CN108841959B - Kit and system for predicting susceptibility of oral cavity and head and neck malignant tumors - Google Patents
Kit and system for predicting susceptibility of oral cavity and head and neck malignant tumors Download PDFInfo
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- CN108841959B CN108841959B CN201810765719.2A CN201810765719A CN108841959B CN 108841959 B CN108841959 B CN 108841959B CN 201810765719 A CN201810765719 A CN 201810765719A CN 108841959 B CN108841959 B CN 108841959B
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Abstract
The invention relates to a kit for predicting susceptibility of oral cavity and head and neck malignant tumors, 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 still includes: PCR amplification reaction liquid, LIZ-500 molecular weight internal standard and deionized formamide. The kit for predicting susceptibility of the oral cavity and the head and neck malignant tumors can be used for diagnosing and predicting susceptibility of the oral cavity and the head and neck malignant tumors. The invention also provides a system for predicting the susceptibility of the malignant tumors of the oral cavity and the head and neck.
Description
Technical Field
The present invention relates to the field of biomedicine. In particular to a susceptibility prediction kit and a susceptibility prediction system for oral cavity and head and neck malignant tumors. More specifically, the invention relates to a kit for detecting STR of oral cavity and head and neck malignant tumor susceptibility related genes by Short Tandem Repeat (STR) site fragment analysis method, and early warning of oral cavity and head and neck malignant tumor susceptibility of a detected object can be performed by combining with a discriminant analysis statistical method, and the tumor benign and malignant of oral cavity and head and neck tumor patients can also be identified.
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.
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 benign and malignant tumors needs to be improved.
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 oral cavity and head and neck malignant tumors by an STR locus fragment analysis method, and early warning on susceptibility of the oral cavity and the head and neck malignant tumors by combining a discriminant analysis statistical method.
The present invention has been completed based on the following findings of the inventors: the inventor discovers that the repetition times of short tandem sequences of each independent STR locus has no significant correlation with the oral cavity and head and neck malignant tumor of the detected object, and the combination of the repetition times of the short tandem sequences of certain specific STR loci has close relation with the oral cavity and head and neck malignant tumor of the detected object by analyzing STRs of the oral cavity and head and neck malignant tumor detected objects and healthy control detected objects and verifying a large number of oral cavity and head and neck malignant tumor samples and control samples.
To this end, the present invention proposes a set of isolated STR sites that have a high association with the development of oral and head and neck malignancies. 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 the separated STR loci as reference, the susceptibility of oral cavity and head and neck malignant tumors can be effectively predicted, or the benign and malignant tumors of the oral cavity and the head and neck can be identified.
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 have surprisingly found that by analyzing the cell genome of the subject to obtain the number of times of repeat of the short tandem sequence at each STR locus and performing a statistical analysis such as discriminant analysis using the number of times of repeat as an argument, early warning of susceptibility to oral and head and neck malignant tumors and identification of benign and malignant tumors in oral and head and neck can be achieved.
On the basis, one of the technical problems solved by the invention is to provide a kit for predicting susceptibility to oral and head and neck malignant tumors, 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 oral cavity and head and neck malignant tumor 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 susceptibility to oral and head and neck malignant tumors 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 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 oral and head and neck malignant tumors 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 oral and head and neck malignant tumors of the invention, preferably, the LIZ-500 molecular weight internal standard can be stored at-20 ℃;
in the kit for predicting susceptibility to oral and head-neck malignant tumors of the present invention, preferably, the deionized formamide can be stored at 2-8 ℃.
Preferably, the kit for predicting susceptibility to oral and head-neck malignant tumors of the present invention further comprises instructions for use.
