CN113817831A - Primer and kit for diagnosis and detection of lung adenocarcinoma and detection method of methylation region - Google Patents
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Abstract
Compared with the prior art, the primer, the kit and the detection method for the methylation region for the lung adenocarcinoma diagnosis and detection can effectively judge the occurrence condition of early lung cancer by detecting two ctDNA methylation sites of lung cancer, are simple in technology, can be developed in primary hospitals, and compared with NGS, the detection cost is greatly reduced, and the detection efficiency is improved.
Description
Technical Field
The application relates to the field of lung adenocarcinoma diagnosis and detection, in particular to a primer and a kit for lung adenocarcinoma diagnosis and detection and a detection method of a methylation region.
Background
According to the '2018 global cancer statistics' report published by the american cancer society official journal of clinicians, lung cancer remains the first malignancy of global morbidity (11.6%) and mortality (18.4%). At present, the number of people with pulmonary nodules found by chest radiographs or CT examination is increasing every year.
Pulmonary nodules are space occupying lesions in the lungs. Defined according to the diameter, the similar round or irregular focus with the lung inner diameter less than or equal to 3cm can be singly or multiply called as a pulmonary nodule. The lung nodules have a diameter of 0.5cm-1cm and belong to small lung nodules, and the lung nodules with a diameter of more than 3cm are called lumps. According to the representation of CT imaging, the lung nodules can be divided into pure ground glass nodules, mixed ground glass nodules and solid nodules. The pure ground glass nodules are like ground glass and present cloud-like shadows; the solid nodules are the shadows with higher density in the lung; the mixed ground glass nodules include both the actual component and the ground glass component, with the actual component generally being intermediate the ground glass components. Changes in CT images such as pleural depression, drainage line, blood vessel cluster, burr, spinous process, lobular division, vacuole and bronchiole inflation are all signs of pulmonary nodule malignant change. Wherein, the ranking order of the malignant change probability should be: mixed ground glass nodules > solid nodules.
The international association for the study of lung cancer (IASLC) advocates the use of fluid biopsies for the diagnosis of non-small cell lung cancer (NSCLC). The greatest advantage of plasma free tumor dna (ctdna) methylated liquid biopsy is the possibility of detecting whether there is a cancerous change in cells at an ultra-early stage that cannot be detected by conventional means. DNA methylation has the characteristic of cell tissue specificity, has extremely high specificity, and is suitable for early diagnosis of diseases.
At present, most of detection means of lung cancer ctDNA methylation are NGS, the method is long in time consumption and high in cost, and needs professional personnel to perform letter generation analysis, so that the method is not beneficial to clinical popularization in primary hospitals.
Therefore, how to improve the detection efficiency of the ctDNA methylation of lung cancer has become an urgent problem to be solved by those skilled in the art.
Disclosure of Invention
In order to solve the technical problems, the application provides a primer, a kit and a detection method of a methylation region for diagnosing and detecting lung adenocarcinoma, which can improve the detection efficiency of lung cancer ctDNA methylation.
The technical scheme provided by the application is as follows:
the application provides a primer for diagnosing and detecting lung adenocarcinoma based on plasma ctDNA, which comprises the following components: an internal reference primer and a detection primer, wherein the detection primer is used for detecting a lung adenocarcinoma specific DNA methylation marker, and the lung adenocarcinoma specific DNA methylation marker comprises: ALX3 gene, HOXC13 gene.
Further, in a preferred mode of the present invention, the detection primer is used for amplifying a sequence of the lung cancer specific DNA methylation marker.
Further, in a preferred mode of the present invention, the primer sequence of the ALX3 gene is as follows:
primer 1 a: GGCGTAGTTGCGCGTTTATTC, respectively; AAACCCGCCTTATTTCCCG
Probe 1 a: TGGATTGGAAACGTTTCGAGTCGGTTATTT
Primer 1 b: CGAAGAGGGAATTCGTTTTATTTTTC, respectively; CAACTACGCGCTCACTCG
Probe 1 b: TTAAAATAATCGGTTCGGAGCGTTTTTAGT are provided.
Further, in a preferred embodiment of the present invention, the primer sequences of the HOXC13 gene are as follows:
primer 2 a: GATTTGGGGGATTCGAGTTGC, respectively; GAACGAACGATAACTAAAAAACGCG
The probe 2 a: CAACTAAACCCGCTCGTTTCCCGCCA
And (3) primer 2 b: GTGGTTAGGGGACGC, respectively; AAAATCTAAAAAACCCGAACTACG
And (3) probe 2 b: TAGTTGAGTTCGTTCGTTTTTCGTTATTGT are provided.
