CN110628900A - Method for detecting methylation of multiple genes of non-small cell lung cancer - Google Patents

Method for detecting methylation of multiple genes of non-small cell lung cancer Download PDF

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CN110628900A
CN110628900A CN201810654035.5A CN201810654035A CN110628900A CN 110628900 A CN110628900 A CN 110628900A CN 201810654035 A CN201810654035 A CN 201810654035A CN 110628900 A CN110628900 A CN 110628900A
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primer
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陈琦
梁昊原
谷东风
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Shenzhen Shengbizhi Technology Development Co Ltd
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Abstract

The invention provides a methylation detection method of multiple genes of non-small cell lung cancer, which comprises the following steps: constructing 6 primer pairs of methylation markers of the non-small cell lung cancer; extracting free ctDNA in a blood sample to be detected, and purifying and converting the ctDNA; adding 4ul ctDNA with the concentration of 27ng into a 25ul PCR reaction system; putting the PCR reaction system into a PCR instrument for PCR amplification reaction; performing gel electrophoresis on the PCR reaction product; staining the gel and performing fluorescence analysis on the gel by using a gel imager; judging whether 2 or more than 2 fluorescent color bands appear in the gel; if yes, the blood sample to be detected contains the gene mutation DNA segment of the non-small cell lung cancer; if not, the result shows that the blood sample to be tested does not contain the gene mutation DNA segment of the non-small cell lung cancer. The invention utilizes one-time PCR reaction to detect whether a plurality of methylation markers of the non-small cell lung cancer exist or not, and improves the accuracy, specificity and sensitivity of the detection of the non-small cell lung cancer.

Description

Method for detecting methylation of multiple genes of non-small cell lung cancer
Technical Field
The invention relates to the technical field of EB virus detection, in particular to a methylation detection method for multiple genes of non-small cell lung cancer.
Background
DNA methylation is an important epigenetic mechanism and is one of the key mechanisms for oncogene inactivation, and in some cases may be the only mechanism. A summary of methylation in several protocols has been demonstrated for non-small cell lung cancer, suggesting that non-small cell lung cancer displays a CpG island methylation phenotype. As an early event of tumorigenesis, detection of abnormal methylation of tumor suppressor gene DNA allows molecular diagnosis before clinical manifestations or imaging evidence appear in patients.
ctdna (circulating tumor dna): DNA fragments from tumor genomes that carry certain characteristics (including mutations, deletions, insertions, rearrangements, copy number abnormalities, methylation, etc.) are constantly circulating in the human blood circulation. The concentration of the polypeptide in blood is low, the polypeptide is highly fragmented, the extraction difficulty is high, and the sensitivity and specificity of clinical detection are reduced. In the prior art, one methylation specific PCR reaction can only detect the methylation level of a single gene, and the possibility of lung cancer occurrence cannot be accurately predicted. Meanwhile, the uncertainty of the cancer suppressor gene of the non-small cell lung cancer and the limitation of ctDNA cause that the detection of the single methylation marker of the non-small cell lung cancer cannot meet the clinical requirement.
Disclosure of Invention
The invention mainly aims to provide a method for detecting methylation of multiple genes of non-small cell lung cancer, which can detect whether a plurality of methylation markers of the non-small cell lung cancer exist or not at the same time in one-time PCR reaction detection and improves the accuracy, specificity and sensitivity of the detection of the non-small cell lung cancer.
In order to achieve the above object, the present invention provides a method for detecting methylation of multiple genes of non-small cell lung cancer, the method comprising the steps of: selecting 6 genes as methylation markers of the non-small cell lung cancer; constructing 6 primer pairs corresponding to 6 methylation markers respectively; extracting free ctDNA in a blood sample to be detected, and purifying and converting the ctDNA; adding the ctDNA with the concentration of 4ul and the concentration of 27ng after conversion treatment into a 25ul PCR reaction system; putting the PCR reaction system into a PCR instrument, and carrying out PCR amplification reaction on the PCR reaction system according to a set PCR reaction program to obtain a PCR reaction product; performing gel electrophoresis on the PCR reaction product; dyeing the gel after electrophoresis by using a dyeing agent, and performing fluorescence analysis on the dyed gel by using a gel imager; the gel imager judges whether 2 or more than 2 fluorescent color bands appear in the gel; if 2 or more than 2 fluorescent color bands appear in the gel, the gel imager displays that the blood sample to be detected contains the gene mutation DNA fragment of the non-small cell lung cancer; if 2 strips or no fluorescence color band appears in the gel, the gel imager shows that the blood sample to be detected does not contain the gene mutation DNA fragment of the non-small cell lung cancer.