The application instruction describes a use method of the kit for predicting susceptibility to oral and head and neck malignant tumors, 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:
FHNC＝8.338X1-5.839X2+9.039X3+82.475X4+1.717X5+3.121X6+0.525X7+4.394X8+1.202X9-5.911X10+2.864X11+7.707X12-2.238X13-1262.984
FHNN＝8.432X1-5.903X2+8.883X3+83.361X4+1.452X5+3.054X6+0.502X7+4.228X8+1.046X9-5.766X10+2.850X11+7.293X12-4.865X13-1265.756
wherein, if the subject is female, X is13When the subject is male, X is 013＝1；
(4-4) prediction of susceptibility to oral and head and neck malignancies:
comparison FHNCValue sum FHNNValue if FHNC>FHNNPredicting the probability of the tested object suffering from oral cavity and head and neck malignant tumor is more than or equal to 75.0%; if FHNC≤FHNNAnd predicting that the probability that the detected object does not suffer from oral cavity and head and neck malignant tumors is more than or equal to 75.0%.
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, buccal swab or buccal tissue of a subject, preferably 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 oral cavity and head and neck malignant tumors is the sum of the probability of suffering from oral cavity and head and neck malignant tumors and the probability of suffering from oral cavity and head and neck malignant tumors in the future. Therefore, the kit can be used for diagnosing malignant tumors of oral cavity and head and neck; the system can also be used for risk early warning of malignant tumors of oral cavities and head and neck parts in the future, can assist a detected object to carry out risk prevention, and reduces the disease probability of the malignant tumors of the oral cavities and the head and neck parts through the modes of medicine conditioning, life and rest change, eating rules, regular physical examination and the like.
The second technical problem to be solved by the invention is to provide a method for predicting susceptibility to oral and head-neck malignant tumors, namely, the method is operated by using the kit according to the instruction.
The invention solves the third technical problem by providing the application of the oral cavity and head and neck malignant tumor susceptibility pre-test kit in preparing oral cavity and head and neck malignant tumor diagnosis products.
The fourth technical problem to be solved by the present invention is to provide a system for predicting susceptibility to oral and head and neck malignant tumors, 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:
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 oral cavity and the head and neck malignant tumors and output the results.
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 FHNC＝8.338X1-5.839X2+9.039X3+82.475X4+1.717X5+3.121X6+0.525X7+4.394X8+1.202X9-5.911X10+2.864X11+7.707X12-2.238X13-1262.984
Second discrimination function FHNN＝8.432X1-5.903X2+8.883X3+83.361X4+1.452X5+3.054X6+0.502X7+4.228X8+1.046X9-5.766X10+2.850X11+7.293X12-4.865X13-1265.756
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 outputAn output module for outputting the first discriminant function FHNCAnd a second discrimination function FHNNIf F is the result of the calculation ofHNC>FHNNOutputting a prediction result that the probability of the examined object suffering from the malignant tumors of the oral cavity and the head and neck is more than or equal to 75.0%; if FHNC≤FHNNAnd outputting a prediction result that the probability that the detected object does not suffer from oral cavity and head and neck malignant tumors is more than or equal to 75.0 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 oral cavity and head and neck malignant tumor susceptibility prediction system in the preparation of oral cavity and head and neck malignant tumor prediction products, oral cavity and head and neck malignant tumor diagnosis products and oral cavity and head and neck health auxiliary products.
The sixth technical problem to be solved by the invention is to provide a product for predicting oral cavity and head and neck malignant tumors, a product for diagnosing oral cavity and head and neck malignant tumors, or an auxiliary product for oral cavity and head and neck health, which comprises the system for predicting susceptibility of oral cavity and head and neck malignant tumors.
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 susceptibility of oral and head and neck malignant tumors and identifying the oral and head and neck malignant tumors.
Drawings
Fig. 1 is a schematic diagram of modules included in a data processing and determining device in the oral cavity and head and neck malignant tumor susceptibility prediction system according to 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 system for predicting susceptibility to oral and head and neck malignancies
A system for predicting susceptibility to oral and head-neck malignancies 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 oral cavity and the head and neck malignant tumors and output the results.
