CN117467763A - Nucleic acid combination and kit for detecting methylation level of non-small cell lung cancer biomarker - Google Patents
Nucleic acid combination and kit for detecting methylation level of non-small cell lung cancer biomarker Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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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- C12Q2600/154—Methylation markers
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes
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Abstract
The invention belongs to the field of biological medicine, and in particular relates to a nucleic acid combination and a kit for detecting methylation level of a non-small cell lung cancer biomarker. The biomarker can be used for early detection of non-small cell lung cancer, can detect early non-small cell lung cancer by detecting the methylation level of polynucleotide molecules in a sample, has excellent detection sensitivity on both stage I and stage II non-small cell lung cancer, has the sensitivity of 85.7% for detecting a stage I non-small cell lung cancer blood sample, has the sensitivity of 93.8% for detecting a stage II non-small cell lung cancer blood sample, has the specificity of 93.7% for detecting a healthy human blood sample, and can effectively distinguish early non-small cell lung cancer patients from healthy human.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and in particular relates to a nucleic acid combination and a kit for detecting methylation level of a non-small cell lung cancer biomarker.
Background
Lung cancer is one of the most common malignant tumors in the world, with morbidity and mortality accounting for the first place among malignant tumors. Non-small cell lung cancer (NSCLC) accounts for about 80% of all lung cancers, including squamous cell carcinoma (squamous carcinoma), adenocarcinoma, large cell carcinoma. The incidence of non-small cell lung cancer is high and the survival rate of 5 years is low. The main reason for this is that early non-small cell lung cancer lacks typical clinical symptoms, and about 75% of non-small cell lung cancer patients are at middle and late stages when diagnosed, or have metastasized, so early diagnosis of non-small cell lung cancer is a key factor in reducing death and improving prognosis. Currently, common methods for lung cancer diagnosis include imaging examination, sputum shedding cytology and bronchoscopy, which are easy to cause missed diagnosis and misdiagnosis, and expensive in equipment.
Therefore, it is of great importance to explore early detection markers for non-small cell lung cancer. There is currently no effective biomarker that can be used for early detection of non-small cell lung cancer.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a nucleic acid combination and a kit for detecting the methylation level of a non-small cell lung cancer biomarker, so as to solve the technical problems that the detection rate of the non-small cell lung cancer biomarker in the prior art is low, and especially the biomarker for early detection of the non-small cell lung cancer (stage I and stage IIa) is lacking.
To achieve the above object, the present invention provides a nucleic acid combination for detecting methylation level of a non-small cell lung cancer biomarker selected from at least one of the polynucleotide molecules shown in (a) to (c):
(a) A full length or a partial region of the nucleotide sequence shown in SEQ ID No.1, said partial region comprising at least one CpG dinucleotide site; and/or the number of the groups of groups,
a full length or a partial region of the nucleotide sequence shown in SEQ ID No.2, said partial region comprising at least one CpG dinucleotide site;
(b) A polynucleotide molecule having more than 85% sequence identity to (a) wherein the CpG dinucleotide site is identical to (a);
(c) A polynucleotide molecule in which the CpG dinucleotide site of (a) or (b) is partially or fully methylated.
Preferably, the polynucleotide molecule is selected from at least one of the polynucleotide molecules shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 5.
Preferably, the nucleic acid combination comprises a pair of methylation primers for detecting the methylation level of the polynucleotide molecule.
Preferably, the methylation primer pair is selected from at least one of the first methylation primer pair of SEQ ID NOS.7-8, the second methylation primer pair of SEQ ID NOS.11-12, and the third methylation primer pair of SEQ ID NOS.15-16.
Preferably, the nucleic acid combination further comprises a pair of unmethylated primers for detecting the methylation level of the polynucleotide molecule.
Further preferably, the combination of nucleic acids comprises at least one set of the following combinations:
a first combination comprising a first pair of methylated primers shown in SEQ ID NOS.7-8 and a first pair of unmethylated primers shown in SEQ ID NOS.9-10;
a second combination comprising a second pair of methylated primers shown in SEQ ID NOS.11-12 and a second pair of unmethylated primers shown in SEQ ID NOS.13-14;
a third combination comprising a third pair of methylated primers shown in SEQ ID NOS.15-16 and a third pair of unmethylated primers shown in SEQ ID NOS.17-18.
Preferably, the nucleic acid combination further comprises a detection probe.
Further preferably, the combination of nucleic acids comprises at least one set of the following combinations:
a fourth combination comprising a first pair of methylation primers shown in SEQ ID NOS.7-8, and a first detection probe shown in SEQ ID NO. 19;
a fifth combination comprising a second pair of methylation primers shown in SEQ ID NOS.11-12, and a second detection probe shown in SEQ ID NO. 20;
a sixth combination comprising a third pair of methylation primers shown in SEQ ID NOS.15-16, and a third detection probe shown in SEQ ID NO. 21.
Preferably, the 5 'end of the detection probe comprises a fluorescence reporting group, and the 3' end comprises a fluorescence quenching group.
The invention also provides a kit for detecting the non-small cell lung cancer, which comprises the nucleic acid combination and other reagents, wherein the other reagents comprise one or more of a DNA extraction reagent, a DNA purification reagent, a methylation conversion reagent, an amplification reagent, a primer pair for amplifying an internal reference gene, and a probe and a quality control product corresponding to the primer pair.
The invention also provides application of the nucleic acid combination or the kit in preparation of non-small cell lung cancer diagnosis products.
Preferably, the non-small cell lung cancer comprises at least one of stage i non-small cell lung cancer and stage iia non-small cell lung cancer.
