CN116024349A - Primer probe combination and kit for methylation detection of cervical cancer - Google Patents
Primer probe combination and kit for methylation detection of cervical cancer Download PDFInfo
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Abstract
The invention provides a primer probe combination and a kit for detecting methylation of cervical cancer, which comprise a PAX1 primer probe combination for detecting methylation of a PAX1 gene and an EPB41L3 primer probe combination for detecting methylation of an EPB41L3 gene, wherein methylation specific fluorescent quantitative PCR (qMSP) is adopted to detect methylation target genes PAX1 and EPB41L3, the primer probes meeting detection requirements are screened to obtain an optimal combination, and simultaneous detection of 2 methylation target genes is realized through multichannel fluorescent probe marking.
Description
Technical Field
The application relates to the field of cervical cancer methylation detection, in particular to a primer probe combination and a kit for cervical cancer methylation detection.
Background
Cervical cancer is one of the main cancers threatening the life health of women in China, and the onset age of cervical cancer is approaching to younger in recent years. According to statistics, the new cervical cancer cases and the death cases of China in 2020 are about 11 ten thousand and 5.9 ten thousand, and the cervical cancer is the fifth female cancer susceptible species at present.
Cervical cancer is taken as a cancer species with definite cause, and early screening is an effective method for preventing cervical cancer. The currently mainly used detection means mainly comprise cytology examination and HPV detection, and the cytology detection comprises traditional Papanicolaou smear method and liquid-based cytology examination. The traditional Papanicolaou smear method has low cost and higher repeatability, but the film-making method can cause missed diagnosis or misdiagnosis to a certain extent, has lower accuracy and sensitivity, and is stronger in subjective factors of doctors; the liquid-based cytology examination is influenced by factors such as subjective experience of doctors, sampling, tabletting and the like, the automation application degree is low, meanwhile, the sensitivity and the specificity are not obviously advantageous, and the false negative condition exists; HPV detection has higher sensitivity but poorer specificity, has a certain degree of false positive, and often carries out repeated examination to increase the risk of re-infection, and is easy to cause mental panic of HPV patients; VIA inspection, while low cost, requires less infrastructure conditions, is less specific and sensitive. In other words, the conventional cervical cancer screening methods at present have more or less technical defects.
DNA methylation is an important epigenetic modification, and with the progressive development of molecular biology, early events of cancer occurrence have been found to be associated with epigenetic abnormalities, and the literature has shown that cancer occurrence is associated with DNA methylation of promoters and other regions of tumor suppressor genes, and research on epigenetic studies have demonstrated that DNA methylation can be used for early recognition and analysis of cervical cancer, and that DNA methylation has a fairly broad prospect in cancer diagnosis. The current common DNA methylation detection methods include: methylation Specific PCR (MSP), bisulfite Sequencing (BSP), high resolution melting curve methods, and the like. Sulfite sequencing is considered a gold standard for DNA methylation analysis, and is a method of amplifying a target fragment by PCR through sulfite treatment, sequencing a PCR product, and judging whether CpG sites are methylated. The method is reliable and high in accuracy, but the process is complex and expensive. The high resolution melting curve method can judge the methylation degree according to the melting temperature and the peak type change, but the method has higher requirement on instruments. Compared with the methylation specific PCR detection method, the methylation specific PCR detection method is economical and practical, no special instrument is needed, the method is high in sensitivity, free of restriction of endonuclease, low in detection cost and short in time consumption, and is the most widely used methylation detection method at present, however, different schemes aim at the CpG sites involved, and the final detection specificity and sensitivity of the different schemes are different.
Disclosure of Invention
The invention aims to provide a primer probe combination and a kit for detecting cervical cancer methylation, and provides a primer probe combination which is high in sensitivity and high in specificity and is used for detecting cervical cancer gene methylation, so that the joint detection of polygene methylation is realized, and further early screening and diversion of cervical cancer are realized.