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:
the discriminant function includes:
first discriminant function FHNC＝8.338X1-5.839X2+9.039X3+82.475X4+1.717X5+3.121X6+0.525X7+4.394X8+1.202X9-5.911X10+2.864X11+7.707X12-2.238X13-1262.984
Second discrimination function FHNN＝8.432X1-5.903X2+8.883X3+83.361X4+1.452X5+3.054X6+0.502X7+4.228X8+1.046X9-5.766X10+2.850X11+7.293X12-4.865X13-1265.756
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 FHNCAnd a second discrimination function FHNNIf F is the result of the calculation ofHNC>FHNNThen, the pre-treatment that the probability of the tested object suffering from the malignant tumor of the oral cavity and the head and neck is more than or equal to 75.0 percent is outputMeasuring a result; if FHNC≤FHNNAnd outputting a prediction result that the probability that the detected object does not suffer from oral cavity and head and neck malignant tumors is more than or equal to 75.0 percent.
Example 2 kit for predicting susceptibility to oral and head and neck malignant tumors
A kit for predicting susceptibility to oral and head and neck malignant tumors comprises 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 oral and head and neck malignancies in the subject
The detected object is: a male, 64 years old, visits the ear-nose-throat department of the Mirabilis-friendship Hospital of Jilin university, signs an informed consent on the premise of fully informing the examination purpose and the purpose, and collects 1mL of anticoagulation blood through peripheral veins.
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 resultDegree: 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＝268.23，L2＝301.26，L3＝404.18，L4＝404.18，L5＝229.04，L6＝246.20，L7＝404.57，L8＝404.57，L9＝312.08，L10＝324.94，L11＝260.53，L12＝264.09。
(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]＝26；X2＝round[(L2-191)/3]＝37；
X3＝round(L3-379)＝25；X4＝round(L4-379)＝25；
X5＝round[(L5-202)/2]＝14；X6＝round[(L6-202)/2]＝22；
X7＝round[(L7-359)/2]＝23；X8＝round[(L8-359)/2]＝23；
X9＝round[(L9-278)/2]＝17；X10＝round[(L10-278)/2]＝23；
X11＝round[(L11-200)/4]＝15；X12＝round[(L12-200)/4]＝16；
the patient is male, X13＝1。
Using a computer running the system for predicting susceptibility to oral and head and neck malignancies described in example 1, a subject is predicted to have a susceptibility to oral and head and neck malignancies:
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 FHNC＝8.338X1-5.839X2+9.039X3+82.475X4+1.717X5+3.121X6+0.525X7+4.394X8+1.202X9-5.911X10+2.864X11+7.707X12-2.238X13-1262.984＝1279.963
Second discrimination function FHNN＝8.432X1-5.903X2+8.883X3+83.361X4+1.452X5+3.054X6+0.502X7+4.228X8+1.046X9-5.766X10+2.850X11+7.293X12-4.865X13-1265.756＝1277.208
Analyzed, determined and result output module compared FHNCValue sum FHNNValue, FHNC>FHNNAnd outputting a prediction result that the probability of the tested object suffering from oral cavity and head and neck malignant tumors is more than or equal to 75.0 percent.
The examined object performs throat tumor tissue biopsy after the examination, the pathological examination confirms that the examined object is the laryngeal squamous cell carcinoma, and the clinical diagnosis result of the examined object 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 oral and head and neck malignancies in the subject
The detected object is: female, age 62, visiting the otorhinolaryngology department of the Mirabilis-friendship Hospital, Jilin university, signed an informed consent on the premise of his own accord, and collected 1mL of anticoagulated blood via the peripheral vein, with full informed examination purpose and use.
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 system for predicting susceptibility to oral and head and neck malignancies described in example 1, a subject is predicted to have a susceptibility to oral and head and neck malignancies:
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 FHNC＝8.338X1-5.839X2+9.039X3+82.475X4+1.717X5+3.121X6+0.525X7+4.394X8+1.202X9-5.911X10+2.864X11+7.707X12-2.238X13-1262.984＝1160.663
Second discrimination function FHNN＝8.432X1-5.903X2+8.883X3+83.361X4+1.452X5+3.054X6+0.502X7+4.228X8+1.046X9-5.766X10+2.850X11+7.293X12-4.865X13-1265.756＝1163.504
Analyzed, determined and result output module compared FHNCValue sum FHNNValue, FHNC≤FHNNAnd outputting a prediction result that the probability that the detected object does not suffer from oral cavity and head and neck malignant tumors is more than or equal to 75.0 percent.
