CN110452984A - A kind of methylated genes combination for cervical carcinoma DNA methylation assay, primer and probe combination, kit and its application method - Google Patents

A kind of methylated genes combination for cervical carcinoma DNA methylation assay, primer and probe combination, kit and its application method Download PDF

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CN110452984A
CN110452984A CN201910687592.1A CN201910687592A CN110452984A CN 110452984 A CN110452984 A CN 110452984A CN 201910687592 A CN201910687592 A CN 201910687592A CN 110452984 A CN110452984 A CN 110452984A
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fam19a4
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韩林志
肖芳
李翔
李书
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Abstract

The present invention provides a kind of for the methylated genes combination of cervical carcinoma DNA methylation assay, primer and probe combination, kit and its application method, belongs to cma gene detection technique field.The present invention is based on real-time fluorescent PCR technologies, in the primer and probe of the DNA methylation assay of the promoter region design specificity of FAM19A4, has-miR-124-2, ZNF671 gene, and it is configured to the sample DNA after PCR reaction solution goes amplification to vulcanize, by controlling with quality of the reference gene β-actin to sample, whether judgement sample methylates.Compared with conventional method, this method has the characteristics that quick, high-throughput, sensitive and specific good, plays an important role to the early diagnosis and Index for diagnosis of cervical carcinoma.

Description

A kind of methylated genes combination for cervical carcinoma DNA methylation assay, primer and probe Combination, kit and its application method
Technical field
The present invention relates to cma gene detection technique field more particularly to a kind of methyl for cervical carcinoma DNA methylation assay Change the assortment of genes, primer and probe combination, kit and its application method.
Background technique
Cervical carcinoma is always to perplex the important malignant tumour of women.Normal cervical cell develops by precancerous lesion Cervical carcinoma may be up to decades.In this whole process the infection of part HPV viruse can by self immune system reparation and from Turn is classified as normally, and another part and cervical carcinoma contact closer precancerous lesion then and need to be intervened in time to prevent cancer Preceding pathological development becomes cervical carcinoma.
The main screening means that China clinically applies at present are cytology detection method, including conventional smear and liquid-based Cytology (TCT).But the testing process of cytology detection method includes multiple rings such as sampling-smear preparation-observation-result interpretation Section, vulnerable to man's activity a possibility that, are larger, cause the probability of missing inspection higher.Also, since cervical carcinoma is attributed to HPV viruse The HPV-DNA detection to grow up after persistent infection.Because what it was detected is only the presence or absence of HPV viruse, and HPV viruse Presence, be largely transient, that is, mean that these can be also judged as by the HPV infection that autoimmunity is disposed The positive leads to over-treatment.
Summary of the invention
The purpose of the present invention is to provide a kind of methylated genes combination, primer and spies for cervical carcinoma DNA methylation assay Needle combination, kit and its application method, the kit have the advantages that detection sensitivity is high, specificity is good.
In order to achieve the above-mentioned object of the invention, the present invention the following technical schemes are provided:
The present invention provides a kind of methylated genes combinations for cervical carcinoma DNA methylation assay, including following methylation base Cause: FAM19A4, has-miR-124-2 and ZNF671.
The present invention also provides a kind of primer and probe combination for cervical carcinoma DNA methylation assay, the primer and probes For detecting FAM19A4, has-miR-124-2 and ZNF671 described in above scheme；The primer and probe combines FAM19A4 forward primer F, FAM19A4 reverse primer R, FAM19A4 probe P, has-miR-124-2 forward primer F, has- MiR-124-2 reverse primer R, has-miR-124-2 probe P, ZNF671 forward primer F, ZNF671 reverse primer R and ZNF671 Probe P；
Described FAM19A4 forward primer F, FAM19A4 reverse primer R, FAM19A4 probe P, has-miR-124-2 are positive Primers F, has-miR-124-2 reverse primer R, has-miR-124-2 probe P, ZNF671 forward primer F, ZNF671 reversely draw The nucleotide sequence of object R and ZNF671 probe P is respectively as shown in SEQ ID NO:1~SEQ ID NO:9.
Preferably, the 5 ' ends of FAM19A4 probe P and has-miR-124-2 probe P, ZNF671 probe P use FAM Label；3 ' the ends of FAM19A4 probe P, ZNF671 probe P and has-miR-124-2 probe P are marked using MGB.