Specifically, the primer probe combination for cervical cancer methylation detection provided by the scheme comprises a PAX1 primer probe combination for detecting PAX1 gene methylation and an EPB41L3 primer probe combination for detecting EPB41L3 gene methylation, wherein the PAX1 primer probe combination is selected from any one of the following combinations:
PAX1 primer probe combination 1: the sequence is shown in SEQ ID NO:1, and the PAX1 forward primer 1 has a sequence shown as SEQ ID NO:2, and the sequence of the PAX1 negative primer 1 is shown as a PAX1 probe 1 shown as SEQ ID NO. 3;
PAX1 primer probe combination 2: the sequence is shown in SEQ ID NO:4, and the PAX1 forward primer 2 has a sequence shown as SEQ ID NO:5, the PAX1 negative primer 2 has a sequence shown as SEQ ID NO.6, and a PAX1 probe 2;
PAX1 primer probe combination 3: the sequence is shown in SEQ ID NO:7, the PAX1 forward primer 3 has a sequence shown as SEQ ID NO:8, and a PAX1 negative primer 3 with a sequence shown as SEQ ID NO. 9 and a PAX1 probe 3;
wherein the EPB41L3 primer probe combination is selected from any one of the following combinations:
EPB41L3 primer probe combination 1: EPB41L3 forward primer 1 shown in SEQ ID NO. 10, EPB41L3 reverse primer 1 shown in SEQ ID NO. 11, and EPB41L3 reverse primer 1 shown in SEQ ID NO:12, EPB41L3 probe 1;
EPB41L3 primer probe combination 2: EPB41L3 forward primer 2 shown in SEQ ID NO. 13, EPB41L3 reverse primer 2 shown in SEQ ID NO. 14, and EPB41L3 reverse primer 2 shown in SEQ ID NO:15 EPB41L3 probe 2;
EPB41L3 primer probe combination 3: EPB41L3 forward primer 3 shown in SEQ ID NO. 16, EPB41L3 reverse primer 3 shown in SEQ ID NO. 17, and EPB41L3 reverse primer 3 shown in SEQ ID NO:18, EPB41L3 probe 3.
The sequences of the PAX1 primer probe combination and the EPB41L3 primer probe combination are shown in the following table one:
surface PAX1 primer probe combination and EPB41L3 primer probe combination sequence
In some embodiments, the PAX1 primer pair detects the No.238, 239, 241 site of the PAX1 gene.
The following italics are in place: CGGGGGGGTGGTGGAAGAGGGTCGAGGGGGGAGGATAGAAGGAGGGGGTAGAGTTTTAGGGCGGGGAGGGGGGCGTTGGGGCGTAGTGACGGGAATTAATGAGTTGTTAATTCGCGCGTTTTCGGCGTGATTGTCGAGATTGACGTGGAGGATACGTTAAATTGATTTTCGTACGTTGTAGTTTTTCGGTTAGACGAATTTTTTTTAATCGGATGAAGTTTATTTTGGGTTTGGGGTCGCGGGCGTGGAGAGTGTTTTGGGAGGGGGTAGTAGCGGCGGCGGTAGGTTTTGGAGCGGGCGGTAGCGCGTTTCGTTGTCGCGTATAGCGCGTTTTTAGTTCGCGGTTGGGTCGTCGCGGTTTTCGGTTTTCGGGCGTTTTTTTTTTATGTTTTTTACGCGGCGGCGGCGGCGTTTAAGTTTTTTCGGATTGCGTCGGGTTTAGTTTCGGTTATTTCGGTTATTTCGGCGTTAGGTAGTTGGTCGGTTCGTTCGTTATGGGT。
In some embodiments, the EPB41L3 primer pair detects positions No.65,71,75 of the EPB41L3 gene.
In some embodiments, the primer probe combinations comprise an internal reference gene primer probe combination, the most common ATCB gene is selected as the internal reference gene, wherein the internal reference gene primer probe combination is selected from any one of the following combinations:
internal reference gene primer probe combination 1: the sequence is shown in SEQ ID NO:19, and the sequence of the reference gene forward primer 1 is shown as SEQ ID NO:20, and an internal reference gene probe 1 with a sequence shown as SEQ ID NO. 21;
internal reference gene primer probe combination 2: the sequence is shown in SEQ ID NO:22, and the sequence of the reference gene forward primer 2 is shown as SEQ ID NO:23, and an internal reference gene negative primer 2 with a sequence shown as SEQ ID NO. 24;
internal reference gene primer probe combination 3: the sequence is shown in SEQ ID NO:25, and the sequence of the reference gene forward primer 3 is shown as SEQ ID NO:26, and an internal reference gene negative primer 3 with a sequence shown as SEQ ID NO. 27.
