CN111549129B - Kit for detecting gastric cancer and application thereof - Google Patents
Kit for detecting gastric cancer and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/154—Methylation markers
Abstract
The kit for detecting gastric cancer and the application thereof are characterized by comprising a plasma free DNA methylation treatment reagent, a PCR detection premix, a PCR detection nucleotide premix and a quality control agent, wherein the PCR detection nucleotide premix contains a nucleotide sequence and specifically comprises the following components: nucleotide sequence 01, nucleotide sequence 02, nucleotide sequence 03, nucleotide sequence 04, nucleotide sequence 05, nucleotide sequence 06, nucleotide sequence 07, nucleotide sequence 08, nucleotide sequence 09, nucleotide sequence 10, nucleotide sequence 11, nucleotide sequence 12, nucleotide sequence 13, nucleotide sequence 14. The method has the advantages of high accuracy of the measurement result, small sample size, simple operation, stable detection, capability of analyzing methylation abnormality of a plurality of gastric cancer markers at one time, high detection sensitivity, and capability of realizing early detection and real-time monitoring of gastric cancer and diagnosis of gastric cancer recurrence.
Description
Technical Field
The application belongs to the technical field of tumor student detection, and particularly relates to a kit for detecting gastric cancer and application thereof.
Background
Stomach cancer is a characteristic cancer in east Asia, the incidence rate can reach 32.1/10 ten thousand people, and the death rate is 13.2/10 ten thousand people, wherein the incidence rate of Japan and Korea is high in the top of the world list. According to the report of world health organization, 100 tens of thousands of new stomach cancers are found worldwide in 2018, and more than 78 tens of thousands of new stomach cancers die at the same time, and the stomach cancers are cancers with fifth highest incidence and third highest death rate. The incidence rate of the gastric cancer is the first in various malignant tumors in China, the incidence rate of the gastric cancer has obvious regional difference, and the incidence rate of the gastric cancer is obviously higher in northwest and eastern coastal areas of China than in southern areas. The good incidence age is over 50 years old, and the incidence rate of men and women is 2:1.
gastric cancer tends to be younger due to unreasonable dietary structure, disturbed life work and rest, excessive working pressure, infection by pathogenic microorganisms (such as helicobacter pylori), and the like. Gastric cancer can occur in any part of the stomach, with more than half occurring in the antrum, the greater curvature, lesser curvature and anterior and posterior walls of the stomach all affected. Most stomach cancers belong to adenocarcinoma, have no obvious symptoms in early stage, or have nonspecific symptoms such as epigastric discomfort, eructation and the like, are often similar to symptoms of chronic gastric diseases such as gastritis, gastric ulcer and the like, and are easy to ignore.
The 5-year survival rate of gastric cancer patients in China is lower, and the data of the article 2000-2014 global cancer survival rate change trend monitoring research report published by the medical journal "Lancet", show that the 5-year survival rate of gastric cancer is 60.3% in Japan, 68.9% in Korea and only 35.9% in China. The survival rate of early gastric cancer in 5 years is up to more than 90%, and the survival rate of late gastric cancer is greatly reduced. The early gastric cancer has similar symptoms with common digestive tract diseases such as superficial gastritis, dyspepsia and the like, such as abdominal distension, eructation, acid regurgitation, appetite reduction and the like, and has obvious specific symptoms such as emaciation, hemorrhage and black stool, and the middle and late stages are reached.
Current methods for detecting gastric cancer occurrence or recurrence and related features include the following: (1) CT examination: it can be shown whether stomach cancer involves gastric wall growth into and out of the cavity, etc., but has limited clinical significance. (2) endoscopic ultrasound: the layers of the stomach wall can be directly seen, the overall appearance of the tumor is known, the diagnosis of gastric cancer and TNM staging are facilitated, but the clinical significance is effective. (3) endoscopic diagnosis: the fiber gastroscope has important significance for diagnosing gastric cancer, can find early gastric cancer, identify benign and malignant ulcers, determine the type of gastric cancer and the focus infiltration range, and can carry out follow-up examination on precancerous lesions. The disadvantage is that patient dependence is poor and there is a certain risk of infection. (4) Carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA 19-9), carbohydrate antigen 72-4 (CA 72-4), pepsinogen, etc.: the conventional tumor marker has an auxiliary effect on tumor diagnosis, and for a certain tumor marker to be raised, the sensitivity of detection can be improved by continuously monitoring dynamic change and combining detection, and the early diagnosis of the tumor can be facilitated by combining other detection. But has low detection sensitivity, poor specificity and limited clinical utility.
