Reagent and kit for diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesion and application of reagent and kit
阅读说明：本技术 一种食管癌或癌前病变的诊断或辅助诊断的试剂、试剂盒及其应用 (Reagent and kit for diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesion and application of reagent and kit ) 是由 张良禄 周俊 董兰兰 熊杨辉 李婷婷 于 2021-06-30 设计创作，主要内容包括：本发明公开了一种食管癌或癌前病变的诊断或辅助诊断的试剂、试剂盒及其应用,涉及基因检测技术领域。以人RAPGEFL1基因的CpG岛的甲基化作为标志物,通过检测其甲基化水平的增加可以对食管癌进行诊断或辅助诊断,具有较高的灵敏度和特异性。采用本发明提供的检测试剂和试剂盒能有效地提高食管癌的检出率,从而满足食管癌/癌前病变早筛早诊的临床需求。(The invention discloses a reagent and a kit for diagnosing or assisting in diagnosing esophageal cancer or precancerous lesions and application thereof, and relates to the technical field of gene detection. Methylation of CpG island of human RAPGEFL1 gene is used as marker, and diagnosis or auxiliary diagnosis of esophageal cancer can be carried out by detecting increase of methylation level, and the method has high sensitivity and specificity. The detection reagent and the kit provided by the invention can effectively improve the detection rate of esophageal cancer, thereby meeting the clinical requirements of early screening and early diagnosis of esophageal cancer/precancerous lesions.)
1. Use of an agent for the manufacture of a product for the diagnosis or the aided diagnosis of esophageal cancer or a precancerous condition, wherein the agent is an agent for detecting methylation of a target region on an esophageal cancer-associated gene, wherein the esophageal cancer-associated gene is RAPGEFL1 gene, and the target region is selected from the group consisting of a full-length region and a partial region of at least one of the following regions of RAPGEFL1 gene: region 1, region 2, and region 3;
wherein, the region 1 is selected from the positive chains of Chr17:40191282-40191349, the region 2 is selected from the positive chains of Chr17:40191371-40191476, and the region 3 is selected from the positive chains of Chr17: 40191544-40191653.
2. The use according to claim 1, wherein the target region is selected from the full length or partial region of region 3 of the RAPGEFL1 gene: the region 3 is selected from the positive strand of Chr17: 40191544-40191653.
3. The use according to claim 1, wherein the reagent is used for detecting methylation of cytosine at least one position of Chr7:40191282, Chr7:40191291, Chr7:40191299, Chr7:40191305, Chr7:40191324, Chr7:40191333, Chr7:40191342 and Chr7:40191349 on the positive strand of region 1 of the RAPGEFL1 gene;
the reagent is used for detecting the methylation of cytosine at least one position of Chr7:40191391, Chr7:40191399, Chr7:40191407, Chr7:40191456 and Chr7:40191458 on the positive chain of the region 2 on the RAPGEFL1 gene;
the reagent is used for detecting methylation of cytosine at least one position of Chr7:40191545, Chr7:40191548, Chr7:40191552, Chr7:40191564, Chr7:40191615, Chr7:40191618, Chr7:40191639, Chr7:40191642 and Chr7:40191652 on a region 3 positive chain on the RAPGEFL1 gene;
preferably, the esophageal cancer diagnosis or auxiliary diagnosis product is selected from at least one of the following products: kits, chips and sequencing libraries.
4. The use according to claim 1, wherein the agent is selected from at least one of the following nucleic acid combinations: a nucleic acid set 1 for detecting the region 1, a nucleic acid set 2 for detecting the region 2, and a nucleic acid set 3 for detecting the region 3;
the base sequence of the nucleic acid combination 1 is shown as SEQ ID NO.1-3, the base sequence of the nucleic acid combination 2 is shown as SEQ ID NO.4-6, and the base sequence of the nucleic acid combination 3 is shown as SEQ ID NO. 7-9.
5. A nucleic acid combination for detecting the methylation level of a CpG island of the RAPGEFL1 gene, wherein the nucleic acid combination for detecting the CpG island of the RAPGEFL1 gene is at least one of the following nucleic acid combinations: nucleic acid set 1, nucleic acid set 2, and nucleic acid set 3;
the base sequence of the nucleic acid combination 1 is shown as SEQ ID NO.1-3, the base sequence of the nucleic acid combination 2 is shown as SEQ ID NO.4-6, and the base sequence of the nucleic acid combination 3 is shown as SEQ ID NO. 7-9.
