CN113774129A - Composition for detecting liver cancer, kit and application thereof - Google Patents
Composition for detecting liver cancer, kit and application thereof Download PDFInfo
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Abstract
The invention provides a composition for detecting liver cancer, a kit and application thereof. The liver cancer gene methylation detection kit provided by the invention takes important biological index DNA methylation abnormality of cancer diagnosis, early screening and prognosis as a detection object, can realize non-invasive detection by detecting the gene methylation state of peripheral blood, and simultaneously adopts a fluorescence quantitative PCR technology to obtain methylation chip data related to liver cancer through TCGA data for analysis, so as to screen out 3 gene methylation detection sites such as SGIP1, SCAND3, MYO1G and the like, and obtain the liver cancer detection kit with higher sensitivity and better specificity by establishing liver cancer methylation detection based on fluorescence quantitative PCR, so that the early screening and diagnosis of liver cancer are realized, and the early diagnosis and early treatment of liver cancer are facilitated.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a composition and application thereof in disease detection, and particularly relates to a composition for detecting liver cancer, a corresponding kit and application thereof.
Background
Liver cancer is one of the common malignant tumor diseases in China, and the mortality rate is the second place of malignant tumor. The liver cancer is well developed in middle-aged men, the liver cancer generally has no symptoms or atypical symptoms at the early stage, non-specific digestive tract symptoms such as anorexia, abdominal distension, vomiting and the like can be generated, when patients feel obvious discomfort or clinical symptoms are very obvious, most of the conditions enter the middle and late stages, the late-stage liver cancer treatment is not ideal, and the survival time is usually only half a year to half a year. Therefore, establishing a method for early warning and early screening of liver cancer is very important for preventing and treating liver cancer, and early discovery, early diagnosis, early treatment and early operation are effective means for preventing and controlling liver cancer.
The current liver cancer diagnosis technology comprises: 1) detection of alpha-fetoprotein: generally, the increase is more in primary liver cancer, but hepatitis lesion or other tumors can also cause the increase, and the specificity is not high; 2) imaging technology: MRI examination, B-ultrasonic examination and CT examination, but the small tumor is not sensitive enough and can not be diagnosed clearly; 3) liver puncture biopsy: liver biopsy under ultrasound or CT guidance is currently the most reliable method for determining liver cancer. However, the method belongs to invasive examination, has a certain false negative rate, causes physical discomfort to patients needing long-term tracking and observation, and has heavy economic burden. Therefore, there is a need to develop a novel sensitive and specific liver cancer marker and detection technology, which can improve the detection rate of early cancer of liver cancer, improve the treatment effect of liver cancer and reduce the death rate of liver cancer.
Epigenetics is a hot field of tumor research in recent years, epigenetic changes such as DNA methylation, histone modification, chromatin remodeling, and non-coding RNA regulation are considered to be closely related to tumor occurrence, wherein DNA methylation is the most common epigenetic change that can regulate cell proliferation, apoptosis, and differentiation, and the level is closely related to the biological characteristics of tumors. Various researches prove that the pathological process of liver cancer is a complex process of multi-gene variation accumulation, and relates to abnormal methylation of various oncogenes and cancer suppressor genes, wherein most abnormal methylation is hypermethylation of the cancer suppressor genes, and the hypermethylation often causes transcriptional silencing of the cancer suppressor genes. DNA methylation abnormality usually occurs in early cancer, and the methylation state of the DNA methylation abnormality changes once the DNA methylation abnormality is formed and needs to be continuously stimulated by external environment for a long time through the occurrence and development processes of the cancer, so that the detection of the DNA methylation index can be used as an important biological index for cancer diagnosis, early screening and prognosis.
The main detection methods for DNA methylation are numerous and can be roughly divided into two types from the application: whole genome methylation analysis and specific site methylation detection. The whole genome methylation analysis has higher detection cost and is often used as a high-throughput means for screening and discovering target genes; the specific site methylation detection method comprises a sodium bisulfite-associated restriction endonuclease analysis method (COBRA), a Methylation Specificity PCR (MSP), a methylation fluorescence quantitative method (MethyLight), a methylation sensitive high-resolution melting curve analysis method and the like, the restriction endonuclease analysis method can only obtain the methylation condition of a special enzyme cutting site, the methylation specificity PCR method is based on common PCR and electrophoretic analysis, the operation is complicated and the sample pollution is easy to cause, the methylation sensitive high-resolution melting curve analysis method has high requirements on instruments, a fluorescence quantitative PCR instrument with a high-resolution melting (HRM) module is required, and the methylation fluorescence quantitative method is based on high flux and high sensitivity, does not need operations such as electrophoresis and hybridization after PCR, reduces the pollution and operation errors, and is widely applied to the detection of DNA methylation. At present, methods for detecting liver cancer DNA methylation based on a methylation fluorescence quantitative method are few, and related detection is only directed at a single gene, so that the detection accuracy is not ideal, and the diagnosis effect is limited.