Numerous studies have reported that cancer cells, upon rupture and death, release circulating tumor DNA (circulating tumor DNA, ctDNA). ctDNA is an extracellular DNA, which is mainly present in body fluids such as blood (serum or plasma), synovial fluid, and cerebrospinal fluid. The mechanism by which ctDNA is released into the blood is currently not clearly understood, and apoptosis and necrosis of cells are now widely recognized as the major sources of ctDNA in the blood. In recent years, a large number of studies on ctDNA detection in tumor diagnosis and treatment have been reported. The ctDNA fragmentation is serious, and a dispersion band with a main peak of about 160-180bp can be found by using capillary electrophoresis detection. At the same time, the ctDNA content in the blood is low, and studies report 5-20ng per ml of blood, although higher content samples are reported in the literature. However, such a general content range causes a great difficulty in detection, and detection with higher sensitivity is required to have a good detection effect. In order to achieve the ideal detection effect, some companies develop the detection, blood collection amounts as much as 10ml and even 20ml are used, and patients can reject the mind of detecting the large blood collection amount, so that the detection is very important to be completed by using a smaller sample amount.
Disclosure of Invention
Aiming at the defects in the prior art, the application provides the kit for detecting the gastric cancer, which has the advantages of high accuracy of a measurement result, small sample size, simplicity in operation and stability in detection, can analyze methylation abnormality of a plurality of gastric cancer markers at one time, has high detection sensitivity, and can realize early detection and real-time monitoring of the gastric cancer and diagnosis of gastric cancer recurrence.
In order to solve the technical problems, the application adopts the following technical scheme: the kit for detecting gastric cancer comprises a plasma free DNA methylation treatment reagent, a PCR detection premix, a PCR detection nucleotide premix and a quality control reagent, wherein the PCR detection nucleotide premix contains a nucleotide sequence and specifically comprises the following components: nucleotide sequence 01, nucleotide sequence 02, nucleotide sequence 03, nucleotide sequence 04, nucleotide sequence 05, nucleotide sequence 06, nucleotide sequence 07, nucleotide sequence 08, nucleotide sequence 09, nucleotide sequence 10, nucleotide sequence 11, nucleotide sequence 12, nucleotide sequence 13, nucleotide sequence 14.
Preferably, in the above kit, the specific sequences of nucleotide sequences 01 to 14 are shown in the following table:
TABLE 1 nucleotide premix composition
Preferably, in the kit, 3' end of the nucleotide sequence 04 and the nucleotide sequence 11 is provided with a partition wall 3 modification or a phosphorylation modification.
Preferably, in the kit, adenine nucleotides at positions 5, 14, 22 and 32 of the nucleotide sequence 04 are modified by a locked nucleic acid. The locked nucleic acid modification may increase the binding capacity of the repressor fragment (blocker) to the template, making the negative template more susceptible to repression.
Preferably, in the above kit, the free DNA methylation treatment reagent (solvent) includes a bisulfite modification reagent, a DNA protection solution, a desulfonation solution (strong alkali solution), a binding solution, a rinsing solution, an eluent (ph=8.0), and/or a silica magnetic bead suspension.
Further, the bisulphite modification reagent is dissolved and used according to 40-60g/ml deionized water when in use.
Further, the DNA protection solution may contain water-soluble VE dissolved in dimethyl ether, and the concentration of the water-soluble VE in the protection solution may be 2.5-4.5M (mol/L), and the optimal concentration is 3.4M (concentration of the water-soluble VE in the protection solution).
Further, the binding solution may include guanidine bisulfate, and the concentration of guanidine bisulfate in the binding solution may be 5-8M, and the optimal concentration may be 6.8M.
Further, the desulfonation liquid can comprise sodium hydroxide, the concentration of the sodium hydroxide in the desulfonation liquid is 1.3-1.8M, and the optimal concentration can be 1.7M.
Further, the rinse solution may include 75% absolute ethanol or Tri-HCl at a concentration of 9-11mM (9-11 mM is the concentration of Tri-HCl in the rinse solution), and the optimal concentration may be 10.6mM.
Further, the eluent may include Tri-Hcl, and the concentration of Tri-Hcl in the eluent may be 10mmol/L.