The tested object is diagnosed as chronic pharyngitis after the visit, and the clinical diagnosis result of the tested object is consistent with the prediction result of the kit.
Example 5 Using the System of example 1 and the kit of example 2 to predict the risk of oral and head and neck malignancies in the subject
The detected object is: for men, at age 39, visit department of the department of stomatology in the Mitsuga-Riidng Hospital, Jilin university, perform biopsy on swollen matter in the tongue, sign an informed consent on the premise of fully informing the purpose and application of examination, and collect 1mL of anticoagulation blood through peripheral veins.
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＝260.25，L2＝260.25，L3＝402.1，L4＝402.1，L5＝229.03，L6＝246.15，L7＝386.85，L8＝400.5，L9＝312.16，L10＝318.59，L11＝256.99，L12＝260.51。
Calculated according to the fragment length and the following formula, denoted X1-X12Where round stands for rounded integer:
X1＝round[(L1-191)/3]＝23；X2＝round[(L2-191)/3]＝23；
X3＝round(L3-379)＝23；X4＝round(L4-379)＝23；
X5＝round[(L5-202)/2]＝14；X6＝round[(L6-202)/2]＝22；
X7＝round[(L7-359)/2]＝14；X8＝round[(L8-359)/2]＝21；
X9＝round[(L9-278)/2]＝17；X10＝round[(L10-278)/2]＝20；
X11＝round[(L11-200)/4]＝14；X12＝round[(L12-200)/4]＝15；
the patient is male, X13＝1。
Using a computer running the system for predicting susceptibility to oral and head and neck malignancies described in example 1, a subject is predicted to have a susceptibility to oral and head and neck malignancies:
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 FHNC＝8.338X1-5.839X2+9.039X3+82.475X4+1.717X5+3.121X6+0.525X7+4.394X8+1.202X9-5.911X10+2.864X11+7.707X12-2.238X13-1262.984＝1147.316
Second discrimination function FHNN＝8.432X1-5.903X2+8.883X3+83.361X4+1.452X5+3.054X6+0.502X7+4.228X8+1.046X9-5.766X10+2.850X11+7.293X12-4.865X13-1265.756＝1144.247
Analyzed, determined and result output module compared FHNCValue sum FHNNValue, FHNC>FHNNAnd outputting a prediction result that the probability of the tested object suffering from oral cavity and head and neck malignant tumors is more than or equal to 75.0 percent.
The pathological examination of the examined object confirms that the examined object is tongue squamous cell carcinoma, and the clinical diagnosis result of the examined object is consistent with the prediction result of the kit.
Example 6 Using the System of example 1 and the kit of example 2 to predict the risk of oral and head and neck malignancies in the subject
The detected object is: female, age 48, visiting otorhinolaryngology department of the Mirabilis-friendship Hospital, Jilin university, performing a throat-tumor tissue biopsy, signing an informed consent on the premise of fully informing the purpose and the purpose of examination, and collecting 1mL of anticoagulation blood through peripheral veins.
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＝260.10，L2＝260.10，L3＝404.22，L4＝404.22，L5＝228.59，L6＝228.59，L7＝385.03，L8＝388.90，L9＝311.78，L10＝324.52，L11＝259.24，L12＝263.80。
Calculated according to the fragment length and the following formula, denoted X1-X12Where round stands for rounded integer:
X1＝round[(L1-191)/3]＝23；X2＝round[(L2-191)/3]＝23；
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]＝13；X8＝round[(L8-359)/2]＝15；
X9＝round[(L9-278)/2]＝17；X10＝round[(L10-278)/2]＝23；
X11＝round[(L11-200)/4]＝15；X12＝round[(L12-200)/4]＝16；
the patient is female, X13＝0。
Using a computer running the system for predicting susceptibility to oral and head and neck malignancies described in example 1, a subject is predicted to have a susceptibility to oral and head and neck malignancies:
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 FHNC＝8.338X1-5.839X2+9.039X3+82.475X4+1.717X5+3.121X6+0.525X7+4.394X8+1.202X9-5.911X10+2.864X11+7.707X12-2.238X13-1262.984＝1268.725
Second discrimination function FHNN＝8.432X1-5.903X2+8.883X3+83.361X4+1.452X5+3.054X6+0.502X7+4.228X8+1.046X9-5.766X10+2.850X11+7.293X12-4.865X13-1265.756＝1271.637
Analyzed, determined and result output module compared FHNCValue sum FHNNValue, FHNC≤FHNNAnd outputting a prediction result that the probability that the detected object does not suffer from oral cavity and head and neck malignant tumors is more than or equal to 75.0 percent.