The sequence of the internal reference gene primer probe combination is shown in the following table two:
sequence of exterior two internal reference gene primer probe combination
In some embodiments, FAM, JOE, VIC, HEX, ROX, FITC, CY or CY5 is selected as the fluorescent moiety at the 5 'end of the PAX1 gene probe and EPB41L3 gene probe, and one of BHQ1, DABCYL, BHQ2, BHQ3, TAMRA, DABCYL, SQ1, or SQ2 is selected as the quenching moiety at the 3' end of the PAX1 gene probe and EPB41L3 gene probe.
In some embodiments, FAM, JOE, VIC, HEX, ROX, FITC, CY3 or CY5 is selected as the fluorophore at the 5 'end of the reference gene probe and BHQ1, DABCYL, BHQ2, BHQ3, TAMRA, DABCYL, SQ1 or SQ2 is selected as the quencher at the 3' end of the reference gene probe.
In some embodiments, the PAX1 gene probe has FAM as the luminophore at the 5 'end and BHQ1 as the quencher at the 3' end; the 5 '-end of the reference gene ACTB probe uses VIC as a luminous group, and the 3' -end uses TAMRA as a quenching group.
In some embodiments, the EPB41L3 gene probe has FAM as the luminophore at the 5 'end and DABCYL as the quencher at the 3' end; the 5 '-end of the reference gene ACTB probe uses VIC as a luminous group, and the 3' -end uses TAMRA as a quenching group.
The sequence NCBI database numbers of the PAX1 gene detected by the scheme are NC_000020.11 and NC_000018.10 respectively, and the sequence NCBI database numbers of the EPB41L3 gene.
In some embodiments, the primer probe combination for cervical cancer methylation detection detects PAX1 gene methylation and EPB41L3 gene methylation simultaneously.
In some embodiments, primer probe combinations for cervical cancer methylation detection detect PAX1 gene methylation, EPB41L3 gene methylation, and reference genes simultaneously, and corresponding gene methylation degree judgment is performed by detecting differences between target Ct values and reference Ct values of the reference genes through PAX1 gene methylation and/or EPB41L3 gene methylation.
In some embodiments, the cell preservation solution of the cervical exfoliated cells is centrifuged to remove the supernatant, the cervical exfoliated cells in the cell preservation solution are extracted with DNA nucleic acid, the DNA nucleic acid is subjected to sulfite conversion to obtain a test object, and the test object is subjected to PAX1 gene methylation detection and EPB41L3 methylation detection by using a diagnostic reagent or a kit for cervical cancer methylation detection.
In a second aspect, the present solution provides the use of the above-mentioned primer probe combination for cervical cancer methylation detection in the preparation of a cervical cancer methylation diagnostic reagent or kit.
In a third aspect, the present disclosure provides a diagnostic reagent or kit for methylation detection of cervical cancer, comprising a primer probe combination for methylation detection of cervical cancer.
In some embodiments, the diagnostic reagent or kit for methylation detection of cervical cancer comprises a PCR reaction solution and a hot start enzyme, wherein the PCR reaction solution comprises a PAX1 gene methylation PCR reaction solution comprising a PAX1 primer probe combination, an internal reference gene primer pair, an internal reference gene probe, and a base solution, and an EPB41L3 gene methylation PCR reaction solution comprising an EPB41L3 primer probe combination, an internal reference gene primer pair, an internal reference gene probe, and a base solution.
In some embodiments, the hot start enzyme is a faststat PCR start enzyme.
In some embodiments, the base fluid comprises a Tris solution, (NH) 4 ) 2 SO 4 Solution, mgCl 2 Solution, dNTP solution.
In some embodiments, the final concentration of Tris solution is 10-40mM.
In some embodiments, the final concentration of the (NH 4) 2SO4 solution is 10-40mM.
In some embodiments, the final concentration of MgCl2 solution is 4-10mM.
