CN108676889B - Gastric adenocarcinoma susceptibility prediction kit and system - Google Patents
Gastric adenocarcinoma susceptibility prediction kit and system Download PDFInfo
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- CN108676889B CN108676889B CN201810766318.9A CN201810766318A CN108676889B CN 108676889 B CN108676889 B CN 108676889B CN 201810766318 A CN201810766318 A CN 201810766318A CN 108676889 B CN108676889 B CN 108676889B
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- 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
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Abstract
The invention relates to a reagent kit for predicting susceptibility of gastric adenocarcinoma, which comprises the following components: STR-1 primer, STR-2 primer, STR-3 primer, STR-4 primer, STR-5 primer and STR-6 primer; further, it may further include: PCR amplification reaction liquid, LIZ-500 molecular weight internal standard and deionized formamide. The gastric adenocarcinoma susceptibility prediction kit can be used for diagnosis and susceptibility prediction of gastric adenocarcinoma. The invention also provides a gastric adenocarcinoma susceptibility prediction system.
Description
Technical Field
The present invention relates to the field of biomedicine. In particular to a gastric adenocarcinoma susceptibility prediction kit and a gastric adenocarcinoma susceptibility prediction system. More specifically, the invention relates to a kit for detecting STR of gastric adenocarcinoma susceptibility related genes by Short Tandem Repeat (STR) site fragment analysis method, and early warning is carried out on gastric adenocarcinoma susceptibility of a detected object by combining with a discriminant analysis statistical method.
Background
The tumor is a disease closely related to genetic genes, the molecular genetics basis of the tumor is researched, and further a tumor specific genetics marker is provided, so that the tumor specific genetics marker is expected to provide a simple and feasible method for common detection, clinical diagnosis, personalized treatment, disease tracking after recovery and the like. However, the individual differences of patients, the intercrossing of related biomolecular events at different stages of development, etc. all bring great difficulties to the work.
Gastric adenocarcinoma is a type of gastric cancer, which is a malignant change of gastric gland somatic cells, and is called adenocarcinoma. The incidence of gastric adenocarcinoma accounts for 95% of gastric malignancies. The incidence of gastric cancer increases with age, and over 75% of patients are older than 50 years, but the pathogenesis is not clear. The gastric adenocarcinoma susceptibility of the examined population is predicted, the risk awareness of the disease is favorably improved, the prediction result shows that the population with higher gastric adenocarcinoma disease probability can reduce the disease probability or discover and treat as soon as possible by controlling diet and other modes.
A large number of studies have shown that genetic polymorphisms of tumor-associated genes play a key role in the development of malignant tumors. However, the development of tumors is a very complex process, and the diagnosis of the disease using changes in a single molecular genetic marker is clearly impossible and not scientific. In the prior art, accurate early warning on tumor susceptibility cannot be performed only through genetic information, and the current early identification and prediction method for tumors needs to be improved. The invention relates to a kit for early warning the susceptibility of gastric adenocarcinoma by jointly detecting a plurality of STR loci with high relevance to the occurrence of gastric adenocarcinoma through an STR locus fragment analysis method and combining a discriminant analysis statistical method.
Disclosure of Invention
In order to solve the problems in the prior art, the invention relates to a method for jointly detecting a plurality of STR loci with high relevance to the occurrence of gastric adenocarcinoma by an STR locus fragment analysis method and early warning the susceptibility of gastric adenocarcinoma by combining a discriminant analysis statistical method.
The present invention has been completed based on the following findings of the inventors: the inventor finds that the short tandem sequence repetition times of each independent STR locus have no significant correlation with the stomach adenocarcinoma suffering from the detected object and the combination of the short tandem sequence repetition times of certain specific STR loci has close relation with the stomach adenocarcinoma suffering from the detected object by analyzing STRs of the genome DNA of the detected object of the stomach adenocarcinoma and a healthy control detected object and verifying the STRs in a large number of stomach adenocarcinoma samples and a control sample.
To this end, the present invention proposes a set of isolated STR loci that have a high association with the development of gastric adenocarcinoma. According to an embodiment of the present invention, these isolated STR loci comprise the nucleotide sequences shown as STR-1 to STR-6 (Table 1). By using these isolated STR loci as references, the susceptibility to gastric adenocarcinoma can be predicted efficiently.
TABLE 1
| Locus code | Starting position | Belonging gene | Short tandem sequence |
| STR-1 | X chromosome, position 66657655 | AR | CAG |
| STR-2 | Chromosome 4, position 55633758 | Bat-25 | T |
| STR-3 | Chromosome 5, position 111646983 | D5S346 | GT |
| STR-4 | Chromosome 6, position 151806531 | ER1 | TA |
| STR-5 | Chromosome 14, position 64253561 | ER2 | TG |
| STR-6 | Chromosome 4, position 154587748 | FGA | AAAG |
For the above detailed description of STR sites, those skilled in the art can log in relevant databases (such as GeneBank, Nucleotide, etc.) to obtain the details, which are not described herein. The inventors surprisingly found that a statistical analysis method, such as a discriminant analysis using the number of times of repetition of the short tandem sequence of each STR locus obtained by analyzing the cell genome of the subject as described above as an independent variable, can early warn about the susceptibility to gastric adenocarcinoma.
On the basis, the invention solves the technical problem of providing a gastric adenocarcinoma susceptibility prediction kit, which comprises the following components: STR-1 primer, STR-2 primer, STR-3 primer, STR-4 primer, STR-5 primer and STR-6 primer, wherein the primers are respectively used for amplifying target fragments containing short tandem sequences listed in Table 1 so as to determine the repetition times of the short tandem sequences.
Preferably, the gastric adenocarcinoma susceptibility prediction kit of the present invention further comprises: PCR amplification reaction liquid, LIZ-500 molecular weight internal standard and deionized formamide.
