CN108753979B - Kit for early screening of liver cancer and use method thereof - Google Patents
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Abstract
The invention belongs to the field of molecular biology, and particularly relates to a kit for early screening of liver cancer and a using method thereof. The invention discovers that SCAND3 can be used as a methylation marker for detecting liver cancer for the first time. The primer sequences for detecting SCAND3 methylation are as follows: the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2; the sequence of the fluorescent probe is SEQ ID NO.3, the 5 'end of the probe is marked with FAM, and the 3' end of the probe is marked with MGB. The invention takes SCAND3 as a methylation marker for early screening of liver cancer, is used for diagnosing liver cancer, has the advantages of high sensitivity and strong specificity, and particularly has the methylation incidence rate of SCAND3 reaching 60 percent in patients with AFP less than 400 mug/L; therefore, the invention takes SCAND3 as a methylation marker, and is suitable for diagnosing liver cancer, particularly early liver cancer.
Description
Technical Field
The invention belongs to the field of molecular biology, and particularly relates to a kit for early screening of liver cancer and a using method thereof.
Background
Hepatocellular carcinoma (HCC) is one of common malignant tumors threatening human life, while the early symptoms of most primary liver cancer patients are not obvious, and most patients reach middle and late stages when diagnosis is confirmed, so that the realization of early diagnosis and radical resection is the only hope for long-term survival of the patients. Due to the lack of specific tumor markers, most patients have been diagnosed at an advanced stage and the chance of radical resection is lost.
Currently, liver cancer diagnosis methods include imaging examination, pathology examination, and laboratory examination. However, the imaging performance of early liver cancer is not typical, and partial examination has the defects of radiation and high price. Liver biopsy is the gold standard for diagnosing HCC, but the focus of early liver cancer is small, the difficulty of biopsy is high, and the liver biopsy is traumatic to a certain extent and is not easy to be accepted by patients. The detection of the tumor marker has important supplementary value for liver cancer imaging and pathological diagnosis, and the recurrence and metastasis of the tumor or the tumor can be found in advance compared with other methods by monitoring the liver cancer marker, so that the accuracy of prediction and diagnosis is improved, the curative effect is improved, and the prognosis is improved. In the diagnosis of primary liver cancer, the tumor marker which is most widely used clinically is Alpha Fetoprotein (AFP), but the diagnosis sensitivity and specificity are not satisfactory, and the sensitivity and specificity for diagnosing liver cancer are 41-65% and 80-94% respectively.
In recent years, researches show that free tumor DNA exists in peripheral plasma of tumor patients and has the same gene change with tumor cell DNA, so that the detection of the peripheral circulating DNA of the tumor patients provides a new means for early minimally invasive diagnosis of tumors.
AFP is still the most common tumor marker for diagnosing liver cancer in clinical application, and 400 mug/L is taken as the critical value for clinically diagnosing liver cancer; when AFP is more than or equal to 400 mug/L, the diagnosis rate is higher; for some early stage patients with resectable disease, AFP < 400 μ g/L is easy to be missed. Therefore, the search for tumor markers with higher sensitivity and specificity has important significance for the early diagnosis of liver cancer, especially for patients with AFP < 400 mug/L.
Disclosure of Invention
The invention mainly aims to provide a kit for early screening of liver cancer and a using method thereof. The invention discovers that the methylation degree of SCAND3 is related to liver cancer occurrence for the first time, and the invention can be used for early detection and diagnosis of liver cancer by detecting the methylation degree of SCAND3 in peripheral blood.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a methylation marker for early screening of liver cancer, and the methylation marker is SCAND 3.
The second purpose of the invention is to provide the application of the methylation marker in the preparation of a reagent for screening and diagnosing liver cancer at an early stage.
The third object of the present invention is to provide a set of primers and probes for detecting the above methylation markers, wherein the sequences of the primers are as follows: the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2; the sequence of the fluorescent probe is SEQ ID NO.3, the 5 'end of the probe is marked with FAM, and the 3' end of the probe is marked with MGB.
The fourth purpose of the invention is to provide the application of the primer and the probe in the preparation of a kit for early screening of liver cancer.
