WO2019242753A1

WO2019242753A1 - Primer pair for detecting non-small cell lung cancer multiplex gene methylation, and reagent kit

Info

Publication number: WO2019242753A1
Application number: PCT/CN2019/092334
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: CN110628899A

Abstract

A primer pair for detecting non-small cell lung cancer multiplex gene methylation, and a reagent kit, the primer pair comprising 6 sets of primer sequences corresponding to 6 genes serving as methylation markers of non-small cell lung cancer, the methylation markers respectively being: CALCA, DLEC1, HOXA9, TBX5, PITX2, and RASSF1a, and the 6 sets of primer sequences comprising CALCA forward primer, CALCA reverse primer, DLEC1 forward primer, DLEC1 reverse primer, HOXA9 forward primer, HOXA9 reverse primer, TBX5 forward primer, TBX5 reverse primer, PITX2 forward primer, PITX2 reverse primer, RASSF1a forward primer, and RASSF1a reverse primer. The primer pairs also comprise one set of primer sequences corresponding to one representative marker β-ACTIN, specifically comprising β-ACTIN forward primer and β-ACTIN reverse primer. A single PCR reaction detection can be used to simultaneously detect the presence of multiple non-small cell lung cancer methylation markers, improving the accuracy, specificity, and sensitivity of non-small cell lung cancer detection.

Description

Primer pair and kit for detecting multiple gene methylation of non-small cell lung cancer

Technical field

The invention relates to the technical field of EB virus detection, in particular to a primer pair and a kit 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 purpose of the present invention is to provide a primer pair and a kit for detecting multiple gene methylation of non-small cell lung cancer, which aims to solve the existing methylation-specific PCR reaction that can only detect single gene methylation and cannot The problem of accurately predicting the likelihood of lung cancer.

Technical solutions

To achieve the above object, the present invention provides a primer pair for detecting multiple gene methylation of non-small cell lung cancer, including 6 sets of primer sequences corresponding to 6 genes as methylation markers of non-small cell lung cancer. The methylation markers are: CALCA, DLEC1, HOXA9, TBX5, PITX2, RASSF1a, and the six sets of primer sequences are specifically represented 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 primer pair used for detecting multiple gene methylation of non-small cell lung cancer further includes a set of primer sequences corresponding to a reference marker β-ACTIN, which is specifically expressed as follows:

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

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

In another aspect, the present invention also provides an application of a primer pair for detecting multiple gene methylation of non-small cell lung cancer in preparing a non-small cell lung cancer detection reagent.

In another aspect, the present invention also provides a kit for detecting multiple gene methylation of non-small cell lung cancer, including the primer pair and a PCR reaction system, wherein:

The PCR reaction system includes a 1.8 × PCR solution, 5 mM MgCl2, 0.3 nM deoxyribonucleoside triphosphate, and 2.5 units of DNA polymerase;

The concentrations of the primer pairs are 40 nM CALCA forward primer, 40 nM CALCA reverse primer, 40 nM DLEC1 forward primer, 40 nM DLEC1 reverse primer, 120 nM HOXA9 forward primer, 120 nM 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.

Preferably, the present invention also provides a kit for detecting multiple gene methylation of non-small cell lung cancer, comprising the primer pair and a PCR reaction system, wherein:

The concentrations of the primer pairs are 40 nM CALCA forward primer, 40 nM CALCA reverse primer, 40 nM DLEC1 forward primer, 40 nM DLEC1 reverse primer, 120 nM HOXA9 forward primer, 120 nM 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.

Preferably, the DNA polymerase is Taq Platinum DNA polymerase.

In another aspect, the present invention also provides an application of a kit for detecting multiple gene methylation of non-small cell lung cancer in a marker for detecting multiple gene methylation of non-small cell lung cancer.

Beneficial effect

Compared with the prior art, the primer pair and kit for detecting multiple gene methylation of non-small cell lung cancer according to the present invention adopt the above technical scheme, and achieve the following technical effects: multiple non-small cell lung cancers can be detected simultaneously in one PCR reaction The presence of methylation markers in small cell lung cancer improves the accuracy, specificity, and sensitivity of non-small cell lung cancer detection. Detection of multiple gene methylation in non-small cell lung cancer by using the primer pair and the kit according to the present invention can solve a methylation-specific PCR reaction that can only detect the methylation level of a single gene and cannot accurately predict the occurrence of lung cancer. Possibility, and the uncertainty of non-small cell lung cancer tumor suppressor genes and the limitation of ctDNA lead to the problem that single non-small cell lung cancer methylation marker detection cannot meet 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;

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.