In the kit for predicting gastric adenocarcinoma susceptibility of the present invention, preferably, the sequences of the STR-1 primer, STR-2 primer, STR-3 primer, STR-4 primer, STR-5 primer and STR-6 primer are as shown in table 2 below, and more preferably, the concentrations of the primers are all 10 μ M:
TABLE 2
In table 2, HEX, FAM, and ROX are all fluorophores labeling the 5' end, HEX is hexachloro-6-methylfluorescein, FAM is 6-carboxyfluorescein, and ROX is ROX reference dye.
In the kit for predicting susceptibility to gastric adenocarcinoma of the present invention, preferably, the PCR amplification reaction solution is a mixture of the following reagents: TaqDNA polymerase (5U/. mu.L), Tris-HCl (100mM, pH 8.8 at 25 ℃), KCl (500mM), ethylphenylpolyethyleneglycol (0.8% (v/v)), MgCl2(25mM), dNTP (10mM), deionized water.
More preferably, the PCR amplification reaction solution is stored at-20 ℃.
In the kit for predicting susceptibility to gastric adenocarcinoma, preferably, the LIZ-500 molecular weight internal standard can be stored at-20 ℃;
in the kit for predicting susceptibility to gastric adenocarcinoma of the present invention, preferably, the deionized formamide may be stored at 2-8 ℃.
Preferably, the gastric adenocarcinoma susceptibility prediction kit of the present invention further comprises instructions for use.
The instruction describes a method for using the gastric adenocarcinoma susceptibility prediction kit, which comprises the following steps:
(1) extracting sample DNA;
(2) PCR reaction
(2-1) taking out the STR-1 primer, the STR-2 primer, the STR-3 primer, the STR-4 primer, the STR-5 primer, the STR-6 primer and the PCR amplification reaction solution from a refrigerator, balancing to room temperature, fully dissolving each component, and respectively and rapidly centrifuging for 10 seconds;
(2-2) adding 30-300ng of sample DNA into 60 mu L of PCR amplification reaction solution, adding deionized water to supplement to 115.2 mu L, fully and uniformly mixing, quickly centrifuging for 10 seconds, and subpackaging the mixed solution into 6 PCR reaction tubes according to 19.2 mu L/hole;
(2-3) respectively adding an STR-1 primer, an STR-2 primer, an STR-3 primer, an STR-4 primer, an STR-5 primer and an STR-6 primer into the 6 PCR reaction tubes in the step (2-2) according to 0.8 mu L/hole; covering a PCR reaction tube cover, recording the sample adding condition, quickly centrifuging for 10 seconds, then transferring the PCR reaction tube to a corresponding position of a sample groove of a PCR amplification instrument, recording the placing sequence, and starting the PCR amplification reaction; the amplification reaction conditions are as follows: 3 minutes at 95 ℃; 30 seconds at 95 ℃, 30 seconds at 60 ℃ and 30 seconds at 72 ℃ for 10 cycles; 30 seconds at 95 ℃, 30 seconds at 55 ℃, 30 seconds at 72 ℃ and 20 cycles; 6 groups of PCR amplification products are obtained at 72 ℃ for 6 minutes;
(3) STR fragment analysis
(3-1) adding 990 mu L of deionized formamide into 10 mu L of LIZ-500 molecular weight internal standard, fully and uniformly mixing, quickly centrifuging for 10 seconds, respectively adding into a sequencing reaction tube according to 10 mu L/hole, and quickly centrifuging for 10 seconds;
(3-2) adding the 6 groups of PCR amplification products into 6 sequencing reaction tubes according to 1 mu L/hole respectively, and quickly centrifuging for 10 seconds; then transferring the sequencing reaction tube to a corresponding position of a sample tank of a PCR (polymerase chain reaction) amplification instrument, heating at 98 ℃ for 5 minutes, immediately placing the sequencing reaction tube on an ice-water mixture after the program is finished, rapidly cooling to 0 ℃, and rapidly centrifuging for 10 seconds; then transferring the sequencing reaction tube to a corresponding position of a sample groove of an STR locus fragment analyzer, recording the placement sequence, and performing fragment analysis detection;
(4) analysis and determination of results
(4-1) respectively recording the fragment lengths of two alleles at each site of STR-1, STR-2, STR-3, STR-4, STR-5 and STR-6 according to the fragment analysis result:
the length of the smaller of the two STR-1 alleles is recorded as L1And the length of the larger fragment of the two STR-1 alleles is designated as L2；
The length of the smaller fragment of the two STR-2 alleles is recorded as L3And the length of the larger fragment of the two STR-2 alleles is designated as L4；
The length of the smaller fragment of the two STR-3 alleles is recorded as L5And the length of the larger fragment of the two STR-3 alleles is marked as L6；
The length of the smaller fragment of the two STR-4 alleles is recorded as L7And the length of the larger fragment of the two STR-4 alleles is marked as L8；
The length of the smaller of the two STR-5 alleles is recorded as L9And the length of the larger fragment of the two STR-5 alleles is designated as L10；
The length of the smaller fragment of the two STR-6 alleles was designated L11And the length of the larger fragment of the two STR-6 alleles is marked as L12；
(4-2) the number of repetitions of the short tandem sequence is calculated from the fragment length and the following formula, and is denoted as X1-X12Where round stands for rounded integer:
X1＝round[(L1-191)/3]；X2＝round[(L2-191)/3]；
X3＝round(L3-379)；X4＝round(L4-379)；
X5＝round[(L5-202)/2]；X6＝round[(L6-202)/2]；
X7＝round[(L7-359)/2]；X8＝round[(L8-359)/2]；
X9＝round[(L9-278)/2]；X10＝round[(L10-278)/2]；
X11＝round[(L11-200)/4]；X12＝round[(L12-200)/4]；
(4-3) substituting the number of the short tandem sequence repetitions into a preset discriminant function:
FGC＝10.528X1+0.564X2+15.752X3+121.525X4+2.662X5+0.174X6+1.845X7+4.898X8-0.049X9-8.142X10+2.514X11+9.819X12-21.742X13-1913.157
FGN＝10.625X1+1.269X2+15.685X3+121.518X4+1.444X5+0.411X6+2.186X7+4.306X8-0.708X9-6.965X10+2.859X11+9.767X12-27.582X13-1926.622
wherein, if the subject is female, X is13When the subject is male, X is 013＝1；
(4-4) prediction of gastric adenocarcinoma susceptibility:
comparison FGCValue sum FGNValue if FGC>FGNPredicting the probability of gastric adenocarcinoma of the detected object to be more than or equal to 93.2%; if FGC≤FGNAnd predicting that the probability that the detected object does not suffer from gastric adenocarcinoma is more than or equal to 91.7 percent.
