CN117305446A - Methylation molecular marker for detecting benign and malignant lung nodules and application thereof - Google Patents
Methylation molecular marker for detecting benign and malignant lung nodules and application thereof Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/686—Polymerase chain reaction [PCR]
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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Abstract
The present invention provides a DNA methylation molecular marker for detecting benign and malignant lung nodules, the DNA methylation molecular marker comprising SEQ ID No.6 or its full complement, or a continuous fragment of at least 55% of the full length of SEQ ID No.6 or its full complement. The invention also provides application of the DNA methylation molecular marker, and a corresponding detection reagent box and a detection method. The DNA methylation molecular marker combination is highly relevant to lung cancer, has high sensitivity and specificity for detecting benign and malignant lung nodules, can improve the detection rate of malignant lung nodules, and reduces the false positive rate of detection.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a methylation molecular marker for detecting benign and malignant lung nodules and application thereof, wherein the application comprises a detection reagent box and a detection method.
Background
The lung nodule (isolated lung nodule) is a solid or sub-solid lesion with a circular shadow like image, single, clear boundary, diameter less than or equal to 3cm and high and low density surrounded by air-containing lung tissue, and does not accompany the symptoms of non-expanded lung, phylum or pleural effusion. It often invades organs such as lung, bilateral portal lymph node, eye, skin, etc., and the chest invasion rate is as high as 80% -90%. Considerable lung nodules cannot rule out the possibility of early malignant tumors. Pulmonary nodules are an important indicator of primary lung cancer
Lung nodules are generally classified as benign and malignant, but are often asymptomatic, whether benign or malignant, and benign nodules require treatment for the etiology, and malignant require early surgery. The etiology of benign lung nodules is often associated with autoimmune diseases or various infections, and the etiology of malignant lung nodules is often associated with lung cancer.
The clinical manifestations of lung cancer are complex, and the presence, severity and appearance of symptoms and signs depend on the location of the tumorigenesis, the type of pathology, the presence or absence of metastasis and complications, and the differences in the extent of the patient's response and tolerance. Early symptoms of lung cancer are usually mild, and even without any discomfort. The symptoms of central lung cancer appear early and heavy, and the symptoms of peripheral lung cancer appear late and lighter, even without symptoms. Thus, once clinical symptoms are present or routinely detected, advanced stages of lung cancer are reached. Early screening is therefore crucial. Early lung cancer generally manifests as malignant lung nodules in the lungs, and therefore early screening generally begins with the detection of lung nodules.
Compared with tissue biopsy, the liquid biopsy has the advantages of simple operation, non-invasiveness, strong repeatability, contribution to dynamic monitoring of diseases and the like. The lung cancer liquid biopsy takes blood, spittoon, alveolar lavage and the like of a patient as samples, and detects and analyzes tumor cell DNA and modification levels thereof, such as DNA methylation and the like. For cancer patients, the improvement of sensitivity is a great challenge for the detection method due to the low ctDNA content in the blood and the large individual difference. And the sputum and the alveolar lavage liquid are collected, the sputum can be collected through atomization-induced expectoration in clinic, and the alveolar lavage liquid is obtained through a fiber branched gas tube mirror, so that the sample is directly from the lung, and the signal detection sensitivity is more advantageous than that of a blood sample. In the sample collection mode, the collection of the spittoon is noninvasive operation, so that the collection is safer; fiber bronchoscope collection alveolar lavage (BAL) is a method of infusing saline into the bronchopulmonary alveoli and then aspirating the saline, collecting the effective fluid on the alveolar surface, and examining its cellular components and soluble substances. Compared with percutaneous lung puncture and surgical biopsy, the method is a minimally invasive biopsy method with high safety.
In the previous research, the applicant and the inventor of the application always screen DNA methylation molecular markers which can well detect benign and malignant lung nodules, and in the gradually deepened research, further search for some DNA methylation molecular markers which can be well suitable for detecting benign and malignant lung nodules or combinations thereof, so as to realize the detection of benign and malignant lung nodules with high sensitivity and specificity, and particularly realize the detection of high sensitivity aiming at respiratory tract liquid samples, thereby providing technical support for noninvasive diagnosis of lung cancer, particularly early lung cancer.
Disclosure of Invention
Based on the above, an object of the present invention is to provide a DNA methylation molecular marker for detecting benign and malignant lung nodules, wherein the DNA methylation molecular marker combination has excellent sensitivity and specificity for detecting benign and malignant lung nodules, and can effectively improve the detection rate of malignant lung nodules.
The technical scheme for achieving the above purpose comprises the following steps.
In a first aspect of the invention, there is provided a DNA methylation molecular marker useful for detecting benign and malignant lung nodules.
A DNA methylation molecular marker useful for detecting benign and malignant pulmonary nodules comprising SEQ ID No.6 or its full complement, or a continuous fragment of at least 55% of the full length of SEQ ID No.6 or its full complement.
In a second aspect, the invention provides the use of a DNA methylation molecular marker and/or a reagent for detecting the methylation level thereof in the preparation of a kit for detecting benign and malignant lung nodules and/or lung cancer.
In a third aspect of the invention, there is provided a kit for detecting benign and malignant pulmonary nodules comprising reagents for detecting the methylation level of a DNA methylation molecular marker as described above.
In a fourth aspect of the present invention, there is provided a method for detecting methylation level of the above DNA methylation molecular marker combination, comprising the steps of:
(1) Extracting genomic DNA from a sample to be tested;
(2) Performing hydrogen sulfite treatment on the extracted genomic DNA to obtain converted DNA;
(3) And (3) carrying out multiplex fluorescence quantitative PCR detection on the transformation product obtained in the step (2) by using a probe aiming at the DNA methylation molecular marker.
In a fifth aspect of the present invention, there is also provided a method of detecting benign and malignant lung nodules, comprising the steps of:
(1) Extracting genomic DNA from a sample to be tested;
(2) Performing hydrogen sulfite treatment on the extracted genomic DNA to obtain converted DNA;
(3) Detecting by using the reagent box; or also include
(4) By reference gene C T Judging whether the sample is effective or not by using the value, and then using the reference gene C T C for each molecular marker detected in the valid sample T Correcting the value;
(5) And (5) carrying out model analysis on the corrected data, and finally judging the benign and malignant lung nodules.
The invention finds the combination of specific molecular markers with DNA methylation specificity highly related to benign and malignant lung nodules, can better detect benign and malignant lung nodules by detecting the methylation level of the markers, has higher sensitivity and specificity, improves the sensitivity and specificity of detecting benign and malignant lung nodules, can effectively improve the detection rate of early malignant lung nodules, and can treat and intervene early, thereby improving the survival rate of patients; meanwhile, the detected false positive rate is reduced, and excessive diagnosis and treatment of benign lung nodules are avoided. The preferred DNA methylation molecular marker combination provided by the invention is highly correlated with benign and malignant lung nodules, is particularly suitable for respiratory tract samples, comprises respiratory tract liquid samples obtained through minimally invasive or noninvasive means, and can realize noninvasive detection of lung nodules.
According to the primer and the probe for the fluorescent quantitative PCR detection of the DNA methylation molecular marker, the primer is used for carrying out multiple fluorescent quantitative PCR detection on the DNA treated by hydrogen sulfite, and then the conversion product is subjected to multiple fluorescent quantitative PCR detection.
Drawings
FIG. 1 is a ROC graph of the methylation molecular marker combinations of the present invention described in example 4 for the identification of benign and malignant nodules in 134 lung tissue samples.
FIG. 2 is a ROC graph of the methylation molecular marker combinations of the invention described in example 5 for the identification of benign and malignant nodules for 173 respiratory tract liquid samples.
FIG. 3 is a ROC graph of the methylation molecular marker combinations of the invention described in example 6 for the identification of benign and malignant nodules for 61 respiratory tract liquid samples.
