CN106521006A - Reagent system and kit for NDRG4 gene methylation detection and application thereof - Google Patents
Reagent system and kit for NDRG4 gene methylation detection and application thereof Download PDFInfo
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Abstract
The invention relates to a reagent system and kit for NDRG4 gene methylation detection and application thereof. The reagent system or the kit comprises specific primers SEQ ID NO.1 and SEQ NO.2 and a specific probe SEQ ID NO.3, and a report group and a quenching group are connected to the probe. The reagent system further comprises a positive standard substance, a negative standard substance, a buffer solution, dNTPs and DNA polymerase. A use method of the kit comprises the steps that 1, methylation treatment is conducted on a sample DNA; 2, a fluorescent quantitative PCR reaction is conducted on the sample DNA obtained after methylation treatment in the step 1 by means of the specific primers SEQ ID NO.1 and SEQ NO.2 and the specific probe SEQ ID NO.3, a fluorescent quantitative PCR reaction is conducted on the positive standard substance and the negative standard substance simultaneously, and the methylation state of a gene is judged by analyzing fluorescence alteration of the PCR process. The preparation method of the kit is simple, the kit is convenient to use and high in speed, and the detection result is accurate, visual and high in sensitivity.
Description
Technical field
The present invention relates to genetic engineering field, and in particular to a kind of NDRG4 gene methylations detectable system and reagent
Box and its application.
Background technology
Colorectal cancer is one of mankind's major malignant tumor, and its M ＆ M is respectively positioned at the 3rd and the 4th
Position.There are 1,200,000 cases newly made a definite diagnosis every year, and have more than 600,000 patients and die from colorectal cancer.Colorectal cancer incidence rate exists
The right side of fifty age bracket is relatively low, but can increase with the increase at age.The Colorectal Cancer the median age of developed country is
70 years old.Colorectal cancer early symptom is not obvious, show with the increase of tumor bowl evacuation habit change, have blood in stool, suffering from diarrhoea, suffer from diarrhoea with
The symptoms such as constipation replaces, local stomachache, late period such as then show anemia, lose weight at the General Symptomies.Colorectal cancer can occur
Any position of colon or rectum, but it is the most common with rectum, sigmoid colon, and remaining sees caecum, ascending colon, descending colon successively
And transverse colon.Carcinoma great majority are adenocarcinoma, and minority is squamous cell carcinoma and mucinous carcinoma.Primary disease can pass through lymph, blood circulation
And the approach such as direct extension, send out its hetero-organization and internal organs.At present the diagnostic method of colorectal cancer mainly have fecal occult blood detection,
X-ray barium enema or fibercolonscopy, but said method accuracy and sensitivity are relatively low, especially fibro-colonoscope
Checking and very big pain is brought to patient, the compliance of patient is poor.Therefore a kind of new detection method is extremely urgent.
In tumor research, the generation of many tumors is all accompanied by methylating for gene, the demethylation of oncogene and suppression
The methylation state of oncogene, can cause activation, the inactivation of antioncogene of oncogene.The hypomethylation and suppression cancer base of oncogene
The hyper-methylation change of cause is a key character of tumor cell.
NDRG4 genes are antioncogene NDRG gene family members, and the gene family is in the various other normal structures of human body
Middle high expression, does not express or low expression in some tumor tissues, and low expression then may be related to promoter hyper-methylation.In recent years
Come, research shows that NDRG4 genes are present in colorectal cancer tumor tissues and body fluid, and stability is high, the high first of NDRG4 genes
Baseization can cause the low expression of NDRG4 genes, i.e. antioncogene inactivation, indicate the generation of colorectal cancer.Research shows, in feces
NDRG4 aberrant methylations can be used as the tumor markerses of colorectal cancer early diagnosiss.
Methylating for NDRG4 genes is determined through a large amount of clinical trials can cause the generation of colorectal cancer, typically pass through
The NDRG4 gene methylation states come off in cancerous cell in a large number in detection patient's intestinal, so as to diagnose the generation of colorectal cancer.It is existing
In having technology, there is lot of documents to elaborate the relation of NDRG4 genes and NDRG4 gene methylations and colorectal cancer, but be a lack of
The method that quick, easy, sensitive, specific detection is carried out to NDRG4 gene methylations, it is impossible to timely and effectively diagnose Colon and rectum
Cancer.
The content of the invention
Present invention solves the technical problem that being：Colorectal cancer early diagnosiss are difficult, the detection method sensitivity of prior art
Low, accuracy is low and inconvenient, not yet has the reagent system that can quickly, easily detect NDRG4 gene methylation states
And test kit.
It is an object of the invention to provide a kind of NDRG4 gene methylations detectable system and test kit, using the reagent
The generation of box combined with fluorescent quantitative PCR method quickly and easily qualitative detection colorectal cancer；Detected using the test kit
The sensitivity of NDRG4 gene methylation states is high, accuracy is high, high specificity, easy to operate, cycle is short.
