CN108103197B - Primer group, reagent, kit and application for detecting RPRM gene and PRDM5 gene methylation - Google Patents
Primer group, reagent, kit and application for detecting RPRM gene and PRDM5 gene methylation Download PDFInfo
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- CN108103197B CN108103197B CN201810127514.1A CN201810127514A CN108103197B CN 108103197 B CN108103197 B CN 108103197B CN 201810127514 A CN201810127514 A CN 201810127514A CN 108103197 B CN108103197 B CN 108103197B
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Abstract
The invention provides a primer group, a reagent, a kit and an application for detecting the methylation of an RPRM gene and a PRDM5 gene, and relates to the technical field of biology. The method for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene is simple and convenient to operate and easy to master. The invention also provides application of the primer group, the reagent or the kit in preparation of products for auxiliary diagnosis and/or early screening of gastric cancer.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a primer group, a reagent, a kit and application for detecting RPRM gene and PRDM5 gene methylation.
Background
Gastric cancer is one of the most common tumors worldwide, with the incidence rate at the 4 th highest among all malignancies, the mortality rate at the 2 nd highest among malignancies, and over 70% of gastric cancer cases present in developing countries. About 40 million new cases of gastric cancer in China each year account for 40 percent of the total diseases in the world. The early diagnosis rate of the gastric cancer in China is less than 10 percent, once the gastric cancer is diagnosed mostly in the middle and late stages, and the 5-year survival rate is less than 20 percent; meanwhile, after national screening is carried out in developed countries, the early diagnosis rate of the gastric cancer is up to 50%, and the survival rate in 5 years can reach more than 60%. For this reason, how to achieve early detection of gastric cancer is critical to reducing its mortality rate.
More than 90% of gastric cancers are adenocarcinoma, and in 1965, Lauren classifies adenocarcinoma into intestinal type and diffuse type according to the tissue structure and biological behavior of gastric cancer. The occurrence and development processes of the two mainly comprise the following stages: normal-gastritis-chronic atrophic gastritis-intestinal metaplasia-dysplasia-mucosal cancer-invasive cancer. In 1978, the world health organization classified chronic atrophic gastritis as a precancerous state of gastric cancer, with intestinal metaplasia and dysplasia called chronic atrophic gastritis precancerous lesion or precancerous lesion of gastric cancer, and incomplete intestinal metaplasia and (or) moderate and severe dysplasia accompanied by chronic atrophy were regarded as real precancerous lesions. The cure rate of the gastric cancer precancerous lesion and even early gastric cancer is as high as 90 percent, so the discovery of the early gastric cancer, the precancerous lesion and even precancerous diseases is very important.
The main approaches for screening the early gastric cancer include natural population census, outpatient opportunistic screening and high risk population screening. At present, outpatient opportunistic screening is mainly adopted in China, people who participate in outpatient physical examination are mainly used as screening objects, and gastric cancer is examined through detection diagnosis of serum and gastric fluid markers, electron microscope diagnosis and imaging diagnosis sections to confirm the occurrence of gastric cancer. The detection of the PG level in the serum is a good index for reflecting the function and the state of the gastric mucosa, and has the advantages of no wound, simplicity, convenience, quickness and the like; the gastroscope detection is considered as the 'gold standard' for detecting the gastric cancer, the sensitivity and the specificity of the gastroscope detection exceed 90 percent, and the gastroscope detection is the most intuitive and effective early detection means for the gastric cancer at present; before the use of endoscopes, barium meal contrast is the main means for diagnosis and screening of gastric cancer, but is gradually replaced by endoscopic detection because pathological diagnosis cannot be obtained. When the clinical routine diagnosis is applied to early diagnosis of the gastric cancer, the gastric tissue has substantial pathological changes, cells have canceration, early screening of the gastric cancer is really required, and the gene diagnosis is expected.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the present invention is to provide a primer set for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene, so as to alleviate the technical problems of the prior art that a primer which has strong specificity and high sensitivity and can simultaneously detect the methylation of the RPRM gene and the PRDM5 gene is absent.
