CN117568480A - Reagent and kit for breast cancer or precancerous lesion detection and application - Google Patents

Abstract

The invention belongs to the field of molecular biology detection, and particularly relates to a reagent for detecting breast cancer or precancerous lesions, a kit and application thereof. The reagent and the kit for detecting the breast cancer or the precancerous lesions can detect or diagnose the breast cancer or the precancerous lesions by detecting the methylation level of a target sequence (the full length or partial region of the Chr10: 101140210-101140778) in a sample, effectively distinguish breast cancer patients from healthy people, and have important significance for early screening of the breast cancer, in particular for early screening of the precancerous lesions.

Description

Reagent, kit and application for detecting breast cancer or precancerous lesions

Technical field

The invention belongs to the field of molecular biology detection, and more specifically, relates to a reagent, a kit and an application for detecting breast cancer or precancerous lesions.

Background technique

Breast cancer is one of the most common malignant tumors in women. Lobular carcinoma in situ (LCIS) is a non-invasive breast cancer in situ. Data show that women diagnosed with lobular carcinoma in situ have an 18-fold increased risk of developing invasive lobular cancer, and a 3- to 4-fold increased risk of invasive ductal carcinoma. Among women diagnosed with lobular dysplasia and lobular carcinoma in situ, the probability of developing invasive cancer is 5% to 32%. Lobular carcinoma in situ is considered a precancerous lesion of invasive breast cancer that deserves attention.

Lobular carcinoma in situ usually does not have any clinical symptoms, such as breast lumps, nipple discharge, nipple swelling and skin changes, and sometimes only hyperplasia-like changes. Therefore, lobular carcinoma in situ is usually difficult to detect and diagnose, and most patients go to the hospital only when they have obvious clinical symptoms. But at the same time, lobular carcinoma in situ has the characteristics of long cancer progression cycle, bilateral breast and multiple quadrant onset. If lobular carcinoma in situ can be detected and diagnosed as early as possible, it will be of great significance for breast-conserving treatment and preventing deterioration into invasive cancer.

At present, there is no mature research on methylation detection of breast cancer precancerous lesions, especially lobular carcinoma in situ.

Contents of the invention

In view of the shortcomings of the existing technology, the purpose of the present invention is to provide a reagent, a kit and an application for detecting breast cancer or precancerous lesions, so as to improve the existing technology's insufficient diagnostic methods for breast cancer and the detection of breast cancer. The detection rate of anterior lesions is low, especially the poor detection sensitivity of lobular carcinoma in situ.

In order to achieve the above object, the present invention provides a reagent for detecting breast cancer or precancerous lesions, which is a reagent for detecting the methylation level of a target sequence in a sample, using GRCh38.p14 as the reference genome, the The target sequence includes the full length or partial region of Chr10:101140210-101140778.

Preferably, the partial region of Chr10:101140210-101140778 is selected from at least one of target sequences 1 to 4; wherein the target sequence 1 is selected from the positive chain of Chr10:101140230-101140416, and the target sequence 2 is selected from Chr10: 101140374-101140492 negative chain, the target sequence 3 is selected from the positive chain of Chr10:101140639-101140778, and the target sequence 4 is selected from the negative chain of Chr10:101140210-101140348.

Preferably, the target sequence is selected from at least one of the nucleotide sequences shown in SEQ ID NO. 1 to 4.

Further preferably, the target sequence is selected from any combination of the following:

The combination of any one of SEQ ID NO.2~4 and SEQ ID NO.1;

A combination of any one of SEQ ID NO.3 or SEQ ID NO.4 and SEQ ID NO.2;

A combination of SEQ ID NO.3 and SEQ ID NO.4.

Preferably, the reagents include a primer pair for detecting the methylation level of the target sequence.

Further preferably, the primer pair is selected from at least one group of the following primer pairs:

A first primer pair for detecting the methylation level in the SEQ ID NO.1 region, a second primer pair for detecting the methylation level in the SEQ ID NO.2 region, and a second primer pair for detecting the SEQ ID NO.3 The third primer pair is used to detect the methylation level in the region, and the fourth primer pair is used to detect the methylation level in the SEQ ID NO. 4 region.

Preferably, the primer pair is selected from at least one of the following (1)-(4) groups of primer pairs:

(1) The first primer pair shown in SEQ ID NO. 9-10;

(2) The second primer pair shown in SEQ ID NO. 12-13;

(3) The third primer pair shown in SEQ ID NO. 15-16;

(4) The fourth primer pair shown in SEQ ID NO. 18 to 19.