The present invention also provides a kind of kit for cervical carcinoma DNA methylation assay, the kit includes above scheme The described primer and probe combination, β-actin forward primer F, β-actin reverse primer R and β-actin probe P；The β- Nucleotide sequence such as SEQ ID NO:10~SEQ of actin forward primer F, β-actin reverse primer R and β-actin probe P Shown in ID NO:12.
Preferably, the 5 ' ends of the β-actin probe P are marked using VIC；3 ' the ends of the β-actin probe P use MGB label.
Preferably, the kit further includes 10 × PCR Buffer, Taq enzyme, dNTPs, nuclease-free water, positive control And negative control.
Preferably, the positive control includes cervical cancer cell lines CaSki；The negative control includes cervical cancer cell lines C33A。
The present invention also provides the application methods of kit described in above scheme, comprising the following steps:
1) sample to be examined DNA is extracted；
2) DNA by the sample to be examined DNA through sulphite conversion processing, after being converted；
3) it using the DNA after the conversion as template, is combined using primer and probe described in above scheme and carries out PCR amplification Reaction, obtains fluorescence detection result；
4) according to the fluorescence detection as a result, determining sample to be examined, when-actin≤35 β, and testing result is FAM19A4 or miR-124-2 any gene Δ Ct≤9, then determine sample to be examined for cervical carcinoma methylation positive；As β-actin ≤ 35, and testing result is FAM19A4, has-miR-124-2, ZNF671 gene Δ Ct > 9, then determines that sample to be examined is FAM19A4, has-miR-124-2, ZNF671 methylation are negative；As β-actin > 35, then determine that sample is insufficient or has mortifier In the presence of PCR reaction is invalid, need to repeat to test or sample；
The Δ Ct=FAM Ct-VIC Ct.
Preferably, the reaction system that pcr amplification reaction described in step 3) uses includes FAM19A4 amplification system, has- MiR-124-2 amplification system and ZNF671 amplification system；
The FAM19A4 amplification system includes: 18.3 μ L of FAM19A4PCR reaction solution, 0.2 μ L of Taq enzyme in terms of 20 μ L and turns DNA1.5 μ L after change；The FAM19A4PCR reaction solution includes the component of following concentration: PCRbuffer 1 ×, dNTP 0.25mM, FAM19A4 forward primer F 0.4uM, FAM19A4 reverse primer R 0.4uM, FAM19A4 probe P 0.4uM, β- Actin-F 0.4uM, β-actin-R 0.4uM and β-actin-MP 0.4uM；
The has-miR-124-2 amplification system include: in terms of 20 μ L 18.3 μ L of has-miR-124-2 PCR reaction solution, 0.2 μ L of the Taq enzyme and DNA1.5 μ L after conversion；The has-miR-124-2 PCR reaction solution includes the component of following concentration: PCRbuffer 1 ×, dNTP 0.25mM, has-miR-124-2 forward primer F 0.4uM, has-miR-124-2 reverse primer R 0.4uM, has-miR-124-2 probe P0.4uM, β-actin-F 0.4uM, β-actin-R 0.4uM and β-actin-MP 0.4uM；
The ZNF671 amplification system includes: 18.3 μ L of ZNF671PCR reaction solution, 0.2 μ L of Taq enzyme and conversion in terms of 20 μ L DNA1.5 μ L afterwards；The ZNF671PCR reaction solution includes the component of following concentration: PCRbuffer 1 ×, dNTP 0.25mM, ZNF671 forward primer F 0.4uM, ZNF671 reverse primer R 0.4uM, ZNF671 probe P 0.4uM, β-actin-F 0.4uM, β-actin-R 0.4uM and β-actin-MP 0.4uM.
Preferably, the program of pcr amplification reaction described in step 3) are as follows: 95 DEG C of 5min, [95 DEG C of 15s, 60 DEG C of 30s, 50 Circulation], reaction terminates；Fluorescence is acquired during 60 DEG C of 30s.
Beneficial effects of the present invention: the present invention provides a kind of methylated genes groups for cervical carcinoma DNA methylation assay It closes, including following methylated genes: FAM19A4, has-miR-124-2 and ZNF671.In the present invention, described FAM19A4, has- MiR-124-2 and ZNF671 gene is closely related with the process of cervical carcinoma.The equal needle of methylation region detection of each target gene CpGs multiple to core regulatory region, the detection of relatively single base reduce the randomness of base variation.The methyl of target gene Change judgement and be not limited to one or two of base, high density methylation causes protein expression obviously to lower.