The pathological examination of the detected object confirms that the detected object is the laryngeal fibroma and is a benign tumor, and the clinical diagnosis result of the detected object 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> kit and system for predicting susceptibility of oral cavity and head and neck malignant tumors
<130> DI18-8161-XC47
<160> 12
<170> PatentIn version 3.3
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<212> DNA
<213> Artificial
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<221> primer_bind
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Claims (7)
1. A kit for predicting susceptibility to oral and head and neck malignant tumors 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 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 use method of the kit for predicting susceptibility to oral and head and neck malignant tumors, 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:
FHNC＝8.338X1-5.839X2+9.039X3+82.475X4+1.717X5+3.121X6+0.525X7+4.394X8+1.202X9-5.911X10+2.864X11+7.707X12-2.238X13-1262.984
FHNN＝8.432X1-5.903X2+8.883X3+83.361X4+1.452X5+3.054X6+0.502X7+4.228X8+1.046X9-5.766X10+2.850X11+7.293X12-4.865X13-1265.756
wherein, if the subject is female, X is13When the subject is male, X is 013＝1；
(4-4) prediction of susceptibility to oral and head and neck malignancies:
comparison FHNCValue sum FHNNValue if FHNC>FHNNPredicting the probability of the tested object suffering from oral cavity and head and neck malignant tumor is more than or equal to 75.0%; if FHNC≤FHNNAnd predicting that the probability that the detected object does not suffer from oral cavity and head and neck malignant tumors is more than or equal to 75.0%.
2. The kit for predicting susceptibility to oral and head and neck malignancies 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 method according to claim 1, wherein the susceptibility to oral and head neck malignancies is pre-determinedThe test kit is characterized in that: 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 oral and head and neck malignancies of claim 1, wherein: the sample is whole blood, buccal swab or buccal tissue of a subject.
5. Use of the oral cavity and head and neck malignant tumor susceptibility pre-test kit according to any one of claims 1 to 4 in the preparation of an oral cavity and head and neck malignant tumor diagnosis product.
6. A system for predicting susceptibility to oral and head-neck malignancies, 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;
an analysis, discrimination and result output module for comparing the discrimination function results to predict susceptibility to oral and head neck malignant tumors and outputting the results,
wherein the number of times X of repetition of STR site short tandem sequences of sample DNA obtained using the kit of any one of claims 1 to 51-X12(ii) a And
wherein:
the discriminant function includes:
first discriminant function FHNC＝8.338X1-5.839X2+9.039X3+82.475X4+1.717X5+3.121X6+0.525X7+4.394X8+1.202X9-5.911X10+2.864X11+7.707X12-2.238X13-1262.984
Second discrimination function FHNN＝8.432X1-5.903X2+8.883X3+83.361X4+1.452X5+3.054X6+0.502X7+4.228X8+1.046X9-5.766X10+2.850X11+7.293X12-4.865X13-1265.756
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;
X6is the larger of the two alleles of STR-3The number of repetitions of the short tandem sequence of segments;
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 STR-1 two, etcA larger fragment length value in the allele;
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 FHNCAnd a second discrimination function FHNNIf F is the result of the calculation ofHNC>FHNNOutputting a prediction result that the probability of the examined object suffering from the malignant tumors of the oral cavity and the head and neck is more than or equal to 75.0%; if FHNC≤FHNNAnd outputting a prediction result that the probability that the detected object does not suffer from oral cavity and head and neck malignant tumors is more than or equal to 75.0 percent.
7. The use of the system for predicting susceptibility to oral and head and neck malignancies of claim 6 in the manufacture of a product for predicting oral and head and neck malignancies or a product for diagnosing oral and head and neck malignancies.
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