Further, the magnetic bead suspension may be a silica magnetic bead suspension, and the silica magnetic bead suspension may contain 70-90mg/ml silica magnetic beads, and the optimal concentration may be 82mg/ml (here, the concentration is the concentration of silica magnetic beads in the silica magnetic bead suspension).
Further preferably, in the above kit, the quality control product includes a positive quality control product, a negative quality control product, and/or a blank quality control product.
Further preferably, in the above kit, the positive quality control (PC) is a cell line gDNA after transfer of methyl and a simulated plasma, the negative quality control (NC) is a human blood cell gDNA after demethylation and a simulated plasma, and the blank quality control (NTC) is a simulated plasma.
Preferably, the kit further comprises a PCR detection premix solution comprising polymerase buffer, DNA heat shock polymerase, magnesium ions, dNTPs, and/or betaine, and/or dimethyl sulfoxide.
Further, in the above-mentioned kit, the polymerase buffer in the PCR detection premix is a 2 x polymerase buffer; the Taq polymerase may be present in an amount of 0.26U/. Mu.l; the divalent cation may be MgCl 2 The concentration thereof was 25mM; dNTPs may be at a concentration of 3.8mM.
The application also provides application of the kit for detecting gastric cancer in preparing medicines for treating gastric cancer diagnosis, gastric cancer real-time monitoring and postoperative recurrence diagnosis.
Preferably, the combination of multiplex PCR assays comprises: ACTB (β -action), RN180; and/or the combination mode of the double PCR detection is NDRG4 and ACTB; and/or the combination mode of triple PCR detection is RN180, NDRG4 and ACTB.
Preferably, three fluorescence signal channels detected by PCR are 513-523nm, 543-553nm and 662-672nm respectively; and/or quadruple 513-523nm, 543-553nm, 585-610nm, 662-672nm.
The application has the advantages and beneficial effects that:
1. the kit can be used for auxiliary diagnosis of gastric cancer and recurrence diagnosis.
2. In the detection process of the kit, only smaller blood consumption is needed: when the blood volume is large, the patient dependence is poor, and when 4 target genes are detected, the blood collection volume can be reduced from 10ml to 5ml, so that the patient dependence is effectively improved.
3. The kit has the advantage of low false positive rate: conventional tumor markers may also be elevated under non-tumor pathological or physiological conditions, whereas only patients with cancerous and precancerous conditions may be detected as positive by the detection markers of the kit of the application.
4. The kit provided by the application can reflect the tumor dynamics in real time: conventional tumor markers can exist in blood for several weeks, and the half-life of ctDNA is less than two hours, so that the detection kit can reflect the real-time progress of tumor.
5. The kit has the advantage of higher sensitivity: the detection kit can quantitatively and qualitatively analyze single copy ctDNA depending on a high-sensitivity detection technology and a polygene methylation marker detection technology, thereby realizing higher detection sensitivity.
6. The method for detecting the occurrence of gastric cancer by detecting the ctDNA in the blood plasma can analyze the methylation abnormality of a plurality of gastric cancer markers by using 2ml of blood plasma (5 ml of whole blood) at one time through detecting the methylation abnormality of the circulating tumor DNA (ctDNA) with high sensitivity, has the detection sensitivity as low as 0.1 percent on the methylation abnormality of the ctDNA, can realize early detection of gastric cancer and diagnosis of gastric cancer recurrence, and has the advantage of detection.
Drawings
FIG. 1 is a graph of sample detection results of SEPT9 gene detection results in 4-fold PCR according to some embodiments of the present application;
FIG. 2 is a graph of sample detection results of RN180 gene detection results in 4-fold PCR according to some embodiments of the present application;
FIG. 3 is a graph of sample detection results of NDRG4 gene detection results in 4-fold PCR according to some embodiments of the present application;
FIG. 4 is a graph of sample detection results of ACTB gene detection in 4-fold PCR, according to some embodiments of the application;
Detailed Description
The following will further describe the specific embodiments of the present application in conjunction with examples, so that the technical solution is easier to understand and grasp. But the application is not limited thereto. The experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified, and the following examples are not intended to limit the scope of the present application and are intended to be included in the scope of the present application without departing from the equivalent implementations or modifications to which the present application pertains.