Preferably, the reagent further includes a detection probe corresponding to the primer pair, and the detection probe is selected from at least one of the following detection probes:

The detection probe used to detect the methylation level in the SEQ ID NO.1 region is shown in SEQ ID NO.11; the detection probe used to detect the methylation level in the SEQ ID NO.2 region is shown in SEQ ID NO.14 is shown; the detection probe used to detect the methylation level in the SEQ ID NO.3 region is shown in SEQ ID NO.17; the detection probe used to detect the methylation level in the SEQ ID NO.4 region The probe is shown in SEQ ID NO. 20.

The present invention also provides a kit for detecting breast cancer or precancerous lesions, which includes the reagent.

Preferably, the kit also includes one or more of nucleic acid extraction reagents, nucleic acid purification reagents, methylation conversion reagents, PCR reaction reagents, quality control products, detection primer pairs of internal reference genes, and detection probes of internal reference genes. kind.

The present invention also provides the use of the reagent or the kit in preparing diagnostic products for breast cancer or precancerous lesions.

Preferably, the breast cancer includes at least one of lobular invasive carcinoma, ductal invasive carcinoma and lobular carcinoma in situ.

Preferably, the breast cancer diagnostic product includes one or more of primers, probes, kits, chips, sequencing libraries, membrane strips and protein arrays.

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

(1) The present invention provides a reagent and kit for detecting breast cancer or precancerous lesions. By detecting the methylation level of the target sequence (full length or partial region of Chr10:101140210-101140778) in the sample, it can Detection or diagnosis of breast cancer or precancerous lesions, effectively distinguishing breast cancer patients from healthy people, is of great significance for early screening of breast cancer, especially early screening of precancerous lesions.

(2) The kit provided by the present invention has good detection effects on breast cancer and breast cancer precancerous lesions. The sensitivity for detecting breast cancer plasma samples can reach 92.86%, and the sensitivity for detecting lobular carcinoma in situ plasma samples can reach 89.29%. , can effectively avoid the detection of false positive results, and can be used for the auxiliary diagnosis of lobular carcinoma in situ. In addition, the specificity of this kit for detecting plasma samples from healthy people is up to 97.89%, which can effectively avoid the detection of false negative results and improve detection accuracy.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with examples. It should be understood that the specific embodiments described here are only used to explain the present invention and are not intended to limit the present invention.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which the invention belongs. The terminology used herein in the description of the invention is for the purpose of describing specific embodiments only and is not intended to limit the invention.

The term "diagnosis" refers to determining the health status of a subject, covering aspects such as detecting the presence or absence of disease, response to treatment, assessment of recurrence risk, assessment of cancer risk and degree of cancer, and prognosis judgment. In some cases, the term "diagnosis" refers to being used as a single factor to determine, verify or confirm a patient's clinical status, and "auxiliary diagnosis" is used to provide various information to assist judgment in the process of determining or verifying a patient's clinical status. Not as the only determining indicator. In some embodiments, "detecting" breast cancer refers to detecting the presence or absence of the disease, that is, determining whether the subject has breast 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 usually more than ten. The exact size will depend on many factors, which in turn will depend on the ultimate function or use of the oligonucleotide. Oligonucleotides can be produced by any means, 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" refers to a form of chemical modification of DNA that can alter genetic expression without changing the DNA sequence. DNA methylation refers to the covalent binding of a methyl group at carbon position 5 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 interaction between DNA and proteins, thereby regulating gene expression.

The term "methylation level" refers to whether cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/ratio/percentage of methylation, which represents a qualitative concept. It also represents 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, comparisons can be made based on the Ct values detected in the samples; in some cases, the proportion of gene methylation in the sample can be calculated, that is, the number of methylated molecules/(number of methylated molecules + non-methylated Number of molecules) × 100, and then compare; in some cases, statistical analysis and integration of each indicator is required to obtain the final judgment indicator.

The term "primer" refers to an oligonucleotide that can be used in an amplification method (such as polymerase chain reaction, PCR) to amplify a target sequence based on a polynucleotide sequence corresponding to a target gene or a partial region 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, source of primer, and method used. For example, for diagnostic and prognostic applications, oligonucleotide primers typically contain at least 10, 15, 20, 25, or more nucleotides, but may contain fewer nucleotides, depending on the complexity of the target sequence. In the present disclosure, the term "primer" refers to a pair of primers capable of hybridizing to both strands of a target DNA molecule or to regions of the target DNA molecule flanking the nucleotide sequence to be amplified. A "primer pair" refers to a set of upstream primers and downstream primers.

