.
- 9. the method for detection stomach cancer according to claim 8, which is characterized in that extracted using magnetic bead absorption method through lysate The DNA in plasma sample after cracking, and the DNA is subjected to sulphite conversion, then purified using paramagnetic particle method, it obtains The plasma sample DNA after sulphite converts；It preferably, will after the mixed liquor of application fetches rinsing liquid and absolute ethyl alcohol washs the DNA in the plasma sample The DNA carries out sulphite conversion.
- 10. the method for detection stomach cancer according to claim 8, which is characterized in that will be mixed with the combination liquid of magnetic bead with it is described After plasma sample DNA mixings after sulphite converts are adsorbed, using purifying rinsing liquid and the mixed liquor of absolute ethyl alcohol Magnetic bead is washed, then using the plasma sample DNA described in elution after sulphite converts.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810127514.1A CN108103197B (en) | 2018-02-08 | 2018-02-08 | Primer group, reagent, kit and application for detecting RPRM gene and PRDM5 gene methylation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810127514.1A CN108103197B (en) | 2018-02-08 | 2018-02-08 | Primer group, reagent, kit and application for detecting RPRM gene and PRDM5 gene methylation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108103197A true CN108103197A (en) | 2018-06-01 |
| CN108103197B CN108103197B (en) | 2022-03-04 |
Family
ID=62222013
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810127514.1A Active CN108103197B (en) | 2018-02-08 | 2018-02-08 | Primer group, reagent, kit and application for detecting RPRM gene and PRDM5 gene methylation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108103197B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101443462A (en) * | 2006-05-15 | 2009-05-27 | 北海道公立大学法人札幌医科大学 | Method for detecting disease-related marker using gastric mucosal lavage fluid |
| WO2011035453A2 (en) * | 2009-09-28 | 2011-03-31 | Pontificia Unversidad Católica De Chile | Non-invasive method for the early detection of stomach cancer |
| CN105274100A (en) * | 2015-10-27 | 2016-01-27 | 苏州工业园区为真生物医药科技有限公司 | Human TWIST1/Vimentin gene methylation detection marker and kit |
| CN105567850A (en) * | 2016-02-26 | 2016-05-11 | 福建师范大学 | Reagent kit and method for quantitatively detecting RPRM gene DNA methylation |
-
2018
- 2018-02-08 CN CN201810127514.1A patent/CN108103197B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101443462A (en) * | 2006-05-15 | 2009-05-27 | 北海道公立大学法人札幌医科大学 | Method for detecting disease-related marker using gastric mucosal lavage fluid |
| WO2011035453A2 (en) * | 2009-09-28 | 2011-03-31 | Pontificia Unversidad Católica De Chile | Non-invasive method for the early detection of stomach cancer |
| CN105274100A (en) * | 2015-10-27 | 2016-01-27 | 苏州工业园区为真生物医药科技有限公司 | Human TWIST1/Vimentin gene methylation detection marker and kit |
| CN105567850A (en) * | 2016-02-26 | 2016-05-11 | 福建师范大学 | Reagent kit and method for quantitatively detecting RPRM gene DNA methylation |
Non-Patent Citations (4)
| Title |
|---|
| DANIELLE QUEIROZ CALCAGNO等: "DNA and histone methylation in gastric carcinogenesis", 《WORLD J GASTROENTEROL》 * |
| KURT BUCHEGGER等: "Reprimo as a modulator of cell migration and invasion in the MDA-MB-231 breast cancer cell line", 《BIOL RES》 * |
| XING-SHENG SHU等: "The Epigenetic Modifier PRDM5 Functions as a Tumor Suppressor through Modulating WNT/b-Catenin Signaling and Is Frequently Silenced in Multiple Tumors", 《PLOS ONE》 * |
| 刘晶晶等: "PRDM5 基因甲基化及在HTB-182、A549 中的表达", 《现代生物医学进展》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108103197B (en) | 2022-03-04 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Wang et al. | Automated quantitative RNA in situ hybridization for resolution of equivocal and heterogeneous ERBB2 (HER2) status in invasive breast carcinoma | |
| CN105671181B (en) | Gene marker, primer, probe and kit for detecting lung cancer | |
| CN105256014B (en) | Breast cancer combined diagnosis marker and detection kit | |
| Davis et al. | A novel urine-based assay for bladder cancer diagnosis: multi-institutional validation study | |
| Du et al. | Establishment and development of the personalized criteria for microscopic review following multiple automated routine urinalysis systems | |
| CN107955836A (en) | For the primer pair of lung cancer related gene SHOX2 DNA methylation assays, kit and method | |
| Wu et al. | The analysis of cell-free DNA concentrations and integrity in serum of initial and treated of lymphoma patients | |
| CN114460311B (en) | Reagent composition for B-ALL/LBL immunophenotyping and application thereof | |
| CN109355406B (en) | A kind of kit of the detection mycobacterium tuberculosis based on blood free nucleic acid | |
| CN106148501A (en) | A kind of kit detecting Septin9 gene methylation and application thereof | |
| JP7187081B2 (en) | Methods for early diagnosis and post-treatment monitoring of breast cancer using liquid biopsy multiplex oncogene biomarkers | |
| CN106244675B (en) | Kit for adult AML risk stratification and clinical prognosis evaluation and application of CPNE3 | |
| CN108660209A (en) | Colorectal cancer early detection method based on BMP3 gene methylation sequences | |
| CN108103197A (en) | For detecting the primer sets of RPRM genes and PRDM5 gene methylations, reagent, kit and application | |
| CN102212621B (en) | Detection kit and detection method for hepatitis B virus genotyping | |
| CN114517233B (en) | Primer probe combination for early warning and clinical diagnosis of colorectal cancer | |
| CN115851958A (en) | Primer, probe, kit and method for detecting pancreatic cancer related gene methylation | |
| Mogensen et al. | Measurement of the ovarian cancer-associated antigen CA 125 prior to second look operation | |
| CN108728543B (en) | MiRNA combination for detecting lung cancer brain metastasis and kit containing same | |
| CN112501306A (en) | Kit for CpG island methylation phenotype detection and application thereof | |
| Ingle et al. | Fine Needle Aspiration Cytology [FNAC] | |
| CN107447029B (en) | Primer, probe and method for screening leukemia MLL-SEPT6 fusion gene by real-time fluorescence PCR technology | |
| Ta et al. | Molecular subtyping of diffuse large B-cell lymphoma using a novel quantitative RT-PCR assay | |
| WO2024045170A1 (en) | Combination of differentially methylated regions, kit, and use thereof | |
| JP2024520100A (en) | Liver cancer diagnostic device |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |