CN113774129B - 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 cancer diagnosis, early screening and prognosis important biological index DNA methylation abnormality as detection objects, can realize noninvasive detection by detecting the gene methylation state of peripheral blood, simultaneously adopts a fluorescent quantitative PCR technology, obtains methylation chip data related to liver cancer through TCGA data for analysis, screens out 3 gene methylation detection sites of SGIP1, SCAND3, MYO1G and the like, and obtains the liver cancer detection kit with higher sensitivity and better specificity by establishing liver cancer basal methylation detection based on fluorescent quantitative PCR, thereby realizing early screening and diagnosis of liver cancer and facilitating early diagnosis and early treatment of liver cancer.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a composition and application thereof in disease detection, and in particular relates to a composition for detecting liver cancer, and a corresponding kit and application thereof.
Background
Liver cancer is one of common malignant tumor diseases in China, and mortality is the second most serious malignant tumor. The liver cancer is good for middle-aged men, the early liver cancer generally has no symptoms or atypical symptoms, and can have non-specific digestive tract symptoms such as anorexia, abdominal distension, vomiting and the like, when patients feel obvious discomfort or clinical symptoms are obvious, the patients mostly enter middle and late stages, the treatment of the late liver cancer is not ideal, and the survival time is often only half a year to half a year. Therefore, the 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 the following steps: 1) Alpha fetoprotein detection: generally, the primary liver cancer shows a relatively large increase, but hepatitis lesions or other tumors are possibly increased, and the specificity is not high; 2) Imaging technology: MRI, B-ultrasonic and CT, but is not sensitive enough to smaller tumors and cannot be clearly diagnosed; 3) Liver biopsy: liver biopsy under ultrasound or CT guidance is currently the most reliable method for diagnosing liver cancer. However, the method belongs to invasive examination, has a certain false negative rate, causes uncomfortable body for patients needing long-term tracking and observation, and has heavy economic burden. Therefore, it is necessary to develop sensitive and specific novel liver cancer markers and detection techniques, to improve the early cancer detection rate of liver cancer, to improve the treatment effect of liver cancer and to reduce the death rate of liver cancer.
Epigenetic is a hot field of recent tumor research, and methylation of DNA, histone modification, chromatin remodeling, non-coding RNA regulation and other epigenetic changes are considered to have close relation with tumor occurrence, wherein the methylation of DNA is the most common epigenetic change, can regulate cell proliferation, apoptosis and differentiation, and the level is closely related with the biological characteristics of the tumor. Several studies have demonstrated that the hepatoma lesion process is a complex process of multiple genetic variation accumulation involving abnormal methylation of multiple oncogenes and cancer suppressor genes, most of which are hypermethylated for cancer suppressor genes, which often lead to transcriptional silencing of cancer suppressor genes. DNA methylation abnormalities usually occur in early cancer and throughout the course of cancer development and progression, the methylation state of which changes once it is established that it requires prolonged continuous stimulation by the external environment, so that the detection of DNA methylation indicators can be used as important biological indicators for diagnosis, early screening and prognosis of cancer.
The main detection methods for DNA methylation are numerous and can be broadly divided into two categories: whole genome methylation analysis and specific site methylation detection. The whole genome methylation analysis has higher detection cost, and is often used as a means for screening and finding target genes with high flux; specific site methylation detection methods include a restriction enzyme analysis method (COBRA) combined with sodium bisulphite, a methylation specific PCR method (methylation specific PCR, MSP), a methylation fluorescent quantitative method (methyl light), a methylation sensitivity high-resolution melting curve analysis method and the like, wherein the restriction enzyme analysis method can only obtain methylation conditions of specific enzyme cutting sites, the methylation specific PCR method is complicated in operation and easy to cause sample pollution based on common PCR and electrophoresis analysis, the methylation sensitivity high-resolution melting curve analysis method has high requirements on instruments, a fluorescent quantitative PCR instrument with a high-resolution melting (HRM) module is needed, and the methylation fluorescent quantitative method is based on high flux and high sensitivity, does not need operations such as electrophoresis and hybridization after PCR, pollution and operation errors are reduced, so that the methylation detection method is widely applied to DNA methylation detection. At present, methylation fluorescent quantitative method based liver cancer DNA methylation detection methods are few, and meanwhile, related detection is often only aimed at a single gene, so that detection accuracy is often not ideal, and diagnosis effect is limited.