The present invention also provides a kind of primer and probe combination for cervical carcinoma DNA methylation assay, the spy that the present invention uses Needle set has the advantage of high specific and hypersensitivity, can accurate stable detect down to concentration 1ng/ μ L, 5% methyl rate； And the probe has single base resolution ratio, can specifically distinguish methylation and unmethylated DNA.
The present invention also provides a kind of kits and its application method for cervical carcinoma DNA methylation assay.It is of the present invention Kit uses house-keeping gene β-actin to control as quality of the reference gene to sample, it is contemplated that house-keeping gene may The case where methylation, selects the position on no island CpG when designing internal control primer probe.Guarantee house-keeping gene both can to sample into The control of row quality, and the methylation level of sample can be evaluated to a certain extent.Also, cervical carcinoma is a more The process of inducement multi-step, the present invention evaluate polygenes methylation joint-detection by unique algorithm synthesis, sensitivity, Specificity is better than single-gene detection.In addition, the application method of kit of the present invention is not necessarily in PCR rear electrophoresis, hybridization Deng operation, reduce pollution and operating error, while entire PCR process is completed in 1h.Its quick, convenient feature is suitable for Clinical detection.
Detailed description of the invention:
Fig. 1 is cervical cancer tissues FAM19A4, has-miR-124-2, ZNF671 gene methylation distribution situation.
Specific embodiment
The present invention provides a kind of methylated genes combinations for cervical carcinoma DNA methylation assay, including following methylation base Cause: FAM19A4, has-miR-124-2 and ZNF671.
The present invention also provides a kind of primer and probe combination for cervical carcinoma DNA methylation assay, the primer and probes For detecting FAM19A4, has-miR-124-2 and ZNF671 described in above scheme；The primer and probe combines FAM19A4 forward primer F, FAM19A4 reverse primer R, FAM19A4 probe P, has-miR-124-2 forward primer F, has- MiR-124-2 reverse primer R, has-miR-124-2 probe P, ZNF671 forward primer F, ZNF671 reverse primer R and ZNF671 Probe P；
Described FAM19A4 forward primer F, FAM19A4 reverse primer R, FAM19A4 probe P, has-miR-124-2 are positive Primers F, has-miR-124-2 reverse primer R, has-miR-124-2 probe P, ZNF671 forward primer F, ZNF671 reversely draw The nucleotide sequence of object R and ZNF671 probe P is respectively as shown in SEQ ID NO:1~SEQ ID NO:9, referring specifically to table 1:
The primer and probe of 1 cervical carcinoma DNA methylation assay of table combines
Title Specific nucleotide sequence Number
FAM19A4 forward primer F CGGCGGGTTCGGTTT SEQ ID NO:1
FAM19A4 reverse primer R CGCAACTAACAATACGAAACCG SEQ ID NO:2
FAM19A4 probe P TACGAAACCGAACCCA SEQ ID NO:3
Has-miR-124-2 forward primer F TAATTTGGATTTACGTCGTTATTTGA SEQ ID NO:4
Has-miR-124-2 reverse primer R GTAAAAATATAAACGATACGTATACCTACGTA SEQ ID NO:5
Has-miR-124-2 probe P TACAACACACGCCTAA SEQ ID NO:6
ZNF671 forward primer F GTTTTCGGTAGTTGTTCGCGTT SEQ ID NO:7
ZNF671 reverse primer R AAAATAACTAAAAACCCGAAAAACG SEQ ID NO:8
ZNF671 probe P TCCGCCATAAAACCGTAAA SEQ ID NO:9
In the present invention, the primer and probe combination is disclosed according to NCBI (US National Biotechnology Information center) Mankind's whole genome sequence is designed using Primer Premier3.0 and Methyl Primerpress v1.0, by the victory of the English Weihe River The synthesis of base (Shanghai) trade Co., Ltd.