Detailed Description
The experimental methods of the present invention, in which the specific conditions are not specified in the following examples, are generally followed by conventional conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the Laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. The various chemicals commonly used in the examples are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In the present invention, the term "plurality" means two or more. "and/or", describes an association relationship of the association object, and indicates that three relationships may exist, for example, a and/or B may indicate: a exists alone, A and B exist together, and B exists alone. The character "/" generally indicates that the associated object is an or relationship.
The DNA methylation molecular marker for detecting benign and malignant lung nodules provided by the invention is specific to 6 methylation regions in 4 detection genes HOXB4, PTGER4, LHX9 and ZCAN 31 or a combination thereof. Wherein the DNA methylation molecular marker comprises SEQ ID NO.6 or a complete complementary sequence thereof, or a continuous fragment of at least 55% of the full length of SEQ ID NO.6 or a complete complementary sequence thereof.
In some embodiments, the kit further comprises at least one selected from the sequences shown in SEQ ID NO.1 to SEQ ID NO.5 or the complete complement thereof, and the obtained combination of molecular markers; or at least one selected from the group consisting of a sequence represented by SEQ ID NO.1 to SEQ ID NO.5 or a continuous fragment thereof having at least 55% of the full length of the complete complement thereof.
In some embodiments, the DNA methylation molecular marker comprises SEQ ID No.6 and SEQ ID No.4, or comprises the complete complement of SEQ ID No.6 and SEQ ID No. 4.
In some embodiments, the DNA methylation molecular marker further comprises SEQ ID No.2 or a complete complement thereof;
or further comprises a contiguous segment of at least 55% of the total length of each of the sequences described above, and combinations thereof.
In some embodiments, the DNA methylation molecular marker further comprises SEQ ID No.5 or a complete complement thereof, in addition to the above-described sequence containing SEQ ID No.6, or SEQ ID No.6 and SEQ ID No. 4.
In some embodiments, in the above combinations comprising SEQ ID No.5 or its full complement, SEQ ID No.2 and/or SEQ ID No.3, or its full complement;
or further comprises a contiguous segment of at least 55% of the total length of each of the sequences described above, and combinations thereof.
In some embodiments, in the above combinations comprising SEQ ID No.5 or its full complement, SEQ ID No.1 and/or SEQ ID No.3, or its full complement;
or further comprises a contiguous segment of at least 55% of the total length of each of the sequences described above, and combinations thereof.
In some embodiments, the DNA methylation molecular marker comprises SEQ ID No.6 and SEQ ID No.1, the sequences shown or their complete complements; or (b)
The DNA methylation molecular marker comprises SEQ ID NO.6 and SEQ ID NO.2, and the sequence shown or the complete complementary sequence thereof; or (b)
The DNA methylation molecular marker comprises SEQ ID NO.6 and SEQ ID NO.3, and the sequence shown or the complete complementary sequence thereof; or (b)
The DNA methylation molecular marker comprises SEQ ID NO.6 and SEQ ID NO.4, and the sequence shown or the complete complementary sequence thereof; or (b)
The DNA methylation molecular marker comprises sequences shown in SEQ ID NO.6, SEQ ID NO.1 and SEQ ID NO.2 or complete complementary sequences thereof; or (b)
Or a contiguous fragment comprising at least 55% of the full length of each of the sequences described above, and combinations thereof.
In some embodiments thereof, the DNA methylation molecular marker comprises SEQ ID No.6, SEQ ID No.2, and SEQ ID No.4, or comprises the complete complement of SEQ ID No.6, SEQ ID No.2, and SEQ ID No. 4;
or a contiguous fragment comprising at least 55% of the entire length of the above sequence, and combinations thereof.
In some embodiments thereof, the DNA methylation molecular marker comprises SEQ ID No.6, SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, and SEQ ID No.4, or comprises the complete complement of SEQ ID No.6, SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, and SEQ ID No. 4;
or a contiguous fragment comprising at least 55% of the entire length of the above sequence, and combinations thereof.
In some embodiments thereof, the DNA methylation molecular marker comprises SEQ ID No.6, SEQ ID No.1, SEQ ID No.3, SEQ ID No.4, and SEQ ID No.5, or comprises the complete complement of SEQ ID No.6, SEQ ID No.1, SEQ ID No.3, SEQ ID No.4, and SEQ ID No. 5;
Or a contiguous fragment comprising at least 55% of the entire length of the above sequence, and combinations thereof.
In some embodiments thereof, the DNA methylation molecular marker comprises SEQ ID No.1 to SEQ ID No.6, or comprises the complete complement of SEQ ID No.1 to SEQ ID No. 6;
or a contiguous fragment comprising at least 55% of the entire length of the above sequence, and combinations thereof.
In some embodiments, the DNA methylation molecular marker is a continuous fragment of at least 55% of the full length of the sequences shown in SEQ ID No.1 to SEQ ID No.6, respectively:
the primers are SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.10 and SEQ ID NO.11, SEQ ID NO.13 and SEQ ID NO.14, and the sequence of the amplified fragment in any group of SEQ ID NO. 1;
and/or the sequence of the amplified fragment of any one group of SEQ ID NO.16 and SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.22 and SEQ ID NO.23 in SEQ ID NO. 2;
and/or the sequence of the amplified fragment of any one group of SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.28 and SEQ ID NO.29, SEQ ID NO.31 and SEQ ID NO.32 in SEQ ID NO. 3;
and/or the sequence of the amplified fragment of any one group of SEQ ID NO.34 and SEQ ID NO.35, SEQ ID NO.37 and SEQ ID NO.38, SEQ ID NO.409 and SEQ ID NO.41 in SEQ ID NO. 4;
And/or the sequence of the amplified fragment of any one group of SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.46 and SEQ ID NO.47, SEQ ID NO.49 and SEQ ID NO.50 in SEQ ID NO. 5;
and/or the sequence of the amplified fragment of any one group of SEQ ID NO.52 and SEQ ID NO.53, SEQ ID NO.55 and SEQ ID NO.56, SEQ ID NO.58 and SEQ ID NO.59 in SEQ ID NO. 5.
In some embodiments, the DNA methylation molecular marker is a continuous fragment of at least 55% of the full length of the sequences shown in SEQ ID No.1 to SEQ ID No.6, respectively:
the primers are the sequences corresponding to the fragments amplified by SEQ ID NO.7 and SEQ ID NO.8 in SEQ ID NO. 1;
and/or the sequences of the amplified fragments of SEQ ID NO.16 and SEQ ID NO.17 in SEQ ID NO. 2;
and/or the sequences of the amplified fragments of SEQ ID NO.28 and SEQ ID NO.29 in SEQ ID NO. 3;
and/or the sequences of the amplified fragments of SEQ ID NO.37 and SEQ ID NO.38 in SEQ ID NO. 4;
and/or the sequences of the amplified fragments of SEQ ID NO.43 and SEQ ID NO.44 in SEQ ID NO. 5;
And/or the sequences of the amplified fragments of SEQ ID NO.52 and SEQ ID NO.53 in SEQ ID NO. 6.
In some embodiments, the DNA methylation molecular marker combination is a molecular marker for a respiratory tract sample, preferably a lung tissue sample, or a respiratory tract fluid sample.
The DNA methylation molecular marker is a combination of any two or more than two of sequences shown in SEQ ID NO. 1-SEQ ID NO.6 or complete complementary sequences thereof; or a combination of any two or more of consecutive fragments selected from the full length of the sequences shown in SEQ ID No.1 to SEQ ID No.6 or at least 55% of the complete complementary sequences thereof.
In some embodiments, there is also provided a DNA methylation molecular marker useful for detecting benign and malignant lung nodules, the DNA methylation molecular marker comprising the sequences shown in SEQ ID No.2 and SEQ ID No.4 or the complete complement thereof; or (b)
The DNA methylation molecular marker comprises sequences shown in SEQ ID NO.1 to SEQ ID NO.4 or complete complementary sequences thereof; or (b)
The DNA methylation molecular marker comprises sequences shown in SEQ ID NO.1 to SEQ ID NO.5 or complete complementary sequences thereof; or (b)
The DNA methylation molecular marker comprises sequences shown in SEQ ID NO.3 and SEQ ID NO.6 or complete complementary sequences thereof;
Or a continuous fragment comprising at least 55% of the full length of each of the sequences described above.
