CN117587127A - Primer pair, primer probe combination, kit and application for lung cancer detection - Google Patents

Abstract

The invention belongs to the field of biological medicine, and in particular relates to a primer pair, a primer probe combination, a kit and application for lung cancer detection. The kit for detecting lung cancer provided by the invention can effectively distinguish lung cancer subjects from healthy subjects by detecting the methylation level of the target region on the FLJ32063 gene CpG island in a sample, and provides a new thought for noninvasive, high-sensitivity and high-specificity detection of lung cancer.

Description

Primer pair, primer probe combination, kit and application for lung cancer detection

Technical Field

The present invention belongs to the field of biomedicine technology, and more specifically, relates to a primer pair, a primer-probe combination, a kit and applications for lung cancer detection.

Background Art

Lung cancer is the leading cause of cancer death worldwide and has become a prominent global public health issue, resulting in multiple unmet medical needs for lung cancer patients. Although the complementary integration of surgical resection, chemotherapy, radiotherapy, immunotherapy, and targeted therapy has greatly improved the survival rate of lung cancer patients, the prognosis is still poor. One of the main reasons is that approximately 75% of lung cancer patients are diagnosed with advanced cancer (stage III-IV), and the 5-year survival rate of stage IV is less than 10%. In contrast, the 5-year survival rate of stage I cancer has increased significantly, ranging from 68% to 92%. Lung cancer is difficult to detect in these early stages due to the lack of clinical symptoms and sensitive technology. Low-dose computed tomography (LDCT) is widely used for early screening of high-risk groups for lung cancer, but the high false positive rate of screening can lead to the problem of overdiagnosis of patients, which will cause certain difficulties in the subsequent diagnosis of patients. It is imperative to develop a new screening method to solve the problems of LDCT.

Studies have shown that the CpG islands in the promoter regions of tumor suppressor genes in specific areas where tumors occur are abnormally hypermethylated. Combining DNA methylation analysis technology to detect the methylation status of circulating tumor DNA (ctDNA) in the human body can be used as a non-invasive, screening and early diagnosis method for lung cancer. Therefore, the development of a highly accurate, sensitive, specific, and non-invasive lung cancer diagnosis method is the key to improving the early diagnosis and treatment of lung cancer.

Summary of the invention

In view of the defects of the prior art, the purpose of the present invention is to provide a primer pair, a primer-probe combination, a kit and an application for lung cancer detection, so as to improve the technical problems of the prior art such as insufficient means for detecting lung cancer, low sensitivity, specificity and accuracy in detecting lung cancer.

To achieve the above object, the present invention provides a primer pair for lung cancer detection, which includes a first primer pair for detecting the methylation level of a target region on the CpG island of the FLJ32063 gene.

Preferably, the above primer pair further comprises a second primer pair for detecting the methylation level of the target region on the CpG island of the SHOX2 gene.

Preferably, GRCh38.p14 is used as a reference genome, and the target region on the CpG island of the FLJ32063 gene is selected from the full length or partial region of the negative chain of Chr2:199470636-199471036.

Preferably, GRCh38.p14 is used as a reference genome, and the target region on the CpG island of the SHOX2 gene is selected from the full length or partial region of the positive chain of Chr3:158103418-158103818.

Further preferably, the partial region of the above-mentioned Chr3:158103418-158103818 positive chain includes Chr3:158103480-158103619 positive chain.

Preferably, the above-mentioned first primer pair includes a first methylation primer pair for detecting the methylation level of the full length or partial region of the Chr2:199470636-199471036 negative chain; the above-mentioned second primer pair includes a second methylation primer pair for detecting the methylation level of the full length or partial region of the Chr3:158103418-158103818 positive chain.

More preferably, the nucleotide sequence of the first methylation primer pair is shown as SEQ ID NOs. 7-8, and the nucleotide sequence of the second methylation primer pair is shown as SEQ ID NOs. 11-12.

Preferably, the above-mentioned first primer pair also includes a first non-methylated primer pair for detecting the methylation level of the full length or partial region of the Chr2:199470636-199471036 negative chain; the above-mentioned second primer pair also includes a second non-methylated primer pair for detecting the methylation level of the full length or partial region of the Chr3:158103418-158103818 positive chain.

More preferably, the nucleotide sequence of the first non-methylated primer pair is shown as SEQ ID NOs. 9-10, and the nucleotide sequence of the second non-methylated primer pair is shown as SEQ ID NOs. 13-14.

The present invention also provides a primer-probe combination for lung cancer detection, which comprises the above primer pair and a detection probe.

Preferably, the primer-probe combination comprises at least one of the following combinations:

A first primer-probe combination, comprising a first methylation primer pair shown in SEQ ID NOs. 7 to 8 and a first probe shown in SEQ ID NO. 21;

A second primer-probe combination, comprising a first methylation primer pair shown in SEQ ID NOs. 7 to 8 and a second probe shown in SEQ ID NO. 22;

A third primer-probe combination, comprising a second methylation primer pair shown in SEQ ID NOs. 11 to 12 and a third probe shown in SEQ ID NO. 23;

A fourth primer-probe combination, comprising the second methylated primer pair shown in SEQ ID NOs. 11 to 12 and the fourth probe shown in SEQ ID NO. 24;

The fifth primer-probe combination comprises the second methylated primer pair shown in SEQ ID NOs. 11 to 12 and the fifth probe shown in SEQ ID NO. 25.