6. The nucleic acid combination for detecting the methylation level of the CpG island of the RAPGEFL1 gene according to claim 5, wherein the nucleic acid combination 1 is used for detecting the full-length region or the partial region of the region 1, the nucleic acid combination 2 is used for detecting the full-length region or the partial region of the region 2, and the nucleic acid combination 3 is used for detecting the full-length region or the partial region of the region 3;
wherein, the region 1 is selected from the positive chains of Chr17:40191282-40191349, the region 2 is selected from the positive chains of Chr17:40191371-40191476, and the region 3 is selected from the positive chains of Chr17: 40191544-40191653.
7. A reagent for the diagnosis or the auxiliary diagnosis of esophageal cancer or precancerous lesions, which comprises the nucleic acid composition for detecting the methylation level of CpG islands of RAPGEFL1 gene according to any one of claims 5 to 6.
8. The reagent for detecting the diagnosis or the auxiliary diagnosis of esophageal cancer or precancerous lesion according to claim 7, wherein the reagent for detecting the methylation state of CpG island of RAPGEFL1 gene comprises at least one of the following methods: methylation specificity PCR method, bisulfite sequencing method, methylation specificity microarray method, whole genome methylation sequencing method, pyrosequencing method, methylation specificity high performance liquid chromatography, digital PCR method, methylation specificity high resolution dissolution curve method, methylation sensitivity restriction endonuclease method and flap endonuclease method;
preferably, the esophageal cancer is esophageal squamous carcinoma.
9. A kit for diagnosing or aiding diagnosis of esophageal cancer or precancerous lesions, which comprises the nucleic acid combination for detecting the methylation level of CpG island of rapgofl 1 gene according to any one of claims 5 to 6 or the detection reagent for diagnosing or aiding diagnosis of esophageal cancer or precancerous lesions according to any one of claims 7 to 8.
10. The detection kit for the diagnosis or the auxiliary diagnosis of esophageal cancer or precancerous lesion according to claim 9, wherein the detection sample of the kit is a plasma sample, a serum sample, a saliva sample, a tissue sample or a cell sample of esophageal origin.
Technical Field
The invention relates to the technical field of gene detection, in particular to a reagent and a kit for diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesions and application thereof.
Background
Esophageal cancer is a malignant tumor that occurs in the esophageal mucosal epithelium and esophageal gland epithelium, and is one of the most common malignant tumors of the digestive system. According to global cancer statistical data, the incidence rate of esophageal cancer in 2018 is eighth of malignant tumors, and the mortality rate is seventh.
Early diagnosis of esophageal cancer can obviously improve the treatment effect and the prognosis of patients, and the detection rate of esophageal cancer in high risk groups is effectively improved by combining the current technologies such as endoscopy and cell biopsy, but the technologies have the problems of low patient acceptance, inflexible operation and the like.
Recent advances in molecular biology have provided new avenues for the development of simple and effective early diagnosis methods, with multiple genes and multiple sites of methylation in esophageal cancer tissues, and with changes in DNA methylation occurring at early stages of esophageal carcinogenesis, and therefore, methylation changes can be used as markers for early diagnosis of esophageal cancer, and it has become a new idea to explore esophageal cancer diagnosis strategies from the viewpoint of DNA methylation.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a reagent and a kit for diagnosing or assisting in diagnosing esophageal cancer or precancerous lesions and application thereof so as to solve the technical problems.
The invention is realized by the following steps:
the invention provides an application of a reagent for diagnosing or assisting in diagnosing esophageal cancer or precancerous lesions in preparation of products for diagnosing or assisting in diagnosing esophageal cancer or precancerous lesions, wherein the reagent is a reagent for detecting methylation of a target region on an esophageal cancer-related gene, the esophageal cancer-related gene is RAPGEFL1 gene, and the target region is selected from a full-length region or a partial region in at least one of the following regions of RAPGEFL1 gene: region 1, region 2, and region 3;
wherein, the region 1 is selected from the positive chains of Chr17:40191282-40191349, the region 2 is selected from the positive chains of Chr17:40191371-40191476, and the region 3 is selected from the positive chains of Chr17: 40191544-40191653.
The RAPGEFL1 gene was located on human chromosome 17 at position 40177010-. The RAPGEFL1 gene encodes the Rap-type guanylate exchange factor 1(Rap guanine nucleotide exchange factor like 1).