Preferably, the 6 methylation markers are CALCA, DLEC1, HOXA9, TBX5, PITX2 and RASSF1a, and the 6 primer pairs comprise 6 groups of primer sequences, which are specifically represented as follows:
CALCA forward primer: 5'-CGGAATTTTTTCGATTTATAGC-3', respectively;
CALCA reverse primer: 5'-AAAACCCTATAAAAACGACGAC-3', respectively;
forward primer of DLEC 1: 5'-GATTAAGCGATGACGGGATTC-3', respectively;
DLEC1 reverse primer: 5'-ACCCGACTAATAACGAAATTAACG-3', respectively;
HOXA9 forward primer: 5'-GGTTAATGGGGGCGCGGGCGTC-3', respectively;
HOXA9 reverse primer: 5'-TCATATAACAACTTAATAACACCG-3', respectively;
TBX5 forward primer: 5'-GGGACGCGTAAAATTTAGAATC-3', respectively;
TBX5 reverse primer: 5'-AACACAAAACCGAAAAACGTC-3', respectively;
PITX2 forward primer: 5'-CGTTATTAGTTGAAGGTAAGGTCG-3', respectively;
PITX2 reverse primer: 5'-AACACCGAAAAATACAATCCG-3', respectively;
RASSF1a forward primer: 5'-GTGTTAACGCGTTGCGTATC-3', respectively;
RASSF1a reverse primer: 5'-AACCCCGCGAACTAAAAACGA-3' are provided.
Preferably, the PCR reaction system comprises 1.8 XPCR solution, 5mM MgCl20.3nM deoxyribonucleoside triphosphate, 40nM CALCA forward primer, 40nM CALCA reverse primer, 40nM DLEC1 forward primer, 40nM DLEC1 reverse primer, 120nM HOXA9 forward primer, 120nM HOXA9 reverse primer, 20nM PITX2 forward primer, 20nM PITX2 reverse primer, 20nM TBX5 forward primer, 20nM TBX5 reverse primer, 280nM RASSF1a forward primer, 280nM RASSF1a reverse primer and 2.5 units of DNA polymerase.
Preferably, the DNA polymerase is Taq Platinum DNA polymerase.
Preferably, the present invention also provides a method for detecting methylation of multiple genes of non-small cell lung cancer, which comprises the following steps: selecting 6 genes as methylation markers and 1 reference marker of the non-small cell lung cancer; constructing 6 primer pairs corresponding to 6 methylation markers and 1 primer pair corresponding to 1 reference marker respectively; extracting free ctDNA in a blood sample to be detected, and purifying and converting the ctDNA; adding the ctDNA with the concentration of 4ul and the concentration of 27ng after conversion treatment into a 25ul PCR reaction system; putting the PCR reaction system into a PCR instrument, and carrying out PCR amplification reaction on the PCR reaction system according to a set PCR reaction program to obtain a PCR reaction product; performing gel electrophoresis on the PCR reaction product; dyeing the gel after electrophoresis by using a dyeing agent, and performing fluorescence analysis on the dyed gel by using a gel imager; the gel imager judges whether 3 or more than 3 fluorescent color bands appear in the gel; if 3 or more than 3 fluorescent color bands appear in the gel, the gel imager displays that the blood sample to be detected contains the gene mutation DNA fragment of the non-small cell lung cancer; if 2 or 1 fluorescent color band appears in the gel, the gel imager shows that the blood sample to be detected does not contain the gene mutation DNA segment of the non-small cell lung cancer; and if no fluorescence color band appears in the gel, the gel imager judges that the ctDNA conversion and the PCR reaction are invalid.
Preferably, the 6 methylation markers are CALCA, DLEC1, HOXA9, TBX5, PITX2 and RASSF1a, and the 6 primer pairs comprise 6 groups of primer sequences, which are specifically represented as follows:
CALCA forward primer: 5'-CGGAATTTTTTCGATTTATAGC-3', respectively;
CALCA reverse primer: 5'-AAAACCCTATAAAAACGACGAC-3', respectively;
forward primer of DLEC 1: 5'-GATTAAGCGATGACGGGATTC-3', respectively;
DLEC1 reverse primer: 5'-ACCCGACTAATAACGAAATTAACG-3', respectively;
HOXA9 forward primer: 5'-GGTTAATGGGGGCGCGGGCGTC-3', respectively;
HOXA9 reverse primer: 5'-TCATATAACAACTTAATAACACCG-3', respectively;
TBX5 forward primer: 5'-GGGACGCGTAAAATTTAGAATC-3', respectively;
TBX5 reverse primer: 5'-AACACAAAACCGAAAAACGTC-3', respectively;
PITX2 forward primer: 5'-CGTTATTAGTTGAAGGTAAGGTCG-3', respectively;
PITX2 reverse primer: 5'-AACACCGAAAAATACAATCCG-3', respectively;
RASSF1a forward primer: 5'-GTGTTAACGCGTTGCGTATC-3', respectively;
RASSF1a reverse primer: 5'-AACCCCGCGAACTAAAAACGA-3', respectively;
the 1 reference marker is beta-ACTIN, and 1 primer pair corresponding to the reference marker beta-ACTIN comprises 1 group of primer sequences, which are specifically expressed as follows:
β -ACTIN forward primer: 5'-TTTTTAGGGAGGAGT TGGAAGTAGT-3', respectively;
beta-ACTIN reverse primer: 5'-AAAATATACC CTCCCCCATA CC-3' are provided.