In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:
the nucleic acid combination and the kit for detecting the methylation level of the non-small cell lung cancer biomarker can detect early non-small cell lung cancer by detecting the methylation level of polynucleotide molecules in a sample, and provide the biomarker for early detection of non-small cell lung cancer. The kit provided by the invention has excellent detection sensitivity for the non-small cell lung cancer in the I stage and the IIa stage, the sensitivity for detecting the blood sample of the non-small cell lung cancer in the I stage can reach 85.7%, the sensitivity for detecting the blood sample of the non-small cell lung cancer in the IIa stage can reach 93.8%, the specificity for detecting the blood sample of a healthy person can reach 93.7%, and the kit can effectively distinguish early non-small cell lung cancer patients from healthy persons.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention.
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 herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
The term "diagnosis" refers to determining the health status of a subject, and encompasses detecting the presence or absence of a disease, responding to a therapeutic regimen, assessing risk of recurrence, assessing risk and extent of cancerous lesions, prognostic assays, and the like. In some cases, the term "diagnosis" refers to the use of a single factor in determining, validating or confirming a clinical state of a patient. In some embodiments, "detecting" non-small cell lung cancer refers to detecting the presence or absence of a disease, i.e., determining whether a subject has non-small cell lung cancer.
The term "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refers to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and typically more than ten. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotides may be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine and guanine.
The term "methylation" is a form of chemical modification of DNA that can alter genetic manifestations without altering the DNA sequence. DNA methylation refers to covalent binding of a methyl group at the 5 th carbon position of cytosine of a genomic CpG dinucleotide under the action of a DNA methyltransferase. DNA methylation can cause alterations in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby regulating gene expression.
The term "methylation level" refers to whether or not cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/proportion/percentage of methylation, representing both qualitative and quantitative concepts. In practical application, different detection indexes can be adopted to compare the DNA methylation level according to practical conditions. As in some cases, the comparison may be made based on Ct values detected by the sample; in some cases, the ratio of gene methylation in the sample, i.e., number of methylated molecules/(number of methylated molecules+number of unmethylated molecules). Times.100, can be calculated and then compared; in some cases, statistical analysis and integration of each index is also required to obtain a final decision index.
The term "biomarker" refers to a biochemical marker that can label changes in system, organ, tissue, cell and subcellular structure or function, or changes that may occur, such as proteins, DNA or RNA, etc., for a very broad range of uses. Biomarkers can be used for disease diagnosis, for judging disease stage or for evaluating the safety and effectiveness of new drugs or new therapies in a target population. Screening biomarkers for disease screening and early diagnosis can greatly improve the clinical treatment effect of patients.
The term "primer" refers to an oligonucleotide that can be used in an amplification method (e.g., polymerase chain reaction, PCR) to amplify a sequence of interest based on a polynucleotide sequence corresponding to a gene of interest or a portion thereof. Typically, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence specific for that polynucleotide sequence. The exact length of the primer will depend on many factors, including temperature, source of primer, and method used. For example, for diagnostic and prognostic applications, the oligonucleotide primers will typically contain at least 10, 15, 20, 25 or more nucleotides, but may also contain fewer nucleotides, depending on the complexity of the target sequence. In the present disclosure, the term "primer" refers to a pair of primers that hybridize to the double strand of a target DNA molecule or to regions of the target DNA molecule that flank the nucleotide sequence to be amplified. "primer pair" refers to a group of an upstream primer and a downstream primer.
The term "methylation-specific PCR" is one of the most sensitive experimental techniques currently studied for methylation, and a minimum of about 50pg of DNA methylation can be found. After the single-stranded DNA is subjected to bisulfite conversion, all unmethylated cytosines are deaminated to uracil, and methylated cytosines in CpG sites are kept unchanged, so that two pairs of primers aiming at methylated and unmethylated sequences are respectively designed, and the methylated and unmethylated DNA sequences can be distinguished through PCR amplification. In the present disclosure, methylation primers are added when performing real-time quantitative methylation-specific PCR, and if the Ct value meets the requirement (e.g., ct.ltoreq.38 in a tissue sample), it indicates that the target sequence is methylated.
The term "methylation specific fluorescent quantitative PCR (qMSP)" is an experimental technique combining fluorescent quantitative PCR technology and methylation specific PCR technology. In the technology, proper primer pairs are designed based on sequence differences of DNA in different methylation states after bisulfite conversion, so that methylated sequences and unmethylated sequences are distinguished, but the final detection index of the qMSP is a fluorescent signal, so that a fluorescent probe or a fluorescent dye is required to be added in addition to a methylation detection primer in a qMSP reaction system. Compared with the traditional methylation specific PCR technology, the qMSP detection DNA methylation level has higher sensitivity and specificity, is more suitable for detecting trace amounts of DNA fragments with abnormal methylation mixed in the DNA of patients in early cancer, does not need gel electrophoresis detection, and is simpler and more convenient to operate.
The term "TaqMan probe" refers to a stretch of oligonucleotide sequences comprising a 5 'fluorescent group and a 3' quenching group. When the probe binds to the corresponding site on the DNA, the probe does not fluoresce because of the presence of a quenching group near the fluorescent group. During amplification, if the probe binds to the amplified strand, the 5'-3' exonuclease activity of the DNA polymerase (e.g., taq enzyme) digests the probe and the fluorescent group is far from the quenching group, its energy is not absorbed, i.e., a fluorescent signal is generated. The fluorescence signal is also identical to the target fragment with a synchronous exponential increase per PCR cycle.