Further, in a preferred embodiment of the present invention, there is provided a kit for the diagnostic detection of lung adenocarcinoma based on plasma ctDNA, comprising: the primer for lung adenocarcinoma diagnosis and detection based on plasma ctDNA, a washing solution, a purification solution, a DNA extraction solution, a positive control and a negative control.
Preferably, the detergent is purified water.
Preferably, the purification solution is isopropanol or ethanol.
Further, in a preferred mode of the invention, the kit is a fluorescent PCR kit, and the kit is used for detecting the lung cancer specific DNA methylation sites.
Further, in a preferred mode of the present invention, the conversion solution is a sulfite solution.
Further, in a preferred mode of the invention, the kit is used for early diagnosis, screening and detection of the micro-lesions after treatment by detecting the lung cancer specific DNA methylation region in plasma and other body fluids and combining the expression condition of an internal reference.
Further, in a preferred mode of the present invention, there is also provided a method for detecting a region of methylation of lung adenocarcinoma-specific DNA, comprising the steps of:
s1, extracting a blood sample, and separating plasma;
s2, extracting a ctDNA sample;
s3, carrying out sulfite conversion treatment on the ctDNA sample;
s4, methylation specific PCR: using the kit for diagnosing and detecting the lung adenocarcinoma to perform real-time fluorescence methylation specific PCR amplification on the transformed ctDNA so as to detect the methylation region of the ALX3 gene or the HOXC13 gene;
s5, comparing the detected methylation region results.
Further, in a preferred mode of the invention, the method is used for distinguishing between tumor and normal tissue.
Compared with the prior art, the technical scheme provided by the invention has the advantages that the occurrence condition of early lung cancer can be effectively judged by detecting two ctDNA methylation sites of the lung cancer, the technology is simple, the detection can be carried out in primary hospitals, the detection cost is greatly reduced and the detection efficiency is improved compared with NGS.
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In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present application, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
Fig. 1 is a comparison graph of methylation regions of ALX3 gene or HOXC13 gene detected by the kit for plasma ctDNA-based lung adenocarcinoma diagnostic test provided by the embodiment of the invention;
FIG. 2 is a flowchart of the steps of a method for detecting a region of methylation of DNA specific to lung adenocarcinoma according to an embodiment of the present invention.
Detailed Description
In order to make those skilled in the art better understand the technical solutions in the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application, and it is obvious that the described embodiments are only a part of the embodiments of the present application, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
It will be understood that when an element is referred to as being "fixed" or "disposed" on another element, it can be directly on the other element or be indirectly disposed on the other element; when an element is referred to as being "connected to" another element, it can be directly connected to the other element or be indirectly connected to the other element.
It will be understood that the terms "length," "width," "upper," "lower," "front," "rear," "first," "second," "vertical," "horizontal," "top," "bottom," "inner," "outer," and the like, as used herein, refer to an orientation or positional relationship indicated in the drawings that is solely for the purpose of facilitating the description and simplifying the description, and do not indicate or imply that the referenced device or element must have a particular orientation, be constructed and operated in a particular orientation, and thus should not be considered as limiting the present application.
Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. In the description of the present application, "plurality" or "a plurality" means two or more unless specifically limited otherwise.
It should be understood that the structures, ratios, sizes, and the like shown in the drawings are only for illustrative purposes and are not intended to limit the scope of the present disclosure, so that those skilled in the art can understand and read the present disclosure, and the present disclosure is not limited to the conditions and conditions that can be implemented in the present disclosure.
As shown in fig. 1-2, the present application provides a primer for plasma ctDNA-based lung adenocarcinoma diagnostic test, comprising: an internal reference primer and a detection primer, wherein the detection primer is used for detecting a lung adenocarcinoma specific DNA methylation marker, and the lung adenocarcinoma specific DNA methylation marker comprises: ALX3 gene, HOXC13 gene.
