WO2019242754A1

WO2019242754A1 - Method for detecting methylation of multiplex gene for non-small cell lung cancer

Info

Publication number: WO2019242754A1
Application number: PCT/CN2019/092335
Authority: WO
Inventors: 陈琦; 梁昊原; 谷东风
Original assignee: 深圳市圣必智科技开发有限公司
Priority date: 2018-06-22
Filing date: 2019-06-21
Publication date: 2019-12-26
Also published as: CN110628900A

Abstract

Provided is a method for detecting the methylation of multiplex genes for non-small cell lung cancer, comprising the following steps: constructing six primer pairs of methylation markers for non-small cell lung cancer; extracting free ctDNA from the blood sample to be tested and performing the purification and transformation of ctDNA; adding 4 μl of 27 ng (concentration) ctDNA into 25 μl of a PCR reaction system; putting the PCR reaction system into a PCR instrument for a PCR amplification reaction; performing gel electrophoresis on PCR reaction products; dyeing the gel and carrying out fluorescence analysis of the gel by using a gel imager; judging whether there are two or more fluorescent bands in the gel; if so, this indicates that the blood sample to be tested comprises gene mutant DNA fragments for non-small cell lung cancer; and if not, this indicates that the blood sample to be tested does not comprise gene mutant DNA fragments for non-small cell lung cancer.

Description

Non-small cell lung cancer multiple gene methylation detection method

Technical field

The invention relates to the technical field of EB virus detection, in particular to a method for detecting multiple gene methylation of non-small cell lung cancer.

Background technique

DNA methylation is an important epigenetic mechanism, one of the key mechanisms for inactivation of tumor suppressor genes, and may be the only mechanism in some cases. Methylation of non-small cell lung cancer with multiple motions has been confirmed, 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 can make molecular diagnosis before clinical manifestations or imaging evidence appears.

ctDNA (circulating tumor DNA): DNA fragments from the tumor genome that carry certain characteristics (including mutations, deletions, insertions, rearrangements, abnormal copy number, methylation, etc.) in the human blood circulation system. Its low concentration in blood and high fragmentation make it difficult to extract and reduce the sensitivity and specificity of clinical detection. In the prior art, one methylation-specific PCR reaction can only detect the methylation level of a single gene and cannot accurately predict the possibility of lung cancer. At the same time, the uncertainty of tumor suppressor genes and the limitation of ctDNA in non-small cell lung cancer have caused the detection of methylation markers in single non-small cell lung cancer to meet clinical needs.

technical problem

The main object of the present invention is to provide a method for detecting multiple gene methylation of non-small cell lung cancer, which can detect whether there are multiple non-small cell lung cancer methylation markers at the same time in one PCR reaction detection, thereby improving the detection of non-small cell lung cancer. Accuracy, specificity, and sensitivity.

Technical solutions

To achieve the above object, the present invention provides a method for detecting multiple gene methylation in non-small cell lung cancer. The method includes the steps of: selecting 6 genes as methylation markers of non-small cell lung cancer; and constructing 6 methylations respectively. 6 primer pairs corresponding to the markers; extract free ctDNA from the blood sample to be tested, and purify and transform the ctDNA; take 4ul of 27ng converted ctDNA and add it to 25ul PCR reaction system; PCR reaction system Put it into the PCR instrument, and perform PCR amplification reaction on the PCR reaction system to obtain the PCR reaction product according to the set PCR reaction program; perform gel electrophoresis on the PCR reaction product; use a stain to stain the gel after electrophoresis, and Use a gel imager to perform fluorescence analysis on the stained gel; the gel imager determines whether two or more fluorescent bands appear in the gel; if two or more fluorescent colors appear in the gel The gel imager shows that the blood sample to be tested contains non-small cell lung cancer gene mutation DNA fragments; if two or no fluorescent bands appear in the gel, the gel imager Display test blood sample does not contain non-small cell lung cancer gene mutant DNA fragment.

