CN106755491A - Primer pair, kit and method based on the detection of cervical carcinoma specific methylation - Google Patents
Primer pair, kit and method based on the detection of cervical carcinoma specific methylation Download PDFInfo
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Abstract
The present invention relates to a kind of primer pair based on the detection of cervical carcinoma specific methylation.The primer pair includes PAX1 forward primers, reverse primer and detection probe, ACTB forward primers, reverse primer and detection probe.The present invention relates to a kind of kit based on the detection of cervical carcinoma specific methylation.The kit includes the PCR reaction solutions containing above-mentioned primer and probe, it includes PAX1 forward primers, PAX1 reverse primers, PAX1 detection probes, ACTB forward primers, ACTB reverse primers, ACTB detection probes, 10 × PCR buffer, dNTP, nuclease free water and Ex Taq enzymes.The invention further relates to a kind of method based on the detection of cervical carcinoma specific methylation.The kit and its detection method that the present invention is provided have the advantages that testing result is accurate, high, the specific good, sensitivity of detection flux is good, detection time is quick, easy to use and can effectively meet clinical requirement.
Description
Technical field
The present invention relates to vitro diagnostic techniques field, particularly, it is related to a kind of based on the detection of cervical carcinoma specific methylation
Primer pair, kit and method.
Background technology
Cervical carcinoma is one of most commonly seen gynecologic malignant tumor, is the principal disease for endangering women's health and life.Stream
Row disease learns investigation display, and there are 46.5 ten thousand cervical carcinoma new cases in the annual whole world, wherein China there are about 130,000 new cases every year.Mesh
The age of onset of preceding cervical carcinoma tends to rejuvenation, and the gold for bearing children and managing family is in the women encroached on by cervical carcinoma more
Period.
Cervical carcinoma is different from other tumours, and natural history of disease clearly, has a series of precancerous lesion, its occurrence and development
It is by quantitative change to qualitative change, is gradient to mutation, experiences several years processes to the more than ten years.Therefore, the preventing and treating of cervical carcinoma it is critical only that
Found in time by examination and treat cervical lesionses, terminate its development to cervical carcinoma.Thus, it is early to find, early diagnose and control early
Treat and be significant for improving treatment of human cervical cancer effect.
The cervical carcinoma routine screening technology for using at present includes：
Conventional smear：The sensitivity is low, and specificity is general, and detection method artificial disturbance factor is larger, as a result accurate
True property is limited and low cost；
ThinPrep liquid-based cytology test (Thin-Cytologic Test, TCT)：The sensitivity is low, and specificity is high,
Detection method needs manual observation to analyze and easily interference, not high to ASC diagnosis, it is impossible to check adenocarcinoma of the uterine cervix；
HPV-DNA is detected：The sensitivity is high, and specificity is low, and detection method flux is high, it is adaptable to which larger scale clinical is sieved
Look into, but false positive rate is high, can only early warning rather than early detection；
HPV-E6E7mRNA is detected：The sensitivity is high, and specificity is general, and detection method parting system is complicated, and RNA is not
Stabilization, detection is viral rather than genes of individuals, also can only early warning rather than early detection.
Above-mentioned these methods have that cumbersome, false positive rate is high, the low shortcoming of sensitivity, limit it clinical real
Test the accurate examination of room.
Recent cervical carcinoma DNA methylation research shows that the DNA methylation of PAX1 changes related to the disease progression of cervical carcinoma
Connection.PAX1 genes play potential cancer suppressing action in the mechanism of cervical carcinogenesis, and the hyper-methylation of PAX1 genes can promote
Enter the generation of cervical carcinoma.Therefore the methylation state of cervical carcinoma PAX1 gene promoters is detected, there is weight to the examination of cervical carcinoma
The meaning wanted.
At present, traditional methylation detecting method includes：Methylation status of PTEN promoter and bisulfite sequencing.But
These methods have the shortcomings that cumbersome, accuracy is low and sensitiveness is not high, limit its extensively should in clinical labororatory
With.
Therefore, it is necessary to provide a kind of methylation detecting method of new cervical carcinoma PAX1 genes to overcome drawbacks described above.
The content of the invention
There is the technology that cumbersome, accuracy is low and sensitiveness is not high to solve above-mentioned traditional methylation detecting method
Problem, a kind of simple to operate, accuracy of present invention offer is high and sensitiveness is high and special based on cervical carcinoma based on Fluorescence PCR assay
The primer pair of different in nature DNA methylation assay, kit and its method.