The inventors found that the methylation level of the three regions of RAPGEFL1 gene is significantly higher than that of the normal sample, based on which, the esophageal cancer diagnosis or the auxiliary diagnosis can be realized by detecting the methylation level of the three regions, if the methylation level of at least one of the three regions is higher than that of the normal sample, the sample can be diagnosed or the auxiliary diagnosis is suffered from the esophageal cancer/precancerous lesion.
In a preferred embodiment of the present invention, the target region is selected from the full length region or a partial region of region 3 of the RAPGEFL1 gene: region 3 is selected from the positive strands of Chr17:40191544 and 40191653.
The inventors found that the detection sensitivity and specificity of region 3 for the sample was superior to the detection sensitivity and specificity of regions 1 and 2 for the sample.
In a preferred embodiment of the invention, the reagent is used for detecting methylation of cytosine at least one position of Chr7:40191282, Chr7:40191291, Chr7:40191299, Chr7:40191305, Chr7:40191324, Chr7:40191333, Chr7:40191342 and Chr7:40191349 on the positive strand of the region 1 of the RAPGEFL1 gene;
the reagent is used for detecting the methylation of cytosine at least one position of Chr7:40191391, Chr7:40191399, Chr7:40191407, Chr7:40191456 and Chr7:40191458 on the positive chain of the region 2 on the RAPGEFL1 gene;
the reagent is used for detecting methylation of cytosine at least one position of Chr7:40191545, Chr7:40191548, Chr7:40191552, Chr7:40191564, Chr7:40191615, Chr7:40191618, Chr7:40191639, Chr7:40191642 and Chr7:40191652 on the positive chain of the region 3 of the RAPGEFL1 gene.
In a preferred embodiment of the present invention, the above-mentioned esophageal cancer diagnosis or diagnosis-assisting product is at least one selected from the following products: kits, chips and sequencing libraries.
It should be noted that the diagnosis or auxiliary diagnosis product for esophageal cancer or precancerous lesion may be any in vitro diagnosis product form, and is not limited to the product types of the kit, the chip and the sequencing library, and the invention is within the protection scope as long as the requirements for diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesion can be met.
In a preferred embodiment of the present invention, the reagent is selected from at least one of the following nucleic acid combinations: a nucleic acid set 1 for detecting region 1, a nucleic acid set 2 for detecting region 2, and a nucleic acid set 3 for detecting region 3;
the base sequence of the nucleic acid combination 1 is shown as SEQ ID NO.1-3, the base sequence of the nucleic acid combination 2 is shown as SEQ ID NO.4-6, and the base sequence of the nucleic acid combination 3 is shown as SEQ ID NO. 7-9.
The invention also provides a nucleic acid combination for detecting the methylation level of the CpG island of the RAPGEFL1 gene, which is selected from at least one of the following nucleic acid combinations: nucleic acid set 1, nucleic acid set 2, and nucleic acid set 3.
The base sequence of the nucleic acid combination 1 is shown as SEQ ID NO.1-3, the base sequence of the nucleic acid combination 2 is shown as SEQ ID NO.4-6, and the base sequence of the nucleic acid combination 3 is shown as SEQ ID NO. 7-9.
It is also within the scope of the present invention that a nucleic acid combination has a sequence identity of at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the base sequence indicated by the above-mentioned nucleic acid combination (nucleic acid combinations 1 to 3), and that the nucleic acid combination also has a certain diagnostic function for esophageal cancer or a precancerous lesion (such as a specificity or sensitivity that is substantially or slightly lower or slightly higher or greatly higher than that of nucleic acid combinations 1 to 3 of the present invention).
In a preferred embodiment of the present invention, the CpG island is selected from the full-length region or the partial region of the RAPGEFL1 gene as follows:
region 1, region 2 and region 3. The CpG islands of the present invention are equivalent to the target regions described above.
Wherein, the region 1 is selected from the positive chains of Chr17:40191282-40191349, the region 2 is selected from the positive chains of Chr17:40191371-40191476, and the region 3 is selected from the positive chains of Chr17: 40191544-40191653.
Nucleic acid set 1 was used for detection of region 1, nucleic acid set 2 was used for detection of region 2, and nucleic acid set 3 was used for detection of region 3.
In the canceration process, DNA methylation level abnormality mostly occurs in CpG islands which are mainly located in promoter and exon regions of genes and are regions rich in CpG dinucleotide, the length of the regions is between 200 and 3000bp, and the content of G + C is over 50 percent.