Example 1 kit composition for detecting gastric cancer
The kit for detecting gastric cancer in the present application may comprise the following components as shown in table 2:
TABLE 2 composition of the kit
Example 2 kit composition for detection of gastric cancer
The kit for detecting gastric cancer in the present application may comprise the following components as shown in table 3:
TABLE 3 composition of the kit
The respective compositions used in example 1 and example 2 will be specifically described below:
plasma free DNA modifying reagents include: (1) The bisulphite modification reagent contains 100g of sodium bisulphite solid, and is dissolved in 2ml of deionized water when in use; (2) The DNA protection solution may comprise water-soluble VE dissolved in dimethyl ether, which may be in a concentration of 2.5-4.5M. Wherein, in some embodiments, its optimal concentration is 3.4M; (3) The binding fluid may include guanidine hydrogen sulfate, at a concentration of 5-8M. Wherein, in some embodiments, its optimal concentration may be 6.8M; (4) The desulfonation liquid may comprise sodium hydroxide at a concentration of 1.3-1.8M. Wherein, in some embodiments, its optimal concentration may be 1.7M; (5) The rinse solution may include 75% absolute ethanol, tri-Hcl, and the concentration of Tri-Hcl may be 9-11mM. Wherein, in some embodiments, its optimal concentration may be 10.6mM; (6) The eluent may include Tri-HCl, and the concentration thereof may be 10mmol/L; (7) The magnetic bead suspension may be a silica magnetic bead suspension, and in some embodiments, the silica magnetic bead suspension may contain 70-90mg/ml silica magnetic beads. In some embodiments, the optimal concentration may be 82mg/ml.
The PCR detection premix may include: polymerase buffer, which in some embodiments may be a 2 x polymerase containing buffer; taq polymerase, in some embodiments, may be present in an amount of 0.26U/. Mu.l; divalent cations, in some embodiments, the divalent cations may be MgCl 2 The concentration thereof was 25mM; dNTPs may be at a concentration of 3.8mM.
The nucleotide sequences shown in Table 4 and the content thereof (the concentration in the PCR detection nucleotide premix) are included in the PCR detection nucleotide premix:
TABLE 4 PCR detection nucleotide premix
| Nucleotide fragment | Sequence number | Optimum content of |
| RN180-F | Nucleotide 01 | 18μM |
| RN180-R | Nucleotide 02 | 18μM |
| RN180-Pro | Nucleotide 03 | 3μM |
| RN180-Blocker | Nucleotide 04 | 3μM |
| SEPT9-F | Nucleotide 05 | 18μM |
| SEPT9-R | Nucleotide 06 | 18μM |
| SEPT9-PRO | Nucleotide 07 | 3μM |
| NDRG4-F | Nucleotide 08 | 18μM |
| NDRG4-R | Nucleotide 09 | 18μM |
| NDRG4-PRO | Nucleotide 10 | 3μM |
| NDRG4-Blocker | Nucleotide 11 | 3μM |
| ACTB-F | Nucleotide 12 | 9μM |
| ACTB-R | Nucleotide 13 | 9μM |
| ACTB-Pro | Nucleotide 14 | 3μM |
Wherein the 3' end of the nucleotide sequence 04 and the nucleotide sequence 11 is provided with a partition wall 3 modification or a phosphorylation modification. Adenine nucleotides 5, 14, 22 and 32 of nucleotide sequence 04 carry a locked nucleic acid modification.
The quality control agent comprises: (1) cationic quality control (PC): the cell line DNA after the transfer of methyl and simulated plasma have the DNA concentration of 30ng/ml; (2) negative quality control (NC): the DNA concentration of the demethylated human blood cells and the simulated blood plasma is 30ng/ml; (3) white matter control (NTC) is simulated plasma.
Example 3 detection method of the kit of the present application
1. Enriching nucleic acid of a sample to be detected and a quality control product, eluting the sample by using eluent, and then carrying out bisulphite modification and purification to obtain a sample template to be detected and a quality control product template;
2. performing PCR detection on the sample template to be detected and the quality control template, and adding the sample template to be detected and the quality control template into a PCR detection tube;
3. dividing a sample template to be detected and a quality control template into detection tubes;
the test tube composition is shown in table 5 below:
TABLE 5 detection tube I composition
| PCR premix | 15μl |
| Nucleotide premix | 1μl |
| ddH2O | 6μl |
| DNA/quality control product | 8μl |
4. The PCR detection tube is put into a QPCR (real-time fluorescence quantitative nucleic acid amplification detection system) instrument, and the related procedures are as follows:
step 1: reacting for 30 minutes at the temperature of between 35 and 39 ℃; step 2: reacting at 92-96 ℃, preferably 94 ℃ for 2 minutes; step 3: 54-58 ℃, 40 seconds of reaction, then 94 ℃ of reaction for 30 seconds, 16 times of circulation: step 4: the reaction is carried out for 2 seconds at 65 ℃, 40 seconds at 56 ℃ (fluorescence is collected), 20 seconds at 94 ℃, and the cycle is 32 times; step 5: reaction at 40 ℃): 30 seconds.
