CN116287229A - Reagent and kit for detecting esophagus cancer - Google Patents

Reagent and kit for detecting esophagus cancer Download PDF

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CN116287229A
CN116287229A CN202211239235.7A CN202211239235A CN116287229A CN 116287229 A CN116287229 A CN 116287229A CN 202211239235 A CN202211239235 A CN 202211239235A CN 116287229 A CN116287229 A CN 116287229A
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王洛伟
周俊
张良禄
董兰兰
李兆申
边岩
高野
林寒
庞亚南
辛磊
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Wuhan Aimisen Life Technology Co ltd
First Affiliated Hospital of Naval Military Medical University of PLA
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First Affiliated Hospital of Naval Military Medical University of PLA
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Abstract

The invention relates to the technical field of medical biological detection, provides a PCR primer pair and a probe combination for detecting the methylation level of genes, and further provides an esophageal cancer diagnosis reagent and a kit containing the combination. Experiments prove that the sensitivity of the scheme provided by the invention for distinguishing early esophageal adenocarcinoma, advanced esophageal adenocarcinoma, early esophageal squamous carcinoma and advanced esophageal squamous carcinoma can reach 59.38%, 77.78%, 80% and 90%, respectively, and the specificity of the scheme in healthy people can reach 94%. Therefore, the primer pair, the probe combination, the reagent or the kit provided by the invention have good sensitivity and specificity in diagnosis of esophageal cancer, and more importantly, the primer pair, the probe combination, the reagent or the kit improves the diagnosis rate of early esophageal cancer, is convenient, quick and noninvasive, and has wide market prospect.

Description

Reagent and kit for detecting esophagus cancer
Technical Field
The invention belongs to the technical field of medical biological detection, relates to a diagnosis marker for esophageal cancer, in particular to application of a reagent for detecting KCNA3 and OTOP2 gene methylation levels in preparation of an esophageal cancer diagnosis reagent or kit, and also relates to a primer pair and probe combination for detecting esophageal cancer, a diagnosis reagent and a diagnosis kit.
Background
Esophageal cancer is one of the most common malignant tumors in the world, and in all malignant tumors, the incidence of esophageal cancer is located at the 7 th position, and the mortality rate is located at the 6 th position. Esophageal cancer is highly invasive and often results in a poor prognosis. Based on global cancer statistics, 1 out of 20 cancer-related deaths in 2018 had esophageal cancer as a cause. Esophageal cancer mainly includes two histological subtypes: esophageal Squamous Cell Carcinoma (ESCC) and Esophageal Adenocarcinoma (EAC). Esophageal Squamous Cell Carcinoma (ESCC) has a higher prevalence in east asia, east africa, south africa and south europe, but has a lower prevalence in north america and other regions of europe, exhibiting a distinct geographic distribution profile. Esophageal squamous cell carcinoma occurs primarily in the squamous cells in the upper approximately 2/3 region of the esophagus. Risk factors for esophageal cancer include chronic irritation, inflammation, smoking, drinking, continuous ingestion of hot drinks, high temperature cooking, and the like. Esophageal squamous cell carcinoma is the most common cancer species in china, accounting for about 90% of the total number of esophageal cancer cases.
The morbidity and mortality of esophageal cancer in China are respectively at the 6 th and the 5 th positions in all malignant tumors, and the disease burden is heavy. Esophageal cancer does not characteristically manifest in early stages and often has progressed to mid-to late stages at the time of diagnosis. Radiation therapy and drug therapy are the primary treatments for unresectable locally advanced esophageal cancer and metastatic esophageal cancer. Although the diagnosis and treatment of esophageal cancer have progressed in recent years, prognosis of esophageal cancer is not ideal, and overall survival rate of esophageal cancer patients is less than 30% for 5 years, which may be related to atypical symptoms of early stage esophageal cancer and rapid progress, so early diagnosis and treatment are of great importance for esophageal cancer patients.
Endoscopy and pathology biopsy are gold standards for diagnosing esophageal cancer, but more patients have lower compliance with endoscopy, so that a non-invasive, high-cost-performance early diagnosis method is urgently needed to improve the detection rate of early esophageal cancer.