The invention also provides a detection reagent for diagnosing or assisting in diagnosing esophageal cancer or precancerous lesion, which comprises a nucleic acid combination for detecting the methylation level of the CpG island of the RAPGEFL1 gene.
The detection reagent may be in the form of a lyophilized powder or a solution, for example, a nucleic acid composition dissolved in ultrapure water or a buffer solution.
In a preferred embodiment of the present invention, the detection reagent is a reagent for detecting the methylation state of the CpG island of the RAPGEFL1 gene, and the means for detecting the methylation state includes 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 melting curve, methylation-sensitive restriction endonuclease, and flap endonuclease.
In one embodiment, the esophageal cancer is esophageal squamous carcinoma.
The invention also provides a detection kit for diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesions, which comprises a nucleic acid combination for detecting the methylation level of the CpG island of the RAPGEFL1 gene or a detection reagent for diagnosis or auxiliary diagnosis of esophageal cancer.
In a preferred embodiment of the present invention, the detection sample of the kit includes, but is not limited to, a plasma sample, a serum sample, a saliva sample, a tissue sample, or a cell sample of esophageal origin.
In one embodiment, the detection kit further comprises a PCR buffer.
The invention has the following beneficial effects:
the esophageal cancer diagnosis or auxiliary diagnosis reagent, the detection kit and the nucleic acid composition provided by the invention take methylation of CpG island of human RAPGEFL1 gene as a marker, can be used for diagnosing or auxiliary diagnosing esophageal cancer by detecting the increase of methylation level, and have higher sensitivity and specificity. The detection reagent and the kit provided by the invention can effectively improve the detection rate of esophageal cancer, thereby meeting the clinical requirements of early screening and early diagnosis of esophageal cancer/precancerous lesions.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a ROC curve for distinguishing between high grade neoplastic and normal samples of the esophagus for regions 1-3 of interest;
FIG. 2 is a ROC curve for regions of interest 1-3 distinguishing esophageal cancer samples from normal samples.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The present example provides a kit for the diagnosis or aided diagnosis of esophageal cancer or a precancerous lesion, comprising nucleotide combination 1. The nucleic acid combination 1 comprises nucleotides shown in SEQ ID NO. 1-3. The nucleotide combination 1 can detect methylation of the positive strand (target region 1) of the Chr17:40191282-40191349 region on the RAPGEFL1 gene.
The positive strand base sequence of region 1 is as follows (5 '-3'):
CGCCCTGGCCGCCCCTCCGCTGCCGTGGGTGCAGCTGGAG TTCGTGGACTACGTGTTCCACGGGGAGC。
the sequence of the completely methylated region 1 after bisulfite conversion is as follows (5 '-3'):
CGTTTTGGTCGTTTTTTCGTTGTCGTGGGTGTAGTTGGAGT TCGTGGATTACGTGTTTTACGGGGAGC。
the sequence of the upstream primer of region 1 methylation-specific PCR is (5 '-3'):
CGTTTTGGTCGTTTTTTCG(SEQ ID NO.1)；
the sequence of the downstream primer of region 1 methylation-specific PCR is (5 '-3'):
GCTCCCCGTAAAACACGTAAT(SEQ ID NO.2)；
the probe sequence for region 1 methylation specific PCR was (5 '-3'):
TGTCGTGGGTGTAGTTGGAGTTCGT(SEQ ID NO.3)。
the nucleotides shown in SEQ ID NO.1-3 can detect the methylation of cytosine at the positions of Chr7:40191282, Chr7:40191291, Chr7:40191299, Chr7:40191305, Chr7:40191324, Chr7:40191333, Chr7:40191342 and Chr7:40191349 on the positive strand of the region.
Example 2
The embodiment provides a kit for diagnosing or assisting in diagnosing esophageal cancer or precancerous lesions, which comprises a nucleotide combination 2, wherein the nucleotide combination 2 comprises nucleotides shown in SEQ ID NO. 4-6. The nucleotide combination 2 can detect methylation of the plus chain of the Chr17:40191371-40191476 region (target region 2) on the RAPGEFL1 gene.