Preferably, the PCR reaction system comprises 1.8 XPCR solution, 5mM MgCl20.3nM deoxyribonucleoside triphosphate, 40nM CALCA forward primer, 40nM CALCA reverse primer, 40nM DLEC1 forward primer, 40nM DLEC1 reverse primer, 120nM HOXA9 forward primer, 120nM HOXA9 reverse primer, 20nM PITX2 forward primer, 20nM PITX2 reverse primer, 20nM TBX5 forward primer, 20nM TBX5 reverse primer, 280nM RASSF1a forward primer, 280nM RASSF1a reverse primer, 10nM beta-ACTIN forward primer, 10nM beta-ACTIN reverse primer and 2.5 units of DNA polymerase.
Preferably, the DNA polymerase is Taq Platinum DNA polymerase.
Preferably, the PCR reaction procedure comprises the steps of: step 1: at a temperature of 95 ℃ for 3 minutes; step 2: a total of 4 cycles of step 2 performed at a temperature of 94 ℃ for 1 minute, at a temperature of 60 ℃ for 30 seconds, and at a temperature of 65 ℃ for 45 seconds; and step 3: a total of 36 amplification cycles of step 3 performed at a temperature of 94 ℃ for 1 minute, 56 ℃ for 1 minute, 65 ℃ for 45 seconds; and 4, step 4: extension reaction at 65 ℃ for 4 minutes; and 5: the PCR reaction was stopped at a temperature of 4 ℃ and the PCR reaction product was stored at a temperature of 4 ℃.
Preferably, the purification treatment adopts a magnetic bead method or a centrifugal column method to extract free ctDNA in the blood sample to be detected so as to remove impurities and purify the ctDNA; in the conversion treatment, sulfite or bisulfite is used as a reagent to carry out sulfite conversion treatment or bisulfite conversion treatment on ctDNA; performing gel electrophoresis on the PCR reaction product by using agarose gel or polyacrylamide gel; the stain is ethidium bromide, SYBR Green I, GelRed or GoldView stain.
Compared with the prior art, the method for detecting methylation of multiple genes of non-small cell lung cancer adopts the technical scheme, and the following technical effects are achieved: the method can detect the existence of a plurality of non-small cell lung cancer methylation markers simultaneously in one-time PCR reaction detection, and improves the accuracy, specificity and sensitivity of non-small cell lung cancer detection. The invention solves the problems that the possibility that the occurrence of lung cancer cannot be accurately predicted because only the methylation level of a single gene can be detected by one methylation specific PCR reaction and the detection of a single methylation marker of non-small cell lung cancer cannot meet the clinical requirement due to the uncertainty of a cancer suppressor gene of non-small cell lung cancer and the limitation of ctDNA.
Drawings
FIG. 1 is a diagram of a first preferred embodiment of the primer pair for detecting methylation of multiple genes of non-small cell lung cancer according to the present invention;
FIG. 2 is a diagram of a second preferred embodiment of the primer pair for detecting methylation of multiple genes of non-small cell lung cancer according to the present invention;
FIG. 3 is a flowchart of a first preferred embodiment of the methylation detection method of multiple genes of non-small cell lung cancer according to the present invention;
FIG. 4 is a flowchart of a method for detecting methylation of multiple genes of non-small cell lung cancer according to a second preferred embodiment of the present invention.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
To further explain the technical means and effects of the present invention adopted to achieve the predetermined objects, the following detailed description of the embodiments, structures, features and effects of the present invention will be given with reference to the accompanying drawings and preferred embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a primer pair for detecting methylation of multiple genes of non-small cell lung cancer, which comprises 6 groups of primer sequences corresponding to methylation markers of the non-small cell lung cancer, wherein the methylation markers can be used for detecting the non-small cell lung cancer by using blood as a sample. As a preferred embodiment, the methylation markers are respectively: CALCA, DLEC1, HOXA9, TBX5, PITX2, RASSF1 a.