The invention provides a nucleic acid combination for detecting methylation level of a non-small cell lung cancer biomarker selected from at least one of the polynucleotide molecules shown in (a) - (c):
(a) A full length or a partial region of the nucleotide sequence shown in SEQ ID No.1, said partial region comprising at least one CpG dinucleotide site; and/or the number of the groups of groups,
a full length or a partial region of the nucleotide sequence shown in SEQ ID No.2, said partial region comprising at least one CpG dinucleotide site;
(b) A polynucleotide molecule having more than 85% sequence identity to (a) wherein the CpG dinucleotide site is identical to (a);
(c) A polynucleotide molecule in which the CpG dinucleotide site of (a) or (b) is partially or fully methylated.
In some embodiments, the polynucleotide molecule is selected from at least one of the polynucleotide molecules set forth in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO. 5.
In some embodiments, the nucleic acid combinations described above include methylation primer pairs for detecting the methylation level of the polynucleotide molecules described above.
In a preferred embodiment, the methylation primer pairs include at least one of the first methylation primer pairs of SEQ ID NOS.7-8, the second methylation primer pairs of SEQ ID NOS.11-12, and the third methylation primer pairs of SEQ ID NOS.15-16.
In some embodiments, the above nucleic acid combinations further comprise a pair of unmethylated primers for detecting the methylation level of the above polynucleotide molecule.
In a preferred embodiment, the unmethylated primer pair includes at least one of the first unmethylated primer pair shown in SEQ ID NOS.9-10, the second unmethylated primer pair shown in SEQ ID NOS.13-14, and the third unmethylated primer pair shown in SEQ ID NOS.17-18.
In a more preferred embodiment, the above combination of nucleic acids comprises at least one of the following combinations:
a first combination comprising a first pair of methylated primers shown in SEQ ID NOS.7-8 and a first pair of unmethylated primers shown in SEQ ID NOS.9-10;
a second combination comprising a second pair of methylated primers shown in SEQ ID NOS.11-12 and a second pair of unmethylated primers shown in SEQ ID NOS.13-14;
a third combination comprising a third pair of methylated primers shown in SEQ ID NOS.15-16 and a third pair of unmethylated primers shown in SEQ ID NOS.17-18.
It is to be noted that, if one primer pair has at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.) or more sequence identity with the nucleotide sequence indicated by the primer pair (first methylated primer pair, first unmethylated primer pair, second methylated primer pair, second unmethylated primer pair, third unmethylated primer pair) and the primer pair has a certain non-small cell lung cancer diagnosis function (the specificity or sensitivity is reduced, slightly increased, or greatly increased, etc.) as compared with the primer pair of the present application, the primer pair is also within the scope of the present invention.
In some embodiments, the above nucleic acid combinations further comprise a detection probe. In a preferred embodiment, the detection probe includes at least one of a first detection probe shown in SEQ ID NO.19, a second detection probe shown in SEQ ID NO.20, and a third detection probe shown in SEQ ID NO. 21.
In a more preferred embodiment, the above combination of nucleic acids comprises at least one of the following combinations:
a fourth combination comprising a first pair of methylation primers shown in SEQ ID NOS.7-8, and a first detection probe shown in SEQ ID NO. 19;
a fifth combination comprising a second pair of methylation primers shown in SEQ ID NOS.11-12, and a second detection probe shown in SEQ ID NO. 20;
a sixth combination comprising a third pair of methylation primers shown in SEQ ID NOS.15-16, and a third detection probe shown in SEQ ID NO. 21.
In some embodiments, the detection probe comprises a fluorescent reporter group at the 5 'end and a fluorescence quenching group at the 3' end.
The invention also provides a kit for detecting the non-small cell lung cancer, which comprises the nucleic acid combination and other reagents, wherein the other reagents comprise one or more of a DNA extraction reagent, a DNA purification reagent, a methylation conversion reagent, an amplification reagent, a primer pair for amplifying an internal reference gene, and a probe and a quality control product corresponding to the primer pair.
In some embodiments, the methylation conversion reagent is used to deaminate unmethylated cytosines in DNA to uracil while methylated cytosines remain unchanged. In some embodiments, the methylation conversion reagent is bisulfite or bisulfite.
In some embodiments, the amplification reagents include, but are not limited to, amplification buffers, dNTPs, DNA polymerase, and Mg 2+ One or more of the following.
In some embodiments, the reference gene may be but is not limited to ACTB, and the primer pair for amplifying the reference gene ACTB includes an upstream primer and a downstream primer, wherein the nucleotide sequence of the upstream primer is: 5'-AAGGTGGTTGGGTGGTTGTTTTG-3' (SEQ ID NO. 22), the nucleotide sequence of the downstream primer is: 5'-AATAACACCCCCACCCTGC-3' (SEQ ID NO. 23), the nucleotide sequence of the probe of the internal reference gene ACTB is: 5'-GGAGTGGTTTTTGGGTTTG-3' (SEQ ID NO. 24).
In some embodiments, the 5 'end of the detection probe and the probe of the reference gene comprises a fluorescence reporter group, the 3' end comprises a fluorescence quenching group, and the fluorescence reporter groups of the probes are independently selected from one or more of FAM, ROX, CY and VIC; the fluorescence quenching groups of the probes are independently selected from one or more of TAMRA, MGB, BHQ, BHQ, BHQ2 and BHQ 3. Examples of the fluorescent reporter group and the fluorescent quencher group of each probe each independently include, but are not limited to, those listed above.
In some embodiments, the quality control comprises a positive control and a negative control. The positive control refers to a biomarker comprising methylation therein for monitoring the detection performance of the reagents in the kit. The negative control mentioned above refers to a biomarker that does not contain methylation therein, and is used to monitor whether the experiment is contaminated.
In some embodiments, the above-described kits can also include any article of manufacture (e.g., packaging or container) of at least one device. The containers may include at least one vial, test tube, flask, bottle, syringe, and/or other container. The kit may further comprise suitable means for receiving a biological sample, instructions for carrying out the methods described herein or the steps thereof.