In the present invention, the nucleotide sequence of the target gene after the FAM19A4 amplification is as shown in SEQ ID NO:13, specifically Are as follows: CGGCGGGTTCGGTTTTTTTTGGTTAGCGCGTTAGTTGGGTTCGGTTTCGTATTGTT AGTTGCG；The has- MiR-124-2 amplification after target gene nucleotide sequence as shown in SEQ ID NO:14, specifically: TAATTAATTTGGATT TACGTCGTTATTTGAAAATTAGATTTATAGGTTTATGTATGTTTTTAGGCGTGTGTTGTAAATGGTATGGAGATAT ATGTATATGTATACGTAGGTATACGTATCGTTTATATTTTTAC；The nucleotide of target gene after the ZNF671 amplification Sequence as shown in SEQ ID NO:15, specifically: GTTTTCGGTAGTTGTTCGCGTTTACGGTTTTATGGCGGAGTTAACG GATTTCGCGCGGGTGGGTGTTGCGTTTTTCGGGTTTTTAGTTATTTT。
In the present invention, the 5 ' ends of FAM19A4 probe P and has-miR-124-2 probe P, ZNF671 probe P are preferred Luminophore is used as using FAM label；The 3 ' of FAM19A4 probe P and has-miR-124-2 probe P, ZNF671 probe P End is preferably used as quenching group using MGB label.
The present invention also provides a kind of kit for cervical carcinoma DNA methylation assay, the kit includes above scheme The described primer and probe combination, β-actin forward primer F, β-actin reverse primer R and β-actin probe P；The β- Nucleotide sequence such as SEQ ID NO:10~SEQ of actin forward primer F, β-actin reverse primer R and β-actin probe P It is specific as follows shown in ID NO:12: β-actin forward primer F:GGTTAGGAAGGAGGTTGTTTGTTTT；β-actin is reversed Primer R:ATCATCTTTCCCACCAAACTATAACC；β-actin probe P:CCCATTAACTAAACACAACCT；
In the present invention, the 5 ' ends of the β-actin probe P are preferably used as luminophore using VIC label；The β- 3 ' the ends of actin probe P are preferably used as quenching group using MGB label.
In the present invention, the kit preferably further includes 10 × PCR Buffer, Taq enzyme, dNTPs, nuclease-free water, Positive control and negative control；The positive control preferably includes that (people's Cervical squamous carcinoma is thin by cervical cancer cell lines CaSki Born of the same parents system, FAM19A4, has-miR-124-2 and ZNF671 methyl rate 100%)；The negative control preferably includes cervical carcinoma (in 273 bit codons point mutation occurs cell strain C33A for p53+, pRB+.Papillomavirus DNA, RNA are negative, FAM19A4, Has-miR-124-2 and ZNF671 methyl rate 0%).
In the present invention, the nuclease-free water, 10 × PCR Buffer, Taq enzyme and dNTPs are preferably purchased from precious biology (Dalian) Co., Ltd；The Taq enzyme is preferably HS Taq enzyme.
The present invention also provides the application methods of kit described in above scheme, comprising the following steps:
1) sample to be examined DNA is extracted；
2) DNA by the sample to be examined DNA through sulphite conversion processing, after being converted；
3) it using the DNA after the conversion as template, is combined using primer and probe described in above scheme and carries out PCR amplification Reaction, obtains fluorescence detection result；
4) according to the fluorescence detection as a result, determining sample to be examined, when-actin≤35 β, and testing result is FAM19A4 or miR-124-2 any gene Δ Ct≤9, then determine sample to be examined for cervical carcinoma methylation positive；As β-actin ≤ 35, and testing result is FAM19A4, has-miR-124-2, ZNF671 gene Δ Ct > 9, then determines that sample to be examined is FAM19A4, has-miR-124-2, ZNF671 methylation are negative；As β-actin > 35, then determine that sample is insufficient or has mortifier In the presence of PCR reaction is invalid, need to repeat to test or sample；
The Δ Ct=FAM Ct-VIC Ct.
The present invention extracts sample to be examined DNA first；The sample to be tested includes tissue samples, FFPE (fix by formalin Paraffin-embedded tissue) and cervical exfoliated cell；The sample to be tested is preferably from the refined medical test institute in Hunan；The present invention is to institute The method for extracting sample to be examined DNA is stated to be not particularly limited, using conventional method in that art, specific implementation process of the present invention In preferably extracted using " the nucleic acid extraction purified reagent " of the primary gene technology Co., Ltd in Hunan hundred.
After obtaining sample to be examined DNA, the present invention through sulphite conversion processing, is turned the sample to be examined DNA DNA after change；The present invention is not particularly limited to by the sample to be examined DNA through the method for sulphite conversion processing, is used Conventional method in that art preferably uses EZ DNA Methylation in specific implementation process of the present inventionTMKits conversion Kit is handled.
