CN108841960B - Reagent box and system for colon adenocarcinoma susceptibility prediction - Google Patents
Reagent box and system for colon adenocarcinoma susceptibility prediction Download PDFInfo
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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 colon 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 colon adenocarcinoma susceptibility prediction kit can be used for diagnosis and susceptibility prediction of colon adenocarcinoma. The invention also provides a colon adenocarcinoma susceptibility prediction system.
Description
Technical Field
The present invention relates to the field of biomedicine. In particular to a colon adenocarcinoma susceptibility prediction kit and a colon adenocarcinoma susceptibility prediction system. More specifically, the invention relates to a kit for detecting STR of colon adenocarcinoma susceptibility related genes by Short Tandem Repeat (STR) site fragment analysis method, and early warning of colon adenocarcinoma susceptibility of a detected object is carried out 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.
Colonic adenocarcinoma is a common digestive tract malignant tumor of colonic glandular epithelium origin, and belongs to one of various pathological types of colon cancer. The etiology is not clear, but the occurrence of the disease is related to diet with high fat and little fiber, adenomatous polyp, schistosoma coli, nonspecific ulcerative colitis, colon adenocarcinoma bacillary dysentery, amebic enteropathy and the like are also closely related to the occurrence of the disease. About 40% of colon cancer is distributed in the rectum and rectum sigmoid flexure, and the rest is distributed in the sigmoid, cecum, ascending colon, descending colon, transverse colon, and hepatic and splenic flexure. The colon adenocarcinoma susceptibility of the tested population is predicted, the risk awareness of the colon adenocarcinoma is favorably improved, the prediction result shows that the population with higher colon adenocarcinoma incidence rate can reduce the incidence rate 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 of colon adenocarcinoma susceptibility by jointly detecting a plurality of STR loci with high relevance to colon adenocarcinoma occurrence 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 colon adenocarcinoma by an STR locus fragment analysis method, and early warning is carried out on the susceptibility of the colon 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 colon adenocarcinoma suffering from the detected object and the short tandem sequence repetition times of certain specific STR loci have close relation with the colon adenocarcinoma suffering from the detected object by analyzing STRs of the genome DNA of the colon adenocarcinoma detected object and healthy control detected objects and verifying the STRs in a large number of colon adenocarcinoma samples and control samples.
To this end, the present invention proposes a set of isolated STR loci that have a high association with the development of colon 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 of colon adenocarcinoma can be predicted effectively.
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 by analyzing the cell genome of the tested object to obtain the repeat times of the short tandem sequence of each STR locus, and performing a statistical analysis method such as discriminant analysis by using the repeat times as independent variables, early warning on the susceptibility of the colon adenocarcinoma can be performed.
On the basis, one of the technical problems solved by the invention is to provide a kit for predicting susceptibility of colon 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, 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 kit for predicting colon adenocarcinoma susceptibility of the present invention further comprises: PCR amplification reaction liquid, LIZ-500 molecular weight internal standard and deionized formamide.
In the kit for predicting colon 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 colon adenocarcinoma of the present invention, preferably, 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.
More preferably, the PCR amplification reaction solution is stored at-20 ℃.
In the kit for predicting colon adenocarcinoma susceptibility of the present invention, preferably, the LIZ-500 molecular weight internal standard can be stored at-20 ℃;
in the kit for predicting susceptibility to colon adenocarcinoma of the present invention, preferably, the deionized formamide can be stored at 2-8 ℃.
Preferably, the colon adenocarcinoma susceptibility prediction kit of the present invention further comprises instructions for use.
The application instruction describes a method for using the colon 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:
STR-1 smaller length of fragment of two allelesIs marked 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:
FCC＝11.227X1-3.019X2+16.464X3+106.046X4-0.739X5-0.535X6-1.158X7+5.333X8+0.837X9-0.899X10+5.151X11+0.555X12-28.523X13-1672.409
FCN＝11.492X1-2.895X2+16.658X3+107.047X4-0.858X5–0.871X6-0.784X7+4.983X8+1.020X9-0.826X10+5.057X11-0.759X12-29.399X13-1685.324
wherein, if the subject is female, X is13When the subject is male, X is 013＝1；
(4-4) prediction of colon adenocarcinoma susceptibility:
comparison FCCValue sum FCNValue if FCC>FCNPredicting the probability of the tested object suffering from the colon adenocarcinoma to be more than or equal to 83.3 percent; if FCC≤FCNAnd predicting that the probability that the tested object does not suffer from the colon adenocarcinoma is more than or equal to 86.1 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 colon adenocarcinoma in the invention is the sum of the probability of already suffering from colon adenocarcinoma and the probability of suffering from colon adenocarcinoma in the future. Therefore, the method can be used for diagnosing the colon adenocarcinoma; the method can also be used for risk early warning of colon adenocarcinoma in the future, can assist a detected object in risk prevention, and reduces the incidence of the colon adenocarcinoma by means of medicine conditioning, change of daily work and rest, diet rules, regular physical examination and the like.
