CN113308522A - Method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level - Google Patents

Method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level Download PDF

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CN113308522A
CN113308522A CN202110757522.6A CN202110757522A CN113308522A CN 113308522 A CN113308522 A CN 113308522A CN 202110757522 A CN202110757522 A CN 202110757522A CN 113308522 A CN113308522 A CN 113308522A
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孙成铭
杨新
姜慧慧
彭绒雪
孙茁凯
刘杰
杜江东
孙枝红
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Abstract

The invention discloses a method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level, which comprises the following steps: s1, selecting a CpG island region of a PER2 promoter: 1) inquiring a PER2 promoter sequence according to a gene database; 2) predicting a GpG island enrichment region contained in a PER2 promoter sequence through a CpG island online prediction website and determining a target sequence; s2, designing a primer and a probe for detecting methylation of a PER2 promoter according to a target sequence; s3, designing a target sequence methylated sequence and a target sequence unmethylated sequence according to the conversion rule of the bisulfite to the gene sequence, artificially synthesizing corresponding sequences, and storing the sequences in a plasmid form; s4, establishing a real-time fluorescence quantitative PCR method for detecting PER2 promoter methylation and confirming the performance. The invention can accurately and quantitatively detect the methylation level of the PER2 promoter and provides an effective tool and method for researching PER2 methylation.

Description

Method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level
Technical Field
The invention relates to the technical field of detection of PER2 promoter methylation level, and particularly relates to a method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level.
Background
Many studies prove that PER2 genes of leukemia patients, especially myeloid leukemia patients, are hypermethylated, and PER2 methylation can be an important epigenetic marker for early diagnosis, prognosis and treatment of leukemia. Currently, methylation-specific MSP (MSP) method is mostly adopted for methylation detection of PER2 promoters, is a qualitative detection method, and is not easy to be used for dynamic monitoring and level comparison. At present, a method for quantitatively detecting PER2 gene promoter methylation which is strictly confirmed does not exist, and a reference substance for quantitatively detecting the PER2 promoter methylation level does not exist.
H Peng (PMID:31031806), M Pienkowska (PMID:31409384) and the like detect the methylation of the PER2 promoter by sulfite pyrosequencing. However, the sulfite pyrophosphate sequencing method is relatively expensive.
F Hernandez-Rosas (PMID:30008892) and the like adopt methylation-specific PCR (MS-PCR) to detect the methylation of the PER2 promoter, but the method can only carry out qualitative detection, cannot realize accurate quantification and cannot dynamically detect the change of the methylation level.
Disclosure of Invention
In view of the above, in order to solve the above technical problems, the present invention aims to establish a reliable and stable real-time fluorescence quantitative MSP method for detecting the methylation level of the PER2 gene promoter based on a Taqman probe, that is, a method for establishing real-time fluorescence quantitative detection of the methylation level of the PER2 promoter.
The adopted technical scheme is as follows:
a method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level comprises the following steps:
s1, selecting a CpG island region of a PER2 promoter:
1) inquiring a PER2 promoter sequence according to a gene database;
2) predicting a GpG island enrichment region contained in a PER2 promoter sequence through a CpG island online prediction website and determining a target sequence;
s2, designing a primer and a probe for detecting methylation of a PER2 promoter according to a target sequence;
s3, designing a target sequence methylated sequence and a target sequence unmethylated sequence according to the conversion rule of the bisulfite to the gene sequence, artificially synthesizing corresponding sequences, and storing the sequences in a plasmid form;
s4, establishing a real-time fluorescence quantitative PCR method for detecting PER2 promoter methylation and confirming the performance: screening proper primers, selecting a primer probe combination, and evaluating the performance of the detection method by taking corresponding plasmids as templates so as to confirm the detection performance of the established method.
Further, in S4, the evaluating the performance of the detection method includes evaluating each of the sensitivity, specificity, accuracy and amplification efficiency of the detection method.
Further, in S4, in evaluating the performance of the detection method, the copy concentration of the pseudo reference substance is calculated as follows: the concentration of plasmid DNA was 20 ng/uL. The plasmid concentration is converted into a copy number calculation formula: copy number (copy/. mu.L) × (plasmid concentration ng/. mu.L × 10-9) × (6.02 × 1023)/(plasmid base number × 660), the plasmid copy number calculated according to this formula is 6 × 109 copies/uL; plasmid MSP-5 was then serially diluted 10-fold to 0.6 copies/uL.