Preferably, the six methylation markers are CALCA, DLEC1, HOXA9, TBX5, PITX2, RASSF1a, and the six primer pairs include six sets of primer sequences, which are specifically expressed as follows:

CALCA forward primer: 5'-CGGAATTTTTTCGATTTATAGC-3 ';

CALCA reverse primer: 5'-AAAACCCTATAAAAACGACGAC-3 ';

DLEC1 forward primer: 5'-GATTAAGCGATGACGGGATTC-3 ';

DLEC1 reverse primer: 5'-ACCCGACTAATAACGAAATTAACG-3 ';

HOXA9 forward primer: 5'-GGTTAATGGGGGCGCGGGCGTC-3 ';

HOXA9 reverse primer: 5'-TCATATAACAACTTAATAACACCG-3 ';

TBX5 forward primer: 5'-GGGACGCGTAAAATTTAGAATC-3 ';

TBX5 reverse primer: 5'-AACACAAAACCGAAAAACGTC-3 ';

PITX2 forward primer: 5'-CGTTATTAGTTGAAGGTAAGGTCG-3 ';

PITX2 reverse primer: 5'-AACACCGAAAAATACAATCCG-3 ';

RASSF1a forward primer: 5'-GTGTTAACGCGTTGCGTATC-3 ';

RASSF1a reverse primer: 5'-AACCCCGCGAACTAAAAACGA-3 '.

Preferably, the PCR reaction system includes a 1.8 × PCR solution, 5 mM MgCl ₂ , 0.3 nM deoxyribonucleoside triphosphate, 40 nM CALCA forward primer, 40 nM CALCA reverse primer, 40 nM 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 multiple gene methylation in non-small cell lung cancer. The method includes the steps of: selecting 6 genes as a methylation marker and a reference marker for non-small cell lung cancer; 6 primer pairs corresponding to 6 methylation markers and 1 primer pair corresponding to a reference marker; extract free ctDNA from the blood sample to be tested, and purify and transform the ctDNA; take 4ul to 27ng The transformed ctDNA is added to the 25ul PCR reaction system; the PCR reaction system is placed in a PCR instrument, and the PCR reaction system is subjected to a PCR amplification reaction according to a set PCR reaction program to obtain a PCR reaction product; the PCR reaction product is subjected to Gel electrophoresis; stain the gel after electrophoresis with a stain, and use a gel imager to perform fluorescence analysis on the stained gel; the gel imager determines whether three or more fluorescences appear in the gel Color bands; if three or more fluorescent bands appear in the gel, the gel imager shows that the blood sample to be tested contains genetically modified DNA fragments of non-small cell lung cancer; if the gel contains Two or one fluorescent bands appear, and the gel imager shows that the blood sample to be tested does not contain genetically modified DNA fragments of non-small cell lung cancer; if no fluorescent bands appear in the gel, the gel imager determines ctDNA transformation and PCR reactions failed.

CALCA forward primer: 5'-CGGAATTTTTTCGATTTATAGC-3 ';

CALCA reverse primer: 5'-AAAACCCTATAAAAACGACGAC-3 ';

DLEC1 forward primer: 5'-GATTAAGCGATGACGGGATTC-3 ';

DLEC1 reverse primer: 5'-ACCCGACTAATAACGAAATTAACG-3 ';

HOXA9 forward primer: 5'-GGTTAATGGGGGCGCGGGCGTC-3 ';

HOXA9 reverse primer: 5'-TCATATAACAACTTAATAACACCG-3 ';

TBX5 forward primer: 5'-GGGACGCGTAAAATTTAGAATC-3 ';

TBX5 reverse primer: 5'-AACACAAAACCGAAAAACGTC-3 ';

PITX2 forward primer: 5'-CGTTATTAGTTGAAGGTAAGGTCG-3 ';

PITX2 reverse primer: 5'-AACACCGAAAAATACAATCCG-3 ';

RASSF1a forward primer: 5'-GTGTTAACGCGTTGCGTATC-3 ';

RASSF1a reverse primer: 5'-AACCCCGCGAACTAAAAACGA-3 ';

The one reference marker is β-ACTIN, and one primer pair corresponding to the reference marker β-ACTIN includes a set of primer sequences, which are specifically expressed as follows:

β-ACTIN forward primer: 5'-TTTTTAGGGAGGAGT TGGAAGTAGT-3 ';

β-ACTIN reverse primer: 5'-AAAATATACC CTCCCCCATA CC-3 '.