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

The term "methylation-specific fluorescence quantitative PCR (qMSP)" is an experimental technology that combines fluorescence quantitative PCR technology and methylation-specific PCR technology. This technology is also based on the sequence differences of DNA with different methylation states after bisulfite conversion to design appropriate primer pairs to distinguish methylated and unmethylated sequences, but the final detection index of qMSP is a fluorescent signal. Therefore, in the qMSP reaction system, in addition to adding methylation detection primers, it is also necessary to add fluorescent probes or fluorescent dyes. 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-stage cancer patients. Moreover, this technology does not require gel electrophoresis detection and is easier to operate. In the present invention, methylation primers are added when performing real-time quantitative methylation-specific PCR. If the Ct value meets the requirements (for example, Ct≤38 in tissue samples), it indicates that the target sequence is methylated.

The term "TaqMan probe" refers to an oligonucleotide sequence containing a 5' fluorophore and a 3' quencher group. When the probe binds to the corresponding site on the DNA, the probe does not fluoresce due to the presence of a quenching group near the fluorophore. During the amplification process, if the probe binds to the amplified strand, the 5'-3' exonuclease activity of DNA polymerase (such as Taq enzyme) will digest the probe, and the fluorescent group will move away from the quenching group. Its energy is not absorbed, that is, a fluorescent signal is generated. After each PCR cycle, the fluorescence signal also has a synchronous exponential growth process like the target fragment.

The present invention provides a reagent for detecting breast cancer or precancerous lesions. It is a reagent for detecting the methylation level of a target sequence in a sample. GRCh38.p14 is used as a reference genome. The target sequence includes Chr10:101140210. The full length or part of -101140778. It can be understood that when detecting the methylation level of the above-mentioned Chr10:101140210-101140778, its full-length region can be detected, and its partial region can also be detected.

In some embodiments, the partial region of the above-mentioned Chr10:101140210-101140778 is selected from at least one of the target sequences 1 to 4, wherein the above-mentioned target sequence 1 is selected from the positive chain of Chr10:101140230-101140416, and the above-mentioned target sequence 2 is selected from the group consisting of Chr10:101140374 -101140492 negative chain, the above target sequence 3 is selected from the positive chain of Chr10:101140639-101140778, and the above target sequence 4 is selected from the negative chain of Chr10:101140210-101140348.

In some embodiments, the above target sequence is selected from at least one of the following sequences: target sequence 1, target sequence 2, target sequence 3 and target sequence 4. Taking GRCh38.p14 as the reference genome, the specific information of target sequences 1 to 4 is shown in the table below.

target sequence Chromosomal location (GRCh38.p14) Nucleotide sequence target sequence 1 Chr10:101140230-101140416 positive chain SEQ ID NO.1 target sequence 2 Chr10:101140374-101140492 negative chain SEQ ID NO.2 Target sequence 3 Chr10:101140639-101140778 positive chain SEQ ID NO.3 target sequence 4 Chr10:101140210-101140348 negative chain SEQ ID NO.4

The inventor found that target sequences 1 to 4 can achieve better differentiation between breast cancer samples and normal samples. Further experiments found that when the above target sequences in the present invention were combined and the methylation levels of the target sequence combinations were detected, some targets The performance of sequence combinations in detecting breast cancer or precancerous lesions is improved relative to detection of a single target sequence.

The preferred target sequence is selected from any of the following combinations: a combination of any one of target sequences 2 to 4 and target sequence 1; a combination of any one of target sequence 3 or target sequence 4 and target sequence 2; target sequence 3 in combination with target sequence 4.

In some embodiments, the preferred target sequence is selected from any combination of the following:

The combination of any one of SEQ ID NO.2~4 and SEQ ID NO.1;

A combination of any one of SEQ ID NO.3 or SEQ ID NO.4 and SEQ ID NO.2;

A combination of SEQ ID NO.3 and SEQ ID NO.4.

It can be understood that when detecting the methylation level of a target sequence, the full-length region of any of the above target sequences can be detected, or a partial region of any of the above target sequences can be detected.

In some embodiments, the above-mentioned reagents include a primer pair for detecting the methylation level of the above-mentioned target sequence.

Optionally, the above primer pair is selected from at least one group of the following primer pairs: a first primer pair for detecting the methylation level in the SEQ ID NO.1 region, a first primer pair for detecting the methylation level in the SEQ ID NO.2 region A second primer pair for methylation level, a third primer pair for detecting methylation level in SEQ ID NO.3 region, and a fourth primer pair for detecting methylation level in SEQ ID NO.4 region right.

Optionally, the above-mentioned primer pairs are selected from at least one group of the following (1)-(4) groups of primer pairs:

(1) The first primer pair shown in SEQ ID NO. 9-10;

(2) The second primer pair shown in SEQ ID NO. 12-13;

(3) The third primer pair shown in SEQ ID NO. 15-16;

(4) The fourth primer pair shown in SEQ ID NO. 18 to 19.