Further, in S1, the sequence of the PER2 promoter is shown as SEQ ID NO: 1.
further, in S1, the target sequence is determined as shown in SEQ ID NO: 2.
furthermore, in S3, the artificially synthesized corresponding sequences are obtained by designing methylated and unmethylated sequences of the sequences respectively, and then carrying out chemical modification with bisulfite, and the sequences are respectively used as a positive reference substance MSP-5 and a negative reference substance MSP-6.
Further, in S3, MSP-5 and MSP-6 were ligated to pUC57 vector, transformed into Escherichia coli Top10 competent cells, cultured with shaking at 37 ℃ for 1 hour, spread on Amp + agar plates, screened for positive clones and expanded for culture, and identified by PCR using methylated primers and unmethylated primers, respectively, for MSP-5 and MSP-6, with the methylated primer sequences as shown in SEQ ID NO: 3 and SEQ ID NO: 4, the sequence of the non-methylated primer is shown as SEQ ID NO: 5 and SEQ ID NO: 6.
further, in S3, the MSP-5 sequence is shown in SEQ ID NO: 7, the MSP-6 sequence is shown as SEQ ID NO: 8.
further, the sequence of MSP-5 linked to pUC57 vector is shown in SEQ ID NO: 9, the sequence of the MSP-6 connected with pUC57 vector is shown in SEQ ID NO: 10.
further, in S4, the primers are a pair of primers, and the sequences are shown in SEQ ID NO: 11 and SEQ ID NO: 12, the probe is a probe with a sequence of SEQ ID NO: 13.
the invention has the beneficial effects that:
the invention can accurately and quantitatively detect the methylation level of the PER2 promoter and provides an effective tool and method for researching PER2 methylation.
Drawings
FIG. 1 is a diagram showing the CpG island distribution of a target sequence.
FIG. 2 is a graph showing the results of PCR amplification experiments for a target gene (target sequence). In the figure, M: DNA marker; 1: MSP-5; 2: MSP-6; 3: blank control.
FIG. 3 is a diagram showing the results of a lower limit test in a conventional MSP method. In the figure, M is DNA marker. Labels1-9: 6X 107copies/uL,6×106copies/uL,6×105copies/uL,6×104copies/uL,6×103copies/uL,6×102copies/uL,6×10copies/uL,6copies/uL,MSP-6.
FIG. 4 is a diagram showing the results of a lower limit test in a real-time fluorescent quantitative MSP method. In the figure, 1-9: 6X 107copies/uL,6×106copies/uL,6×105copies/uL,6×104copies/uL,6×103copies/uL,6×102copies/uL,6×10copies/uL,6copies/uL,MSP-6.
FIG. 5 is a diagram showing the results of the specificity experiment of Taqman real-time fluorescence quantitative MSP method.
FIG. 6 is a standard graph of an amplification reaction.
Detailed Description
The following examples are illustrative of preferred embodiments of the present invention and are not to be construed as limiting the invention in any way.
The invention discloses a method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level, which is a method for establishing real-time fluorescence quantitative PCR detection of PER2 promoter methylation level based on Taqman probe, and comprises the following steps:
s1, selecting a CpG island region of a PER2 promoter:
1) inquiring a PER2 promoter sequence according to a gene database Genebank; PER2 promoter sequence (generally, 2kb upstream region of gene is considered to be promoter region of the gene, so 2kb upstream sequence of gene is selected, each gene has multiple promoter sequences, the method is a research aiming at the first promoter sequence): homo sapiens period circular algorithm regulator 2(PER2), RefSeqGene on chromosome 2, Sequence ID: NG _012146.1:3465to 5464
Specifically, the PER2 promoter sequence is shown as SEQ ID NO: 1.
2) predicting a GpG island enrichment region contained in a PER2 promoter sequence through a CpG island online prediction website and determining a target sequence; the target Sequence rich in CpG islands selected by PER2 (Homo sapiens period circular promoter 2(PER2), RefSeqGene on chromosome 2, Sequence ID: NG-012146.1: 4857 and 5156) is specifically shown in SEQ ID NO: 2.
the CpG island distribution of the target sequence is shown in FIG. 1.