The present invention also provides a kit for lung cancer detection, comprising the above-mentioned primer pair or the above-mentioned primer-probe combination, and also comprising one or more of a detection primer pair and probe for an internal reference gene, a nucleic acid extraction reagent, a nucleic acid purification reagent, a methylation conversion reagent, an amplification reagent, a positive control substance and a negative control substance.

The present invention also provides the use of the primer pair or the primer-probe combination or the kit in preparing a lung cancer diagnosis product.

Preferably, the above diagnosis includes but is not limited to early differential diagnosis of lung cancer, assessment and dynamic monitoring of residual microlesions, auxiliary judgment of lung cancer recurrence and prognosis, drug efficacy evaluation and drug resistance monitoring.

Preferably, the above-mentioned lung cancer diagnosis product includes but is not limited to at least one of a kit, a chip, a membrane strip, and a protein array.

In general, the above technical solution conceived by the present invention has the following beneficial effects compared with the prior art:

(1) The primer pairs and primer probe combinations for lung cancer detection provided by the present invention can effectively distinguish lung cancer subjects from healthy subjects by detecting the methylation level of the target region on the CpG island of the FLJ32063 gene in the sample, and provide lung cancer gene markers that can be used for lung cancer detection. The above primer probe combination can detect the methylation level of the target region on the CpG island of the FLJ32063 gene, and the sensitivity of diagnosing lung cancer plasma samples can reach 83.6%, and the specificity of detecting healthy human plasma samples can reach 92.9%, with high detection accuracy.

(2) In a preferred embodiment, the kit for lung cancer detection provided by the present invention detects the methylation levels of the target regions on the CpG island of the FLJ32063 gene and the target regions on the CpG island of the SHOX2 gene, and the sensitivity of diagnosing lung cancer plasma samples is 86.6%, and the specificity of detecting healthy human plasma samples is 90.9%, which provides a new idea for non-invasive, highly sensitive and highly specific detection of lung cancer.

DETAILED DESCRIPTION

In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

The term "and/or" includes any and all combinations of one or more of the associated listed items.

The term "diagnosis" refers to determining the health status of a subject, including the presence or absence of a disease, response to treatment, recurrence risk assessment, assessment of the risk and extent of canceration, and prognosis. In some cases, the term "diagnosis" refers to a single factor used to determine, verify, or confirm the clinical status of a patient. In some embodiments, "detecting" lung cancer refers to detecting the presence or absence of a disease, that is, determining whether a subject has lung cancer.

The term "subject" refers to an object that is observed, tested, or experimented on. In some embodiments, the subject can be a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates) and rodents (e.g., mice and rats). In some embodiments, the mammal can be a human.

The term "methylation" is a form of chemical modification of DNA that can change genetic expression without changing the DNA sequence. DNA methylation refers to the covalent attachment of a methyl group to the 5th carbon position of cytosine in genomic CpG dinucleotides under the action of DNA methyltransferase. DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and the way DNA interacts with proteins, thereby controlling gene expression.

The term "methylation level" refers to whether the cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/proportion/percentage of methylation. It represents both a qualitative and a quantitative concept. In practical applications, different detection indicators can be used to compare DNA methylation levels according to actual conditions. For example, in some cases, the comparison can be made based on the Ct value of the sample test; in some cases, the proportion of gene methylation in the sample can be calculated, that is, the number of methylated molecules/(the number of methylated molecules + the number of unmethylated molecules) × 100%, and then compared; in some cases, it is also necessary to statistically analyze and integrate the various indicators to obtain the final judgment indicator.

The term "primer" refers to an oligonucleotide that can be used in an amplification method (e.g., polymerase chain reaction, PCR) to amplify a target sequence based on a polynucleotide sequence corresponding to a target gene or a portion thereof. Typically, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence-specific for the polynucleotide sequence. The exact length of the primer depends on many factors, including temperature, primer source, and the method used. For example, for diagnostic and prognostic applications, oligonucleotide primers 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 can hybridize with the double strands of a target DNA molecule or can hybridize with a region of a target DNA molecule that is located on both sides of a nucleotide sequence to be amplified. A "primer pair" refers to a group consisting of an upstream primer and a downstream primer.

The term "bisulfite sequencing PCR (BSP)" refers to the process of treating genomic DNA with bisulfite, in which non-methylated cytosine is deaminated and converted into uracil. In the subsequent PCR reaction, uracil is converted into thymine, while methylated cytosine cannot be deaminated and is retained when the reaction is completed. Primers are then designed in the non-methylated region for PCR amplification, the amplified PCR products are cloned and sequenced, the measured sequences are compared with the original sequences, the methylation sites and quantities are counted, and the degree of methylation is analyzed.

The term "methylation-specific PCR" is one of the most sensitive experimental techniques for studying methylation, and can detect methylation in as little as about 50 pg of DNA. After single-stranded DNA is converted by bisulfite, all unmethylated cytosines are deaminated and converted to uracil, while methylated cytosines in CpG sites remain unchanged. Therefore, two pairs of primers are designed for methylated and unmethylated sequences respectively, and methylated and unmethylated DNA sequences can be distinguished through PCR amplification.