It should be noted that if a primer pair has at least 85% (for example, 85%) of the nucleotide sequence shown in the above primer pair (first primer pair, second primer pair, third primer pair, fourth primer pair) %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, etc.) sequence identity or above , and the primer pair also has a certain diagnostic function for breast cancer or precancerous lesions (the specificity or sensitivity is equivalent to or slightly lowered or slightly improved or significantly improved compared with the primer pair of the present application), and is also protected by the present invention. within the range.

In some embodiments, the above reagents use methods to detect the methylation level of target sequences in samples, including but not limited to methylation-specific PCR (MSP), sulfide sequencing (BSP), and methylation-sensitive random primer polymerase chain reaction. (MS AP-PCR), restriction enzyme-based microarray analysis, methylation-sensitive single nucleotide primer extension (Ms-SNuPE), methylation-sensitive DNA restriction enzyme 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), methylation specificity 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 microarray analysis based on methylation-specific giant magnetoresistive sensors.

In some embodiments, the above-mentioned samples include, but are not limited to, individual cells, tissues, organs and/or biological fluids obtained by any method known to those skilled in the art. In some embodiments, the sample is selected from the group consisting of histological sections, tissue biopsies, paraffin-embedded tissue, body fluids, surgical resection samples, isolated blood cells, cells isolated from blood, and any combination thereof. In some embodiments, the body fluid is selected from the group consisting of whole blood, serum, plasma, and any combination thereof.

In some embodiments, the above-mentioned reagent further includes a detection probe corresponding to the above-mentioned primer pair.

Optionally, the above-mentioned detection probe is selected from at least one of the following detection probes: the detection probe used to detect the methylation level in the SEQ ID NO.1 region is as shown in SEQ ID NO.11; The detection probe used to detect the methylation level in the SEQ ID NO.2 region is shown in SEQ ID NO.14; the detection probe used to detect the methylation level in the SEQ ID NO.3 region is shown in SEQ ID NO. 17; the detection probe used to detect the methylation level in the SEQ ID NO.4 region is shown in SEQ ID NO.20.

The present invention also provides a kit for detecting breast cancer or precancerous lesions, which includes the above reagents.

In some embodiments, the above kit also includes one of nucleic acid extraction reagents, nucleic acid purification reagents, methylation conversion reagents, PCR reaction reagents, quality control products, detection primer pairs of internal reference genes, detection probes of internal reference genes, or Various.

The present invention has no special limitations on the above-mentioned nucleic acid extraction reagents and nucleic acid purification reagents. Any reagent reported in the prior art that can realize nucleic acid extraction and nucleic acid purification can be used. In some embodiments, the above-mentioned nucleic acid extraction reagents may include, but are not limited to, any one or more of lysis buffer, binding buffer, washing buffer and elution buffer.

In some embodiments, the above-mentioned methylation conversion reagent is used to deaminate and convert unmethylated cytosine in DNA into uracil, while the methylated cytosine remains unchanged. The present invention has no special restrictions on the methylation conversion reagent conversion agent. Any reagent reported in the prior art that can realize the conversion of cytosine to uracil can be used, such as hydrazine salt, bisulfite (such as sodium bisulfate, potassium bisulfate). , ammonium bisulfite) and bisulfite (such as sodium bisulfite, potassium bisulfite, calcium bisulfite, magnesium bisulfite, aluminum bisulfite, cesium bisulfite, ammonium bisulfite, etc.) one or more.

In some embodiments, the above-mentioned PCR reaction reagents include but are not limited to amplification buffer, dNTPs, DNA polymerase and Mg ²⁺ . In some embodiments, the above-mentioned quality control products include positive reference products and negative reference products, where the positive reference product refers to a target sequence containing methylation, which is used to monitor the detection performance of the reagents in the kit. The above-mentioned negative reference refers to a target sequence that does not contain methylation and is used to monitor whether the experiment is contaminated.

In some embodiments, the above-mentioned internal reference gene can be but is not limited to ACTB. In specific embodiments of the present invention, the nucleotide sequence of the detection primer pair of the internal reference gene ACTB is shown in SEQ ID NO. 21-22, and the nucleotide sequence of the detection probe is shown in SEQ ID NO. 23. It can be understood that in other embodiments, other genes can also be selected as internal reference genes. In this case, the internal reference primer pairs can be designed accordingly.