The invention provides a kind of primer pair based on the detection of cervical carcinoma specific methylation, including for PAX1 genes and
The specific primer and probe of the promoter region of ACTB genes, it is as follows：
PAX1 forward primers：5'-GTGGAGAGTGTTTTGGGAGGG-3'(SEQ ID NO.1),
PAX1 reverse primers：5'-CTACCRCCCRCTCCAAAACC-3'(SEQ ID NO.2),
PAX1 detection probes：5'FAM-TAGTAGCGGCGGCGGT-3'MGB(SEQ ID NO.3)；
ACTB forward primers：5'-GGTTAGGAAGGAGGTTGTTTGTTTT-3'(SEQ ID NO.4),
ACTB reverse primers：5'-ATCATCTTTCCCACCAAACTATAACC-3'(SEQ ID NO.5),
ACTB detection probes：5'VIC-CCCATTAACTAAACACAACCT-3'MGB(SEQ ID NO.6).
Present invention also offers a kind of kit based on the detection of cervical carcinoma specific methylation, including containing drawing as described above
The PCR reaction solutions of thing and probe, the PCR reaction solutions include：PAX1 forward primers, PAX1 reverse primers, PAX1 detection probes,
ACTB forward primers, ACTB reverse primers, ACTB detection probes, 10 × PCR buffer, dNTP and nuclease free water.
In a kind of preferred embodiment of the kit that the present invention is provided, the kit also includes Ex Taq enzymes.
In a kind of preferred embodiment of the kit that the present invention is provided, the composition final concentration of the PCR reaction solutions
For：1 × PCR buffer, 0.4 μM of PAX1 forward primers, 0.4 μM of PAX1 reverse primers, 0.4 μM of PAX1 detection probe, 0.4
μM ACTB forward primers, 0.4 μM of ACTB reverse primers, 0.4 μM of ACTB detection probe, 0.25mM dNTP.
In a kind of preferred embodiment of the kit that the present invention is provided, the kit also includes positive reference substance
And negative controls, the positive reference substance is the standard items DNA that methylates, and the negative controls are the non-standard items that methylate
DNA。
The wherein described standard items DNA that methylates is as shown in SEQ ID NO.7：
AAATTGATTttCGtACGtTGtAGttTttCGGTtAGACGAATTTtTtttAATCGGATGAAGTTtAtttTGGGttTGGG
GTCGCGGGCGTGGAGAGTGTttTGGGAGGGGGtAGtAGCGGCGGCGGtAGGtttTGGAGCGGGCGGtAGCGCGtTtC
GtTGtCGCGtAtAGCGCGTtTttAGttCGCGGtTGGGtCGtCGCGGtTtTCG；
The non-standard items DNA that methylates is as shown in SEQ ID NO.8：
AAATTGATTtttGtAtGtTGtAGttTtttGGTtAGAtGAATTTtTtttAATtGGATGAAGTTtAtttTGGGttTGGG
GTtGtGGGtGTGGAGAGTGTttTGGGAGGGGGtAGtAGtGGtGGtGGtAGGtttTGGAGtGGGtGGtAGtGtGtTtt
GtTGttGtGtAtAGtGtGTtTttAGtttGtGGtTGGGttGttGtGGtTtTtGGt。
Present invention also offers a kind of method based on the detection of cervical carcinoma specific methylation, comprise the following steps：
Step one：Take the DNA of sample to be detected extracting, conversion processing carried out to it, the DNA after conversion as PCR mould
Plate；
Step 2：The above-mentioned kit based on the detection of cervical carcinoma specific methylation is provided, performing PCR expansion is entered to the template
Increase；
Step 3：Sample to be detected is determined with the relative fluorescence CT values of ACTB gene PCR amplifications according to PAX1
Methyl rate, i.e., ratio of the ratio of PAX1/ACTB divided by PAX1/ACTB in positive reference substance in sample to be detected.
In a kind of preferred embodiment of the methods described that the present invention is provided, conversion processing is used in the step one
Reagent is bisulfites or bisulfite.
In a kind of preferred embodiment of the methods described that the present invention is provided, pcr amplification reaction program in the step 2
For：95 DEG C of denaturation 10min；50cycles, 95 DEG C of 15sec, 60 DEG C of 30sec.