In the present invention,
preferably, the sample DNA extracted in step (1) can be performed using a commercially available genomic DNA extraction kit according to the kit instructions. The sample may be whole blood of a subject.
Preferably, the rotational speed of all the centrifuges in the method of use is preferably 3000 g/min.
The probability of suffering from gastric adenocarcinoma in the invention is the sum of the probability of suffering from gastric adenocarcinoma already and the probability of suffering from gastric adenocarcinoma in the future. Therefore, the method can be used for diagnosing gastric adenocarcinoma; the method can also be used for risk early warning of gastric adenocarcinoma in the future, can assist the detected object in risk prevention, and reduces the incidence of gastric adenocarcinoma through drug conditioning, changes of daily work and rest, diet rules, regular physical examination and other modes.
The second technical problem solved by the invention is to provide a method for predicting gastric adenocarcinoma susceptibility, namely, the method is operated by using the kit according to the instruction.
The invention solves the technical problem of providing the application of the gastric adenocarcinoma susceptibility prediction kit in preparing gastric adenocarcinoma diagnosis products.
The fourth technical problem to be solved by the present invention is to provide a gastric adenocarcinoma susceptibility prediction system, which comprises:
a device for obtaining the repeat times of the STR locus short tandem sequence of the sample DNA;
data processing and decision device, comprising the following modules:
the data input module is used for inputting the age, the sex and the STR locus short tandem sequence repetition times of the detected object;
the database management module is used for the operation management of data storage, modification, deletion, inquiry and printing;
the data calculation module is used for calculating a discrimination function result according to the repeat times of the STR locus short serial sequence in the data input module;
and the analysis, discrimination and result output module is used for comparing the discrimination function results so as to predict the susceptibility of the gastric adenocarcinoma and outputting the result.
Wherein the content of the first and second substances,
the number of times of the STR locus short tandem sequence repetition is 6 pairs of the number of times of the STR locus short tandem sequence repetition:
| locus code | Starting position | Belonging gene | Short tandem sequence |
| STR-1 | X chromosome, position 66657655 | AR | CAG |
| STR-2 | Chromosome 4, position 55633758 | Bat-25 | T |
| STR-3 | Chromosome 5, position 111646983 | D5S346 | GT |
| STR-4 | Chromosome 6, position 151806531 | ER1 | TA |
| STR-5 | Chromosome 14, position 64253561 | ER2 | TG |
| STR-6 | Chromosome 4, position 154587748 | FGA | AAAG |
The discriminant function includes:
first discriminant function FGC＝10.528X1+0.564X2+15.752X3+121.525X4+2.662X5+0.174X6+1.845X7+4.898X8-0.049X9-8.142X10+2.514X11+9.819X12-21.742X13-1913.157
Second discrimination function FGN＝10.625X1+1.269X2+15.685X3+121.518X4+1.444X5+0.411X6+2.186X7+4.306X8-0.708X9-6.965X10+2.859X11+9.767X12-27.582X13-1926.622
In the case of the discriminant function,
X1the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-1;
X2the number of repeats of the short tandem sequence for the larger of the two alleles of STR-1;
X3the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-2;
X4the number of repeats of the short tandem sequence for the larger of the two alleles of STR-2;
X5the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-3;
X6the number of repeats of the short tandem sequence for the larger of the two alleles of STR-3;
X7the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-4;
X8the number of repeats of the short tandem sequence for the larger of the two alleles of STR-4;
X9the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-5;
X10the number of repeats of the short tandem sequence for the larger of the two alleles of STR-5;
X11the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-6;
X12the number of repeats of the short tandem sequence for the larger of the two alleles of STR-6;
if the subject is female, X13When the subject is male, X is 013＝1。
Wherein, X1 -X12Calculated from the fragment length and the following formula, where round stands for rounded integer:
X1＝round[(L1-191)/3]；X2＝round[(L2-191)/3]；
X3＝round(L3-379)；X4＝round(L4-379)；
X5＝round[(L5-202)/2]；X6＝round[(L6-202)/2]；
X7＝round[(L7-359)/2]；X8＝round[(L8-359)/2]；
X9＝round[(L9-278)/2]；X10＝round[(L10-278)/2]；
X11＝round[(L11-200)/4]；X12＝round[(L12-200)/4]；
X1 -X12in, L1Is the smaller segment length value, L, of the two alleles of STR-12Is the larger fragment length value of the two alleles of STR-1;
L3is the smaller segment length value, L, of the two alleles of STR-24Is the larger fragment length value of the two alleles of STR-2;
L5is the smaller segment length value, L, of the two alleles of STR-36Is the larger fragment length value of the two alleles of STR-3;
L7is the smaller segment length value, L, of the two alleles of STR-48Is the larger fragment length value of the two alleles of STR-4;
L9is the smaller segment length value, L, of the two alleles of STR-510Is the larger fragment length value of the two alleles of STR-5;
L11is the smaller segment length value, L, of the two STR-6 alleles12Is the larger fragment length value of the two alleles of STR-6;
the analysis discrimination and result output module outputs a first discrimination function FGCAnd a second discrimination function FGNIf F is the result of the calculation ofGC>FGNThen outputThe prediction result that the probability of gastric adenocarcinoma of the detected object is more than or equal to 93.2 percent is obtained; if FGC≤FGNAnd outputting a prediction result that the probability that the detected object does not suffer from the gastric adenocarcinoma is more than or equal to 91.7 percent.