The term "methylation-specific fluorescent quantitative PCR (qMSP)" is an experimental technique that combines fluorescent quantitative PCR technology with methylation-specific PCR technology. This technology is also based on the sequence differences of DNA in different methylation states after bisulfite conversion to design appropriate primer pairs, so as to distinguish methylated and unmethylated sequences, but the final detection index of qMSP is the fluorescent signal. Therefore, in the qMSP reaction system, in addition to adding methylation detection primers, fluorescent probes or fluorescent dyes need to be added. Compared with traditional methylation-specific PCR technology, qMSP has higher sensitivity and specificity in detecting DNA methylation levels, and is more suitable for detecting trace amounts of abnormally methylated DNA fragments mixed in the DNA of early cancer patients, and this technology does not require gel electrophoresis detection, and the operation is simpler. In the disclosure of the present application, a methylation primer pair is added when performing real-time quantitative methylation-specific PCR. If the Ct value meets the requirements (for example, Ct≤45 in a plasma sample), it indicates that the target sequence is methylated.

The term "TaqMan probe" refers to an oligonucleotide sequence containing a 5' fluorescent group and a 3' quencher group. When the probe binds to the corresponding site on the DNA, the probe will not emit fluorescence because of the presence of a quencher group near the fluorescent group. During the amplification process, if the probe binds to the amplified chain, the 5'-3' exonuclease activity of the DNA polymerase (such as Taq enzyme) will digest the probe, and the fluorescent group will be away from the quencher group, and its energy will not be absorbed, thus generating a fluorescent signal. After each PCR cycle, the fluorescent signal also has a synchronous exponential growth process like the target fragment.

The present invention provides a primer pair for lung cancer detection, which comprises a first primer pair for detecting the methylation level of a target region on a CpG island of a FLJ32063 gene.

In some embodiments, the primer pair further comprises a second primer pair for detecting the methylation level of the target region on the CpG island of the SHOX2 gene.

In some embodiments, taking GRCh38.p14 as the reference genome, the target region on the CpG island of the FLJ32063 gene is selected from the full length or partial region of the negative chain of Chr2:199470636-199471036; the target region on the CpG island of the SHOX2 gene is selected from the full length or partial region of the positive chain of Chr3:158103418-158103818.

In some embodiments, the first primer pair includes a first methylation primer pair for detecting the methylation level of the full length or partial region of the Chr2:199470636-199471036 negative chain; the second primer pair includes a second methylation primer pair for detecting the methylation level of the full length or partial region of the Chr3:158103418-158103818 positive chain.

It is understandable that the second primer pair can detect the methylation level of the positive chain of Chr3:158103418-158103818 by targeting the full-length region or a partial region thereof. In some embodiments, the partial region of the positive chain of Chr3:158103418-158103818 includes the positive chain of Chr3:158103480-158103619.

In a preferred embodiment, the nucleotide sequence of the first methylated primer pair is shown as SEQ ID NOs. 7-8, and the nucleotide sequence of the second methylated primer pair is shown as SEQ ID NOs. 11-12.

In some embodiments, the first primer pair also includes a first non-methylated primer pair for detecting the methylation level of the full length or partial region of the Chr2:199470636-199471036 negative chain; the second primer pair also includes a second non-methylated primer pair for detecting the methylation level of the full length or partial region of the Chr3:158103418-158103818 positive chain.

In a preferred embodiment, the nucleotide sequence of the first non-methylated primer pair is shown as SEQ ID NOs. 9-10, and the nucleotide sequence of the second non-methylated primer pair is shown as SEQ ID NOs. 13-14.

It should be noted that if a primer pair has at least 85% (for example, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%) sequence identity with the nucleotide sequence shown by the above-mentioned primer pairs (the first methylated primer pair, the first unmethylated primer pair, the second methylated primer pair, the second unmethylated primer pair), and the primer pair also has a certain lung cancer diagnosis function (the specificity or sensitivity is equivalent to or slightly decreased or slightly improved or greatly improved compared with the primer pair of the present application), it is also within the scope of protection of the present invention.

The present invention also provides a primer-probe combination for lung cancer detection, which comprises the above primer pair and a detection probe.

In some embodiments, the primer-probe combination comprises at least one of the following combinations:

A first primer-probe combination, comprising a first methylation primer pair shown in SEQ ID NOs. 7 to 8 and a first probe shown in SEQ ID NO. 21;

A second primer-probe combination, comprising a first methylation primer pair shown in SEQ ID NOs. 7 to 8 and a second probe shown in SEQ ID NO. 22;

A third primer-probe combination, comprising a second methylation primer pair shown in SEQ ID NOs. 11 to 12 and a third probe shown in SEQ ID NO. 23;

A fourth primer-probe combination, comprising the second methylated primer pair shown in SEQ ID NOs. 11 to 12 and the fourth probe shown in SEQ ID NO. 24;

The fifth primer-probe combination comprises the second methylated primer pair shown in SEQ ID NOs. 11 to 12 and the fifth probe shown in SEQ ID NO. 25.

In a preferred embodiment, the primer-probe combination includes the second primer-probe combination and the fifth primer-probe combination.