In some embodiments, the final concentration of dNTP solution is 0.4-0.8mM.
In some embodiments, the PCR reaction system is 15 μl of reaction solution, 15 μl of post-conversion DNA template; the reaction amplification procedure was 94℃for 10min; [55.5 ℃ 35s,93 ℃ 30s,45 cycles ],40 ℃ 5s. The collection of fluorescent signals was performed at 55.5 ℃.
In some embodiments, the diagnostic reagent or kit for methylation detection of cervical cancer uses CpG methylated genomic DNA and human genomic DNA as positive controls and water as negative controls.
In some embodiments, diagnostic reagents or kits for methylation detection of cervical cancer employ methylation-specific fluorescent quantitative PCR methods for detection of PAX1 gene methylation and EPB41L3 gene methylation.
In some embodiments, the primer probe combination for cervical cancer methylation detection detects PAX1 gene methylation and EPB41L3 gene methylation simultaneously.
In some embodiments, primer probe combinations for cervical cancer methylation detection detect PAX1 gene methylation, EPB41L3 gene methylation, and reference genes simultaneously, and corresponding gene methylation degree judgment is performed by detecting differences between target Ct values and reference Ct values of the reference genes through PAX1 gene methylation and/or EPB41L3 gene methylation.
Specifically, in one embodiment, the components of the diagnostic reagent or the kit for methylation detection of cervical cancer provided in the present embodiment are shown in table three:
three-purpose diagnostic reagent or kit for methylation detection of cervical cancer
Compared with the prior art, the technical scheme has the following characteristics and beneficial effects:
1. the invention adopts a methylation specific fluorescent quantitative PCR method (qMSP method) to detect methylation target genes PAX1 and EPB41L3, and obtains the optimal combination after screening primer probes meeting detection requirements, and realizes simultaneous detection of 2 methylation target genes through multichannel fluorescent probe labeling.
2. ACTB is selected as an internal reference gene for two target genes, the target genes and the internal reference genes are detected simultaneously according to the multi-channel fluorescent probe marks, and finally, the methylation degree is judged according to delta Ct values (target Ct value-internal reference Ct value) of the two genes.
The scheme provides a reaction solution and a detection method for methylation PCR of cervical cancer, provides a matched method for extracting sample nucleic acid and converting sulfite, takes DNA converted by sulfite as a template for PCR reaction, and simultaneously realizes detection of two target genes PAX1 and EPB41L 3.
Drawings
FIG. 1 is a schematic diagram of DNA methylation.
FIG. 2 is a typical methylation PCR amplification curve of cervical cancer shed cell samples.
FIG. 3 is a typical methylation PCR amplification curve of a normal cervical exfoliated cell sample.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which are derived by a person skilled in the art based on the embodiments of the invention, fall within the scope of protection of the invention.
It will be appreciated by those skilled in the art that in the present disclosure, the terms "longitudinal," "transverse," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," etc. refer to an orientation or positional relationship based on that shown in the drawings, which is merely for convenience of description and to simplify the description, and do not indicate or imply that the apparatus or elements referred to must have a particular orientation, be constructed and operated in a particular orientation, and therefore the above terms should not be construed as limiting the present invention.
1. Designing and synthesizing a primer probe:
aiming at PAX1, EPB41L3 and internal reference gene ACTB loci, using Primer preimar 5.0 software, designing a PAX1 Primer probe combination, an EPB41L3 Primer probe combination and an internal reference gene Primer probe combination, wherein the PAX1 Primer probe combination and the EPB41L3 Primer probe combination are designed as shown in the following table I and table II:
surface PAX1 primer probe combination and EPB41L3 primer probe combination sequence
Sequence of exterior two internal reference gene primer probe combination
2. Selecting a quality control product:
2.1 negative quality control was water, used to monitor the whole PCR reaction.
2.2 cationic quality control consists of two DNA: cpG methylation genome derived from DNA extracted from blood of healthy individuals, treatment with CpG methyltransferase (M.SssI), purification and dissolution in TE buffer, and detection of CpG methylation level of 98% or more by sulfite sequencing, wherein the human genome DNA is derived from a plurality of anonymous donors, and methylation level of less than 1% is mixed at a methylation ratio of 100%, 1%, 0%.