The fifth purpose of the invention is to provide a kit, which comprises the primers and the probes, and also comprises a forward primer and a probe for detecting GAPDH, a PCR Master Mix, a lysate, proteinase K, a rinsing solution, a bisulfite solution and ddH2O。
The sixth object of the present invention is to provide a method for using the above kit, comprising the steps of:
(1) extraction of genomic DNA from the sample: taking a patient sample, and using lysis solution, proteinase K, rinsing solution and ddH2O, extracting to obtain a genome DNA solution;
(2) DNA methylation modification: modifying the genomic DNA solution extracted in the step (1) with a bisulfite solution to obtain methylated modified DNA;
(3) and (3) carrying out PCR amplification on the DNA modified in the step (2).
Preferably, the sample in step (1) is peripheral blood cells.
Preferably, the PCR reaction solution is: 20 mu L of PCR Master Mix, 1 mu L of each primer and probe, 3 mu L of fluorescent quantitative reaction solution, and 26 mu L of total amount per person; the PCR reaction conditions are as follows:
the seventh purpose of the invention is to provide the application of the kit in the preparation of the liver cancer early screening reagent.
The eighth purpose of the invention is to provide the application of the kit in preparing a reagent for detecting SCAND3 methylation
Free tumor DNA exists in peripheral blood, and liver cancer is diagnosed by detecting the methylation degree of genes in the peripheral blood, so that the diagnosis pain of a liver cancer patient can be relieved, the early and rapid diagnosis of the liver cancer can be realized, and the early detection and early treatment of the patient are facilitated. However, the degree of methylation of different genes in peripheral blood does not necessarily reflect the actual methylation of tissues. The inventor screens and obtains the SCAND3 gene in the research of the relationship between gene methylation and liver cancer occurrence, and finds that the methylation degree of the gene is related to the liver cancer occurrence for the first time through experiments. In further research, the methylation degree of the SCAND3 gene in liver cancer patients with AFP < 400 mug/L is higher than that of the known liver cancer marker genes SLIT2 and DAPK, so that the SCAND3 has more potential in early diagnosis of liver cancer.
Compared with the prior art, the invention has the following advantages:
(1) the invention takes SCAND3 as a target gene, is used for liver cancer diagnosis, has the advantages of high sensitivity and strong specificity, and particularly has the SCAND3 methylation incidence rate reaching 60 percent in patients with AFP less than 400 mug/L; therefore, the invention takes SCAND3 as a target gene, and is suitable for diagnosing liver cancer, particularly early liver cancer.
(2) The invention takes the peripheral blood of the patient as a sample, is used for minimally invasive diagnosis, can greatly reduce the pain degree of the patient, reduces the diagnosis cost of the patient and can realize rapid diagnosis.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a graph of RT-PCR amplification: 1-7 is sample No. 1-7 in liver cancer patient sample with AFP more than or equal to 400 mug/L, and 8-11 is sample No. 11-14 in liver cancer patient with AFP less than 400 mug/L.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of the stated features, steps, operations, and/or combinations thereof, unless the context clearly indicates otherwise.
In order to make the technical solution of the present invention more clearly understood by those skilled in the art, the technical solution of the present invention will be described in detail below with reference to specific examples and comparative examples.
Example 1SCAND3 and primers and probes therefor
The invention discovers for the first time that SCAND3 methylation can be used for early screening and diagnosis of liver cancer.
The primer sequences for detecting SCAND3 methylation are as follows:
the forward primer sequence is: 5'-GTTATAAATTGAGCGGTAAGATATTTGC-3', as shown in SEQ ID NO: 1 sequence is shown in the specification;
the reverse primer sequence is as follows: 5'-CCTCGCCCAAACTACTCCG-3', as shown in SEQ ID NO: 2 sequence is shown in the specification; ,
the fluorescent probe sequence is as follows: 5'-AATCGAGGTTATGACGGATA-3', as shown in SEQ ID NO: 3 sequence is shown; the 5 'end of the fluorescent probe is marked with FAM (fluorescent reporter group), and the 3' end is marked with MGB (fluorescent quencher group).
Other primers and probes used in the screening process of the invention are as follows:
first set of primers and probes:
the sequence of the forward primer is shown as SEQ ID NO.4 (5'-GCGAGATAGAAAATCGAGGTTATGAC-3'),
the reverse primer sequence is shown as SEQ ID NO.5 (5'-TATAAACCCCTCTCCCTCTCCG-3'),
the sequence of the fluorescent probe is shown as SEQ ID NO.6 (5'-AAGGAAACGAAGATCGGAGTA-3'), the 5 'end of the fluorescent probe is marked with FAM (fluorescent reporter group), and the 3' end of the fluorescent probe is marked with MGB (fluorescent quencher group).