Compared with the prior art, the primer, the kit and the method for detecting the methylation region for diagnosing and detecting the lung adenocarcinoma, which are provided by the invention, based on the ctDNA of the plasma, comprise the following steps: an internal reference primer and a detection primer, wherein the detection primer is used for detecting a lung adenocarcinoma specific DNA methylation marker, and the lung adenocarcinoma specific DNA methylation marker comprises: ALX3 gene and HOXC13 gene, the invention relates to a reagent kit for diagnosing and detecting lung adenocarcinoma based on plasma ctDNA, which comprises: the invention relates to a method for detecting a specific DNA methylation region of lung adenocarcinoma, which comprises the following steps of: s1, extracting a blood sample, and separating plasma; s2, extracting a ctDNA sample; s3, carrying out sulfite conversion treatment on the ctDNA sample; s4, the ctDNA obtained after conversion is amplified by using the kit for diagnosing and detecting the lung adenocarcinoma through real-time fluorescence methylation specificity PCR so as to detect the methylation region of the ALX3 gene or the HOXC13 gene, therefore, the occurrence condition of early lung cancer can be effectively judged by detecting two lung cancer ctDNA methylation sites, the technology is simple, the kit can be developed in primary hospitals, compared with NGS, the detection cost is greatly reduced, and the detection efficiency is improved.
Specifically, in an embodiment of the present invention, there is provided a primer for plasma ctDNA-based lung adenocarcinoma diagnostic detection, comprising: an internal reference primer and a detection primer, wherein the detection primer is used for detecting a lung adenocarcinoma specific DNA methylation marker, and the lung adenocarcinoma specific DNA methylation marker comprises: ALX3 gene, HOXC13 gene.
Specifically, in the embodiment of the present invention, the detection primer is used for amplifying a sequence of the lung cancer specific DNA methylation marker.
Specifically, in the embodiment of the present invention, the primer sequence of the ALX3 gene is as follows:
primer 1 a: GGCGTAGTTGCGCGTTTATTC, respectively; AAACCCGCCTTATTTCCCG
Probe 1 a: TGGATTGGAAACGTTTCGAGTCGGTTATTT
Primer 1 b: CGAAGAGGGAATTCGTTTTATTTTTC, respectively; CAACTACGCGCTCACTCG
Probe 1 b: TTAAAATAATCGGTTCGGAGCGTTTTTAGT are provided.
Specifically, in the present embodiment, the primer sequences of the HOXC13 gene are as follows:
primer 2 a: GATTTGGGGGATTCGAGTTGC, respectively; GAACGAACGATAACTAAAAAACGCG
The probe 2 a: CAACTAAACCCGCTCGTTTCCCGCCA
And (3) primer 2 b: GTGGTTAGGGGACGC, respectively; AAAATCTAAAAAACCCGAACTACG
And (3) probe 2 b: TAGTTGAGTTCGTTCGTTTTTCGTTATTGT are provided.
Specifically, in an embodiment of the present invention, there is also provided a kit for plasma ctDNA-based lung adenocarcinoma diagnostic detection, comprising: the primer for lung adenocarcinoma diagnosis and detection based on plasma ctDNA, a washing solution, a purification solution, a DNA extraction solution, a positive control and a negative control.
It should be noted that, in the present embodiment, the positive control solution is a 100% methylation control solution, and the negative control solution is a 0% methylation control solution.
Specifically, in the embodiment of the invention, the kit is a fluorescent PCR kit, and the kit is used for detecting the lung cancer specific DNA methylation sites.
Specifically, in the embodiment of the present invention, the conversion solution is a sulfite solution.
Specifically, in the embodiment of the invention, the kit is used for detecting the lung cancer specific DNA methylation region in plasma and other body fluids, and is combined with the expression condition of an internal reference, so that the kit is used for early diagnosis, screening and detection of the micro-focus after treatment.
It should be added that, in the embodiment of the present invention, the kit detects two lung cancer specific DNA methylation regions by a fluorescent probe PCR method, the two lung adenocarcinoma specific DNA methylation markers are ALX3 and HOXC13 genes, and one or both of the ALX3 and HOXC13 genes can be used as a detection target.
More specifically, the kit detects a primer sequence of ALX3 gene or a variant thereof, and a primer sequence of HOXC13 gene or a variant thereof by a fluorescent probe PCR method.
It should be added that, in the embodiment of the present invention, when the kit detects two lung cancer specific DNA methylation regions by a fluorescent probe PCR method, an internal reference detection of an input sample is also required.
Specifically, in the embodiment of the present invention, there is also provided a method for detecting a lung adenocarcinoma specific DNA methylation region, comprising the steps of:
s1, extracting a blood sample, and separating plasma;
s2, extracting a ctDNA sample;
s3, carrying out sulfite conversion treatment on the ctDNA sample;
s4, methylation specific PCR: using the kit for diagnosing and detecting the lung adenocarcinoma to perform real-time fluorescence methylation specific PCR amplification on the transformed ctDNA so as to detect the methylation region of the ALX3 gene or the HOXC13 gene;
s5, comparing the detected methylation region results.
