阅读说明：本技术 人pcdh10基因甲基化检测试剂盒 (Human PCDH10 gene methylation detection kit ) 是由 许嘉森 吴诗扬 彭璨璨 刘志明 刘芳 于 2019-11-07 设计创作，主要内容包括：本发明提供了一种人PCDH10基因甲基化检测试剂盒及其使用方法,包括针对PCDH10基因的两个CGI1岛和CGI2岛设计的引物及探针,所述引物和探针的组成为,针对CGI1岛的SEQ ID NO.1-3,针对CGI2岛的SEQ ID NO.4-6、SEQ ID NO.7-9和SEQ ID NO.10-12。本发明所述检测试剂盒具有很好的特异性和准确性,检测效果好。(The invention provides a human PCDH10 gene methylation detection kit and a using method thereof, comprising primers and probes designed aiming at two CGI1 islands and CGI2 islands of a PCDH10 gene, wherein the primers and the probes comprise SEQ ID NO.1-3 aiming at the CGI1 island, SEQ ID NO.4-6, SEQ ID NO.7-9 and SEQ ID NO.10-12 aiming at the CGI2 island. The detection kit disclosed by the invention has good specificity and accuracy and a good detection effect.)

1. The human PCDH10 gene methylation detection kit is characterized by comprising primers and fluorescent probes designed aiming at two CGI1 islands and CGI2 islands of a PCDH10 gene, wherein the primers and the fluorescent probes comprise the following components: SEQ ID Nos. 1-3 for island CGI1, SEQ ID Nos. 4-6, SEQ ID Nos. 7-9 and SEQ ID Nos. 10-12 for island CGI 2.

2. The kit for detecting methylation of the human PCDH10 gene according to claim 1, further comprising an internal standard gene primer and a probe, preferably, the composition is SEQ ID NO. 13-15.

3. The kit for detecting methylation of human PCDH10 gene according to claim 1 or 2, wherein a tag sequence is added to the 5' end of the primer.

4. The kit for detecting methylation of human PCDH10 gene according to claim 3, wherein the tag sequence consists of SEQ ID NO 16.

5. The kit for detecting methylation of human PCDH10 gene according to claim 1 or 2, wherein the fluorescent probe has LNA modified base at the appropriate CpG position, more preferably the probe with LNA modification isThe bases in italics are LNA modified bases.

6. The kit for detecting the methylation of the human PCDH10 gene, according to claim 1, further comprising a magnetic bead mixture for purifying the converted DNA, preferably, the magnetic bead mixture is an aqueous solution containing nano-magnetic particles with a concentration of 50 +/-1 mg/ml.

7. The kit for detecting the methylation of the human PCDH10 gene, according to claim 1, further comprising a washing solution, wherein the washing solution is an aqueous solution containing 2.5M guanidine hydrochloride and 50% absolute ethyl alcohol, and the pH value is 5.0-7.0; and/or further comprises an eluent, wherein the eluent is an aqueous solution containing 10mM Tris-HCl and has a pH value of 7.5-8.5.

8. The kit for detecting the methylation of the human PCDH10 gene, according to claim 1, further comprising a transformation solution, wherein the transformation solution is an aqueous solution containing 75% ammonium bisulfite, 0.1% disodium edetate and 0.1g/ml anhydrous sodium sulfite, and the pH value is 5.3-5.5.

9. The methylation detection kit for the human PCDH10 gene, according to claim 1, wherein the kit further comprises a negative quality control substance and a negative quality control substance, preferably, the negative quality control substance is composed of bovine serum albumin and human genome DNA of which the PCDH10 gene is not methylated; the positive quality control product consists of bovine serum albumin, human genome DNA of PCDH10 gene non-methylation and human genome DNA of PCDH10 gene methylation, and further preferably: the negative quality control material consists of 0.1 mug bovine serum albumin and 1ng non-methylated human genome DNA of PCDH10 gene; the positive quality control product consists of 0.1 mug of bovine serum albumin, 0.9ng of unmethylated human genomic DNA of PCDH10 gene and 0.1ng of methylated human genomic DNA of PCDH10 gene.