The second purpose of the present invention is to provide a reagent for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene, so as to alleviate the technical problems of the prior art that the reagent which has strong specificity and high sensitivity and can simultaneously detect the methylation of the RPRM gene and the PRDM5 gene is lacked.
The third purpose of the present invention is to provide a kit for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene, so as to alleviate the technical problems of the prior art that the kit has strong specificity and high sensitivity and can simultaneously detect the methylation of the RPRM gene and the PRDM5 gene.
The fourth purpose of the present invention is to provide the application of the primer set, the reagent or the kit in the preparation of products for auxiliary diagnosis and/or early screening of gastric cancer, so as to alleviate the technical problem of the prior art that an effective means for early screening and diagnosis of gastric cancer is lacked.
A fifth object of the present invention is to provide a method for detecting gastric cancer, which alleviates the technical problems of the prior art that an accurate and effective method for early detection of gastric cancer is lacking.
The invention provides a primer group for simultaneously detecting the methylation of an RPRM gene and a PRDM5 gene, which comprises a primer pair for detecting the RPRM gene and a primer pair for detecting the PRDM5 gene;
the primer pair for detecting the RPRM gene comprises an upstream primer of the RPRM gene shown as SEQ ID NO.1 and a downstream primer of the RPRM gene shown as SEQ ID NO. 2;
the primer pair for detecting the PRDM5 gene comprises an upstream primer of the PRDM5 gene shown as SEQ ID NO.3 and a downstream primer of the PRDM5 gene shown as SEQ ID NO. 4.
The invention also provides a reagent for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene, which comprises the primer group.
The invention also provides a kit for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene, which comprises the primer group or the reagent.
Further, the kit comprises a PCR Mix and a Taq enzyme Mix;
the PCR Mix comprises the primer set of claim 1 or the reagent of claim 2 and dntps;
the Taq enzyme Mix comprises Taq enzyme and a probe.
Further, the kit also comprises a negative control substance and a positive control substance;
preferably, the negative control comprises genomic DNA in which the TE buffer is unmethylated both the RPRM gene and the PRDM5 gene;
the positive control included TE buffer and genomic DNA methylated at both the RPRM gene and the PRDM5 gene.
Further, the kit also comprises a DNA treatment article;
preferably, the DNA treatment product comprises a product for extracting DNA and a product for performing sulfite conversion on the DNA;
preferably, the article for extracting DNA comprises lysis solution, magnetic beads and extraction rinsing solution, and the article for performing sulfite conversion on the DNA comprises a sulfuration reagent, binding solution, purification rinsing solution and eluent.
The invention also provides application of the kit in preparation of products for auxiliary diagnosis and/or early screening of gastric cancer.
In addition, the invention also provides a method for detecting gastric cancer by using the primer group, the reagent or the kit, wherein the method comprises the following steps:
performing a fluorescent quantitative PCR reaction on the sulfite-converted plasma sample DNA and the primer set of claim 1 or the reagent of claim 2, and judging whether the gastric cancer is suffered according to the Cp value of the sulfite-converted plasma sample DNA;
when the Cp value of the RPRM gene is less than or equal to 44.76, judging that the risk of gastric cancer is high; and/or the presence of a gas in the gas,
when the Cp value of the PRDM5 gene is less than or equal to 38.85, the risk of gastric cancer is judged to be high.
Further, extracting DNA in the plasma sample cracked by the cracking solution by adopting a magnetic bead adsorption method, carrying out sulfite conversion on the DNA, and purifying by adopting a magnetic bead method to obtain the sulfite-converted plasma sample DNA;
preferably, after washing the DNA in the plasma sample with a mixture of an extraction rinse and absolute ethanol, the DNA is subjected to sulfite conversion.
Further, after uniformly mixing the binding solution mixed with the magnetic beads and the plasma sample DNA converted by the sulfite for adsorption, washing the magnetic beads by using a mixed solution of a purified rinsing solution and absolute ethyl alcohol, and then eluting the plasma sample DNA converted by the sulfite by using an eluent.
The primer group for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene, provided by the invention, comprises a primer pair for detecting the RPRM gene and a primer pair for detecting the PRDM5 gene, has strong specificity and high sensitivity, and can be used for simply, quickly and accurately detecting the methylation levels of the RPRM gene and the PRDM5 gene simultaneously. The reagent for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene, provided by the invention, comprises the primer group, has strong specificity and high sensitivity, and can be used for simply, quickly, accurately and simultaneously detecting the methylation levels of the RPRM gene and the PRDM5 gene. The kit for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene, provided by the invention, comprises the primer group or the reagent, and can be used for simply, conveniently, quickly, accurately and simultaneously detecting the methylation states of the RPRM gene and the PRDM5 gene. According to the method for detecting the gastric cancer, the plasma sample DNA converted by the sulfite and the primer group reagent are subjected to fluorescent quantitative PCR reaction, and the risk of suffering from the gastric cancer is judged according to the Cp value of the plasma sample DNA converted by the sulfite. The invention also provides application of the primer group, the reagent or the kit in preparation of products for auxiliary diagnosis and/or early screening of gastric cancer, the application of the primer group, the reagent or the kit provided by the invention in early screening and diagnosis of gastric cancer has the advantages of high sensitivity and high accuracy, the screening method is simple and easy to master, the occurrence of gastric cancer can be accurately detected in precancerous lesion of gastric cancer or early gastric cancer period, the cure rate of gastric cancer can be greatly improved, and the method has great popularization value.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be understood that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides a primer group for simultaneously detecting the methylation of an RPRM gene and a PRDM5 gene, which comprises a primer pair for detecting the RPRM gene and a primer pair for detecting the PRDM5 gene;
the primer pair for detecting the RPRM gene comprises:
upstream primer of RPRM gene: 5'-GCGAGTGAGCGTTTAGTTC-3' (SEQ ID NO. 1);
downstream primer of RPRM gene: 5'-ACCCGCCACGTCCGTC-3' (SEQ ID NO. 2).
The primer pair for detecting the PRDM5 gene comprises:
upstream primer of PRDM5 gene: 5'-AGTTTTGTTTCGGGTTTCGC-3' (SEQ ID NO. 3);
downstream primer of PRDM5 gene: 5'-CATTCCTACTACGAAAACGC-3' (SEQ ID NO. 4).
The primer group for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene provided by the invention has the advantages of strong specificity and high sensitivity, and can be used for simply, conveniently, quickly and accurately detecting the methylation levels of the RPRM gene and the PRDM5 gene.
The invention also provides a reagent for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene, which comprises the primer group.
The reagent for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene provided by the invention has strong specificity and high sensitivity, and can be used for simply, conveniently, quickly and accurately detecting the methylation levels of the RPRM gene and the PRDM5 gene.
The invention also provides a kit for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene, which comprises the primer group or the reagent.
The kit for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene can simply, quickly and accurately detect the methylation levels of the RPRM gene and the PRDM5 gene simultaneously.
In a preferred embodiment, the kit comprises a PCR Mix and a Taq enzyme Mix.
The PCR Mix comprises the primer group or reagent and dNTP; taq enzyme Mix includes Taq enzyme and probe.
Wherein, the probe sequence of the RPRM is as follows: FAM-CGTCGTTTTTTTATTTTTCG-MGB (SEQ ID NO. 5);
the probe sequence of PRDM5 was: FAM-AGCGTTTAGGTTCGCGTTTT-MGB (SEQ ID NO. 6);
the probe sequence for ACTB is: VIC-AGTAAGTTTTTTGGATTGTG-MGB (SEQ ID NO. 7).
The conventional methylation detection method uses a dye method, and in the preferred embodiment of the invention, the MGB probe method is used, so that the sensitivity is higher and the specificity is stronger.
The PCR Mix premix solution is composed of components containing the primers and dNTP, the Taq enzyme and the probe are composed of the Taq enzyme Mix premix solution, on the basis that the reagent for detection is guaranteed not to lose efficacy before use, only 2 kinds of premix solution are required to be mixed when the kit is used, the PCR pre-reaction solution is prepared, then the PCR pre-reaction solution is subpackaged into a PCR reaction container, a sample template to be detected with the same volume is added, and sealing is carried out. Simple operation and easy mastering.