Specifically, for the deficiencies in the prior art, the invention provides following technical scheme：
On the one hand, the invention provides a kind of NDRG4 gene methylations detection reagent system, it is characterised in that described
Reagent system includes specific primer and specific probe, the specific primer such as SEQ ID NO.1 and SEQ ID NO.2 institutes
Show, the specific probe is as shown in SEQ ID NO.3.
Preferably, in the reagent system, fluorescent reporter group and fluorescent quenching on the specific probe, are connected with
Group, wherein, the fluorescent reporter group is selected from 6- CF 5(6)-Carboxyfluorescein (FAM), chlordene -6- methylfluoresceins (HEX), four chloro-
One kind in 6- CF 5(6)-Carboxyfluorescein (TET), Fluorescein isothiocyanate (FITC) or VIC, preferred 6- CF 5(6)-Carboxyfluorescein；The fluorescence
Quenching group is selected from BHQ or tetramethylrhodamine (TAMRA).
Preferably, the reagent system also includes positive criteria product, contains and insert NDRG4 bases in the positive criteria product
Because of the recombiant plasmid of methylation-specific sequence SEQ ID NO.4.
Preferably, the reagent system also includes negative standards' product, contains and be not inserted into NDRG4 in negative standards' product
The plasmid of gene methylation specific sequence.
Preferably, the recombiant plasmid concentration in the positive criteria product be 1.0 × 103copies/ μ L, the negative standards
Plasmid concentration in product is 1.0 × 103copies/ μ L.
Preferably, the recombiant plasmid is identical with plasmid, in pETs, pUCs, pGM-T, pBR322 or pMD-T
Kind, preferred pGM-T or pBR322.
The preparation method of the plasmid in recombiant plasmid and negative standards' product preferably, in the positive criteria product is such as
Under：
(1) with sequence SEQ ID NO.4 as template, expanded using primer sequence SEQ ID NO.5 and SEQ ID NO.6
Increase, obtain genes of interest；
(2) genes of interest is connected on plasmid vector, obtains connection product；
(3) connection product is transformed in host cell；
(4) recombiant plasmid containing genes of interest is screened as positive criteria product；Plasmid without genes of interest is used as the moon
Property standard substance.
Preferably, the reagent system also includes buffer and dNTPs, and the composition of the buffer includes trihydroxy methyl ammonia
Methylmethane, potassium chloride and magnesium chloride.
Preferably, the concentration of the trishydroxymethylaminomethane is 50～200mM, preferably 100～150mM；Potassium chloride
Concentration be 300～800mM, preferably 400～600mM；The concentration of magnesium chloride is 10～30mM, preferably 10～20mM.
Preferably, the reagent system also includes archaeal dna polymerase, preferably Taq DNA polymerase.
On the other hand, the invention provides a kind of NDRG4 gene methylations detection test kit, it is characterised in that described
Test kit includes aforesaid reagent system.
Another further aspect, the invention provides specific primer SEQ ID NO.1 and SEQ ID NO.2 and specific probe
SEQ ID NO.3 are preparing NDRG4 gene methylation detection reagents or the application in test kit, are preferably preparing NDRG4 bases
Because of methylate colorectal cancer detection reagent or the application in test kit.
Preferably, the using method of the NDRG4 gene methylations detection reagent or test kit is comprised the following steps：
(1) methylate process sample DNA；
(2) using specific primer SEQ ID NO.1 and SEQ ID NO.2 and specific probe SEQ ID NO.3 couple
Step (1) methylate process after sample DNA carry out quantitative fluorescent PCR reaction.
Preferably, aforementioned using method also includes：Will be containing inserting NDRG4 gene methylation specific sequence SEQ ID
The recombiant plasmid of NO.4 carries out quantitative fluorescent PCR reaction as positive criteria product；
The plasmid for being not inserted into NDRG4 gene methylation specific sequence SEQ ID NO.4 will be contained as negative standards
Product, carry out quantitative fluorescent PCR reaction.
Preferably, the step of the processing sample DNA that methylate includes：(1) genomic DNA is extracted from fecal sample；
(2) using bisulf iotate-treated genomic DNA, the sample DNA of the process that obtains methylating.
Preferably, in the application, the quantitative fluorescent PCR reaction condition is：94～95 DEG C of 2～5min of denaturation, 1
Individual circulation；94～95 DEG C of 15～20s, 53～55 DEG C of 30～50s, 58～60 DEG C of 30～50s, totally 40～50 circulations.
Compared with prior art, effect of the invention and it has an advantage that：
1) kit of the present invention accurately, has the high sensitivity of PCR, the specificity of DNA hybridization and spectral technique essence concurrently
It is determined that the advantage of amount, visual result, the change during energy direct detection PCR, compared with regular-PCR, its result can be seen in real time
Examine, product does not need detected through gel electrophoresis, complete stopped pipe operation to significantly reduce pollution.