In some embodiments, the detection probe used in the present invention is a fluorescent probe, such as a TaqMan probe. In the present invention, the detection probe corresponding to the above-mentioned primer pair and the detection probe of the above-mentioned internal reference gene contain a fluorescent reporter group and a fluorescent quenching group, wherein the 5' end of the detection probe contains a fluorescent reporter group, and the above-mentioned The fluorescent reporter group of each probe is independently selected from any one of FAM, ROX, VIC, HEX, NED, TET, JOE, Cy3 and Cy5; the 3' end of the detection probe contains a fluorescence quenching group, The fluorescence quenching group of each of the above probes is independently selected from any one of TAMRA, MGB, BHQ, BHQ1, BHQ2 and BHQ3. Examples of the fluorescent reporting group and the fluorescent quenching group of each probe independently include, but are not limited to, those listed above.

The present invention also provides the use of the above-mentioned reagents or the above-mentioned kits in preparing diagnostic products for breast cancer or precancerous lesions. Breast cancer diagnostic products include, but are not limited to, one or more of primers, probes, kits, chips, sequencing libraries, membrane strips and protein arrays. Alternatively, the above-mentioned products may be in the form of freeze-dried powder, solution, suspension, emulsion and other product forms.

In some embodiments, the breast cancer includes at least one of lobular invasive carcinoma, ductal invasive carcinoma, and lobular carcinoma in situ.

In addition, the present invention also provides a method for detecting breast cancer of different pathological stages by detecting the methylation level of the target sequence in the sample, which includes the following steps: extracting DNA from the subject's plasma sample, using a methylation conversion reagent Transform it, then purify the transformed DNA, and use the kit provided by the invention to perform qPCR amplification, and then determine whether the sample to be tested is breast cancer or precancerous lesions by detecting the methylation level of the target sequence. Positive.

Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available or can be prepared by methods known in the art. The above technical solution will be described in detail below with reference to specific embodiments.

Example 1

Using GRCh38.p14 as the reference genome and the DNA molecules of the four target sequences of the target sequence Chr10:101140210-101140778 as templates, a methylation primer pair and detection probe suitable for fluorescence quantitative PCR amplification were designed. The specific experimental steps are as follows:

1. Sample DNA extraction

The tissue samples were all formalin-soaked and paraffin-embedded samples, and QIAamp DNA FFPE TissueKit (Cat: 56404) was used to extract DNA from the tissue samples.

Plasma samples were extracted from plasma cfDNA using the magnetic bead serum/plasma cell-free DNA (cfDNA) extraction kit (DP709) of Tiangen Biochemical Technology (Beijing) Co., Ltd.

Please refer to the kit instructions for specific DNA extraction operations.

2. Transformation and purification of sample DNA

The kits used for the transformation and purification of sample DNA were all nucleic acid conversion reagents from Wuhan Amison Life Technology Co., Ltd. (Ehan Mechanical Preparation No. 20200843). For specific operating steps, please refer to the kit instructions.

3. Methylation-specific fluorescence quantitative PCR

1) Methylation primer pair and detection probe design:

Using the bisulfite-converted DNA sequence as a template, methylation primer pairs and detection probes were designed to detect the methylation level of specific regions. The DNA sequence of the target sequence and the sequence after bisulfite conversion are shown in Table 1. The methylation primer pair and detection probe sequence used to amplify the target sequence are shown in Table 2.

Table 1 Base sequences of target sequences 1 to 4

Table 2 Primer pairs of target sequences 1 to 4 and nucleotide sequences of detection probes

2) PCR amplification:

The primer pairs and detection probes used in the PCR reaction system are shown in Table 2. The templates used are DNA extracted from various samples and converted by bisulfite, as shown in Table 1. When detecting the methylation level of a single target sequence, in each PCR tube, in addition to the primer pair and detection probe of the target sequence, it is also necessary to add the primer pair and detection probe of the internal reference gene ACTB. Among them, the upstream amplification primer SEQ ID NO.21 of the internal reference gene ACTB is: 5'-AAGGGTGGTTGGGTGGTTGTTTTG-3', the downstream amplification primer SEQ ID NO.22 is: 5'-AATAACACCCCCACCCTGC-3', and the detection probe SEQ ID NO .23 is: 5'-GGAGTGGTTTTTGGGTTTG-3'.

If the methylation level of a dual target sequence combination is detected, the detection primer pair and detection probe corresponding to the dual target sequence combination need to be added to amplify the two target sequences simultaneously. In this case, there are 6 combination methods according to the different combinations of dual-target sequences detected, as shown in Table 3. During amplification, in addition to the necessary reaction components and templates, each PCR tube also needs to add two detection primer pairs and detection probes corresponding to specific sequences, as well as a detection primer pair and detection probe for the internal reference gene ACTB.