In a preferred embodiment, the PCR Mix and the Taq enzyme Mix further comprise a solvent, respectively. The solvent contained in the PCR Mix may be, for example, but not limited to, one or more of sterilized purified water, water for injection, or double distilled water; the solvent contained in the Taq enzyme Mix may be, for example, but not limited to, one or more of sterilized purified water, water for injection, double distilled water, or TE buffer.
In a preferred embodiment, the kit further comprises a negative control and a positive control.
Preferably, the negative control comprises TE buffer and genomic DNA that is unmethylated for both the RPRM gene and the PRDM5 gene; the positive control included TE buffer and genomic DNA methylated at both the RPRM gene and the PRDM5 gene.
The kit directly contains a negative reference substance and a positive reference substance, and can ensure that the detection result of a sample to be detected has standard control when the kit provided by the invention is applied to detection. The detection result of the sample to be detected can be effectively judged through the detection results of the negative reference substance and the positive reference substance, and meanwhile, the accuracy and the effectiveness of the experiment are ensured.
In a preferred embodiment, the kit further comprises a DNA treatment device.
Preferably, the DNA treatment product comprises a product for extracting DNA and a product for converting the extracted DNA with sulfite.
The kit directly contains DNA treatment articles, and only one kit provided by the invention is needed, so that a sample to be detected can be detected, the pretreatment of extraction, sulfite conversion and the like before detection is met, and the aims of multiple purposes and convenience in use can be fulfilled.
Preferably, the article for extracting DNA comprises lysis solution, magnetic beads and extraction rinse solution, and the article for performing sulfite conversion on the extracted DNA comprises a sulfuration reagent, a binding solution, a purification rinse solution and an eluent.
Preferably, the lysis solution comprises guanidinium isothiocyanate; the magnetic beads comprise silicon-based magnetic beads; the extraction rinsing liquid comprises guanidinium isothiocyanate; the vulcanizing agent comprises a sulfite solution, preferably sodium bisulfite; the binding solution comprises guanidine hydrochloride; the purification rinsing solution comprises one or more of Tris-HCl solution, sodium hydroxide solution or acetic acid solution.
The invention also provides application of the primer group or the reagent or the kit in preparation of products for auxiliary diagnosis and/or early screening of gastric cancer.
DNA methylation is closely associated with tumors, and aberrant methylation status is a key factor in carcinogenesis. RPRM, located at chromosome 2q23, whose abnormal hypermethylation is closely related to the occurrence and development of gastric cancer; PRDM5 is located at chromosome 4q25-26, is highly methylated in dysplasia and early gastric cancer, and the combination of the two can be used as an important marker for auxiliary diagnosis and early screening of gastric cancer. The primer group, the reagent or the kit provided by the invention are used for carrying out early screening diagnosis on the gastric cancer, have the advantages of high sensitivity and high accuracy, are simple in screening method and easy to master, can accurately detect the occurrence of the gastric cancer in the precancerous lesion of the gastric cancer or early gastric cancer period, can greatly improve the cure rate of the gastric cancer, and have great popularization value.
In addition, the invention also provides a method for detecting gastric cancer by using the primer group or the reagent or the kit, which comprises the following steps:
performing fluorescent quantitative PCR reaction on the plasma sample DNA converted by the sulfite and the primer group or the reagent, and judging whether the gastric cancer is suffered according to the Cp value of the plasma sample DNA converted by the sulfite;
when the Cp value of the RPRM gene is less than or equal to 44.76, judging that the risk of gastric cancer is high; and/or judging that the risk of gastric cancer is high when the Cp value of the PRDM5 gene is less than or equal to 38.85.
The method for detecting the gastric cancer provided by the invention is simple and convenient to operate, easy to master and capable of quickly and accurately detecting the cancer.