The present invention also provides a kit for lung cancer detection, which includes the above-mentioned primer pair or the above-mentioned primer-probe combination, and also includes one or more of a detection primer pair and probe for an internal reference gene, a nucleic acid extraction reagent, a nucleic acid purification reagent, a methylation conversion reagent, an amplification reagent, a positive control substance, and a negative control substance.

In some embodiments, the internal reference gene may be, but is not limited to, ACTB. In an optional specific example, the nucleotide sequence of the detection primer pair of the internal reference gene ACTB is shown in SEQ ID NO.19-20, and the nucleotide sequence of the probe of the internal reference gene ACTB is shown in SEQ ID NO.26. It is understood that in other embodiments, other internal reference genes may be selected, in which case the detection primer pair and probe of the internal reference gene may be designed accordingly.

In a preferred embodiment, the 5' end of the first probe, the second probe, the third probe, the fourth probe, the fifth probe, and the probe of the internal reference gene ACTB all contain a fluorescent reporter group, and the 3' end contains a fluorescent quencher group. The fluorescent reporter group of each of the above-mentioned probes is independently selected from any one of FAM, VIC, HEX, NED, ROX, TET, JOE, CY3 and CY5; the fluorescent quencher group of each of the above-mentioned probes is independently selected from any one of TAMRA, MGB, BHQ, BHQ1, BHQ2 and BHQ3. Examples of the fluorescent reporter group and the fluorescent quencher group of each probe independently include but are not limited to those listed above.

In some embodiments, the above-mentioned methylation conversion reagent is used to deaminize unmethylated cytosine in DNA into uracil, while the methylated cytosine remains unchanged. The present invention has no particular restrictions on the methylation conversion reagent, and any reagent reported in the prior art that can achieve the conversion of cytosine to uracil can be used, such as one or more of hydrazine salts, bisulfates (e.g., sodium bisulfate, potassium bisulfate, ammonium bisulfate) and bisulfites (e.g., sodium metabisulfite, potassium bisulfite, calcium bisulfite, cesium bisulfite, ammonium bisulfite, etc.).

In some embodiments, the amplification reagents include but are not limited to one or more of amplification buffer, dNTPs, DNA polymerase and Mg ²⁺ . In some embodiments, the positive control refers to a methylated lung squamous cell carcinoma marker, which is used to monitor the detection performance of the reagents in the kit; the negative control refers to a methylated lung squamous cell carcinoma marker, which is used to monitor whether the experiment is contaminated.

In some embodiments, the test samples of the above kit include, but are not limited to, compositions of organs, tissues, cells and/or body fluids separated from a subject. In some embodiments, the above samples can be tissue or body fluid samples, and further, the above tissues include lung tissue; the above body fluids include blood, plasma, serum, extracellular fluid, tissue fluid, lymph fluid, etc., and also include sputum, urine, saliva, feces, etc. Furthermore, in a specific embodiment of the present invention, the sample to be tested is a plasma sample.

The present application does not specifically limit the method for detecting the methylation level of a sample by the methylation detection kit. The method used may 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, combined bisulfite restriction analysis (COBRA), methylated CpG island amplification (MCA), methylated CpG island amplification and microarray (MCAM), HpaII small fragment enrichment by ligation-mediated PCR (HELP), bisulfite sequencing (BSP), bisulfite microarray analysis, methylation-specific pyrosequencing, HELP sequencing (HELP-seq), TET-assisted pyridine borane sequencing (TAPS), Gal hydrolysis and ligation adapter-dependent PCR (GLAD-PCR), methylated DNA immunoprecipitation sequencing (MeDIP-Seq) or methylated DNA immunoprecipitation-microarray analysis (MeDIP-chip), Southern blotting using methyl-sensitive restriction enzymes, and methylation-specific giant magnetoresistance sensor-based microarray analysis.

Based on the contents disclosed in the present invention, those skilled in the art can use any technology well known in the art to detect the methylation level of the sample to diagnose lung cancer. No matter which technology is used, it falls within the protection scope of the present invention.

The present invention also provides a method for detecting lung cancer by detecting the methylation level of the target region of the FLJ32063 gene in the sample. In some embodiments, the present invention uses the following method for detection, which includes the following steps: extracting DNA from the blood sample of the subject, converting it using a nucleic acid conversion reagent, then purifying the converted DNA, and performing qPCR amplification using the kit of the present invention, detecting the methylation level of the target region on the CpG island of the FLJ32063 gene by the qMSP method, and then judging whether the blood sample to be tested is negative or positive for lung cancer.

The present invention also provides the use of the primer pair or the primer-probe combination or the kit in preparing a lung cancer diagnosis product.

In some embodiments, the above-mentioned diagnosis includes but is not limited to early differential diagnosis of lung cancer, residual micro-lesions assessment and dynamic monitoring, auxiliary judgment of lung cancer recurrence and prognosis, drug efficacy evaluation and drug resistance monitoring, etc. Among them, the above-mentioned early differential diagnosis of lung cancer can be specifically used to confirm whether an individual has lung cancer or is more likely to have lung cancer; the above-mentioned residual micro-lesions assessment and dynamic monitoring can be specifically used to confirm whether there are residual micro-lesions in an individual; the above-mentioned auxiliary judgment of lung cancer recurrence and prognosis refers to the risk of lung cancer recurrence and the prediction of prognosis, which can be specifically used to confirm the possibility of recurrence or deterioration of individual lung cancer, and can be used to guide clinical diagnosis and treatment; the above-mentioned drug efficacy evaluation and drug resistance monitoring can be specifically used to confirm whether a certain drug or treatment is effective for an individual.