Compared to prior art, primer pair, kit based on the detection of cervical carcinoma specific methylation that the present invention is provided
And the beneficial effect of method is：The primer and probe high by designing specificity, and it is configured to easy to use and testing result
Reliable kit, the rational PCR reaction systems of the science of redesigning out so that the present invention has quick, high flux, sensitive and spy
The characteristics of opposite sex is good, realizes quickly and accurately measuring the methylation of cervical carcinoma PAX1 genes, so as to indirect to cervical carcinoma
Timely, effective diagnosis and treatment are carried out, medical treatment cost is reduced, social resources are saved.
Brief description of the drawings
Fig. 1 is the PCR amplification curve diagrams for different methyl rate samples；
Fig. 2 is the PCR amplification curve diagrams of the minimum detectability that be can be detected out using primer pair of the present invention and kit.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with the accompanying drawings and embodiment, it is right
The present invention is described in further detail.It should be appreciated that specific embodiment described herein is only used to explain the present invention, and
It is not used in the restriction present invention.
Embodiment 1：The preparation of kit
First, the design of primer and probe and synthesis
For the promoter region of PAX1 genes in human genome and internal standard gene ACTB (β-actin), (sequence is referring to NCBI
Mankind's whole genome sequence disclosed in database), use Primer Premier 3.0 and Methyl Primer Express
V1.0 softwares, separately design a pair of specific primers and probe.
Specific primer and probe sequence, it is as shown in the table：
2nd, reference substance selection
Using the standard items DNA that methylates for positive reference substance, its sequence is：
AAATTGATTttCGtACGtTGtAGttTttCGGTtAGACGAATTTtTtttAATCGGATGAAGTTtAtttTGGGttTGGG
GTCGCGGGCGTGGAGAGTGTttTGGGAGGGGGtAGtAGCGGCGGCGGtAGGtttTGGAGCGGGCGGtAGCGCGtTtC
GtTGtCGCGtAtAGCGCGTtTttAGttCGCGGtTGGGtCGtCGCGGtTtTCG；
For negative controls, its sequence is the non-standard items DNA that methylates：
AAATTGATTtttGtAtGtTGtAGttTtttGGTtAGAtGAATTTtTtttAATtGGATGAAGTTtAtttTGGGttTGGG
GTtGtGGGtGTGGAGAGTGTttTGGGAGGGGGtAGtAGtGGtGGtGGtAGGtttTGGAGtGGGtGGtAGtGtGtTtt
GtTGttGtGtAtAGtGtGTtTttAGtttGtGGtTGGGttGttGtGGtTtTtGGt。
3rd, PCR reaction solutions composition
Including the PCR reaction solutions containing above-mentioned specific primer and probe, the PCR reaction solutions include：
PAX1 forward primers：5'-GTGGAGAGTGTTTTGGGAGGG-3',
PAX1 reverse primers：5'-CTACCRCCCRCTCCAAAACC-3',
PAX1 detection probes：5'FAM-TAGTAGCGGCGGCGGT-3'MGB,
ACTB forward primers：5'-GGTTAGGAAGGAGGTTGTTTGTTTT-3',
ACTB reverse primers：5'-ATCATCTTTCCCACCAAACTATAACC-3',
ACTB detection probes：5'VIC-CCCATTAACTAAACACAACCT-3'MGB；
10 × PCR buffer,
DNTP and nuclease free water.
Wherein, described 10 × PCR buffer, dNTP and nuclease free water are purchased from precious biological (the precious biology (Dalian) in Dalian
Engineering Co., Ltd)；The nuclease free water is that (Nuclease-Free Water) uses DEPC (Diethyl
Pyrocarbonate, pyrocarbonic acid diethyl ester) treat and through the ultra-pure water of autoclave sterilization.
The PCR reaction solutions composition it is final concentration of：
1 × PCR buffer,
0.4 μM of (μm ol/L) PAX1 forward primer,
0.4 μM of PAX1 reverse primer,
0.4 μM of PAX1 detection probe；
0.4 μM of ACTB forward primer,
0.4 μM of ACTB reverse primer,
0.4 μM of ACTB detection probe；
0.25mM(mmol/L)dNTP。
The kit also includes Ex Taq enzymes, precious biological (precious biology (Dalian) Engineering Co., Ltd) purchased from Dalian；Institute
The activity for stating Ex Taq enzymes remains more preferable, and longer fragment can be effectively expanded relative to general T aq.