The positive strand base sequence of region 2 is as follows (5 '-3'):
ACTTGGAGCTGCTGCTGCAGCGCTGCAGCGAGGTCACGCA CTGGGTGGCCACCGAAGTGCTGCTCTGCGAGGCCCCGGGCAA GCGCGCGCAGCTGCTCAAGAAGTT。
the sequence of the completely methylated region 2 after bisulfite conversion is as follows (5 '-3'):
ATTTGGAGTTGTTGTTGTAGCGTTGTAGCGAGGTTACGTAT TGGGTGGTTATCGAAGTGTTGTTTTGCGAGGTTTCGGGTAAGC GCGCGTAGTTGTTTAAGAAGTT。
the sequence of the upstream primer of region 2 methylation-specific PCR was (5 '-3'):
ATTTGGAGTTGTTGTTGTAGCG(SEQ ID NO.4)；
the sequence of the downstream primer of region 2 methylation-specific PCR was (5 '-3'):
AACTTCTTAAACAACTACGCGC(SEQ ID NO.5)；
probe sequence for methylation specific PCR for region 2 was (5 '-3'):
TGTAGCGAGGTTACGTATTGGGTG(SEQ ID NO.6)。
the nucleotides shown in SEQ ID NO.4-6 can detect the methylation of cytosine at the positions of Chr7:40191391, Chr7:40191399, Chr7:40191407, Chr7:40191456 and Chr7:40191458 on the positive strand of the region.
Example 3
This example provides a kit for diagnosis or aided diagnosis of esophageal cancer or precancerous lesions, comprising nucleotide set 3, wherein nucleic acid set 3 comprises nucleotides shown in SEQ ID nos. 7-9. The nucleotide combination 3 can detect the methylation of the plus strand of the Chr17:40191544-40191653 region (target region 3) of the RAPGEFL1 gene.
The plus strand base sequence of region 3 is as follows (5 '-3'):
CCGCCGCCCGCCCCTGACCCCGCCTCCCACCCCCGCAGCT GCAAGCAGAACCAGGACCTGCTGTCTTTCTACGCCGTGGTCAT GGGGCTGGACAACGCCGCTGTCAGCCG。
the sequence of the completely methylated region 3 after bisulfite conversion was as follows (5 '-3')
TCGTCGTTCGTTTTTGATTTCGTTTTTTATTTTCGTAGTTGTA AGTAGAATTAGGATTTGTTGTTTTTTTACGTCGTGGTTATGGGGT TGGATAACGTCGTTGTTAGTCG。
The sequence of the upstream primer of the region 3 methylation specific PCR is (5 '-3'):
TCGTCGTTCGTTTTTGATTTCG(SEQ ID NO.7)；
the sequence of the downstream primer of the region 3 methylation specific PCR is (5 '-3'):
CCTCCCAAATAAATCGAAAACG(SEQ ID NO.8)；
the probe sequence for region 3 methylation specific PCR was (5 '-3'):
CGACTAACAACGACGTTATCCAA(SEQ ID NO.9)。
the nucleotides shown in SEQ ID NO.7-9 can detect methylation of cytosine at the positions of Chr7:40191545, Chr7:40191548, Chr7:40191552, Chr7:40191564, Chr7:40191615, Chr7:40191618, Chr7:40191639, Chr7:40191642 and Chr7:40191652 on the positive strand of the region.
Example 4
The present embodiment provides a method for diagnosing or assisting in diagnosing esophageal cancer/precancerous lesion, which includes:
methylation of sample region 1-3 (see regions 1-3 shown in examples 1-3) was detected by methylation specific PCR, comprising the steps of:
(1) cfDNA extraction:
blood samples were collected with BCT blood collection tubes (Streck) and plasma was separated by centrifugation at 1900g for 10 minutes at 4 ℃ over 24 hours. The cfDNA in plasma was extracted with QIAamp MinElute cfDNA Mini Kit, see manufacturer's instructions for specific procedures.
(2) Bisulfite conversion:
the nucleic acid transformation Kit is EZ DNA Methylation-Gold (TM) Kit of ZYMO RESEARCH, and the specific experimental operation is described in the Kit specification. In this process, unmethylated cytosine (C) is converted to uracil (U), methylated cytosine is unchanged, uracil pairs with adenine (A) and cytosine pairs with guanine (G) in the subsequent PCR step, thereby achieving a distinction between methylated and unmethylated sequences.