In some embodiments, the test samples of the above kit include, but are not limited to, plasma samples, serum samples, blood samples, and tissue samples.
Based on the present disclosure, one of ordinary skill in the art can detect the methylation level of the above-described non-small cell lung cancer biomarkers by any technique known in the art, and diagnosis of non-small cell lung cancer is within the scope of the present invention, regardless of the technique used.
The method for detecting methylation of non-small cell lung cancer biomarkers in a sample by using the kit is not particularly limited, and the method used can be but is not limited to methylation-sensitive random primer polymerase chain reaction (MS AP-PCR), methylation-sensitive single nucleotide primer extension (Ms-SNuPE), methylation-specific PCR (qMSP), methylation-sensitive DNA restriction enzyme analysis, restriction enzyme-based sequencing, restriction enzyme-based microarray analysis, joint bisulfite restriction analysis (COBRA), methylation CpG island amplification (MCA), methylation CpG island amplification and microarray (MCAM), hpaII small fragment enrichment (HELP) by ligation-mediated PCR, bisulfite sequencing, bisulfite microarray analysis, methylation-specific pyrophosphate sequencing, HELP sequencing (HELP-Seq), TET-assisted pyridine borane sequencing (TAPS), gal hydrolysis and ligation adapter-dependent PCR (GLAD-PCR), methylation DNA immunoprecipitation (MeP-Seq) or methylation DNA immunoprecipitation (MeP-Seq) or methylation DNA immunoprecipitation-amplification (MCA), and methylation-mediated DNA amplification (MCAM), and magneto-restriction enzyme-based magneto-sensitive microarray analysis (MCAM), and magneto-dependent magneto-restriction analysis using a magneto-resistive array.
The invention also provides application of the nucleic acid combination or the kit in preparation of non-small cell lung cancer detection products. In some embodiments, the non-small cell lung cancer detection product described above comprises at least one of a kit, a chip, and a sequencing library.
In some embodiments, the non-small cell lung cancer comprises at least one of stage i non-small cell lung cancer and stage iia non-small cell lung cancer.
The invention also provides application of the nucleic acid combination or the kit to detection of non-small cell lung cancer or detection of a subject with increased risk of non-small cell lung cancer, suspected of having non-small cell lung cancer or already having non-small cell lung cancer.
Based on the complexity of human disease diagnosis, the results obtained by the kit are only used as intermediate results of non-small cell lung cancer diagnosis or the possibility or risk of prompting the patient to suffer from the non-small cell lung cancer, and the conclusion of whether the patient suffers from the non-small cell lung cancer is finally obtained by combining the clinical manifestations and other physiological indexes of the individual.
The following describes the above technical scheme in detail with reference to specific embodiments.
Example 1 screening of detection regions for non-small cell lung cancer biomarkers based on DNA methylation
And (3) carrying out pyrophosphoric acid sequencing by taking 54 tissue samples of early non-small cell lung cancer patients and 54 tissue samples of paracancerous controls as training sets, analyzing the sequencing result, and selecting a detection area with sensitivity not less than 85% and specificity not less than 85% for next verification. And further verifying the screened differential methylation sites in 30 cases of early non-small cell lung cancer and 30 cases of healthy control blood sample test sets by utilizing a pyrosequencing method, and finally obtaining the biomarker with optimal early diagnosis efficacy on the early non-small cell lung cancer through screening verification. The specific process is as follows:
the non-small cell lung cancer biomarker provided by the invention is an isolated polynucleotide molecule, and the polynucleotide molecules are SEQ ID NO.1 and/or SEQ ID NO.2, SEQ ID NO.1 and SEQ ID NO.2 are located on the 199462477 ～ 199462642 th and 199470701 ～ 199470940 th bases of the negative strand of the 2 nd chromosome of SATB2 gene, and the nucleotide sequence of the polynucleotide molecule has multiple methylation sites, which occur at C-5 position of cytosine, and the product is called 5-methylcytosine (5-mC). SEQ ID NO.1 sequence has 12 methylation sites from 5'-3' and SEQ ID NO.2 sequence has 19 methylation sites from 5'-3', all of which have been usedThe identification is as follows:
SEQ ID NO.1 sequence (5 '-3'):
SEQ ID NO.2 sequence (5 '-3'):
TGCCAAGTGGGTGCTGCTC。
the "C" in the double strand of DNA is converted to "U" after the bisulfite or bisulfite treatment of the above polynucleotide molecule, and the "U" is converted to "T" by subsequent PCR, but the bisulfite does not allow the above conversion of the "C" of the DNA which has been methylated. Thus, the polynucleotide molecules obtained after bisulfite or bisulfite treatment of SEQ ID NO.1 include SEQ ID NO.3 (fully methylated sequence) and SEQ ID NO.4 (unmethylated sequence), and the polynucleotide molecules obtained after bisulfite or bisulfite treatment of SEQ ID NO.2 include SEQ ID NO.5 (fully methylated sequence) and SEQ ID NO.6 (unmethylated sequence). These polynucleotide molecules are used in the design of detection primers or detection kits.
When designing primers based on bisulfite treated polynucleotide molecules, the original DNA sequence is first entered and the program will show 2 sequences: one is the original DNA sequence that was input; the other is a sulfurized DNA sequence, all unmethylated "C" are converted to "T" except for 5 methylcytosine (5 mC) on CpG islands, and primers are designed based on the converted sequence.