The device for obtaining the repetition times of the STR locus short tandem sequence of the sample DNA can comprise an STR locus fragment analyzer, a PCR amplification instrument and the like; the data processing and determining device may be a computer or the like.
The fifth technical problem to be solved by the invention is to provide the application of the gastric adenocarcinoma susceptibility prediction system in preparing gastric adenocarcinoma prediction products, gastric adenocarcinoma diagnosis products and stomach health auxiliary products.
The sixth technical problem to be solved by the invention is to provide a gastric adenocarcinoma prediction product, a gastric adenocarcinoma diagnosis product or a stomach health auxiliary product, which comprises the gastric adenocarcinoma susceptibility prediction system.
The test material used in the present invention is human genomic DNA, which theoretically does not change during the life of a human. The human genome DNA encodes all life activities of human, so theoretically, the risk of a detected object suffering from a certain disease can be predicted at an early stage by detecting the genome DNA, and even the detected object can be predicted at birth.
The development of tumors is a very complex process. The molecular genetics basis of tumor research is expected to provide a simple and feasible method for common detection, clinical diagnosis, personalized treatment, disease tracking after recovery and the like. However, the individual differences of patients, the intercrossing of related biomolecular events at different stages of development, etc. all bring great difficulties to the work. It is clearly impossible and not scientific to use single molecular genetic changes to diagnose tumors. The inventor applies modern molecular biology technology to carry out combined analysis on a plurality of STRs of the genome DNA of a detected object, and combines statistical analysis methods such as discriminant analysis and the like, thereby inventing a kit for early warning of gastric adenocarcinoma susceptibility.
Drawings
FIG. 1 is a schematic diagram of modules included in a data processing and determining device in the gastric adenocarcinoma susceptibility predicting system of the present invention.
Detailed Description
The invention will be better understood from the following description of specific embodiments thereof, taken in conjunction with the accompanying drawings and examples. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the accompanying drawings are illustrative only for the purpose of explaining the present invention, and are not to be construed as limiting the present invention.
The PCR amplification apparatus in the examples was a Mastercycler nexus amplification apparatus (purchased from eppendorf, USA);
the STR locus fragment analyzer in the examples was a 3730XL sequencing analyzer (purchased from ABI, usa);
the DNA extraction kit in the examples was a blood DNAout kit (purchased from engze, beijing);
the rotational speed of all the centrifuges in the examples was 3000 g/min.
Example 1 gastric adenocarcinoma susceptibility prediction system
A gastric adenocarcinoma susceptibility prediction system comprising:
a device for obtaining the repeat times of the STR locus short tandem sequence of the sample DNA;
data processing and decision device, comprising the following modules (fig. 1):
the data input module is used for inputting the age, the sex and the STR locus short tandem sequence repetition times of the detected object;
the database management module is used for the operation management of data storage, modification, deletion, inquiry and printing;
the data calculation module is used for calculating a discrimination function result according to the repeat times of the STR locus short serial sequence in the data input module;
and the analysis, discrimination and result output module is used for comparing the discrimination function results so as to predict the susceptibility of the gastric adenocarcinoma and outputting the result.
Wherein the content of the first and second substances,
the number of times of the STR locus short tandem sequence repetition is 6 pairs of the number of times of the STR locus short tandem sequence repetition:
| locus code | Starting position | Belonging gene | Short tandem sequence |
| STR-1 | X chromosome, position 66657655 | AR | CAG |
| STR-2 | Chromosome 4, position 55633758 | Bat-25 | T |
| STR-3 | Chromosome 5, position 111646983 | D5S346 | GT |
| STR-4 | Number 6Chromosome, position 151806531 | ER1 | TA |
| STR-5 | Chromosome 14, position 64253561 | ER2 | TG |
| STR-6 | Chromosome 4, position 154587748 | FGA | AAAG |
The discriminant function includes:
first discriminant function FGC＝10.528X1+0.564X2+15.752X3+121.525X4+2.662X5+0.174X6+1.845X7+4.898X8-0.049X9-8.142X10+2.514X11+9.819X12-21.742X13-1913.157
Second discrimination function FGN＝10.625X1+1.269X2+15.685X3+121.518X4+1.444X5+0.411X6+2.186X7+4.306X8-0.708X9-6.965X10+2.859X11+9.767X12-27.582X13-1926.622
In the case of the discriminant function,
X1the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-1;
X2the number of repeats of the short tandem sequence for the larger of the two alleles of STR-1;
X3the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-2;
X4the number of repeats of the short tandem sequence for the larger of the two alleles of STR-2;
X5the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-3;
X6the number of repeats of the short tandem sequence for the larger of the two alleles of STR-3;
X7the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-4;
X8the number of repeats of the short tandem sequence for the larger of the two alleles of STR-4;
X9the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-5;
X10the number of repeats of the short tandem sequence for the larger of the two alleles of STR-5;
X11the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-6;
X12the number of repeats of the short tandem sequence for the larger of the two alleles of STR-6;
if the subject is female, X13When the subject is male, X is 013＝1。
Wherein, X1 -X12Calculated from the fragment length and the following formula, where round stands for rounded integer:
X1＝round[(L1-191)/3]；X2＝round[(L2-191)/3]；
X3＝round(L3-379)；X4＝round(L4-379)；
X5＝round[(L5-202)/2]；X6＝round[(L6-202)/2]；
X7＝round[(L7-359)/2]；X8＝round[(L8-359)/2]；
X9＝round[(L9-278)/2]；X10＝round[(L10-278)/2]；
X11＝round[(L11-200)/4]；X12＝round[(L12-200)/4]；
X1 -X12in, L1Is the smaller segment length value, L, of the two alleles of STR-12Is the larger fragment length value of the two alleles of STR-1;
L3is the smaller segment length value, L, of the two alleles of STR-24Is the larger fragment length value of the two alleles of STR-2;
L5is the smaller segment length value, L, of the two alleles of STR-36Is the larger fragment length value of the two alleles of STR-3;
L7is the smaller segment length value, L, of the two alleles of STR-48Is the larger fragment length value of the two alleles of STR-4;
L9is the smaller segment length value, L, of the two alleles of STR-510Is the larger fragment length value of the two alleles of STR-5;
L11is the smaller segment length value, L, of the two STR-6 alleles12Is the larger fragment length value of the two alleles of STR-6;
the analysis discrimination and result output module outputs a first discrimination function FGCAnd a second discrimination function FGNIf F is the result of the calculation ofGC>FGNOutputting a prediction result that the probability of gastric adenocarcinoma of the detected object is more than or equal to 93.2%; if FGC≤FGNAnd outputting a prediction result that the probability that the detected object does not suffer from the gastric adenocarcinoma is more than or equal to 91.7 percent.
