CN118086509A - Primer pair, kit and application of kit for detecting Gong Gengliang malignant lesion gene methylation - Google Patents
Primer pair, kit and application of kit for detecting Gong Gengliang malignant lesion gene methylation Download PDFInfo
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Abstract
The invention provides a primer pair for detecting Gong Gengliang malignant lesion gene methylation and a kit thereof, and relates to the technical field of Gong Gengliang malignant lesion detection, wherein the primer pair comprises at least any pair of biomarkers as a primer sequence corresponding to a methylation marker, and the methylation markers are respectively selected from PAX1 genes, GYPC genes and ZIK genes. According to the invention, cpG island analysis is carried out on promoter regions of PAX1 genes, GYPC genes and ZIK genes, the related regions of different CpG islands are used as target genes for primer probe design, and amplification conditions are optimized, so that a primer pair which is mutually combined and can be used for methylation detection of cervical cancer is obtained, and the technical problem that the sensitivity and the specificity of final detection are different due to the primer probes which are designed for CpG sites in the prior art is solved.
Description
Technical Field
The invention relates to the technical field of Gong Gengliang malignant lesion detection, in particular to a primer pair, a kit and application of the kit for detecting Gong Gengliang malignant lesion gene methylation.
Background
Cervical cancer is the most common gynaecological malignancy, and its onset has been a trend of more and more younger in recent years. Persistent infection with high-risk human papilloma virus (hrHPV) is the main cause of cervical cancer and precancerous lesions, more than 90% of cervical cancers are accompanied by hrHPV infection, but most women infected with HPV can clear them through their own immune system and do not develop into cervical cancer precancerous lesions or cervical cancer.
Currently, the methods used for cervical cancer screening are mainly HPV detection and cervical cytology. Because the HPV detection method has poor specificity, the detection result has high proportion of false positives, and the single HPV positive detection result cannot distinguish whether the HPV is continuously infected or not, and in addition, the risk of HPV infection is increased by repeated detection; liquid-based cytology examination (TCT) is greatly influenced by factors such as subjective experience of examination doctors, sampling, film-making and the like, has low accuracy and poor repeatability, and is easy to cause phenomena such as missed diagnosis, misdiagnosis and the like.
Gene methylation, a common epigenetic modification, is one of the most deeply studied epigenetic regulatory mechanisms. The gene promoter region is located in the region 1000bp before the start coding codon, and is rich in a large number of CpG sites and a large number of CpG sites are rich in CpG islands. Methylation of C in promoter region CpG affects the binding of transcription factors to transcription factor binding sites, and thus the normal expression of genes. Methylation of a gene promoter region can change along with change of lesion degree, and change of tumor progress can be monitored better. The method is used for detecting the methylation condition of human genome DNA, and has higher sensitivity and specificity; and the TCT test is used for detecting the residual sample, so that the panic of a patient can be avoided, and the test error caused by human factors can be reduced. However, the analysis of CpG islands in the promoter region of a gene revealed that there are a plurality of CpG islands in the promoter region of a gene, and that the sensitivity and specificity of the final detection are different due to the different design of primer probes for CpG sites.
Disclosure of Invention
The invention aims to provide a primer pair, a kit and an application of the kit, which have high sensitivity and specificity and are suitable for clinical molecular detection, so as to solve the technical problem that the sensitivity and the specificity of final detection are different due to primer probes which are designed for CpG sites in the prior art.
The primer pair for detecting Gong Gengliang malignant lesion gene methylation provided by the invention comprises at least any pair of biomarkers as primer sequences corresponding to methylation markers, wherein the methylation markers are respectively selected from PAX1 genes, GYPC genes and ZIK genes;
The primer sequences are specifically expressed as follows:
Forward primer of PAX1 gene: 5'-GTATTTCGCGTTCGACGAAGTTA-3', or 5'-GTGCGAGATTTTATCGTTAGGG-3', or 5'-TAGAGGGGTGGGTTTCGGG-3';
Reverse primer of PAX1 gene: 5'-ACACGCCTAAACCAAAAATTCCG-3', or 5'-AAAAATCCCGATCTACTAC-3', or 5'-ATTAATTCCCGTCACTACGC-3';
Forward primer of GYPC gene: 5'-CGTCGAGGGTTAGGAGTTCGGG-3', or 5'-AGTTACGGTTACGGACGTTTTG-3', or 5'-TCGATCGGTTAGGTTTCGGTG-3';
reverse primer of GYPC gene: 5'-TAACTACTACGAACCGCGACCT-3', or 5'-ACGAACTAACGACTTCTCCAATC-3', or 5'-ACGAAAAATTACAAACCCC-3';
forward primer of ZIK gene: 5'-AGTCGCGACGGGATTATAGA-3', or 5'-GTTGAGTTAATTCGCGTTGA-3', or 5'-AGTGGCGGTTATTTTTGTAGCG-3';
reverse primer of ZIK gene: 5'-ATACTACCGCCCAACGTCACC-3', or 5'-ACTATCGCCAAACAAAACGC-3', or 5'-CCGCCGACCCTTAAAACC-3'.