As shown in FIG. 1, FIG. 1 is a schematic diagram of a first preferred embodiment of the primer pair for detecting methylation of multiple genes of non-small cell lung cancer according to the present invention. In a first preferred embodiment, the primer pair for detecting methylation of the lung cancer multiple gene comprises 6 sets of primer sequences corresponding to 6 methylation markers, each methylation marker corresponds to one primer pair, and each primer pair comprises a forward primer (MF) and a reverse primer (MR), which are specifically represented as follows:
(1) and CALCA forward primer: 5'-CGGAATTTTTTCGATTTATAGC-3' (SEQ ID NO. 1);
(2) and CALCA reverse primer: 5'-AAAACCCTATAAAAACGACGAC-3' (SEQ ID NO. 2);
(3) DLEC1 forward primer: 5'-GATTAAGCGATGACGGGATTC-3' (SEQ ID NO. 3);
(4) DLEC1 reverse primer: 5'-ACCCGACTAATAACGAAATTAACG-3' (SEQ ID NO. 4);
(5) HOXA9 forward primer: 5'-GGTTAATGGGGGCGCGGGCGTC-3' (SEQ ID NO. 5);
(6) HOXA9 reverse primer: 5'-TCATATAACAACTTAATAACACCG-3' (SEQ ID NO. 6);
(7) TBX5 forward primer: 5'-GGGACGCGTAAAATTTAGAATC-3' (SEQ ID NO. 7);
(8) TBX5 reverse primer: 5'-AACACAAAACCGAAAAACGTC-3' (SEQ ID NO. 8);
(9) PITX2 forward primer: 5'-CGTTATTAGTTGAAGGTAAGGTCG-3' (SEQ ID NO. 9);
(10) PITX2 reverse primer: 5'-AACACCGAAAAATACAATCCG-3' (SEQ ID NO. 10);
(11) RASSF1a forward primer: 5'-GTGTTAACGCGTTGCGTATC-3' (SEQ ID NO. 11);
(12) RASSF1a reverse primer: 5'-AACCCCGCGAACTAAAAACGA-3' (SEQ ID NO. 12).
As another preferred embodiment, in order to verify the correctness of the ctDNA transformation and PCR reaction process in the blood sample to be tested and ensure the accuracy of the detection of the non-small cell lung cancer, the invention adds a primer pair (comprising a beta-ACTIN forward primer and a beta-ACTIN reverse primer) corresponding to a reference marker beta-ACTIN into the PCR reaction system.
As shown in FIG. 2, FIG. 2 is a diagram of a second preferred embodiment of the primer pair for detecting methylation of multiple genes of non-small cell lung cancer according to the present invention. In a second embodiment, the primer pair for detecting methylation of a lung cancer multiple gene according to the present invention not only includes 6 sets of primer sequences corresponding to the above 6 methylation markers, but also includes 1 set of primer sequences corresponding to 1 reference marker. Wherein, 1 group of primer sequences corresponding to 1 reference marker comprises:
(13) β -ACTIN forward primer: 5'-TTTTTAGGGAGGAGT TGGAAGTAGT-3' (SEQ ID NO. 13);
(14) beta-ACTIN reverse primer: 5'-AAAATATACC CTCCCCCATA CC-3' (SEQ ID NO. 14).
Referring to FIG. 3, FIG. 3 is a flowchart of a first preferred embodiment of the methylation detection method of multiple genes of non-small cell lung cancer according to the present invention. In a first preferred embodiment, the method for detecting methylation of multiple genes of non-small cell lung cancer comprises the following steps S30 to S39.
S30, selecting 6 genes as methylation markers of the non-small cell lung cancer; specifically, based on the lung cancer early-stage research and the existing research results, 6 genes are selected as methylation markers related to the non-small cell lung cancer, and the methylation markers are respectively as follows: CALCA, DLEC1, HOXA9, TBX5, PITX2, RASSF1 a.
Step S31, respectively constructing 6 primer pairs corresponding to 6 methylation markers; in this example, as shown in fig. 1, fig. 1 lists 6 sets of primer sequences corresponding to 6 methylation markers, which are DLEC1 forward and reverse primers, PITX2 forward and reverse primers, TBX5 forward and reverse primers, CALCA forward and reverse primers, RASSF1a forward and reverse primers, and HOXA9 forward and reverse primers, respectively.
Step S32, extracting free ctDNA from the blood sample to be tested, in this embodiment, it is considered that the concentration of ctDNA in serum of the blood sample is 3-24 times of that in plasma, but the coagulation process is easily contaminated by impurities, so it is preferable to extract ctDNA from plasma of the blood sample, and purify and convert the ctDNA. In this embodiment, ctDNA is easily decomposed by dnase in blood, and the purification of ctDNA needs to be performed as soon as possible, the purification method may be a magnetic bead method or a centrifugal column method commonly used in the industry to extract free ctDNA in a blood sample to be tested to remove impurities and purify ctDNA, and the reagent used in the conversion treatment may be sulfite or bisulfite, that is, a sulfite or bisulfite conversion treatment is performed on ctDNA.
Step S33, adding 4ul (27ng) of ctDNA subjected to conversion treatment into a 25ul PCR reaction system; in this example, the PCR reaction system included 1.8 XPCR solution, 5mM MgCl20.3nM deoxyribonucleoside triphosphate, 40nM CALCA forward primer, 40nM CALCA reverse primer, 40nM DLEC1 forward primer, 40nM DLEC1 reverse primer, 120nM HOXA9 forward primer, 120nM HOXA9 reverse primer, 20nM PITX2 forward primer, 20nM PITX2 reverse primer, 20nM TBX5 forward primer, 20nM TBX5 reverse primer, 280nM RASSF1a forward primer, 280nM RASSF1a reverse primer and 2.5 units of DNA polymerase, such as preferably Taq Platinum DNA polymerase.