The second technical problem solved by the invention is to provide a method for predicting susceptibility to colon adenocarcinoma, namely, the kit is used and the method is operated according to the instruction.
The invention solves the technical problem of providing the application of the reagent kit for predicting the susceptibility of the colon adenocarcinoma in preparing colon adenocarcinoma diagnostic products.
The fourth technical problem to be solved by the present invention is to provide a colon 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 colon 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 FCC＝11.227X1-3.019X2+16.464X3+106.046X4-0.739X5-0.535X6-1.158X7+5.333X8+0.837X9-0.899X10+5.151X11+0.555X12-28.523X13-1672.409
Second discrimination function FCN＝11.492X1-2.895X2+16.658X3+107.047X4-0.858X5–0.871X6-0.784X7+4.983X8+1.020X9-0.826X10+5.057X11-0.759X12-29.399X13-1685.324
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;
X11repeat times of short tandem sequence for the smaller of two alleles of STR-6Counting;
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 of two alleles of STR-5Value of fragment length, L10Is 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 FCCAnd a second discrimination function FCNIf F is the result of the calculation ofCC>FCNOutputting a prediction result that the probability of the tested object suffering from the colon adenocarcinoma is more than or equal to 83.3 percent; if FCC≤FCNAnd outputting a prediction result that the probability that the detected object does not suffer from the colon adenocarcinoma is more than or equal to 86.1 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 colon adenocarcinoma susceptibility prediction system in the preparation of colon adenocarcinoma prediction products, colon adenocarcinoma diagnosis products and intestinal health auxiliary products.
The invention solves the technical problem of providing a colon adenocarcinoma prediction product, a colon adenocarcinoma diagnosis product or an intestinal tract health auxiliary product, which comprises the colon 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 genomic 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 susceptibility to colon adenocarcinoma.
Drawings
FIG. 1 is a schematic diagram of modules included in a data processing and determining device of the colon 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 1A colon adenocarcinoma susceptibility prediction system
A system for predicting susceptibility to colon adenocarcinoma, 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 colon 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 FCC＝11.227X1-3.019X2+16.464X3+106.046X4-0.739X5-0.535X6-1.158X7+5.333X8+0.837X9-0.899X10+5.151X11+0.555X12-28.523X13-1672.409
Second discrimination function FCN＝11.492X1-2.895X2+16.658X3+107.047X4-0.858X5–0.871X6-0.784X7+4.983X8+1.020X9-0.826X10+5.057X11-0.759X12-29.399X13-1685.324
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 FCCAnd a second discrimination function FCNIf F is the result of the calculation ofCC>FCNOutputting a prediction result that the probability of the tested object suffering from the colon adenocarcinoma is more than or equal to 83.3 percent; if FCC≤FCNAnd outputting a prediction result that the probability that the detected object does not suffer from the colon adenocarcinoma is more than or equal to 86.1 percent.
Example 2 kit for predicting susceptibility to colon adenocarcinoma
A kit for colon adenocarcinoma susceptibility prediction 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 Using the System of example 1 and the kit of example 2 to predict the risk of colon adenocarcinoma in the subject
The detected object is: a male, age 74, attending the colorectal anal surgery of the second hospital of Jilin university, with full informed examination purpose and use, signed an informed consent and collected 1mL of anticoagulated blood via the peripheral vein, on the premise of his own accord.
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＝271.02，L2＝271.02，L3＝404.25，L4＝404.25，L5＝229.13，L6＝248.70，L7＝388.76，L8＝392.61，L9＝322.83，L10＝329.29，L11＝256.92，L12＝269.36。
(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]＝27；X2＝round[(L2-191)/3]＝27；
X3＝round(L3-379)＝25；X4＝round(L4-379)＝25；
X5＝round[(L5-202)/2]＝14；X6＝round[(L6-202)/2]＝23；
X7＝round[(L7-359)/2]＝15；X8＝round[(L8-359)/2]＝17；
X9＝round[(L9-278)/2]＝22；X10＝round[(L10-278)/2]＝26；
X11＝round[(L11-200)/4]＝14；X12＝round[(L12-200)/4]＝17；
the patient is male, X13＝1。
(4-3) predicting susceptibility of a subject to colon adenocarcinoma using a computer running the system for predicting susceptibility to colon 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 FCC＝11.227X1-3.019X2+16.464X3+106.046X4-0.739X5-0.535X6-1.158X7+5.333X8+0.837X9-0.899X10+5.151X11+0.555X12-28.523X13-1672.409＝1710.663
Second discrimination function FCN＝11.492X1-2.895X2+16.658X3+107.047X4-0.858X5–0.871X6-0.784X7+4.983X8+1.020X9-0.826X10+5.057X11-0.759X12-29.399X13-1685.324＝1709.786
Analyzed, determined and result output module compared FCCValue sum FCNValue, FCC>FCNAnd outputting a prediction result that the probability of the tested object suffering from the colon adenocarcinoma is more than or equal to 83.3 percent.