Preferably, the PCR reaction system includes a 1.8 × PCR solution, 5 mM MgCl ₂ , 0.3 nM deoxyribonucleoside triphosphate, 40 nM CALCA forward primer, 40 nM CALCA reverse primer, 40 nM 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 β-ACTIN forward primer, 10nM β-ACTIN reverse primer and 2.5 units of DNA polymerase.

Preferably, the DNA polymerase is Taq Platinum DNA polymerase.

Preferably, the PCR reaction program includes the following steps: Step 1: Lasting for 3 minutes at 95 ° C; Step 2: Lasting for 1 minute at 94 ° C, 30 seconds at 60 ° C, and 65 ° C For 45 seconds, this step 2 executes a total of 4 cycles; Step 3: At 94 ° C for 1 minute, at 56 ° C for 1 minute, and at 65 ° C for 45 seconds, this step 3 is performed 36 times Amplification cycle; Step 4: Continue for 4 minutes at 65 ° C as an extension reaction; Step 5: Stop the PCR reaction at 4 ° C and store the PCR reaction product at 4 ° C.

Preferably, the purification process uses magnetic bead method or spin column method to extract free ctDNA in the blood sample to be tested to remove impurities to purify ctDNA; the conversion process uses sulfite or bisulfite as reagent to perform ctDNA Sulfite conversion treatment or bisulfite conversion treatment; the gel electrophoresis uses agarose gel or polyacrylamide gel to perform gel electrophoresis on the PCR reaction product; the stain is ethidium bromide, SYBR Green I. GelRed or GoldView stain.

Beneficial effect

Compared with the prior art, the method for detecting multiple gene methylation of non-small cell lung cancer according to the present invention adopts the above technical scheme, and achieves the following technical effects: it can detect multiple non-small cell lung cancer methylation markers in one PCR reaction detection at the same time The presence of substances improves the accuracy, specificity and sensitivity of non-small cell lung cancer detection. The invention solves the possibility that a methylation-specific PCR reaction can only detect the methylation level of a single gene and cannot accurately predict the occurrence of lung cancer, and the uncertainty of non-small cell lung cancer tumor suppressor genes and the limitations of ctDNA The problem is that the detection of methylation markers in single non-small cell lung cancer cannot meet the clinical needs.

BRIEF DESCRIPTION OF THE DRAWINGS

1 is a schematic diagram of a first preferred embodiment of a primer pair for detecting multiple gene methylation of non-small cell lung cancer according to the present invention;

2 is a schematic diagram of a second preferred embodiment of a primer pair for detecting multiple gene methylation of non-small cell lung cancer according to the present invention;

3 is a flowchart of a first preferred embodiment of a method for detecting multiple gene methylation in non-small cell lung cancer according to the present invention;

4 is a flowchart of a second preferred embodiment of a method for detecting multiple gene methylation in non-small cell lung cancer according to the present invention.

The realization of the purpose, functional characteristics and advantages of the present invention will be further described with reference to the embodiments and the drawings.

Embodiments of the invention

In order to further explain the technical means and effects adopted by the present invention to achieve the intended purpose of the present invention, the specific implementation, structure, features, and effects of the present invention are described in detail below with reference to the drawings and preferred embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

The invention provides a primer pair for detecting multiple gene methylation of non-small cell lung cancer, comprising 6 sets of primer sequences corresponding to 6 genes as methylation markers of non-small cell lung cancer, the methylation markers Can be used to detect non-small cell lung cancer using blood as a sample. As a preferred embodiment, the methylation markers are: CALAC, DLEC1, HOXA9, TBX5, PITX2, RASSF1a.

As shown in FIG. 1, FIG. 1 is a schematic diagram of a first preferred embodiment of a primer pair for detecting multiple gene methylation of non-small cell lung cancer according to the present invention. In a first preferred embodiment, the primer pair for detecting multiple gene methylation of lung cancer includes 6 sets of primer sequences corresponding to 6 methylation markers, and each methylation marker corresponds to one primer pair, each A primer pair includes a forward primer (MF) and a reverse primer (MR), which are specifically expressed as follows:

(1) CALCA forward primer: 5'-CGGAATTTTTTCGATTTATAGC-3 '(SEQ ID NO.1);

(2) 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 conversion and PCR reaction process in the blood sample to be tested to ensure the accuracy of the detection of non-small cell lung cancer, the present invention will refer to the primer pair corresponding to the marker β-ACTIN ( Including β-ACTIN forward primer and β-ACTIN reverse primer) were added to the PCR reaction system.