And step S34, placing the PCR reaction system into a PCR instrument, and carrying out PCR amplification reaction on the PCR reaction system according to a set PCR reaction program to obtain a PCR reaction product. In this example, the PCR reaction procedure includes the following steps: (1) at a temperature of 95 ℃ for 3 minutes; (2) a total of 4 cycles of 1 minute at a temperature of 94 ℃, 30 seconds at a temperature of 60 ℃ and 45 seconds at a temperature of 65 ℃; (3) a total of 36 amplification cycles performed at a temperature of 94 ℃ for 1 minute, at a temperature of 56 ℃ for 1 minute, and at a temperature of 65 ℃ for 45 seconds; (4) at 65 ℃ for 4 minutes as extension reaction; (5) the PCR reaction was stopped at a temperature of 4 ℃ and the PCR reaction product was stored at a temperature of 4 ℃.
Step S35, performing gel electrophoresis on the PCR reaction product; specifically, the product after PCR reaction is subjected to gel electrophoresis, in this embodiment, the gel electrophoresis may be agarose gel electrophoresis, and the concentrations of the gel electrophoresis are: agarose concentration in agarose gel was 2.5%), or polyacrylamide gel electrophoresis (PAGE).
Step S36, dyeing the gel after electrophoresis by using a coloring agent, and performing fluorescence analysis on the dyed gel by using a gel imager; specifically, the gel after electrophoresis is stained with a stain, such as Ethidium Bromide (EB), SYBR Green I, GelRed, or GoldView stain, as a preferred embodiment, and the stained gel is subjected to fluorescence analysis using a gel imager, and a light fluorescence band in the stained gel is read by the gel imager.
Step S37, judging whether 2 or more than 2 fluorescence bands appear in the gel; in this embodiment, whether the gel imager reads 2 or more fluorescence bands from the dyed gel is determined, and if yes, step S38 is executed; if not, step S39 is executed.
And step S38, if 2 or more than 2 fluorescent color bands appear in the gel, the gel imager displays that the blood sample to be detected contains the gene mutation DNA fragment of the non-small cell lung cancer.
And step S39, if 1 strip or no fluorescence band appears in the gel, the gel imager shows that the blood sample to be detected does not contain the gene mutation DNA fragment of the non-small cell lung cancer.
Referring to FIG. 4, FIG. 4 is a flowchart illustrating a method for detecting methylation of multiple genes of non-small cell lung cancer according to a second preferred embodiment of the present invention. In a second preferred embodiment, the method for detecting methylation of multiple genes of non-small cell lung cancer comprises the following steps S40 to S50:
s40, selecting 6 genes as methylation markers of the non-small cell lung cancer and 1 gene as reference markers; specifically, based on the lung cancer early-stage research and the existing research results, 6 genes are selected as methylation markers of the non-small cell lung cancer, and the methylation markers are respectively as follows: CALCA, DLEC1, HOXA9, TBX5, PITX2, RASSF1a, and a gene serving as a reference marker is different from a gene serving as a methylation marker, and the reference marker is beta-ACTIN.
Step S41, constructing 6 primer pairs corresponding to 6 methylation markers and 1 primer pair corresponding to 1 reference marker respectively; in this example, each methylation marker corresponds to a primer pair, and each primer pair comprises a forward primer (MF) and a reverse primer (MR). In this example, the reference marker β -ACTIN forward primer is a positive control, and the β -ACTIN forward primer sequence is: 5'-TTTTTAGGGAGGAGT TGGAAGTAGT-3', the reverse primer of the reference marker is a negative control, the sequence of the beta-ACTIN reverse primer is: 5'-AAAATATACC CTCCCCCATACC-3' are provided. As shown in FIG. 2, FIG. 2 lists 7 primer pairs, including 6 primer sequences corresponding to 6 methylation markers and 1 primer sequence corresponding to 1 reference marker.
Step S42, extracting free ctDNA from the blood sample to be tested, in this embodiment, it is considered that the concentration of ctDNA in serum of the blood sample is 3-24 times of that in plasma, but the coagulation process is easily contaminated by impurities, so it is preferable to extract ctDNA from plasma of the blood sample, and purify and convert the ctDNA. In this embodiment, ctDNA is easily decomposed by dnase in blood, and the purification of ctDNA needs to be performed as soon as possible, the purification method may be a magnetic bead method or a centrifugal column method commonly used in the industry to extract free ctDNA in a blood sample to be tested to remove impurities and purify ctDNA, and the reagent used in the conversion treatment may be sulfite or bisulfite, that is, a sulfite or bisulfite conversion treatment is performed on ctDNA.