In the examples below, markers described correspondingly for the DNA methylation molecular markers described in SEQ ID No.1 to SEQ ID No.6, and corresponding markers amplified by the corresponding primers.
In one embodiment of the present invention, a combination of two or more of any of the methylation detection regions comprising 6 DNA methylation molecular markers suitable for detecting benign and malignant lung nodules in a respiratory tract sample is included.
In one embodiment, the present invention relates to the use of a detection reagent for the above-described combination of DNA methylation molecular markers for the preparation of a kit for detecting benign and malignant pulmonary nodules.
In one embodiment of the present invention, the DNA methylation molecular marker is used for detecting benign and malignant lung nodules and/or lung cancer.
In some embodiments of the invention, a kit for detecting benign and malignant pulmonary nodules comprises reagents for detecting the methylation level of the DNA methylation molecular marker described above.
In some embodiments, the kit comprises reagents used with PCR amplification, fluorescent quantitative PCR, digital PCR, liquid chip, generation sequencing, second generation sequencing, pyrophosphate sequencing, bisulfite conversion sequencing, methylation chip, simplified hydrogen sulfite sequencing techniques, or combinations thereof. That is, any platform that can realize high-throughput detection may be used.
In some embodiments, the reagents include primers and probes for fluorescent quantitative PCR detection of DNA methylation molecular markers, the primers and probes being:
the primers and probes for SEQ ID NO.1 are selected from at least one of the following groups: primers shown as SEQ ID NO.7 and SEQ ID NO.8, and a probe shown as SEQ ID NO. 9; primers shown as SEQ ID NO.10 and SEQ ID NO.11, and a probe shown as SEQ ID NO. 12; primers shown as SEQ ID NO.13 and SEQ ID NO.14, and a probe shown as SEQ ID NO. 15;
and/or the primers and probes directed against SEQ ID No.2 are selected from at least one of the following groups: primers shown as SEQ ID No.16 and SEQ ID No.17, and a probe shown as SEQ ID No. 18; primers shown as SEQ ID NO.19 and SEQ ID NO.20, and a probe shown as SEQ ID NO. 21; primers shown as SEQ ID No.22 and SEQ ID No.23, and a probe shown as SEQ ID No. 24;
and/or the primers and probes directed against SEQ ID No.3 are selected from at least one of the following groups: primers shown as SEQ ID No.25 and SEQ ID No.26, and a probe shown as SEQ ID No. 27; primers shown as SEQ ID No.28 and SEQ ID No.29, and a probe shown as SEQ ID No. 30; primers shown as SEQ ID NO.31 and SEQ ID NO.32, and a probe shown as SEQ ID NO. 33;
And/or the primers and probes directed against SEQ ID No.4 are selected from at least one of the following groups: primers shown as SEQ ID No.34 and SEQ ID No.35, and a probe shown as SEQ ID No. 36; primers shown as SEQ ID No.37 and SEQ ID No.38, and a probe shown as SEQ ID No. 39; primers shown as SEQ ID No.40 and SEQ ID No.41, and a probe shown as SEQ ID No. 42;
and/or the primers and probes directed against SEQ ID No.5 are selected from at least one of the following groups: primers shown as SEQ ID No.43 and SEQ ID No.44, and a probe shown as SEQ ID No. 45; primers shown as SEQ ID No.46 and SEQ ID No.47, and a probe shown as SEQ ID No. 48; primers shown as SEQ ID NO.49 and SEQ ID NO.50, and a probe shown as SEQ ID NO. 51;
and/or the primers and probes directed against SEQ ID No.6 are selected from at least one of the following groups: primers shown as SEQ ID NO.52 and SEQ ID NO.53, and a probe shown as SEQ ID NO. 54; primers shown as SEQ ID No.55 and SEQ ID No.56, and a probe shown as SEQ ID No. 57; primers shown as SEQ ID No.58 and SEQ ID No.59, and a probe shown as SEQ ID No. 60;
or selected from primers and probes having at least 70%, 80%, 90%, 95% or 99% sequence identity to a plurality of consecutive nucleotides of the sequence.
In some embodiments, the primers and probes are:
the primers and probes for SEQ ID NO.1 are: primers shown as EQ ID No.7 and SEQ ID No.8, and a probe shown as SEQ ID No. 9;
and/or the primers and probes for SEQ ID No.2 are: primers shown as SEQ ID No.16 and SEQ ID No.17, and a probe shown as SEQ ID No. 18;
and/or the primers and probes for SEQ ID No.3 are: primers shown as SEQ ID No.28 and SEQ ID No.29, and a probe shown as SEQ ID No. 30;
and/or the primers and probes for SEQ ID No.4 are: primers shown as SEQ ID No.37 and SEQ ID No.38, and a probe shown as SEQ ID No. 39;
and/or the primers and probes for SEQ ID No.5 are: primers shown as SEQ ID No.43 and SEQ ID No.44, and a probe shown as SEQ ID No. 45;
and/or the primers and probes for SEQ ID No.6 are: primers shown as SEQ ID No.52 and SEQ ID No.53, and a probe shown as SEQ ID No. 54.
In some embodiments, the kit further comprises primers and probes for fluorescent quantitative PCR detection of the reference gene ACTB. The primers and probes for the reference gene ACTB are as follows: primers shown as SEQ ID No.61 and SEQ ID No.62, and a probe shown as SEQ ID No. 63.
The invention designs primers and probes for each marker of the DNA methylation molecular marker combination of the specific methylation region, and utilizes the amplification primers of the DNA methylation molecular marker combination to extract genomic DNA (gDNA) extracted from a respiratory tract sample and subjected to hydrogen sulfite treatment; and (3) carrying out multiplex fluorescence quantitative PCR detection on methylation signals of the detection region by utilizing a specific probe of the DNA methylation molecular marker, then establishing a benign and malignant prediction model by adopting a naive Bayes algorithm, and finally diagnosing benign and malignant of the lung nodule by the established model.
In some embodiments of the present invention, there is also provided a method for detecting methylation level of the above-described DNA methylation molecular marker combination, comprising the steps of:
(1) Extracting genomic DNA from a sample to be tested;
(2) Performing hydrogen sulfite treatment on the extracted genomic DNA to obtain converted DNA;
(3) And (3) carrying out multiplex fluorescence quantitative PCR detection on the transformation product obtained in the step (2) by using a probe aiming at the DNA methylation molecular marker.
In some of these embodiments, the multiplex fluorescent quantitative PCR reaction conditions are as follows:
Polymerase Activation：95℃，5min；
amplification I:95 ℃ for 15s; 10-20 cycles, 30S at 60-66 ℃;
Amplification II:95 ℃ for 15s; 40-60 cycles, 60-64 deg.C for 30s.
In some embodiments, the reference gene C in step (2) T The value is between 10 and 25, and the sample is judged to be a valid sample; otherwise, the sample is an invalid sample; then using reference gene C T Value C for each DNA methylation molecular marker in an effective sample T Correcting the value; if target DNA methylation molecular target C T Value of<50 determining that the DNA methylation molecular marker is detected to obtain the relative cycle number delta C of the target DNA methylation molecular marker T ：ΔC T Target DNA methylation molecular marker C T Value-reference gene C T A value; if target DNA methylation molecular target C T A value of "uncovered" indicates that the DNA methylation molecular marker is not detected and that ΔC is assigned thereto T ＝40。
In some embodiments, the step (3) is based on corrected ΔC T After the data analysis is carried out on the values, a Logistic Regression (Logistic Regression) algorithm is adopted to build a lung nodule benign and malignant prediction model. In the model construction process, a Cross-validation method is used, a data set is randomly divided into 3 equal parts, any 2 parts of the data set are taken and combined to serve as a training set, the rest 1 part of the data set serves as a test set, 3 different training-test set combinations can be obtained according to a combination principle for any one 3 equal part of the data set, a good and malignant prediction model is built according to combinations containing different DNA methylation molecular markers in the training set by adopting a logistic regression algorithm, and the classification capability of the model is evaluated in the test set containing the specific DNA methylation molecular marker combinations. 100 random and independent tests were performed according to the above procedure, and the classification ability of the model finally containing the specific DNA methylation molecular marker was determined by the average classification ability of 100 models.