2) test kit detection sensitivity of the present invention and specificity are high, as the dual fail-safe for taking specific primer and probe sets
Meter, sensitivity and specificity improve a lot, and can detect colorectal cancer before clinical symptoms occur.
3) test kit detection speed of the present invention is fast, only needs altogether 2 hours, and step is simple, can carry out high flux sample simultaneously
This detection.
Description of the drawings
Fig. 1 is the fluorescence curve of positive criteria product and negative standards' product in fluorescent PCR amplification procedure；
Fig. 2 is fluorescence curve of the sample in fluorescent PCR amplification procedure.
Wherein, the curve 1 in Fig. 1 is fluorescence curve of negative standards' product in PCR amplification procedures, and curve 2 is positive criteria
Fluorescence curve of the product in PCR amplification procedures；
Fluorescence curve of the curve 1 in Fig. 2 for sample 1, fluorescence curve of the curve 2 for sample 2, curve 3 are glimmering for sample 3
Light curve, fluorescence curve of the curve 4 for sample 4, fluorescence curve of the curve 5 for sample 5；Wherein, the supplier of sample 1 is not knot
Rectal cancer patient, the supplier of sample 2-5 suffer from colorectal cancer.
Specific embodiment
Fluorescence PCR assay took the lead in succeeding in developing by PE companies of the U.S. in nineteen ninety-five, and it is miscellaneous that it has the high sensitivity of PCR, DNA concurrently
The advantage of the specificity and spectral technique accurate quantification of friendship, visual result, the change during energy direct detection PCR.With it is common
PCR is compared, its result can Real Time Observation, product do not need detected through gel electrophoresis, and complete stopped pipe operation significantly reduces dirt
Dye.
Fluorescence PCR assay is applied to detect the methylation state of NDRG4 genes by the present invention, so as to qualitative detection Colon and rectum
The generation of cancer.Specifically, the invention provides the reagent system and test kit of a kind of detection of NDRG4 gene methylations, institute
Contain specific primer SEQ ID NO.1 and SEQ ID NO.2, and specific probe SEQ ID NO.3 in stating reagent system；
The 5 ' of specific probe and 3 ' ends contain fluorescent reporter group and fluorescent quenching group respectively, and the fluorescent reporter group is selected from
FAM, HEX, TET, the one kind in FITC or VIC, the fluorescent quenching group is selected from BHQ or TAMRA.When containing on probe simultaneously
When having fluorescent reporter group and fluorescent quenching group, fluorescence is not sent；When specific probe is degraded by archaeal dna polymerase, fluorescence
Reporter group and fluorescent quenching group separate, and send fluorescence.Reagent system also includes positive criteria product, negative standards' product, also contains
The solvent for having polymerase chain reaction to need, such as buffer and dNTPs.
In a preferred embodiment, the buffer in reagent system consists of 50～200mM trishydroxymethylaminomethane,
300～800mM potassium chloride and 10～30mM magnesium chlorides.Archaeal dna polymerase in reagent system is Taq DNA polymerase.The present invention is carried
A kind of recombiant plasmid containing NDRG4 gene methylation sequences, the NDRG4 gene methylations sequence such as SEQ ID are supplied
Shown in NO.4, the one kind of the plasmid in pETs, pUCs, pGM-T, pBR322 and pMD-T.In recombiant plasmid screening process
In, the recombiant plasmid successfully containing NDRG4 gene methylation sequences is converted as positive criteria product, i.e., as positive control；
The empty plasmid of work(is not converted into as negative standards' product, i.e., as negative control.
In another preferred embodiment, the invention provides a kind of NDRG4 gene methylations detection test kit, described
Test kit includes positive criteria product, negative standards' product, reactant liquor and archaeal dna polymerase.The reactant liquor contains specific primer
SEQ ID NO.1 and SEQ ID NO.2, and specific probe SEQ ID NO.3, reactant liquor are consisted of：Every 18 μ L reactions
The SEQ ID NO.1 that contain in liquid, SEQ ID NO.2, the amount of SEQ ID NO.3 and dNTP are followed successively by 200-300nM, 200-
300nM, 200-300nM and 100-200mM.The screening side of positive criteria product (recombiant plasmid) and negative standards' product (empty plasmid)
Method is comprised the following steps：
(1) with sequence SEQ ID NO.4 as template, expanded using primer sequence SEQ ID NO.5 and SEQ ID NO.6
Increase, obtain genes of interest；
(2) genes of interest is connected on plasmid vector, obtains connection product；
(3) connection product is transformed in host cell；
(4) recombiant plasmid containing genes of interest is screened as positive criteria product；Plasmid without genes of interest is used as the moon
Property standard substance.