Epigenetic changes, such as DNA methylation and histone modification, are events in the course of cancer occurrence and pathogenesis. DNA methylation refers to the addition of a methyl group at the 5-position carbon of the cytosine base of a CpG dinucleotide without altering the nucleotide sequence. Abnormal methylation of a promoter region of a gene can silence gene expression by inhibiting gene transcription. Studies have shown that abnormal DNA methylation, particularly methylation of cancer suppressor genes, is intimately involved in the occurrence and progression of esophageal cancer, and thus early diagnosis of esophageal cancer can be achieved by detecting changes in the methylation state of DNA. DNA methylation has wide clinical application prospect as a biomarker for noninvasive diagnosis.
Disclosure of Invention
The invention is carried out by relying on the research, provides a PCR primer pair and probe combination for detecting the esophageal cancer, further provides an esophageal cancer diagnosis reagent and a kit containing the PCR primer pair and the probe, and has a certain application value in early diagnosis of the esophageal cancer.
In a first aspect of the invention, there is provided a PCR primer pair and probe combination for detecting the methylation level of a gene comprising a first primer pair, a first probe, a second primer pair and a second probe. The nucleotide sequence of the first primer pair is shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequence of the first probe is shown as SEQ ID NO. 3; the nucleotide sequences of the second primer pair are shown as SEQ ID NO.10 and 11, and the nucleotide sequence of the second probe is shown as SEQ ID NO. 12.
The first primer pair and the first probe are used for detecting the methylation level of the KCNA3 gene, and the second primer pair and the second probe are used for detecting the methylation level of the OTOP2 gene.
Preferably, the 5 'end of the probe contains a fluorescent reporter group such as any one of FAM, HEX, VIC, CY, ROX, texsaRed, JOE and Quasar 705, and the 3' end contains a fluorescent quenching group such as any one of MGB, BHQ-1, BHQ-2 and BHQ-3.
For the KCNA3 gene, three primer pairs and probe nucleotide combinations 1 to 3 were designed in total, and were used to detect methylation levels of region 1 (position Ch1: 110674528-110674658, plus strand), region 2 (position Ch1: 110674719-110674845, minus strand) and region 3 (position Ch1: 110674827-110674964, plus strand) of the KCNA3 gene, respectively.
For the OTOP2 gene, two primer pairs and probe nucleotide combinations 4 and 5 were designed together to detect methylation levels in region 4 (positions Chr17:74924472-74924577, minus strand) and region 5 (positions Chr17:74924277-74924416, minus strand) of the OTOP2 gene, respectively.
In addition, nucleotide combination 6 is also provided, including an upstream primer, a downstream primer and a fluorescent probe for detecting the ACTB gene of the internal reference gene. The primer sequences, probe sequences and gene sequences of the corresponding detection regions in the nucleotide combinations 1 to 6 are listed in Table 1.
And selecting any one from the nucleotide combinations 1-3, selecting any one from the nucleotide combinations 4-5, trying all 6 nucleotide combinations for simultaneously detecting methylation levels of the KCNA3 gene and the OTOP2 gene on a sample to be detected, and analyzing and comparing diagnostic performances of different nucleotide combinations. The primer pair with the highest detection rate and the probe combination, namely the combination of the nucleotide combination 1 and the nucleotide combination 4, detect the methylation level of the KCNA3 gene and the OTOP2 gene in the sample, and can effectively diagnose early esophagus cancer including esophageal cancer adenocarcinoma.
In a second aspect, the invention provides application of the PCR primer pair and probe combination for detecting the methylation level of genes in preparation of esophageal cancer diagnostic reagents.
The esophageal cancer diagnostic reagent is used for detecting the methylation level of the KCNA3 gene and the methylation level of the OTOP2 gene in a biological sample, wherein the first primer pair and the first probe are used for detecting the methylation level of the KCNA3 gene, and the second primer pair and the second probe are used for detecting the methylation level of the OTOP2 gene.
Preferably, the biological sample is an esophageal tissue sample, an esophageal desquamation cell sample or a blood sample; the blood sample includes plasma, serum, whole blood, isolated blood cells, or a combination thereof.
Preferably, the methylation level of KCNA3 gene and the methylation level of OTOP2 gene in the biological sample are detected by at least one of the following methods by the esophageal cancer diagnostic reagent: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole genome methylation sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, digital PCR, methylation-specific high resolution dissolution profile, methylation-sensitive restriction endonuclease, and methylation-specific fluorescent quantitative PCR.