Example 2 kit for early screening of liver cancer
The kit comprises a forward primer with a sequence shown as SEQ ID NO.1, a reverse primer with a sequence shown as SEQ ID NO.2 and a fluorescent probe with a sequence shown as SEQ ID NO. 3; the 5 'end of the probe is marked with FAM, and the 3' end is markedRecording MGB; in addition, the kit also comprises a forward primer and a probe for detecting GAPDH, a PCR Master Mix, a lysate, proteinase K, a rinsing solution, a bisulfite solution and ddH2O; the forward primer and probe for detecting GAPDH can be any known primer and probe in the art.
Method of use of the kit described in example 3
The use method of the kit in the embodiment 2 comprises the following specific steps:
(1) extraction of peripheral blood cell genomic DNA: taking peripheral blood cells, and using lysis solution, proteinase K, rinsing solution and ddH2O, extracting to obtain a genome DNA solution;
the method comprises the following specific steps:
and (3) sucking 200 mu L of peripheral blood sample, placing the sample in a 1.5mL centrifuge tube, adding 1mL lysate 1, continuously vortexing, shaking and uniformly mixing for 1min, fully mixing, and placing the 1.5mL centrifuge tube in a 70 ℃ water bath for 5 min.
The 1.5mL centrifuge tube was removed from the water bath, centrifuged at 12,000rpm for 5min, and 300. mu.L of the supernatant was slowly pipetted into a new 1.5mL centrifuge tube.
Add 200. mu.L lysate 2 and 20. mu.L proteinase K to a new 1.5mL centrifuge tube in sequence, vortex and mix well.
A new 1.5mL centrifuge tube was placed in a 70 ℃ water bath for 10 min.
Add 200. mu.L of isopropanol and vortex and mix.
The whole amount of the solution obtained in the previous step was put into an adsorption column (the adsorption column was put into a collection tube), centrifuged at 12,000rpm for 30sec, and the waste liquid was discarded.
To the adsorption column, 600. mu.L of the rinse solution was added, centrifuged at 12,000rpm for 30sec, and the waste solution was discarded.
Repeating the above operation, centrifuging at 12,000rpm for 2min, and discarding the waste liquid; the adsorption column was allowed to stand at room temperature for 2min to completely volatilize the residual rinse solution in the adsorption column.
Transferring the adsorption column into a 1.5mL centrifuge tube, suspending and dropwise adding 50 μ L of eluent into the middle position of the adsorption membrane, standing at room temperature for 2min, centrifuging at 12,000rpm for 2min, and collecting the solution into the centrifuge tube.
(2) DNA methylation modification: modifying the genomic DNA solution extracted in the step (1) with a bisulfite solution to obtain methylated modified DNA;
transferring all the DNA eluent extracted in the step (1) into a 0.2mL centrifuge tube, and adding 150 mu L of bisulfite solution; and (4) gently blowing and beating the mixture by a flick centrifugal tube or a liquid transfer device, and centrifuging the mixture for a short time after the mixture is uniformly mixed.
Placing the centrifugal tube in a temperature cycling temperature changer and setting according to the following conditions:
①98℃10min
②64℃1.5h
③4℃(≦20h)
the binding solution was added to 500. mu.L of the adsorption column, the mixture from the previous step was transferred to the adsorption column, the column cap was closed and the mixture was inverted and mixed, centrifuged at 12,000rpm for 30sec, and the waste solution was discarded.
To the adsorption column, 600. mu.L of the rinse solution was added, centrifuged at 12,000rpm for 30sec, and the waste solution was discarded.
Repeating the above operation once, centrifuging at 12,000rpm for 2min, and discarding the waste liquid; the adsorption column was allowed to stand at room temperature for 2min to completely volatilize the residual rinse solution in the adsorption column.
Transferring the adsorption column into a 1.5mL centrifuge tube, suspending and dropwise adding 30 mu L of eluent into the middle position of the adsorption film, standing at room temperature for 2min, centrifuging at 12,000rpm for 2min, and collecting the solution into the centrifuge tube.
(3) And (3) carrying out PCR amplification on the DNA modified in the step (2).
The PCR reaction solution was prepared according to the following table:
TABLE 1
And after the PCR reaction solution is fully and uniformly mixed, the PCR reaction solution is divided into PCR eight-connected tubes according to the volume of 26 mu L of each tube, and the PCR eight-connected tubes are labeled and transferred to a sample processing area.