The invention is further illustrated by the following examples, which are not intended to limit the scope of the invention.
Example 1
The DNA methylation molecular markers for detecting benign and malignant lung nodules provided by the invention are detection primers and probes for DNA methylation molecular markers 1 to Marker6 of 6 methylation regions in 4 detection genes HOXB4, PTGER4, LHX9 and ZCAN 31, the detection region sequences and sequence numbers of the DNA methylation molecular markers are specifically shown in table 1 (wherein the underlined part of each region is the Marker of the corresponding sequence of the amplified fragment of the primer preferably used in the following embodiments of the invention):
TABLE 1 molecular marker detection region sequence
The reagent kit designs three pairs of primers and three probes for specific methylation sites in Marker1 to Marker5 of 6 molecular markers for detecting benign and malignant lung nodules of respiratory tract samples (the fluorescent markers of the probes can adopt fluorescent group markers such as FAM, VIC and NED) and marks as combinations 1, 2 and 3 respectively. Wherein, the selected primer and probe combination in each molecular marker can be arbitrarily selected to be combined with the primer and probe combinations 1, 2 and 3 in other molecular markers and detected on the same platform. Specific primer and probe sequences corresponding to each molecular marker are shown in table 2:
TABLE 2 primer and probe sequences for related molecular markers
In both the examples and the following examples, the primer and probe combinations preferably used are as follows: primer and probe combination 1 for Marker1, primer and probe combination 1 for Marker2, primer and probe combination 2 for Marker3, primer and probe combination 2 for Marker4, primer and probe combination 1 for Marker5, primer and probe combination 1 for Marker 6.
The kit also comprises primers and probes of an internal reference gene ACTB, and the sequences of the primers and probes are shown in Table 3:
TABLE 3 reference gene ACTB primer and probe
Example 2
The methylation levels of markers 1 through 6 in the respiratory tract samples were tested using the kit described in example 1.
A method for detecting the methylation level of a DNA methylation molecular marker, comprising the steps of:
1. extraction of gDNA in the respiratory tract sample:
1) Extracting gDNA of a respiratory tract liquid sample: firstly, carrying out low-speed centrifugation on a respiratory tract liquid sample, wherein the temperature is 4 ℃, and the time is 5000g and 5min; removing supernatant and collecting precipitate; then according to Qiagen companyBlood&Extracting gDNA by performing an operation according to the Tissue Kit instruction;
2) If the gDNA extraction of the paraffin section sample of the lung tissue is carried out, the following method can be adopted: specific procedures for paraffin tissue gDNA extraction were performed according to the ALLPrep DNA/RNA FFPE Kit instructions from Qiagen.
2. Sulfite conversion of extracted gDNA
The extracted gDNA is subjected to hydrogen sulfite conversion, 40-80ng of gDNA is added, and the actual embodiment is preferably 50ng, and the operation is carried out according to the description of the Zymo DNA methyl-Direct MagPrep, so that unmethylated cytosine in the DNA is deaminated and converted into uracil, and methylated cytosine is kept unchanged.
3. Multiplex fluorescent quantitative PCR assay of conversion products
All DNA products after hydrogen sulfite conversion were used for multiplex fluorescent quantitative PCR. The fluorescent quantitative PCR reaction comprises the following components: primer probe mixtures, wherein each primer concentration is 100-500nM, in this embodiment 200nM; the probe concentration is 50-150nM, and 100nM is preferred in this embodiment. Each reaction was performed using a methyl Tect Taq HS PCR kit (Ai Kerui organism, cat#AG 11209) as a 25ul system.
The specific reaction conditions are as follows: pre-denaturation, 95 ℃ for 5min; amplification I:95 ℃ for 15s; 10-20 cycles, annealing at 60-66 ℃ for 30S, 15 cycles are preferred in this embodiment, 65 ℃; amplification II:95 ℃ for 15s; 40-60 cycles, annealing 60-64 ℃ for 30s, 50 cycles in the practical embodiment, 62 ℃. The qPCR fluorescent quantitative reaction system was formulated as in Table 5:
TABLE 5 qPCR fluorescent quantitative reaction System
The embodiment further provides a method for detecting benign and malignant lung nodules, which further comprises the following steps:
5. c of reference gene in sample according to fluorescent quantitative PCR reaction T Judging whether the detection sample is an effective sample, if so, detecting C of the reference gene in the sample T A value between 10 and 25, the sample is judged to be a valid sample; no detection or analysis was included for the initial plunge samples judged to be invalid samples.
6. If the target DNA methylation molecule target C is under the premise that the sample is judged to be an effective sample T Value of<50 determining that the DNA methylation molecular marker is detected to obtain the relative cycle number delta C of the target DNA methylation molecular marker T ：ΔC T Target DNA methylation molecular marker C T Value-reference gene C T A value; if target DNA methylation molecular target C T A value of "uncovered" indicates that the DNA methylation molecular marker is not detected and that ΔC is assigned thereto T ＝40。
7. And (3) carrying out data analysis according to the corrected delta CT value, and establishing a lung nodule benign and malignant prediction model by adopting a Logistic Regression (Logistic Regression) algorithm. In the model construction process, a Cross-validation method is used, a lung tissue sample and a lung respiratory tract liquid sample are combined, a data set is randomly divided into 3 equal parts, any 2 parts of the data set are combined to be used as a training set, the rest 1 part of the data set is used as a test set, 3 different training-test set combinations can be obtained according to a combination principle for any one of the 3 equal parts of the data set, a benign and malignant prediction model is built according to the combination containing different DNA methylation molecular markers in the training set by adopting a logistic regression algorithm, and the classification capability of the model is evaluated in the test set containing the specific DNA methylation molecular marker combination. 100 random and independent tests were performed according to the above procedure, and the classification ability of the model finally containing the specific DNA methylation molecular marker was determined by the average classification ability of 100 models.
Example 3
The embodiment provides a detection method of a molecular marker in a standard substance, which comprises the following detection steps:
1. standard preparation
1) Preparation of 0% methylation standard:
by REPLI-Single Cell Kit (Qiagen, cat# 150343) and Mung Bean nucleic (NEB, cat#M0250L) treated NA12878 DNA to prepare 0% methylation standard;
2) 100% methylation standard preparation:
treatment of the prepared 0% methylation standard with CpG methylation ferrase (M.SssI) gives 100% methylation standard.
2. Standard preparation of different methylation ratios:
the methylation standards were mixed in a gradient of 0% to 100% according to the desired methylation ratio to give a 0.2%,0.4% and 1% methylation ratio standard.
3. Standard DNA of different methylation ratios was subjected to hydrogen sulfite conversion: the procedure is as in example 2, with a conversion input of 40-80ng, preferably 50ng.
4. The converted standard DNA was subjected to fluorescent quantitative PCR assay, and the procedure was as in example 2.
5. C of reference gene ACTB in sample according to fluorescent quantitative PCR reaction T Judging whether the detection sample is an effective sample, if so, detecting C of the reference gene in the sample T A value of between 10 and 25, then Judging the sample as an effective sample;
6. if the target DNA methylation molecule target C is under the premise that the sample is judged to be an effective sample T Value of<50 determining that the DNA methylation molecular marker is detected, if the target DNA methylation molecular marker C T A value of "unlabeled" indicates that the DNA methylation molecular marker is not detected.
In the examples and examples below, the primer probe combinations for each molecular marker are as preferred in example 1.
In the examples and the following examples, a negative control was set up for each test, and the negative control was subjected to fluorescent quantitative PCR measurement of each specific molecular marker using water as a template. If the negative control has no detection signal, the whole experimental operation is judged to have no exogenous pollution.