In view of the above, the invention establishes polygene combined detection based on methylation fluorescence quantification by screening liver cancer related methylation genes and performing polygene combination, and is expected to obtain detection reagents with higher sensitivity, specificity and accuracy so as to realize early screening and diagnosis of liver cancer.
Disclosure of Invention
In order to achieve the aim, the invention provides a composition for detecting liver cancer, a kit and application thereof, wherein the composition acquires methylation chip data related to the liver cancer through TCGA data for analysis, screens out 3 methylation detection sites of genes such as SGIP1, SCAND3 and MYO1G, and the like, and obtains a liver cancer detection kit with higher sensitivity and better specificity through establishing fluorescence quantitative PCR-based liver cancer methylation detection, thereby realizing early screening and diagnosis of the liver cancer and facilitating early diagnosis and early treatment of the liver cancer.
The first aspect of the present invention provides a PCR primer and probe combination for methylation detection of liver cancer genes, comprising one or more of the nucleic acid sequence combinations shown in the following 1) -3):
1) 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, wherein 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 the probe for SCAND3 methylation detection 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 the probe for MYO1G methylation detection 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 as SEQ ID NO.22, a downstream primer shown as SEQ ID NO.23 and a fluorescent probe shown as SEQ ID NO.24, and the primer probe combination 9 comprises an upstream primer shown as SEQ ID NO.25, a downstream primer shown as SEQ ID NO.26 and a fluorescent probe shown as SEQ ID NO. 27.
In an embodiment of the invention, the combination of the PCR primer and the probe for methylation detection of liver cancer genes further comprises a PCR primer and a probe for detection of reference gene GAPDH, including an upstream primer shown as SEQ ID No.28, a downstream primer shown as SEQ ID No.29, and a fluorescent probe shown as SEQ ID No. 30.
In an embodiment of the present invention, the 5' end of the fluorescent probe includes a fluorescent reporter group, including any one of FAM, HEX, NED, ROX, TET, JOE, TAMRA, CY and 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 invention, the fluorescence quenching group is MGB.
The second aspect of the invention provides a liver cancer gene methylation detection kit, which comprises the PCR primer and probe combination according to 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 substance is human liver cancer cell line DNA.
In an embodiment of the invention, the negative quality control product is human peripheral blood leukocyte DNA.
In an embodiment of the invention, the final concentration composition of the liver cancer gene methylation detection kit reaction system includes: 0.1-1 mu M PCR primer, 0.1-1 mu M probe, 0.001-10 ng/mu l DNA template to be detected.
In a preferred embodiment of the present invention, the final concentration composition of the liver cancer gene methylation detection kit reaction system includes: 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 invention, the PCR reaction conditions of the liver cancer gene methylation detection kit are as follows:
in a preferred embodiment of the present invention, the PCR reaction conditions of the liver cancer gene methylation detection kit are as follows:
the third aspect of the present invention provides a method for detecting methylation of liver cancer genes, comprising the steps of:
1) Isolating nucleic acid of a target gene in a biological sample to be tested;
2) Subjecting the nucleic acid obtained in the step 1) to bisulfite conversion treatment to obtain bisulfite converted DNA (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 invention, the biological sample is peripheral blood.
The fourth aspect of the invention provides a PCR primer and probe combination for liver cancer gene methylation detection 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 liver cancer gene methylation detection method according to the third aspect of the invention in preparation of a liver cancer detection kit.