In a third aspect of the present invention, there is provided an esophageal cancer diagnostic reagent comprising at least a PCR primer pair and a probe combination for detecting methylation levels of KCNA3 and OTOP2 genes, the PCR primer pair and probe combination being as described above.
The diagnostic esophageal cancer reagent also includes a reagent that converts unmethylated cytosine to uracil. Preferably, the reagent is bisulphite.
In a fourth aspect of the present invention, there is provided a kit for diagnosing esophageal cancer, comprising a PCR primer pair and a probe combination for detecting methylation levels of KCNA3 and OTOP2 genes, the kit further comprising one or more of an amplification reagent, a reagent for converting unmethylated cytosine into uracil, a DNA extraction reagent, a DNA purification reagent and a quality control product.
Wherein the PCR primer pair and probe combination are as described above.
Preferably, a pair ofThe amplification reagent comprises amplification buffer solution, dNTPs, DNA polymerase and Mg 2+ One or more of the following.
The positive quality control is a plasmid containing a fully methylated KCNA3 gene fragment and an OTOP2 gene fragment.
The primer pair and the probe combination, the detection reagent or the kit provided by the invention detect the DNA sequence after bisulfite conversion, unmethylated cytosine in DNA CpG dinucleotide is converted into uracil after bisulfite conversion, and methylated cytosine is unchanged.
In a fifth aspect of the present invention, there is provided a method for detecting methylation level of an esophageal cancer-associated gene, comprising the steps of:
A. DNA extraction
Extracting DNA from samples such as tissues, blood and the like;
B. DNA transformation
Performing bisulfite conversion and purification on the extracted DNA;
C. methylation quantitative PCR reaction
Taking DNA of a sample to be detected after bisulphite conversion as a template, simultaneously using a primer pair and a probe for detecting the methylation level of the KCNA3 gene, and a primer pair and a probe for detecting the methylation level of the OTOP2 gene (the sequences are as described in the second aspect), and configuring a PCR reaction system according to a formula to carry out methylation quantitative PCR reaction.
D. Methylation outcome determination
And judging the methylation level of the sample to be tested according to the Ct values of the KCNA3 gene and the OTOP2 gene. For a tissue sample, if the Ct value of a certain gene is amplified to be less than or equal to 38, the gene in the sample is considered to be methylation positive, and if the Ct value of a certain gene is amplified to be more than 38, the gene in the sample is considered to be methylation negative. For a blood sample, if the Ct value of a certain gene is amplified to be less than or equal to 48, the gene in the sample is considered to be methylation positive, and if the Ct value of a certain gene is amplified to be more than 48, the gene in the sample is considered to be methylation negative.
E. Diagnosis of esophageal cancer or determination of auxiliary diagnosis result
If at least one gene in the sample to be detected is methylation positive, the sample is a cancer positive sample; only if both genes in the sample to be tested are methylation negative, the sample is a cancer negative sample.
The beneficial effects of the invention are as follows:
in esophageal cancer samples, methylation levels of KCNA3 genes and OTOP2 genes are essentially detected by a qPCR amplification and probe hybridization method by using a methylation primer pair, and the kit has the characteristics of simplicity and convenience in operation, sensitivity in detection, good specificity, high repeatability and the like. The methylation levels of the KCNA3 gene and the OTOP2 gene can be detected by using the primer pair, the probe combination, the reagent or the kit and using a fluorescence quantitative PCR method, so that whether a subject suffers from esophageal cancer or not can be judged according to the Ct value. By adopting the specific primer pair and probe combination provided by the invention, the sensitivity for distinguishing early esophageal adenocarcinoma, advanced esophageal adenocarcinoma, early esophageal squamous carcinoma and advanced esophageal squamous carcinoma can reach 59.38%, 77.78%, 80% and 90%, respectively, and the specificity in healthy people can reach 94%. Therefore, the primer pair, the probe combination, the reagent or the kit provided by the invention have good sensitivity and specificity, and more importantly, the primer pair, the probe combination, the reagent or the kit improves the diagnosis rate of early esophageal cancer, is convenient, quick and noninvasive, and has wide market prospect.