Step S43, adding 4ul (27ng) of ctDNA subjected to conversion treatment into a 25ul PCR reaction system; in this example, the PCR reaction system included 1.8 XPCR solution, 5mM MgCl20.3nM deoxyribonucleoside triphosphate, 40nM CALCA forward primer, 40nM CALCA reverse primer, 40nM DLEC1 forward primer, 40nM DLEC1 reverse primer, 120nM HOXA9 forward primer, 120nM HOXA9 reverse primer, 20nM PITX2 forward primer, 20nM PITX2 reverse primer, 20nM TBX5 forward primer, 20nM TBX5 reverse primer, 280nM RASSF1a forward primer, 280nM RASSF1a reverse primer, 10nM beta-ACTIN forward primer, 10nM beta-ACTIN reverse primer and 2.5 units of DNA polymerase, such as preferably Taq Platinum DNA polymerase.
And step S44, placing the PCR reaction system into a PCR instrument, and carrying out PCR amplification reaction on the PCR reaction system according to a set PCR reaction program to obtain a PCR reaction product. In this example, the PCR reaction procedure includes the following steps: (1) at a temperature of 95 ℃ for 3 minutes; (2) a total of 4 cycles of 1 minute at a temperature of 94 ℃, 30 seconds at a temperature of 60 ℃ and 45 seconds at a temperature of 65 ℃; (3) a total of 36 amplification cycles performed at a temperature of 94 ℃ for 1 minute, at a temperature of 56 ℃ for 1 minute, and at a temperature of 65 ℃ for 45 seconds; (4) at 65 ℃ for 4 minutes as extension reaction; (5) the PCR reaction was stopped at a temperature of 4 ℃ and the PCR reaction product was stored at a temperature of 4 ℃.
Step S45, performing gel electrophoresis on the PCR reaction product; specifically, the product after PCR reaction is subjected to gel electrophoresis, in this embodiment, the gel electrophoresis may be agarose gel electrophoresis, and the concentrations of the gel electrophoresis are: agarose concentration in agarose gel was 2.5%), or polyacrylamide gel electrophoresis (PAGE).
Step S46, dyeing the gel after electrophoresis by using a coloring agent, and performing fluorescence analysis on the dyed gel by using a gel imager; specifically, the gel after electrophoresis is stained with a stain, such as Ethidium Bromide (EB), SYBR Green I, GelRed, or GoldView stain, as a preferred embodiment, and the stained gel is subjected to fluorescence analysis using a gel imager, and a light fluorescence band in the stained gel is read by the gel imager.
Step S47, judging whether 3 or more than 3 fluorescence bands appear in the gel; in this embodiment, whether the gel imager reads 3 or more fluorescence bands from the dyed gel, and if 3 or more fluorescence bands appear in the gel, step S48 is executed; if 2 or 1 fluorescent color bands appear in the gel, step S49 is performed. If no fluorescence bands appear in the gel, step S50 is performed. Wherein, 1 fluorescence band is a PCR reaction band of a reference marker beta-ACTIN, and the other fluorescence bands are PCR reaction bands of a mutant gene marker.
And step S48, if 3 or more than 3 fluorescent color bands appear in the gel, the gel imager displays that the blood sample to be detected contains the gene mutation DNA fragment of the non-small cell lung cancer.
And step S49, if 2 or 1 fluorescent color bands appear in the gel, the gel imager shows that the blood sample to be tested does not contain the gene mutation DNA fragment of the non-small cell lung cancer.
And step S50, if no fluorescent color band appears in the gel, the gel imager judges that the ctDNA conversion and the PCR reaction are invalid in the lung cancer characteristic methylation detection process, and can not verify whether the blood sample to be detected contains the gene mutation DNA fragment of the non-small cell lung cancer. Therefore, in this embodiment, the reference markers β -ACTIN forward primer and reverse primer are added to the PCR reaction system, mainly to verify whether the ctDNA transformation and PCR reaction process are correct, so as to ensure the accuracy of the non-small cell lung cancer detection.
The method for detecting the methylation of the multiple genes of the non-small cell lung cancer can detect the existence of a plurality of methylation markers of the non-small cell lung cancer simultaneously by one-time PCR reaction detection, and improves the accuracy, specificity and sensitivity of the lung cancer detection. The invention solves the problem that the methylation level of a single gene can only be detected by one methylation specific PCR reaction, and the possibility of lung cancer occurrence cannot be accurately predicted. Meanwhile, the uncertainty of the non-small cell lung cancer suppressor gene and the limitation of ctDNA are solved, so that the detection of the single non-small cell lung cancer methylation marker cannot meet the clinical requirement.
The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and all modifications of equivalent structures and equivalent processes, which are made by using the contents of the present specification and the accompanying drawings, or directly or indirectly applied to other related technical fields, are included in the scope of the present invention.