10. A method for obtaining a DNA to be detected, comprising the steps of:

(1) DNA transformation: adding a conversion solution and a DNA protection solution into a DNA solution to be detected, uniformly mixing, and reacting at a constant temperature of 80 +/-3 ℃ for 1 +/-0.1 hour to obtain a converted DNA solution, wherein the DNA protection solution is a tetrahydrofuran solution of vitamin E, and the concentration of the vitamin E is 0.125 +/-0.01 g/ml;

(2) DNA purification: cooling the obtained converted DNA solution to room temperature, adding the binding solution and the magnetic bead mixed solution, uniformly mixing, and standing at room temperature; after the magnetic beads are separated, removing supernatant; washing for 1-3 times by adding washing liquid, adding eluent for elution after the magnetic beads are dried, transferring supernatant into a new DNase and RNase-free centrifugal tube after the magnetic beads are separated, and obtaining the DNA to be detected.

Technical Field

The invention belongs to the technical field of biology, relates to a DNA methylation detection kit, and particularly relates to a human PCDH10 gene methylation detection kit based on a fluorescence PCR method.

Technical Field

Epigenetic alterations, and in particular aberrant methylation of DNA molecules, play an important role in the development of a variety of solid tumors. Procadherin 10(Protocadherin 10, PCDH10), a member of the Protocadherin Cell Adhesion molecule family, is an important Oncogene, located in chromosome 4q28.3, consisting of 5 exons, about 42.26kb (Kim SY et al, Cell addition & differentiation, 2011,5,97-105), whose aberrant methylation has been shown to be important in the development of a variety of solid and hematological tumors (Ying J et al, Oncogene,2006,25, 1070-1080; Ying J et al, Brit J Haematol,2007,136, 829-832).

In the cervical cancer research, the detection rates of the methylation of the PCDH10 promoter in normal cervical tissues, atypical squamous cells with unknown significance, mild squamous intraepithelial neoplasia, high squamous intraepithelial neoplasia and invasive cervical cancer tissues are respectively 0%, 5.7%, 13.1%, 46.0% and 90.9% (Narayan G et al, Genes, Chromosomees and cancer,2009,48,983 and 992.), and the methylation of the PCDH10 promoter is suggested to be closely related to the development of cervical cancer.

In gastric Cancer studies, PCDH10 was found to be widely expressed in normal gastric gland epithelial cells and stromal cells, but expression was down-regulated or silenced in most gastric Cancer cell lines and Cancer tissues due to methylation of the PCDH10 gene promoter, and patients with hypermethylation of the PCDH10 gene promoter had poor prognosis (Yu J et al, Gastroenterology,2009,136, 640-.

In liver cancer research, it is found that the expression of PCDH10 is down-regulated in 69.2% of liver cancer cell lines, the down-regulation of the expression is closely related to the methylation state of a PCDH10 promoter, the detection rates of PCDH10 methylation in liver cancer tissues, para-cancer tissues and normal liver tissues are respectively 76%, 40% and 0%, and the methylation state of PCDH1 is closely related to the tumor size, the serum AFP level, the metastasis condition and the TNM stage (Fan S et al, Clin Exp Med,2013,13, 127-charge 134.).

In colorectal Cancer studies, it was found that PCDH10 was unmethylated in adjacent normal colorectal tissues, but hypermethylated in 85% of primary colorectal tumors, and that abnormal methylation of PCDH10 was closely associated with down-regulation or silencing of PCDH10 expression (Zhong X et al, J Cancer Res Clin Oncol,2013,139, 485-490); the down-regulation or silencing of PCDH10 expression is closely related to tumor progression and distant metastasis (Jao TM et al, Int J Cancer,2014,135, 2593-2603).