(3) Methylation-specific PCR reaction:
and carrying out methylation specific PCR reaction on the DNA after bisulfite conversion to detect the methylation state of the 1-3 regions of the RAPGEFL1 gene, wherein each region is separately detected, namely, only one region of detection primers and probes are added into one PCR tube each time, and simultaneously, the detection probes of the internal reference gene are added. The PCR reaction system using ACTB as an internal reference gene is shown in Table 1. ACTB is used as an internal reference gene, wherein the upstream primer of the ACTB is as follows: AAGGTGGTTGGGTGGTTGTTTTG (SEQ ID NO. 10); the downstream primers for ACTB were: AATAACACCCCCACCCTGC (SEQ ID NO. 11); the probe for ACTB was: GGAGTGGTTTTTGGGTTTG (SEQ ID NO. 12).
The 5 'end of the probe in the target region is marked with a report group FAM, the 3' end is marked with a quenching group MGB, the 5 'end of the ACTB probe is marked with a report group VIC, and the 3' end of the ACTB probe is marked with a quenching group BHQ 1.
Table 1 PCR reaction system table.
Components
Specification of
Volume (μ L)
Buffer solution
5×
5
dNTPs
2.5mM each
2
Region upstream primer
10μM
0.5
Region downstream primer
10μM
0.5
Area probe
10μM
0.5
ACTB upstream primer
10μM
0.5
ACTB downstream primer
10μM
0.5
ACTB probes
10μM
0.5
DNA enzyme
5U/μL
0.3
DNA of sample to be tested
/
2
Purified water
/
Supply to 25
As shown in Table 1, when detecting the methylation state of any one of the regions 1 to 3 of RAPGEFL1 in a sample, the primer probe corresponding to the region, ACTB primer probe, buffer, dNTP, DNase, sample DNA, etc. are added to the reaction system in the volume indicated in the table, buffer, dNTP and DNA. When methylation of the detection regions 1-3 is detected, the DNA of the sample to be detected is added by converting the cfDNA extracted in the step (1) into bisulfite (step (2)).
The PCR reaction conditions are shown in Table 2 below.
Table 2 PCR reaction procedure.
Ct value reading: and after the PCR is finished, adjusting a base line, setting a fluorescence value of the sample in the primary PCR before the minimum Ct value is advanced by 1-2 cycles as a base line value, and setting a threshold value at an inflection point of an S-shaped amplification curve to obtain the Ct value of the target gene to be detected and the Ct value of the reference gene ACTB of each sample.
Quality control: the negative control and the positive control are synchronously detected at each detection.
The negative control was purified water.
The preparation method of the positive control comprises the following steps: and (3) artificially synthesizing a bisulfite converted sequence corresponding to the amplified region of the ACTB, and cloning the bisulfite converted sequence onto a vector to form an artificially synthesized plasmid. The bisulfite converted sequences corresponding to the completely methylated regions 1-3 were synthesized and cloned into an artificially synthesized plasmid. Positive control for zones 1-3 was 103Copy/microliter ACTB Artificial Synthesis plasmid and 103Copy/microliter of regions 1-3 of synthetic plasmid 1: 1, e.g. zone 1 positive control 103Copy/microliter ACTB Artificial Synthesis plasmid and 103Copy/microliter of region 1 synthetic plasmid 1: 1 are mixed.
And (3) the negative control needs no amplification, the Ct value of the positive control is 26-30, the Ct value of the reference gene of the sample to be detected is not more than 35, and the negative control, the positive control and the reference gene all meet the requirements, so that the experiment is effective and the next sample result can be judged. Otherwise, when the experiment is invalid, the detection is required to be carried out again.
Results analysis and interpretation methods: 2-ΔΔCtROC analysis is carried out on a gastric cancer/high-grade tumor sample and a healthy human sample, and the sensitivity, specificity and AUC value of the maximum Yoden index are recorded, wherein delta Ct (Ct) isArea to be examined-CtACTB)Sample(s)-(CtArea to be examined-CtACTB)Positive control。
And carrying out ROC analysis by using SPSS 22.0 software to verify the diagnosis efficiency of each region to be detected on the esophageal cancer sample and the normal sample and the diagnosis efficiency of each region to be detected on the esophageal precancerous lesion sample and the normal sample. The delta Ct at the time of maximum john's index was selected as cut-off value, and the sensitivity, specificity and AUC value at that time were recorded.
Example 5
This example provides a comparison of the detection effects of the regions 1-3 provided by the present invention and a control region, wherein the sequence of the upstream primer of the control group is: GCGAGTTTAAGTTTCGTGC, the sequence of the downstream primer of the control group is: GCAAAACTAAAAAAACGCG are provided.