Embodiment 2：The method that cervical carcinoma DNA methylation assay is carried out using mentioned reagent box
First, know-why
In human genome the promoter region of PAX1 genes and internal standard gene ACTB (b-actin) separately design a pair it is special
The primer and probe of the DNA methylation assay of property.Then amplification is gone through the sample DNA of sulphite conversion, root with the primer and probe
The methyl rate of sample to be tested is determined according to the relative fluorescence CT values of PAX1 and ACTB gene PCR amplifications, according to methylating
Rate judges the risk of cervical carcinogenesis.
2nd, detection method
Step one：Take the DNA of sample to be detected extracting, conversion processing carried out to it, the DNA after conversion as PCR mould
Plate；
Wherein, the reagent that conversion processing is used is bisulfites or bisulfite, and other auxiliary (corresponding reagents
Box is purchased from the EpiTect Fast DNA BisuLfite Kit of German QIAGEN companies).
Step 2：The above-mentioned kit based on the detection of cervical carcinoma specific methylation is provided, performing PCR expansion is entered to the template
Increase；
Wherein, pcr amplification reaction program is：95 DEG C of denaturation 10min；50cycles, 95 DEG C of 15sec, 60 DEG C of 30sec.
Step 3：Sample to be detected is determined with the relative fluorescence CT values of ACTB gene PCR amplifications according to PAX1
Methyl rate, i.e., ratio of the ratio of PAX1/ACTB divided by PAX1/ACTB in positive reference substance in sample to be detected.
Specific detection method is as follows：
One) biological specimen is collected：
During selected from the 3-6 months in 2016 Hunan Provincial Tumour Hospital go to a doctor 30 C1N1 cervical exfoliated cells, 30
C1N2 patient's cervical exfoliated cell, 30 C1N3 cervical cancer patients cervical exfoliated cells, the uterine neck of 90 normal populations come off carefully
Born of the same parents.
Two) tissue DNA is extracted：
Hereinafter extract the agent formulations used by sample tissue DNA and be selected from Hunan Honghao Genetic Biology Technology Co., Ltd
《Human peripheral genome DNA extracting reagent kit (filtration post method)》(the number of putting on record：Hunan tool long is for 20150166).
1) the sample fully shaking 1min that will be fetched, takes out Uterine neck bush.
2) aseptic 1.5mL EP pipes are taken, 1.5ml samples, 12000rpm centrifugations 1.5min is added；Top waste liquid is outwelled, is received
Collection ttom of pipe precipitation.
3) repeat step 2) all of sample process is complete.
4) 20 μ L Proteinase Ks, 250 μ L lysate ABL are sequentially added, vortex oscillation 10s, 65 DEG C of water-bath 15min, period shakes
Swing mixing 2-3 times.
5) 250 μ L absolute ethyl alcohols, vortex oscillation 10sec, of short duration centrifugation 5sec are added after taking out.
6) by adsorption column insertion collecting pipe, previous step gained mixed liquor is transferred in adsorption column.10,000rpm is centrifuged
1min。
7) waste liquid in collecting pipe is abandoned, adsorption column is reentered into collecting pipe；Add 500 μ L washing lotions I, 10,000rpm, centrifugation
1min。
8) waste liquid in collecting pipe is abandoned, 700 μ L washing lotions II, 10,000rpm, centrifugation 1min are added,.Abandon waste liquid.(washing lotion II makes
Absolute ethyl alcohol need to be added before to 80%)
9) repeat step 8) once.
10) efflux is abandoned, adsorption column is turned back into collecting pipe, idle running centrifugation, 13,000rpm, 2min.
11) adsorption column is inserted into new EP pipes.(65 DEG C of preheatings can improve DNA to add 30-100 μ L DNA lysates
Pick-up rate), it is stored at room temperature 5min.
12) again 10,000rpm, 1min is centrifuged.Adsorption column is abandoned, EP liquid in pipe is DNA solution.2-8 DEG C of preservation, if
Need to for a long time preserve, be placed in -20 DEG C or lower temperature.
Three) DNA conversion process：
1) the DNA configuration sulphite transformation system configurations that will be extracted：
2) operation architecture of sulphite conversion：
| Reaction temperature | Reaction time |
| 95℃ | 5min |
| 60℃ | 10min |
| 95℃ | 5min |
| 60℃ | 10min |
| 20℃ | ∞ |
3) DNA purifying after sulphite conversion：
DNA purifying agent formulations used are selected from German QIAGEN companies after being converted to sulphite below
EpiTect Fast DNA sodium hydrogensulfite kits.