In view of the above, the invention establishes the multi-gene joint detection based on methylation fluorescence quantification by screening the liver cancer-related methylated genes and combining the multiple genes, and expects to obtain a detection reagent with higher sensitivity, specificity and accuracy, thereby realizing the early screening and diagnosis of liver cancer.
Disclosure of Invention
In order to achieve the purpose, the invention provides a composition for detecting liver cancer, a kit and application thereof, wherein TCGA data is used for acquiring methylation chip data related to liver cancer for analysis, 3 gene methylation detection sites such as SGIP1, SCAND3 and MYO1G are screened out, and liver cancer methylation detection kit with higher sensitivity and better specificity is obtained by establishing fluorescence quantitative PCR-based liver cancer methylation detection, so that early screening and diagnosis of liver cancer are realized, and early diagnosis and early treatment of liver cancer are facilitated.
The invention provides a PCR primer and probe combination for detecting liver cancer gene methylation, which is one or more of the following nucleic acid sequence combinations shown in 1) -3):
1) the PCR primer and probe for detecting SGIP1 methylation comprise one of a primer probe combination 1 and a primer probe combination 3, wherein the primer probe combination 1 comprises an upstream primer shown as SEQ ID No.1, a downstream primer shown as SEQ ID No.2 and a fluorescent probe shown as SEQ ID No.3, and the primer probe combination 3 comprises an upstream primer shown as SEQ ID No.4, a downstream primer shown as SEQ ID No.5 and a fluorescent probe shown as SEQ ID No. 6;
2) the PCR primer and probe for detecting the methylation of SCAND3 comprise one of a primer probe combination 5 and a primer probe combination 6, wherein the primer probe combination 5 comprises an upstream primer shown as SEQ ID NO.13, a downstream primer shown as SEQ ID NO.14 and a fluorescent probe shown as SEQ ID NO.15, and the primer probe combination 5 comprises an upstream primer shown as SEQ ID NO.16, a downstream primer shown as SEQ ID NO.17 and a fluorescent probe shown as SEQ ID NO. 18.
3) The PCR primer and probe for methylation detection of MYO1G comprise one of a primer probe combination 8 and a primer probe combination 9, wherein the primer probe combination 8 comprises an upstream primer shown in SEQ ID No.22, a downstream primer shown in SEQ ID No.23 and a fluorescent probe shown in SEQ ID No.24, and the primer probe combination 9 comprises an upstream primer shown in SEQ ID No.25, a downstream primer shown in SEQ ID No.26 and a fluorescent probe shown in SEQ ID No. 27.
In an embodiment of the present invention, the PCR primer and probe combination for detecting liver cancer gene methylation further includes a PCR primer and probe for detecting internal reference gene GAPDH, including an upstream primer shown in SEQ ID No.28, a downstream primer shown in SEQ ID No.29, and a fluorescent probe shown in SEQ ID No. 30.
In one embodiment of the invention, the 5' end of the fluorescent probe comprises a fluorescent reporter group, including any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY3, CY 5.
In one embodiment of the invention, the 3' end of the fluorescent probe comprises a fluorescence quenching group, including any one of MGB, BHQ-1, BHQ-2 and BHQ-3.
In a preferred embodiment of the present invention, the fluorescence quenching group is MGB.
The second aspect of the invention provides a kit for detecting liver cancer gene methylation, which comprises the PCR primer and probe combination of the first aspect of the invention, and further comprises a positive quality control product and a negative quality control product.
In one embodiment of the invention, the positive quality control product is human liver cancer cell line DNA.
In one embodiment of the present invention, the negative quality control material is human peripheral blood leukocyte DNA.