In one embodiment of the invention, the application comprises 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, progress monitoring and prognosis evaluation of liver cancer: the liver cancer gene methylation detection kit provided by the invention takes DNA methylation abnormality as a detection object, the DNA methylation abnormality usually occurs in early cancer and penetrates through the occurrence and development processes of the cancer, and the methylation state of the liver cancer gene methylation detection kit can be changed once the methylation state is formed and needs to be continuously stimulated by the external environment for a long time, so that the detection of the DNA methylation index can be used as an important biological index for early screening, process monitoring and prognosis evaluation of liver cancer;
2) Non-invasive detection is possible: the liver cancer gene methylation detection kit 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 fluorescence quantitative PCR technology, and through screening liver cancer related methylation genes and carrying out polygene combination, polygene joint detection based on methylation fluorescence quantification is established, and detection reagents with higher sensitivity, specificity and accuracy are obtained through system optimization and experimental verification, so that early screening and diagnosis of liver cancer are realized.
Drawings
FIG. 1 is a diagram showing the screening result of a single PCR primer probe combination for methylation detection of liver cancer genes according to an embodiment of the present invention;
FIG. 2 is a diagram showing the result of screening by multiplex PCR primer probe combinations for methylation detection of liver cancer genes according to an embodiment of the present invention.
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way.
Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art. Reagents and materials used in the following examples are commercially available unless otherwise specified. Experimental methods, in which specific conditions are not specified, are generally performed under conventional conditions, or under conditions recommended by the manufacturer.
Example 1: sample DNA extraction and bisulfite conversion
1. Serum sample processing and DNA extraction
1) Obtaining a sample to be tested: selecting a serum sample of a clinical patient: taking venous blood of a tested person, extracting 2mL by using a sterile injection needle, collecting the venous blood in a sterile collecting tube, standing for 30min at room temperature, and allowing a blood sample to spontaneously and completely coagulate and separate out serum, or directly using a horizontal centrifuge for 5min at 3000rpm, and transferring the extracted serum into a 1.5mL centrifuge tube for later use.
2) DNA extraction:
the extraction was performed using the Guangzhou Dajian Biotechnology Co., ltd nucleic acid extraction kit, and the procedure was as follows:
(a) Taking a 1.5mLEP tube, sequentially adding 200 mu L of sample to be detected and 20 mu L of pancreatic lipase, carrying out vortex vibration, fully mixing, and standing at room temperature for 5min;
(b) Adding 20 mu L of proteinase K,360 mu L of lysate into an EP tube, vortex shaking, fully mixing, centrifuging briefly, and carrying out water bath at 70 ℃ for 10min;
(c) Adding 200 mu L of precooled isopropanol into an EP tube, carrying out vortex shaking, fully and uniformly mixing, carrying out short centrifugation to remove liquid drops on the inner wall of a tube cover, and standing for 5min at-20 ℃;
(d) C, adding the solution and flocculent precipitate in the step c into an adsorption column (the adsorption column is placed into a collecting pipe), centrifuging at 13000rpm for 1min, pouring out waste liquid, and recycling the collecting pipe;
(e) Adding 600 mu L of rinsing liquid I into an adsorption column for rinsing (precooling), centrifuging at 13000rpm for 1min, and discarding waste liquid;
(f) Adding 600 mu L of rinsing liquid II into an adsorption column for rinsing (precooling), centrifuging at 13000rpm for 1min, and discarding 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 the waste liquid;
(h) Placing the adsorption column into a clean 1.5mL EP tube, opening the tube cover, and air drying for 3min;
(i) And (3) suspending and dripping 100 mu L of eluent (absorption Buffer) into the middle position of the adsorption column, standing for 3min at room temperature, centrifuging for 2min at 13000rpm, collecting DNA into a centrifuge tube, and preserving at-20 ℃.