In a preferred embodiment, performing a fluorescent quantitative PCR reaction comprises the steps of:
a) uniformly mixing the PCR Mix and the Taq enzyme Mix to obtain a PCR pre-reaction solution;
b) subpackaging the PCR pre-reaction solution into a PCR reaction container, adding a sample template to be detected with the same volume, and sealing;
c) the PCR procedure used for detection was as follows: first, denaturation at 95 ℃ for 5 minutes, then denaturation at 95 ℃ for 20 seconds, annealing at 61 ℃ for 40 seconds, and 50 cycles are carried out, wherein the temperature rise and fall speed of the instrument is set to be 1.3 ℃/S in the PCR cycle process, and finally the temperature of the instrument is maintained at 40 ℃ for 30 seconds.
In a preferred embodiment, ACTB is used as an internal control for PCR amplification.
Wherein, the upstream primer of the internal reference ACTB is as follows: 5'-TGGGGTGGTGATGGAGGAGG-3' (SEQ ID NO. 8);
the downstream primers of the internal reference ACTB are as follows: 5'-CCAACACACAATAACAAACA-3' (SEQ ID NO. 9).
In a preferred embodiment, the DNA in the plasma sample after lysis by the lysis solution is extracted by a magnetic bead adsorption method, and the DNA is subjected to sulfite conversion, and then purified by a magnetic bead method to obtain sulfite-converted plasma sample DNA.
Preferably, after the DNA in the plasma sample is washed by using a mixed solution of an extraction rinsing solution and absolute ethyl alcohol, the DNA is subjected to sulfite conversion.
The method specifically comprises the following steps:
a) adding 1.0-4.0mL of lysate into 0.5-2.0mL of plasma sample, and performing shaking incubation at 10-30 ℃ for 10-30 min;
b) extracting free DNA in the step a) by adopting a magnetic bead adsorption method;
c) washing the DNA obtained in the step b) by using a mixed solution of an extraction rinsing solution and absolute ethyl alcohol;
d) and c) carrying out sulfite conversion on the DNA washed in the step c), and purifying by using a magnetic bead method to obtain bisDNA (DNA vulcanized by sulfite).
In a preferred embodiment, the binding solution containing the magnetic beads is mixed with the bisDNA and adsorbed, the magnetic beads are washed with a mixture of a purification rinse and absolute ethanol, and the bisDNA is eluted with an eluent.
The method specifically comprises the following steps:
A) mixing the bisDNA with the binding solution and the magnetic beads uniformly, and adsorbing the bisDNA onto the magnetic beads;
B) and mixing the purified rinsing liquid with absolute ethyl alcohol, washing the magnetic beads with the bisDNA adsorbed in the step A), and then eluting by using an eluent to obtain the bisDNA.
To facilitate a clearer understanding of the contents of the present invention, reference will now be made in detail to the following specific embodiments.
EXAMPLE 1 kit composition and treatment of samples to be tested
1. The kit for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene comprises a DNA treatment kit and a methylation gene detection kit. The composition of the DNA treatment kit is shown in Table 1, and the composition of the methylated gene detection kit is shown in Table 2.
TABLE 1 DNA treatment kit
TABLE 2 methylated Gene detection kit
| Reagent | Principal Components |
| PCR Mix | dNTP, primer, buffer salt |
| Taq enzyme Mix | Taq enzyme, Probe, buffer salt |
| Negative control | TE buffer, unmethylated human genomic DNA |
| Positive control | TE buffer solution, methylated human genomic DNA |
2. The instrument equipment comprises: the instrumentation required for the detection is as follows:
vortex oscillation instrument, rotary mixer, 15mL magnetic frame, 2.0mL magnetic frame, metal bath, PCR instrument, palm centrifuge, micropipette, superclean bench, Lightcycler 480, etc.
3. Extraction and sulfuration purification of plasma free DNA
The specific method comprises the following steps:
1. reagent preparation
1) Extracting a rinsing liquid: adding equal volume of anhydrous ethanol before first use, shaking and mixing.
2) Purifying the rinsing liquid: adding 4 times volume of absolute ethyl alcohol before the first use, and shaking and mixing uniformly.