In some embodiments, the lung cancer diagnostic product can be in any suitable product form, including but not limited to primers, probes, kits, chips, membrane strips, protein arrays, etc.

The present invention also provides a method for using the above kit to monitor whether a subject suffers from lung cancer, or to monitor whether a lung cancer patient has lung cancer recurrence, comprising the following steps: extracting DNA from a blood sample of the subject, converting it using a nucleic acid conversion reagent, then purifying the converted DNA, and performing qPCR amplification using the above kit, detecting the methylation level of the target region on the CpG island of the FLJ32063 gene by the qMSP method, and then judging whether the blood sample to be tested is negative or positive for lung cancer.

The above technical solution is described in detail below in conjunction with specific embodiments.

In the examples of the present invention, all reagents used are commercially available products unless otherwise specified.

The following are examples:

Example 1 Collection and processing of lung cancer samples

Training set samples (tissue samples): A total of 58 cancer tissue samples and corresponding adjacent normal tissue samples from patients diagnosed with lung cancer by pathological biopsy were collected. All the cancer tissue samples were samples of middle and late stage lung cancer, and all tissue samples were formalin-fixed and paraffin-embedded.

Blood samples: A total of 265 blood samples were collected, including 97 blood samples from patients diagnosed with lung cancer (including 39 cases of lung adenocarcinoma, 29 cases of lung squamous cell carcinoma, 10 cases of large cell lung cancer, and 19 cases of small cell lung cancer), and 228 blood samples from healthy people. After excluding the subjects' previous history of cancer, autoimmune diseases, acute inflammation, etc., 8mL to 10mL of venous blood was collected with EDTA anticoagulation tubes before surgery and radiotherapy and chemotherapy. The collected anticoagulated blood samples were centrifuged after standing at room temperature to obtain plasma samples and blood leukocyte samples. The collection process of all tissue and blood samples was approved by the Ethics Committee. 30 lung cancer plasma samples (including 12 cases of lung adenocarcinoma, 9 cases of lung squamous cell carcinoma, 4 cases of large cell lung cancer, and 5 cases of small cell lung cancer), 30 plasma samples from healthy people, and 30 blood leukocyte samples from healthy people were used as test set samples. The extraction, transformation and purification process of the above samples are as follows:

1) Extract DNA samples

When the DNA samples are lung tissue samples and blood leukocyte samples, the blood/cell/tissue genomic DNA extraction kit (catalog number: DP304) of Tiangen Biochemical Technology (Beijing) Co., Ltd. is used to extract the cell genomic DNA of each sample. For specific operations, please refer to the kit manual.

When the DNA sample is a blood sample, the collected blood sample is centrifuged to separate the plasma layer and the white blood cells for later use. The plasma layer is extracted from plasma cfDNA using the magnetic bead serum/plasma free DNA extraction kit (catalog number: DP709) of Tiangen Biochemical Technology (Beijing) Co., Ltd. The plasma volume used is 1.5 mL. For specific operations, refer to the kit instructions. After the extraction is completed, 50 μL of purified water is used for elution. The white blood cells are extracted from the cell genomic DNA of each sample using the blood/cell/tissue genomic DNA extraction kit (catalog number: DP304) of Tiangen Biochemical Technology (Beijing) Co., Ltd. For specific operations, refer to the kit instructions.

2) Bisulfite conversion and purification

The extracted genomic DNA of each sample was subjected to bisulfite conversion. The nucleic acid conversion kit used was the nucleic acid purification reagent of Wuhan Aimison Life Science Technology Co., Ltd. (Ehan Xiebei 20200843). For specific experimental operations, please refer to the instructions of the kit. After the conversion is completed, 30 μL of purified water was used for elution and stored at -20°C for later use.

Example 2 Screening of methylation regions of lung cancer gene markers based on training set samples

By detecting some high-methylation status regions in the FLJ32063 gene and SHOX2 gene in tissue samples, the differentially methylated regions that can distinguish lung cancer patients from healthy people were screened out, and primers for the multiplex fluorescent PCR system were designed to preliminarily screen out target regions with specificity and sensitivity greater than or equal to 85% on tissue samples.

Taking GRCh38.p14 as the reference genome, the present invention provides isolated polynucleotides, wherein polynucleotide SEQID NO.1 is located in the base fragment of 199470636-199471036 of the negative strand of chromosome 2 of the FLJ32063 gene, and polynucleotide SEQ ID NO.2 is located in the base fragment of 158103418-158103818 of the positive strand of chromosome 3 of the SHOX2 gene.