In this example, 3 completely independent replicates were performed, with 6 molecular markers each having a detection signal in the 100% methylation standard, and no detection signal in both the negative control and the unmethylated detection standard. In each standard with the methylation ratio of more than or equal to 0.2%, all the three tests of Marker5 and Marker6 have detection signals, which shows that the detection rate of the molecular markers on samples with the methylation ratio of more than or equal to 0.2% reaches 100%; the 6 molecular markers all have detection signals in three tests in the standard with the methylation proportion of 1%, which indicates that the molecular markers can detect the signals with the methylation proportion of 1%, and the signals are shown in the table 6:
TABLE 6 test results of molecular markers in respective methylation proportion standards
Example 4 detection of molecular markers on samples of benign and malignant Lung nodule tissue
The experimental example detects 6 molecular markers in 134 lung tissue samples respectively. Among them, the samples were determined as benign samples by surgical biopsy in 57 cases, and malignant samples in 77 cases, wherein the malignant samples include 66 cases of phase I samples, 4 cases of phase II samples, and 5 cases of phase III samples. Specific test kits and assay methods and data determination processes are described in example 2. The primer and probe combination is the preferred combination as in example 1.
Single molecular markers were detected in lung tissue samples at markers 1, 2, 3, 4, 5, 6. The 6 molecular markers have high correlation with lung cancer, and the single detection performance in lung tissue samples is shown in table 7, wherein each Marker has an individual AUC ranging from 0.77 to 0.92. The detection results are shown in Table 7.
TABLE 7
The examples also tested combinations of 6 molecular markers, samples and test methods as described above.
The specific molecular marker combinations tested are shown in Table 8.
TABLE 8 combinations of different molecular markers
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The combination of molecular marker combination 1 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.91 (specificity: 91%; sensitivity: 82%), a sensitivity of 82% for stage I malignant samples, a stage II sensitivity of 100%, a stage III sensitivity of 100%, and ROC as shown in FIG. 1.
The combination of molecular marker combinations 2 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.91 (specificity: 96%; sensitivity: 75%), a sensitivity to stage I malignant samples of 74%, a stage II sensitivity of 100%, a stage III sensitivity of 100%, a stage IV sample sensitivity of 100%, and ROC as shown in FIG. 1.
The combination of molecular marker combinations 3 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.87 (specificity: 91%; sensitivity: 75%), a sensitivity of 74% for stage I malignant samples, a stage II sensitivity of 100%, a stage III sensitivity of 100%, and ROC as shown in FIG. 1.
The combination of molecular marker combinations 4 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.91 (specificity: 96%; sensitivity: 79%), a sensitivity to stage I malignancy of 79%, a stage II sensitivity of 100%, a stage III sensitivity of 100%, and ROC as shown in FIG. 1.
The combination of molecular marker combinations 5 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.91 (specificity: 91%; sensitivity: 82%), a sensitivity of 82% for stage I malignant samples, a stage II sensitivity of 100%, a stage III sensitivity of 100%, and ROC as shown in FIG. 1. The performance of the combination is similar to that of the combination 1, but Marker1 is less than that of the combination 1, which indicates that the performance of the combination of 6 molecular markers can be achieved by the 5 molecular markers.
The combination of molecular marker combinations 6 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.91 (specificity: 96%; sensitivity: 75%), a sensitivity to stage I malignancy of 74%, a stage II sensitivity of 100%, a stage III sensitivity of 100%, and ROC as shown in FIG. 1. The combination and the combination 1 only differ from marker2, and the combination 1 containing marker2 has better performance than the combination without marker 1.
The combination of molecular marker combinations 7 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.90 (specificity: 96%; sensitivity: 78%), a sensitivity to stage I malignancy of 77%, a stage II sensitivity of 100%, a stage III sensitivity of 100%, and ROC as shown in FIG. 1. The combination and the combination 1 only differ from marker6, and the performance of the combination 1 containing marker6 is better than that of the combination without marker 6.
The combination of molecular marker combinations 8 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.91 (specificity: 95%; sensitivity: 81%), a sensitivity of 80% for stage I malignant samples, a stage II sensitivity of 100%, a stage III sensitivity of 100%, and ROC as shown in FIG. 1. The combination and the combination 1 only differ from marker5, and the sensitivity of the combination 1 containing the marker5 to the malignancy in the I phase is slightly better than that of the combination without the marker1, but the overall performance of the combination is similar.
The combination of molecular marker combinations 9 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.92 (specificity: 96%; sensitivity: 81%), a sensitivity to stage I malignancy of 80%, a stage II sensitivity of 100%, and a stage III sensitivity of 100%.
The combination of molecular marker combinations 10 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.92 (specificity: 96%; sensitivity: 81%), a sensitivity to stage I malignancy of 80%, a stage II sensitivity of 100%, and a stage III sensitivity of 100%.
The combination of molecular marker combination 11 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.88 (specificity: 81%; sensitivity: 86%), 86% sensitivity to stage I malignant samples, 100% sensitivity II, 100% sensitivity III.
The combination of molecular marker combinations 12 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.81 (specificity: 82%; sensitivity: 75%), a sensitivity to stage I malignancy of 74%, a stage II sensitivity of 100% and a stage III sensitivity of 100%.
The combination of molecular marker combinations 13 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.79 (specificity: 82%; sensitivity: 75%), a sensitivity to stage I malignancy of 75%, a stage II sensitivity of 100% and a stage III sensitivity of 100%.
The combination of molecular marker combinations 14 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.82 (specificity: 79%; sensitivity: 81%), a sensitivity of 80% for stage I malignant samples, a sensitivity of 100% for stage II, and a sensitivity of 100% for stage III.
The combination of molecular marker combinations 15 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.91 (specificity: 96%; sensitivity: 79%), a sensitivity to stage I malignant samples of 79%, a stage II sensitivity of 100% and a stage III sensitivity of 100%.
The combination of molecular marker combinations 16 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.81 (specificity: 75%; sensitivity: 81%), a sensitivity to stage I malignancy of 80%, a stage II sensitivity of 100% and a stage III sensitivity of 100%.
The combination of molecular marker combinations 17 was selected for modeling analysis of lung nodule tissue samples with an average AUC of 0.92 (specificity: 96%; sensitivity: 81%), a sensitivity to stage I malignancy of 80%, a stage II sensitivity of 100% and a stage III sensitivity of 100%.
The ROC of the combination of the above markers is shown in fig. 1. From the above experimental results, it is known that the combined use of specific molecular markers may be due to the properties of the individual molecular markers. The overall AUC, SP or SN of the molecular marker combinations 1-2, 4-11, 14, 15 and 17 as described in the examples were higher than that of the marker alone. The molecular marker screened by the invention has better sensitivity and specificity when combined with a proper marker, and has potential value in clinical application.
Example 5 molecular markers and combination of manifestations of benign and malignant lung nodule detection on respiratory tract fluid samples
The experimental example detects the combination of 6 molecular markers in 173 respiratory tract liquid samples. The samples are 65 samples and 108 samples, wherein the samples comprise 33 samples of phase I, 4 samples of phase II, 4 samples of phase III, 10 samples of IV and 57 samples of other malignant samples. Specific test kits and assay methods and data determination processes are described in example 2. The primer and probe combination is the preferred combination as in example 1.
Single molecular markers are detected on markers 1, 2, 3, 4, 5 and 6 in the respiratory tract liquid sample. The 6 molecular markers have a high correlation with lung cancer, and their single detection in a respiratory fluid sample is shown in Table 7, wherein each Marker alone has an AUC ranging from 0.66 to 0.79 (< 0.8). The detection results are shown in Table 9.
TABLE 9
The combination of 6 molecular markers was also tested in the examples, and the sample and test methods and data analysis methods set up as described in example 2.
The specific molecular marker combinations tested are shown in Table 8.
The combination of molecular marker sets 1 was selected for modeling analysis on respiratory tract liquid samples with an average AUC of 0.84 (specificity: 80%; sensitivity: 77%), a sensitivity to stage I malignant samples of 68%, a stage II sensitivity of 75%, a stage III sensitivity of 100%, a stage IV sample sensitivity of 88%, and ROC as shown in FIG. 2.