S2, designing a primer and a probe for detecting methylation of a PER2 promoter according to a target sequence;
s3, designing a target sequence methylated sequence and a target sequence unmethylated sequence according to the conversion rule of the bisulfite to the gene sequence, artificially synthesizing corresponding sequences, and storing the sequences in a plasmid form;
wherein corresponding sequences are artificially synthesized as positive and negative reference substances:
according to a PER2 sequence searched by GenBank, a section of CpG enrichment region of a PER2 gene promoter region is selected as an original template sequence. According to the rule that DNA is chemically modified by bisulfite, sequences of the sequence after methylation and unmethylated bisulfite chemical modification are respectively designed as positive (MSP-5) and negative (MSP-6) reference substances. The two sequences are respectively connected with a pUC57 vector, then transformed into escherichia coli Top10 competent cells, cultured for 1h under oscillation at 37 ℃, coated on an Amp + agar plate, screened for positive clones and cultured in an amplification way, and respectively identified by a methylated primer and an unmethylated primer for PCR products of a bacterial liquid of MSP-5 and MSP-6, wherein the primer sequences are shown in Table 1. Extracting positive plasmids, sending the positive plasmids to Shanghai bio-corporation for sequencing, and carrying out BLAST comparison analysis on the sequences by using NCBI.
TABLE 1 primer sequences for methylated and unmethylated plasmid identification
Figure BDA0003148373360000051
Two fragments were synthesized, the mock post-methylated bisulfite conversion sequence (MSP-5) and the non-methylated post-bisulfite conversion sequence (MSP-6):
MSP-5 has a sequence shown in SEQ ID NO: 7, the MSP-6 sequence is shown as SEQ ID NO: 8.
wherein, the PCR identification result is as follows:
PCR product identification and recombinant plasmid sequencing identification
The PCR amplification product was identified by agarose gel electrophoresis, and the results showed that the size of the fragment was consistent with the size of the fragment shown on the electrophoretogram, which is in line with the expected results, as shown in FIG. 2. Through sequencing identification, the sequence of the recombinant plasmids MSP-5 and MSP-6 is completely matched with the reference sequence.
After the plasmid is inserted, the complete sequence of PUC-57-MSP-5, namely the sequence of MSP-5 connected with pUC57 vector is shown as SEQ ID NO: 9, pUC57-MSP-6 complete sequence, namely the sequence of MSP-6 after being respectively connected with pUC57 vector is shown as SEQ ID NO: 10.
s4, establishing a real-time fluorescence quantitative PCR method for detecting PER2 promoter methylation and confirming the performance: screening proper primers, selecting an optimal primer probe combination, and evaluating the sensitivity, specificity, accuracy, amplification efficiency and other performances of the detection method by taking corresponding plasmids as templates so as to confirm the detection performance of the established method.
Wherein the synthesis of methylation specific primers and probes: a pair of specific primers and a probe are designed according to the CpG distribution characteristics of MSP-5 and MSP-6, the distance between the upstream primer and the downstream primer and the Tm value. As shown in table 2.
TABLE 2 methylation specific primer and probe sequences
Figure BDA0003148373360000052
Specific performance evaluation of the detection method:
copy concentration calculation method of simulated reference substance: the concentration of plasmid DNA was 20 ng/uL. The plasmid concentration is converted into a copy number calculation formula: copy number (copy/. mu.L) ═ plasmid concentration ng/. mu.L × 10-9)×(6.02×1023) V (plasmid base number. times.660), the plasmid copy number calculated according to this formula is 6X 109copies/uL. Plasmid MSP-5 was then serially diluted 10-fold to 0.6 copies/uL.
(1) Sensitivity of analysis (sensitivity)
And performing 20 times of Taqman real-time fluorescence quantitative MSP detection on the positive standard substance diluted by 10 times according to an optimal reaction system and reaction conditions, and determining the lowest concentration which can be detected by at least 95% of samples in a detection result as a lower detection limit according to an MIQE guideline. Meanwhile, a common MSP method and a fluorescence quantitative PCR method (SYBR GREEN) are adopted to amplify the same template, MSP-6 is set as negative control in each experiment, and finally, the lower detection limits of 3 methods are determined, and the detection results of various methods are compared to evaluate the detection sensitivity of the method.
The result shows that the detection limit of the common MSP method is 60copies/uL, as shown in FIG. 3, the detection limit of the fluorescent quantitative PCR method (SYBR GREEN) is 60copies/uL, and the detection limit of the method is 6copies/uL, as shown in FIG. 4. The detection sensitivity of TaqMan real-time fluorescence quantitative MSP is 10 times higher than that of common MSP and fluorescence quantitative PCR (SYBR GREEN).