In terms of the detection mode, the detection result can be obtained only by collecting blood of a detector, the operation is simple and noninvasive, and the acceptance degree and the compliance of patients are high.
Detailed Description
The following examples of the present invention are provided to illustrate the detailed embodiments and specific operation procedures, but the scope of the present invention is not limited to the following examples.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein in the description of the application is for the purpose of describing the embodiments and examples only and is not intended to be limiting of the application.
Unless otherwise indicated or contradicted, terms or phrases used herein have the following meanings:
the term "and/or", "or" as used herein includes a selection of any one of two or more of the listed items and also includes any and all combinations of the listed items, including any two or more of the listed items, or all combinations of the listed items. It should be noted that, when at least three items are connected by at least two conjunctions selected from "and/or", "or/and", it is understood that, in the present application, the technical solutions certainly include technical solutions all connected by "logical and", and also certainly include technical solutions all connected by "logical or". For example, "a and/or B" includes three parallel schemes A, B and a+b. For another example, the technical schemes of "a, B, C, and/or D" include any one of A, B, C, D (i.e., the technical schemes all connected by "logical or"), and also include any and all combinations of A, B, C, D, i.e., any two or three of A, B, C, D, and further include four combinations of A, B, C, D (i.e., the technical schemes all connected by "logical and").
The terms "plurality", "plural", "multiple", and the like are used herein, and refer to a number of 2 or more, unless otherwise specified. For example, "one or more" means one kind or two or more kinds. "above" includes the present number, for example "two or more" includes two, three or more.
In the present application, "at least one" and "at least one" mean any one of the listed items, or a combination of any two or more thereof.
As used herein, "a combination thereof," "any combination thereof," and the like include all suitable combinations of any two or more of the listed items.
In the present application, "suitable" is described in "suitable combination mode", "suitable mode", "any suitable mode", etc., so as to implement the technical scheme of the present application, solve the technical problem of the present application, and achieve the technical effect expected in the present application.
In this application, "preferred," "better," "preferred," and "preferred" are merely examples of better performing implementations or examples, and it should be understood that they are not limiting the scope of the application.
In this application, "further," "still further," "particularly," and the like are used for descriptive purposes and are not to be construed as limiting the scope of the present application.
In this application, the terms "first," "second," "third," "fourth," and the like are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying an importance or quantity of a technical feature being indicated. Moreover, the terms "first," "second," "third," "fourth," and the like are used for non-exhaustive list description purposes only, and are not to be construed as limiting the number of closed forms.
In the present application, the technical features described in an open manner include a closed technical scheme composed of the listed features, and also include an open technical scheme including the listed features.
As used herein, an "esophageal cancer" is a malignancy of the digestive tract from the hypopharynx to the esophageal epithelium between the esophageal and gastric junctions. Esophageal cancer mainly includes esophageal squamous cell carcinoma and esophageal adenocarcinoma.
In this application, the term "diagnosis" includes auxiliary diagnosis, recurrence risk assessment, assessment of risk and extent of cancerous lesions, prognosis, and the like.
The term "gene" refers to a segment of DNA encoding a polypeptide chain that produces amino acids, and includes sequences located in coding and non-coding regions, as well as exon and intron sequences involved in gene transcription/translation and transcriptional/translational regulation.
The term "oligonucleotide" or "polynucleotide" or "nucleotide" or "nucleic acid" refers to a molecule having two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and typically more than ten. The exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. The oligonucleotides may be produced in any manner, including chemical synthesis, DNA replication, reverse transcription, or a combination thereof. Typical deoxyribonucleotides of DNA are thymine, adenine, cytosine and guanine. Typical ribonucleotides of RNA are uracil, adenine, cytosine and guanine.
The term "methylation" is a form of chemical modification of DNA that can alter genetic manifestations without altering the DNA sequence. DNA methylation refers to covalent binding of a methyl group at the 5 th carbon position of cytosine of a genomic CpG dinucleotide under the action of a DNA methyltransferase. DNA methylation can cause alterations in chromatin structure, DNA conformation, DNA stability, and the manner in which DNA interacts with proteins, thereby controlling gene expression.