The situation of NDRG4 gene methylations is detected using test kit of the present invention, detection method is comprised the following steps：Sample
Product pretreatment, the sample after process is added in sample cell.The step of preparing solution to be measured includes, takes new reaction tube and number, so
After be separately added into positive criteria product, negative standards' product and sample, then be separately added into reactant liquor and archaeal dna polymerase thereto, mix；
Sample size or positive criteria product content or negative standards' product content in each group solution to be measured is identical, and reactant liquor and DNA are poly-
The content of synthase is also identical；Generally, the positives standard substance of solution to be measured, negative standards' product or sample are polymerized with reactant liquor, DNA
The ratio of enzyme is：(1-2)μL：(18-20)μL：(5-20)U.Solution to be measured is put in quantitative real time PCR Instrument and is measured, PCR
The condition of measure is：94～95 DEG C of 2～5min of denaturation, 1 circulation；94～95 DEG C of 15～20s, 53～55 DEG C of 30～50s, 58
～60 DEG C of 30～50s, totally 40～50 circulations；Whether ultimate analysis quantitative fluorescent PCR measurement result, judgement sample supplier are suffered from
There is colorectal cancer.
Sample treatment before detection includes, is extracted first with genome DNA extracting reagent kit from fecal sample
DNA, recycles genomic DNA methylation level treatment kits to process and extracts the DNA for obtaining.The principle of the treatment kits that methylate is
Bisulfite method methylates process, and the DNA Jing after the treatment kits that methylate are processed is used as sample.
Sample genomic dna Jing methylate treatment kits process after, if in the NDRG4 gene orders in sample genome
Cytosine (C) it is methylated, then Jing methylate treatment kits process after NDRG4 gene orders it is constant, such as SEQ
Shown in ID NO.4, specific primer SEQ ID NO.1 and SEQ ID NO.2 and specific probe SEQ ID NO.3 can be with
SEQ ID NO.4 are combined, in quantitative fluorescent PCR course of reaction, in the presence of archaeal dna polymerase on probe SEQ ID NO.3
5 ' fluorescent reporter groups are separated with 3 ' quenching groups, send fluorescence；If the born of the same parents in the NDRG4 gene orders in sample genome are phonetic
Pyridine (C) is no methylated, then, Jing after the treatment kits that methylate are processed, the C in NDRG4 gene orders becomes uracil (U),
The NDRG4 gene orders methylated after processing are different from SEQ ID NO.4, specific primer SEQ ID NO.1 and SEQ ID
NO.2 and specific probe SEQ ID NO.3 cannot be combined with the sample DNA after process, in quantitative fluorescent PCR course of reaction,
Probe SEQ ID NO.3 keep complete, due to being connected with 5 ' fluorescent reporter groups and 3 ' quenching groups, Fluorescence PCR simultaneously
During do not send fluorescence；I.e. Jing after the treatment kits that methylate, methylated NDRG4 genes are in fluorescence quantitative PCR detection mistake
Cheng Zhonghui sends fluorescence, does not fluoresce without methylated NDRG4 genes.Therefore, reagent system of the present invention and examination
After agent box is combined with fluorescence quantitative PCR detection, can be used for detecting the methylation state of NDRG4 genes.NDRG4 genes height
It is the important molecule mark of colorectal cancer to methylate, therefore reagent system of the present invention and test kit and its use are in Colon and rectum
It is significant in cancer diagnosis.
The method that NDRG4 gene methylation states are detected using test kit of the present invention is simple, and sensitivity is high, specificity
By force；By detecting whether NDRG4 gene methylations can judgment sample donor be effectively colorectal cancer patients.
Below by specific embodiment, the present invention is described further, the factory of agents useful for same and instrument in embodiment
Family is as follows：
Trishydroxymethylaminomethane, producer：Lark prestige Science and Technology Ltd.；
Potassium chloride, producer：Lark prestige Science and Technology Ltd.；
Magnesium chloride, producer：Lark prestige Science and Technology Ltd.；
DNTPs, producer：TAKARA, Japan；
Taq archaeal dna polymerases, producer：TAKARA, Japan；
SAP enzyme EF0511, producer：Thermo Fisher Scientific Inc.；
ExoI enzymes, producer：TAKARA, Japan；
Quantitative real time PCR Instrument, producer：American AB I real-time fluorescence quantitative PCR instrument 7500, the U.S.；
Genome DNA extracting reagent kit, purchased from QIAGEN companies；
Genomic DNA methylation level treatment kits, purchased from QIAGEN companies；
PGM-T plasmid vectors, are purchased from TIANGEN Biotech (Beijing) Co., Ltd.；
The specific primer used in the present invention, and specific probe sequence is by the limited public affairs of Shanghai English fine horse biotechnology
Department's synthesis, wherein, the fluorescent reporter group connected on specific probe and fluorescent quenching group are by the handsome biotechnology in Shanghai
Company limited synthesizes.