3) Bisulfite conversion:
transformation was performed using the Guangzhou Dajian Biotechnology Co., ltd, as follows:
(a) Taking 45 mu L of DNA sample to be detected in a new 1.5mL centrifuge tube, adding 5 mu L of conversion buffer solution, and placing in a metal bath for incubation at a constant temperature of 37 ℃ for 15min;
(b) After the incubation is completed, 100 mu L of a pre-prepared conversion solution is added into each sample, the mixture is uniformly mixed and centrifuged for a short time, and the metal bath is incubated for 12 to 16 hours at 50 ℃ in a dark place;
(c) Placing the sample on ice (0-4 ℃) and incubating for 10min;
(d) Placing the adsorption column in a collecting pipe, and adding 400 mu L of binding solution into the adsorption column;
(e) C, adding the sample in the step into an adsorption column (containing a binding solution), covering a tube cover, uniformly mixing the sample with the binding solution in an upside down manner for a plurality of times, centrifuging the sample at full speed (14000 rpm) for 30s, and discarding waste liquid;
(f) Adding 100 mu L of rinsing liquid into the adsorption column, centrifuging at full speed for 30s, and discarding waste liquid;
(g) Adding 200 mu L of desulfonation liquid into an 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, discarding waste liquid and collecting the tube;
(i) Placing the adsorption column into a 1.5mL sterile centrifuge tube, suspending and dripping 30 mu L of eluent into the middle part of the adsorption film, eluting and transforming DNA, centrifuging at full speed for 1min, collecting Bis-DNA, and preserving at-20 ℃.
Example 2: liver cancer tissue hypermethylation candidate gene, specific primer and probe screening
1. Screening of serum hypermethylation candidate genes of liver cancer patients
Methylation chip data related to liver cancer are obtained through a TCGA database (http:// cancetrgenome. Nih. Gov /), and are analyzed, so that 9 candidate gene loci hypermethylated in the liver cancer are obtained: SGIP1, SCAND3, MYO1G, CDKN2A, slit2, DAPK, PSD4, KLF3, ATXN1. Finally we screen out SGIP1, SCAND3, MYO1G.
2. Specific primer and probe screening for liver cancer methylation detection
1) Specific primer and probe design screening:
according to the nucleic acid sequences of SGIP1, SCAND3 and MYO1G, designing methylation primers and probes on Methyl primer Express v 1.0.0 software, repeatedly designing and knocking by the applicant, screening to obtain related gene methylation fluorescent quantitative PCR probes and primers, and sending the designed primers and probes to Beijing Rui Boxing family biotechnology Co., ltd for synthesis, wherein the specific sequences are shown in the following table:
meanwhile, a specific primer and a probe aiming at the reference gene GAPDH are arranged, and the specific sequence is as follows:
| name of the name | 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, CY and CY 5; the 3' end is marked with fluorescence quenching group selected from any one of MGB, BHQ-1, BHQ-2 and BHQ-3, preferably MGB.
2) PCR amplification further screening primer probe combinations:
PCR reaction system: in a 25. Mu.L PCR reaction system, the premixed solution of 2 XPCR reaction was 12.5. Mu.L, 10. Mu.M GAPDH primer and probe were each 0.1. Mu.L, 10. Mu.M each 0.5. Mu.L of the primer in the above primer probe combination, and 5. Mu.L of Bis-DNA were each 0.2. Mu.L of the probe, and water was added to 25. Mu.L.
The PCR reaction was performed as follows:
the DNA samples of serum of a patient with a definite diagnosis of liver cancer and a healthy person are taken as templates, and the primer probe combination screened in the step 1) is screened through PCR amplification, and the result is shown in a figure 1, wherein the CT values of SGIP1-2, SCAND3-1 and MYO1G-1 are obviously larger than those of the rest primer probe combinations, so that the PCR identification primers for liver cancer are screened by SGIP1-1, SGIP1-3, SCAND3-2, SCAND3-3, MYO1G-2 and MYO 1G-3.