In lung Cancer studies, it was found that PCDH10 expression was down-regulated in non-small cell lung Cancer (NSCLC) tissues compared to paracancerous tissues, that PCDH10 promoter methylation could be detected in 50% of NSCLC tissues but not in paracancerous or normal tissues, and that PCDH10 promoter methylation was associated with smoking (Tang X et al, Cancer Biomarkers,2013,12, 11-19). In patients with pathological stage I NSCLC who received radical resection, patients with PCDH10 methylation had no relapse survival, overall survival, and disease-specific survival worse than patients with PCDH10 unmethylated, with PCDH10 indicating a poor prognosis (Harada H et al.,. Cancer Medicine,2015,4, 1536-.

In the study of tumors in the blood system, the detection rate of PCDH10 promoter hypermethylation in B-cell acute lymphoblastic leukemia, T-cell acute lymphoblastic leukemia, most subtypes of myeloid leukemia and chronic myelogenous leukemia is 81.9%, 80%, 25.9% and 2.2%, respectively, PCDH10 expression in primary acute lymphoblastic leukemia samples and leukemia cell lines is down-regulated by its promoter hypermethylation, and acute lymphoblastic leukemia cell lines carrying methylation mediated inactivation of PCDH10 are less sensitive to commonly used leukemia specific drugs, suggesting that PCDH10 methylation may be a biomarker of acute lymphoblastic leukemia chemotherapy response (Narayan G et al, Genes, Chromosomes and Cancer, 50, 1043-20111053).

In view of the close relationship between the methylation of the PCDH10 promoter and tumors, the development of a human PCDH10 gene methylation detection kit is promoted, and the kit is helpful for further and deeply researching the clinical significance of the methylation of the PCDH10 promoter in the aspects of the occurrence, development and prognosis of various systemic tumors.

At present, a plurality of DNA methylation detection methods exist, and the methods can be divided into three types according to different pretreatment means of target DNA: a DNA methylation detection technology based on Methylation Sensitive Restriction Enzyme (MSRE); DNA methylation detection technology based on affinity enrichment; and detecting DNA methylation based on bisulfite conversion. Among them, the detection technique of DNA methylation based on bisulfite conversion is most widely used. The commonly used DNA methylation detection technology based on bisulfite conversion mainly comprises a bisulfite sequencing method, a methylation specificity PCR method, a fluorescence PCR method and the like.

Bisulfite sequencing is a gold standard for DNA methylation detection. The method comprises the steps of firstly treating DNA by using bisulfite, then designing a primer to amplify a target fragment, and distinguishing methylated cytosine from other bases through sequencing to obtain the site information of the methylated cytosine. The method has accurate detection result and high accuracy, can detect the methylation state of each CpG locus of the target fragment, but has more complicated sample preparation process, needs a large amount of clone sequencing and has higher cost.

The methylation specificity PCR method is to design two pairs of primers which are respectively in complementary pairing with a methylated DNA chain and an unmethylated DNA chain after bisulfite treatment, carry out primer specificity PCR on a DNA sample after bisulfite treatment by utilizing the two pairs of primers, and detect a PCR amplification product through electrophoresis to judge the methylation state. The method is the most economical and practical DNA methylation detection method, does not need special instruments, has high cost-benefit ratio, but is of great importance in primer design, is not easy to generate false positive results due to poor design, and is easy to pollute due to electrophoresis separation detection. Furthermore, this method can only be used for qualitative studies.

The fluorescence PCR method is to design specific primers and fluorescent probes for the DNA fragments to be detected after bisulfite treatment, perform fluorescence PCR on the DNA samples after bisulfite treatment by using the specific primers and the fluorescent probes, and judge the methylation state according to the detected fluorescence signals. The method has the characteristics of high sensitivity, strong specificity, repeatability, less required sample amount and no need of electrophoretic separation, and is also suitable for quantitative research. This method is one of the methods widely used in clinical laboratory research related to DNA methylation at present. However, the method requires a probe with two ends labeled with fluorescein for measuring each site, and is unreliable in detection result of heterogeneous DNA methylation, and the design requirements for primers and probes are high. The unmethylated cytosine in the bisulfite converted DNA sample is eventually converted to thymine, reducing the complexity of the DNA sequence, which increases the difficulty and requirements of primer and probe design, since the possibility of non-specific amplification and non-specific binding is increased if the requirements of primer and probe design are not high. In addition, because there may be multiple pairs of primers and multiple probes in the detection system, the probability of primer dimer and non-specific amplification increases, which in turn affects the sensitivity and specificity of amplification. It can be seen that the difficulties in designing primers and probes are: firstly, designing primers and probes with high specificity and high sensitivity aiming at a DNA sequence with low complexity; secondly, non-specific binding between a plurality of groups of primers and probes in a detection system is avoided, and the occurrence probability of primer dimer and non-specific amplification is reduced.