In an embodiment of the present invention, the final concentration of the reaction system of the kit for detecting liver cancer gene methylation comprises: 0.1-1 μ M PCR primer, 0.1-1 μ M probe, 0.001-10ng/μ l DNA template to be detected.
In a preferred embodiment of the present invention, the final concentration of the reaction system of the kit for detecting liver cancer gene methylation comprises: 0.1-0.5. mu.M PCR primer, 0.1-0.5. mu.M probe, 0.001-10 ng/. mu.l DNA template to be detected.
In an embodiment of the present invention, the PCR reaction conditions of the kit for detecting liver cancer gene methylation are as follows:
in a preferred embodiment of the present invention, the PCR reaction conditions of the kit for detecting liver cancer gene methylation are as follows:
the third aspect of the present invention provides a method for detecting methylation of a liver cancer gene, comprising the steps of:
1) separating nucleic acid of a target gene in a biological sample to be detected;
2) subjecting the nucleic acid obtained in the step 1) to bisulfite conversion treatment to obtain bisulfite converted DNA, namely Bis-DNA;
3) detecting the methylation state of the Bis-DNA obtained in the step 2) by adopting a PCR technology.
In one embodiment of the present invention, the biological sample in step 1) includes peripheral blood, fresh pathological tissue, paraffin-embedded tissue, and liver cancer cells.
In a preferred embodiment of the present invention, the biological sample is peripheral blood.
In a fourth aspect, the invention provides a PCR primer and probe combination for detecting liver cancer gene methylation according to the first aspect of the invention, a liver cancer gene methylation detection kit according to the second aspect of the invention, or an application of a detection method for detecting liver cancer gene methylation according to the third aspect of the invention in preparing a kit for detecting liver cancer.
In one embodiment of the invention, the applications include early screening, progress monitoring and prognosis evaluation of liver cancer.
The invention has the following beneficial effects:
1) can be used as an important index for early screening, process monitoring and prognosis evaluation of liver cancer: the kit for detecting liver cancer gene methylation provided by the invention takes DNA methylation abnormality as a detection object, the DNA methylation abnormality usually occurs in the early stage of cancer and runs through the occurrence and development processes of the cancer, and the methylation state of the DNA methylation abnormality changes once the DNA methylation abnormality is formed and needs to be continuously stimulated by external environment for a long time, so that the detection of DNA methylation indexes can be used as important biological indexes for early screening, process monitoring and prognosis evaluation of liver cancer;
2) the noninvasive detection can be realized: the kit for detecting liver cancer gene methylation provided by the invention can detect various samples, and can realize noninvasive detection by detecting the gene methylation state of peripheral blood;
3) the accuracy is high: the liver cancer gene methylation detection kit provided by the invention is based on a fluorescent quantitative PCR technology, establishes multi-gene joint detection based on methylation fluorescence quantification by screening liver cancer related methylation genes and combining multiple genes, obtains a detection reagent with higher sensitivity, specificity and accuracy by system optimization and experimental verification, and realizes early screening and diagnosis of liver cancer.
Drawings
FIG. 1 is a diagram showing the screening results of the primer-probe combination for single PCR for methylation detection of liver cancer genes according to the present invention;
FIG. 2 is a diagram showing the results of multiplex PCR primer probe combination screening for liver cancer gene methylation detection provided in the embodiments of the present invention.
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way.
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. Unless otherwise indicated, reagents and materials used in the following examples are commercially available. The experimental method not specified for the specific conditions is usually carried out under the conventional conditions or the conditions recommended by the manufacturer.
Example 1: sample DNA extraction and bisulfite conversion
1. Treatment of serum samples and DNA extraction
1) Obtaining a sample to be detected: selecting a serum sample of a clinical patient: taking venous blood of a subject, extracting 2mL by using a sterile syringe needle, collecting in a sterile collection tube, standing at room temperature for 30min, and spontaneously and completely condensing to separate out serum, or directly using a horizontal centrifuge, centrifuging at 3000rpm for 5min, and sucking the serum into a 1.5mL centrifuge tube for later use.