As shown in FIG. 2, FIG. 2 is a schematic diagram of a second preferred embodiment of a primer pair for detecting multiple gene methylation of non-small cell lung cancer according to the present invention. In a second embodiment, the primer pair for detecting multiple gene methylation of lung cancer according to the present invention includes not only the six sets of primer sequences corresponding to the above six methylation markers, but also 1 corresponding to 1 reference marker. Set of primer sequences. Among them, a set of primer sequences corresponding to a reference marker includes:

(13) β-ACTIN forward primer: 5’-TTTTTAGGGAGGAGT TGGAAGTAGT-3 '(SEQ ID NO. 13);

(14) β-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 a method for detecting multiple gene methylation of non-small cell lung cancer according to the present invention. In a first preferred embodiment, the non-small cell lung cancer multiple gene methylation detection method includes the following steps S30 to S39.

Step S30, selecting 6 genes as methylation markers of non-small cell lung cancer; specifically, based on the pre-lung cancer research and existing research results, selecting 6 genes as methylation markers related to non-small cell lung cancer, They are: CALCA, DLEC1, HOXA9, TBX5, PITX2, RASSF1a.

In step S31, six primer pairs corresponding to the six methylation markers are respectively constructed. In this embodiment, as shown in FIG. 1, FIG. 1 lists six sets of primer sequences corresponding to the six methylation markers. They are DLEC1 forward and reverse primers, PITX2 forward and reverse primers, TBX5 forward and reverse primers, CALAC forward and reverse primers, RASSF1a forward and reverse primers, HOXA9 Forward and reverse primers.

In step S32, the free ctDNA in the blood sample to be tested is extracted. In this embodiment, it is considered that the concentration of ctDNA in the serum of the blood sample is 3-24 times that in the plasma, but the coagulation process is easily contaminated by impurities. CtDNA was extracted from plasma, and ctDNA was purified and transformed. In this embodiment, ctDNA is easily decomposed by DNase in the blood. The purification of ctDNA needs to be performed as soon as possible. The purification method can use the industry's common magnetic bead method or spin column method to extract free ctDNA in the blood sample to be tested to remove impurities. The ctDNA is purified, and the reagent used for the conversion treatment may be sulfite or bisulfite, that is, the ctDNA is subjected to sulfite or bisulfite conversion treatment.

In step S33, 4ul (27ng) of the transformed ctDNA is added to a 25ul PCR reaction system. In this embodiment, the PCR reaction system includes a 1.8 × PCR solution, 5mM MgCl ₂ , and 0.3nM 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 unit DNA polymerase, for example, Taq Platinum is preferred DNA polymerase.

In step S34, the PCR reaction system is put into a PCR instrument, and a PCR amplification reaction is performed on the PCR reaction system according to a set PCR reaction program to obtain a PCR reaction product. In this embodiment, the PCR reaction procedure includes the following steps: (1), lasting for 3 minutes at a temperature of 95 ° C; (2), continuing for 1 minute at a temperature of 94 ° C, for 30 seconds at a temperature of 60 ° C, This step is performed for 4 seconds at a temperature of 65 ° C, and a total of 4 cycles are performed in this step; (3) This step is performed for 1 minute at a temperature of 94 ° C, for 1 minute at a temperature of 56 ° C, and for 45 seconds at a temperature of 65 ° C. A total of 36 amplification cycles were performed; (4), at 65 ° C for 4 minutes, as an extension reaction; (5), the PCR reaction was stopped at 4 ° C, and the PCR reaction product was stored at 4 ° C.

In step S35, gel electrophoresis is performed on the PCR reaction product. Specifically, gel electrophoresis is performed on the product after the PCR reaction. In this embodiment, the gel electrophoresis may be agarose gel electrophoresis, and the concentration may be selected as: The agarose gel has an agarose concentration of 2.5%) or polyacrylamide gel electrophoresis (PAGE).