Disclosure of Invention

Based on this, one of the objects of the present invention is to provide a methylation detection kit (fluorescent PCR method) for human PCDH10 gene, which has the advantages of improving the sensitivity and specificity of PCR amplification and thus improving the accuracy of the detection result.

In order to achieve the purpose, the technical scheme of the invention is as follows:

a human PCDH10 gene methylation detection kit (fluorescence PCR method) comprises primers and a fluorescent probe designed aiming at a CGI1 island and a CGI2 island of a PCDH10 gene, wherein the primers and the fluorescent probe comprise SEQ ID NO.1-3 aiming at the CGI1 island, SEQ ID NO.4-6, SEQ ID NO.7-9 and SEQ ID NO.10-12 aiming at the CGI2 island.

The primers and the fluorescent probes comprise four groups of methylation detection specific primers and probes designed aiming at two CpG islands (CGI1 and CGI2) of the PCDH10 gene and one group of internal standard gene primers and probes. Wherein, the four groups of PCDH10 gene methylation detection specific primers and the corresponding detection target areas of the probes are respectively as follows: the transcription start site is-1805 to-931 bp, +905 to +1101bp, +1294 to +1438bp, +1726 to +1826bp, and the other three target areas belong to CGI2 except that the target area of the transcription start site is-1805 to-931 bp which belongs to CGI 1.

In some embodiments, the kit further comprises internal standard gene primers and probes, preferably consisting of SEQ ID NO. 13-15.

In some of these embodiments, a tag sequence is added to the 5' end of the primer. Preferably, the tag sequence consists of SEQ ID NO 16.

In some of these embodiments, the probe has LNA modified bases at appropriate CpG sites.

In some of these embodiments, the probe with LNA modification is CGTTTCGTTCGGTTGTCGCGTGAC,

TTAACGTCGTGTTTGCGTATTG, GAACGATAACGCGTCGCGTT, CGTCGTGGACGCGGACGACGGC, the base in italics is an LNA modified base.

In some embodiments, the kit further comprises a magnetic bead mixture for purifying the converted DNA, and preferably, the magnetic bead mixture is an aqueous solution containing nano-magnetic particles with a concentration of 50 ± 1 mg/ml.

In some embodiments, the washing solution is an aqueous solution containing 2.5M guanidine hydrochloride and 50% absolute ethyl alcohol, and the pH value is 5.0-7.0.

In some of these embodiments, the eluent is an aqueous solution containing 10mM Tris-HCl and has a pH of 7.5 to 8.5.

In some embodiments, the kit further comprises that the conversion solution is an aqueous solution containing 75% of ammonium bisulfite, 0.1% of disodium ethylenediaminetetraacetic acid (EDTA-2Na) and 0.1g/ml of anhydrous sodium sulfite, and the pH value is 5.3-5.5. The transformation process using the transforming agent only needs to react for 1 hour at 80 ℃ without high-temperature treatment and transformation at 98 ℃ overnight, thus effectively reducing the degradation of DNA while greatly shortening the transformation operation time and ensuring the quality of the transformed DNA. The transforming agent contains 0.1% of EDTA-2Na as a stabilizing agent, so that the service life of the transforming solution can be prolonged, and the stability and the availability of the transforming solution are greatly improved.

In some of these embodiments, the kit further comprises a negative quality control and a negative quality control, preferably, the negative quality control consists of 0.1 μ g bovine serum albumin and 1ng unmethylated human genomic DNA of PCDH10 gene per reaction; the positive quality control product consists of 0.1 mug of bovine serum albumin, 0.9ng of unmethylated human genomic DNA of PCDH10 gene and 0.1ng of methylated human genomic DNA of PCDH10 gene.