Furthermore, the primer pair for detecting Gong Gengliang malignant lesion gene methylation provided by the invention also comprises a group of primer sequences corresponding to a pair of reference markers COL2A1 genes, and the primer sequences are specifically expressed as follows:
forward primer of COL2A1 gene: 5'-ATTAGTGATTTGTATATGTGTG-3';
reverse primer of COL2A1 gene: 5'-AATTAAACTCTCCACCCACTAT-3'.
Furthermore, the invention also provides an application of the primer pair for detecting Gong Gengliang malignant lesion gene methylation in preparing a cervical cancer detection kit.
Further, the invention provides a kit for detecting Gong Gengliang malignant lesion gene methylation, which comprises a primer pair as described above.
Furthermore, the invention also provides an application of the kit for detecting Gong Gengliang malignant lesion gene methylation in preparing the kit for detecting Gong Gengliang malignant lesion gene methylation marker.
Further, in the process of detecting the Gong Gengliang malignant lesion biomarker by using the kit, the result of diagnosing cervical cancer by gene methylation is interpreted as follows:
the methylation level of a target region of a particular gene is reflected in a Δcp value of:
ΔCp(PAX1)= Cp(PAX1)- Cp(COL2A1)，
ΔCp(GYPC)= Cp(GYPC)- Cp(COL2A1)，
ΔCp(ZIK1)= Cp(ZIK1)- Cp(COL2A1)；
wherein COL2A1 is an internal reference gene.
Furthermore, in the process of detecting the Gong Gengliang malignant lesion biomarker by using the kit, the result interpretation mode of diagnosing the cervical cancer by adopting the PAX1 gene and GYPC gene combination is as follows:
。
Furthermore, in the process of detecting the Gong Gengliang malignant lesion biomarker by using the kit, the result interpretation mode of diagnosing the cervical cancer by using the PAX1 gene, GYPC gene and ZIK1 gene combination is as follows:
。
Compared with the prior art, the invention has the following beneficial effects:
According to the invention, cpG island analysis is carried out on promoter regions of PAX1 genes, GYPC genes and ZIK genes, and primer probe design is carried out by taking relevant regions of different CpG islands as target genes, so that amplification conditions are optimized, and a primer pair which is mutually combined and can be used for methylation detection of cervical cancer is obtained. The primer is applied to preparing a cervical cancer detection kit, and the kit has higher clinical value for detecting cervical lesions.
In addition to the objects, features and advantages described above, the present invention has other objects, features and advantages. The present invention will be described in further detail with reference to the drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:
FIG. 1 is a CpG island analysis of the PAX1 gene of example 1 of the present invention;
FIG. 2 is a GYPC gene CpG island analysis in example 1 of the present invention;
FIG. 3 is a ZIK gene CpG island analysis in example 1 of the present invention;
FIG. 4 is a graph showing the methylation of the first CpG island of the PAX1 gene in experimental example 1 of the present invention for diagnosing cervical cancer;
FIG. 5 is a graph showing the methylation of the second CpG island of the PAX1 gene in experimental example 1 of the present invention for diagnosing cervical cancer;
FIG. 6 is a graph of a subject diagnosed with cervical cancer by methylation of the third CpG island of the PAX1 gene in Experimental example 1 of the present invention;
FIG. 7 is a graph of a subject diagnosed with cervical cancer by methylation of a first CpG island-first segment-related site of the GYPC gene of Experimental example 1 of the present invention;
FIG. 8 is a graph of a subject diagnosed with cervical cancer by methylation of a site associated with the second segment of the first CpG island of the GYPC gene of Experimental example 1 of the present invention;
FIG. 9 is a graph of a subject diagnosed with cervical cancer by methylation of the GYPC gene of Experimental example 1 of the present invention;
FIG. 10 is a graph of a subject diagnosed with cervical cancer by methylation of the first CpG island of ZIK gene in Experimental example 1 of the present invention;
FIG. 11 is a graph of a subject diagnosed with cervical cancer by methylation of a first segment of relevant site of the ZIK gene of Experimental example 1 of the present invention;
FIG. 12 is a graph of the methylation of the relevant site of the second segment of the second CpG island of ZIK gene of experimental example 1 of the present invention for diagnosing cervical cancer.
Detailed Description
The following are specific embodiments of the present invention and the technical solutions of the present invention will be further described with reference to the accompanying drawings, but the present invention is not limited to these embodiments.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Example 1:
The primer pair for detecting Gong Gengliang malignant lesion gene methylation provided by the invention comprises at least any pair of biomarkers which are selected from a target gene PAX1 gene, a GYPC gene, a ZIK1 gene and a sequence about 1000bp before a start codon in an internal reference gene COL2A1, and CpG island analysis is carried out on a gene sequence fragment selected by the target gene by utilizing METHPRIMER on-line design software. The methylation sequence regions of the PAX1 gene, GYPC gene and ZIK gene are shown in Table 1.