The positions of the control regions on the chromosome are: chr17: 40177371-.
The primers for the upstream and downstream regions 1-3 are shown in examples 1-3.
Detecting methylation of the sample region by a methylation-specific PCR method, comprising the steps of:
(1) cfDNA extraction:
blood samples were collected with BCT blood collection tubes (Streck) and plasma was separated by centrifugation at 1900g for 10 minutes at 4 ℃ over 24 hours. The cfDNA in plasma was extracted with QIAamp MinElute cfDNA Mini Kit, see manufacturer's instructions for specific steps.
(2) Bisulfite conversion
The nucleic acid transformation Kit is EZ DNA Methylation-Gold (TM) Kit of ZYMO RESEARCH, and the specific experimental operation is described in the manufacturer's instruction. In this process, unmethylated cytosine (C) is converted to uracil (U), methylated cytosine is unchanged, uracil pairs with adenine (A) and cytosine pairs with guanine (G) in the subsequent PCR step, thereby achieving a distinction between methylated and unmethylated sequences.
(3) PCR reaction
And carrying out methylation specific PCR reaction on the DNA after bisulfite conversion by using a SYBR Green method to detect the methylation state of the RAPGEFL1 gene regions 1-3 and the control region in the sample, wherein each region is separately detected, namely, only one region of detection primers is added into one PCR tube at a time, and simultaneously, the detection primers of the reference gene are added. ACTB as internal reference gene, upstream and downstream primer sequence of ACTBAs shown in SEQ ID NO.10 and SEQ ID NO.11, using SYBRTMPCR was carried out using the Green PCR premix (cat. 4309155), and the PCR reaction system is shown in Table 3.
TABLE 3 SYBRTMAnd (3) performing a PCR reaction system by using the Green PCR premixed solution.
The PCR reaction conditions are shown in Table 4 below.
TABLE 4 SYBRTMPerforming a PCR reaction program by using the Green PCR premix.
Ct value reading: and after the PCR is finished, adjusting a base line, setting a fluorescence value of the sample in the primary PCR before the minimum Ct value is advanced by 1-2 cycles as a base line value, and setting a threshold value at an inflection point of an S-shaped amplification curve to obtain the Ct value of the target gene to be detected and the Ct value of the reference gene ACTB of each sample.
Quality control: the negative control and the positive control are synchronously detected in each detection, the negative control is purified water, the positive control is artificially synthesized plasmid containing ACTB gene and target gene sequence, and the concentration is 103Copying/microliter, wherein negative control needs no amplification, positive control needs obvious exponential growth, Ct value of reference gene of sample to be detected is less than or equal to 35, and the negative control, the positive control and the reference gene all meet the requirements, which shows that the experiment is effective and can be used for judging sample result in next step. Otherwise, when the experiment is invalid, the detection is required to be carried out again.
Results analysis and interpretation methods: and when the Ct value of a certain region is less than or equal to 38, determining that the region is detected to be methylated in the sample, and calculating the methylation detection rates of the target region 1-3 and the control region in the esophageal precancerous lesion sample, the esophageal cancer sample and the normal sample.
Example 6
This example provides a reagent for detecting or aiding diagnosis of esophageal cancer or precancerous lesions, which comprises the nucleic acid composition 1 of example 1.
Example 7
This example provides a reagent for detecting or aiding diagnosis of esophageal cancer or precancerous lesions, which comprises the nucleic acid composition 2 of example 2.
Example 8
This example provides a reagent for detecting or aiding diagnosis of esophageal cancer or precancerous lesions, which comprises the nucleic acid composition 3 of example 3.
Example 9
The present example provides a detection reagent for diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesions, which comprises nucleic acid combination 1 in example 1, nucleic acid combination 2 in example 2, and nucleic acid combination 3 in example 3.
Example 10
This example provides a reagent for the diagnosis or diagnosis-assisting of esophageal cancer or precancerous lesions, which comprises a combination of nucleic acids (the same as in example 1) for detecting the methylation level of cytosine at least one position of Chr7:40191282, Chr7:40191291, Chr7:40191299, Chr7:40191305, Chr7:40191324, Chr7:40191333, Chr7:40191342 and Chr7:40191349 in region 1.