After DNA after being converted, the present invention is using the DNA after the conversion as template, using drawing described in above scheme Object and probe combinations carry out pcr amplification reaction, obtain fluorescence detection result；The reaction system packet that the pcr amplification reaction uses Include FAM19A4 amplification system, has-miR-124-2 amplification system and ZNF671 amplification system；
The FAM19A4 amplification system includes: 18.3 μ L of FAM19A4PCR reaction solution, 0.2 μ L of Taq enzyme in terms of 20 μ L and turns DNA1.5 μ L after change；The FAM19A4PCR reaction solution includes the component of following concentration: PCRbuffer 1 ×, dNTP 0.25mM, FAM19A4 forward primer F 0.4uM, FAM19A4 reverse primer R 0.4uM, FAM19A4 probe P 0.4uM, β- Actin-F 0.4uM, β-actin-R 0.4uM and β-actin-MP 0.4uM；
The has-miR-124-2 amplification system include: in terms of 20 μ L 18.3 μ L of has-miR-124-2 PCR reaction solution, 0.2 μ L of the Taq enzyme and DNA1.5 μ L after conversion；The has-miR-124-2 PCR reaction solution includes the component of following concentration: PCRbuffer 1 ×, dNTP 0.25mM, has-miR-124-2 forward primer F 0.4uM, has-miR-124-2 reverse primer R 0.4uM, has-miR-124-2 probe P0.4uM, β-actin-F 0.4uM, β-actin-R 0.4uM and β-actin-MP 0.4uM；
The ZNF671 amplification system includes: 18.3 μ L of ZNF671PCR reaction solution, 0.2 μ L of Taq enzyme and conversion in terms of 20 μ L DNA1.5 μ L afterwards；The ZNF671PCR reaction solution includes the component of following concentration: PCRbuffer 1 ×, dNTP 0.25mM, ZNF671 forward primer F 0.4uM, ZNF671 reverse primer R 0.4uM, ZNF671 probe P 0.4uM, β-actin-F 0.4uM, β-actin-R 0.4uM and β-actin-MP 0.4uM.
In the present invention, the program of the pcr amplification reaction is preferred are as follows: 95 DEG C of 5min, [95 DEG C of 15s, 60 DEG C of 30s, 50 are followed Ring], reaction terminates；Fluorescence is acquired during 60 DEG C of 30s.
After obtaining fluorescence detection result, the present invention according to the fluorescence detection as a result, determine sample to be examined, When-actin≤35 β, and testing result is FAM19A4 or miR-124-2 any gene Δ Ct≤9, then determines that sample to be examined is Cervical carcinoma methylation positive；When-actin≤35 β, and testing result is FAM19A4, has-miR-124-2, ZNF671 gene Δ It is negative then to determine that sample to be examined methylates for FAM19A4, has-miR-124-2, ZNF671 by Ct > 9；As β-actin > 35, then Determine sample deficiency or with the presence of mortifier, PCR reaction is invalid, need to repeat to test or sample；The Δ Ct=FAM Ct-VIC Ct；The threshold value that the threshold value of the FAM is 120000, VIC is 80000.
Technical solution provided by the invention is described in detail below with reference to embodiment, but they cannot be understood For limiting the scope of the present invention.
Application of the kit of the invention of embodiment 1 in diagnosis human cervical carcinoma
Nuclease-free water that the present invention uses, 10 × PCR Buffer, HS Taq enzyme, dNTPs are (big purchased from precious biology Even) Co., Ltd；
Positive control (cervical cancer cell lines (CaSki)), negative control (cervical cancer cell lines (C33A)).
DNA extraction kit are as follows: " the nucleic acid extraction purified reagent " of the primary gene technology Co., Ltd in Hunan hundred；
Conversion reagent box is EZ DNA MethylationTMKits。
Biomaterial of the present invention is all from the refined medical test institute in Hunan.
Method:
One, biological sample:
For during 1-2017 June in 2017 in the refined hospital admission in Hunan 33 C1N1 cervical exfoliated cells, 30 C1N2 patient's cervical exfoliated cell, 30 C1N3+ cervical cancer patient cervical exfoliated cells, 90 normal populations uterine neck fall off carefully Born of the same parents.