TABLE 1 methylation sequence region in PAX1 Gene, GYPC Gene, ZIK Gene
Specifically, primer probe design was performed for the above target genes (PAX 1 gene, GYPC gene, ZIK gene, and internal reference gene COL2 A1):
And (one) performing CpG island analysis on gene sequence fragments selected from the target gene PAX1 gene, GYPC gene, ZIK gene and internal reference gene COL2A1 by utilizing METHPRIMER on-line design software.
Wherein, the promoter region sequence of the PAX1 gene (namely the gene sequence fragment selected by the PAX1 gene) is as follows:
TTTATCTCAGAGACTCCAAGGTCTGGAAATCAACCTTTCTTGGTATCTCGCGCCCGACGAAGCTAGCAGATTCCGCAATGGAGAGCACTGGGCAGGGTGCGTCCCCACTCCCCGCACCCCCGGAGCCCCGAGCATACTGTGCGTGCTTAGTCCTCTCTTCGCTGCCTTCGCGGAACTCCTGGCTTAGGCGTGCCCACTTTAATCCAAACCATTCCCTTTCCCTGTTCCTTACTCCAAACCTACGCTGTCATGGACAGGCCTAATCTTCTACATAGCTTCCCCTGCTCCCACTCTCGCACCCTCCTGCCAGTGCACTGACCCTCAAACCTCCTCCAGGTCTCCAGGACCTCCCAGAGCCAGAGACTGTACTGTGTTCATGCCGCCGGAGAGGAAGAGTTCACTTCTTTTAAACCTGATTTTTCTTTTTCCAGGAGTAATTCTTGTCTGTGGCCCCACCGCGTGCTCTCTCTAGAGAGTCCAGGAATTATGAGCCAGTCACTCCTGACAGATACATTTAGACAGGTGGATTAGGAGCCAGGGATGCTCTCTGGCCCTACAGCGGAGGCAAAGGGGTCGGGGCCTCCGCAGCCATGGCTCCCCAGGGTGGGCTGATAGGGTCCATGTGCTCCTCAGCCACTGTCACCGCGCTGGCGCCATACTCAACTCTGGGTGGCTAAGGAGAGCTAGACCCTTGGGGTGGGCAGGAGGGAGGGGAAGAGGGTTCTCTCCTCCAGGGGAGCGCTGGGAGTCTCAGAAACCTCGGCTGTTCCTACTCTCTCCCCTATGCCCTCTTCCTGGTCTAGCACATTCAGCCCAATGCCTTTGGTGCTTACTGGGCTCAACCTCAGAGCTCTCAGATCCTGACCCCCTCAAATCAGTTCATGTTCAAAACCACGAGTTTCCTCCTCTTGGCCCTCACCGCATGGCTCCCGGACACAGTTAGCTGCACAGGTTGGGGAGTTCAGAACTGCTTTGGTGCAGGGTAGAAACAGAGAAATTGTTGATGTTCAATCTCAGGGGTGAATAATTCAGGGCTGCGCAAAACCTCATTTAAATGAAGGAGAAATGACCATTCTGTGGTTTAACAGAGACTTCCTGAAGCTGCAGGTGCTGGGAGAAAGACGAGGCCAGGCCACCTGGGATAGAGTGGTGCGAGATTCCACCGCCAGGGAGAAAGGAACTTGTCCTTCAGACCTAGAGCTGGAGCTATGCATTTGGCCTCCCGCCGGTCGCGCTTGGGGACAGGAGGGCGCCGGATCTATGCGCCCCTTAGCAGCAGATCGGGATCCTTTTGCCTCCTGCCCCTTCTGTCACTGCTTGGAGAGGGATGAGTTCTGGTGGCTGGGCCTGGCTGTAGGAGACAGGATTTGGACCGTGCCCCTCTCGCATCACCGAAATCACCCCCACTATTCCAAGAGTGGTTGGCTATTAAACGTGAAGATTTCCTGAGAGAAGGATTGAGGACCTGGCCAGGAATGGGACACAAGTTCCGCCTTGTGTCTTCCTGACAGGAGCCCTGCACCGCGCTGGACGCTCACCTTGACACTCCCAGCCAGCTGGGGTACTGATCCCACCCTTCCCCGGCCGCTGCCCCGGGAGTGGGGAGGTAGAGAGAGCCACACCCGAAACACCTTTCCACGATAAACTTTTATTCTCTATCTTATTATTAATGGTGGCGGAAATAAAACTAAAACCAAAACGAAAACGAGTACTAGTACTAACACACTAATCAATTTGAGATGACTTCCCCCTCATTCCCAAAGCTAGAGGAGGAAGGGGGCTGAAAGGGGCTCAGAGCAGTGGAAGGTCCCAGGCCCAGCTGGGGTTGGGACGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTAGAGGGGTGGGTTCCGGGGGGGTGGTGGAAGAGGGCCGAGGGGGGAGGATAGAAGGAGGGGGTAGAGTTTCAGGGCGGGGAGGGGGGCGCTGGGGCGCAGTGACGGGAACCAATGAGCTGCCAACTCGCGCGTCTCCGGCGTGACTGCCGAGATTGACGTGGAGGACACGT
the fragment was analyzed for CpG islands using METHPRIMER in-line design software and found to have three CpG islands (the underlined regions in the promoter region sequence described above are CpG islands).