2) DNA extraction:
extracting by adopting a nucleic acid extraction kit of Guangzhou Dajian biological technology limited company, and operating as follows:
(a) taking a 1.5mLEP tube, sequentially adding 200 mu L of a sample to be detected and 20 mu L of pancreatic lipase, carrying out vortex oscillation, fully and uniformly mixing, and standing at room temperature for 5 min;
(b) adding 20 mu L of proteinase K and 360 mu L of lysate into an EP tube, shaking in a vortex, fully and uniformly mixing, centrifuging for a short time, and carrying out water bath at 70 ℃ for 10 min;
(c) centrifuging for a short time, adding 200 μ L of precooled isopropanol into an EP tube, performing vortex oscillation, mixing well, centrifuging for a short time to remove liquid drops on the inner wall of the tube cover, and standing for 5min at-20 ℃;
(d) c, completely adding the solution and the flocculent precipitate in the step c into an adsorption column (putting the adsorption column into a collecting pipe), centrifuging at 13000rpm for 1min, pouring out waste liquid, and recycling the collecting pipe;
(e) adding 600 μ L of rinsing solution I into the adsorption column, rinsing (precooling), centrifuging at 13000rpm for 1min, and discarding the waste liquid;
(f) adding 600 μ L of rinsing liquid II into the adsorption column, rinsing (precooling), centrifuging at 13000rpm for 1min, and discarding the waste liquid;
(g) placing the adsorption column into a clean 1.5mL EP tube, centrifuging at 13000rpm for 3min, and discarding the EP tube and waste liquid;
(h) putting the adsorption column into a clean 1.5mL EP tube, opening the tube cover, and air-drying for 3 min;
(i) suspending 100 μ L of eluent (Elution Buffer) at the middle position of the adsorption column, standing at room temperature for 3min, centrifuging at 13000rpm for 2min, collecting DNA, and storing at-20 deg.C.
3) Bisulfite conversion:
the transformation is carried out by adopting a transformation kit of Guangzhou Dajian biological technology limited company, and the operation is as follows:
(a) taking 45 mu L of DNA sample to be detected, putting the sample into a new 1.5mL centrifuge tube, adding 5 mu L of transformation buffer solution, and placing the sample in a metal bath for incubation for 15min at the constant temperature of 37 ℃;
(b) after the incubation is finished, adding 100 mu L of the prepared transformation liquid into each sample, uniformly mixing, centrifuging for a short time, and incubating for 12-16 hours in a metal bath at 50 ℃ in a dark place;
(c) placing the sample on ice (0-4 ℃) and incubating for 10 min;
(d) placing the adsorption column in a collecting tube, and adding 400 μ L binding solution into the adsorption column;
(e) c, adding the sample in the step c into an adsorption column (containing binding liquid), covering a tube cover tightly, turning upside down, uniformly mixing for a plurality of times, centrifuging for 30s at full speed (14000rpm), and discarding waste liquid;
(f) adding 100 mu L of rinsing liquid into the adsorption column, centrifuging at full speed for 30s, and discarding the waste liquid;
(g) adding 200 mu L of desulfonation liquid into the adsorption column, incubating for 20min at room temperature (20-30 ℃), centrifuging for 30s at full speed, and discarding waste liquid;
(h) adding 200 mu L of rinsing liquid into the adsorption column, centrifuging at full speed for 30s, repeatedly adding 200 mu L of rinsing liquid, centrifuging at full speed for 30s, and discarding the waste liquid and the collecting pipe;
(i) placing the adsorption column into a 1.5mL sterile centrifuge tube, suspending and dropwise adding 30 μ L eluent into the middle part of the adsorption membrane, eluting and converting DNA, centrifuging at full speed for 1min, collecting Bis-DNA, and storing at-20 ℃.
Example 2: liver cancer tissue hypermethylation candidate gene, specific primer and probe screening
1. Screening of candidate gene for hypermethylation of serum of liver cancer patient
The methylation chip data related to the liver cancer is obtained through a TCGA database (http:// cancer. nih. gov /), and is analyzed, 9 candidate gene loci which are highly methylated in the liver cancer are obtained: SGIP1, SCAND3, MYO1G, CDKN2A, Slit2, DAPK, PSD4, KLF3 and ATXN 1. Finally, SGIP1, SCAND3 and MYO1G are screened out.