The DNA sequence of SEQ ID NO.1 is: 5’-GGCCTTCGAAATCAAGGACTAAGGTGAGCAGAGGAGTCCCCCAGCCCCTGGCTGTAGCCTTTGGGACTTCTCTCCCGCGTCTTGGGTCAGAGCAGCGTCCGCAGCAAAGTCCTTTGGGGTGAAAGACCCGGGTGCGAATTAGTTTCAAAGTGGGAGGTTGCTTACACGTGAGCGGGGTCGCCAGCTCTTTGAAGGGCGCACGGTGCGGAGGCTCCGGCAAGGG TGCAATTCCCGTGCGCGTGTTAGCGCAATGCGCACTCCGCTGGGCCCAAGCAGTCATCAGTTCTGCCAAGTGGGTGCTGCTCCGGGTACCCACTGGTGTGGTGGGTAAGTCGGGAGCAGAATAATGAAGCGCATTACCTGGGAAGCCAAATTTGCAAAGCTTAACCCAATCCTCCTCG-3’.

The DNA sequence of SEQ ID NO.2 is: 5’-CTGGTCTAATTTAGGAACAATTGGGCCGAAAGGTATCAGCGAGAGCAACAGACCCCGGTTGTGCCGCACAGGGAGCCGCATCCGCAGACGCCCCTCGCTGCCCCTGGGCTCGGGCCAAACCCTGCATAAGGTCCCCTGGACAGCCAGGTAATCTCCGTCCCGCCTGCCCGACCGGGGTCGCACGAGCACAGGCGCCCACGCCATGTTGGCTGCCCAAAGGGCTCGCCGCCCAA GCCGGGCCAGAAGGCAGGAGGCGGAAAACCAGCCTCCGGTGGCGGGCGAAAGCAACCGCTCTTTCTGTTCTCTCTTCGCCCTCCCTCGTGGAAACGCAGACTCGACCCTAAACGCTTAACCCACAGAGATCAACAGGTTCAAGCGGAATATTCGCGATCCTCGGTT-3’.

The sequences of SEQ ID NO.1 and SEQ ID NO.2 after bisulfite treatment are shown in Table 1. Methylated primer pairs and non-methylated primer pairs specific for lung cancer gene markers are designed (Table 2), respectively, that is, the methylated primer pair will not amplify the non-methylated template, and the non-methylated primer pair will not amplify the methylated template. The primers are artificially synthesized and diluted to a suitable working concentration for later use.

Table 1 Sequences of SEQ ID NO.1 and SEQ ID NO.2 after bisulfite treatment

Table 2 Nucleotide sequences of methylated primer pairs and non-methylated primer pairs

Using bisulfite-converted tissue sample DNA as a template, SYBR Green PCR Mix, methylation primer pairs and non-methylation primer pairs of the target region of lung cancer gene markers, and upstream and downstream primers of the internal reference gene ACTB were added for PCR amplification. The upstream primer sequence of the internal reference gene ACTB is SEQ ID NO.19: AAGGTGGTTGGGTGGTTGTTTTG, and the downstream primer sequence is SEQ ID NO.20: AATAACACCCCACCCTGC. The PCR reaction system is shown in Table 3, and the PCR reaction conditions are shown in Table 4. The PCR amplification product was sent to a sequencing company for Sanger sequencing, and the sequencing peak graph was analyzed, and the methylation status of a single CpG site in the target region of the target gene measured by Sanger was used as the basis.

Table 3 SYBR Green PCR reaction system

Table 4SYBR Green PCR reaction program

Result analysis: Samples that failed sequencing were eliminated, and samples that successfully sequenced were retained for result analysis. According to the methylation of CpG sites in the sequencing peak graphs of different amplicons, the methylation values of all CpG sites were calculated. If the sequencing result of cytosine in CpG dinucleotide is thymine, it is unmethylated; if the sequencing result of cytosine in CpG dinucleotide is cytosine, it is completely methylated; if the sequencing result of cytosine in CpG dinucleotide has both cytosine and thymine (double peak), it is partially methylated. In this embodiment, if more than 95% of the cytosine in CpG dinucleotides in a polynucleotide is methylated, the lung cancer gene marker in this sample is considered to be methylation positive. Calculate the sensitivity and specificity of different lung cancer gene markers. Sensitivity (positive coincidence rate) = the proportion of samples with positive pathological results that detect methylation positive; specificity (negative coincidence rate) = the proportion of samples with negative pathological results that detect methylation negative. The methylation levels of the target area are shown in Table 5, and the area with sensitivity ≥85% and specificity ≥85% is selected for the next step of verification.

Table 5 Detection sensitivity and specificity of regions 1, 2, and 3 in tissue samples

As shown in Table 5, the detection sensitivity (positive coincidence rate) of region 1 and region 2 on tissue samples is 91.4% and 87.9% respectively, and the detection specificity (negative coincidence rate) is 87.9% and 86.2%, both of which meet the screening conditions of sensitivity ≥ 85% and specificity ≥ 85%, and can be verified in the next step. The detection sensitivity (positive coincidence rate) of region 3 on tissue samples is 84.5%, and the detection specificity (negative coincidence rate) is 81.0%, which does not meet the above screening conditions.