Another object of the present invention is to provide a method for using the above kit.

The use method of the kit comprises the following steps:

(1) obtaining DNA to be detected;

(2) and (3) fluorescent PCR reaction: pre-denaturation at 95 ℃ for 5 min for 1 cycle; denaturation at 95 ℃ for 30 seconds, annealing at 58 ℃ for 30 seconds,

45 cycles; and cooling at 40 ℃ for 30 s.

The invention also provides a method for obtaining a DNA to be detected, comprising DNA transformation: adding a conversion solution and a DNA protection solution into a DNA solution to be detected, uniformly mixing, and reacting at 80 +/-3 ℃ for 1 +/-0.1 hour to obtain a converted DNA solution, wherein the DNA protection solution is a tetrahydrofuran solution of vitamin E, the tetrahydrofuran is used as a solvent, and the concentration of the vitamin E is 0.125 +/-0.01 g/ml;

the method for obtaining the DNA to be detected also comprises the following steps of DNA purification: cooling the obtained converted DNA solution to room temperature, adding the binding solution and the magnetic bead mixed solution, uniformly mixing, and standing at room temperature; after the magnetic beads are separated, removing supernatant; washing for 1-3 times by adding washing liquid, adding eluent for elution after the magnetic beads are dried, transferring supernatant liquid into a new DNase and RNase-free centrifugal tube after the magnetic beads are separated, and obtaining the converted and purified DNA.

The invention has the following advantages:

(1) by designing four groups of corresponding and appropriate primers and probes for the selected detection target region, the detection accuracy is well ensured, and on the basis, the primers with the tag sequences are further matched to obtain uniquely designed primers. The 5' end of each primer of the four groups of primers is connected with a label sequence with the same sequence, the designed primers can reduce the occurrence of primer dimers, eliminate the interference caused by different initial concentrations of different templates in the same system and improve the sensitivity and specificity of PCR amplification so as to improve the accuracy of a detection result, and correspondingly, LNA (low noise amplifier) modification is carried out at a proper position by matching with the designed probes, so that the LNA-modified TaqMan probe can reduce the length of the probe and simultaneously keep a higher Tm value of a hybrid formed by the probe and a DNA template, the specificity of the probe can be effectively enhanced, and the accuracy of the detection result is improved. The design of the primers and the probes brings good detection effect to the detection kit.

(2) The detection kit provided by the invention can detect the methylation state of four regions of two CpG islands (CGI1 and CGI2) of the PCDH10 gene by one-time reaction in a single tube by adopting quintuple fluorescent PCR reaction, thereby greatly saving reagent consumables, shortening detection time and comprehensively detecting the methylation state of the PCDH10 gene.

(3) Furthermore, the invention is designed with a negative quality control product and a positive quality control product, which can better prevent the generation of false positive results and false negative results, thereby ensuring the accuracy and reliability of the detection result.

(4) Furthermore, the invention combines the magnetic bead method to purify the converted DNA, utilizes the high-salt low-pH value to separate and purify the DNA, and then uses the low-salt high-pH value to elute, thereby having the characteristics of high purity and high recovery efficiency, and the whole purification process does not use sodium hydroxide commonly used in the market to remove sulfonation treatment, the operation is simpler and more convenient, and the whole purification operation time can be saved.

Drawings

FIG. 1 is a graph showing the fluorescence PCR amplification of non-methylated human genomic DNA of PCDH10 gene detected by 4 specific primers with and without tag sequences. Among them, in the detection results using 4 specific primers with tag sequences, no amplification curve rises in FAM, VIC, ROX and CY5 channels (curve 7, overlapping baseline), S-type amplification curve is detected in TAMARA channel, corresponding Ct values of TAMARA channel are respectively between 28-29.5 (curve 3, specific primer with B1 tag sequence), between 31-32.5 (curve 4, specific primer with B2 tag sequence), between 32-33 (curve 1, specific primer with B3 tag sequence), between 30-31.6 (curve 2, specific primer with B1 tag sequence), the baseline is flat without rising; the detection results of the specific primers without the tag sequences show that the FAM, VIC, ROX and CY5 channels have no rising amplification curves, S-type amplification curves are detected in the TAMARA channel, the Ct value of the TAMARA channel is between 32 and 33.5 (curve 5), and the baseline is slightly raised (curve 6).