In some specific embodiments, the procedure may be as follows: step 1: reacting for 30 minutes at 37 ℃; step 2: reacting at 94 ℃ for 2 minutes; step 3: reaction at 56 ℃ for 40 seconds and 94 ℃ for 30 seconds, and circulation for 16 times: step 4: the reaction is carried out for 2 seconds at 65 ℃, 40 seconds at 56 ℃ (fluorescence is collected), 20 seconds at 94 ℃, and the cycle is 32 times; step 5: reaction at 40 ℃): 30 seconds.
Wherein, the fluorescence signal detected by the detection tube is three channels, which is characterized in that the three fluorescence signal channels detected by PCR are 513-523nm, 543-553nm and 662-672nm respectively; and/or quadruple 513-523nm, 543-553nm, 585-610nm, 662-672nm;
the sample to be tested may include one or any combination of tumor tissue genomic DNA, peripheral blood episomal DNA, urine episomal DNA, hydrothorax and ascites episomal DNA, or fecal exfoliated cell DNA.
5. Analyzing the detection data:
the interpretation is divided into gene negative and positive interpretation and negative and positive interpretation detection.
Negative and positive gene interpretation:
(1) The Ct value of (2) is less than or equal to 23.2, positive, ct value is more than 23.2, and negative;
(2) The Ct value of RN180 is less than or equal to 24.1, positive, ct value is more than 24.1, and negative;
(3) The Ct value of NDRG4 is less than or equal to 23.4, positive, ct value is more than 23.4, and negative;
(4) The Ct value of ACTB is less than or equal to 21.0, positive, ct value is more than 21.0, and negative.
Detecting yin-yang interpretation:
(1) Detecting an invalid interpretation: amplified Ct values > 21.0 for ACTB, or amplified delta Rn > 0.1 for blank (NTC), or amplified SEPT9, rn180, NDRG4, ACTB in Negative Control (NC).
(2) Detecting positive interpretation: on the premise of effective judgment, any one gene or two genes or three genes of the three genes SEPT9, RN180 and NDRG4 are positive.
(3) Detecting a negative interpretation: on the premise of effective judgment, the genes in the three genes SEPT9, RN180 and NDRG4 are all negative, and no gene is positive.
EXAMPLE 4 bisulfite treatment DNA recovery, conversion
Reference: from tumor tissue DNA, using ultrasonic wave to break into 50-300bp fragments, using a Qubit quantitative instrument: agilent 2200TapeStation, covaris S220, high-speed centrifuge, PCR instrument, vortex oscillator, instantaneous centrifuge, refrigerator, etc
The steps are as follows:
1. taking three 200ul EP pipes, sequentially adding the reagents in Table 6, tightly covering a pipe cover, vortex uniformly mixing, instantly centrifuging, and then adding reference substances, wherein the amounts of the added reference substances are 10ng, 20ng and 40ng in sequence;
TABLE 6 reaction system
2. The EP tube was placed in a PCR apparatus and the bisulfite treatment reaction was performed according to the following procedure.
Table 7 reaction procedure
| Temperature (temperature) | Reaction time |
| 98℃ | 10min |
| 64℃ | 120min |
| 4℃ | Indefinite |
3. Purification of DNA
A. Adding the mixed solution obtained in the previous step into a 1.5ml EP tube, adding 1.8ml of combined solution, mixing by vortex, and centrifuging instantly; B. adding 50ul of magnetic bead suspension, rotating for 30 minutes, placing the mixture in a magnetic rack, and pouring out the liquid after 30 seconds; C. adding 100ul of rinsing liquid, oscillating for 30s on a vortex oscillator, placing the mixture on a magnetic rack, and pouring out the liquid after 30 seconds; D. adding 200 μl of the desulfonation liquid, standing at room temperature (20-25) for 20min, placing on a magnetic rack, and pouring out the liquid after 30 seconds; E. adding 200ul of rinsing liquid, oscillating for 30s on a vortex oscillator, placing the mixture on a magnetic rack, and pouring out the liquid after 30 seconds; F. repeating the above steps; G. adding 40ul of eluent, placing the eluent in a magnetic rack, and sucking out liquid after 30 seconds; namely, the purified DNA solution.