Wherein the promoter region sequence of the GYPC gene (namely the gene sequence fragment selected by the GYPC gene) is as follows:
GAAAAAAAATAAATGAAGTCTGCCTATCTCCGGGCCAGAGCCCCTCCCCTCGGCCCGCGCGGGAGGAGTGTGACCCAGGTGCCGCTTCCTCTCGCCGCCGAGGGTCAGGAGCCCGGGAGCGCGACCCTCCCCCGGCCCGGCCTGGCCCGGCCTGGCCAGTCCCCGCGGTCTCTGCCCGGGCTGACGCCCAGGAATGTGGTCGACGAGAAGCCCCAACAGCACGGCGTGGCCTCTCAGCCTCGGTGAGTACCCGCCGTGGGGAAGGGTCCTGGGGACCCACTGGAGGCCGCGGCCCGCAGCAGCCAGGGGCCGAGCCACGGCCACGGACGCCCTGGTGTCCCGGTCCGTGCCGGGCCTCCAGGCGGAGGAGGCGTCCGCTGGGCTCAGATCCCCGACTCCAGCCCCGGTTCCCCGGCGCCTGGGCTGCGCGGAGTCCCTGTCCCGCGTCCCGGACCCCTACGCGCAGCCTCCACGCGCTCCGAGCTGGAGAAGCCGCCAGCCCGCCCTCCCAGGGCGTGTCGCCCGCTTTCTGTTTCTTTGTGCGGGGCATGTGAAATGTGTGGGGCCAGAATTGTCTCCCTAAAGAACAGTTAGGTGAGAGTTCACATCACAAGTTGCTTCTGTGCTGCTCCCCAGCCAGGGTCCGACCGGCCAGGCTCCGGTGAACTCCAGGCAACTCCAGCCTTGCCCAGGGACTTGCTCGCTCCGCTGGGCGCCGCAAAGGCACACAGATGCGAGGGAATCCTGCGGCCTGGATCCTTTGGGGATTTGTAATTCCTCGCTGCTGGTTTTCTCTTTTAAACCATCACGTTTTCTTGCTGAGAGGAAATCATCTGTGATGAAAGTCTTTGTAGATGCTTGGAAACATTGCTGGGCCTCTCTCTGCCCTCTTCCTCTGCCGTTGGACTTTTGCAATAAAAACTACCATTTGATTTTACCAAAAAACGTAATGTACATCCCTCTTACCCCCACCCTGCCGCGGTCTGATTCTTAGAGTCTGTCAGAGAGGAGATACCCTGAGCCTATCATGTGACAATGATACCTTATTGATATGATAATGATATGGGTATTGGAGACACCAACTCTTTTATAAGTCTTTGAAATCCAGGGGGTTGTGAGGCCTCACCCCTCACCCCGCCTGAGCTTGGAGCTTGGCGTGGAGGAGTGCTGCTTCGGTCTACGTGTGTACACGGCACTCCATGTGCCTGTGGTTGTGCTTGCGTCAGCCTGGGAGTGCGCAGGCATCAGTGGTGCTTGGGAGCGAGG
the fragment was analyzed for CpG islands using METHPRIMER in-line design software and found to have two CpG islands (the underlined regions in the promoter region sequence described above are CpG islands).