The term "methylation level" refers to whether or not cytosine in one or more CpG dinucleotides in a DNA sequence is methylated, or the frequency/proportion/percentage of methylation, representing both qualitative and quantitative concepts. In practical application, different detection indexes can be adopted to compare the DNA methylation level according to practical conditions. As in some cases, the comparison may be made based on Ct values detected by the sample; in some cases, the ratio of gene methylation in the sample, i.e., number of methylated molecules/(number of methylated molecules+number of unmethylated molecules). Times.100, can be calculated and then compared; in some cases, statistical analysis and integration of each index is also required to obtain a final decision index. It is understood that the target region of the gene to be detected herein is a DNA sequence comprising at least one CpG dinucleotide (CG).
The term "primer" refers to an oligonucleotide that can be used in an amplification method (e.g., polymerase chain reaction, PCR) to amplify a sequence of interest based on a polynucleotide sequence corresponding to a gene of interest or a portion thereof. Typically, at least one of the PCR primers used to amplify a polynucleotide sequence is sequence specific for that polynucleotide sequence. The exact length of the primer will depend on many factors, including temperature, source of primer, and method used. For example, for diagnostic and prognostic applications, the oligonucleotide primers will typically contain at least 10, 15, 20, 25 or more nucleotides, but may also contain fewer nucleotides, depending on the complexity of the target sequence. In the present disclosure, the term "primer" refers to a pair of primers that hybridize to the double strand of a target DNA molecule or to regions of the target DNA molecule that flank the nucleotide sequence to be amplified.
The term "Taqman probe" refers to a stretch of oligonucleotide sequences comprising a 5 'fluorescent group and a 3' quenching group. When the probe binds to the corresponding site on the DNA, the probe does not fluoresce because of the presence of a quenching group near the fluorescent group. During amplification, if the probe binds to the amplified strand, the 5'-3' exonuclease activity of the DNA polymerase (e.g., taq enzyme) digests the probe and the fluorescent group is far from the quenching group, its energy is not absorbed, i.e., a fluorescent signal is generated. The fluorescence signal is also identical to the target fragment with a synchronous exponential increase per PCR cycle.
Various aspects of the present application are described in detail below.
Embodiment one, esophageal cancer diagnosis or auxiliary diagnosis detection method
1. Extraction of DNA template:
for tissue samples, QIAamp DNA FFPE Tissue Kit (56404) was used to extract DNA, and the specific procedure was performed according to the kit instructions.
For blood samples, plasma cfDNA extraction was performed using a magnetic bead serum/plasma free DNA (cfDNA) extraction kit (DP 709) from the company of the biochemical technology of the root of the heaven limited company, the specific procedure was performed according to the kit instructions.
In addition, the extraction of the genomic DNA of the leucocytes was carried out using the blood/cell/tissue genomic DNA extraction kit (DP 304) from Tiangen Biochemical technology (Beijing) limited, see kit instructions for specific procedures.
2. Conversion and purification of bisulphite
The extracted sample DNA is subjected to bisulphite conversion and purification, and the kit is a nucleic acid conversion reagent (Huhan mechanical preparation 20200843) of Wuhan Ai Misen life technology Co., ltd, and specific experimental operation is shown in the instruction book of the kit.
3. Primer and probe
For the KCNA3 gene, three primer pairs and probe nucleotide combinations 1-3 were designed to detect methylation levels in region 1, region 2 and region 3 of the KCNA3 gene, respectively. For the OTOP2 gene, two primer pairs and probe nucleotide combinations 4 and 5 were designed together to amplify the methylation levels of two different regions of region 4 and region 5 of the OTOP2 gene, respectively. In addition, nucleotide combination 6 is also provided, including an upstream primer, a downstream primer and a fluorescent probe for detecting the ACTB gene of the internal reference gene. The primer sequences, probe sequences, and gene sequences and positions of the corresponding detection regions in the nucleotide combinations 1 to 6 are listed in Table 1.
TABLE 1 summary of sequences in nucleotide combinations 1-6
Figure BDA0003884395300000081
Figure BDA0003884395300000091
Note that: fluorescent modifying groups in the probe sequence, italicized at the 5 'and 3' ends, are not part of the probe sequence.