2. Procedure for the preparation of the
1) Taking a 15mL centrifuge tube (self-contained), adding 2mL plasma sample, thawing 1 positive control sample and negative control sample at room temperature, adding 4mL lysate, mixing by vortex, and shaking at room temperature for 10-30 min.
2) Adding 100 μ L of magnetic beads, mixing by vortex, and shaking at room temperature for 10-30 min.
3) Placing the centrifugal tube on a magnetic frame, and discarding the supernatant after the magnetic beads are completely adsorbed.
4) Adding 1mL of extraction rinsing liquid, uniformly mixing magnetic beads, transferring the mixture into a 1.5-2mL centrifuge tube, placing the centrifuge tube on a magnetic frame, and removing a supernatant after the magnetic beads are completely adsorbed.
5) Repeat step 4 1 time).
6) Adding 1mL of 80% ethanol, uniformly mixing by vortex, placing the centrifugal tube on a magnetic frame, completely adsorbing by magnetic beads, and discarding the supernatant.
7) Repeat step 61 time).
8) And (5) instantly separating, placing the centrifugal tube on a magnetic rack, carefully removing residual liquid by using a gun after the magnetic beads are completely adsorbed, and placing at room temperature for 5-10 min.
9) Adding 100 mu L of purified water into a centrifuge tube, uniformly mixing by vortex, incubating for 5-10min at 56 ℃, carrying out instantaneous separation, placing the centrifuge tube on a magnetic frame, absorbing DNA solution for storage after magnetic beads are completely adsorbed.
10) 1 piece of 600. mu.L centrifuge tube was added with 100. mu.L of DNA solution, 190. mu.L of sulfuration reagent and 30. mu.L of DNA protective solution, vortexed and mixed, and subjected to flash separation.
10) The tubes were incubated at 98 ℃ for 10min and at 80 ℃ for 1 h.
11) Cooling to room temperature and instantly separating. Adding 1.2mL of binding solution into 12 mL centrifuge tube, transferring into the above vulcanization product, adding 50 μ L of magnetic bead, mixing by vortex, oscillating at room temperature for 10-30min, separating instantaneously, placing the centrifuge tube on magnetic frame, adsorbing magnetic bead completely, and discarding supernatant.
12) Adding 500 μ L of purified rinsing solution 1, mixing by vortex, separating instantaneously, placing the centrifuge tube on a magnetic frame, adsorbing magnetic beads completely, and discarding the supernatant.
13) Adding 500 μ L of purified rinsing solution 2, mixing, shaking at room temperature for 10-30min, separating, placing the centrifuge tube on a magnetic frame, adsorbing with magnetic beads completely, and removing supernatant.
14) Adding 500 μ L of purified rinsing solution 3, mixing by vortex, separating instantaneously, placing the centrifuge tube on a magnetic frame, adsorbing magnetic beads completely, and discarding the supernatant.
15) Adding 500 μ L of purified rinsing solution 1, mixing by vortex, separating instantaneously, placing the centrifuge tube on a magnetic frame, adsorbing magnetic beads completely, and discarding the supernatant.
16) Repeat step 15 1 time).
17) Taking off the centrifugal tube, instantly separating, placing the centrifugal tube on a magnetic frame, completely adsorbing the magnetic beads, carefully removing residual liquid by using a gun, and standing at room temperature for 5-10 min.
18) Adding 30-60 μ L of sterilized water preheated to 56 deg.C into the magnetic beads, covering with a cover, vortex mixing, incubating at 56 deg.C for 5-10min, and mixing again.
19) And (5) instantaneously separating, placing the centrifugal tube on a magnetic frame, and sucking the supernatant bisDNA into a new centrifugal tube for storage after the magnetic beads are completely adsorbed.
Example 2 detection of RPRM and PRDM5 methylated genes
1) Preparing PCR pre-reaction liquid according to a reaction system shown in the table 3, uniformly mixing, subpackaging into PCR tubes, adding a sample to be detected, a reference substance and purified water as templates, wherein the reaction volume is 20 mu L;
TABLE 3PCR reaction System
2) The fluorescent quantitative PCR reaction was carried out according to the reaction conditions shown in Table 4.