In this application, the above-mentioned detection probes are all TaqMan probes. The fluorescence reporter groups at the 5' end of the detection probes for target sequences 1 to 4 are FAM, ROX, Cy5, and Cy3 respectively, and the fluorescence quenching group at the 3' end is MGB. The fluorescent reporter group at the 5' end of the ACTB gene detection probe is VIC, and the fluorescent quenching group at the 3' end is BHQ-1. The reaction system and reaction procedures are shown in Table 4 and Table 5.

Table 3 Combination methods of dual target sequence combinations

Dual target sequence combination Desired combination of primer pairs and probes target sequence 1 + target sequence 2 SEQ ID NO.9～11+SEQ ID NO.12～14 target sequence 1 + target sequence 3 SEQ ID NO.9～11+SEQ ID NO.15～17 target sequence 1 + target sequence 4 SEQ ID NO.9～11+SEQ ID NO.18～20 target sequence 2 + target sequence 3 SEQ ID NO.12～14+SEQ ID NO.15～17 target sequence 2 + target sequence 4 SEQ ID NO.12～14+SEQ ID NO.18～20 target sequence 3 + target sequence 4 SEQ ID NO.15～17+SEQ ID NO.18～20

Table 4 qPCR reaction system

Components Specification Volume(μL) Platinum II PCR Buffer 5× 5 dNTPs 10mM 0.5 Primers upstream of each target sequence 10μM 0.5 Downstream primers for each target sequence 10μM 0.5 Probes for each target sequence 10μM 0.5 ACTB upstream primer 10μM 0.5 ACTB downstream primer 10μM 0.5 ACTB detection probe 10μM 0.5 Platinum ^TM II Taq Hot-Start DNA Polymerase / 0.5 Sample DNA to be tested / 5 purified water / Replenish to 25

Table 5 qPCR reaction program

3) Negative control and positive control:

When performing PCR reaction to detect samples, the negative control and positive control should also be tested at the same time. The DNA template in the negative control tube is TE buffer. The preparation method of the DNA template of the positive control tube is: artificially synthesize the sequence corresponding to the ACTB gene amplification region that has been completely converted by bisulfite, and clone it into the vector to form an artificially synthesized plasmid; The completely transformed target sequences are artificially synthesized and cloned into vectors respectively to form artificial synthetic plasmids. If only the methylation level of a single region target sequence is detected, the positive control DNA template is 10 ³ copies/μl of a synthetic plasmid containing transformed ACTB, and 10 ³ copies/μl of a synthetic plasmid containing the detection region. are mixed at a ratio of 1:1; if the methylation level of the target sequence combination is detected, the positive control DNA template is 10 ³ copies/microliter of a synthetic plasmid containing transformed ACTB and a synthetic plasmid containing each target sequence respectively. For 10 ³ copies/µl, mix equal volumes.

4) Ct value reading:

After the PCR is completed, adjust the baseline. Set the minimum Ct value of the sample in a PCR 1-2 cycles before the fluorescence value as the baseline value. Set the threshold at the inflection point of the S-type amplification curve to obtain the Ct value of each gene in the sample. .

5)Quality control:

The negative control should have no amplification, the positive control should have an obvious exponential growth phase, and the Ct value of each gene in the positive control should be between 26 and 30. The Ct value of the internal reference gene of the sample to be tested should be ≤35. If the negative control, positive control and internal reference gene all meet the above requirements, it means that the experiment is valid and the next step of determining the sample results can be carried out. Otherwise, the current experiment will be invalid and must be retested.

4.PCR result analysis

The methylation level of the sample to be tested is determined based on the Ct value detected by each target sequence. For tissue samples, if the Ct value of a certain target sequence amplified is ≤38, this region in the sample is considered to be methylation positive. If the Ct value of a certain target sequence amplified is >38, this region in the sample is considered to be methylation positive. It is methylation negative. For plasma samples, if the Ct value of a certain target sequence amplified is ≤45, the sample is considered to be methylation positive in this region. If the Ct value of a certain target sequence amplified is >45, the sample is considered to be methylation positive in this region. The region is methylation negative. When detecting a single target sequence, if the sample to be tested is methylation positive in this region, the sample is a cancer positive sample; if the sample to be tested is methylation negative in this region, the sample is a cancer negative sample. When detecting a target sequence combination, if the sample to be tested is methylation-positive in at least one target sequence that constitutes the target sequence combination, the sample is a cancer-positive sample. If the sequences are all negative for methylation, the sample is a cancer-negative sample.