A) product after sulfurous acid is converted is transferred in the EP pipes of 1.5ml；
B) during 1 μ L Carrier RNA to Buffer BL need to be added less than 100ng/ μ L DNA, if concentration is more than 100ng/ μ L
Carrier RNA need not then be added；
C) 310 μ L Buffer BL are added to mix；
D) add 250 μ L absolute ethyl alcohols again, mix 15sec；
E) mixture by more than is transferred to Filter column, 10000rpm centrifugations 1min；
F) filtrate, plus 500 μ L Buffer BW, 10000rpm centrifugations 1min are abandoned；
G) filtrate, plus 500 μ L Buffer BD static 15min, 10000rpm centrifugations 1min are abandoned；
H) filtrate, plus 500 μ L Buffer BW, 10000rpm centrifugations 1min are abandoned；
I) repeat step h)；
J) filtrate, plus 250 μ L absolute ethyl alcohols, 10000rpm centrifugations 1min are abandoned；
K) filtrate, then 12000rpm idle running 1min are abandoned；
L) 15 μ L Buffer EB room temperatures static 1min, 10000rpm centrifugation 1min is added；
M) DNA of collection is stored in -20 DEG C.
Four) PCR flows
1st, the PCR instrument device for using is that the type real-time fluorescence quantitative PCR systems of American AB I 7500 (are purchased from Life Tech
(applied biosystems companies), reaction system is 20 μ L；
2nd, PCR reaction systems are prepared and condition, as shown in the table：
PCR reaction systems：
| Composition | Reaction volume (μ L) |
| PCR reaction solutions | 18.3μL |
| Ex Taq enzymes | 0.2μL |
| DNA profiling after sulphite conversion | 1.5μL |
PCR response procedures：
Five) interpretation of result
With the ratio of PAX1/ACTB in sample to be tested divided by PAX1/ACTB in positive control (methylate standard items DNA)
Ratio methylates ratio calculating PAX1.
Methyl rate computing formula is that the formula for borrowing relative quantification calculates the methyl rate of sample to be detected, and formula is such as
Shown in lower.
2- △ △ CT=2Sample to be tested (PAX1CT-ACTB CT)-permethylated sample (PAX1CT-ACTB CT)
Part sample methyl rate data, it is as shown in the table：
| Sequence number is numbered | Sample | △CT Mean | △△CT | Methyl rate |
| 10 | 0% | 19.857 | 17.793 | 0 |
| 11 | 5% | 9.929 | 7.864 | 0.004 |
| 12 | 15% | 4.811 | 2.747 | 0.149 |
| 13 | 35% | 3.477 | 1.413 | 0.376 |
| 14 | 100% | 2.064 | 0 | 1 |
Based on specific experiment data of the invention referring to Fig. 1 and Fig. 2, wherein Fig. 1 is for different methyl rate samples
PCR amplification curve diagrams, it can be deduced that different methyl rate sample PAX1 gene magnifications curves are bent with the amplification of ACTB house-keeping genes
Line relation；Fig. 2 is the PCR amplification curve diagrams of the minimum detectability that be can be detected out using primer pair of the present invention and kit, this
The primer and kit of invention design can detect that the methyl rate sample that minimum detectability is 10%, have with the non-sample that methylates
Significant difference.
The beneficial effect of primer pair, kit and method based on the detection of cervical carcinoma specific methylation that the present invention is provided
It is：The primer and probe high by designing specificity, and the reliable kit of easy to use and testing result is configured to, then set
Count out the rational PCR reaction systems of science so that the present invention have quick, high flux, it is sensitive and specific good the characteristics of, realize
Quick to the methylation of cervical carcinoma PAX1 genes and accurate measurement, to carry out timely, effective examining indirectly to cervical carcinoma
Disconnected and treatment, reduces medical treatment cost, saves social resources.
Embodiments of the invention are the foregoing is only, the scope of the claims of the invention is not thereby limited, it is every to utilize this hair
The equivalent flow conversion that bright description is made, or other related technical fields are directly or indirectly used in, similarly wrap
Include in scope of patent protection of the invention.