Step S36: Stain the gel after electrophoresis with a staining agent, and perform fluorescence analysis on the stained gel using a gel imager; specifically, stain the gel after electrophoresis with a staining agent as a preferred implementation. Method, the staining agent can be selected such as ethidium bromide (EB), SYBR Green I, GelRed or GoldView staining agent, and use a gel imager to perform fluorescence analysis on the stained gel, and read the gel imager Take the fluorescent band from the stained gel.

In step S37, it is determined whether two or more fluorescent color bands appear in the gel. In this embodiment, whether the gel imager reads two or more fluorescent color bands from the dyed gel. If yes, go to step S38; if not, go to step S39.

In step S38, if two or more fluorescent color bands appear in the gel, the gel imager displays that the blood sample to be tested contains a non-small cell lung cancer gene mutation DNA fragment.

In step S39, if one or no fluorescent color bands appear in the gel, the gel imager shows that the blood sample to be tested does not contain a gene mutation DNA fragment of non-small cell lung cancer.

Referring to FIG. 4, FIG. 4 is a flowchart of a second preferred embodiment of a method for detecting multiple gene methylation in non-small cell lung cancer according to the present invention. In a second preferred embodiment, the non-small cell lung cancer multiple gene methylation detection method includes the following steps S40 to S50:

In step S40, six genes are selected as methylation markers of non-small cell lung cancer and one gene is used as a reference marker. Specifically, based on the pre-lung cancer research and existing research results, six genes are selected as non-small cell lung cancer. The methylation markers are: CALCA, DLEC1, HOXA9, TBX5, PITX2, RASSF1a. In addition, the gene as a reference marker is different from the gene of the methylation marker, and the reference marker is β-ACTIN .

In step S41, six primer pairs corresponding to the six methylation markers and one primer pair corresponding to the one reference marker are respectively constructed. In this embodiment, each methylation marker corresponds to one primer pair. One primer pair includes a forward primer (MF) and a reverse primer (MR). In this example, the forward primer of the reference marker β-ACTIN is a positive control, and the sequence of the β-ACTIN forward primer is: 5′-TTTTTAGGGAGGAGT TGGAAGTAGT-3 ′, and the reverse primer of the reference marker is a negative control. The sequence of β-ACTIN reverse primer is: 5'-AAAATATACC CTCCCCCATA CC-3 '. As shown in FIG. 2, FIG. 2 lists 7 primer pairs, including 6 sets of primer sequences corresponding to 6 methylation markers and 1 set of primer sequences corresponding to 1 reference marker.

In step S42, the free ctDNA in the blood sample to be tested is extracted. In this embodiment, it is considered that the concentration of ctDNA in the serum of the blood sample is 3-24 times that in the plasma, but the coagulation process is easily contaminated by impurities. CtDNA was extracted from plasma, and ctDNA was purified and transformed. In this embodiment, ctDNA is easily decomposed by DNase in the blood. The purification of ctDNA needs to be performed as soon as possible. The purification method can use the industry's common magnetic bead method or spin column method to extract free ctDNA in the blood sample to be tested to remove impurities. The ctDNA is purified, and the reagent used for the conversion treatment may be sulfite or bisulfite, that is, the ctDNA is subjected to sulfite or bisulfite conversion treatment.

In step S43, 4ul (27ng) of the transformed ctDNA is added to a 25ul PCR reaction system. In this embodiment, the PCR reaction system includes a 1.8 × PCR solution, 5mM MgCl ₂ , and 0.3nM 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 β-ACTIN forward primer, 10nM β- The ACTIN reverse primer and a 2.5 unit DNA polymerase, for example, Taq Platinum DNA polymerase is preferred.