The examinee is subjected to laparoscopic colorectal cancer radical surgery after the examination, the diagnosis is confirmed by pathological examination to be differentiated adenocarcinoma in colon, 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 to predict the risk of colon adenocarcinoma in the subject
The detected object is: men, age 71, visit the colorectal anal surgery of the second hospital of Jilin university, sign informed consent for the purpose and use of the examination, and collect 1mL of anticoagulated blood via the peripheral vein, with the voluntary premise.
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＝268.14，L2＝268.14，L3＝404.16，L4＝404.16，L5＝228.62，L6＝228.62，L7＝388.87，L8＝388.87，L9＝317.83，L10＝324.59，L11＝241.56，L12＝260.17。
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]＝26；
X3＝round(L3-379)＝25；X4＝round(L4-379)＝25；
X5＝round[(L5-202)/2]＝13；X6＝round[(L6-202)/2]＝13；
X7＝round[(L7-359)/2]＝15；X8＝round[(L8-359)/2]＝15；
X9＝round[(L9-278)/2]＝20；X10＝round[(L10-278)/2]＝23；
X11＝round[(L11-200)/4]＝10；X12＝round[(L12-200)/4]＝15；
the patient is male, X13＝1。
Using a computer running the system for predicting colon adenocarcinoma susceptibility as described in example 1, a susceptibility prediction for a subject to suffer from colon 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 FCC＝11.227X1-3.019X2+16.464X3+106.046X4-0.739X5-0.535X6-1.158X7+5.333X8+0.837X9-0.899X10+5.151X11+0.555X12-28.523X13-1672.409＝1677.187
Second discrimination function FCN＝11.492X1-2.895X2+16.658X3+107.047X4-0.858X5–0.871X6-0.784X7+4.983X8+1.020X9-0.826X10+5.057X11-0.759X12-29.399X13-1685.324＝1682.519
Analyzed, determined and result output module compared FCCValue sum FCNValue, FCC≤FCNAnd outputting a prediction result that the probability that the detected object does not suffer from the colon adenocarcinoma is more than or equal to 86.1 percent.
The tested object is diagnosed as idiopathic ulcerative colitis after the visit, and the clinical diagnosis result of the tested object is consistent with the predicted 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> reagent kit and system for predicting susceptibility of colon adenocarcinoma
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ggtttccatt gtagcatctt g 21
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Claims (7)
1. A kit for colon adenocarcinoma susceptibility prediction 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 colon 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:
FCC＝11.227X1-3.019X2+16.464X3+106.046X4-0.739X5-0.535X6-1.158X7+5.333X8+0.837X9-0.899X10+5.151X11+0.555X12-28.523X13-1672.409
FCN＝11.492X1-2.895X2+16.658X3+107.047X4-0.858X5–0.871X6-0.784X7+4.983X8+1.020X9-0.826X10+5.057X11-0.759X12-29.399X13-1685.324
wherein, if the subject is female, X is13When the subject is male, X is 013＝1；
(4-4) prediction of colon adenocarcinoma susceptibility:
comparison FCCValue sum FCNValue if FCC>FCNPredicting the probability of the tested object suffering from the colon adenocarcinoma to be more than or equal to 83.3 percent; if FCC≤FCNAnd predicting that the probability that the tested object does not suffer from the colon adenocarcinoma is more than or equal to 86.1 percent.
2. The kit for predicting susceptibility to colon adenocarcinoma 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 kit for predicting susceptibility to colon adenocarcinoma 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 kit for predicting susceptibility to colon adenocarcinoma of claim 1, wherein: the sample is whole blood of a subject.
5. Use of the kit for colon adenocarcinoma susceptibility prediction according to any one of claims 1 to 4 for the preparation of a colon adenocarcinoma diagnostic product.
6. A system for predicting susceptibility to colon adenocarcinoma, 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 colon 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 FCC＝11.227X1-3.019X2+16.464X3+106.046X4-0.739X5-0.535X6-1.158X7+5.333X8+0.837X9-0.899X10+5.151X11+0.555X12-28.523X13-1672.409
Second discrimination function FCN＝11.492X1-2.895X2+16.658X3+107.047X4-0.858X5–0.871X6-0.784X7+4.983X8+1.020X9-0.826X10+5.057X11-0.759X12-29.399X13-1685.324
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 FCCAnd a second discrimination function FCNIf F is the result of the calculation ofCC>FCNOutputting a prediction result that the probability of the tested object suffering from the colon adenocarcinoma is more than or equal to 83.3 percent; if FCC≤FCNAnd outputting a prediction result that the probability that the detected object does not suffer from the colon adenocarcinoma is more than or equal to 86.1 percent.
7. Use of the colon adenocarcinoma susceptibility prediction system of claim 6 for the preparation of a colon adenocarcinoma prediction product or a colon adenocarcinoma diagnosis product.
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