Two pairs of primers need to be designed for MSP, one pair being for bisulfite treated methylated sequences; the other pair is directed to bisulfite treated unmethylated sequences. Depending on the fact that the methylated primer specifically amplifies only methylated sequences, the unmethylated primer specifically amplifies only unmethylated sequences. Primers were designed based on the polynucleotide molecular sequences SEQ ID No.3 and SEQ ID No.4, including the methylation primer pair shown in SEQ ID No.7 and SEQ ID No.8, and the unmethylation primer pair shown in SEQ ID No.9 and SEQ ID No.10, and the amplified product of the above-mentioned nucleic acid combinations (SEQ ID No. 7-8 and SEQ ID No. 9-10) contained CpG sites No. 03-11 in SEQ ID No. 1.
SEQ ID NO.3 sequence (5 '-3'):
SEQ ID NO.4 sequence (5 '-3'):
the sequences of the nucleic acid combinations (SEQ ID NOS.7 to 8 and SEQ ID NOS.9 to 10) are as follows:
methylation of the upstream primer: ATAGTTTTTATTTTCGGCGGTC (SEQ ID NO. 7);
methylation of the downstream primer: AACAAATCCCAACCCGCG (SEQ ID NO. 8);
unmethylated upstream primer: GCATAGTTTTTATTTTTGGTGGTT (SEQ ID NO. 9);
unmethylated downstream primer: CAAACAAATCCCAACCCACA (SEQ ID NO. 10).
Designing primers according to polynucleotide sequences SEQ ID NO.5 and SEQ ID NO.6, wherein the primers comprise a methylation primer pair shown in SEQ ID NO.11 and SEQ ID NO.12 and a non-methylation primer pair shown in SEQ ID NO.13 and SEQ ID NO. 14; and/or, a methylation primer pair as shown in SEQ ID NO.15 and SEQ ID NO.16, and a non-methylation primer pair as shown in SEQ ID NO.17 and SEQ ID NO. 18. The amplified products of the above nucleic acid combinations (SEQ ID NO. 11-12 and SEQ ID NO. 13-14) contain CpG sites of 03-12 in SEQ ID NO.2, and the amplified products of the above nucleic acid combinations (SEQ ID NO. 15-16 and SEQ ID NO. 17-18) contain CpG sites of 07-19 in SEQ ID NO. 2.
SEQ ID NO.5 sequence (5 '-3'):
SEQ ID NO.6 sequence (5 '-3'):
the sequences of the nucleic acid combinations (SEQ ID NOS.11 to 12 and SEQ ID NOS.13 to 14) are as follows:
methylation of the upstream primer: GTTTTGGGTTAGAGTAGCGTTC (SEQ ID NO. 11);
methylation of the downstream primer: AAACCTCCGCACCGTACG (SEQ ID NO. 12);
unmethylated upstream primer: TAAAGTGGGAGGTTGTTTATAC (SEQ ID NO. 13);
unmethylated downstream primer: CAACGAAATACGCATTACG (SEQ ID NO. 14).
The sequences of the nucleic acid combinations (SEQ ID NOS.15 to 16 and SEQ ID NOS.17 to 18) are as follows:
methylation of the upstream primer: GTGTTTTGGGTTAGAGTAGTGTTT (SEQ ID NO. 15);
methylation of the downstream primer: CAAAACCTCCACACCATACA (SEQ ID NO. 16);
unmethylated upstream primer: CAAAGTGGGAGGTTGTTTATAT (SEQ ID NO. 17);
unmethylated downstream primer: CCCAACAAAATACACATTACA (SEQ ID NO. 18).
The methylation primer pair and the unmethylation primer pair which are designed according to the sequence of the candidate region polynucleotide molecules converted by SEQ ID NO.1 and/or SEQ ID NO.2 are detected on samples of a training set and a testing set, and the specific detection method is as follows:
1) Extraction of DNA samples
When the DNA sample is a lung tissue sample, a blood/cell/tissue genome DNA extraction kit (catalog number: DP 304) of Tiangen biochemical technology (Beijing) limited company is adopted to extract the cell genome DNA of each sample, and the specific operation is described in the kit specification.
When the DNA sample is a blood sample, the blood plasma cfDNA is extracted by using a magnetic bead method serum/blood plasma free DNA extraction kit (catalog number: DP 709) of Tiangen biochemical technology (Beijing) limited company, the volume of the blood plasma is 1.5mL, the specific operation is shown in the kit instruction, and 50 mu L of purified water is used for eluting after the extraction is completed.
2) Bisulphite conversion and purification treatments
The extracted genome DNA of each sample is respectively subjected to bisulphite conversion, the nucleic acid conversion kit is a nucleic acid purification reagent (Huhan mechanical equipment 20200843) of the life technology Co., ltd., wuhan Ai Misen, specific experimental operation is described in the kit instruction, and 30 mu L of purified water is used for eluting after the conversion is completed.
3) PCR reaction
And (3) taking the DNA after bisulfite conversion as a template, and simultaneously adding SYBR Green PCR Mix, a methylation primer pair and a non-methylation primer pair of the non-small cell lung cancer biomarker to perform PCR amplification. Meanwhile, a primer pair for amplifying the reference gene ACTB is added, wherein the nucleotide sequence of an upstream primer is as follows: AAGGTGGTTGGGTGGTTGTTTTG (SEQ ID NO. 22), the nucleotide sequence of the downstream primer is: AATAACACCCCCACCCTGC (SEQ ID NO. 23). The PCR reaction system is shown in Table 1, and the PCR reaction conditions are shown in Table 2.