Example 3 Screening of probes for detecting methylation regions of lung cancer gene markers based on test set samples According to the above results, probes were designed in the methylation regions of FLJ32063 gene and SHOX2 gene (Table 6). All probes used were TaqMan probes, and the 5' end of the probe was a fluorescent reporter group, such as FAM, VIC, HEX, NED, ROX, TET, JOE, CY3, CY5, etc., and the 3' end was a fluorescent quencher group, such as TAMRA, MGB, BHQ, BHQ1, BHQ2, BHQ3, etc. In this embodiment, the 5' end fluorescent reporter group of the probe of the methylation region (region 1) of the FLJ32063 gene was ROX, and the 3' end fluorescent quencher group was BHQ1. The 5' end fluorescent reporter group of the probe of the methylation region (region 2) of the SHOX2 gene was VIC, and the 3' end fluorescent quencher group was MGB. The 5' end fluorescent reporter group of the probe of the internal reference gene ACTB was FAM, and the 3' end fluorescent quencher group was MGB.

Table 6 Primer probe combinations for methylation regions of lung cancer gene markers

The primer and probe combination with the best specificity and sensitivity was screened out on the test set samples. The methylation primer pairs and probes in Table 6 were synthesized artificially and diluted to a suitable concentration for standby use. Using the tissue sample DNA converted and purified by bisulfite as a template, the specific methylation primer pairs and probes in Table 6 were added respectively, and the detection primers and probes of the internal reference gene ACTB were added at the same time. The probe sequence SEQ ID NO.26: GGAGTGGTTTTTGGGTTTG was used to monitor the sample quality. The Ct value of the target sequence amplified by the detection primers and probes of ACTB was less than or equal to 34, indicating that the sample quality was qualified.

The TaqMan PCR amplification system was configured according to Table 7, and negative and positive controls were also required. The template for the negative control was TE buffer, and the template for the positive control was a mixture of 10 ³ copies/μL of a plasmid containing the target region sequence (sequence that was fully methylated and converted by bisulfite) and 10 ³ copies/μL of a plasmid containing the ACTB sequence that was fully methylated and converted by bisulfite. Then, qMSP was performed according to the procedure set in Table 8.

After the PCR reaction is completed, the baseline needs to be manually adjusted and the appropriate threshold value needs to be set. The baseline is usually 3 to 15 cycles of fluorescence signal. Samples that failed to be tested are eliminated according to the requirements of quality control, and the Ct value of qualified samples is read. The quality control standards are: 1) There is no amplification in the negative control; 2) The amplification curve of the positive control tube is S-shaped, and the Ct value of all genes is between 26 and 30; 3) The Ct value of the reference gene in the experimental tube is less than or equal to 34. If the above quality control requirements are not met, the sample needs to be retested. If the samples meet the above requirements, the results of the samples to be tested can be analyzed.

Table 7 TaqMan PCR reaction system

Table 8 TaqMan PCR program

For blood leukocyte samples and plasma samples, the positive judgment value is 45. If the Ct value of amplification using a pair of methylation primers and probes is ≤45, the sample is considered to be methylation-positive in this amplification region; if the Ct value of amplification using a pair of methylation primers and probes is >45, the sample is considered to be methylation-negative in this amplification region. The specific test results are shown in Table 9.

Table 9 Sensitivity and specificity of different primer-probe combinations on test set samples

In the target area where lung cancer is highly methylated, interference in blood leukocytes needs to be eliminated. In Table 9, the specificity of primer probe combination 2 and primer probe combination 5 in detecting healthy human blood leukocyte samples is 96.7% and 100%, respectively, indicating that primer probe combination 2 and primer probe combination 5 have good specificity in blood leukocyte samples. At the same time, when amplifying the methylated region (region 1) of the FLJ32063 gene, the sensitivity and specificity of primer probe combination 2 in detecting lung cancer on plasma samples are 83.3% and 96.7%, respectively, which is better than nucleic acid combination 1. When amplifying the methylated region (region 2) of the SHOX2 gene, compared with primer probe combination 3 and primer probe combination 4, primer probe combination 5 has the best performance in detecting lung cancer on plasma samples, with a sensitivity and specificity of 80.0% and 93.3%. Therefore, primer probe combination 2 and primer probe combination 5 are further verified to test clinical detection performance.

Example 4 Kit for detecting lung cancer plasma samples based on qMSP method

In order to achieve non-invasive detection, further verify and improve the sensitivity of the kit for diagnosing lung cancer blood samples, this embodiment blinded 67 lung cancer blood samples (including 27 lung adenocarcinomas, 20 lung squamous cell carcinomas, 6 large cell lung cancers, and 14 small cell lung cancers) and 198 healthy human blood samples according to the in vitro diagnostic reagent clinical trial protocol, and then the test operator conducted the test, interpreted it according to the interpretation criteria, and compared the obtained test results with the pathological results (gold standard), thereby verifying the clinical effectiveness of the lung cancer gene markers. The specific detection process is the same as in Example 3, and the test results are shown in Table 10.

Table 10 Performance of primer-probe combination for diagnosing lung cancer plasma samples by qMSP

It can be seen from Table 10 that primer probe combination 2 and primer probe combination 5 both have good detection effects on lung cancer plasma samples. Among them, primer probe combination 2 has a total sensitivity of 83.6% in diagnosing lung cancer plasma samples by detecting the methylation level of region 1, and a specificity of 92.9% in detecting healthy human plasma samples, which is better than primer probe combination 5.