In step S44, the PCR reaction system is put into a PCR instrument, and a PCR amplification reaction is performed on the PCR reaction system according to a set PCR reaction program to obtain a PCR reaction product. In this embodiment, the PCR reaction procedure includes the following steps: (1), lasting for 3 minutes at a temperature of 95 ° C; (2), continuing for 1 minute at a temperature of 94 ° C, for 30 seconds at a temperature of 60 ° C, This step is performed for 4 seconds at a temperature of 65 ° C, and a total of 4 cycles are performed in this step; (3) This step is performed for 1 minute at a temperature of 94 ° C, for 1 minute at a temperature of 56 ° C, and for 45 seconds at a temperature of 65 ° C. A total of 36 amplification cycles were performed; (4), at 65 ° C for 4 minutes, as an extension reaction; (5), the PCR reaction was stopped at 4 ° C, and the PCR reaction product was stored at 4 ° C.

In step S45, gel electrophoresis is performed on the PCR reaction product. Specifically, gel electrophoresis is performed on the product after the PCR reaction. In this embodiment, the gel electrophoresis may be agarose gel electrophoresis, and the concentration may be selected as follows: The agarose gel has an agarose concentration of 2.5%) or polyacrylamide gel electrophoresis (PAGE).

In step S46, the gel after electrophoresis is stained with a staining agent, and the gelled imager is used for fluorescence analysis; specifically, the gel after electrophoresis is stained with a staining agent as a preferred implementation. Method, the staining agent can be selected such as ethidium bromide (EB), SYBR Green I, GelRed or GoldView staining agent, and use a gel imager to perform fluorescence analysis on the stained gel, and read through the gel imager Take the fluorescent band from the stained gel.

In step S47, it is determined whether three or more fluorescent color bands appear in the gel. In this embodiment, whether the gel imager reads three or more fluorescent color bands from the dyed gel. If three or more fluorescent color bands appear in the gel, step S48 is performed; if two or one fluorescent color bands appear in the gel, step S49 is performed. If no fluorescent color bands appear in the gel, step S50 is performed. Among them, one fluorescent band is the PCR reaction band of the reference marker β-ACTIN, and the other fluorescent band is the PCR reaction band of the mutant gene marker.

In step S48, if three or more fluorescent color bands appear in the gel, the gel imager displays that the blood sample to be tested contains a non-small cell lung cancer gene mutation DNA fragment.

In step S49, if there are 2 or 1 fluorescent color bands in the gel, the gel imager shows that the blood sample to be tested does not contain a gene mutation DNA fragment of non-small cell lung cancer.

In step S50, if there is no fluorescent color band in the gel, the gel imager determines that the ctDNA conversion and PCR reaction are invalid during the methylation detection of lung cancer characteristics, and it is impossible to verify whether the blood sample to be tested contains non-small cell lung cancer. Gene mutation DNA fragment. Therefore, in this embodiment, the reference marker β-ACTIN forward primer and reverse primer are added to the PCR reaction system, mainly to verify whether the ctDNA conversion and the PCR reaction process are correct to ensure the accuracy of the detection of non-small cell lung cancer.

The non-small cell lung cancer multiple gene methylation detection method can detect the presence of multiple non-small cell lung cancer methylation markers at the same time by one PCR reaction detection, thereby improving the accuracy, specificity and sensitivity of lung cancer detection. The invention solves the possibility that a methylation-specific PCR reaction can only detect the methylation level of a single gene and cannot accurately predict the occurrence of lung cancer. At the same time, addressing the uncertainty of tumor suppressor genes and the limitations of ctDNA in non-small cell lung cancer has led to the detection of methylation markers in single non-small cell lung cancer that cannot meet clinical needs.

The above are only preferred embodiments of the present invention, and thus do not limit the patent scope of the present invention. Any equivalent structure or equivalent process transformation made by using the description and drawings of the present invention, or directly or indirectly used in other related technical fields All are included in the patent protection scope of the present invention.

Industrial applicability

Compared with the prior art, the method for detecting multiple gene methylation of non-small cell lung cancer according to the present invention adopts the above technical scheme, and achieves the following technical effects: it can detect multiple non-small cell lung cancer methylation markers in one PCR reaction detection The presence of substances improves the accuracy, specificity and sensitivity of non-small cell lung cancer detection. The invention solves the possibility that a methylation-specific PCR reaction can only detect the methylation level of a single gene and cannot accurately predict the occurrence of lung cancer, and the uncertainty of non-small cell lung cancer tumor suppressor genes and the limitations of ctDNA The problem is that the detection of methylation markers in single non-small cell lung cancer cannot meet the clinical needs.