The combination of molecular marker combinations 2 was selected for modeling analysis of respiratory tract liquid samples with an average AUC of 0.80 (specificity: 83%; sensitivity: 60%), a sensitivity to stage I malignant samples of 42%, a stage II sensitivity of 50%, a stage III sensitivity of 100%, a stage IV sample sensitivity of 81%, and ROC as shown in FIG. 2.
The combination of molecular marker combinations 3 was selected for modeling analysis on respiratory tract liquid samples with an average AUC of 0.77 (specificity: 71%; sensitivity: 75%), a sensitivity to stage I malignant samples of 58%, a stage II sensitivity of 100%, a stage III sensitivity of 88%, a stage IV sample sensitivity of 88%, and ROC as shown in FIG. 2.
The combination of molecular marker combinations 4 was selected for modeling analysis on respiratory tract liquid samples with an average AUC of 0.81 (specificity: 69%; sensitivity: 81%), a sensitivity to stage I malignant samples of 74%, a stage II sensitivity of 75%, a stage III sensitivity of 88%, a stage IV sample sensitivity of 88%, and ROC as shown in FIG. 2.
The combination of molecular marker combinations 5 was selected for modeling analysis of respiratory tract liquid samples with an average AUC of 0.83 (specificity: 82%; sensitivity: 71%), a sensitivity to stage I malignant samples of 47%, a stage II sensitivity of 100%, a stage III sensitivity of 100%, a stage IV sample sensitivity of 88%, and ROC as shown in FIG. 2.
The combination of molecular marker combinations 6 was selected for modeling analysis of respiratory tract liquid samples with an average AUC of 0.80 (specificity: 88%; sensitivity: 61%), 45% for stage I malignant samples, 50% for stage II, 100% for stage III, 79% for stage IV, and ROC as shown in FIG. 2.
The combination of molecular marker combination 7 was selected for modeling analysis of respiratory tract liquid samples with an average AUC of 0.82 (specificity: 82%; sensitivity: 71%), a sensitivity to stage I malignant samples of 58%, a stage II sensitivity of 75%, a stage III sensitivity of 88%, a stage IV sample sensitivity of 86%, and a ROC as shown in FIG. 2
The combination of molecular marker combinations 8 was selected for modeling analysis of respiratory tract fluid samples with an average AUC of 0.84 (specificity: 86%; sensitivity: 68%), a sensitivity to phase I malignant samples of 53%, a phase II sensitivity of 50%, a phase III sensitivity of 88%, a phase IV sample sensitivity of 83%, and a ROC as shown in FIG. 2.
The combination of molecular marker combinations 9 was selected for modeling analysis of respiratory tract liquid samples with an average AUC of 0.80 (specificity: 82%; sensitivity: 70%), a sensitivity to stage I malignant samples of 50%, a stage II sensitivity of 75%, a stage III sensitivity of 100%, a stage IV sample sensitivity of 86%, and a ROC as shown in FIG. 2.
The combination of molecular marker combinations 10 was selected for modeling analysis on respiratory tract liquid samples with an average AUC of 0.81 (specificity: 71%; sensitivity: 80%), a sensitivity to phase I malignant samples of 66%, a phase II sensitivity of 50%, a phase III sensitivity of 100%, and a phase IV sample sensitivity of 93%.
The combination of the molecular marker combination 11 was selected for modeling analysis on a respiratory tract liquid sample, the average AUC was 0.79 (specificity: 55%; sensitivity: 85%), the sensitivity to phase I malignant samples was 71%, phase II sensitivity was 100%, phase III sensitivity was 88%, and phase IV sample sensitivity was 95%.
The combination of molecular marker combinations 12 was selected for modeling analysis on respiratory tract liquid samples with an average AUC of 0.78 (specificity: 54%; sensitivity: 91%), a sensitivity to phase I malignant samples of 87%, a phase II sensitivity of 100%, a phase III sensitivity of 88%, and a phase IV sample sensitivity of 95%.
The combination of molecular marker combination 13 was selected for modeling analysis on respiratory tract liquid samples with an average AUC of 0.67 (specificity: 86%; sensitivity: 57%), a sensitivity to stage I malignant samples of 63%, a stage II sensitivity of 75%, a stage III sensitivity of 75%, and a stage IV sample sensitivity of 79%.
The combination of molecular marker combinations 14 was selected for modeling analysis on respiratory tract liquid samples with an average AUC of 0.73 (specificity: 86%; sensitivity: 60%), a sensitivity to stage I malignant samples of 39%, a stage II sensitivity of 75%, a stage III sensitivity of 75%, and a stage IV sample sensitivity of 81%.
The combination of molecular marker combinations 15 was selected for modeling analysis on respiratory tract liquid samples with an average AUC of 0.82 (specificity: 68%; sensitivity: 81%), a sensitivity to stage I malignant samples of 74%, a stage II sensitivity of 75%, a stage III sensitivity of 88%, and a stage IV sample sensitivity of 88%.
The combination of molecular marker combinations 16 was selected for modeling analysis on respiratory tract fluid samples with an average AUC of 0.79 (specificity: 52%; sensitivity: 93%), a sensitivity to phase I malignant samples of 87%, a phase II sensitivity of 100%, a phase III sensitivity of 88%, and a phase IV sample sensitivity of 93%.
The combination of molecular marker combinations 17 was selected for modeling analysis on respiratory tract liquid samples with an average AUC of 0.84 (specificity: 60%; sensitivity: 90%), a sensitivity to stage I malignant samples of 79%, a stage II sensitivity of 100%, a stage III sensitivity of 100%, and a stage IV sample sensitivity of 95%.
The ROC of the above marker combinations is shown in fig. 2. From the above experimental results, it is known that the combined use of specific molecular markers may be due to the properties of the individual molecular markers. The combination of molecular markers 1-11, and 15-17 as described in the examples, used in combination with multiple markers, all had higher overall performance (e.g., AUC, SP or SN) than when used with markers alone. The molecular marker screened by the invention has better sensitivity and specificity when combined with a proper marker, and has potential value in clinical application.
Example 6 molecular markers and combinations for the performance of Lung nodule benign and malignant detection on independently validated fluid samples of the collecting respiratory tract
The experimental example detects the combination of 6 molecular markers in 61 naturally-in-set aspiration channel liquid samples. Among them, the surgical biopsy was determined to be benign samples in 19 cases and malignant samples in 42 cases (the stage of malignant samples is unknown). Specific test kits and assay methods and data determination processes are described in example 2. The primer and probe combination is the preferred combination as in example 1.
Single molecular markers are detected on markers 1, 2, 3, 4, 5 and 6 in the respiratory tract liquid sample. The individual detection of the 6 molecular markers in the respiratory fluid sample is shown in table 10, wherein the AUC range for each Marker alone is 0.75-0.86 (< 0.87).
The test results are shown in Table 10.
Table 10
The combination of 6 molecular markers was also tested in the examples, and the sample and test methods and data analysis methods set up as described in example 2.
The specific molecular marker combinations tested are shown in Table 8.
The combination of molecular marker sets 1 was selected for modeling analysis on a fluid sample of the respiratory tract with an average AUC of 0.919 (specificity: 94.4%; sensitivity: 79.5%) and ROC as shown in FIG. 3.
The combination of molecular marker combinations 7 was selected for modeling analysis on a fluid sample of the respiratory tract with an average AUC of 0.915 (specificity: 94.4%; sensitivity: 72.7%) and ROC as shown in FIG. 3.
The combination of molecular marker combinations 10 was selected for modeling analysis on a fluid sample of the respiratory tract with an average AUC of 0.878 (specificity: 94.4%; sensitivity: 72.7%) and ROC as shown in FIG. 3.
The combination of molecular marker combinations 11 was selected for modeling analysis on a fluid sample of the respiratory tract with an average AUC of 0.876 (specificity: 91.4%; sensitivity: 70.5%) and ROC as shown in FIG. 3.