SEQUENCE LISTING
<110>Han Linzhi
<120>Primer pair, kit and method based on the detection of cervical carcinoma specific methylation
<130> 2017
<160> 8
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Artificial sequence
<400> 1
gtggagagtg ttttgggagg g 21
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ctaccrcccr ctccaaaacc 20
<210> 3
<211> 16
<212> DNA
<213>Artificial sequence
<400> 3
tagtagcggc ggcggt 16
<210> 4
<211> 25
<212> DNA
<213>Artificial sequence
<400> 4
ggttaggaag gaggttgttt gtttt 25
<210> 5
<211> 26
<212> DNA
<213>Artificial sequence
<400> 5
atcatctttc ccaccaaact ataacc 26
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
cccattaact aaacacaacc t 21
<210> 7
<211> 206
<212> DNA
<213>Artificial sequence
<400> 7
aaattgattt tcgtacgttg tagtttttcg gttagacgaa ttttttttaa tcggatgaag 60
tttattttgg gtttggggtc gcgggcgtgg agagtgtttt gggagggggt agtagcggcg 120
gcggtaggtt ttggagcggg cggtagcgcg tttcgttgtc gcgtatagcg cgtttttagt 180
tcgcggttgg gtcgtcgcgg ttttcg 206
<210> 8
<211> 208
<212> DNA
<213>Artificial sequence
<400> 8
aaattgattt ttgtatgttg tagttttttg gttagatgaa ttttttttaa ttggatgaag 60
tttattttgg gtttggggtt gtgggtgtgg agagtgtttt gggagggggt agtagtggtg 120
gtggtaggtt ttggagtggg tggtagtgtg ttttgttgtt gtgtatagtg tgtttttagt 180
ttgtggttgg gttgttgtgg tttttggt 208
Claims (8)
1. a kind of primer pair based on the detection of cervical carcinoma specific methylation, it is characterised in that including for PAX1 genes and
The specific primer and probe of the promoter region of ACTB genes, it is as follows：
PAX1 forward primers：5'-GTGGAGAGTGTTTTGGGAGGG-3',
PAX1 reverse primers：5'-CTACCRCCCRCTCCAAAACC-3',
PAX1 detection probes：5'FAM-TAGTAGCGGCGGCGGT-3'MGB；
ACTB forward primers：5'-GGTTAGGAAGGAGGTTGTTTGTTTT-3',
ACTB reverse primers：5'-ATCATCTTTCCCACCAAACTATAACC-3',
ACTB detection probes：5'VIC-CCCATTAACTAAACACAACCT-3'.
2. it is a kind of based on cervical carcinoma specific methylation detection kit, it is characterised in that including containing such as claim 1 institute
The specific primer and the PCR reaction solutions of probe stated, the PCR reaction solutions include：PAX1 forward primers, PAX1 reverse primers,
PAX1 detection probes, ACTB forward primers, ACTB reverse primers, ACTB detection probes, 10 × PCRbuffer, dNTP and seedless
Sour enzyme water.
3. it is according to claim 2 based on cervical carcinoma specific methylation detection kit, it is characterised in that the examination
Agent box also includes Ex Taq enzymes.
4. it is according to claim 2 based on cervical carcinoma specific methylation detection kit, it is characterised in that it is described
The composition of PCR reaction solutions is final concentration of：1 × PCR buffer, 0.4 μM of PAX1 forward primer, 0.4 μM of PAX1 reverse primer,
0.4 μM of PAX1 detection probe, 0.4 μM of ACTB forward primers, 0.4 μM of ACTB reverse primers, 0.4 μM of ACTB detection probe,
0.25mM dNTP。
5., according to any described kit detected based on cervical carcinoma specific methylation in claim 2-4, its feature exists
In also including positive reference substance and negative controls, the positive reference substance is methylate standard items DNA, the negative control
Product are the non-standard items DNA that methylates.
6. it is a kind of based on cervical carcinoma specific methylation detection method, it is characterised in that comprise the following steps：
Step one：Take the DNA of sample to be detected extracting, conversion processing carried out to it, the DNA after conversion as PCR template；
Step 2：Kit as claimed in claim 5 is provided, performing PCR amplification is entered to the template；
Step 3：The methyl of sample to be detected is determined with the relative fluorescence CT values of ACTB gene PCR amplifications according to PAX1
Rate, i.e., ratio of the ratio of PAX1/ACTB divided by PAX1/ACTB in positive reference substance in sample to be detected.
7. it is according to claim 6 based on cervical carcinoma specific methylation detection method, it is characterised in that the step
The reagent that conversion processing is used in one is bisulfites or bisulfite.
8. it is according to claim 7 based on cervical carcinoma specific methylation detection method, it is characterised in that the step
Pcr amplification reaction program is in two：95 DEG C of denaturation 10min；50cycles, 95 DEG C of 15sec, 60 DEG C of 30sec.
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