3. Methylation specific PCR reaction solution composition
The cervical cancer methylation detection kit composition comprises PAX1 gene methylation PCR reaction liquid and EPB41L3 gene methylation PCR reaction liquid, wherein the PAX1 gene methylation PCR reaction liquid is used for detecting methylation of the PAX1 gene, and the EPB41L3 gene methylation PCR reaction liquid is used for detecting methylation of the EPB41L3 gene.
Wherein the PAX1 gene methylation PCR reaction solution comprises the following components: PAX1 primer probe combination, reference gene ACTB forward primer, reference gene ACTB reverse primer, reference gene ACTB probe, tris, (NH 4) 2SO4, mgCl2 and dNTP.
Wherein the PAX1 primer probe combination is selected from any one of the following combinations:
PAX1 primer probe combination 1: the sequence is shown in SEQ ID NO:1, and the PAX1 forward primer 1 has a sequence shown as SEQ ID NO:2, and the sequence of the PAX1 negative primer 1 is shown as a PAX1 probe 1 shown as SEQ ID NO. 3;
PAX1 primer probe combination 2: the sequence is shown in SEQ ID NO:4, and the PAX1 forward primer 2 has a sequence shown as SEQ ID NO:5, the PAX1 negative primer 2 has a sequence shown as SEQ ID NO.6, and a PAX1 probe 2;
PAX1 primer probe combination 3: the sequence is shown in SEQ ID NO:7, the PAX1 forward primer 3 has a sequence shown as SEQ ID NO:8, and the sequence of the PAX1 negative primer 3 is shown as a PAX1 probe 3 shown as SEQ ID NO. 9.
Wherein the EPB41L3 gene methylation PCR reaction solution comprises the following components: EPB41L3 primer probe combination, reference gene ACTB forward primer, reference gene ACTB reverse primer, reference gene ACTB probe, tris, (NH 4) 2SO4, mgCl2 and dNTP.
Wherein the EPB41L3 primer probe combination is selected from any one of the following combinations:
EPB41L3 primer probe combination 1: EPB41L3 forward primer 1 shown in SEQ ID NO. 10, EPB41L3 reverse primer 1 shown in SEQ ID NO. 11, and EPB41L3 reverse primer 1 shown in SEQ ID NO:12, EPB41L3 probe 1;
EPB41L3 primer probe combination 2: EPB41L3 forward primer 2 shown in SEQ ID NO. 13, EPB41L3 reverse primer shown in SEQ ID NO. 14, 2, EPB41L3 reverse primer shown in SEQ ID NO:15 EPB41L3 probe 2;
EPB41L3 primer probe combination 3: EPB41L3 forward primer 3 shown in SEQ ID NO. 16, EPB41L3 reverse primer shown in SEQ ID NO. 17, and EPB41L3 reverse primer 3 shown in SEQ ID NO:18, EPB41L3 probe 3.
Wherein the 5 'end of the EPB41L3 gene probe uses FAM as a luminous group and the 3' end uses DABCYL as a quenching group; the 5 '-end of the reference gene ACTB probe uses VIC as a luminous group, and the 3' -end uses TAMRA as a quenching group.
Wherein FAM is used as a luminous group at the 5 'end of the PAX1 gene probe, and BHQ1 is used as a quenching group at the 3' end; the 5 '-end of the reference gene ACTB probe uses VIC as a luminous group, and the 3' -end uses TAMRA as a quenching group.
Wherein all primers were 0.6. Mu.M in final concentration, probes were 0.3. Mu.M in final concentration, tris was 20mM in final concentration, the (NH) 4 ) 2 SO 4 Final concentration of 20mM, wherein the MgCl 2 The final concentration is 4-10mM, and the final concentration of dNTP is 0.4-0.8mM.
4. Cervical cancer methylation detection: 4.1 collection of clinical samples:
sample design: clinical TCT sample preservation fluid was selected, 12 positive samples, 103 negative samples.