2. Specific primer and probe screening for liver cancer methylation detection
1) Designing and screening specific primers and probes:
designing methylation primers and probes on Methyl primer Express v1.0 software according to the nucleic acid sequences of SGIP1, SCAND3 and MYO1G, repeatedly designing and knocking by an applicant, screening to obtain fluorescence quantitative PCR probes and primers for related gene methylation, and synthesizing the designed primers and probes by Beijing Rui Boxing Biotech Co., Ltd, wherein the specific sequences are shown in the following table:
specific primers and probes for an internal reference gene GAPDH are arranged at the same time, and the specific sequences are as follows:
| name (R) | Sequence (5 '-3') |
| Methy-GAPDH-F | GTGGAGAGAAATTTGGGAGGTTAG(SEQ ID NO.28) |
| Methy-GAPDH-R | CAACACAAACACATCCAACCTACA(SEQ ID NO.29) |
| Methy-GAPDH-P | ATGGTTTGAAGGTGGTAGGG(SEQ ID NO.30) |
Wherein the 5' end of the probe sequence is modified with a fluorescent group selected from any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY3 and CY 5; the 3' end is marked with a fluorescence quenching group, and is selected from one of MGB, BHQ-1, BHQ-2 and BHQ-3, preferably MGB.
2) PCR amplification further screens primer probe combinations:
and (3) PCR reaction system: the 25. mu.L PCR reaction system contained 12.5. mu.L of 2 XPCR reaction premix, 0.1. mu.L of each of 10. mu.M GAPDH primer and probe, 0.5. mu.L of each of 10. mu.M of each of the primer and probe combination, 0.2. mu.L of each of probe, 5. mu.L of Bis-DNA, and 25. mu.L of water.
The extent of the PCR reaction was as follows:
DNA samples of liver cancer patients and healthy human serum are used as templates, the primer probe combinations screened in the step 1) are screened through PCR amplification, and the results are shown in figure 1, the CT values of SGIP1-2, SCAND3-1 and MYO1G-1 are obviously larger than those of the other primer probe combinations, so that SGIP1-1, SGIP1-3, SCAND3-2, SCAND3-3, MYO1G-2 and MYO1G-3 are screened as PCR identification primers for liver cancer.
3) Multiplex PCR primer combination optimization
Performing combined pairing according to the primer probe combination screened in the step 2), and performing further screening through PCR amplification, wherein the combined pairing is shown in the following table:
as shown in FIG. 2, when PCR amplification was carried out using the multiplex PCR primer combinations of P1 and P7, the combinations P1 and P7 were significantly inferior in amplification efficiency to the other combinations in the case of significant deviation of the total CT values of the three genes, and thus the combinations P2, P3, P4, P5, P6 and P8 were selected as the multiplex PCR primer combinations.
Example 3: clinical sample detection and verification kit effect
1. The blood sample liver cancer gene methylation detection result is as follows:
in order to evaluate that the probe and the primer provided by the invention can be used for detecting methylation of liver cancer SGIP1, SCAND3 and MYO1G genes in a sample by fluorescence PCR, DNA templates derived from the same sample are divided into 12 parts, and the fluorescence PCR detection is completed under different detection systems of T1-T12, wherein the detection systems are shown in the following table:
| numbering | Primer probe combination |
| T1 | SGIP1-1 |
| T2 | SGIP1-3 |
| T3 | SCAND3-2 |
| T4 | SCAND3-3 |
| T5 | MYO1G-2 |
| T6 | MYO1G-3 |
| T7 | SGIP1-1、SCAND3-2、MYO1G-3 |
| T8 | SGIP1-1、SCAND3-3、MYO1G-2 |
| T9 | SGIP1-1、SCAND3-3、MYO1G-3 |
| T10 | SGIP1-3、SCAND3-2、MYO1G-2 |
| T11 | SGIP1-3、SCAND3-2、MYO1G-3 |
| T12 | SGIP1-3、SCAND3-3、MYO1G-3 |
The methylation detection of 92 liver cancer patients and 18 healthy human blood samples SGIP1, SCAND3 and MYO1G is respectively completed in a T1-T12 fluorescent PCR detection system, and the detection results are interpreted as follows, wherein target genes, namely SGIP1, SCAND3 and MYO1G, EI is a methylation index, and delta CT is a CT value of the target gene-a CT value of an internal reference gene:
A) the target gene has no amplification curve, and CT is 0; EI is 0. When the CT value of the target gene is less than or equal to 20, the CT value of the target gene is less than that of the reference gene, and the methylation index (EpiIndex, EI) EI is 5; if the CT value of the target gene is greater than the reference gene, the delta CT is less than or equal to 3, and the methylation index EI is 5; delta CT is 4-5, EI is 2; delta CT >5, EI ═ 0
B) And (3) summing the EIs of the target genes, and if the sigma EI value is greater than 5, determining that the sample detection result is positive, and if the sigma EI value is less than or equal to 5, determining that the sample detection result is negative.