3) Multiplex PCR primer combination optimization
The primer probe combinations screened in the step 2) are combined and paired, and further screened through PCR amplification, and the combinations are provided in the following table:
as a result, as shown in FIG. 2, when PCR amplification was performed with the multiplex PCR primer combinations of P1 and P7, the overall CT values of the three genes were significantly biased, and the amplification efficiencies of the combinations P1 and P7 were significantly inferior to those of the remaining combinations, so that the combinations P2, P3, P4, P5, P6 and P8 were selected as multiplex PCR primer combinations.
Example 3: clinical sample detection and verification kit effect
1. Liver cancer gene methylation detection results of blood samples:
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, dividing a DNA template from the same sample into 12 parts, and completing detection of fluorescence PCR under different detection systems of T1-T12, wherein the detection systems are shown in the following table:
| numbering device | 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 SGIP1, SCAND3 and MYO1G genes of 92 liver cancer definite patients and 18 healthy human blood samples is completed in a T1-T12 fluorescence PCR detection system, and the detection results are interpreted as follows, wherein target genes, namely SGIP1, SCAND3 and MYO1G, EI are methylation indexes, and DeltaCT=target gene CT value-internal reference gene CT value:
a) No amplification curve of target gene, ct=0; ei=0. Under the condition that the CT value of the target gene is less than or equal to 20, the CT value of the target gene is smaller than the CT value of the reference gene, and the methylation index (EI) EI=5; if the CT value of the target gene is larger than that of the reference gene, the delta CT is less than or equal to 3, and the methylation index EI=5; Δct=4-5, ei=2; Δct >5, ei=0
B) And summing EI of the target gene, judging that the sample detection result is positive if the value of the Sigma EI is greater than 5, and judging that the sample detection result is negative if the value of the Sigma EI is less than or equal to 5.
As shown in tables 1-12, in 92 cases of liver cancer diagnosis patients, when SGIP1 gene is used for single amplification, the detection rate of T1 and T2 on the liver cancer of stage I is more than 65%, and the detection rate of all the liver cancer diagnosis patients is more than 73%; when the SCAND3 gene is used for single amplification, the detection rate of the T3 and the T4 on the liver cancer in stage I is more than 70%, and the detection rate of the liver cancer in all definite diagnosis is more than 76%; when MYO1G genes are singly amplified, the detection rate of T5 and T6 on the liver cancer in stage I reaches more than 65%, and the detection rate of all the diagnosed liver cancers reaches more than 77%; the detection rate of the multiplex PCR on the liver cancer in stage I reaches more than 85 percent, and the detection rate of the multiplex PCR on all the liver cancers in definite diagnosis reaches more than 89 percent. In 18 healthy person control samples, 1 case of T8 and T12 respectively has false positive detection results, the detection specificity is 94%, and in other detection combinations, the healthy person control samples are not detected, and the detection specificity is 100%. The result shows that the liver cancer gene methylation detection kit provided by the invention has higher detection sensitivity and detection specificity when detecting liver cancer blood sample gene methylation.