Example 2 gastric adenocarcinoma susceptibility prediction kit
A gastric adenocarcinoma susceptibility prediction kit, comprising the following components: STR-1 primer, STR-2 primer, STR-3 primer, STR-4 primer, STR-5 primer, STR-6 primer, PCR amplification reaction liquid, LIZ-500 molecular weight internal standard, deionized formamide and an instruction book.
The concentrations of the STR-1 primer, the STR-2 primer, the STR-3 primer, the STR-4 primer, the STR-5 primer and the STR-6 primer are all 10 mu M, and the primer sequences are shown in the following table:
the PCR amplification reaction solution is a mixed solution of the following reagents: TaqDNA polymerase (5U/. mu.L), Tris-HCl (100mM, pH 8.8 at 25 ℃), KCl (500mM), ethylphenylpolyethyleneglycol (0.8% (v/v)), MgCl2(25mM), dNTP (10mM), deionized water.
Storing the PCR amplification reaction solution at-20 ℃; LIZ-500 molecular weight internal standard is preserved at-20 ℃; storing deionized formamide at 2-8 deg.C.
The kit further comprises instructions for use.
Example 3 prediction of the risk of gastric adenocarcinoma in the subject Using the System of example 1 and the kit of example 2
The detected object is: for men, 43 years old, visit the second hospital Jilin university for gastrointestinal nutrition and hernia surgery, with full informed examination purpose and use, and on the premise of their own wishes, signed an informed consent, and collected 1mL of anticoagulated blood via the peripheral vein.
The following procedure was carried out using the kit of example 2 according to the method described in the kit instructions:
(1) extracting sample DNA: extracting blood genome DNA by using a DNA extraction kit;
(2) PCR reaction
(2-1) taking out the STR-1 primer, the STR-2 primer, the STR-3 primer, the STR-4 primer, the STR-5 primer, the STR-6 primer and the PCR amplification reaction solution from a refrigerator, balancing to room temperature, fully dissolving each component, and respectively and rapidly centrifuging for 10 seconds;
(2-2) adding 100ng of sample DNA into 60 mu L of PCR amplification reaction solution, adding deionized water to supplement to 115.2 mu L, fully and uniformly mixing, quickly centrifuging for 10 seconds, and subpackaging the mixed solution into 6 PCR reaction tubes according to 19.2 mu L/hole;
(2-3) respectively adding an STR-1 primer, an STR-2 primer, an STR-3 primer, an STR-4 primer, an STR-5 primer and an STR-6 primer into the 6 PCR reaction tubes in the step (2-2) according to 0.8 mu L/hole; covering a PCR reaction tube cover, recording the sample adding condition, quickly centrifuging for 10 seconds, then transferring the PCR reaction tube to a corresponding position of a sample groove of a PCR amplification instrument, recording the placing sequence, and starting the PCR amplification reaction; the amplification reaction conditions are as follows: 3 minutes at 95 ℃; 30 seconds at 95 ℃, 30 seconds at 60 ℃ and 30 seconds at 72 ℃ for 10 cycles; 30 seconds at 95 ℃, 30 seconds at 55 ℃, 30 seconds at 72 ℃ and 20 cycles; 6 groups of PCR amplification products are obtained at 72 ℃ for 6 minutes;
(3) STR fragment analysis
(3-1) adding 990 mu L of deionized formamide into 10 mu L of LIZ-500 molecular weight internal standard, fully and uniformly mixing, quickly centrifuging for 10 seconds, respectively adding into a sequencing reaction tube according to 10 mu L/hole, and quickly centrifuging for 10 seconds;
(3-2) adding the 6 groups of PCR amplification products into 6 sequencing reaction tubes according to 1 mu L/hole respectively, and quickly centrifuging for 10 seconds; then transferring the sequencing reaction tube to a corresponding position of a sample tank of a PCR (polymerase chain reaction) amplification instrument, heating at 98 ℃ for 5 minutes, immediately placing the sequencing reaction tube on an ice-water mixture after the program is finished, rapidly cooling to 0 ℃, and rapidly centrifuging for 10 seconds; then transferring the sequencing reaction tube to a corresponding position of a sample groove of an STR locus fragment analyzer, recording the placement sequence, and performing fragment analysis detection;
(4) analysis and determination of results