Sequence listing
<110> Shenzhen Shengbizhi science and technology development Limited
<120> method for detecting methylation of multiple genes of non-small cell lung cancer
<130> 2018
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Claims (10)

1. A method for detecting methylation of multiple genes of non-small cell lung cancer, which is characterized by comprising the following steps:
selecting 6 genes as methylation markers of the non-small cell lung cancer;
constructing 6 primer pairs corresponding to 6 methylation markers respectively;
extracting free ctDNA in a blood sample to be detected, and purifying and converting the ctDNA;
adding the ctDNA with the concentration of 4ul and the concentration of 27ng after conversion treatment into a 25ul PCR reaction system;
putting the PCR reaction system into a PCR instrument, and carrying out PCR amplification reaction on the PCR reaction system according to a set PCR reaction program to obtain a PCR reaction product;
performing gel electrophoresis on the PCR reaction product;
dyeing the gel after electrophoresis by using a dyeing agent, and performing fluorescence analysis on the dyed gel by using a gel imager;
the gel imager judges whether 2 or more than 2 fluorescent color bands appear in the gel;
if 2 or more than 2 fluorescent color bands appear in the gel, the gel imager displays that the blood sample to be detected contains the gene mutation DNA fragment of the non-small cell lung cancer;
if 2 strips or no fluorescence color band appears in the gel, the gel imager shows that the blood sample to be detected does not contain the gene mutation DNA fragment of the non-small cell lung cancer.
2. The method for detecting methylation of multiple genes of non-small cell lung cancer according to claim 1, wherein the 6 methylation markers are CALCA, DLEC1, HOXA9, TBX5, PITX2 and RASSF1a, and the 6 primer pairs comprise 6 sets of primer sequences, which are specifically expressed as follows:
CALCA forward primer: 5'-CGGAATTTTTTCGATTTATAGC-3', respectively;
CALCA reverse primer: 5'-AAAACCCTATAAAAACGACGAC-3', respectively;
forward primer of DLEC 1: 5'-GATTAAGCGATGACGGGATTC-3', respectively;
DLEC1 reverse primer: 5'-ACCCGACTAATAACGAAATTAACG-3', respectively;
HOXA9 forward primer: 5'-GGTTAATGGGGGCGCGGGCGTC-3', respectively;
HOXA9 reverse primer: 5'-TCATATAACAACTTAATAACACCG-3', respectively;
TBX5 forward primer: 5'-GGGACGCGTAAAATTTAGAATC-3', respectively;
TBX5 reverse primer: 5'-AACACAAAACCGAAAAACGTC-3', respectively;
PITX2 forward primer: 5'-CGTTATTAGTTGAAGGTAAGGTCG-3', respectively;
PITX2 reverse primer: 5'-AACACCGAAAAATACAATCCG-3', respectively;
RASSF1a forward primer: 5'-GTGTTAACGCGTTGCGTATC-3', respectively;
RASSF1a reverse primer: 5'-AACCCCGCGAACTAAAAACGA-3' are provided.
3. The method of claim 2, wherein the PCR reaction system comprises 1.8 XPCR solution and 5mM MgCl20.3nM deoxyribonucleoside triphosphate, 40nM CALCA forward primer, 40nM CALCA reverse primer, 40nM DLEC1 forward primer, 40nM DLEC1 reverse primer, 120nM HOXA9 forward primer, 120nM HOXA9 reverse primer, 20nM PITX2 forward primer, 20nM PITX2 reverse primer, 20nM TBX5 forward primer, 20nM TBX5 reverse primer, 280nM RASSF1a forward primer, 280nM RASSF1a reverse primer and 2.5 units of DNA polymerase.
4. The method for detecting methylation of a plurality of genes in non-small cell lung cancer according to claim 3, wherein the DNA polymerase is Taq Platinum DNA polymerase.
5. A method for detecting methylation of multiple genes of non-small cell lung cancer, which is characterized by comprising the following steps:
selecting 6 genes as methylation markers and 1 reference marker of the non-small cell lung cancer;
constructing 6 primer pairs corresponding to 6 methylation markers and 1 primer pair corresponding to 1 reference marker respectively;
extracting free ctDNA in a blood sample to be detected, and purifying and converting the ctDNA;
adding the ctDNA with the concentration of 4ul and the concentration of 27ng after conversion treatment into a 25ul PCR reaction system;
putting the PCR reaction system into a PCR instrument, and carrying out PCR amplification reaction on the PCR reaction system according to a set PCR reaction program to obtain a PCR reaction product;
performing gel electrophoresis on the PCR reaction product;
dyeing the gel after electrophoresis by using a dyeing agent, and performing fluorescence analysis on the dyed gel by using a gel imager;
the gel imager judges whether 3 or more than 3 fluorescent color bands appear in the gel;
if 3 or more than 3 fluorescent color bands appear in the gel, the gel imager displays that the blood sample to be detected contains the gene mutation DNA fragment of the non-small cell lung cancer;
if 2 or 1 fluorescent color band appears in the gel, the gel imager shows that the blood sample to be detected does not contain the gene mutation DNA segment of the non-small cell lung cancer;
and if no fluorescence color band appears in the gel, the gel imager judges that the ctDNA conversion and the PCR reaction are invalid.