TABLE 1 methylation detection of SGIP1-1 Gene in serum
| Clinical staging of liver cancer patients | Sample size/number of samples detected | Detection rate (%) | Detection system |
| Phase 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 diagnosed | 68/92 | 73.91 | T1 |
| Control | 0/18 | 0 | T1 |
TABLE 2 methylation detection of SGIP1-3 genes in serum
| Clinical staging of liver cancer patients | Sample size/number of samples detected | Detection rate (%) | Detection system |
| Phase I | 14/20 | 70.00 | T2 |
| Stage II | 18/24 | 75.00 | T2 |
| Stage III | 22/28 | 78.57 | T2 |
| Stage IV | 17/20 | 85.00 | T2 |
| All patients diagnosed | 70/92 | 76.09 | T2 |
| Control | 0/18 | 0 | T2 |
TABLE 3 methylation detection of SCAND3-2 Gene in serum
TABLE 4 methylation detection of SCAND3-3 genes in serum
| Clinical staging of liver cancer patients | Sample size/number of samples detected | Detection rate (%) | Detection system |
| Phase I | 14/20 | 70.00 | T4 |
| Stage II | 17/24 | 70.83 | T4 |
| Stage III | 22/28 | 78.57 | T4 |
| Stage IV | 17/20 | 85.00 | T4 |
| All patients diagnosed | 70/92 | 76.09 | T4 |
| Control | 0/18 | 0 | T4 |
TABLE 5 methylation detection of MYO1G-2 Gene in serum
| Clinical staging of liver cancer patients | Sample size/number of samples detected | Detection rate (%) | Detection system |
| Phase I | 13/20 | 65.00 | T5 |
| Stage II | 18/24 | 75.00 | T5 |
| Stage III | 23/28 | 82.14 | T5 |
| Stage IV | 18/20 | 90.00 | T5 |
| All patients diagnosed | 72/92 | 78.26 | T5 |
| Control | 0/18 | 0 | T5 |
TABLE 6 methylation detection of MYO1G-3 Gene in serum
| Clinical staging of liver cancer patients | Sample size/number of samples detected | Detection rate (%) | Detection system |
| Phase 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 diagnosed | 71/92 | 77.17 | T6 |
| Control | 0/18 | 0 | T6 |
TABLE 7 methylation detection results of SGIP1-1, SCAND3-2, MYO1G-3 genes in serum
TABLE 8 methylation detection results of SGIP1-1, SCAND3-3, MYO1G-2 genes in serum
| Clinical staging of liver cancer patients | Sample size/number of samples detected | Detection rate (%) | Detection system |
| Phase I | 17/20 | 85.00 | T8 |
| Stage II | 22/24 | 91.67 | T8 |
| Stage III | 25/28 | 89.29 | T8 |
| Stage IV | 18/20 | 90.00 | T8 |
| All patients diagnosed | 82/92 | 89.13 | T8 |
| Control | 1/18 | 5.56 | T8 |
TABLE 9 methylation detection results of SGIP1-1, SCAND3-3, MYO1G-3 genes in serum
| Clinical staging of liver cancer patients | Sample size/number of samples detected | Detection rate (%) | Detection system |
| Phase I | 18/20 | 90.00 | T9 |
| Stage II | 22/24 | 91.67 | T9 |
| Stage III | 26/28 | 92.86 | T9 |
| Stage IV | 19/20 | 95.00 | T9 |
| All patients diagnosed | 85/92 | 92.39 | T9 |
| Control | 0/18 | 0 | T9 |
TABLE 10 methylation detection results of SGIP1-3, SCAND3-2, MYO1G-2 genes in serum
| Clinical staging of liver cancer patients | Sample size/number of samples detected | Detection rate (%) | Detection system |
| Phase 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 diagnosed | 88/92 | 95.65 | T10 |
| Control | 0/18 | 0 | T10 |
TABLE 11 methylation detection results of SGIP1-3, SCAND3-2, MYO1G-3 genes in serum
| Clinical staging of liver cancer patients | Sample size/number of samples detected | Detection rate (%) | Detection system |
| Phase I | 18/20 | 90.00 | T11 |
| Stage II | 22/24 | 91.67 | T11 |
| Stage III | 27/28 | 96.43 | T11 |
| Stage IV | 20/20 | 100.00 | T11 |
| All patients diagnosed | 87/92 | 94.57 | T11 |
| Control | 0/18 | 0 | T11 |
TABLE 12 methylation detection results of SGIP1-3, SCAND3-3, MYO1G-3 genes in serum
| Clinical staging of liver cancer patients | Sample size/number of samples detected | Detection rate (%) | Detection system |
| Phase 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 diagnosed | 85/92 | 92.39 | T12 |
| Control | 1/18 | 5.56 | T12 |
2) Liver cancer gene methylation detection results of tissue samples:
the methylation detection of SGIP1, SCAND3 and MYO1G genes of 14 liver cancer diagnosis patients and 6 healthy human tissue samples are respectively completed in a T1-T12 fluorescence PCR detection system, and the results are shown in the following table:
the results show that in 14 cases of liver cancer definite diagnosis patients, 11-14 cases of liver cancer gene methylation positive samples are detected altogether, the detection rate is 78% -100%, and the total detection rate is more than 78% under all detection conditions; in 6 healthy person control samples, the specificity of the detection method is 100%. The result shows that the liver cancer gene methylation detection kit provided by the invention has higher detection sensitivity and detection specificity when detecting liver cancer tissue sample gene methylation.