(4-1) respectively recording the fragment lengths of two alleles at each site of STR-1, STR-2, STR-3, STR-4, STR-5 and STR-6 according to the fragment analysis result: the length of the smaller of the two STR-1 alleles is recorded as L1And the length of the larger fragment of the two STR-1 alleles is designated as L2(ii) a The length of the smaller fragment of the two STR-2 alleles is recorded as L3And the length of the larger fragment of the two STR-2 alleles is designated as L4(ii) a The length of the smaller fragment of the two STR-3 alleles is recorded as L5And the length of the larger fragment of the two STR-3 alleles is marked as L6(ii) a The length of the smaller fragment of the two STR-4 alleles is recorded as L7And the length of the larger fragment of the two STR-4 alleles is marked as L8(ii) a The length of the smaller of the two STR-5 alleles is recorded as L9And the length of the larger fragment of the two STR-5 alleles is designated as L10(ii) a The length of the smaller fragment of the two STR-6 alleles was designated L11And the length of the larger fragment of the two STR-6 alleles is marked as L12(ii) a The results show that: l is1＝273.82，L2＝273.82，L3＝404.27，L4＝404.27，L5＝224.75，L6＝229.08，L7＝388.88，L8＝400.52，L9＝313.94，L10＝318.15，L11＝249.65，L12＝264.01。
(4-2) the length of the fragment is calculated from the following formula, and is denoted as X1-X12Where round stands for rounded integer:
X1＝round[(L1-191)/3]＝28；X2＝round[(L2-191)/3]＝28；
X3＝round(L3-379)＝25；X4＝round(L4-379)＝25；
X5＝round[(L5-202)/2]＝11；X6＝round[(L6-202)/2]＝14；
X7＝round[(L7-359)/2]＝15；X8＝round[(L8-359)/2]＝21；
X9＝round[(L9-278)/2]＝18；X10＝round[(L10-278)/2]＝20；
X11＝round[(L11-200)/4]＝12；X12＝round[(L12-200)/4]＝16；
the patient is male, X13＝1。
(4-3) predicting susceptibility to gastric adenocarcinoma in a subject using a computer running the system for predicting susceptibility to gastric adenocarcinoma described in example 1:
inputting the age, the sex and the STR locus short tandem sequence repetition times of the detected object into a system through a data input module, and calculating the result of a discriminant function through a data calculation module:
first discriminant function FGC＝10.528X1+0.564X2+15.752X3+121.525X4+2.662X5+0.174X6+1.845X7+4.898X8-0.049X9-8.142X10+2.514X11+9.819X12-21.742X13-1913.157＝1993.403
Second discrimination function FGN＝10.625X1+1.269X2+15.685X3+121.518X4+1.444X5+0.411X6+2.186X7+4.306X8-0.708X9-6.965X10+2.859X11+9.767X12-27.582X13-1926.622＝1992.293
Analyzed, determined and result output module compared FGCValue sum FGNValue, FGC>FGNAnd outputting a prediction result that the probability of gastric adenocarcinoma of the detected object is more than or equal to 93.2 percent.
After the examination, the examinee is subjected to a gastrotomy, the pathological examination confirms that the examinee is the gastric adenocarcinoma, and the clinical diagnosis result of the examinee is consistent with the prediction result of the kit.
Example 4 using the system of example 1 and the kit of example 2, a subject at risk of developing gastric adenocarcinoma was predicted: female, age 79, attending the second hospital of Jilin university, gastrointestinal nutrition and hernia surgery, with full informed examination purposes and uses, signed an informed consent and collected 1mL of anticoagulated blood via the peripheral vein, on the premise of his own accord.
The same treatments and tests were carried out on blood samples, with reference to the prediction method of example 3, and the results show that: l is1＝268.24，L2＝273.70，L3＝403.20，L4＝403.20，L5＝228.74，L6＝228.74，L7＝384.97，L8＝388.57，L9＝311.79，L10＝322.41，L11＝256.61，L12＝256.61。
Calculated according to the fragment length and the following formula, denoted X1-X12Where round stands for rounded integer:
X1＝round[(L1-191)/3]＝26；X2＝round[(L2-191)/3]＝28；
X3＝round(L3-379)＝24；X4＝round(L4-379)＝24；
X5＝round[(L5-202)/2]＝13；X6＝round[(L6-202)/2]＝13；
X7＝round[(L7-359)/2]＝13；X8＝round[(L8-359)/2]＝15；
X9＝round[(L9-278)/2]＝17；X10＝round[(L10-278)/2]＝22；
X11＝round[(L11-200)/4]＝14；X12＝round[(L12-200)/4]＝14；
the patient is female, X13＝0。
Using a computer running the gastric adenocarcinoma susceptibility prediction system described in example 1, a susceptibility prediction for a subject to develop gastric adenocarcinoma:
inputting the age, the sex and the STR locus short tandem sequence repetition times of the detected object into a system through a data input module, and calculating the result of a discriminant function through a data calculation module:
first discriminant function FGC＝10.528X1+0.564X2+15.752X3+121.525X4+2.662X5+0.174X6+1.845X7+4.898X8-0.049X9-8.142X10+2.514X11+9.819X12-21.742X13-1913.157＝1798.039
Second discrimination function FGN＝10.625X1+1.269X2+15.685X3+121.518X4+1.444X5+0.411X6+2.186X7+4.306X8-0.708X9-6.965X10+2.859X11+9.767X12-27.582X13-1926.622＝1806.653
Analyzed, determined and result output module compared FGCValue sum FGNValue, FGC≤FGNAnd outputting a prediction result that the probability that the detected object does not suffer from gastric adenocarcinoma is more than or equal to 91.7 percent.
The tested object is diagnosed as the gastric neurofibroma after the visit, and the clinical diagnosis result is consistent with the prediction result of the kit.