4. Methylation quantitative PCR reaction
The DNA of the sample to be tested after bisulphite conversion is used as a template, and a primer pair and a probe for detecting the KCNA3 gene and a primer pair and a probe for detecting the OTOP2 gene are simultaneously used (see Table 1). A PCR reaction system was prepared according to the formulation of Table 2, and a methylation quantitative PCR reaction was performed using Invitrogen Platinum II Taq Hot-Start DNA polymerase under the conditions shown in Table 3. Thus, each PCR reaction can simultaneously detect the methylation levels of the KCNA3 gene and the OTOP2 gene in a certain sample to be detected. In addition, a detection primer pair and a probe of an internal reference gene ACTB are added into the PCR reaction system to amplify the gene sequence of the ACTB, so that whether a PCR inhibitor exists in the reaction system and whether the reaction system (reagents, PCR programs, fluorescence collection and the like) is normal or not is monitored.
The primer pair and the probe for detecting the methylation level of the KCNA3 gene comprise a nucleotide combination 1, a nucleotide combination 2 and a nucleotide combination 3, and the primer pair and the probe for detecting the methylation level of the OTOP2 gene comprise a nucleotide combination 4 and a nucleotide combination 5. When detecting a certain sample to be detected, selecting any one from the nucleotide combinations 1-3, selecting any one from the nucleotide combinations 4-5, and trying all 6 nucleotide combinations (shown in table 4) to simultaneously detect methylation levels of KCNA3 gene and OTOP2 gene on the sample, and analyzing and comparing diagnostic performances of different nucleotide combinations.
TABLE 2 PCR reaction System
Figure BDA0003884395300000092
Figure BDA0003884395300000101
TABLE 3 PCR reaction conditions
Figure BDA0003884395300000102
TABLE 4 primer set for KCNA3 Gene and OTOP2 Gene and method of combining probes
Figure BDA0003884395300000103
Negative and positive controls: in detecting samples in the different combinations described in Table 4, negative and positive controls should also be detected simultaneously, and the DNA template of the negative control is TE buffer. Positive controlThe preparation method of the DNA template of the tube comprises the following steps: artificially synthesizing the sequence which corresponds to the ACTB gene amplified region and is subjected to complete conversion by bisulphite, and cloning the sequence onto a vector to form an artificially synthesized plasmid; the sequence of the target region, which corresponds to SEQ ID NO. 19-23, after bisulfite conversion is artificially synthesized and cloned to a vector respectively to form an artificially synthesized plasmid. For example, the positive control DNA template of combination A is 10 3 Copy/microliter of synthetic plasmid containing post-transformation ACTB, 10 3 Copy/microliter of synthetic plasmid containing the sequence SEQ ID NO.19 after transformation and 10 3 Copy/microliter of the synthetic plasmid containing the sequence SEQ ID No.22 after transformation.
Ct value reading: after the PCR is completed, a baseline is adjusted, a fluorescence value before a minimum Ct value of a sample in one PCR is advanced by 1-2 cycles is set as a baseline value, and a threshold value is set at the inflection point of an S-type amplification curve to obtain Ct values of all genes of the sample.
And (3) quality control: the negative control needs no amplification, the positive control needs a significant exponential growth period, and the Ct value of the positive control is between 26 and 30. The Ct value of the reference gene of the sample to be detected is less than or equal to 35, and after the negative control, the positive control and the reference gene meet the requirements, the experiment is effective, and the next sample result can be judged. Otherwise, when the experiment is invalid, the detection is needed again.
5. Analysis of PCR results
And judging the methylation level of the sample to be tested according to the Ct values of the KCNA3 gene and the OTOP2 gene. For a tissue sample, if the Ct value of a certain gene is amplified to be less than or equal to 38, the gene in the sample is considered to be methylation positive, and if the Ct value of a certain gene is amplified to be more than 38, the gene in the sample is considered to be methylation negative. For a blood sample, if the Ct value of a certain gene is amplified to be less than or equal to 48, the gene in the sample is considered to be methylation positive, and if the Ct value of a certain gene is amplified to be more than 48, the gene in the sample is considered to be methylation negative. If at least one gene in the sample to be tested is methylation positive, the sample is a cancer positive sample, and only if two genes in the sample to be tested are methylation negative, the sample is a cancer negative sample. Specific criteria are shown in table 5.