4. Recovery calculation
Quantification of purified recovered DNA was performed using Qubit, and recovery was calculated:
TABLE 8 DNA recovery
| Input (ng) | Recovery (ng) | Recovery rate |
| 10 | 6.239 | 62.3％ |
| 20 | 13.67 | 68.35％ |
| 40 | 28.55 | 71.375％ |
5. Conversion calculation
Using the purified DNA, sequencing primers were designed for sanger sequencing and conversion of unmethylated C to U was obtained after alignment with standard sequences. In total 544 carbons were counted, only one of which was not converted to U, with a conversion of 99.82%.
Example 5 analysis of PCR detection System Performance
Instrument: ABI7500, high-speed centrifuge, water bath, vortex oscillator, refrigerator, etc.
Positive references were prepared at different concentrations: 297copies/10ng, 99copies/10ng, 33
copies/10ng、11copies/10ng。
The PCR detection method comprises the following steps:
A. detection tube: a1.5 ml EP tube was taken and 15. Mu.l of the PCR detection premix, 1. Mu.l of the nucleotide premix and 6. Mu.l of dd H were added sequentially 2 O, vortex mixing, instantaneous centrifugation.
B. And subpackaging the premix of the detection tube into a 96-well plate, and then adding template DNA and quality control substances.
C. The 96-well plate was subjected to instantaneous centrifugation, placed in an ABI7500 instrument, and the fluorescence quantitative PCR instrument was set up to perform the reaction as follows:
step 1: reacting for 30 minutes at 37 ℃; step 2: reacting at 94 ℃ for 2 minutes; step 3: reaction at 56 ℃ for 40 seconds and 94 ℃ for 30 seconds, and circulation for 16 times: step 4: the reaction is carried out for 2 seconds at 65 ℃, 40 seconds at 56 ℃ (fluorescence is collected), 20 seconds at 94 ℃, and 30 times of circulation are carried out; step 5: reaction at 40 ℃): 30 seconds.
Wherein, the fluorescence signals detected by the detection tube are four channels, namely 513-523nm, 543-553nm and 662-672nm; and/or quadruple 513-523nm, 543-553nm, 585-610nm, 662-672nm.
Detection result:
the 4-fold PCR amplification in the detection tube has no interference, and the gene amplification efficiencies are respectively as follows: SEPT9:102%; RN180:99.9%;
NDRG4：105％；ACTB：103％。
example 6 application of the kit of the present application in gastric cancer diagnosis and postoperative diagnosis
Sample to be measured: the details of the samples to be tested are shown in Table 9, wherein 40 samples of plasma samples to be tested (2 ml), positive quality control (2 ml), negative quality control (2 ml) and blank quality control (2 ml) are shown in the following.
TABLE 9 basic information of samples to be tested
Instrument: ABI7500, high-speed centrifuge, PCR instrument, water bath, vortex oscillator and refrigerator.
The operation method comprises the following steps: 1. enrichment of nucleic acid: plasma free DNA was extracted according to the instructions using QIAamp Circulating Nucleic Acid (cat No. 55114). QC quality control was performed on cfDNA using an Agilent 2200TapeStation electrophoresis platform.