Example 11
This example provides a reagent for detection of diagnosis or aided diagnosis of esophageal cancer or precancerous lesions, which comprises a combination of nucleic acids (the same as the nucleic acid combination 2 in example 2) for detecting the methylation level of cytosine at least one position of Chr7:40191391, Chr7:40191399, Chr7:40191407, Chr7:40191456 and Chr7:40191458 in the region 2.
Example 12
This example provides a reagent for the diagnosis or diagnosis-assisting of esophageal cancer or precancerous lesions, which comprises a nucleic acid combination (the same as in example 3) for detecting the methylation level of cytosine at least one position of Chr7:40191545, Chr7:40191548, Chr7:40191552, Chr7:40191564, Chr7:40191615, Chr7:40191618, Chr7:40191639, Chr7:40191642 and Chr7:40191652 in region 3.
Experimental example 1
59 samples of high grade esophageal neoplasia and 65 samples of normal blood were selected, the samples were obtained from the first subsidiary hospital of Zheng State university, methylation of the target region 1-3 in each sample was detected by the method of example 1-3, and the diagnostic efficacy, output sensitivity, specificity and AUC value of the target region 1-3 for the high grade esophageal neoplasia and normal samples were analyzed by the method of example 4, and the results are shown in Table 5 below, and the ROC curves of the target region 1-3 are shown in FIG. 1.
Table 5 sensitivity, specificity and AUC values of samples under different regional conditions.
Area name
Sensitivity of the probe
Specificity of
AUC
Destination area 1
67.8％
95.4％
0.824
Destination area 2
64.4％
93.8％
0.801
Destination area 3
76.3％
95.4％
0.872
The results show that the detection sensitivity of the regions 1-3 for the high-grade esophageal neoplasia is above 60%, the specificity is above 90%, the AUC values are above 0.8, the AUC value of the target region 3 is the highest and is obviously superior to that of the target region 1 and the target region 2, the detection sensitivity of the target region 3 for the high-grade esophageal neoplasia sample is 76.3%, and the specificity is 95.4%.
Experimental example 2
59 samples of esophageal cancer and 65 samples of normal blood (the normal samples were the same as those in Experimental example 1) were selected, methylation of the target regions 1-3 in each sample was detected by the method of examples 1-3, and the diagnostic efficacy, output sensitivity, specificity and AUC value of the target regions 1-3 for the esophageal cancer samples and the normal samples were analyzed by the method described in example 4, and the results are shown in Table 6 below, and the ROC curves of the target regions 1-3 are shown in FIG. 2.
Table 6 sensitivity, specificity and AUC values of samples under different regional conditions.
Area name
Sensitivity of the probe
Specificity of
AUC
Destination area 1
86.4％
93.8％
0.911
Destination area 2
84.7％
92.3％
0.902
Destination area 3
91.5％
95.4％
0.948
The results show that the detection sensitivity of the regions 1-3 for esophageal cancer is over 80%, the specificity is over 90%, the AUC values are over 0.9, the AUC value of the target region 3 is the highest and is obviously superior to that of the target region 1 and the target region 2, the detection sensitivity of the target region 3 for esophageal high-grade neoplastic samples is 91.5%, and the specificity is 95.4%.
Comparative example 1
59 cases of high grade neoplastic disease of esophagus (same as experimental example 1), 59 cases of esophageal cancer (same as experimental example 2) and 65 cases of normal blood samples (same as experimental example 1 and experimental example 2) were selected, and the methylation detection rates of the target regions 1 to 3 and the control region in the high grade neoplastic sample, the esophageal cancer sample and the normal sample were measured by the method described in example 5, and the results are shown in Table 7.
Table 7 sensitivity, specificity and AUC values of esophageal cancer high-grade neoplastic sample, esophageal cancer sample and normal sample under different regional conditions.
As shown in Table 7, the methylation detection rates of the target areas 1-3 in the esophageal cancer high-grade neoplastic samples and the esophageal cancer samples are significantly higher than those of the control areas, and the methylation detection rates of the target areas 1-3 in the normal samples are lower than those of the control areas, which indicates that the detection effects of the target areas 1-3 are better than those of the control areas. In addition, the detection effect of the target region 3 is superior to that of the target regions 1 and 2, which is consistent with the results of experimental example 1 and experimental example 2.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
SEQUENCE LISTING
<110> Wuhan Amisen Life technologies Ltd
<120> reagent and kit for diagnosis or auxiliary diagnosis of esophageal cancer or precancerous lesion and application thereof
<160> 12
<170> PatentIn version 3.5
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