The results are shown in table 1-table 12, when the SGIP1 gene was amplified alone in 92 patients with confirmed liver cancer, the detection rates of T1 and T2 for stage i liver cancer were all above 65%, and the detection rates for all confirmed liver cancers were above 73%; when the SCAND3 gene is used for independent amplification, the detection rate of T3 and T4 to liver cancer of stage I is more than 70%, and the detection rate to all liver cancer diagnosed is more than 76%; when MYO1G gene is used for independent amplification, the detection rate of T5 and T6 to liver cancer in stage I reaches more than 65%, and the detection rate to all diagnosed liver cancer reaches more than 77%; the detection rate of multiple PCR for stage I liver cancer reaches more than 85%, and the detection rate for all liver cancer confirmed diagnosis reaches more than 89%. In 18 healthy human control samples, 1 of T8 and T12 showed false positive detection results, and the detection specificity was 94%, while in other detection combinations, none of the healthy human control samples were detected, and the detection specificity was 100%. The results show that the liver cancer gene methylation detection kit provided by the invention has higher detection sensitivity and detection specificity when detecting the liver cancer blood sample gene methylation.
TABLE 1 detection results of SGIP1-1 gene methylation in sera
| Clinical staging of liver cancer patients | Amount of detected sample/number of detected samples | Percentage of detection (%) | Detection system |
| Stage I | 13/20 | 65.00 | T1 |
| Stage II | 17/24 | 70.83 | T1 |
| Stage III | 21/28 | 75.00 | T1 |
| Stage IV | 17/20 | 85.00 | T1 |
| All patients confirmed diagnosis | 68/92 | 73.91 | T1 |
| Control | 0/18 | 0 | T1 |
TABLE 2 detection results of SGIP1-3 gene methylation in serum
| Clinical staging of liver cancer patients | Amount of detected sample/number of detected samples | Percentage of detection (%) | Detection system |
| Stage I | 14/20 | 70.00 | |
| Stage II | |||
| 18/24 | 75.00 | | |
| Stage III | |||
| 22/28 | 78.57 | T2 | |
| Stage IV | 17/20 | 85.00 | T2 |
| All patients confirmed diagnosis | 70/92 | 76.09 | T2 |
| Control | 0/18 | 0 | T2 |
TABLE 3 detection results of SCAND3-2 gene methylation in serum
TABLE 4 detection results of SCAND3-3 gene methylation in serum
| Clinical staging of liver cancer patients | Amount of detected sample/number of detected samples | Percentage of detection (%) | Detection system |
| Stage I | 14/20 | 70.00 | T4 |
| Stage II | 17/24 | 70.83 | |
| Stage III | |||
| 22/28 | 78.57 | T4 | |
| Stage IV | 17/20 | 85.00 | T4 |
| All patients confirmed diagnosis | 70/92 | 76.09 | T4 |
| Control | 0/18 | 0 | T4 |
TABLE 5 detection results of methylation of MYO1G-2 gene in serum
| Clinical staging of liver cancer patients | Amount of detected sample/number of detected samples | Percentage of detection (%) | Detection system |
| Stage I | 13/20 | 65.00 | |
| Stage II | |||
| 18/24 | 75.00 | T5 | |
| Stage III | 23/28 | 82.14 | |
| Stage IV | |||
| 18/20 | 90.00 | T5 | |
| All patients confirmed diagnosis | 72/92 | 78.26 | T5 |
| Control | 0/18 | 0 | T5 |
TABLE 6 detection results of methylation of MYO1G-3 gene in serum
| Clinical staging of liver cancer patients | Amount of detected sample/number of detected samples | Percentage of detection (%) | Detection system |
| Stage I | 14/20 | 70.00 | T6 |
| Stage II | 17/24 | 70.83 | T6 |