2 sodium bisulfite treatment of free DNA
(1) Taking a 200ul EP tube, adding the following reagents at a time, tightly covering a tube cover, vortex-mixing uniformly, and performing instantaneous centrifugation, wherein the reaction system is shown in Table 6;
(2) The EP tube was placed in a PCR apparatus and the bisulfite treatment reaction was performed according to the following procedure. The procedure is shown in Table 7;
(3) Purification of DNA
A. Adding the mixed solution obtained in the previous step into a 1.5ml EP tube, adding 1.8ml of combined solution, mixing by vortex, and centrifuging instantly; B. adding 50ul of magnetic bead suspension, rotating for 30 minutes, placing the mixture in a magnetic rack, and pouring out the liquid after 30 seconds; C. adding 100ul of rinsing liquid, oscillating for 30s on a vortex oscillator, placing the mixture on a magnetic rack, and pouring out the liquid after 30 seconds; D. adding 200 μl of the desulfonation liquid, standing at room temperature (20-25) for 20min, placing on a magnetic rack, and pouring out the liquid after 30 seconds; E. adding 200ul of rinsing liquid, oscillating for 30s on a vortex oscillator, placing the mixture on a magnetic rack, and pouring out the liquid after 30 seconds; F. repeating the above steps; G. adding 40ul of eluent, placing the eluent in a magnetic rack, and sucking out liquid after 30 seconds; namely, the purified DNA solution.
PCR detection
(1) Detection tube: a1.5 ml EP tube was taken and 15. Mu.l of PCR premix, 1. Mu.l of nucleotide premix, 6. Mu.l of dd H were added sequentially 2 Mixing evenly by O and vortex, and carrying out instantaneous centrifugation;
(2) And (3) subpackaging the premix of the detection tube into a 96-well plate, arranging the plates as follows, and then adding template DNA and quality control substances.
Table 10 detection row board
(3) The 96-well plate is subjected to instantaneous centrifugation, placed into an ABI7500 instrument, and a fluorescent quantitative PCR instrument program is set for reaction; PCR interpretation and results
And after the detection is finished, judging the negative and positive of each gene according to the judging standard.
Gene interpretation criteria:
(1) The Ct value of (2) is less than or equal to 23.2, positive, ct value is more than 23.2, and negative;
(2) The Ct value of RN180 is less than or equal to 24.1, positive, ct value is more than 24.1, and negative;
(3) The Ct value of NDRG4 is less than or equal to 23.4, positive, ct value is more than 23.4, and negative;
(4) The Ct value of ACTB is less than or equal to 21.0, positive, ct value is more than 21.0, and negative.
Detecting yin-yang interpretation:
(1) Detecting an invalid interpretation: the amplified Ct value of ACTB is > 21.0, or the amplified delta Rn of blank control (NTC) is > 0.1, or the amplified RN180, NDRG4 and ACTB are amplified in Negative Control (NC).
(2) Detecting positive interpretation: on the premise of effective judgment, any one gene or two genes or three genes of the three genes SEPT9, RN180 and NDRG4 are positive.
(3) Detecting a negative interpretation: on the premise of effective judgment, the genes in the three genes, the RN180 and the NDRG4 are negative, and no gene is positive.
The detection results are shown in the following table.
Table 11 Gene test results for each sample of the kit
Table 12 test results for each sample of the kit
From the above specific embodiments and applications, it is apparent that: a total of 27 samples were tested using the kit of the application, 11 of which were subjected to gastroscopy. Of the 11 gastric cancer samples, 9 cases detected as positive; of the 16 normal human samples, 15 were detected as negative. The detection sensitivity of the kit is 81.82% and the specificity is 93.75%.
The embodiments described above are only for illustrating the technical solution of the present application, and are not limiting. Although the application has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may be modified or some or all of the technical features may be replaced with other technical solutions, which do not depart from the scope of the technical solutions of the embodiments of the present application.