From the above experimental results, the combined use of specific molecular markers is superior to the performance of the molecular markers alone. The molecular marker combinations 1,7, 10, 11 and 14 (see fig. 3) as described in the examples. The overall performance (such as AUC, SP or SN) of the multi-marker combined use is higher than that of the single marker. The AUC of the combination of multiple markers is higher than that of the single marker. At high specificity (SP > 90), the sensitivity of the combination 1,7, 10 and 14 was higher than that of single marker alone; the overall performance of combination 1 is better than that of combination 7, with the sensitivity improved by 6% over combination 7 while maintaining high specificity (SP > 90). The combination 10, 11 and 14 also achieves better performance than a single marker in the case of a combination of only two markers. The main manifestation is an increase in AUC and sensitivity, and the specificity of the 2Marker combination model at the same sensitivity level (SN on the order of 75 left) is due to the use of Marker5 alone. The molecular marker screened by the invention has better sensitivity and specificity when combined with a proper marker, and has potential value in clinical application.
The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it is possible for a person skilled in the art to make several variants and modifications without departing from the inventive concept, which fall within the scope of protection of the present invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.
Claims (24)
1. A DNA methylation molecular marker useful for detecting benign and malignant pulmonary nodules, wherein the DNA methylation molecular marker comprises SEQ ID No.6 or its full complement, or a contiguous fragment of at least 55% of the full length of SEQ ID No.6 or its full complement.
2. The DNA methylation molecular marker of claim 1, further comprising at least one selected from the group consisting of the sequences shown in SEQ ID No.1 to SEQ ID No.5 or the complete complement thereof; or at least one continuous fragment selected from the sequences shown in SEQ ID No.1 to SEQ ID No.5 or at least 55% of the full length of the complete complement thereof.
3. The DNA methylation molecular marker of claim 2, wherein the DNA methylation molecular marker comprises SEQ ID No.6 and SEQ ID No.4 or comprises the complete complementary sequences of SEQ ID No.6 and SEQ ID No. 4.
Or a continuous fragment comprising at least 55% of the full length of each of the sequences described above.
4. A DNA methylation molecular marker useful for detecting benign and malignant lung nodules according to claim 3, wherein said DNA methylation molecular marker further comprises SEQ ID No.2 or its complete complement;
or further comprises a continuous fragment of at least 55% of the total length of each of the sequences.
5. A DNA methylation molecular marker useful for detecting benign and malignant lung nodules according to claim 1 or 3, wherein said DNA methylation molecular marker further comprises SEQ ID No.5 or its complete complement;
or further comprises a continuous fragment of at least 55% of the total length of each of the sequences.
6. The DNA methylation molecular marker of claim 5, further comprising SEQ ID No.2 and/or SEQ ID No.3, or a complete complement thereof;
or further comprises a continuous fragment of at least 55% of the total length of each of the sequences.
7. The DNA methylation molecular marker of claim 5, further comprising SEQ ID No.1 and/or SEQ ID No.3, or a complete complement thereof;
Or further comprises a continuous fragment of at least 55% of the total length of each of the sequences.
8. The DNA methylation molecular marker of claim 2, wherein the DNA methylation molecular marker comprises the sequences shown in SEQ ID No.6 and SEQ ID No.1 or the complete complements thereof; or (b)
The DNA methylation molecular marker comprises sequences shown in SEQ ID NO.6 and SEQ ID NO.2 or complete complementary sequences thereof; or (b)
The DNA methylation molecular marker comprises sequences shown in SEQ ID NO.6, SEQ ID NO.1 and SEQ ID NO.2 or complete complementary sequences thereof; or (b)
The DNA methylation molecular marker comprises sequences shown in SEQ ID NO.6 and SEQ ID NO.3 or complete complementary sequences thereof; or (b)
The DNA methylation molecular marker comprises sequences shown in SEQ ID NO.6 and SEQ ID NO.5 or complete complementary sequences thereof;
or a continuous fragment comprising at least 55% of the full length of each of the sequences described above.
9. The DNA methylation molecular marker of claim 2, wherein the DNA methylation molecular marker comprises SEQ ID No.6, SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4, or comprises the complete complementary sequences of SEQ ID No.6, SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No. 4;
Or a continuous fragment comprising at least 55% of the full length of each of the sequences described above.
10. The DNA methylation molecular marker of claim 2, wherein the DNA methylation molecular marker comprises SEQ ID No.6, SEQ ID No.1, SEQ ID No.3, SEQ ID No.4 and SEQ ID No.5, or comprises the complete complementary sequences of SEQ ID No.6, SEQ ID No.1, SEQ ID No.3, SEQ ID No.4 and SEQ ID No. 5;
or a continuous fragment comprising at least 55% of the full length of each of the sequences described above.
11. The DNA methylation molecular marker of claim 2, wherein the DNA methylation molecular marker comprises SEQ ID No.1 to SEQ ID No.6 or comprises the complete complement of SEQ ID No.1 to SEQ ID No. 6;
or a continuous fragment comprising at least 55% of the full length of each of the sequences described above.
12. A DNA methylation molecular marker for detecting benign and malignant lung nodules, which is characterized by comprising sequences shown in SEQ ID NO.2 and SEQ ID NO.4 or complete complementary sequences thereof; or (b)
The DNA methylation molecular marker comprises sequences shown in SEQ ID NO.1 to SEQ ID NO.4 or complete complementary sequences thereof;
The DNA methylation molecular marker comprises sequences shown in SEQ ID NO.1 to SEQ ID NO.5 or complete complementary sequences thereof;
the DNA methylation molecular marker comprises sequences shown in SEQ ID NO.3 and SEQ ID NO.6 or complete complementary sequences thereof;
or a continuous fragment comprising at least 55% of the full length of each of the sequences described above.
13. A DNA methylation molecular marker useful for detecting benign and malignant lung nodules according to any one of claim 1 to 12,
the DNA methylation molecular markers are continuous fragments with at least 55% of the total length of sequences shown in SEQ ID NO. 1-SEQ ID NO.6, and the continuous fragments are respectively:
the primers are SEQ ID NO.7 and SEQ ID NO.8, SEQ ID NO.10 and SEQ ID NO.11, SEQ ID NO.13 and SEQ ID NO.14, and the sequence of the amplified fragment in any group of SEQ ID NO. 1;
and/or the sequence of the amplified fragment of any one group of SEQ ID NO.16 and SEQ ID NO.17, SEQ ID NO.19 and SEQ ID NO.20, SEQ ID NO.22 and SEQ ID NO.23 in SEQ ID NO. 2;
and/or the sequence of the amplified fragment of any one group of SEQ ID NO.25 and SEQ ID NO.26, SEQ ID NO.28 and SEQ ID NO.29, SEQ ID NO.31 and SEQ ID NO.32 in SEQ ID NO. 3;
And/or the sequence of the amplified fragment of any one group of SEQ ID NO.34 and SEQ ID NO.35, SEQ ID NO.37 and SEQ ID NO.38, SEQ ID NO.409 and SEQ ID NO.41 in SEQ ID NO. 4;
and/or the sequence of the amplified fragment of any one group of SEQ ID NO.43 and SEQ ID NO.44, SEQ ID NO.46 and SEQ ID NO.47, SEQ ID NO.49 and SEQ ID NO.50 in SEQ ID NO. 5;
and/or the sequence of the amplified fragment of any one group of SEQ ID NO.52 and SEQ ID NO.53, SEQ ID NO.55 and SEQ ID NO.56, SEQ ID NO.58 and SEQ ID NO.59 in SEQ ID NO. 6.