For each tested person, placing cervical brush at cervical orifice, rotating in one direction to obtain sufficient cervical epithelial shedding cells, placing cervical brush in tube filled with cervical epithelial shedding cell preservation solution, wherein the cell preservation solution is JIN DIAN cell preservation solution, hao Luo Jie PreservCyt cell preservation solution, etc., making sample mark, and storing at-20deg.C
4.2 extraction of sample nucleic acids:
taking 0.5-1.5mL of cell preservation solution containing cervical exfoliated cells as a sample, and extracting and purifying DNA:
(1) Mixing the sample with the sample for several times upside down, centrifuging, discarding the supernatant, and retaining the cell sediment;
(2) Adding 350 mu Lg guanidine salt lysate, vortexing, standing for 10min until clear;
(3) Adding 40-50 μl of magnetic beads and 250 μl of absolute ethanol, mixing by vortex, and standing at 1000rpm for 30min; in the embodiment of the scheme, 45 mu L of magnetic beads are added;
(4) Centrifuging for a short time, placing the centrifuge tube on a magnetic rack for 2-4min, and discarding the supernatant;
(5) Adding 800 μl of washing solution A, vortexing, centrifuging briefly, standing in a magnetic rack for 2-4min, and discarding supernatant; in the embodiment of the scheme, the magnetic frame is arranged for 3min;
(6) Adding 800 μl of washing solution B, vortexing, centrifuging briefly, standing in a magnetic rack for 2-4min, and discarding supernatant; in the embodiment of the scheme, the magnetic frame is arranged for 3min;
(7) Adding 400 μl of washing solution B, vortexing, centrifuging briefly, standing in a magnetic rack for 2-4min, and discarding supernatant; in the embodiment of the scheme, the magnetic frame is arranged for 3min;
(8) Centrifuging for a short time, and sucking out residual liquid by using a pipetting gun;
(9) Placing the centrifuge tube in a constant temperature oscillator for drying for 8-10min, and injecting: drying for too long and at too high a temperature can result in a decrease in DNA recovery; drying for 9min in the examples of the present protocol;
(10) Adding 55 mu LTris eluent, mixing by vortex, fully oscillating and re-suspending magnetic beads, and placing the beads in a constant-temperature oscillator at 1000rpm for 10min;
(11) The centrifuge tube was briefly centrifuged, placed in a magnet rack for 2-4min, the eluate was transferred to a new centrifuge tube, and the DNA concentration was determined using NanoDrop.
The eluent a and the eluent B in this embodiment were guanidine salt solutions.
4.3 conversion of sulfite:
taking 500-2000ng DNA to perform sulfite chemical conversion:
(1) Adding 150 mu L of sulfite solution and 25 mu L of DNA protective solution into a centrifuge tube at one time, and carrying out vortex mixing and short centrifugation; placing the centrifuge tube filled with the reaction liquid in a constant temperature oscillator at 80 ℃ for 45min;
(2) Taking out the centrifuge tube filled with the converted reaction liquid, centrifuging briefly, sequentially adding 1mL of washing liquid A and 20 mu L of magnetic beads, mixing uniformly by vortex, and placing the mixture in a constant-temperature oscillator at 23 ℃ at 1000rpm for 10min;
(3) Taking out the centrifuge tube, centrifuging for a short time, placing the centrifuge tube on a magnetic rack for 2-4min, and discarding the supernatant; in the embodiment of the scheme, the magnetic frame is arranged for 3min;
(4) Adding 800 μl of washing solution A, vortexing, centrifuging briefly, standing in a magnetic rack for 2-4min, and discarding supernatant; in the embodiment of the scheme, the magnetic frame is arranged for 3min;
(5) Adding 800 μl of washing solution B, vortexing, centrifuging briefly, standing in a magnetic rack for 2-4min, and discarding supernatant; in the embodiment of the scheme, the magnetic frame is arranged for 3min;
(6) Adding 400 μl of washing solution B, vortexing, centrifuging briefly, standing in a magnetic rack for 2-4min, and discarding supernatant; in the embodiment of the scheme, the magnetic frame is arranged for 3min;
(7) Centrifuging for a short time, and sucking out residual liquid by using a pipetting gun;
(8) Placing the centrifuge tube in a constant temperature oscillator for drying for 8-10min, and injecting: drying for too long and at too high a temperature can result in a decrease in DNA recovery; drying for 9min in the examples of the present protocol;
(9) Adding 50-60 μl of eluent, mixing by vortex, shaking thoroughly to re-suspend magnetic beads, and standing at 1000rpm for 10min; in the examples of this protocol 55. Mu.L of eluent was added
(10) Centrifuging briefly, placing the centrifuge tube in a magnetic rack for 2-4min, and transferring the eluate containing bis DNA into a new centrifuge tube; in the embodiment of the scheme, the magnetic frame is arranged for 3min.