TABLE 4 reaction conditions
3) After the sample is loaded on the machine, the experimental name is saved, PCR reaction is operated, and the sample loading typesetting and the sample name are compiled in time according to the experimental layout.
Example 3 analysis of PCR results
1. In that
"Analysis" is selected in the 480 base software module column.
2. The "Abs Quant/Fit Points" analysis mode was chosen.
3. Select "Filter Comb 465-" 510 ".
4. Selecting a 'Cycle Range' window, and setting 'First Cycle' to '1'; the "Last Cycle" is "50".
5. In the "Cycle Range" window, the Background is set to "5-22", the "Background" button in blue is clicked, the "Min Offset" is set to "4", and the "Max Offset" is set to "21".
6. The "Noise Band" window is selected, the "Noise Band" is set to "Noise Band (fluorescent)", and the value of "Noise Band" is manually changed to "2.0".
Note: if the background value is higher than 2.0, the baseline, i.e., the value of "Noise Band" can be adjusted according to actual conditions, and the amplification curve without significant exponential growth period should be negative.
7. The "Analysis" window is selected, "Threshold (auto)" automatically adjusts the "Threshold" value to coincide with the "Noise Band" value, and "Fit Points" is set to "2".
8. Clicking on "Calculate", the software automatically gives the Cp values of the target genes RPRM and PRDM 5.
9. Select "Filter Comb 533-.
10. Click on "Calculate", the software automatically gives the Cp value of the internal reference gene ACTB.
The specific interpretation of the PCR results is shown in Table 5:
if the interpretation result is negative, the risk of gastric cancer is low; if the interpretation result is positive and the risk of gastric cancer is high, microscopic examination confirmation is recommended.
TABLE 5 interpretation of PCR results
Test of clinical samples of Experimental examples
56 clinical samples are collected and detected, and the kit for simultaneously detecting the methylation of the RPRM gene and the PRDM5 gene is applied to detection, and the detection results are shown in the following table. Among them, 9 healthy persons, 17 non-chronic atrophic gastritis, 14 chronic atrophic gastritis, intestinal metaplasia and dysplasia, 16 gastric cancers (2 cases of stage I, 3 cases of stage III, 1 case of stage IV and 10 cases of stage unknown samples), the results of clinical samples showed 94% gastric cancer detection sensitivity, 86% pre-gastric lesion detection sensitivity, 90% overall sensitivity a/(a + C) 100%, 73% specificity D/(B + D) 100%, 79% positive predictive value a/(a + B) 100%, and 86% negative predictive value D/(C + D) 100%.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Jiangsu is the real biological medicine technology corporation
<120> primer group, reagent and kit for detecting RPRM gene and PRDM5 gene methylation and application
<160> 9
<170> PatentIn version 3.5
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agcgtttagg ttcgcgtttt 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence
<400> 7
agtaagtttt ttggattgtg 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
tggggtggtg atggaggagg 20
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence
<400> 9
ccaacacaca ataacaaaca 20
Claims (4)
1. Use of a kit for simultaneously detecting methylation of an RPRM gene and a PRDM5 gene in the preparation of a product for assisting diagnosis and/or early screening of gastric cancer, wherein the kit comprises a PCR Mix and a Taq enzyme Mix; the Taq enzyme Mix comprises Taq enzyme and a probe,
the PCR Mix comprises a primer pair for detecting an RPRM gene, a primer pair for detecting a PRDM5 gene and dNTP;
the primer pair for detecting the RPRM gene comprises an upstream primer of the RPRM gene shown as SEQ ID NO.1 and a downstream primer of the RPRM gene shown as SEQ ID NO. 2;
the primer pair for detecting the PRDM5 gene comprises an upstream primer of the PRDM5 gene shown as SEQ ID NO.3 and a downstream primer of the PRDM5 gene shown as SEQ ID NO. 4.