The foregoing is a preferred embodiment of the present invention and is not intended to limit the present invention, and it should be understood that any changes, modifications, substitutions and alterations (e.g., addition, subtraction, change of STR sites, use of cells or tissues from other sources, use of other statistical methods, etc.) made without departing from the principles and spirit of the present invention are intended to be included within the scope of the present invention.
SEQUENCE LISTING
<110> Jilin university
<120> gastric adenocarcinoma susceptibility prediction kit and system
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Claims (7)
1. A gastric adenocarcinoma susceptibility prediction kit, comprising the following components: STR-1 primer, STR-2 primer, STR-3 primer, STR-4 primer, STR-5 primer and STR-6 primer, wherein the STR-1 primer, STR-2 primer, STR-3 primer, STR-4 primer, STR-5 primer and STR-6 primer are respectively used for amplifying target fragments containing the following short tandem sequences so as to determine the repetition times of the following short tandem sequences:
The sequences of the STR-1 primer, the STR-2 primer, the STR-3 primer, the STR-4 primer, the STR-5 primer and the STR-6 primer are as follows:
wherein, the kit further comprises: PCR amplification reaction liquid, LIZ-500 molecular weight internal standard, deionized formamide and an instruction,
the application instruction describes a using method of the gastric adenocarcinoma susceptibility prediction kit, which comprises the following steps:
(1) extracting sample DNA;
(2) PCR reaction
(2-1) taking out the STR-1 primer, the STR-2 primer, the STR-3 primer, the STR-4 primer, the STR-5 primer, the STR-6 primer and the PCR amplification reaction solution from a refrigerator, balancing to room temperature, fully dissolving each component, and respectively and rapidly centrifuging for 10 seconds;
(2-2) adding 30-300ng of sample DNA into 60 mu L of PCR amplification reaction solution, adding deionized water to supplement to 115.2 mu L, fully and uniformly mixing, quickly centrifuging for 10 seconds, and subpackaging the mixed solution into 6 PCR reaction tubes according to 19.2 mu L/hole;
(2-3) respectively adding an STR-1 primer, an STR-2 primer, an STR-3 primer, an STR-4 primer, an STR-5 primer and an STR-6 primer into the 6 PCR reaction tubes in the step (2-2) according to 0.8 mu L/hole; covering a PCR reaction tube cover, recording the sample adding condition, quickly centrifuging for 10 seconds, then transferring the PCR reaction tube to a corresponding position of a sample groove of a PCR amplification instrument, recording the placing sequence, and starting the PCR amplification reaction; the amplification reaction conditions are as follows: 3 minutes at 95 ℃; 30 seconds at 95 ℃, 30 seconds at 60 ℃ and 30 seconds at 72 ℃ for 10 cycles; 30 seconds at 95 ℃, 30 seconds at 55 ℃, 30 seconds at 72 ℃ and 20 cycles; 6 groups of PCR amplification products are obtained at 72 ℃ for 6 minutes;
(3) STR fragment analysis
(3-1) adding 990 mu L of deionized formamide into 10 mu L of LIZ-500 molecular weight internal standard, fully and uniformly mixing, quickly centrifuging for 10 seconds, respectively adding into a sequencing reaction tube according to 10 mu L/hole, and quickly centrifuging for 10 seconds;
(3-2) adding the 6 groups of PCR amplification products into 6 sequencing reaction tubes according to 1 mu L/hole respectively, and quickly centrifuging for 10 seconds; then transferring the sequencing reaction tube to a corresponding position of a sample tank of a PCR (polymerase chain reaction) amplification instrument, heating at 98 ℃ for 5 minutes, immediately placing the sequencing reaction tube on an ice-water mixture after the program is finished, rapidly cooling to 0 ℃, and rapidly centrifuging for 10 seconds; then transferring the sequencing reaction tube to a corresponding position of a sample groove of an STR locus fragment analyzer, recording the placement sequence, and performing fragment analysis detection;
(4) analysis and determination of results
(4-1) respectively recording the fragment lengths of two alleles at each site of STR-1, STR-2, STR-3, STR-4, STR-5 and STR-6 according to the fragment analysis result:
the length of the smaller of the two STR-1 alleles is recorded as L1And the length of the larger fragment of the two STR-1 alleles is designated as L2；
The length of the smaller fragment of the two STR-2 alleles is recorded as L3And the length of the larger fragment of the two STR-2 alleles is designated as L4；
The length of the smaller fragment of the two STR-3 alleles is recorded as L5And the length of the larger fragment of the two STR-3 alleles is marked as L6；
The length of the smaller fragment of the two STR-4 alleles is recorded as L7And the length of the larger fragment of the two STR-4 alleles is marked as L8；
The length of the smaller of the two STR-5 alleles is recorded as L9And the length of the larger fragment of the two STR-5 alleles is designated as L10；
The length of the smaller fragment of the two STR-6 alleles was designated L11The larger fragment of the two STR-6 alleles is longThe value is recorded as L12；
(4-2) the number of repetitions of the short tandem sequence is calculated from the fragment length and the following formula, and is denoted as X1-X12Where round stands for rounded integer:
X1＝round[(L1-191)/3]；X2＝round[(L2-191)/3]；
X3＝round(L3-379)；X4＝round(L4-379)；
X5＝round[(L5-202)/2]；X6＝round[(L6-202)/2]；
X7＝round[(L7-359)/2]；X8＝round[(L8-359)/2]；
X9＝round[(L9-278)/2]；X10＝round[(L10-278)/2]；
X11＝round[(L11-200)/4]；X12＝round[(L12-200)/4]；
(4-3) substituting the number of the short tandem sequence repetitions into a preset discriminant function:
FGC＝10.528X1+0.564X2+15.752X3+121.525X4+2.662X5+0.174X6+1.845X7+4.898X8-0.049X9-8.142X10+2.514X11+9.819X12-21.742X13-1913.157
FGN＝10.625X1+1.269X2+15.685X3+121.518X4+1.444X5+0.411X6+2.186X7+4.306X8-0.708X9-6.965X10+2.859X11+9.767X12-27.582X13-1926.622
wherein, if the subject is female, X is13When the subject is male, X is 013＝1；
(4-4) prediction of gastric adenocarcinoma susceptibility:
comparison FGCValue sum FGNValue if FGC>FGNPredicting the probability of gastric adenocarcinoma of the detected object to be more than or equal to 93.2%; if FGC≤FGNAnd predicting that the probability that the detected object does not suffer from gastric adenocarcinoma is more than or equal to 91.7 percent.