Two, tissue DNA is extracted:
" the core that agent formulations used in sample tissue DNA are selected from the primary gene technology Co., Ltd in Hunan hundred is extracted below Sour extraction purification reagent ".
1) 1min is fullyd shake into the sample fetched, takes out Uterine neck bush.
2) it takes sterile 1.5mL EP to manage, 1.5ml sample is added, 12000rpm is centrifuged 1.5min；Top waste liquid is outwelled, is received Collect tube bottom precipitating.
3) step 2) is repeated all sample process is complete.
4) 20 μ L Proteinase Ks, 250 μ L lysate ABL are sequentially added, during which vortex oscillation 10s, 65 DEG C of water-bath 15min shake Swing mixing 2-3 times.
5) 250 μ L dehydrated alcohols, vortex oscillation 10sec, of short duration centrifugation 5sec is added after taking out.
6) adsorption column is inserted into collecting pipe, mixed liquor obtained by previous step is transferred in adsorption column.10,000rpm centrifugation 1min。
7) waste liquid in collecting pipe is abandoned, adsorption column is reentered into collecting pipe；It is added 500 μ L washing lotion I, 10,000rpm, centrifugation 1min。
8) waste liquid in collecting pipe is abandoned, 700 μ L washing lotion II, 10,000rpm, centrifugation 1min are added,.Abandon waste liquid.(washing lotion II makes Dehydrated alcohol need to be added before to 80%)
9) it is primary to repeat step 8).
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 pipe.30-100 μ L DNA lysate is added, and (DNA can be improved in 65 DEG C of preheatings Pick-up rate), it is stored at room temperature 5min.
12) again 10,000rpm, it is centrifuged 1min.Adsorption column is abandoned, EP liquid in pipe is DNA solution.2-8 DEG C of preservation, if Long-term preservation is needed then to be placed in -20 DEG C or lower temperature.
Three, the process of DNA conversion
1. by the DNA configuration sulphite transformation system configuration of extraction:
The DNA of extraction is configured according to the transformation system of table 2: (conversion reaction solution is prepared: being taken 1 pipe Solution 1, is added The 300 μ L of μ LSolution 2 and 790 Solution 3 fully shake and mix 8-10min and be completely dissolved to crystal；160 μ are added LSolution 4, concussion mix, for use；Unspent conversion reaction solution is please saved in 2~8 DEG C, is saved and is no more than 1 month)
The DNA that table 2 extracts configures sulphite transformation system
Ingredient Reaction volume (μ L)
The DNA to be transformed extracted 1~20 μ L (200ng~2ug)
RNase-freeWater Supply 20 μ L
Conversion reaction solution 130μL
It amounts to 150μL
2. sulphite converts operation architecture referring to table 3:
3 sulphite of table converts operation architecture
Reaction temperature Reaction time
98℃ 8min
64℃ 3.5h
20℃ ≤20h
3. DNA is purified after sulphite conversion:
1) it takes purification column that 600 μ L BufferA are added, then converted product is transferred in purification column, be mixed by inversion for several times；
2) 10000rpm is centrifuged 30s, abandons efflux；
3) plus 100 μ LBufferB, 10000rpm are centrifuged 30s, abandon efflux；
4) 200 μ LBufferC are added, are stored at room temperature 15min, 10000rpm is centrifuged 30s, abandons efflux；
5) plus 200 μ LBufferB, 10000rpm are centrifuged 30s, abandon efflux；
6) step 5 is repeated；Ah
7) 10000rpm idle running centrifugation 2min；
8) purification column is inserted into a new EP pipe, 20 μ L Elution Buffer is added, are stored at room temperature 1min；
9) 10000rpm is centrifuged 1min and collects the DNA solution that efflux has as converted, and is stored in -20 DEG C.
Four, PCR process
1, the PCR instrument used is ABI 7500, and reaction system is 20 μ L；
2, PCR reaction system is prepared and condition is as shown in such as the following table 4 and table 5:
The preparation of 4 PCR reaction system of table
Ingredient Reaction volume (μ L)
PCR reaction solution 18.3μL
Taq enzyme 0.2μL
DNA profiling after sulphite conversion 1.5μL
5 PCR response procedures of table
Five, interpretation of result
The threshold value that the threshold value of threshold value FAM is 120000, VIC is 80000, Δ Ct=FAM Ct-VIC Ct.