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 non-small cell lung cancer multiple gene methylation includes six sets of primer sequences corresponding to six methylation markers, and each methylation marker corresponds to one primer. Yes, each primer pair includes forward primer (MF) and 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 non-small cell lung cancer multiple gene methylation according to the present invention includes not only the six sets of primer sequences corresponding to the six methylation markers, but also a reference marker Corresponding 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).

The invention also provides the application of the primer pair for detecting non-small cell lung cancer multiple gene methylation in preparing a non-small cell lung cancer detection reagent, for example, preparing a non-small cell lung cancer multiple gene methylation Kit, this non-small cell lung cancer detection reagent can be used to detect the multiple gene methylation level of non-small cell lung cancer, and improve the accuracy, specificity and sensitivity of lung cancer detection.

The invention also provides a kit for detecting multiple gene methylation of non-small cell lung cancer, which can be applied to detect a specific multiple methylation marker of non-small cell lung cancer. As a preferred embodiment of the present invention, the kit includes the six primers corresponding to the six non-small cell lung cancer-related genes as methylation markers and a PCR reaction system, wherein the PCR reaction system includes 1.8 × PCR solution, 5 mM MgCl ₂ , 0.3 nM deoxyribonucleoside triphosphate, and 2.5 units of DNA polymerase, for example, Taq Platinum DNA polymerase is preferred; the concentration of the primer pair is CALCA forward primer of 40 nM, 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.

As another preferred embodiment of the present invention, the kit includes 6 sets of primer pairs corresponding to the methylation markers, 1 set of primer pairs corresponding to a reference marker, and a PCR reaction system, wherein: the The PCR reaction system includes 1.8 × PCR solution, 5mM MgCl2, 0.3nM deoxyribonucleoside triphosphate, and 2.5 units of DNADNA polymerase, for example, Taq Platinum DNA polymerase is preferred; the concentration of each of the primer pairs is 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-ACTIN reverse primer.

In this embodiment, the present invention also provides an application of a kit for detecting multiple gene methylation of non-small cell lung cancer in detecting a specific multiple methylation marker of non-small cell lung cancer. That is, the kit of the present invention can be used to detect multiple gene methylation markers of non-small cell lung cancer, which is convenient for users to detect non-small cell lung cancer, and can detect the presence of multiple non-small cell lung cancers in a single PCR reaction. Basic markers improve the accuracy, specificity and sensitivity of lung cancer detection.

The following describes a method for detecting multiple gene methylation markers of non-small cell lung cancer using the primer pair and the kit according to the present invention, which specifically include the following steps:

Step 1: Select 6 genes as methylation markers of NSCLC and 1 gene as reference marker; specifically, based on pre-lung cancer research and existing research results, select 6 genes as NSCLC 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 .

Step 2: Construct six primer pairs corresponding to the six methylation markers and one primer pair corresponding to the one reference marker; in this embodiment, each methylation marker corresponds to one primer pair, each 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 seven sets of primer sequences, including six sets of primer sequences corresponding to six methylation markers and one set of primer sequences corresponding to one reference marker.

Step 3: Extract the free ctDNA in the blood sample to be tested. In this example, it is considered that the concentration of ctDNA in the blood 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.

Step 4: Take 4ul (27ng) of the transformed ctDNA and add it to a 25ul PCR reaction system; in this embodiment, the PCR reaction system includes: 1.8 × PCR solution, 5mM MgCl2, 0.3nM deoxyribonucleoside triphosphate , 6 primers corresponding to 6 methylation markers (40 nM of forward and reverse primers for DLEC1, 20 nM of forward and reverse primers for PITX2, 20 nM of forward and reverse primers for TBX5, CALCA forward primer and reverse primer each 40nM, RASSF1a forward primer and reverse primer each 280nM, HOXA9 forward primer and reverse primer each 120nM), 1 reference marker β-ACTIN 1 primer (10-M each of the forward primer and the reverse primer of β-ACTIN) and a 2.5-unit DNA polymerase, for example, Taq Platinum DNA polymerase is preferred).

Step 5: Put the PCR reaction system into a PCR instrument, and perform a PCR amplification reaction 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; (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.