Wherein, ZIK gene promoter region sequences (i.e. fragments of the gene sequence selected by ZIK gene) are:
CCTCAGCTTCCTGAGCCTGAATCAATGCTTCTAATCACGTGGGCGCTCAGCCGCGACGGGACCACAGAAATCGACCAGCGGCCTGTTTAAGGAGCGACAAGGCCCAGCCCTCTCCTGCATTGTGGCGACGCCATTTCCCTGCGCCCCCAGCGCGGCCGCTTGGCTTTTGTTTGAGGTGACGCTGGGCGGCAGCATCCTTCCCTCAGCCCTGGGGACCAGCGGGGACTACAGAACCCAGAAGGTTTTGTCTCCAGCCGCAGGGATGCTGAGCCAATCCGCGCTGAGAAAAGGGTCGTTTCCGCTTTGGGACCAATGGGTCGGGGAGGGACTTCCGGTATCACTTCAGTGGCGGTCATTTTTGCAGCGCTTGGGTGCATCCAGACCGTCAGAGCTTTGGGAGCGCTTTGTTTGGCGACAGTCGGAAGGCGCGAGGGGAGGGGTCCTCCCGCTGAACAGTGGGGGTTCTAAGGGTCGGCGGCGGCGGGGTTGACGGCTTTGCCTAGGTCCCTCCGCCCGTAGCTGTCGGGTCCCGGCCCCGCTCTGCCCACAGACTCCGATGGCTGCGGCCGCGCTGAGGGCCCCGACTCAGGTGAGCGCTGCCTCTACTGGGCCTCACCCTCCATCCCCAAATTAGTGCCTTCTTGGGTCACTACGGTCGAGATCCTCATGTCCAGTACAGTGGGGGCTCGTGGGTGGGGTCCCTATTTCCAGAACTTGGGTGATGGGGCACGGAAGAGGGTTACAGGCAAAGGGACCAGCGTTTCTAAACTCTTGGAGACACAGTGAAGAAGGTTCATACCTGGAGTGCCAAGGTGAGGAGTTTTCCCTCAATTCTTAGGATGCTAGGAGCCGTGGAGGGTTGTGTACAGAAGCGGCATATGGTCTGAGTTAGGTCTTTCGAAGTTTTCCAAAAACCTCATGGAGTTAGACTAGAATAGAATGGAATAACACAGAGGCACAGGGAGGTTGAGTTCTTGTGCAAGGTCCCACAGTTGCTAAGAGTTGGCACTGAGGACTGGAACGCTGAGGCTCAGAAATAATGCAGGCATCCTCAGTCCACCTGGCTCTGGCCTGGACAGGAATCCAGGGAAGCCTGAATCCGTAGAGCCCTGTATGGTCACCTGCCTTTCATGATCTTTGGCTTTCCACAGGTTACTGTGTCTCCAGAAACACATATGGACCTCACAAAGGTGAGTGGAGGGTGTCCCAGGCCCTCACTGGTCCTGGCCCCATCCTGGGGTTTTTTCATGGATCTTTTTG
the fragment was analyzed for CpG islands using METHPRIMER in-line design software and found to have two CpG islands (the underlined regions in the promoter region sequence described above are CpG islands).
Wherein the sequence of the promoter region of the reference gene COL2A1 (namely the gene sequence fragment selected by the reference gene COL2A 1) is as follows:
GGGCATTGACCCTACCCCACTGACCTTCGGCTACCATGTTTTGTATTTGTTGTGTCAACTTCACATTGCTTTTCTGTGTCCTCTCTTACTCTCCCTTTTTCCTGTTCTCATTGTGCTTCATCAACAACTGCTGCTTTCCTATCTCACCTCTAGTTTTCTGCATCTAATTGGTGAGGTCAGATGCCATTACGTGTTAATACAATATCTAAACTATGAATTAGCAGACATGGAGTAAGAAGGACTCTACAGTGCACAAAATGCATAGCTTTATATAACATCAATTCTGGCTATTTAGTGGTTTAGTTCCCCTTCTTTTCAGTTTCTTGGGTATGGTATGGATAGTGAGATTGTTGGACGATCAATTTCCAAATCAGTGATTTGCATATGTGTGCCACATATAGGTCTAATTCCAGTTTGAGTCACAAATTAGAGGTTTTTAATTAGTTGTTTGGAGTCACAGATGGGTAAAATATTAATGTTGGTGTTCAATTGGCTTTAGAATTTTAATAATTTCCATAGTTTATTTTGGCTTTGTCAATTTAAATTTTAAATTTTATTTTTCTTATGTCAATTCCCGTTGCTGGTGAAAAGAAATTTTCCCACAGTGGGTGGAGAGCTTAACTTGTACTCATAGATGGACCATGATTGCTGGAAGGAATCCAAAGCTCTTATATGTAGACACAGTCTATTGGCAAAGATGAATCACAGAGCTAGAGGATGAAATTAAGAACAAATTTGCAACGAGTCTGTACCACTTTGCTACTCACTAAACTGGATCATTTTGAACTAGGCTTCGATTTTTTCCACATACTTTAGTGTGTTCTCCCAAATTCTTTTTTTTTCTGTTGAATTTTTATGACAACTTGTATTTGAAGTCAGTTGTATAAATGCCTTTATAGTTGTATCTAAATTATTAGTCCACAGGGGCTTTCCTAACTCCTATGTGTTAGAATTGTGCTAGGCACAGTTCTTGTCCCCAAGCATCTTCCAAGCTCATTGATGAGATAAAGACCAATGGATATAAGGGGAAAAAATTAAAACTACACAAAAACATATGTAGCTTAGGCTATAAATGCCAGGGGGATTCAGAAAGCCAGGGAGACGTTGTGTTCTAGAGATATCAGAGAAGGCTTCTCAGAGGGGCTTTAACCTAGGAGGTCTTGATAAATGTGTGGAATTTGGTCATTCTATCTTCCAGGTAGAGAATATAGGACCTTCTGAGCCCCAAAGAGGACATGGGGCACTGATGGCAAGAGACCAGCTTTCTTCTAGCAGAGATTGGGCCTGAGGAAGAGCCCAGAGCTTAATGCAAGCCCCTGAATGGGAGCATGGAGACTCAGACAAGGCCCGTGGAAGGTAGGGGAGATGCTGGAAACTGGTTGAATTCTTCCCCATCTCCATCCAGACTGGACAAAGCCTAGAAGCATTTCCTTCAACAAT
CpG island analysis was performed on the fragment using METHPRIMER on-line design software, and it was found that the fragment did not have CpG islands.