(2) Analysis of specificity (specificity)
2 different concentrations (2X 10)-4ng/uL and 2X 10-5ng/uL) MSP-5 and MSP-6 are taken as templates to carry out Taqman real-time fluorescence quantitative MSP reaction, the result is shown in figure 3, an amplification curve appears in the MSP-5, and a specific amplification curve does not appear in the control group MSP-6, as shown in figure 5. According to the results, the method of the present invention has good specificity.
(3) Analytical precision (reproducibility)
Select 6X 107copies/uL，6×106copies/uL，6×105copies/uL，6×104The results of 10 repeated tests of the 4 mspes-5 with different concentrations of copies/uL show that the intra-batch repeated variation coefficients are respectively 0.85%, 0.86%, 1.06% and 1.41% (shown in table 3), and the inter-batch repeated variation coefficients are respectively 2.69%, 2.37%, 2.90% and 2.89% (shown in table 4), and are all less than 3%, which indicates that the method of the invention has good repeatability.
TABLE 3 results of repeated experiments in batches for Taqman real-time fluorescent quantitation of MSP
Figure BDA0003148373360000071
TABLE 4 Taqman real-time fluorescence quantitation of MSP batch-to-batch repeat experiment results
Figure BDA0003148373360000072
(4) Accuracy of
And (3) consistency comparison of the real-time fluorescence quantitative MSP and a common MSP detection method:
the detection results of 81 leukemia specimens are shown in Table 5, 36 positive parts and 45.7 positive parts are detected by TaqMan real-time fluorescence quantitative MSP, and only 17 positive parts and 20.9 positive parts are detected by common MSP.
TABLE 5 TaqMan real-time fluorescence quantitation of MSP vs general MSP detection PER2 methylation detection rates
Figure BDA0003148373360000073
Inconsistent third party validation of samples (pyrosequencing method): the sequencing results of the specimen which is positive by the method and negative by the common MSP method are positive.
(5) Efficiency of amplification
Establishing a Taqman real-time fluorescence quantitative MSP method and a standard curve:
MSP-5 is continuously diluted by 10 times to different concentrations to be used as a template for real-time fluorescence quantitative MSP. In order to determine the optimal reaction system and reaction conditions of the Taqman real-time fluorescence quantitative MSP method, different primer concentrations and probe concentrations are adopted for amplification (the primer concentration is 100nM-400nM, and the probe concentration is 200nM-500nM) in an experiment, and meanwhile, the annealing temperature is gradually increased from 55 ℃ to 65 ℃ so as to screen out the optimal primer and probe concentrations and the optimal annealing temperature, so that the optimal amplification efficiency is high. 3 replicates of each concentration were performed at the time of the experiment. And performing Taqman real-time fluorescence quantitative MSP reaction on the MSP-5 diluted in the gradient according to the established optimal reaction system and reaction conditions, finally analyzing a data result, drawing a standard curve, wherein the Y axis represents the cycle number (Ct value) of the fluorescence signal of the detection sample reaching the set threshold value, the X axis represents the logarithmic value of the DNA copy number, and the correlation coefficient is calculated.
The logarithm of the Ct value and the DNA copy number of the MSP-5, which was continuously diluted 10 times and amplified by the Taqman real-time fluorescence quantitative MSP method, was plotted as a standard curve, as shown in FIG. 6. The DNA copy number is 60-6X 10 as can be seen from the standard curve7The copies/uL has good linear relation and a correlation coefficient R299% and an amplification efficiency of 103% with a slope of-3.23, an intercept of 39.88, and a standard curve equation of-3.23 x + 39.887.
Wherein the amplification efficiency is equal to Eff-10 (-1/slope) -1
In summary, in one aspect, the invention provides a mimetic reference substance that is not formulated using any of the previously reported methods, such as treatment of DNA extracted from a cell line or cord blood with methyltransferase. Since the methyltransferase treatment and the subsequent bisulfite conversion have problems in terms of treatment efficiency and conversion efficiency, a reference substance cannot be specified in an accurate quantitative manner. The method adopts a direct synthesis method, artificially synthesizes sequences after 100 percent methylation and non-methylation of the target sequence and 100 percent conversion of the bisulfite respectively to be used as a simulated reference substance, and can accurately and quantitatively prepare standard products with different copy concentrations. The influence of the treatment efficiency of the methyltransferase and the conversion efficiency of the bisulfite on the performance evaluation is avoided.