Step 6: Perform gel electrophoresis on the PCR reaction product. Specifically, perform gel electrophoresis on the PCR reaction product. 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 7: Stain the gel after electrophoresis using a staining agent, and perform fluorescence analysis on the gel after staining using a gel imager; specifically, stain the gel after electrophoresis by using a staining agent as the preferred In an embodiment, the staining agent can be selected from ethidium bromide (EB), SYBR Green I, GelRed or GoldView staining agent, and a gel imager is used to perform fluorescence analysis on the stained gel and pass the gel imager. Read the fluorescent band in the stained gel.

Step 8: Determine 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, 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.

Step 9: If three or more fluorescent color bands appear in the gel, the gel imager determines that the blood sample to be tested contains a non-small cell lung cancer gene mutation DNA fragment.

Step 10: If there are 2 or 1 fluorescent bands in the gel, the gel imager determines that the blood sample to be tested does not contain a mutant DNA fragment of a non-small cell lung cancer gene.

Step 11: If there is no fluorescent 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. It is not possible 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.

By using the primer pair and the kit of the present invention, the presence of multiple non-small cell lung cancer methylation markers can be detected simultaneously in one PCR reaction detection, which improves the accuracy, specificity, and sensitivity of lung cancer detection. The primer pair and the kit of the invention are widely used for detecting methylation of non-small cell lung cancer genes, and solve a methylation-specific PCR reaction that can only detect the methylation level of a single gene and cannot accurately predict lung cancer. The possibility of occurrence, and the uncertainty of non-small cell lung cancer tumor suppressor genes and the limitations of ctDNA lead to the problem that single non-small cell lung cancer methylation marker detection cannot meet the 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

Sequence Listing Free Content

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

<120> Primer pair and kit for detecting multiple gene methylation of non-small cell lung cancer

<130> 2018

<160> 14

<170> PatentIn version 3.5

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Claims

A primer pair for detecting multiple gene methylation of non-small cell lung cancer, characterized in that it comprises 6 sets of primer sequences corresponding to 6 genes as methylation markers of non-small cell lung cancer, the methylation The markers are: CALCA, DLEC1, HOXA9, TBX5, PITX2, RASSF1a, and the six sets of primer sequences 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 primer pair for detecting multiple gene methylation of non-small cell lung cancer according to claim 1, further comprising a set of primer sequences corresponding to a reference marker β-ACTIN, which are specifically expressed as follows:

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

β-ACTIN reverse primer: 5'-AAAATATACC CTCCCCCATA CC-3 '.
The use of the primer pair for detecting multiple gene methylation of non-small cell lung cancer according to claim 1 or 2 in the preparation of a reagent for detecting non-small cell lung cancer.
A kit for detecting multiple gene methylation of non-small cell lung cancer, comprising a primer pair according to claim 1 and a PCR reaction system, wherein:

The PCR reaction system includes a 1.8 × PCR solution, 5 mM MgCl 2 , 0.3 nM deoxyribonucleoside triphosphate, and 2.5 units of DNA polymerase;

The concentrations of the primer pairs are 40 nM CALCA forward primer, 40 nM CALCA reverse primer, 40 nM DLEC1 forward primer, 40 nM DLEC1 reverse primer, 120 nM HOXA9 forward primer, 120 nM 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.
The kit for detecting multiple gene methylation of non-small cell lung cancer according to claim 4, wherein the DNA polymerase is Taq Platinum DNA polymerase.
A kit for detecting multiple gene methylation of non-small cell lung cancer, comprising a primer pair according to claim 2 and a PCR reaction system, wherein:

The PCR reaction system includes a 1.8 × PCR solution, 5mM MgCl2, 0.3nM deoxyribonucleoside triphosphate, and 2.5 units of DNADNA polymerase;

The concentrations of the primer pairs are 40 nM CALCA forward primer, 40 nM CALCA reverse primer, 40 nM DLEC1 forward primer, 40 nM DLEC1 reverse primer, 120 nM HOXA9 forward primer, 120 nM 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.
The kit for detecting multiple gene methylation of non-small cell lung cancer according to claim 6, wherein the DNA polymerase is Taq Platinum DNA polymerase.
Application of the kit for detecting multiple gene methylation of non-small cell lung cancer according to any one of claims 4 to 7 in detecting a multiple gene methylation marker of non-small cell lung cancer.