Adding a BisDNA (DNA after methylation conversion) sample to be detected into a subpackaged PCR eight-way tube according to the amount of 2 mu L per hole, pressing a tube cover, centrifuging for a short time, and centrifuging tube wall liquid to the bottom of the tube.
And placing the PCR octal tubes at corresponding positions of the sample tanks of the instrument, and recording the placing sequence. Selecting instrument detection channels: reporter Dye 1: FAM, Quencher Dye 1: none; reporter Dye2(GAPDH) Cy5, quecher Dye 2: none; passive Reference: none. Setting of corresponding detection holes: before the amplification reaction begins, the sample to be detected and the quality control product are set as 'Unknown'.
The PCR reaction was set up as follows
TABLE 2
Test example 1
Peripheral blood of liver cancer patients (20 patients, wherein AFP is less than 400 mu g/L, 10 patients have 10 patients, AFP is more than or equal to 400 mu g/L), hepatitis patients (5 patients) and normal people (20 patients) is clinically and pathologically diagnosed in 2017 to 2018 in 5 months from a UpHudajian medical examination laboratory.
The CT value of each sample is shown in Table 3.
TABLE 3 CT value of the results of the fluorescent quantitative PCR assay of each sample
(2) According to table 3, the sensitivity and specificity were calculated as follows:
the sensitivity is the number of liver cancer samples with positive detection results/the total number of corresponding liver cancer samples;
the specificity is the number of non-liver cancer samples with negative detection results/the total number of the non-liver cancer samples;
the results are shown in Table 4.
TABLE 4 specificity and sensitivity of the present invention for detecting liver cancer
As can be seen from the detection results in tables 3 and 4, the detection sensitivity of SCAND3 is 70% in liver cancer patients with AFP more than or equal to 400 mug/L, and 60% in liver cancer patients with AFP less than 400 mug/L. The results show that the SCAND3 has good application potential in early diagnosis of liver cancer.
Test example 2
The sample of the invention described in test example 1 was used for detection, and the first set of primers and probes in example 1 were used for detection of liver cancer, and the detection results are shown in table 5 below.
TABLE 5 first set of primer specificity and sensitivity test results
As can be seen from Table 5, the primers and probes used in the present invention have better sensitivity and specificity than the first set of primers and probes.
Finally, although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the embodiments described above, or equivalents may be substituted for elements thereof. 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. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
SEQUENCE LISTING
<110> Anhui Dajian medicine science and technology Limited
<120> kit for early screening of liver cancer and use method thereof
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<170> PatentIn version 3.3
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Claims (2)
1. The application of primers and probes for screening the methylation marker in the early liver cancer stage in preparing a kit for screening the liver cancer stage is characterized in that the methylation marker is SCAND3, and the sequences of the primers and probes for the methylation marker SCAND3 are as follows: the sequence of the forward primer is shown as SEQ ID NO.1, and the sequence of the reverse primer is shown as SEQ ID NO. 2; the probe sequence is SEQ ID NO.3, the 5 'end of the probe is marked with FAM, and the 3' end of the probe is marked with MGB.
2. The use of claim 1, wherein the kit further comprises a forward primer and probe for detecting GAPDH, PCR Master Mix, lysis buffer, proteinase K, rinse, bisulfite solution and ddH2O。
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| CN109825583B (en) * | 2019-03-01 | 2021-08-17 | 清华大学 | Marker for early diagnosis of liver cancer by DNA methylation of human repeat element and application of marker |
| CN110669831B (en) * | 2019-11-11 | 2023-09-19 | 益善生物技术股份有限公司 | Human SGIP1, SCAND3 and MYO1G gene methylation detection kit |
| CN111549134B (en) * | 2020-05-18 | 2023-03-28 | 浙江大学医学院附属第一医院 | Liver cancer early detection kit based on polygenic mutation |
| CN112063717B (en) * | 2020-09-17 | 2022-09-20 | 山东大学深圳研究院 | Application of MDM2 as marker in early diagnosis of hepatitis B virus-related hepatocellular carcinoma and detection kit |
| CN114634981B (en) * | 2020-12-16 | 2024-01-26 | 广州达健生物科技有限公司 | Liver cancer gene methylation detection primer probe combination, kit and application thereof |
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