The preparation of 1 test kit of embodiment
Test kit manufactured in the present embodiment includes the positive criteria product containing NDRG4 gene methylation sequences, containing blank matter
Negative standards' product of grain, reactant liquor and archaeal dna polymerase.Wherein, the test tube volume for filling archaeal dna polymerase is 500 μ L, and pipe is contained within dense
Spend the 22 μ L of Taq DNA polymerase for 5U/ μ L.
(1) preparation of positive criteria product and negative standards' product
Compared by the DNA sequence to a large amount of clinic colorectal cancer patients and normal population, design is obtained
One sequence relevant with colorectal cancer disease, NDRG4 gene methylation sequence SEQ ID NO.4, SEQ ID NO.4 sequences are such as
Under：
With the sequence of SEQ ID NO.4 as purpose gene, primer sequence SEQ ID NO.5 and SEQ ID NO.6 are designed, is entered
The conventional PCR amplifications of row, PCR reaction systems are shown in Table 1.
Primer sequence SEQ ID NO.5：5’-TAGTATAGTTCGCGCGGC-3’
Primer sequence SEQ ID NO.6：5’-CGAACGAACCGCGATC-3’
PCR response parameters are：
1 PCR reaction systems of table
| Composition | Volume (μ L) |
| Genes of interest | 1(30μg/mL) |
| 10×Buffer | 2.5 |
| dNTP | 0.2 (20mM, each) |
| Taq archaeal dna polymerases | 0.25(5U/μL) |
| Primer sequence SEQ ID NO.5 | 0.25(25μM) |
| Primer sequence SEQ ID NO.6 | 0.25(25μM) |
| ddH2O | 15.55 |
| Cumulative volume | 20 |
Tris-HcL buffer of the buffer in table 1 for pH8.0.
After amplification terminates, purified PCR primer is connected on pGM-T plasmid vectors, obtains connecting purposeful base
The plasmid of cause；The purification system of the PCR primer is as shown in table 2：
2 PCR primer purification system of table
| Component | Volume (μ L) |
| PCR primer | 20 |
| SAP enzymes | 0.85(1U/μL) |
| ExoI enzymes | 0.375(10U/μL) |
| ddH2O | 3.775 |
| Cumulative volume | 25 |
PCR primer purification system is positioned in PCR amplification instrument and is reacted, reaction condition be 37 DEG C, 20min, 73 DEG C,
20min。
Then recombiant plasmid is transformed in escherichia coli TOP10 competent cells by heat shock method, the restructuring that will be built
Plasmid carries out the recombiant plasmid of two-way DNA sequencing identification, genes of interest and plasmid successful connection as positive criteria product, using purple
Outer spectrophotometer is quantitative, and positive criteria product is diluted to 103Load after the concentration of per microlitre of copy in positive criteria QC,
It is stored in -20 DEG C.Two-way DNA sequencing shows that the pGM-T plasmids do not recombinated, as negative standards' product, are diluted to 103Copy
Load in negative standards' QC after the concentration of per microlitre of shellfish.The capacity of positive criteria QC and negative standards' QC is 200 μ L,
Wherein, the volume of positive criteria product and negative standards' product is 50 μ L.
(2) preparation of reactant liquor
Specific primer SEQ ID NO.1 and SEQ are designed according to the hyper-methylation sequence SEQ ID NO.4 of NDRG4 genes
ID NO.2, and specific probe SEQ ID NO.3.Contain described specific primer and probe in reactant liquor；
Wherein, forward primer SEQ ID NO.1 are 5 '-CGGTTTTCGTTCGTTTTT TCG-3 '；
Reverse primer SEQ ID NO.2 are 5 '-GTAACTTCCGCCTTCTACGC-3 '；
Probe SEQ ID NO.3 are 5 '-TCGTTTATCGGGTATTTTAGTCGCG-3 '；
Contain fluorescent reporter group FAM and fluorescent quenching group BHQ on specific probe, FAM and BHQ is connected to spy
5 ' and 3 ' ends of specific probes sequence.
Table 3 lists the formula of reactant liquor, prepares reactant liquor according to the formula.
The formula of reactant liquor in 3 the present embodiment of table
| Composition | Volume (μ L) |
| 10x Buffer | 2 |
| SEQ ID NO.1 | 0.4(200nM) |
| SEQ ID NO.2 | 0.4(200nM) |
| SEQ ID NO.3 | 0.2(200nM) |
| dNTP | 0.8(100mM) |
| H2O | 14.2 |
| Cumulative volume | 18 |
Wherein, buffer (buffer) solution is consisted of：Tris-HCl 100mM、KCl 400mM、
MgCl2The pH value of 10mM, wherein Tris-HCl is 8.5.
Proportioning according to table 3 prepares reactant liquor, and the reactant liquor cumulative volume in test kit described in the present embodiment is 380 μ L.