TABLE 1SYBR Green PCR reaction System
| Component (A) | Dosage (mu L) |
| SYBR Green PCR Mix | 17.5 |
| Methylation primer pair upstream primer (10. Mu.M) | 0.5 |
| Methylation primer pair downstream primer (10. Mu.M) | 0.5 |
| Unmethylated primer pair upstream primer (10. Mu.M) | 0.5 |
| Unmethylated primer pair downstream primer (10. Mu.M) | 0.5 |
| ACTB upstream primer (10 mu M) | 0.5 |
| ACTB downstream primer (10. Mu.M) | 0.5 |
| Template DNA | 5 |
| Ultrapure water | Supplement to 50 |
TABLE 2SYBR Green PCR reaction procedure
4) Pyrosequencing
And sending the PCR product to a sequencing company for pyrosequencing, and analyzing a sequencing peak diagram based on the methylation state of key CpG sites of the pyrosequencing non-small cell lung cancer biomarker. Specifically, methylation of cytosine in a CpG nucleotide is classified into two types: i.e., methylated and unmethylated, where methylation is in turn divided into fully methylated and partially methylated, a CpG dinucleotide site is considered partially methylated if the sequencing result of the cytosine at that site reveals both a C and a T at the position of the cytosine.
If more than 95% of the C's in CpG dinucleotide sites in an amplicon are methylated, the sample is considered methylated in this region.
5) Analysis of results
After the PCR reaction is finished, comparing the sequencing result of the amplified product with the pathological result, and calculating the methylation state of CpG sites of the amplified product to calculate the sensitivity and the specificity of the polynucleotide molecule. Sensitivity = methylation positive sample/sample with positive pathological result; specificity = methylation negative samples/samples with negative pathological results. The detection results are shown in Table 3.
TABLE 3 specificity and sensitivity of different CpG sites on training set samples
In Table 3, when the nucleic acid combinations 1, 2 and 3 were used to detect the non-small cell lung cancer biomarkers (SEQ ID NO.1 and SEQ ID NO. 2), respectively, the methylation levels (number of methylation positive samples) of SEQ ID NO.1 and SEQ ID NO.2 were higher in the non-small cell lung cancer tissues than in the paracancerous normal tissue samples. The nucleic acid combination can effectively distinguish a non-small cell lung cancer tissue sample and a paracancerous normal tissue sample by detecting the methylation level of the non-small cell lung cancer biomarker, the detection sensitivity is more than 88.0%, the detection specificity is more than 85.0%, wherein the sensitivity of the nucleic acid combination 3 for detecting the methylation level of the CpG sites of numbers 07-19 in SEQ ID NO.2 for diagnosing the non-small cell lung cancer tissue sample can reach 92.6%, and the specificity for detecting the paracancerous normal tissue sample can reach 90.7%. The nucleic acid combinations 1, 2 and 3 all meet the primary screening standard with the sensitivity more than or equal to 85% and the specificity more than or equal to 85%, so that the nucleic acid combinations can be further verified on a plasma sample.
TABLE 4 specificity and sensitivity of different CpG sites on test set samples
The analysis of the table 4 shows that the nucleic acid combinations 1, 2 and 3 can be used for detecting the blood samples of early non-small cell lung cancer, the detection sensitivity of the nucleic acid combinations is more than 76.7% and the detection specificity of the nucleic acid combinations is more than 90.0% by detecting the methylation level of the non-small cell lung cancer biomarker, wherein the sensitivity of the nucleic acid combination 3 for detecting the methylation level of the CpG sites of between 07 and 19 in SEQ ID NO.2 can reach 83.3%, and the specificity of the nucleic acid combination for detecting the blood samples of healthy people can reach 93.3%.
Example 2 methylation detection kit for early detection of non-small cell lung cancer blood sample based on MSP method
In the embodiment, after blinding an acquired sample in the test process, a test operator performs the test, and a result interpreter compares the obtained detection result with a pathological result (gold standard) according to the interpretation standard to examine the clinical effectiveness of the non-small cell lung cancer biomarker.
In order to realize noninvasive detection and further verify and improve the sensitivity of the kit for diagnosing the non-small cell lung cancer blood sample, the embodiment performs the grouping and collection of the sample according to the clinical test scheme of the in-vitro diagnostic reagent, comprises 174 cases of healthy human blood samples and 58 cases of patients with pathological diagnosis of the non-small cell lung cancer, and all the patients do not receive radiotherapy or chemotherapy before operation. Of the diagnosed non-small cell lung cancer patients, 9 were squamous cancers and 49 were adenocarcinomas. According to the eighth edition TMN stage criteria, 42 cases are stage I and 16 cases are stage IIa. In this example, stage I/IIa patients were defined as early stage non-small cell lung cancer in combination with clinical experience. The present practice diagnoses non-small cell lung cancer patients by detecting the methylation level of a non-small cell lung cancer biomarker (i.e., the polynucleotide molecule of example 1) in a blood sample from the subject. The specific process is as follows:
1) Extraction of DNA samples
When the DNA sample is a blood sample, the blood plasma cfDNA is extracted by using a magnetic bead method serum/blood plasma free DNA extraction kit (catalog number: DP 709) of Tiangen biochemical technology (Beijing) limited company, the volume of the blood plasma is 1.5mL, the specific operation is shown in the kit instruction, and 50 mu L of purified water is used for eluting after the extraction is completed.
2) Bisulphite conversion and purification treatments
The extracted genome DNA of each sample is respectively subjected to bisulphite conversion, the nucleic acid conversion kit is a nucleic acid purification reagent (Huhan mechanical equipment 20200843) of the life technology Co., ltd., wuhan Ai Misen, specific experimental operation is described in the kit instruction, and 30 mu L of purified water is used for eluting after the conversion is completed.