Sequence Listing Free Content

<110> Shenzhen Shengbizhi Technology Development Co., Ltd.

<120> Non-small cell lung cancer multiple gene methylation detection method

<130> 2018

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<170> PatentIn version 3.5

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Claims

A method for detecting multiple gene methylation in non-small cell lung cancer, characterized in that the method includes steps:

Select 6 genes as methylation markers of non-small cell lung cancer;

Construct 6 primer pairs corresponding to 6 methylation markers, respectively;

Extract the free ctDNA from the blood sample to be tested, and purify and transform the ctDNA;

Add 4ul of 27ng transformed ctDNA to 25ul PCR reaction system;

Put the PCR reaction system into the PCR instrument, and perform PCR amplification reaction on the PCR reaction system according to the set PCR reaction program to obtain the PCR reaction product;

Gel electrophoresis of PCR reaction products;

Stain the gel after electrophoresis with a stain, and perform fluorescence analysis on the stained gel using a gel imager;

The gel imager determines whether two or more fluorescent bands appear in the gel;

If there are two or more fluorescent color bands in the gel, the gel imager will show that the blood sample to be tested contains genetic mutation DNA fragments of non-small cell lung cancer;

If there are two or no fluorescent bands in the gel, the gel imager shows that the blood sample to be tested does not contain genetically modified DNA fragments of non-small cell lung cancer.
The method for detecting multiple gene methylation in non-small cell lung cancer according to claim 1, wherein the six methylation markers are CALCA, DLEC1, HOXA9, TBX5, PITX2, RASSF1a, and the six The primer pair includes 6 sets of primer sequences, which are specifically expressed as follows:

CALCA forward primer: 5'-CGGAATTTTTTCGATTTATAGC-3 ';

CALCA reverse primer: 5'-AAAACCCTATAAAAACGACGAC-3 ';

DLEC1 forward primer: 5'-GATTAAGCGATGACGGGATTC-3 ';

DLEC1 reverse primer: 5'-ACCCGACTAATAACGAAATTAACG-3 ';

HOXA9 forward primer: 5'-GGTTAATGGGGGCGCGGGCGTC-3 ';

HOXA9 reverse primer: 5'-TCATATAACAACTTAATAACACCG-3 ';

TBX5 forward primer: 5'-GGGACGCGTAAAATTTAGAATC-3 ';

TBX5 reverse primer: 5'-AACACAAAACCGAAAAACGTC-3 ';

PITX2 forward primer: 5'-CGTTATTAGTTGAAGGTAAGGTCG-3 ';

PITX2 reverse primer: 5'-AACACCGAAAAATACAATCCG-3 ';

RASSF1a forward primer: 5'-GTGTTAACGCGTTGCGTATC-3 ';

RASSF1a reverse primer: 5'-AACCCCGCGAACTAAAAACGA-3 '.
The method for detecting multiple gene methylation in non-small cell lung cancer according to claim 2, wherein the PCR reaction system comprises a 1.8 × PCR solution, 5 mM MgCl 2 , 0.3 nM deoxyribonucleoside triphosphate, 40 nM 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 unit DNA polymerase.
The method for detecting multiple gene methylation in non-small cell lung cancer according to claim 3, wherein the DNA polymerase is Taq Platinum DNA polymerase.
A method for detecting multiple gene methylation in non-small cell lung cancer, characterized in that the method includes steps:

6 genes were selected as methylation markers and 1 reference marker for non-small cell lung cancer;

Construct 6 primer pairs corresponding to 6 methylation markers and 1 primer pair corresponding to 1 reference marker;

Extract the free ctDNA from the blood sample to be tested, and purify and transform the ctDNA;

Add 4ul of 27ng transformed ctDNA to 25ul PCR reaction system;

Put the PCR reaction system into the PCR instrument, and perform PCR amplification reaction on the PCR reaction system according to the set PCR reaction program to obtain the PCR reaction product;

Gel electrophoresis of PCR reaction products;

Stain the gel after electrophoresis with a stain, and perform fluorescence analysis on the stained gel using a gel imager;

The gel imager determines whether three or more fluorescent bands appear in the gel;