Thus, the positive criteria product for preparing equipped with step () and negative standards' product, reactant liquor prepared by step (two), with
And Taq DNA polymerase constitutes the test kit described in the present embodiment.
The preparation of 2 test kit of embodiment
Positive criteria product and negative standards' condition in the test kit that test kit manufactured in the present embodiment is prepared with embodiment 1
Together, the present embodiment with the difference of embodiment 1 is：The formula of the present embodiment reactant liquor is as shown in table 4, wherein, primer SEQ ID
NO.1 and SEQ ID NO.2, and specific probe SEQ ID NO.3 are same as Example 1.Archaeal dna polymerase in the present embodiment
Enzyme in pipe is Taq DNA polymerase, and its concentration is 4U/ μ L, and volume is 30 μ L.
The formula of reactant liquor in 4 the present embodiment of table
| Composition | Volume (μ L) |
| 10x Buffer | 2 |
| SEQ ID NO.1 | 0.5(300nM) |
| SEQ ID NO.2 | 0.5(300nM) |
| SEQ ID NO.3 | 0.3(300nM) |
| dNTP | 1.0(200mM) |
| H2O | 13.7 |
| Cumulative volume | 18 |
Wherein, buffer (buffer) solution is consisted of：Tris-HCl 150mM、KCl 600mM、
MgCl2The pH value of 20mM, wherein Tris-HCl is 8.3.
Proportioning according to table 4 prepares reactant liquor, and the reactant liquor cumulative volume in test kit described in the present embodiment is 350 μ L.
The preparation of 3 test kit of embodiment
Positive criteria product and negative standards' condition in the test kit that test kit manufactured in the present embodiment is prepared with embodiment 1
Together, the present embodiment with the difference of embodiment 1 is：The formula of the present embodiment reactant liquor is as shown in table 5, wherein, primer SEQ ID
NO.1 and SEQ ID NO.2, and specific probe SEQ ID NO.3 are same as Example 1, but the fluorescence report base on probe
Group is HEX, and fluorescent quenching group is BHQ；Enzyme in the present embodiment in archaeal dna polymerase pipe is Taq DNA polymerase, and its concentration is
5U/ μ L, volume are 30 μ L.
The formula of reactant liquor in 5 the present embodiment of table
| Composition | Volume (μ L) |
| 10x Buffer | 3 |
| SEQ ID NO.1 | 0.4(250nM) |
| SEQ ID NO.2 | 0.4(250nM) |
| SEQ ID NO.3 | 0.3(250nM) |
| dNTP | 1.0(200mM) |
| H2O | 12.9 |
| Cumulative volume | 18 |
Wherein, buffer (buffer) solution is consisted of：Tris-HCl 50mM、KCl 300mM、
MgCl2The pH value of 10mM, wherein Tris-HCl is 8.2.
Proportioning according to table 5 prepares reactant liquor, and the reactant liquor cumulative volume in test kit described in the present embodiment is 400 μ L.
The preparation of 4 test kit of embodiment
Positive criteria product and negative standards' condition in the test kit that test kit manufactured in the present embodiment is prepared with embodiment 1
Together, the present embodiment with the difference of embodiment 1 is：The formula of the present embodiment reactant liquor is as shown in table 6, wherein, primer SEQ ID
NO.1 and SEQ ID NO.2, and specific probe SEQ ID NO.3 are same as Example 1；Archaeal dna polymerase in the present embodiment
For Taq DNA polymerase, its concentration is 5U/ μ L, and volume is 30 μ L.
The formula of reactant liquor in 6 the present embodiment of table
| Composition | Volume (μ L) |
| 10x Buffer | 2 |
| SEQ ID NO.1 | 0.4(250nM) |
| SEQ ID NO.2 | 0.4(250nM) |
| SEQ ID NO.3 | 0.2(250nM) |
| dNTP | 0.9(150mM) |
| H2O | 14.1 |
| Cumulative volume | 18 |
Wherein, buffer (buffer) solution is consisted of：Tris-HCl 200mM、KCl 800mM、
MgCl2The pH value of 30mM, wherein Tris-HCl is 8.5.
Proportioning according to table 6 prepares reactant liquor, and the reactant liquor cumulative volume in test kit described in the present embodiment is 380 μ L.
Embodiment 5
The present embodiment detects the NDRG4 gene methylation states of sample using test kit prepared by embodiment 1.
Positive criteria product, negative standards' product, reactant liquor and Taq DNA polymerase are contained in test kit, using test kit
The methylated steps of detection NDRG4 are as follows：
(1) sample preprocessing
The fecal sample of clinical colorectal cancer patients or potential patient is obtained from Tianjin hospital general, according to genomic DNA
Operating instruction in extracts kit extracts genomic DNA, then processes said extracted using DNA methylation treatment kits and arrives
Genomic DNA, the DNA after process is used as sample.