| Stage III | 23/28 | 82.14 | T6 |
| Stage IV | 17/20 | 85.00 | T6 |
| All patients confirmed diagnosis | 71/92 | 77.17 | T6 |
| Control | 0/18 | 0 | T6 |
TABLE 7 detection results of methylation of SGIP1-1, SCAND3-2, MYO1G-3 genes in serum
TABLE 8 detection results of methylation of SGIP1-1, SCAND3-3, MYO1G-2 genes in serum
| Clinical staging of liver cancer patients | Amount of detected sample/number of detected samples | Percentage of detection (%) | Detection system |
| Stage I | 17/20 | 85.00 | |
| Stage II | |||
| 22/24 | 91.67 | T8 | |
| Stage III | 25/28 | 89.29 | |
| Stage IV | |||
| 18/20 | 90.00 | T8 | |
| All patients confirmed diagnosis | 82/92 | 89.13 | |
| Control | |||
| 1/18 | 5.56 | T8 |
TABLE 9 detection results of methylation of SGIP1-1, SCAND3-3, MYO1G-3 genes in serum
| Clinical staging of liver cancer patients | Amount of detected sample/number of detected samples | Percentage of detection (%) | Detection system |
| Stage I | 18/20 | 90.00 | |
| Stage II | |||
| 22/24 | 91.67 | T9 | |
| Stage III | 26/28 | 92.86 | T9 |
| Stage IV | 19/20 | 95.00 | T9 |
| All patients confirmed diagnosis | 85/92 | 92.39 | T9 |
| Control | 0/18 | 0 | T9 |
TABLE 10 detection results of methylation of SGIP1-3, SCAND3-2, MYO1G-2 genes in serum
| Clinical staging of liver cancer patients | Amount of detected sample/number of detected samples | Percentage of detection (%) | Detection system |
| Stage I | 18/20 | 90.00 | T10 |
| Stage II | 23/24 | 95.83 | T10 |
| Stage III | 27/28 | 96.43 | T10 |
| Stage IV | 20/20 | 100.00 | T10 |
| All patients confirmed diagnosis | 88/92 | 95.65 | T10 |
| Control | 0/18 | 0 | T10 |
TABLE 11 detection results of methylation of SGIP1-3, SCAND3-2, MYO1G-3 genes in serum
| Clinical staging of liver cancer patients | Amount of detected sample/number of detected samples | Percentage of detection (%) | Detection system |
| Stage I | 18/20 | 90.00 | |
| Stage II | |||
| 22/24 | 91.67 | T11 | |
| Stage III | 27/28 | 96.43 | T11 |
| Stage IV | 20/20 | 100.00 | T11 |
| All patients confirmed diagnosis | 87/92 | 94.57 | T11 |
| Control | 0/18 | 0 | T11 |
TABLE 12 detection results of methylation of SGIP1-3, SCAND3-3, MYO1G-3 genes in serum
| Clinical staging of liver cancer patients | Amount of detected sample/number of detected samples | Percentage of detection (%) | Detection system |
| Stage I | 18/20 | 90.00 | T12 |
| Stage II | 21/24 | 87.50 | T12 |
| Stage III | 26/28 | 92.86 | T12 |
| Stage IV | 20/20 | 100.00 | T12 |
| All patients confirmed diagnosis | 85/92 | 92.39 | |
| Control | |||
| 1/18 | 5.56 | T12 |
2) The detection result of the methylation of the liver cancer gene in the tissue sample is as follows:
the methylation detection of 14 liver cancer confirmed patients and 6 healthy human tissue samples SGIP1, SCAND3 and MYO1G is respectively completed in a T1-T12 fluorescent PCR detection system, and the results are shown in the following table:
the result shows that 11-14 liver cancer gene methylation positive samples are detected out of 14 liver cancer patients, the detection rate is 78% -100%, and the total detection rate under all detection conditions is more than 78%; in 6 healthy human control samples, the specificity of the detection method was 100%. The results show that the liver cancer gene methylation detection kit provided by the invention has higher detection sensitivity and detection specificity when detecting the gene methylation of the liver cancer tissue sample.