For lung cancer of different histopathological classifications, primer probe combination 2 and primer probe combination 5 both have certain detection effects, and the sensitivity of detecting plasma samples of lung adenocarcinoma, lung squamous cell carcinoma, large cell lung cancer, and small cell lung cancer can reach 85.2%, 90.0%, 66.7%, and 85.7%, respectively.

In view of the differences in sensitivity of primer probe combination 2 and primer probe combination 5 in detecting different histopathological classifications of lung cancer, combined detection may have a synergistic effect, thereby improving the sensitivity of lung cancer detection. When jointly diagnosing lung cancer, the criteria for judging positive samples are: if at least one of the two target regions in a sample to be tested is methylation-positive, then the sample is a lung cancer-positive sample; if both target regions in a sample to be tested are methylation-negative, then the sample is a lung cancer-negative sample. The specific test results are shown in Table 11.

Table 11 Performance of primer-probe combination 2 and primer-probe combination 5 in combined diagnosis of lung cancer plasma samples

In Table 11, the total sensitivity of primer probe combination 2 and primer probe combination 5 in the joint diagnosis of lung cancer plasma samples is 86.6%. Among them, for lung cancers of different histopathological classifications, the sensitivity of primer probe combination 2 and primer probe combination 5 in the joint diagnosis of lung adenocarcinoma can reach 88.9%, which is significantly better than the sensitivity of a single primer probe combination in detecting lung adenocarcinoma. The specificity of primer probe combination 2 and primer probe combination 5 in the joint detection of healthy human plasma samples is 90.9%, which is slightly lower than a single primer probe combination, but still at a relatively high level.

It will be easily understood by those skilled in the art that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A primer pair for lung cancer detection, characterized in that it includes a first primer pair for detecting the methylation level of a target region on the CpG island of the FLJ32063 gene. 2. The primer pair of claim 1, further comprising a second primer pair for detecting the methylation level of the target region on the CpG island of the SHOX2 gene. 3. The primer pair according to claim 2, characterized in that, taking GRCh38.p14 as the reference genome, the target region on the CpG island of the FLJ32063 gene is selected from the full length or partial region of the negative chain of Chr2:199470636-199471036; The target region on the CpG island of the SHOX2 gene is selected from the full length or partial region of the positive chain of Chr3:158103418-158103818. 4. The primer pair according to claim 2 or 3, wherein the first primer pair includes the first methylation level for detecting the methylation level of the full-length or partial region of the negative chain of Chr2:199470636-199471036. A methylation primer pair; the second primer pair includes a second methylation primer pair for detecting the methylation level of the full-length or partial region of the positive chain of Chr3:158103418-158103818; The nucleotide sequence of the first methylation primer pair is shown in SEQ ID NO.7-8, and the nucleotide sequence of the second methylation primer pair is shown in SEQ ID NO.11-12. 5. The primer pair according to claim 4, wherein the first primer pair further includes a first non-methylation level for detecting the methylation level of the full-length or partial region of the negative chain of Chr2:199470636-199471036. A methylated primer pair; the second primer pair also includes a second non-methylated primer pair for detecting the methylation level of the full length or partial region of the positive chain of Chr3:158103418-158103818; The nucleotide sequence of the first unmethylated primer pair is as shown in SEQ ID NO.9-10, and the nucleotide sequence of the second unmethylated primer pair is as shown in SEQ ID NO.13-14 Show. 6. A primer-probe combination for lung cancer detection, characterized in that it includes the primer pair according to any one of claims 1 to 4, and further includes a detection probe. 7. The primer-probe combination according to claim 6, characterized in that it includes at least one group of the following combinations: A first primer-probe combination, a first methylation primer pair shown in SEQ ID NO.7-8 and a first probe shown in SEQ ID NO.21; A second primer-probe combination, a first methylation primer pair shown in SEQ ID NO.7-8 and a second probe shown in SEQ ID NO.22; A third primer-probe combination, a second methylation primer pair shown in SEQ ID NO. 11-12 and a third probe shown in SEQ ID NO. 23; The fourth primer-probe combination is the second methylation primer pair shown in SEQ ID NO. 11-12 and the fourth probe shown in SEQ ID NO. 24; The fifth primer-probe combination is the second methylation primer pair shown in SEQ ID NO. 11-12 and the fifth probe shown in SEQ ID NO. 25. 8. A kit for lung cancer detection, characterized in that it includes the primer pair according to any one of claims 1 to 5 or the primer-probe combination according to claim 6 or 7, and also includes a reference gene One or more of detection primer pairs and probes, nucleic acid extraction reagents, nucleic acid purification reagents, methylation conversion reagents, amplification reagents, positive control substances and negative control substances. 9. Application of the primer pair according to any one of claims 1 to 5, the primer-probe combination according to claim 6 or 7, or the kit according to claim 8 in the preparation of lung cancer diagnostic products. 10. The application according to claim 9, wherein the diagnosis includes early differential diagnosis of lung cancer, residual assessment and dynamic monitoring of minimal lesions, auxiliary judgment of recurrence and prognosis of lung cancer, drug efficacy evaluation and drug resistance monitoring; The lung cancer diagnostic product includes at least one of a kit, a chip, a membrane strip, and a protein array.