(2) prepare solution to be measured
New reaction tube is taken, positive criteria QC, negative standards' QC and sample cell is respectively labeled as, then toward each pipe
Positive criteria product, negative standards' product and sample are separately added into, then reactant liquor and archaeal dna polymerase are separately added into toward each pipe, respectively
Solution composition to be measured in individual pipe is shown in Table 7.
It should be noted that, multiple samples can be detected simultaneously, i.e., prepare multiple sample cells simultaneously, be equipped with each sample cell
The fecal sample from different donors for processing in the same manner, the present embodiment have detected 5 samples simultaneously, and successively
It is labeled as sample 1-5.
The composition of 7 solution to be measured of table
(3) quantitative fluorescent PCR
Fluorescent reporter group FAM on probe SEQ ID NO.3 sends green fluorescence, when the sequence in probe cannot with treat
When the DNA profiling surveyed in solution is matched, probe keeps complete, i.e., contain fluorescent reporter group FAM and fluorescent quenching group simultaneously
Fluorescence cannot be detected after BHQ, PCR amplification；And when probe can occur base pair complementarity with the DNA in solution to be measured,
Probe is degraded by Taq DNA polymerase, and the reporter group of probe 5 ' is separated with 3 ' quenching group, can be examined in PCR amplification procedures
Measure fluorescence.Probe sequence SEQ ID NO.3 be with the DNA sequence after NDRG4 gene hyper-methylations as stencil design, if
PCR amplification procedures can detect the sequence after fluorescence then has NDRG4 gene hyper-methylations in explanation system, colorectal cancer detection
As a result it is possible for the positive；If fluorescence cannot be detected in PCR amplification procedures, Colon and rectum testing result may be feminine gender；Tool
The colorectal cancer testing result of body judges to also need to the quantitative analyses after expanding with reference to PCR.
The response parameter of quantitative fluorescent PCR is set to：95 DEG C 5 minutes, 1 circulation；95 DEG C 15 seconds, 53 DEG C 40 seconds, 58 DEG C
40 seconds, totally 40 circulations.
(4) interpretation of result
The present embodiment have detected 5 samples and 1 positive criteria product and 1 negative standards' product.Using ABI 7500
Software software records PCR amplification procedures, its result is as depicted in figs. 1 and 2.If fluorescence intensity growth curve is not S-type
Curve or Ct values>=35, then judgement sample is the total content of negative or sample less than detectable limit.If growth curve is S-type
Curve and Ct values<35, then the diagnostic result of sample is positive.
What Fig. 1 was given is the fluorescence curve figure of positive criteria product and negative standards' product in PCR amplification procedures, wherein, it is glimmering
The S-type growth of light intensity growth curve, and curve of the Ct values less than 35 is the testing result of positive criteria product, negative standards' product
Concentration is identical with positive criteria product, in detection range, so that with the increase of period, the very little song of fluorescence intensity change
Testing result of the line for negative standards' product.
What Fig. 2 was given is fluorescence curve figure of 5 samples in PCR amplification procedures, and the concentration of all samples is in detection
In the range of.The fluorescence intensity of curve 1 is almost unchanged, may infer that the testing result of the sample is feminine gender, the sample (sample 1)
Supplier is not colorectal cancer patients；The fluorescence intensity of curve 2, curve 3, curve 4 and curve 5 increases S-type, and Ct values are little
In 35, therefore the testing result of sample 2-5 is the positive, and the supplier of sample 2-5 is PATIENTS WITH LARGE BOWEL.The testing result and sample
Actual source is identical, it was demonstrated that the reliability of test kit of the present invention and detection method.
Present pre-ferred embodiments are the foregoing is only, the limitation present invention is not used to, it is all in the spiritual and former of the present invention
Modification, equivalent and improvement for being made within then etc., are required to be included within the protection domain of invention.
SEQUENCE LISTING
<110>Liu Pengfei
<120>NDRG4 gene methylation detection reagent systems and test kit and its application
<130> OICN160092
<160> 6
<170> PatentIn version 3.5
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<213>Artificial sequence
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cggttttcgt tcgttttttc g 21
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<213>Artificial sequence
<400> 2
gtaacttccg ccttctacgc 20
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<212> DNA
<213>Artificial sequence
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tcgtttatcg ggtattttag tcgcg 25
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<213>Artificial sequence
<400> 4
tagtatagtt cgcgcggcgg agcgggtgag aagtcggcgg gggcgcggat cgatcggggt 60
gttttttagg tttcgcgtcg cggttttcgt tcgttttttc gttcgtttat cgggtatttt 120
agtcgcgtag aaggcggaag ttacgcgcga gggatcgcgg ttcgttcg 168
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Claims (16)
1. a kind of NDRG4 gene methylations detection reagent system, it is characterised in that the reagent system includes specific primer
And specific probe, the specific primer as shown in SEQ ID NO.1 and SEQ ID NO.2, the specific probe such as SEQ
Shown in ID NO.3.