Kit test result meets Quality Control requirement, judges according to testing result sample.Criterion is referring to table 6.- actin≤35 β, pattern detection result are wantonly 2 gene Δs Ct≤9 (Δ Ct=FAM Ct-VIC Ct.), then determine sample For methylation positive；- actin≤35 β, pattern detection result are gene≤1 FAM19A4, has-miR-124-2, ZNF671 Gene Δ Ct≤9；Then sentencing sample is that FAM19A4, has-miR-124-2, ZNF671 methylation are negative, β-actin > 35, then sample This deficiency reacts invalid with the presence of mortifier PCR, need to repeat to test or sample.
6 testing result criterion of table
Six, cervical cancer tissues FAM19A4, has-miR-124-2, ZNF671 gene methylation distribution situation (is shown in Table 7 and figure 1)
FAM19A4, has-miR-124-2, ZNF671 gene methylation are not detected in 99 normal populations, at 24 It is detected in cervical carcinoma cast-off cells 30 (accounting for 90.9%, P < 0.001 compared with normal group), FAM19A4, has-miR-124-2, The methylation ratio of ZNF671 gene is increased with the exacerbation of the cervical carcinoma course of disease.
FAM19A4, has-miR-124-2, ZNF671 gene methylation detection statistics result of 7 cervical carcinoma cast-off cells of table
Norma(99) LSIL(59) HSIL(48) CA(44)
FAM19A4 0.00% 10.17% 36.84% 61.53%
Mir-124-2 2.00% 5.08% 42.11% 76.92%
ZNF671 4.00% 13.56% 68.42% 90.31%
Cervi-safe 4.00% 28.81% 84.21% 100.00%
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered It is considered as protection scope of the present invention.
Sequence table
<110>Han Linzhi
<120>a kind of methylated genes combination for cervical carcinoma DNA methylation assay, primer and probe combination, kit and its Application method
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Claims (10)

1. a kind of methylated genes for cervical carcinoma DNA methylation assay combine, including following methylated genes: FAM19A4, Has-miR-124-2 and ZNF671.
2. a kind of primer and probe for cervical carcinoma DNA methylation assay combines, which is characterized in that the primer and probe is used for Detect FAM19A4, has-miR-124-2 and ZNF671 described in claim 1；The primer and probe combination includes FAM19A4 Forward primer F, FAM19A4 reverse primer R, FAM19A4 probe P, has-miR-124-2 forward primer F, has-miR-124-2 Reverse primer R, has-miR-124-2 probe P, ZNF671 forward primer F, ZNF671 reverse primer R and ZNF671 probe P；
FAM19A4 forward primer F, FAM19A4 reverse primer R, FAM19A4 probe P, has-miR-124-2 forward primer F, has-miR-124-2 reverse primer R, has-miR-124-2 probe P, ZNF671 forward primer F, ZNF671 reverse primer R and The nucleotide sequence of ZNF671 probe P is respectively as shown in SEQ ID NO:1~SEQ ID NO:9.
3. primer and probe combination according to claim 2, which is characterized in that described FAM19A4 probe P, ZNF671 are visited 5 ' the ends of needle P and has-miR-124-2 probe P are marked using FAM；FAM19A4 probe P, ZNF671 probe P and has- 3 ' the ends of miR-124-2 probe P are marked using MGB.
4. a kind of kit for cervical carcinoma DNA methylation assay, which is characterized in that the kit includes Claims 2 or 3 The described primer and probe combination, β-actin forward primer F, β-actin reverse primer R and β-actin probe P；The β- Nucleotide sequence such as SEQ ID NO:10~SEQ of actin forward primer F, β-actin reverse primer R and β-actin probe P Shown in ID NO:12.
5. kit according to claim 4, which is characterized in that the 5 ' ends of the β-actin probe P are marked using VIC； 3 ' the ends of the β-actin probe P are marked using MGB.
6. kit according to claim 4 or 5, which is characterized in that the kit further includes 10 × PCRBuffer, Taq enzyme, dNTPs, nuclease-free water, positive control and negative control.
7. kit according to claim 6, which is characterized in that the positive control includes cervical cancer cell lines CaSki； The negative control includes cervical cancer cell lines C33A.