14. The combination of DNA methylation molecular markers useful for detecting benign and malignant lung nodules of claim 13,
the DNA methylation molecular markers are continuous fragments with at least 55% of the total length of sequences shown in SEQ ID NO. 1-SEQ ID NO.6, and the continuous fragments are respectively:
the primers are the sequences corresponding to the fragments amplified by SEQ ID NO.7 and SEQ ID NO.8 in SEQ ID NO. 1;
and/or the sequences of the amplified fragments of SEQ ID NO.16 and SEQ ID NO.17 in SEQ ID NO. 2;
and/or the sequences of the amplified fragments of SEQ ID NO.28 and SEQ ID NO.29 in SEQ ID NO. 3;
And/or the sequences of the amplified fragments of SEQ ID NO.37 and SEQ ID NO.38 in SEQ ID NO. 4;
and/or the sequences of the amplified fragments of SEQ ID NO.43 and SEQ ID NO.44 in SEQ ID NO. 5;
and/or the sequences of the amplified fragments of SEQ ID NO.52 and SEQ ID NO.53 in SEQ ID NO. 6.
15. The DNA methylation molecular marker of claims 1 to 14, wherein the DNA methylation molecular marker is a molecular marker for a respiratory tract sample, preferably a lung tissue sample, or a respiratory tract fluid sample.
16. Use of a DNA methylation molecular marker according to any one of claims 1 to 15 and/or a reagent for detecting the methylation level thereof for the preparation of a kit for detecting benign and malignant lung nodules, and/or lung cancer.
17. A kit for detecting benign and malignant pulmonary nodules, comprising reagents for detecting the methylation level of the DNA methylation molecular marker of any one of claims 1 to 13.
18. The kit for detecting benign and malignant lung nodules of claim 17, comprising reagents for use in PCR amplification, fluorescent quantitative PCR, digital PCR, liquid chip, substituted sequencing, third generation sequencing, pyrosequencing, bisulfite conversion sequencing, methylation chip, simplified hydrogen sulfite sequencing, or a combination thereof.
19. The kit for detecting benign and malignant lung nodules of claim 18, wherein the reagent includes primer and probe for fluorescent quantitative PCR detection of DNA methylation molecular marker:
the primers and probes for SEQ ID NO.1 are selected from at least one of the following groups: primers shown as SEQ ID NO.7 and SEQ ID NO.8, and a probe shown as SEQ ID NO. 9; primers shown as SEQ ID NO.10 and SEQ ID NO.11, and a probe shown as SEQ ID NO. 12; primers shown as SEQ ID NO.13 and SEQ ID NO.14, and a probe shown as SEQ ID NO. 15;
and/or the primers and probes directed against SEQ ID No.2 are selected from at least one of the following groups: primers shown as SEQ ID No.16 and SEQ ID No.17, and a probe shown as SEQ ID No. 18; primers shown as SEQ ID NO.19 and SEQ ID NO.20, and a probe shown as SEQ ID NO. 21; primers shown as SEQ ID No.22 and SEQ ID No.23, and a probe shown as SEQ ID No. 24;
and/or the primers and probes directed against SEQ ID No.3 are selected from at least one of the following groups: primers shown as SEQ ID No.25 and SEQ ID No.26, and a probe shown as SEQ ID No. 27; primers shown as SEQ ID No.28 and SEQ ID No.29, and a probe shown as SEQ ID No. 30; primers shown as SEQ ID NO.31 and SEQ ID NO.32, and a probe shown as SEQ ID NO. 33;
And/or the primers and probes directed against SEQ ID No.4 are selected from at least one of the following groups: primers shown as SEQ ID No.34 and SEQ ID No.35, and a probe shown as SEQ ID No. 36; primers shown as SEQ ID No.37 and SEQ ID No.38, and a probe shown as SEQ ID No. 39; primers shown as SEQ ID No.40 and SEQ ID No.41, and a probe shown as SEQ ID No. 42;
and/or the primers and probes directed against SEQ ID No.5 are selected from at least one of the following groups: primers shown as SEQ ID No.43 and SEQ ID No.44, and a probe shown as SEQ ID No. 45; primers shown as SEQ ID No.46 and SEQ ID No.47, and a probe shown as SEQ ID No. 48; primers shown as SEQ ID NO.49 and SEQ ID NO.50, and a probe shown as SEQ ID NO. 51;
and/or the primers and probes directed against SEQ ID No.6 are selected from at least one of the following groups: primers shown as SEQ ID NO.52 and SEQ ID NO.53, and a probe shown as SEQ ID NO. 54; primers shown as SEQ ID No.55 and SEQ ID No.56, and a probe shown as SEQ ID No. 57; primers shown as SEQ ID No.58 and SEQ ID No.59, and a probe shown as SEQ ID No. 60;
or selected from primers and probes having at least 70%, 80%, 90%, 95% or 99% sequence identity to a plurality of consecutive nucleotides of the sequence.
20. The kit for detecting benign and malignant lung nodules of claim 19, wherein the primer and probe are:
the primers and probes for SEQ ID NO.1 are: primers shown as EQ ID No.7 and SEQ ID No.8, and a probe shown as SEQ ID No. 9;
and/or the primers and probes for SEQ ID No.2 are: primers shown as SEQ ID No.16 and SEQ ID No.17, and a probe shown as SEQ ID No. 18;
and/or the primers and probes for SEQ ID No.3 are: primers shown as SEQ ID No.28 and SEQ ID No.29, and a probe shown as SEQ ID No. 30;
and/or the primers and probes for SEQ ID No.4 are: primers shown as SEQ ID No.37 and SEQ ID No.38, and a probe shown as SEQ ID No. 39;
and/or the primers and probes for SEQ ID No.5 are: primers shown as SEQ ID No.43 and SEQ ID No.44, and a probe shown as SEQ ID No. 45;
and/or the primers and probes for SEQ ID No.6 are: primers shown as SEQ ID No.52 and SEQ ID No.53, and a probe shown as SEQ ID No. 54.
21. Kit for detecting benign and malignant lung nodules according to any of claims 17 to 20, wherein the kit further comprises primers and probes for the fluorescent quantitative PCR detection of the internal reference gene ACTB.
22. The kit for detecting benign and malignant lung nodules according to claim 21, wherein the primer and probe for reference gene ACTB are: primers shown as SEQ ID No.61 and SEQ ID No.62, and a probe shown as SEQ ID No. 63.
23. A kit for detecting a benign and malignant lung nodule according to any one of claims 17 to 20, wherein the test sample of the kit is a respiratory tract sample, preferably a lung tissue sample, or a respiratory tract fluid sample.
24. A method for detecting benign and malignant lung nodules and/or lung cancer, which is characterized by comprising the following steps:
(1) Extracting genomic DNA from a sample to be tested;
(2) Performing hydrogen sulfite treatment on the extracted genomic DNA to obtain converted DNA;
(3) Detection with a kit according to any one of claims 17 to 21.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/CN2023/098075 WO2024001668A1 (en) | 2022-06-28 | 2023-06-02 | Methylation molecular marker for detecting benign and malignant pulmonary nodules and use thereof |
| US18/337,000 US20240084393A1 (en) | 2020-12-17 | 2023-06-17 | Dna methelation molecular markers for identifying benignity or malignancy of lung nodule and applications of the same |
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| CN202210753404 | 2022-06-28 | ||
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| JP6395131B2 (en) * | 2014-07-10 | 2018-09-26 | シスメックス株式会社 | Method for acquiring information on lung cancer, and marker and kit for acquiring information on lung cancer |
| EP3792363A4 (en) * | 2018-01-23 | 2022-01-12 | Excellen Medical Technology Co., Ltd. | Method and kit for identifying lung cancer status |
| CN109022567A (en) * | 2018-08-06 | 2018-12-18 | 北京艾克伦医疗科技有限公司 | For identifying kit and its application of Lung neoplasm and/or lung cancer status |
| CN111876478A (en) * | 2020-04-28 | 2020-11-03 | 中国科学院微生物研究所 | Pulmonary nodule diagnostic marker and application |
| CN111662980A (en) * | 2020-06-03 | 2020-09-15 | 广州市康立明生物科技有限责任公司 | Lung cancer detection reagent and kit |
| CN114645087B (en) * | 2020-12-17 | 2023-03-24 | 广州市基准医疗有限责任公司 | Methylated molecular markers for detecting benign and malignant lung nodules or combination and application thereof |
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