2. reagent system according to claim 1, it is characterised in that fluorescence report base is connected with the specific probe
Group and fluorescent quenching group, wherein, the fluorescent reporter group selected from 6- CF 5(6)-Carboxyfluorescein, chlordene -6- methylfluoresceins, four
One kind in chloro- 6- CF 5(6)-Carboxyfluorescein, Fluorescein isothiocyanate or VIC, preferred 6- CF 5(6)-Carboxyfluorescein；The fluorescent quenching group
Selected from BHQ or tetramethylrhodamine.
3. reagent system according to claim 1 and 2, it is characterised in that the reagent system also includes positive criteria product,
Containing the recombiant plasmid for inserting NDRG4 gene methylation specific sequence SEQ ID NO.4 in the positive criteria product.
4. the reagent system according to any one of claim 1-3, it is characterised in that the reagent system also includes negative mark
Quasi- product, containing the plasmid for being not inserted into NDRG4 gene methylation specific sequences in negative standards' product.
5. the reagent system according to claim 3 or 4, it is characterised in that the recombiant plasmid in the positive criteria product is dense
Spend for 1.0 × 103Copies/ μ L, the plasmid concentration in negative standards' product are 1.0 × 103copies/μL。
6. the reagent system according to any one of claim 3-5, it is characterised in that the recombiant plasmid is identical with plasmid,
One kind in pETs, pUCs, pGM-T, pBR322 or pMD-T, preferred pGM-T or pBR322.
7. the reagent system according to any one of claim 3-6, it is characterised in that the restructuring matter in the positive criteria product
The preparation method of the plasmid in grain and negative standards' product is as follows：
(1) with sequence SEQ ID NO.4 as template, expanded using primer sequence SEQ ID NO.5 and SEQ ID NO.6,
Obtain genes of interest；
(2) genes of interest is connected on plasmid vector, obtains connection product；
(3) connection product is transformed in host cell；
(4) recombiant plasmid containing genes of interest is screened as positive criteria product；Plasmid without genes of interest is used as negative mark
Quasi- product.
8. the reagent system according to any one of claim 1-7, it is characterised in that the reagent system also includes buffer
And dNTPs, the composition of the buffer includes trishydroxymethylaminomethane, potassium chloride and magnesium chloride.
9. reagent system according to claim 8, it is characterised in that the concentration of the trishydroxymethylaminomethane is 50～
200mM, preferably 100～150mM；The concentration of potassium chloride is 300～800mM, preferably 400～600mM；The concentration of magnesium chloride
For 10～30mM, preferably 10～20mM.
10. the reagent system according to any one of claim 1-9, it is characterised in that the reagent system is also poly- including DNA
Synthase, preferably Taq DNA polymerase.
11. a kind of NDRG4 gene methylations detection test kits, it is characterised in that comprising described in any one of claim 1-10
Reagent system.
12. specific primer SEQ ID NO.1 and SEQ ID NO.2 and specific probe SEQ ID NO.3 are preparing NDRG4
Application in gene methylation detection reagent or test kit, is preferably preparing NDRG4 gene methylations colorectal cancer detection use
Application in reagent or test kit.
13. applications according to claim 12, it is characterised in that the NDRG4 gene methylations detection reagent or examination
The using method of agent box is comprised the following steps：
(1) methylate process sample DNA；
(2) using specific primer SEQ ID NO.1 and SEQ ID NO.2 and specific probe SEQ ID NO.3 to step
(1) sample DNA after processing that methylates carries out quantitative fluorescent PCR reaction.
14. applications according to claim 13, it is characterised in that the using method also includes：
The recombiant plasmid for inserting NDRG4 gene methylation specific sequence SEQ ID NO.4 will be contained as positive criteria product,
Carry out quantitative fluorescent PCR reaction；
, enter containing the plasmid for being not inserted into NDRG4 gene methylation specific sequence SEQ ID NO.4 as negative standards' product
Row quantitative fluorescent PCR reacts.
15. applications according to claim 13 or 14, it is characterised in that the step of the processing sample DNA that methylate is wrapped
Include：
(1) genomic DNA is extracted from fecal sample；
(2) using bisulf iotate-treated genomic DNA, the sample DNA of the process that obtains methylating.
16. applications according to any one of claim 13-15, it is characterised in that the quantitative fluorescent PCR reaction condition
For：94～95 DEG C of 2～5min of denaturation, 1 circulation；94～95 DEG C of 15～20s, 53～55 DEG C of 30～50s, 58～60 DEG C 30～
50s, totally 40～50 circulations.
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Cited By (1)
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| CN114085904A (en) * | 2020-08-24 | 2022-02-25 | 广州达健生物科技有限公司 | Colorectal cancer gene methylation detection primer probe combination, kit and application thereof |
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