In conclusion, the liver cancer gene methylation detection kit provided by the invention has higher detection sensitivity and detection specificity, and is ideal for diagnosis and early screening of liver cancer, and early diagnosis and early treatment of liver cancer are assisted.
While the foregoing description illustrates and describes several preferred embodiments of the invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as limited to other embodiments, and is capable of use in various other combinations, modifications and environments and is capable of changes or modifications within the spirit of the invention described herein, either as a result of the foregoing teachings or as a result of the knowledge or skill of the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
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Claims (9)
1. The PCR primer and probe combination for liver cancer gene methylation detection is characterized by comprising the following components:
1) 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, wherein 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 SCAND3 methylation detection comprises a primer probe combination 5 or 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 MYO1G methylation detection comprises a primer probe combination 8 or a primer probe combination 9, wherein the primer probe combination 8 comprises an upstream primer shown as SEQ ID NO.22, a downstream primer shown as SEQ ID NO.23 and a fluorescent probe shown as SEQ ID NO.24, and the primer probe combination 9 comprises an upstream primer shown as SEQ ID NO.25, a downstream primer shown as SEQ ID NO.26 and a fluorescent probe shown as SEQ ID NO. 27.
2. The combination of PCR primers and probes for methylation detection of liver cancer genes according to claim 1, further comprising PCR primers and probes for detecting GAPDH of a reference gene, wherein the PCR primers for detecting GAPDH of the reference gene comprise an upstream primer shown as SEQ ID No.28 and a downstream primer shown as SEQ ID No.29, and the probes comprise fluorescent probes shown as SEQ ID No. 30.
3. The PCR primer and probe combination for methylation detection of liver cancer genes 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 methylation detection of liver cancer genes 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. The liver cancer gene methylation detection kit is characterized by comprising the PCR primer and probe combination as set forth in claim 1, and further comprising a positive quality control product and a negative quality control product.
6. The liver cancer gene methylation detection kit of claim 5, wherein the final concentration composition of the kit reaction system comprises: 0.1-1 mu M PCR primer, 0.1-1 mu M probe, 0.001-10 ng/mu l DNA template to be detected.
7. The non-disease diagnosis, treatment and detection method for liver cancer gene methylation is characterized by comprising the following steps:
1) Isolating nucleic acid of a target gene in a biological sample to be tested;
2) Subjecting the nucleic acid obtained in the step 1) to bisulfite conversion treatment to obtain bisulfite converted DNA (Bis-DNA);
3) The methylation state of the Bis-DNA obtained in the step 2) is detected by the PCR primer and probe combination and the PCR technology.
8. The method for detecting liver cancer gene methylation non-disease diagnosis and treatment according to claim 7, wherein the biological sample in step 1) comprises peripheral blood, fresh pathological tissue, paraffin-embedded tissue, liver cancer cells.
9. The use of the PCR primer and probe combination for detecting liver cancer gene methylation according to claim 1 in preparing a kit for detecting liver cancer.
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| 肝细胞癌早期诊断血清学标志物的研究进展;朱明玉 等;临床肝胆病杂志;第30卷(第10期);1091-1093 * |
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