3) qPCR reaction
The detection probes were designed using the bisulfite converted polynucleotide sequences as templates, and the nucleic acid combinations comprising the methylation primer pairs and the detection probes are shown in Table 5. The clinical samples were subjected to PCR amplification of polynucleotide molecules using TaqMan probe method. When detecting the methylation level of a single polynucleotide molecule, the PCR reaction tube is added with a primer pair (SEQ ID NO. 22-23) and a probe (SEQ ID NO.24: GGAGTGGTTTTTGGGTTTG) for amplifying an internal reference gene ACTB in addition to the necessary reaction components, a template, a methylation primer pair of the single polynucleotide molecule and a detection probe. Negative and positive controls also need to be provided: the template of the negative control PCR tube is TE buffer solution, and other components are the same as those of the experimental tube; template of positive control PCR tube 10 3 copies/. Mu.L of plasmid containing the sequence ACTB after transformation and 10 3 cobies/. Mu.L of the mixtureThe plasmid-equivalent volume of the mixture of the transformed polynucleotide molecule sequences and the other components are the same as those of the experimental tube. If the methylation level of a combination of double polynucleotide molecules is to be detected, a methylation primer pair and a detection probe for another polynucleotide molecule are added to the PCR reaction tube in addition to the above components.
The detection probes are TaqMan probes, and the 5' end of the polynucleotide molecule detection probes is a fluorescent reporter group, such as FAM, ROX, VIC, CY; the 3' end is a fluorescence quenching group, such as TAMRA, BHQ, MGB and the like. In this example, the fluorescent reporter group at the 5 'end of the polynucleotide molecule detection probe SEQ ID NO.19 (5'-ATAAATAACTCCTAACTCC-3') is ROX, and the fluorescent reporter groups at the 5' ends of the polynucleotide molecule detection probes SEQ ID NO.20 (5'-CCGAATCTTTCACCCCA-3') and 21 (5'-ACGACCCCGCTCAC-3') are VIC; the fluorescence quenching groups at the 3' end are BHQ or BHQ1. The fluorescence report group at the 5 '-end of the detection probe of the reference gene ACTB is FAM, and the fluorescence quenching group at the 3' -end is MGB. The qPCR reaction system is shown in Table 6 (if only a single polynucleotide molecule is detected, no methylation primer pair and no detection probe for another polynucleotide molecule are added, and the remaining volume is complemented with purified water), and the qPCR reaction procedure is shown in Table 7.
TABLE 5 nucleic acid combinations
| Detection of CpG sites | Nucleic acid combinations | Methylation primer pairs | Detection probe |
| CpG sites of 03-11 numbers in SEQ ID NO.1 | 4 | SEQ ID NO.7～8 | SEQ ID NO.19 |
| CpG sites of 03-12 numbers in SEQ ID NO.2 | 5 | SEQ ID NO.11～12 | SEQ ID NO.20 |
| CpG sites of 07-19 numbers in SEQ ID NO.2 | 6 | SEQ ID NO.15～16 | SEQ ID NO.21 |
TABLE 6qPCR reaction System
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TABLE 7qPCR reaction procedure
After qPCR reaction is completed, the baseline, which is typically a fluorescent signal of 3-15 cycles, is manually adjusted and a suitable threshold is set, which is placed in the exponential amplification phase.
4) Analysis of results
Analyzing the result of qPCR reaction, requiring (1) no amplification of negative control PCR tube; (2) the positive control PCR tube has obvious index increasing period, and the Ct value of the target gene of the positive control PCR tube is between 26 and 30; (3) the Ct value of the reference gene of the sample to be detected is less than or equal to 34.
If the positive control, the negative control and the reference gene all meet the requirements, the detection result of the sample to be detected can be analyzed and the result can be interpreted, otherwise, the detection must be carried out again when the experiment is invalid. For a plasma sample, the positive judgment value is 45, and if the Ct value obtained by amplifying a certain pair of methylation primer pairs and detection probes is less than or equal to 45, the sample is judged to be methylation positive in the amplification region; if Ct value > 45 amplified using a certain pair of methylation primers and detection probe, the sample is judged to be methylation negative in this amplified region. When the polynucleotide molecules are detected in a combined way, if any one polynucleotide molecule in the polynucleotide molecule combination in the sample to be detected is methylation positive, the sample is a cancer positive sample, and only if two polynucleotide molecules in the polynucleotide molecule combination in the sample to be detected are methylation negative, the sample is a cancer negative sample. The specific detection results are shown in Table 8.
TABLE 8 verification of CpG sites of Polynucleotide molecules on blood samples
As can be seen from Table 8, the MSP method for detecting the methylation level of a single polynucleotide molecule in a blood sample has good effect in diagnosing early non-small cell lung cancer. The sensitivity of the nucleic acid combination 4 and the nucleic acid combination 5 for detecting the blood sample of the phase I non-small cell lung cancer is 78.6 percent and 81 percent respectively, the sensitivity of the nucleic acid combination 4 and the nucleic acid combination 5 for detecting the blood sample of the phase IIa non-small cell lung cancer is 81.3 percent, and the specificity of the nucleic acid combination 5 for detecting the blood sample of the phase IIa non-small cell lung cancer is 92 percent and 93.1 percent respectively; the sensitivity of the nucleic acid combination 6 for detecting the blood sample of the I-stage non-small cell lung cancer is 83.3%, the sensitivity of the nucleic acid combination 6 for detecting the blood sample of the IIa-stage non-small cell lung cancer is 87.5%, and the specificity of the nucleic acid combination 6 for detecting the blood sample of the healthy human is 93.7%. In combination, when tested using a single nucleic acid combination, the nucleic acid combinations described above are able to distinguish between early non-small cell lung cancer patients and healthy humans, wherein the effect of detection of nucleic acid combination 6 > nucleic acid combination 5> nucleic acid combination 4.