If there are 3 or more fluorescent color bands in the gel, the gel imager will show that the blood sample to be tested contains genetic mutation DNA fragments of non-small cell lung cancer;

If there are 2 or 1 fluorescent color bands in the gel, the gel imager shows that the blood sample to be tested does not contain genetically modified DNA fragments of non-small cell lung cancer;

If no fluorescent bands appear in the gel, the gel imager determines that ctDNA conversion and PCR reactions have failed.
The method for detecting multiple gene methylation in non-small cell lung cancer according to claim 5, wherein the six methylation markers are CALCA, DLEC1, HOXA9, TBX5, PITX2, RASSF1a, and the six The primer pair includes 6 sets of primer sequences, which are specifically expressed as follows:

CALCA forward primer: 5'-CGGAATTTTTTCGATTTATAGC-3 ';

CALCA reverse primer: 5'-AAAACCCTATAAAAACGACGAC-3 ';

DLEC1 forward primer: 5'-GATTAAGCGATGACGGGATTC-3 ';

DLEC1 reverse primer: 5'-ACCCGACTAATAACGAAATTAACG-3 ';

HOXA9 forward primer: 5'-GGTTAATGGGGGCGCGGGCGTC-3 ';

HOXA9 reverse primer: 5'-TCATATAACAACTTAATAACACCG-3 ';

TBX5 forward primer: 5'-GGGACGCGTAAAATTTAGAATC-3 ';

TBX5 reverse primer: 5'-AACACAAAACCGAAAAACGTC-3 ';

PITX2 forward primer: 5'-CGTTATTAGTTGAAGGTAAGGTCG-3 ';

PITX2 reverse primer: 5'-AACACCGAAAAATACAATCCG-3 ';

RASSF1a forward primer: 5'-GTGTTAACGCGTTGCGTATC-3 ';

RASSF1a reverse primer: 5'-AACCCCGCGAACTAAAAACGA-3 ';

The one reference marker is β-ACTIN, and one primer pair corresponding to the reference marker β-ACTIN includes a set of primer sequences, which are specifically expressed as follows:

β-ACTIN forward primer: 5'-TTTTTAGGGAGGAGT TGGAAGTAGT-3 ';

β-ACTIN reverse primer: 5'-AAAATATACC CTCCCCCATA CC-3 '.
The method for detecting multiple gene methylation in non-small cell lung cancer according to claim 6, wherein the PCR reaction system comprises a 1.8 × PCR solution, 5 mM MgCl 2 , 0.3 nM deoxyribonucleoside triphosphate, 40 nM 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 β-ACTIN forward primer, 10nM β-ACTIN reverse primer And 2.5 units of DNA polymerase.
The method for detecting multiple gene methylation in non-small cell lung cancer according to claim 7, wherein the DNA polymerase is Taq Platinum DNA polymerase.
The method for detecting multiple gene methylation in non-small cell lung cancer according to any one of claims 1 to 8, wherein the PCR reaction program comprises the following steps:

Step 1: 3 minutes at 95 ° C;

Step 2: For 1 minute at a temperature of 94 ° C, for 30 seconds at a temperature of 60 ° C, and for 45 seconds at a temperature of 65 ° C, this step 2 is executed for a total of 4 cycles;

Step 3: 1 minute at 94 ° C, 1 minute at 56 ° C, and 45 seconds at 65 ° C. This step 3 performs a total of 36 amplification cycles;

Step 4: The extension reaction is continued at 65 ° C for 4 minutes;

Step 5: Stop the PCR reaction at 4 ° C and store the PCR reaction product at 4 ° C.
The method for detecting multiple gene methylation in non-small cell lung cancer according to claim 9, wherein the purification process uses magnetic bead method or spin column method to extract free ctDNA in the blood sample to be tested to remove impurities and perform ctDNA purification;

The conversion treatment uses sulfite or bisulfite as a reagent to perform sulfite conversion treatment or bisulfite conversion treatment on ctDNA;

The gel electrophoresis uses agarose gel or polyacrylamide gel to perform gel electrophoresis on the PCR reaction product;

The stain is ethidium bromide, SYBR Green I, GelRed or GoldView stain.