In conclusion, the liver cancer gene methylation detection kit provided by the invention has higher detection sensitivity and detection specificity, becomes an ideal choice for liver cancer diagnosis and early screening, and assists in early diagnosis and early treatment of liver cancer.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
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Claims (10)
1. The PCR primer and probe combination for detecting the methylation of the liver cancer gene is characterized by comprising one or more of the following nucleic acid sequence combinations shown in 1) -3):
1) the PCR primer and probe for detecting SGIP1 methylation comprise one of a primer probe combination 1 and a primer probe combination 3, wherein the primer probe combination 1 comprises an upstream primer shown as SEQ ID No.1, a downstream primer shown as SEQ ID No.2 and a fluorescent probe shown as SEQ ID No.3, and the primer probe combination 3 comprises an upstream primer shown as SEQ ID No.4, a downstream primer shown as SEQ ID No.5 and a fluorescent probe shown as SEQ ID No. 6;
2) the PCR primer and probe for detecting the methylation of SCAND3 comprise one of a primer probe combination 5 and a primer probe combination 6, wherein the primer probe combination 5 comprises an upstream primer shown as SEQ ID NO.13, a downstream primer shown as SEQ ID NO.14 and a fluorescent probe shown as SEQ ID NO.15, and the primer probe combination 5 comprises an upstream primer shown as SEQ ID NO.16, a downstream primer shown as SEQ ID NO.17 and a fluorescent probe shown as SEQ ID NO. 18.
3) The PCR primer and probe for methylation detection of MYO1G comprise one of a primer probe combination 8 and a primer probe combination 9, wherein the primer probe combination 8 comprises an upstream primer shown in SEQ ID No.22, a downstream primer shown in SEQ ID No.23 and a fluorescent probe shown in SEQ ID No.24, and the primer probe combination 9 comprises an upstream primer shown in SEQ ID No.25, a downstream primer shown in SEQ ID No.26 and a fluorescent probe shown in SEQ ID No. 27.
2. The PCR primer and probe combination for liver cancer gene methylation detection according to claim 1, further comprising a PCR primer and a probe for detecting an internal reference gene GAPDH, wherein the PCR primer for detecting the internal reference gene GAPDH comprises an upstream primer shown in SEQ ID No.28 and a downstream primer shown in SEQ ID No.29, and the probe comprises a fluorescent probe shown in SEQ ID No. 30.
3. The PCR primer and probe combination for liver cancer gene methylation detection according to claim 1, wherein the 5' end of the fluorescent probe comprises a fluorescent reporter group, and the fluorescent reporter group comprises any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY3, and CY 5.
4. The PCR primer and probe combination for liver cancer gene methylation detection according to claim 1, wherein the 3' end of the fluorescent probe comprises a fluorescence quenching group, and the fluorescence quenching group comprises any one of MGB, BHQ-1, BHQ-2 and BHQ-3.
5. A kit for detecting liver cancer gene methylation, which is characterized by comprising the PCR primer and probe combination of claim 1, and further comprising a positive quality control product and a negative quality control product.
6. The kit for detecting liver cancer gene methylation according to claim 5, wherein the final concentration composition of the reaction system of the kit comprises: 0.1-1 μ M PCR primer, 0.1-1 μ M probe, 0.001-10ng/μ l DNA template to be detected.
7. The kit for detecting liver cancer gene methylation according to claim 5, wherein the PCR reaction conditions of the kit are as follows:
8. a method for detecting liver cancer gene methylation is characterized by comprising the following steps:
1) separating nucleic acid of a target gene in a biological sample to be detected;
2) subjecting the nucleic acid obtained in the step 1) to bisulfite conversion treatment to obtain bisulfite converted DNA, namely Bis-DNA;
3) detecting the methylation state of the Bis-DNA obtained in the step 2) by adopting a PCR technology.
9. The method for detecting liver cancer gene methylation according to claim 8, wherein the biological sample in step 1) comprises peripheral blood, fresh pathological tissue, paraffin-embedded tissue, and liver cancer cells.
10. The use of the PCR primer and probe combination for detecting liver cancer gene methylation according to claim 1, the liver cancer gene methylation detection kit according to claim 5, or the detection method for detecting liver cancer gene methylation according to claim 8 in the preparation of a kit for detecting liver cancer.
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| JILIANG QIU 等: "CpG Methylation Signature Predicts Recurrence in Early-Stage Hepatocellular Carcinoma: Results From a Multicenter Study", JOURNAL OF CLINICAL ONCOLOGY, vol. 35, no. 7, XP055734672, DOI: 10.1200/JCO.2016.68.2153 * |
| 朱明玉 等: "肝细胞癌早期诊断血清学标志物的研究进展", 临床肝胆病杂志, vol. 30, no. 10, pages 1091 - 1093 * |
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