In particular, in embodiments of the invention, the methods are used to differentiate between tumor and normal tissue.
It should be noted that, in the embodiment of the present invention, the blood cells do not affect the result judgment.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
SEQUENCE LISTING
<110> Hunan Point Biotechnology Ltd
<120> primer and kit for diagnosis and detection of lung adenocarcinoma and detection method of methylation region
<130> 20210918
<160> 12
<170> PatentIn version 3.5
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Claims (10)
1. A primer for diagnostic detection of lung adenocarcinoma based on plasma ctDNA, comprising: an internal reference primer and a detection primer, wherein the detection primer is used for detecting a lung adenocarcinoma specific DNA methylation marker, and the lung adenocarcinoma specific DNA methylation marker comprises: ALX3 gene, HOXC13 gene.
2. The primer for plasma ctDNA-based diagnostic detection of lung adenocarcinoma according to claim 1, wherein the detection primer is used for amplifying a sequence of the lung cancer specific DNA methylation marker.
3. The primer for plasma ctDNA-based lung adenocarcinoma diagnostic detection according to claim 2, wherein the primer sequence of the ALX3 gene is as follows:
primer 1 a: GGCGTAGTTGCGCGTTTATTC, respectively; AAACCCGCCTTATTTCCCG
Probe 1 a: TGGATTGGAAACGTTTCGAGTCGGTTATTT
Primer 1 b: CGAAGAGGGAATTCGTTTTATTTTTC, respectively; CAACTACGCGCTCACTCG
Probe 1 b: TTAAAATAATCGGTTCGGAGCGTTTTTAGT are provided.
4. The primer for plasma ctDNA-based lung adenocarcinoma diagnostic test according to claim 3, wherein the primer sequence of HOXC13 gene is as follows:
primer 2 a: GATTTGGGGGATTCGAGTTGC, respectively;
GAACGAACGATAACTAAAAAACGCG
the probe 2 a: CAACTAAACCCGCTCGTTTCCCGCCA
And (3) primer 2 b: GTGGTTAGGGGACGC, respectively; AAAATCTAAAAAACCCGAACTACG
And (3) probe 2 b: TAGTTGAGTTCGTTCGTTTTTCGTTATTGT are provided.
5. A kit for plasma ctDNA-based diagnostic detection of lung adenocarcinoma, comprising: primer, washing solution, purification solution, DNA extraction solution, positive control and negative control for plasma ctDNA-based lung adenocarcinoma diagnostic test of claim 4.
6. The plasma ctDNA-based lung adenocarcinoma diagnostic test kit according to claim 5, wherein the kit is a fluorescent PCR kit for detecting lung cancer specific DNA methylation sites.
7. The kit for plasma ctDNA-based diagnostic detection of lung adenocarcinoma according to claim 6, characterized in that said conversion solution is a sulfite solution.
8. The kit for plasma ctDNA-based lung adenocarcinoma diagnostic test according to claim 7, wherein the kit is used for early diagnosis, screening and detection of micro-lesions after treatment by detecting lung cancer specific DNA methylation regions in plasma and other body fluids in combination with internal reference expression.
9. A method for detecting a region of lung adenocarcinoma specific DNA methylation comprising the steps of:
s1, extracting a blood sample, and separating plasma;
s2, extracting a ctDNA sample;
s3, carrying out sulfite conversion treatment on the ctDNA sample;
s4, methylation specific PCR: amplifying the transformed ctDNA by real-time fluorescence methylation specific PCR using the kit for the diagnostic detection of lung adenocarcinoma according to claim 8 to detect the methylation region of ALX3 gene or HOXC13 gene;
s5, comparing the detected methylation region results.
10. The method of detecting regions of lung adenocarcinoma specific DNA methylation according to claim 9, wherein the method is used to distinguish between tumor and normal tissue.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115725737A (en) * | 2022-08-16 | 2023-03-03 | 山东大学 | Polygene methylation kit for early diagnosis of lung adenocarcinoma |
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| CN112239787A (en) * | 2019-07-19 | 2021-01-19 | 河南远止生物技术有限公司 | Primer, probe, kit and device for detecting human IDH1 gene mutation |
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| 王欣: "细胞周期相关蛋白ZYG11A促进肺腺癌恶性进展的机制研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115725737A (en) * | 2022-08-16 | 2023-03-03 | 山东大学 | Polygene methylation kit for early diagnosis of lung adenocarcinoma |
| CN115725737B (en) * | 2022-08-16 | 2023-09-08 | 山东大学 | Polygene methylation kit for early diagnosis of lung adenocarcinoma |
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