6. The method for detecting methylation of multiple genes of non-small cell lung cancer according to claim 5, wherein the 6 methylation markers are CALCA, DLEC1, HOXA9, TBX5, PITX2 and RASSF1a, and the 6 primer pairs comprise 6 sets of primer sequences, which are specifically expressed as follows:
CALCA forward primer: 5'-CGGAATTTTTTCGATTTATAGC-3', respectively;
CALCA reverse primer: 5'-AAAACCCTATAAAAACGACGAC-3', respectively;
forward primer of DLEC 1: 5'-GATTAAGCGATGACGGGATTC-3', respectively;
DLEC1 reverse primer: 5'-ACCCGACTAATAACGAAATTAACG-3', respectively;
HOXA9 forward primer: 5'-GGTTAATGGGGGCGCGGGCGTC-3', respectively;
HOXA9 reverse primer: 5'-TCATATAACAACTTAATAACACCG-3', respectively;
TBX5 forward primer: 5'-GGGACGCGTAAAATTTAGAATC-3', respectively;
TBX5 reverse primer: 5'-AACACAAAACCGAAAAACGTC-3', respectively;
PITX2 forward primer: 5'-CGTTATTAGTTGAAGGTAAGGTCG-3', respectively;
PITX2 reverse primer: 5'-AACACCGAAAAATACAATCCG-3', respectively;
RASSF1a forward primer: 5'-GTGTTAACGCGTTGCGTATC-3', respectively;
RASSF1a reverse primer: 5'-AACCCCGCGAACTAAAAACGA-3', respectively;
the 1 reference marker is beta-ACTIN, and 1 primer pair corresponding to the reference marker beta-ACTIN comprises 1 group of primer sequences, which are specifically expressed as follows:
β -ACTIN forward primer: 5'-TTTTTAGGGAGGAGT TGGAAGTAGT-3', respectively;
beta-ACTIN reverse primer: 5'-AAAATATACC CTCCCCCATA CC-3' are provided.
7. The method of claim 6, wherein the PCR reaction system comprises 1.8 XPCR solution and 5mM MgCl20.3nM deoxyribonucleoside triphosphate, 40nM CALCA forward primer, 40nM CALCA reverse primer, 40nM DLEC1 forward primer, 40nM DLEC1 reverse primer, 120nM HOXA9 forward primer, 120nM HOXA9 reverse primer, 20nM PITX2 forward primer, 20nM PITX2 reverse primer, 20nM TBX5 forward primer, 20nM TBX5 reverse primer, 280nM RASSF1a forward primer, 280nM RASSF1a reverse primer, 10nM beta-ACTIN forward primer, 10nM beta-ACTIN reverse primer and 2.5 units of DNA polymerase.
8. The method for detecting methylation of a plurality of genes in non-small cell lung cancer according to claim 7, wherein the DNA polymerase is Taq Platinum DNA polymerase.
9. The method for detecting methylation of a plurality of genes of non-small cell lung cancer according to any one of claims 1 to 8, wherein the PCR reaction procedure comprises the following steps:
step 1: at a temperature of 95 ℃ for 3 minutes;
step 2: a total of 4 cycles of step 2 performed at a temperature of 94 ℃ for 1 minute, at a temperature of 60 ℃ for 30 seconds, and at a temperature of 65 ℃ for 45 seconds;
and step 3: a total of 36 amplification cycles of step 3 performed at a temperature of 94 ℃ for 1 minute, 56 ℃ for 1 minute, 65 ℃ for 45 seconds;
and 4, step 4: extension reaction at 65 ℃ for 4 minutes;
and 5: the PCR reaction was stopped at a temperature of 4 ℃ and the PCR reaction product was stored at a temperature of 4 ℃.
10. The method for detecting methylation of multiple genes of non-small cell lung cancer according to claim 9, wherein the purification treatment comprises a magnetic bead method or a centrifugal column method to extract free ctDNA in a blood sample to be detected so as to remove impurities and purify the ctDNA;
in the conversion treatment, sulfite or bisulfite is used as a reagent to carry out sulfite conversion treatment or bisulfite conversion treatment on ctDNA;
performing gel electrophoresis on the PCR reaction product by using agarose gel or polyacrylamide gel;
the stain is ethidium bromide, SYBR Green I, GelRed or GoldView stain.
CN201810654035.5A 2018-06-22 2018-06-22 Method for detecting methylation of multiple genes of non-small cell lung cancer Pending CN110628900A (en)

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