Sequence listing
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<211> 38
<212> DNA
<213> Artificial sequence ()
<400> 4
tgaattggta ttgttgttgt tggagttgta gtgagttt 38
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 5
gtttaggggt tttttcggcg 20
<210> 6
<211> 17
<212> DNA
<213> Artificial sequence ()
<400> 6
aacccgaacc catcgcg 17
<210> 7
<211> 24
<212> DNA
<213> Artificial sequence ()
<400> 7
ttttgtattg taggagcgcg ggcg 24
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 8
atagttcgcg cggcggagcg 20
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 9
cgaacgaaaa ccgcgacgcg 20
<210> 10
<211> 22
<212> DNA
<213> Artificial sequence ()
<400> 10
cggcgggggc gcggatcgat cg 22
<210> 11
<211> 32
<212> DNA
<213> Artificial sequence ()
<400> 11
cgatcggggt gttttttagg tttcgcgtcg cg 32
<210> 12
<211> 23
<212> DNA
<213> Artificial sequence ()
<400> 12
ttttttttgg gtgagtggag att 23
<210> 13
<211> 21
<212> DNA
<213> Artificial sequence ()
<400> 13
aaaacttaac ttccacaaca c 21
<210> 14
<211> 24
<212> DNA
<213> Artificial sequence ()
<400> 14
gatatgaggg ttattttttg gggt 24
Claims (14)
1. A kit for detecting gastric cancer, characterized in that the kit for detecting gastric cancer comprises the following components: the PCR detection nucleotide premix comprises the following nucleotide sequences: nucleotide sequence 01, nucleotide sequence 02, nucleotide sequence 03, nucleotide sequence 04, nucleotide sequence 05, nucleotide sequence 06, nucleotide sequence 07, nucleotide sequence 08, nucleotide sequence 09, nucleotide sequence 10, nucleotide sequence 11, nucleotide sequence 12, nucleotide sequence 13 and nucleotide sequence 14, the specific sequences of nucleotide sequence 01-nucleotide sequence 14 being:
Wherein the 3 'end of the nucleotide sequence 04 is provided with a partition wall 3 modification or phosphorylation modification, and the 3' end of the nucleotide sequence 11 is provided with a partition wall 3 modification or phosphorylation modification; adenine nucleotides 5, 14, 22 and 32 of the nucleotide sequence 04 are modified by a locked nucleic acid; the kit for detecting gastric cancer further comprises the following components: plasma free DNA methylation processing reagents, PCR detection premix and quality control reagents.
2. The kit for detecting gastric cancer of claim 1, wherein the plasma free DNA methylation treatment reagent comprises a bisulphite modification reagent, a DNA protection solution, a desulfonation solution, a binding solution, a rinse solution, an eluent with ph=8.0, and/or a silica magnetic bead suspension.
3. The kit for detecting gastric cancer according to claim 2, wherein the bisulphite modification reagent is dissolved in deionized water at 40-60g/ml for use.
4. The kit for detecting gastric cancer according to claim 2, wherein the DNA protective solution comprises water-soluble VE dissolved in dimethyl ether, and the concentration of the water-soluble VE in the protective solution is 2.5 to 4.5 mol/L.
5. The kit for detecting gastric cancer according to claim 2, wherein the binding solution comprises guanidine bisulfate, and the concentration of guanidine bisulfate in the binding solution is 5-8M.
6. The kit for detecting gastric cancer according to claim 2, wherein the desulfonating liquid comprises sodium hydroxide, and the concentration of the sodium hydroxide in the desulfonating liquid is 1.3-1.8M.
7. The kit for detecting gastric cancer according to claim 2, wherein the rinse solution may comprise 75% absolute ethanol or Tri-Hcl at a concentration of 9-11mM.
8. The kit for detecting gastric cancer according to claim 2, wherein the eluent comprises Tri-Hcl, and the concentration of Tri-Hcl in the eluent is 10mmol/L.
9. The kit for detecting gastric cancer according to claim 2, wherein the silica magnetic bead suspension contains 70-90mg/ml silica magnetic beads.
10. The kit for detecting gastric cancer of claim 1, wherein the quality control comprises a positive quality control, a negative quality control, and/or a blank quality control.
11. The kit for detecting gastric cancer according to claim 10, wherein the positive quality control substance is a cell line gDNA after the transfer of methyl and simulated plasma, the negative quality control substance is a human blood cell gDNA after the removal of methyl and simulated plasma, and the blank quality control substance is simulated plasma.
12. The kit for detecting gastric cancer according to claim 1, wherein the PCR detection pre-mix comprises a polymerase buffer, a DNA heat shock polymerase, magnesium ions, dNTPs, and/or betaine, and/or dimethyl sulfoxide.
13. The kit for detecting gastric cancer of claim 12, wherein the polymerase buffer comprises a 2 x polymerase buffer; the content of the DNA heat shock polymerase is 0.26U/. Mu.l; the magnesium ion compound is MgCl 2 Magnesium ion concentration 25mM; dNTPs concentration was 3.8mM.
14. The application of the kit for detecting gastric cancer is characterized in that the kit for detecting gastric cancer according to any one of claims 1-13 is used for preparing a medicament for monitoring gastric cancer in real time.
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| CN202010234920.5A CN111549129B (en) | 2020-03-30 | 2020-03-30 | Kit for detecting gastric cancer and application thereof |
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| CN202010234920.5A CN111549129B (en) | 2020-03-30 | 2020-03-30 | Kit for detecting gastric cancer and application thereof |
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