And (II) converting the non-CpG C in the sequences of the target genes PAX1, GYPC, ZIK1 and internal reference genes COL2A1 in different CpG island regions into T, wherein the CpG C is unchanged, so as to design a primer probe aiming at the different regions. The method comprises the following steps:
The first CpG island related sequence of the PAX1 gene is as follows:
ATTAATTTTTTTTGGTATTTCGCGTTCGACGAAGTTAGTAGATTTCGTAATGGAGAGTATTGGGTAGGGTGCGTTTTTATTTTTCGTATTTTCGGAGTTTCGAGTATATTGTGCGTGTTTAGTTTTTTTTTCGTTGTTTTCGCGGAATTTTTGGTTTAGGCGTGTTTATTTTAATTTAAATTATTTTTTTTTTTTGTTTTTTA
The second CpG island related sequence of PAX1 gene is:
GAGTGGTGCGAGATTTTATCGTTAGGGAGAAAGGAATTTGTTTTTTAGATTTAGAGTTGGAGTTATGTATTTGGTTTTTCGTCGGTCGCGTTTGGGGATAGGAGGGCGTCGGATTTATGCGTTTTTTAGTAGTAGATCGGGATTTTTTTGTTT
The third CpG island related sequence of PAX1 gene is:
TGTGTGTGTGTGTGTGTGTAGAGGGGTGGGTTTCGGGGGGGTGGTGGAAGAGGGTCGAGGGGGGAGGATAGAAGGAGGGGGTAGAGTTTTAGGGCGGGGAGGGGGGCGTTGGGGCGTAGTGACGGGAATTAATGA
the first CpG island related sequence of GYPC gene is:
TTTCGGTTCGCGCGGGAGGAGTGTGATTTAGGTGTCGTTTTTTTTCGTCGTCGAGGGTTAGGAGTTCGGGAGCGCGATTTTTTTTCGGTTCGGTTTGGTTCGGTTTGGTTAGTTTTCGCGGTTTTTGTTCGGGTTGACGTTTAGGAATGTGGTCGACGAGAAGTTTTAATAGTACGGCGTGGTTTTTTAGTTTCGGTGAGTATTCGTCGTGGGGAAGGGTTTTGGGGATTTATTGGAGGTCGCGGTTCGTAGTAGTTAGGGGTCGAGTTACGGTTACGGACGTTTTGGTGTTTCGGTTCGTGTCGGGTTTTTAGGCGGAGGAGGCGTTCGTTGGGTTTAGATTTTCGATTTTAGTTTCGGTTTTTCGGCGTTTGGGTTGCGCGGAGTTTTTGTTTCGCGTTTCGGATTTTTACGCGTAGTTTTTACGCGTTTCGAGTTGGAGAAGTCGTTAGTTCGTTTTTTTAGGGCGTGTCGTTCGTTTTTTGTTTTTTTGTGCGGGGTATGTGAAATGTGT
the second CpG island related sequence of GYPC gene is:
AGTTAGGGTTCGATCGGTTAGGTTTCGGTGAATTTTAGGTAATTTTAGTTTTGTTTAGGGATTTGTTCGTTTCGTTGGGCGTCGTAAAGGTATATAGATGCGAGGGAATTTTGCGGTTTGGATTTTTTGGGGATTTGTAATTTTTCGTTGTTGGTT
the first CpG island related sequence of ZIK gene is:
AGTCGCGACGGGATTATAGAAATCGATTAGCGGTTTGTTTAAGGAGCGATAAGGTTTAGTTTTTTTTTGTATTGTGGCGACGTTATTTTTTTGCGTTTTTAGCGCGGTCGTTTGGTTTTTGTTTGAGGTGACGTTGGGCGGTAGTATT
The second CpG island related sequence of ZIK gene is:
TTTTGTTTTTAGTCGTAGGGATGTTGAGTTAATTCGCGTTGAGAAAAGGGTCGTTTTCGTTTTGGGATTAATGGGTCGGGGAGGGATTTTCGGTATTATTTTAGTGGCGGTTATTTTTGTAGCGTTTGGGTGTATTTAGATCGTTAGAGTTTTGGGAGCGTTTTGTTTGGCGATAGTCGGAAGGCGCGAGGGGAGGGGTTTTTTCGTTGAATAGTGGGGGTTTTAAGGGTCGGCGGCGGCGGGGTTGACGGTTTTGTTTAGGTTTTTTCGTTCGTAGTTGTCGGGTTTCGGTTTCGTTTTGTTTATAGATTTCGATGGTTGCGGTCGCGTTGAGGGTTTCGATTTAGGTGAGCGTTGTTTTTATTGGGTTTT
specifically, the sequences of the primer pair and probe corresponding to the target gene are shown in tables 2.1 to 2.4.