8. the application method of claim 4~7 any one kit, comprising the following steps:
1) sample to be examined DNA is extracted；
2) DNA by the sample to be examined DNA through sulphite conversion processing, after being converted；
3) it using the DNA after the conversion as template, is combined using primer and probe described in Claims 2 or 33 and carries out PCR amplification Reaction, obtains fluorescence detection result；
4) according to the fluorescence detection as a result, determining sample to be examined, when-actin≤35 β, and testing result is FAM19A4 or miR-124-2 any gene Δ Ct≤9, then determine sample to be examined for cervical carcinoma methylation positive；As β-actin ≤ 35, and testing result is FAM19A4, has-miR-124-2, ZNF671 gene Δ Ct > 9, then determines that sample to be examined is FAM19A4, has-miR-124-2, ZNF671 methylation are negative；As β-actin > 35, then determine that sample is insufficient or has mortifier In the presence of PCR reaction is invalid, need to repeat to test or sample；
The Δ Ct=FAM Ct-VIC Ct.
9. application method according to claim 8, which is characterized in that pcr amplification reaction described in step 3) uses anti- Answering system includes FAM19A4 amplification system, has-miR-124-2 amplification system and ZNF671 amplification system；
The FAM19A4 amplification system includes: 18.3 μ L of FAM19A4 PCR reaction solution, 0.2 μ L of Taq enzyme and conversion in terms of 20 μ L DNA1.5 μ L afterwards；The FAM19A4 PCR reaction solution includes the component of following concentration: PCRbuffer1 ×, dNTP0.25mM, FAM19A4 forward primer F0.4uM, FAM19A4 reverse primer R0.4uM, FAM19A4 probe P0.4uM, β-actin-F0.4uM, β-actin-R0.4uM and β-actin-MP0.4uM；
The has-miR-124-2 amplification system includes: 18.3 μ L of has-miR-124-2 PCR reaction solution, Taq enzyme in terms of 20 μ L The 0.2 μ L and DNA1.5 μ L after conversion；The has-miR-124-2 PCR reaction solution includes the component of following concentration: PCRbuffer1 ×, dNTP0.25mM, has-miR-124-2 forward primer F0.4uM, has-miR-124-2 reverse primer R0.4uM, has-miR-124-2 probe P0.4uM, β-actin-F0.4uM, β-actin-R0.4uM and β-actin- MP0.4uM；
After the ZNF671 amplification system includes: 18.3 μ L of ZNF671 PCR reaction solution, 0.2 μ L of Taq enzyme in terms of 20 μ L and converts DNA1.5 μ L；The ZNF671 PCR reaction solution includes the component of following concentration: PCRbuffer1 ×, dNTP0.25mM, ZNF671 forward primer F0.4uM, ZNF671 reverse primer R0.4uM, ZNF671 probe P0.4uM, β-actin-F0.4uM, β- Actin-R0.4uM and β-actin-MP0.4uM.
10. preparation method according to claim 8, which is characterized in that the program of pcr amplification reaction described in step 3) Are as follows: 95 DEG C of 5min, [95 DEG C of 15s, 60 DEG C of 30s, 50 circulations], reaction terminates；Fluorescence is acquired during 60 DEG C of 30s.
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CN112646882A (en) * 2020-07-03 2021-04-13 苏州凯爱健康科技有限公司 Composition and diagnostic reagent for detecting cervical high-grade lesion and cervical cancer
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CN112195251A (en) * 2020-11-30 2021-01-08 中南大学 Molecular marker, primer, method and kit for early screening of cervical cancer
CN112501297A (en) * 2020-12-03 2021-03-16 广东辉锦创兴生物医学科技有限公司 Fluorescent quantitative PCR detection kit for detecting methylation of Septin9 gene and application thereof
CN113265456A (en) * 2021-03-17 2021-08-17 苏州凯爱健康科技有限公司 Primer and probe combination for detecting cervical high-grade lesion and cervical cancer related gene methylation
CN112941189A (en) * 2021-05-06 2021-06-11 中南大学 Primer probe composition and kit for methylation detection of cervical cancer early-screening molecular marker ZNF671 gene and using method
CN112941189B (en) * 2021-05-06 2023-07-21 中南大学 Primer probe composition and kit for methylation detection of cervical cancer early screening marker ZNF671 gene and use method
WO2022238542A3 (en) * 2021-05-12 2022-12-15 Ecole Polytechnique Federale De Lausanne (Epfl) Krab-containing zinc finger protein and cancer
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