When the single nucleic acid combinations are subjected to pairwise joint detection, the specificity of the joint detection of the nucleic acid combination 4+nucleic acid combination 5, the nucleic acid combination 4+nucleic acid combination 6 and the nucleic acid combination 5+nucleic acid combination 6 of the blood sample of the healthy person is slightly lower than that of the single nucleic acid combination detection, but still is at a higher level, and the joint detection specificity is more than 90%.
The nucleic acid combination 6 with optimal detection sensitivity is detected with the nucleic acid combination 4 and the nucleic acid combination 5 respectively, and the sensitivity of the nucleic acid combination 4+nucleic acid combination 6 and the nucleic acid combination 5+nucleic acid combination 6 for detecting the blood sample of the stage I non-small cell lung cancer is 85.7 percent, which is superior to that of the single nucleic acid combination detection, and the sensitivity of the nucleic acid combination 5+nucleic acid combination 6 for detecting the blood sample of the stage IIa non-small cell lung cancer is 81.3 percent, which is lower than that of the nucleic acid combination 4+nucleic acid combination 6 (the detection sensitivity is 93.8 percent) and even lower than that of the single nucleic acid combination 6. It can be seen that when two nucleic acid combinations with better detection sensitivity are combined for detection, the effect of the combined detection of the IIa stage small cell lung cancer blood sample is not improved compared with that of a single nucleic acid combination.
The combined detection of the nucleic acid combination 4 and the nucleic acid combination 5 shows that the sensitivity of detecting the blood sample of the phase I non-small cell lung cancer is 83.3 percent, which is superior to that of single nucleic acid combination detection; the sensitivity of detecting IIa stage non-small cell lung cancer blood samples is 93.8%, which is obviously superior to single nucleic acid combination detection and is also superior to nucleic acid combination 5+nucleic acid combination 6 combination detection. It can be seen that when the nucleic acid combination 4 and the nucleic acid combination 5 with slightly poorer detection sensitivity are combined for detection, the sensitivity of detecting the IIa stage non-small cell lung cancer blood sample is obviously improved compared with that of a single nucleic acid combination, and is better than that of the nucleic acid combination 6 and the nucleic acid combination 5 with optimal detection sensitivity.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (10)
1. A nucleic acid combination for detecting the methylation level of a non-small cell lung cancer biomarker, wherein the non-small cell lung cancer biomarker is selected from at least one of the polynucleotide molecules set forth in (a) - (c):
(a) A full length or a partial region of the nucleotide sequence shown in SEQ ID No.1, said partial region comprising at least one CpG dinucleotide site; and/or the number of the groups of groups,
a full length or a partial region of the nucleotide sequence shown in SEQ ID No.2, said partial region comprising at least one CpG dinucleotide site;
(b) A polynucleotide molecule having more than 85% sequence identity to (a) wherein the CpG dinucleotide site is identical to (a);
(c) A polynucleotide molecule in which the CpG dinucleotide site of (a) or (b) is partially or fully methylated.
2. The nucleic acid combination of claim 1, wherein the polynucleotide molecule is selected from at least one of the polynucleotide molecules set forth in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3, and SEQ ID No. 5.
3. The nucleic acid combination of claim 2, wherein the nucleic acid combination comprises a pair of methylation primers for detecting the methylation level of the polynucleotide molecule.
4. The nucleic acid combination of claim 3, wherein the methylation primer pair is selected from at least one of the first methylation primer pair of SEQ ID NOS.7-8, the second methylation primer pair of SEQ ID NOS.11-12, and the third methylation primer pair of SEQ ID NOS.15-16.
5. The nucleic acid combination of claim 4, further comprising a pair of unmethylated primers for detecting the methylation level of the polynucleotide molecule;
the combination of nucleic acids comprises at least one set of the following combinations:
a first combination comprising a first pair of methylated primers shown in SEQ ID NOS.7-8 and a first pair of unmethylated primers shown in SEQ ID NOS.9-10;
a second combination comprising a second pair of methylated primers shown in SEQ ID NOS.11-12 and a second pair of unmethylated primers shown in SEQ ID NOS.13-14;
a third combination comprising a third pair of methylated primers shown in SEQ ID NOS.15-16 and a third pair of unmethylated primers shown in SEQ ID NOS.17-18.
6. The nucleic acid combination of claim 4, further comprising a detection probe;
the combination of nucleic acids comprises at least one set of the following combinations:
a fourth combination comprising a first pair of methylation primers shown in SEQ ID NOS.7-8, and a first detection probe shown in SEQ ID NO. 19;
a fifth combination comprising a second pair of methylation primers shown in SEQ ID NOS.11-12, and a second detection probe shown in SEQ ID NO. 20;
a sixth combination comprising a third pair of methylation primers shown in SEQ ID NOS.15-16, and a third detection probe shown in SEQ ID NO. 21.
7. The nucleic acid assembly of claim 6, wherein the detection probe comprises a fluorescent reporter group at the 5 'end and a fluorescent quenching group at the 3' end.
8. A kit for detecting non-small cell lung cancer, comprising the nucleic acid combination of any one of claims 1 to 7 and a remaining reagent comprising one or more of a DNA extraction reagent, a DNA purification reagent, a methylation conversion reagent, an amplification reagent, a primer pair for amplifying an internal reference gene, and a probe and a quality control product corresponding to the primer pair.
9. Use of the nucleic acid combination of any one of claims 1 to 7 or the kit of claim 8 for the preparation of a non-small cell lung cancer diagnostic product.
10. The use of claim 9, wherein the non-small cell lung cancer comprises at least one of stage i non-small cell lung cancer and stage la non-small cell lung cancer.
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