TABLE 2.1 first primer pair and base sequence of first probe for detecting methylation of PAX1 Gene
TABLE 2.2 first primer pair and base sequence of first probe for detecting GYPC Gene methylation
TABLE 2.3 detection of nucleotide sequences of first primer pair and first Probe for ZIK Gene methylation
TABLE 2.4 first primer pair and base sequence of first Probe for detecting methylation of internal control Gene COL2A1
Example 2:
The kit provided by the invention is used for a detection test method for early screening and diagnosing PAX1 gene, GYPC gene, ZIK gene and internal control gene COL2A1 of cervical cancer, and comprises the following steps:
1. Sample DNA extraction
The DNA of the cervical exfoliated cell sample is extracted by adopting a nucleic acid extraction or purification reagent (Hunan Hongya gene technology Co., ltd., hunan Long Instrument No. 20170149), the quality of the extracted DNA is monitored, the total amount of the DNA is 500ng-10ug, and the OD260/280 is 1.7-2.1, so that the next experiment can be carried out.
2. DNA bisulfite conversion
A certain amount of DNA (500 ng-1000 ng) is subjected to bisulfite treatment by using a nucleic acid extraction or purification reagent (Hunan Hongya Gene technology Co., ltd., hunan Long Instrument No. 20170149) as a DNA bisulfite conversion reagent, unmethylated cytosine (C) in the treated DNA is converted into uracil (U), and methylated cytosine (C) is kept unchanged, and then the uracil (U) is converted into thymine (T) through a desulfonation reaction, so that DNA (Bis-DNA) after bisulfite conversion is obtained.
3. Methylation specific fluorescent quantitative PCR amplification
3.1 A Real-Time PCR reaction system was prepared as shown in Table 3.
TABLE 3 PCR reaction system (20 ul/person)
Note that: the DNA is sample DNA after bisulfite conversion, or negative/positive quality control.
3.2 PCR reaction
The invention skillfully designs the primer probes of PAX1, GYPC and ZIK1 genes, the Tm values of the primer probes are close, and the primers are not interfered with each other in the same reaction system. Programming and running on an ABI 7500 real-time fluorescence quantitative PCR instrument was performed as described in Table 4.
Table 4 PCR machine program parameters
4. Gene methylation result interpretation
4.1 Quality control is qualified: the internal control gene COL2A1 is amplified in an S type, and Cp is less than or equal to 34 and is in control.
4.2 The Δcp value is used to reflect the methylation level of a particular gene target region.
ΔCp(PAX1)= Cp(PAX1)- Cp(COL2A1)，
ΔCp(GYPC)= Cp(GYPC)- Cp(COL2A1)，
ΔCp(ZIK1)= Cp(ZIK1)- Cp(COL2A1)。
4.3 Interpretation of results of diagnosis of cervical cancer by gene methylation
TABLE 5 interpretation of results of methylation diagnosis of cervical cancer
Experimental example 1:
70 cervical exfoliated cells with well-known pathological consequences were selected, of which cervical normal or chronic inflammatory patients 5, low-grade squamous intraepithelial lesions 14, high-grade squamous intraepithelial lesions 43, cervical cancer 8.
The methylation of the PAX1, GYPC and ZIK1 genes was performed on DNA from the exfoliated cell samples according to example 1, and the detection performance of the related areas of different CpG islands of each gene on cervical high-grade lesions is shown in Table 6.
TABLE 6 methylation of different CpG island related regions for diagnosing cervical high grade lesions
Different CpG islands of the same gene, or different related regions of the same CpG island, have differences in detection performance for cervical high-level lesions.
The group with the largest area under the curve of the subject in each gene (numbers 3, 6 and 8 in table 6) is selected as the optimal detection group of the gene, and the performance of the combined diagnosis of cervical high-grade lesions of two genes (table 7) and three genes (table 8) is respectively analyzed.
TABLE 7 sensitivity and specificity of double Gene methylation in combination diagnosis of cervical high grade lesions
TABLE 8 sensitivity and specificity of Trigenic methylation in combination diagnosis of cervical high grade lesions
Experimental example 1, with cervical pathology results as references, performance analysis of PAX1, GYPC and ZIK1 in single or combined diagnosis can be used as screening of high-risk population according to clinical requirements, and single gene or multi-gene combination with high specificity can be selected, for example, the specificity of PAX1 gene methylation diagnosis of cervical high-grade lesions is 86.3%; the combination of the genes PAX1 and GYPC has the interpretation mode that when any one of the two genes is positive, the interpretation mode is positive, when the sensitivity is 90.20%, the specificity is highest when the genes are positive when two or three genes are combined, and the interpretation mode is 89.47%. As a clinical auxiliary diagnosis test, a polygene combination with high sensitivity, such as PAX1 and GYPC gene combination, can be selected, and when any one gene is positive and judged to be positive, the sensitivity reaches 90.20 percent when the specificity is higher (78.95 percent).