Example 2

In this example, a total of 63 cancer tissue samples were collected from patients diagnosed with breast cancer through pathological examination (35 cases of lobular invasive cancer, 28 cases of ductal invasive cancer) and 63 corresponding adjacent normal tissue samples. Breast lobular carcinoma in situ samples There were 37 samples and 37 adjacent normal tissues. The collection process of all samples was approved by the Ethics Committee, all volunteers signed informed consent forms, and all samples were anonymized.

The above samples were subjected to genomic DNA extraction and bisulfite conversion according to the method provided in Example 1, and the converted DNA was used as a template to detect the methylation status of target sequences 1 to 4 and the target sequence combination. The test results are shown in Table 6 and Table 7.

Table 6 Methylation status of target sequences 1 to 4 in tissue samples and diagnostic sensitivity and specificity

Table 7 Methylation status of target sequence combinations in tissue samples and diagnostic sensitivity and specificity

As can be seen from Table 6, the methylation level of target sequences 1 to 4 in breast cancer tissue samples (the number of methylation-positive cases) is significantly higher than its methylation level (methylation level) in adjacent normal tissue samples. (number of positive samples), target sequences 1 to 4 were hypermethylated in breast cancer tissue samples. Each target sequence has a good detection effect on breast cancer tissue samples, and the detection sensitivity is greater than 80%, up to 92.06%, and the detection sensitivity is excellent. At the same time, all target sequences have certain detection effects on breast cancer precancerous lesions (lobular carcinoma in situ), and the detection sensitivity can reach 70.27%. All target sequences have good detection specificity for adjacent normal tissue samples, with detection specificities greater than or equal to 81%. Among them, target sequence 1 has the best detection specificity, reaching 91%.

In Table 7, the sensitivity range of any pairwise combination of the above target sequences for detecting breast cancer tissue samples is 88.89% to 95.24%, which is mostly higher than the sensitivity of single target sequence detection; its sensitivity for detecting breast cancer precancerous lesion tissue samples is 67.57% ~78.38%, mostly higher than the sensitivity of single target sequence detection; its specificity range for detecting adjacent normal tissue samples is 80% ~ 86%, which is only slightly lower than single target sequence detection, but still at the higher level. Overall, among the above target sequence combinations, the comprehensive detection performance of the target sequence 2 + target sequence 4 combination is better. Its sensitivity for detecting breast cancer tissue samples is 95.24%, and its sensitivity for detecting breast cancer precancerous lesion tissue samples is 95.24%. 78.38%. Compared with target sequence 2 and target sequence 4, the detection performance of a single target sequence is significantly improved; its specificity range for detecting adjacent normal tissue samples is 83%, which is at a high level.

Example 3

This example uses the methylation fluorescence quantitative PCR method to analyze the performance of a single target sequence and a combination of target sequences in diagnosing plasma samples from patients with breast cancer or precancerous lesions.

A total of 56 plasma samples were collected from patients with invasive breast cancer (30 cases with lobular invasive cancer and 26 cases with ductal invasive cancer), 28 plasma samples from lobular carcinoma in situ, and 95 plasma samples from healthy individuals. All samples were collected by professional medical staff and approved by the ethics committee. All volunteers signed informed consent forms, and all samples were anonymized.

The above plasma samples were subjected to genomic DNA extraction and bisulfite conversion according to the method provided in Example 1, and the converted DNA was used as a template to detect the methylation status of target sequences 1 to 4 and the target sequence combination. The test results are shown in Table 8 and Table 9.

Table 8 Methylation status of target sequences 1 to 4 in plasma samples and diagnostic sensitivity and specificity

Table 9 Methylation status of target sequence combinations in plasma samples and diagnostic sensitivity and specificity

In Table 8, breast cancer patients and healthy people can be effectively distinguished by detecting the methylation levels of target sequences 1 to 4. Specifically, the sensitivities of detecting breast cancer and breast cancer precancerous lesions in plasma samples by detecting the methylation levels of each target sequence are 80.36% to 89.28% and 67.86% to 75% respectively, among which target sequence 2 and target sequence 3 detect breast cancer. The sensitivity of cancer plasma samples is greater than 85%, and the sensitivity of detecting breast cancer precancerous lesions in plasma samples reaches 75%. It can effectively avoid the detection of false positive results, and has better detection performance of breast cancer precancerous lesions. The specificity of detecting healthy human plasma samples by detecting the methylation levels of target sequences 1 to 4 is greater than 90%, up to 97.89%, which can effectively avoid the detection of false negative results and improve detection accuracy.