2. The use of claim 1, wherein the kit further comprises a negative control and a positive control;
the negative control product comprises TE buffer solution and genomic DNA which is not methylated by both RPRM gene and PRDM5 gene;
the positive control included TE buffer and genomic DNA methylated at both the RPRM gene and the PRDM5 gene.
3. The use of claim 1, wherein the kit further comprises a DNA treatment;
the DNA treatment product comprises a product for extracting DNA and a product for performing sulfite conversion and purification on the DNA;
the product for extracting DNA comprises lysis solution, magnetic beads and extraction rinsing solution, and the product for performing sulfite conversion and purification on DNA comprises a vulcanizing reagent, DNA protection solution, binding solution, purification rinsing solution and eluent.
4. A kit for simultaneously detecting methylation of RPRM gene and PRDM5 gene, which comprises the primer pair for detecting methylation of RPRM gene and the primer pair for detecting methylation of PRDM5 gene as claimed in claim 1, wherein the kit is used for detecting DNA of plasma sample, the Cp value of RPRM gene and the Cp value of PRDM5 gene associated with gastric cancer risk are 44.76 and 38.85 respectively, and the quality control Cp value is 37.0.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810127514.1A CN108103197B (en) | 2018-02-08 | 2018-02-08 | Primer group, reagent, kit and application for detecting RPRM gene and PRDM5 gene methylation |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810127514.1A CN108103197B (en) | 2018-02-08 | 2018-02-08 | Primer group, reagent, kit and application for detecting RPRM gene and PRDM5 gene methylation |
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| CN108103197A CN108103197A (en) | 2018-06-01 |
| CN108103197B true CN108103197B (en) | 2022-03-04 |
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Citations (4)
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| CN101443462A (en) * | 2006-05-15 | 2009-05-27 | 北海道公立大学法人札幌医科大学 | Method for detecting disease-related marker using gastric mucosal lavage fluid |
| WO2011035453A2 (en) * | 2009-09-28 | 2011-03-31 | Pontificia Unversidad Católica De Chile | Non-invasive method for the early detection of stomach cancer |
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| CN105567850A (en) * | 2016-02-26 | 2016-05-11 | 福建师范大学 | Reagent kit and method for quantitatively detecting RPRM gene DNA methylation |
-
2018
- 2018-02-08 CN CN201810127514.1A patent/CN108103197B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101443462A (en) * | 2006-05-15 | 2009-05-27 | 北海道公立大学法人札幌医科大学 | Method for detecting disease-related marker using gastric mucosal lavage fluid |
| WO2011035453A2 (en) * | 2009-09-28 | 2011-03-31 | Pontificia Unversidad Católica De Chile | Non-invasive method for the early detection of stomach cancer |
| CN105274100A (en) * | 2015-10-27 | 2016-01-27 | 苏州工业园区为真生物医药科技有限公司 | Human TWIST1/Vimentin gene methylation detection marker and kit |
| CN105567850A (en) * | 2016-02-26 | 2016-05-11 | 福建师范大学 | Reagent kit and method for quantitatively detecting RPRM gene DNA methylation |
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| Title |
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| DNA and histone methylation in gastric carcinogenesis;Danielle Queiroz Calcagno等;《World J Gastroenterol》;20130228;第19卷(第8期);第1182-1192页 * |
| PRDM5 基因甲基化及在HTB-182、A549 中的表达;刘晶晶等;《现代生物医学进展》;20120430;第12卷(第12期);第2285-2289页 * |
| Reprimo as a modulator of cell migration and invasion in the MDA-MB-231 breast cancer cell line;Kurt Buchegger等;《Biol Res》;20160122;第49卷;第2页左栏倒数第2段，第7页右栏第2段至第8页左栏第1段，表1 * |
| The Epigenetic Modifier PRDM5 Functions as a Tumor Suppressor through Modulating WNT/b-Catenin Signaling and Is Frequently Silenced in Multiple Tumors;Xing-sheng Shu等;《PLoS One》;20111130;第6卷(第11期);摘要，第9页左栏第3段 * |
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| CN108103197A (en) | 2018-06-01 |
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