4.4 extraction of sample nucleic acids:
the reaction system for methylation of cervical cancer methylation marker PAX1 gene and EPB41L3 gene is configured according to the following tables four to five:
methylation-specific PCR reaction system of surface four PAX1 genes
Table five EPB41L3 methylation specific PCR reaction system
The prepared 96-well plate is placed on ABI7500 for fluorescence PCR reaction, and the reaction conditions are shown in the following table six:
table six fluorescent PCR reaction condition parameters
And setting the collection of fluorescent signals at the temperature of 50-58 ℃.
Wherein, the PAX1 methylation specific PCR reaction system is used for detecting the PAX1 of the human genome, and the fluorescent channels are set as FAM and VIC and are respectively used for detecting the PAX1 gene and the ACTB reference gene.
The EPB41L3 methylation specific PCR reaction system is used for detecting human genome EPB41L3, and fluorescent channels are set to FAM and VIC and are respectively used for detecting EPB41L3 genes and ACTB reference genes.
5. Analysis of results:
the threshold value of the fluorescence channel FAM for detecting the PAX1, EPB41L3 genes was set to 50000, the threshold value of the fluorescence channel VIC for detecting the reference gene ACTB was 25000, and the DeltaCt calculation method of the PAX1, EPB41L3 genes and the reference gene was as follows:
Δct=pax1ct-ACTB Ct of PAX1 gene;
Δct=epb41L 3 Ct-ACTB Ct of EPB41L3 gene.
Furthermore, the validity interpretation method provided by the invention requires that the amplification curves of all amplification channels of all positive samples are typical S-shaped curves, and Ct of an internal reference gene ACTB is less than or equal to 35.
When one of the PAX1 and EPB41L3 genes is positive, the result of the sample to be tested is positive, and the result judgment standard is shown in the following table seven:
seven table decision criteria
The reaction results of the PCR reaction system of this scheme are shown in Table eight below, with the numbers ΔCt=FAM Ct-VIC Ct. The larger numbers indicate lower methylation ratios.
Table eight reaction results of PCR reaction System
The experimental results of the kit 1 composed of PAX1 primer probe combination 1, EPB41L3 primer probe combination 1 and internal reference gene primer probe 1 are shown in Table nine:
test results of Table nine kit 1
The clinical sample results are analyzed, the sensitivity of detecting cervical high-grade lesions (HSIL) by the two targets of PAX1 and EPB41L3 is 92.3 percent, the specificity is 100 percent, and the detection capability of the invention on cervical high-grade lesions and cervical cancer is fully embodied.
The present invention is not limited to the above-described preferred embodiments, and any person who can obtain other various products under the teaching of the present invention, however, any change in shape or structure of the product is within the scope of the present invention, and all the products having the same or similar technical solutions as the present application are included.