The advantages of the present invention over the prior art include:
1. compared with HPV detection, the gene methylation detection specificity is higher, and misdiagnosis can be effectively reduced;
2. Compared with cytological examination, the invention has higher sensitivity and can effectively reduce missed diagnosis;
3. the methylation state of the genes is positively correlated with the diseases, so that the progression degree of cervical lesions can be predicted better.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. A primer pair for detecting Gong Gengliang malignant lesion gene methylation, characterized by comprising at least any one pair of biomarkers consisting of a 1000bp sequence before the start codon in the PAX1 gene and the GYPC gene or a 1000bp sequence before the start codon in the PAX1 gene, the GYPC gene and the ZIK gene as primer sequences corresponding to the methylation markers;
The primer sequences are specifically expressed as follows:
Forward primer of PAX1 gene: 5'-GTATTTCGCGTTCGACGAAGTTA-3', or 5'-GTGCGAGATTTTATCGTTAGGG-3', or 5'-TAGAGGGGTGGGTTTCGGG-3';
Reverse primer of PAX1 gene: 5'-ACACGCCTAAACCAAAAATTCCG-3', or 5'-AAAAATCCCGATCTACTAC-3', or 5'-ATTAATTCCCGTCACTACGC-3';
Forward primer of GYPC gene: 5'-CGTCGAGGGTTAGGAGTTCGGG-3', or 5'-AGTTACGGTTACGGACGTTTTG-3', or 5'-TCGATCGGTTAGGTTTCGGTG-3';
reverse primer of GYPC gene: 5'-TAACTACTACGAACCGCGACCT-3', or 5'-ACGAACTAACGACTTCTCCAATC-3', or 5'-ACGAAAAATTACAAACCCC-3';
forward primer of ZIK gene: 5'-AGTCGCGACGGGATTATAGA-3', or 5'-GTTGAGTTAATTCGCGTTGA-3', or 5'-AGTGGCGGTTATTTTTGTAGCG-3';
reverse primer of ZIK gene: 5'-ATACTACCGCCCAACGTCACC-3', or 5'-ACTATCGCCAAACAAAACGC-3', or 5'-CCGCCGACCCTTAAAACC-3'.
2. The primer pair for detecting Gong Gengliang malignant disease gene methylation according to claim 1, further comprising a set of primer sequences corresponding to a pair of reference markers COL2A1 gene, specifically expressed as follows:
forward primer of COL2A1 gene: 5'-ATTAGTGATTTGTATATGTGTG-3';
reverse primer of COL2A1 gene: 5'-AATTAAACTCTCCACCCACTAT-3'.
3. The use of a primer pair for detecting Gong Gengliang malignant lesion gene methylation according to claim 1 or 2 in the preparation of a cervical cancer detection kit.
4. A kit for detecting Gong Gengliang malignant lesions gene methylation, comprising a primer pair according to claim 1.
5. The use of the kit for detecting Gong Gengliang malignant tumor gene methylation according to claim 4 in the preparation of a kit for detecting Gong Gengliang malignant tumor gene methylation marker.
6. The use according to claim 5, wherein in the detection of the biomarker of Gong Gengliang malignant lesions using the kit, the result of diagnosing cervical cancer by gene methylation is interpreted as follows:
the methylation level of a target region of a particular gene is reflected in a Δcp value of:
ΔCp(PAX1)= Cp(PAX1)- Cp(COL2A1)，
ΔCp(GYPC)= Cp(GYPC)- Cp(COL2A1)，
ΔCp(ZIK1)= Cp(ZIK1)- Cp(COL2A1)；
wherein COL2A1 is an internal reference gene.
7. The use according to claim 5, wherein in the detection of the biomarker of Gong Gengliang malignant lesions using the kit, the result of diagnosing cervical cancer by gene methylation using the combination of PAX1 gene and GYPC gene is interpreted in the following manner:
。
8. The use according to claim 5, wherein in the detection of the biomarker of Gong Gengliang malignant lesions using the kit, the result of diagnosing cervical cancer by gene methylation using the combination of PAX1 gene, GYPC gene and ZIK gene is interpreted in the following manner:
。
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| CN117701718A (en) * | 2024-02-04 | 2024-03-15 | 湖南宏雅基因技术有限公司 | Gene methylation marker for diagnosing cervical cancer, primer pair and application thereof |
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| CN117701718A (en) * | 2024-02-04 | 2024-03-15 | 湖南宏雅基因技术有限公司 | Gene methylation marker for diagnosing cervical cancer, primer pair and application thereof |
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