In this embodiment, any pairwise combination of the above target sequences is detected. As can be seen from Table 9, the sensitivity range of the dual target sequence combination for detecting breast cancer plasma samples is 85.71% to 92.86%, including target sequence 1 + target sequence 4, target sequence 2 + target sequence 4, and target sequence 3 + target sequence 4 The sensitivity for detecting breast cancer plasma samples was greater than 91%. The sensitivity of dual target sequence combination detection of breast cancer precancerous lesions in plasma samples ranges from 71.43% to 89.29%, which is significantly improved compared to single target sequence detection or is the same as single target sequence detection, in which target sequence 1 + target sequence 3. The sensitivity of target sequence 2 + target sequence 4 in detecting breast cancer precancerous lesions in plasma samples is both greater than 85%. The specificity range of dual-target sequence combination detection of healthy human plasma samples is 87.37% to 94.74%, which is at a relatively high level. Taken together, compared with single target sequence detection, the combination method shown in Table 9 shows better results in the detection of breast cancer, breast cancer precancerous lesions (lobular carcinoma in situ), and healthy human plasma samples. Among them, the target sequence The comprehensive detection performance of 2+target sequence 4 is better.

In summary, this application can detect or diagnose breast cancer or precancerous lesions by detecting the methylation level of at least one target sequence among target sequences 1 to 4. The detection sensitivity for lobular carcinoma in situ can reach 89.29%. It can be used for the auxiliary diagnosis of lobular carcinoma in situ and is of great significance for the early screening of breast cancer, especially the early screening of precancerous lesions.

It is easy for those skilled in the art to understand that the above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements, etc., made within the spirit and principles of the present invention, All should be included in the protection scope of the present invention.

Claims (10)

1. A reagent for breast cancer or precancerous lesion detection, characterized in that it is a reagent for detecting the methylation level of a target sequence in a sample, said target sequence comprising the full length or partial region of Chr10:101140210-101140778, using grch38.p14 as a reference genome.

2. The reagent according to claim 1, wherein the target sequence is at least one nucleotide sequence selected from the group consisting of SEQ ID NO.1 to 4.

3. The reagent of claim 2, wherein the target sequence is selected from any combination of:

a combination of any one of SEQ ID NO. 2-4 with SEQ ID NO. 1;

a combination of any one of SEQ ID No.3 or SEQ ID No.4 with SEQ ID No. 2;

a combination of SEQ ID NO.3 and SEQ ID NO. 4.

4. A reagent according to claim 2 or 3, wherein the reagent comprises a primer pair for detecting the methylation level of the target sequence, the primer pair being selected from at least one of the following primer pairs:

a first primer pair for detecting the methylation level in the region of SEQ ID NO.1, a second primer pair for detecting the methylation level in the region of SEQ ID NO.2, a third primer pair for detecting the methylation level in the region of SEQ ID NO.3, and a fourth primer pair for detecting the methylation level in the region of SEQ ID NO. 4.

5. The reagent according to claim 4, wherein the primer pair is selected from at least one of the following primer pairs (1) to (4):

(1) A first primer pair shown in SEQ ID NO. 9-10;

(2) A second primer pair shown in SEQ ID NO. 12-13;

(3) A third primer pair shown in SEQ ID NOS.15-16;

(4) And a fourth primer pair shown in SEQ ID NOS.18-19.

6. The reagent according to claim 4, further comprising a detection probe corresponding to the primer pair, wherein the detection probe is at least one selected from the group consisting of:

the detection probe for detecting the methylation level in the region of SEQ ID NO.1 is shown as SEQ ID NO. 11; the detection probe for detecting the methylation level in the region of SEQ ID NO.2 is shown as SEQ ID NO. 14; the detection probe for detecting the methylation level in the region of SEQ ID NO.3 is shown as SEQ ID NO. 17; the detection probe for detecting methylation level in the region of SEQ ID NO.4 is shown as SEQ ID NO. 20.

7. Kit for the detection of breast cancer or precancerous lesions, characterized in that it comprises a reagent according to any one of claims 1 to 6.

8. The kit of claim 7, further comprising one or more of a nucleic acid extraction reagent, a nucleic acid purification reagent, a methylation conversion reagent, a PCR reaction reagent, a quality control product, a detection primer pair for an internal reference gene, and a detection probe for an internal reference gene.

9. Use of the reagent of any one of claims 1 to 6 or the kit of claim 7 or 8 for the preparation of a diagnostic product for breast cancer or precancerous lesions.

10. The use of claim 9, wherein the breast cancer comprises at least one of lobular invasive cancer, ductal invasive cancer, and lobular carcinoma in situ;

the breast cancer diagnostic product comprises one or more of a primer, a probe, a kit, a chip, a sequencing library, a membrane strip and a protein array.