Table 5 sample judgment criteria
Figure BDA0003884395300000111
Example two, primer and Probe Screen 1
In a certain hospital, 45 tissue samples of early stage (stage I and II) esophageal adenocarcinoma and 45 corresponding paracancerous tissue samples are collected, 56 tissue samples of advanced stage esophageal adenocarcinoma (stage III and IV) and 56 tissue samples of corresponding paracancerous tissue samples are collected, 68 tissue samples of early stage (stage I and II) esophageal squamous carcinoma and 80 tissue samples of corresponding paracancerous tissue samples are collected, and 80 tissue samples of advanced stage esophageal squamous carcinoma (stage III and IV) and 80 tissue samples of corresponding paracancerous tissue samples are collected, wherein all the samples are formalin-immersed and paraffin embedded tissue samples. All samples were approved by the ethics committee, all volunteers signed informed consent, and all samples were anonymized.
The DNA of the tissue sample was extracted and bisulphite converted according to the method provided in example I, methylation fluorescent quantitative PCR was performed according to the nucleotide combination method shown in Table 4 using the primer pair and the probe shown in Table 1, and whether the sample was pathologically positive was analyzed according to the PCR detection result, and the sensitivity and the specificity of detecting the esophageal cancer tissue sample were calculated, and the results are shown in Table 6.
TABLE 6 sensitivity and specificity of detection of esophageal cancer tissue samples by different nucleotide combinations
Figure BDA0003884395300000121
As can be seen from Table 6, the primer pairs and probes of different combinations are used to detect tissue samples, which have higher detection rates for early stage esophageal squamous cell carcinoma and adenocarcinoma, for advanced stage esophageal squamous cell carcinoma and adenocarcinoma, and have sensitivity of more than 42% for early stage esophageal adenocarcinoma, sensitivity of more than or equal to 75% for advanced stage esophageal adenocarcinoma, specificity of more than 77% in normal tissue beside adenocarcinoma, sensitivity of more than 76% for early stage esophageal squamous cell carcinoma, sensitivity of more than 83% for advanced stage esophageal squamous cell carcinoma, and specificity of more than 85% in normal tissue beside squamous cell carcinoma, wherein the diagnosis effect of combination A (i.e., nucleotide combination 1+nucleotide combination 4) is optimal. For early esophageal adenocarcinoma, the sensitivity of detection in combination mode A (namely nucleotide combination 1+nucleotide combination 4) can reach 68.89%, and for early esophageal squamous carcinoma, the sensitivity of detection can reach 94.12%, which is the highest detection rate in all combination modes.
The results show that the primer pair and the probe in the combination mode A (namely the nucleotide combination 1+the nucleotide combination 4) are used for detecting the methylation levels of the KCNA3 gene and the OTOP2 gene in the tissue sample, so that the early esophageal cancer can be effectively diagnosed.
Third embodiment, primer and probe combination for verifying esophageal cancer detection effect
Blood samples of 32 cases of early stage (stage I and II) esophageal adenocarcinoma patients, 45 cases of early stage (stage III and stage IV) esophageal adenocarcinoma patients, 120 cases of early stage (stage I and II) esophageal squamous carcinoma patients, 150 cases of early stage (stage III and stage IV) esophageal squamous carcinoma patients and 100 cases of healthy people are collected in a certain hospital, all the collecting processes of the samples are approved by ethical committee, all volunteers sign informed consent, and all the samples are anonymized.
Extraction and bisulfite conversion of cfDNA of blood samples were performed according to the method provided in example one, methylation fluorescent quantitative PCR detection was performed according to the nucleotide combination method shown in table 4 using the primer pair and probe shown in table 1, and then whether the samples were pathologically positive was analyzed according to the PCR detection results, and further the sensitivity and specificity of detection of esophageal cancer plasma samples were calculated, and the results are shown in table 7.