On the other hand, conventionally, methylation detection of PER2 was carried out by a qualitative method, and judgment was carried out by agarose gel electrophoresis. According to the method, the synthetic primer and the Taqman probe are designed, and the quantitative standard is prepared, so that the PER2 methylation level can be quantitatively detected, and the change of the PER2 methylation level can be dynamically detected.
The above-listed detailed description is only a specific description of a possible embodiment of the present invention, and they are not intended to limit the scope of the present invention, and equivalent embodiments or modifications made without departing from the technical spirit of the present invention should be included in the scope of the present invention.

Claims (10)

1. A method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level is characterized by comprising the following steps:
s1, selecting a CpG island region of a PER2 promoter:
1) inquiring a PER2 promoter sequence according to a gene database;
2) predicting a GpG island enrichment region contained in a PER2 promoter sequence through a CpG island online prediction website and determining a target sequence;
s2, designing a primer and a probe for detecting methylation of a PER2 promoter according to a target sequence;
s3, designing a target sequence methylated sequence and a target sequence unmethylated sequence according to the conversion rule of the bisulfite to the gene sequence, artificially synthesizing corresponding sequences, and storing the sequences in a plasmid form;
s4, establishing a real-time fluorescence quantitative PCR method for detecting PER2 promoter methylation and confirming the performance: screening proper primers, selecting a primer probe combination, and evaluating the performance of the detection method by taking corresponding plasmids as templates so as to confirm the detection performance of the established method.
2. The method for establishing real-time quantitative fluorescence detection of promoter methylation level of PER2, according to claim 1, wherein in S4, the evaluation of the performance of the detection method comprises the evaluation of the sensitivity, specificity, accuracy and amplification efficiency of the detection method.
3. The method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level according to claim 2, wherein in S4, in the evaluation of the performance of the detection method, the copy concentration of the simulated reference substance is calculated as follows: the concentration of plasmid DNA was 20 ng/uL. The plasmid concentration is converted into a copy number calculation formula: copy number (copy/. mu.L) ═ plasmid concentration ng/. mu.L × 10-9)×(6.02×1023) V (plasmid base number. times.660), the plasmid copy number calculated according to this formula is 6X 109copies/uL; plasmid MSP-5 was then serially diluted 10-fold to 0.6 copies/uL.
4. The method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level according to claim 1, wherein in S1, the sequence of PER2 promoter is shown in SEQ ID NO: 1.
5. the method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level according to claim 1, wherein in S1, the sequence of interest is determined as shown in SEQ ID NO: 2.
6. the method for establishing real-time quantitative fluorescence detection of promoter methylation level of PER2, according to claim 1, wherein the artificially synthesized corresponding sequences in S3 are sequences that are chemically modified with bisulfite after methylation and non-methylation of the sequences, and are used as positive reference substance MSP-5 and negative reference substance MSP-6, respectively.
7. The method for establishing real-time quantitative fluorescence detection of methylation level of PER2 promoter according to claim 6, wherein in S3, MSP-5 and MSP-6 are respectively connected with pUC57 vector, then transformed into competent cells of Escherichia coli Top10, cultured with shaking at 37 ℃ for 1h, spread on Amp + agar plate, screened for positive clone and expanded for culture, and identified by PCR with methylated primer and unmethylated primer respectively for MSP-5 and MSP-6, and the sequence of methylated primer is as shown in SEQ ID NO: 3 and SEQ ID NO: 4, the sequence of the non-methylated primer is shown as SEQ ID NO: 5 and SEQ ID NO: 6.
8. the method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level according to claim 7, wherein in S3, MSP-5 has a sequence as shown in SEQ ID NO: 7, the MSP-6 sequence is shown as SEQ ID NO: 8.
9. the method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level according to claim 8, wherein the sequence of MSP-5 linked to pUC57 vector is as shown in SEQ ID NO: 9, the sequence of the MSP-6 connected with pUC57 vector is shown in SEQ ID NO: 10.
10. the method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level according to claim 1, wherein in S4, the primers are a pair of primers, and the sequence is shown as SEQ ID NO: 11 and SEQ ID NO: 12, the probe is a probe with a sequence of SEQ ID NO: 13.
CN202110757522.6A 2021-07-05 2021-07-05 Method for establishing real-time fluorescence quantitative detection of PER2 promoter methylation level Pending CN113308522A (en)

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