Claims (10)
1. A primer probe combination for cervical cancer methylation detection, comprising a PAX1 primer probe combination for detecting PAX1 gene methylation and an EPB41L3 primer probe combination for detecting EPB41L3 gene methylation, wherein the PAX1 primer probe combination is selected from any one of the following combinations:
PAX1 primer probe combination 1: the sequence is shown in SEQ ID NO:1, and the PAX1 forward primer 1 has a sequence shown as SEQ ID NO:2, and the sequence of the PAX1 negative primer 1 is shown as a PAX1 probe 1 shown as SEQ ID NO. 3;
PAX1 primer probe combination 2: the sequence is shown in SEQ ID NO:4, and the PAX1 forward primer 2 has a sequence shown as SEQ ID NO:5, the PAX1 negative primer 2 has a sequence shown as SEQ ID NO.6, and a PAX1 probe 2;
PAX1 primer probe combination 3: the sequence is shown in SEQ ID NO:7, the PAX1 forward primer 3 has a sequence shown as SEQ ID NO:8, and a PAX1 negative primer 3 with a sequence shown as SEQ ID NO. 9 and a PAX1 probe 3;
wherein the EPB41L3 primer probe combination is selected from any one of the following combinations:
EPB41L3 primer probe combination 1: EPB41L3 forward primer 1 shown in SEQ ID NO. 10, EPB41L3 reverse primer 1 shown in SEQ ID NO. 11, and EPB41L3 reverse primer 1 shown in SEQ ID NO:12, EPB41L3 probe 1;
EPB41L3 primer probe combination 2: EPB41L3 forward primer 2 shown in SEQ ID NO. 13, EPB41L3 reverse primer 2 shown in SEQ ID NO. 14, and EPB41L3 reverse primer 2 shown in SEQ ID NO:15 EPB41L3 probe 2;
EPB41L3 primer probe combination 3: EPB41L3 forward primer 3 shown in SEQ ID NO. 16, EPB41L3 reverse primer 3 shown in SEQ ID NO. 17, and EPB41L3 reverse primer 3 shown in SEQ ID NO:18, EPB41L3 probe 3.
2. The primer probe combination for cervical cancer methylation detection according to claim 1, wherein the primer probe combination comprises an internal reference gene primer probe combination selected as an ATCB gene, wherein the internal reference gene primer probe combination is selected from any one of the following combinations:
internal reference gene primer probe combination 1: the sequence is shown in SEQ ID NO:19, and the sequence of the reference gene forward primer 1 is shown as SEQ ID NO:20, and an internal reference gene probe 1 with a sequence shown as SEQ ID NO. 21;
internal reference gene primer probe combination 2: the sequence is shown in SEQ ID NO:22, and the sequence of the reference gene forward primer 2 is shown as SEQ ID NO:23, and an internal reference gene negative primer 2 with a sequence shown as SEQ ID NO. 24;
internal reference gene primer probe combination 3: the sequence is shown in SEQ ID NO:25, and the sequence of the reference gene forward primer 3 is shown as SEQ ID NO:26, and an internal reference gene negative primer 3 with a sequence shown as SEQ ID NO. 27.
3. The primer probe combination for methylation detection of cervical cancer according to claim 1, wherein the PAX1 primer pair detects the No.238, 239, 241 site of the PAX1 gene.
4. The primer probe combination for cervical cancer methylation detection according to claim 1, wherein the EPB41L3 primer pair detects positions No.65,71,75 of the EPB41L3 gene.
5. Use of a primer probe combination for methylation detection of cervical cancer according to any one of claims 1 to 4 for preparing a cervical cancer methylation diagnostic reagent.
6. A diagnostic reagent or kit for methylation detection of cervical cancer, comprising a primer probe combination for methylation detection of cervical cancer according to any one of claims 1 to 4.
7. The diagnostic reagent or kit for methylation detection of cervical cancer according to claim 6, comprising a PCR reaction solution and a hot start enzyme, wherein the PCR reaction solution comprises a PAX1 gene methylation PCR reaction solution comprising a PAX1 primer probe combination, an internal reference gene primer pair, an internal reference gene probe and a base solution, and an EPB41L3 gene methylation PCR reaction solution comprising an EPB41L3 primer probe combination, an internal reference gene primer pair, an internal reference gene probe and a base solution.
8. The diagnostic reagent or kit for methylation detection of cervical cancer according to claim 7, comprising CpG methylated genomic DNA and human genomic DNA as positive controls, and water as negative controls.
9. The diagnostic reagent or kit for methylation detection of cervical cancer according to claim 6, wherein methylation-specific fluorescent quantitative PCR method is used to detect PAX1 gene methylation and EPB41L3 gene methylation simultaneously.
10. The diagnostic reagent or kit for cervical cancer methylation detection according to claim 7, wherein the corresponding degree of gene methylation is determined by the difference between the Ct value of the target for detection of PAX1 gene methylation and/or EPB41L3 gene methylation and the Ct value of the reference gene.
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