2. The gastric adenocarcinoma susceptibility prediction kit of claim 1, wherein: the using concentration of the STR-1 primer, the STR-2 primer, the STR-3 primer, the STR-4 primer, the STR-5 primer and the STR-6 primer is 10 mu M.
3. The gastric adenocarcinoma susceptibility prediction kit of claim 1, wherein: the PCR amplification reaction solution is a mixed solution of the following reagents: TaqDNA polymerase 5U/. mu. L, Tris-HCl 100mM, KCl 500mM, ethylphenylpolyethylene glycol 0.8 vol%, MgCl225mM, dNTP 10mM and deionized water; wherein Tris-HCl has a pH of 8.8 at 25 ℃.
4. The gastric adenocarcinoma susceptibility prediction kit of claim 1, wherein: the sample is whole blood of a subject.
5. Use of the gastric adenocarcinoma susceptibility prognostic kit according to any one of claims 1 to 4 in the preparation of a gastric adenocarcinoma diagnostic product.
6. A gastric adenocarcinoma susceptibility prediction system, comprising:
means for obtaining the number of repetitions of the following short tandem STR loci of the sample DNA:
；
Data processing and decision device, comprising the following modules:
the data input module is used for inputting the age, the sex and the STR locus short tandem sequence repetition times of the detected object;
the database management module is used for the operation management of data storage, modification, deletion, inquiry and printing;
the data calculation module is used for calculating a discrimination function result according to the repeat times of the STR locus short serial sequence in the data input module;
the analysis, discrimination and result output module is used for comparing the discrimination function results to make gastric adenocarcinoma susceptibility prediction and outputting the results;
obtaining the number of times X of repetition of STR site short tandem sequences of sample DNA using the kit of any one of claims 1 to 51-X12(ii) a And
wherein:
the discriminant function includes:
first discriminant function FGC＝10.528X1+0.564X2+15.752X3+121.525X4+2.662X5+0.174X6+1.845X7+4.898X8-0.049X9-8.142X10+2.514X11+9.819X12-21.742X13-1913.157
Second discrimination function FGN＝10.625X1+1.269X2+15.685X3+121.518X4+1.444X5+0.411X6+2.186X7+4.306X8-0.708X9-6.965X10+2.859X11+9.767X12-27.582X13-1926.622
In the case of the discriminant function,
X1the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-1;
X2the number of repeats of the short tandem sequence for the larger of the two alleles of STR-1;
X3the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-2;
X4the number of repeats of the short tandem sequence for the larger of the two alleles of STR-2;
X5the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-3;
X6the number of repeats of the short tandem sequence for the larger of the two alleles of STR-3;
X7the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-4;
X8the number of repeats of the short tandem sequence for the larger of the two alleles of STR-4;
X9the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-5;
X10the number of repeats of the short tandem sequence for the larger of the two alleles of STR-5;
X11the number of repeats of the short tandem sequence for the smaller of the two alleles of STR-6;
X12the number of repeats of the short tandem sequence for the larger of the two alleles of STR-6;
if the subject is female, X13When the subject is male, X is 013＝1；
Wherein, X1 -X12Calculated from the fragment length and the following, where round stands for rounded integer:
X1＝round[(L1-191)/3]；X2＝round[(L2-191)/3]；
X3＝round(L3-379)；X4＝round(L4-379)；
X5＝round[(L5-202)/2]；X6＝round[(L6-202)/2]；
X7＝round[(L7-359)/2]；X8＝round[(L8-359)/2]；
X9＝round[(L9-278)/2]；X10＝round[(L10-278)/2]；
X11＝round[(L11-200)/4]；X12＝round[(L12-200)/4]；
X1 -X12in, L1Is the smaller segment length value, L, of the two alleles of STR-12Is the larger fragment length value of the two alleles of STR-1;
L3is the smaller segment length value, L, of the two alleles of STR-24Is the larger fragment length value of the two alleles of STR-2;
L5is the smaller segment length value, L, of the two alleles of STR-36Is the larger fragment length value of the two alleles of STR-3;
L7is the smaller segment length value, L, of the two alleles of STR-48Is the larger fragment length value of the two alleles of STR-4;
L9is the smaller segment length value, L, of the two alleles of STR-510Is the larger fragment length value of the two alleles of STR-5;
L11is the smaller segment length value, L, of the two STR-6 alleles12Is the larger fragment length value of the two alleles of STR-6;
the analysis discrimination and result output module outputs a first discrimination function FGCAnd a second discrimination function FGNIf F is the result of the calculation ofGC>FGNOutputting a prediction result that the probability of gastric adenocarcinoma of the detected object is more than or equal to 93.2%; if FGC≤FGNAnd outputting a prediction result that the probability that the detected object does not suffer from the gastric adenocarcinoma is more than or equal to 91.7 percent.
7. Use of the gastric adenocarcinoma susceptibility prediction system of claim 6 for the preparation of gastric adenocarcinoma prediction products or gastric adenocarcinoma diagnosis products.
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