TABLE 7 sensitivity and specificity of detection of esophageal cancer plasma samples by different nucleotide combinations
Figure BDA0003884395300000131
As can be seen from Table 7, the primer pairs and probes of different combinations have different detection effects on blood samples of different pathological states, and in the whole, the sensitivity of the primer pairs and probes of different combinations for detecting early esophageal adenocarcinoma is greater than 37%, the sensitivity for detecting early esophageal squamous carcinoma is greater than 60%, the sensitivity for detecting early esophageal squamous carcinoma is greater than 58%, the sensitivity for detecting early esophageal squamous carcinoma is greater than 77%, and the specificity of the primer pairs and probes of different combinations in a healthy human blood sample is greater than or equal to 88%. It is noted that combination A (i.e., nucleotide combination 1+nucleotide combination 4) provides the best diagnostic effect on blood samples. For early esophageal adenocarcinoma, the sensitivity of detection in the combined mode A (namely nucleotide combination 1+nucleotide combination 4) can reach 59.38, and for early esophageal squamous carcinoma, the sensitivity of detection can reach 80%, and the specificity of detection of healthy human blood samples is 94%.
In conclusion, the methylation levels of the KCNA3 gene and the OTOP2 gene in the blood sample are detected by using the primer pair and the probe in the combination mode 1 (namely the nucleotide combination 1+the nucleotide combination 4), so that the detection rate of early-stage esophageal cancer can be remarkably improved, and the guarantee is provided for saving the life of a patient and improving the life quality of the patient.
While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.

Claims (10)

1. The PCR primer pair and the probe combination for detecting the methylation level of the gene are characterized by comprising a first primer pair, a first probe, a second primer pair and a second probe,
the nucleotide sequences of the first primer pair are shown as SEQ ID NO.1 and 2, and the nucleotide sequence of the first probe is shown as SEQ ID NO. 3;
the nucleotide sequences of the second primer pair are shown as SEQ ID NO.10 and 11, and the nucleotide sequence of the second probe is shown as SEQ ID NO. 12.
2. The PCR primer pair and probe combination for detecting gene methylation level according to claim 1, wherein the first probe and the second probe have a fluorescent reporter group at their 5 'end and a fluorescent quenching group at their 3' end.
3. The use of a PCR primer pair and probe combination for detecting the methylation level of a gene according to claim 1 or 2 in the preparation of a diagnostic reagent for esophageal cancer.
4. The use according to claim 3, wherein the esophageal cancer diagnostic reagent is for detecting the methylation level of KCNA3 gene and the methylation level of OTOP2 gene in a biological sample, wherein the first primer pair and the first probe are for detecting the methylation level of KCNA3 gene and the second primer pair and the second probe are for detecting the methylation level of OTOP2 gene.
5. The use according to claim 4, wherein the biological sample is an esophageal tissue sample, an esophageal exfoliated cell sample or a blood sample,
the blood sample includes plasma, serum, whole blood, isolated blood cells, or a combination thereof.
6. The use according to claim 3, wherein the esophageal cancer diagnostic agent detects the methylation level of KCNA3 gene and the methylation level of OTOP2 gene in the biological sample by at least one of the following methods: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole genome methylation sequencing, pyrosequencing, methylation-specific high performance liquid chromatography, digital PCR, methylation-specific high resolution dissolution profile, methylation-sensitive restriction endonuclease, and methylation-specific fluorescent quantitative PCR.
7. An esophageal cancer diagnostic reagent comprising the PCR primer pair and probe combination for detecting gene methylation level according to claim 1 or 2.
8. The esophageal cancer diagnostic agent of claim 7, further comprising an agent that converts unmethylated cytosine to uracil, said agent comprising bisulfite.
9. A kit for diagnosing esophageal cancer, which comprises the PCR primer pair for detecting the methylation level of genes, the probe combination and the quality control product according to claim 1 or 2,
wherein the positive quality control is a plasmid containing a completely methylated KCNA3 gene target fragment and a plasmid containing a completely methylated OTOP2 gene target fragment.
10. The esophageal cancer diagnostic kit of claim 9, further comprising one or more of amplification reagents, reagents that convert unmethylated cytosine to uracil, DNA extraction reagents, DNA purification reagents, and quality control; alternatively, the amplification reagents include an amplification buffer, dNTPs, a DNA polymerase, and Mg 2+ One or more of the following.
CN202211239235.7A 2022-10-11 2022-10-11 Reagent and kit for detecting esophagus cancer Pending CN116287229A (en)

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CN113308544A (en) * 2021-06-04 2021-08-27 武汉艾米森生命科技有限公司 Reagent for DNA methylation detection and esophageal cancer detection kit
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