FIG. 2 is a graph showing the fluorescent PCR amplification of methylated human genomic DNA of PCDH10 gene with 4 specific primers with and without tag sequences. Wherein the Ct values of 2- (a) specific primers with B1 tag sequences, FAM, VIC, ROX and CY5 channels are all between 27 and 29 (curves 2 to 5 respectively), the Ct value of TAMARA channel is between 26 and 27.5 (curve 1), and the baseline is flat without upward (curve 6); 2- (B) specific primers with the B2 tag sequence, FAM, VIC, ROX and CY5 channels (curves 2-5, respectively), TAMARA channel (curve 1), straight baseline without upward (curve 6); 2- (c) specific primers with B3 tag sequence, FAM, VIC, ROX and CY5 channels (curves 2-5, respectively), TAMARA channel (curve 1), straight baseline without upward (curve 6); 2- (d) specific primers with the B1 tag sequence, AM, VIC, ROX and CY5 channels (curves 2-5, respectively), TAMARA channel (curve 1), straight baseline without upward (curve 6); 2- (e) primers specific for the unlabeled sequence, FAM, VIC, ROX and CY5 channels (curves 1,3-5, respectively), TAMARA channel (curve 2), with some upward baseline (curve 6).

FIG. 3 is a graph showing the fluorescence PCR amplification curve of the non-methylated human genomic DNA of PCDH10 gene detected by the LNA modified probe of the present invention and the general probe without LNA modification; (a) the LNA modified probe of the invention; (b) common probes not modified with LNA.

FIG. 4 is a fluorescent PCR amplification graph of the inventive LNA modified probe and the general probe without LNA modification for detecting PCDH10 gene methylated human genome DNA. (a) The LNA modified probe of the invention; (b) common probes not modified with LNA.

FIG. 5 is a fluorescent PCR amplification plot of the non-methylated human genomic DNA of the PCDH10 gene purified by the purification reagent of the present invention and Qiagen kit.

FIG. 6 is a fluorescent PCR amplification plot of the inventive purification reagent and Qiagen kit purified PCDH10 gene methylated human genomic DNA; (a) purification reagents of the invention in which FAM, VIC, ROX and CY5 channels (curves 1-2, curves 4-5), TAMARA channel (curve 4), curve 6 are baseline; (b) qiagen kit, where FAM, VIC, ROX and CY5 channels (curves 2-5), TAMARA channel (curve 1), and curve 6 are baseline.

FIG. 7 is a graph showing the fluorescence PCR amplification curve of a DNA sample showing negative methylation of the PCDH10 gene of the present invention, showing no curves for FAM, VIC, ROX and CY5 channels, and showing curve 1 for TAMARA channel.

FIG. 8 is a graph showing the fluorescence PCR amplification curves of the DNA samples positive for the methylation of the PCDH10 gene of the present invention, FAM, VIC, ROX and CY5 channel curves 2-5, and TAMARA channel curve 1.

FIG. 9 is a graph showing the fluorescence PCR amplification curve of the negative quality control of the present invention, showing no curves for FAM, VIC, ROX and CY5 channels, and curve 1 for TAMARA channel.

FIG. 10 is a fluorescent PCR amplification curve of the positive quality control of the present invention, FAM, VIC, ROX and CY5 channel curves 2-5, and TAMARA channel curve 1.

Detailed Description

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, cell biology, immunology and recombinant DNA, which are within the skill of the art. See, e.g., Sambrook, Fritsch and maniotis, molecular cloning, a laboratory manual, 3 rd edition (2002). The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out under conventional conditions or conditions recommended by the manufacturers. The various chemicals used in the examples are commercially available.

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 in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

The present invention will be further illustrated with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.