CN108866189B - Kit and system for predicting susceptibility of squamous cell carcinoma of larynx - Google Patents
Kit and system for predicting susceptibility of squamous cell carcinoma of larynx Download PDFInfo
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Abstract
The invention relates to a kit for predicting susceptibility of squamous cell carcinoma of larynx, 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 kit for predicting the susceptibility of the laryngeal squamous cell carcinoma can be used for diagnosing the laryngeal squamous cell carcinoma and predicting the susceptibility. The invention also provides a system for predicting the susceptibility of the squamous cell carcinoma of larynx.
Description
Technical Field
The present invention relates to the field of biomedicine. In particular to a reagent kit for predicting the susceptibility of the laryngeal squamous cell carcinoma and a system for predicting the susceptibility of the laryngeal squamous cell carcinoma. More specifically, the invention relates to a kit for detecting STR of a gene related to laryngeal squamous cell carcinoma susceptibility by a Short Tandem Repeat (STR) locus fragment analysis method, and early warning is carried out on the laryngeal squamous cell carcinoma susceptibility of a detected object by combining 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.
Squamous cell carcinoma of the larynx is the most common malignant tumor of the larynx, and early symptoms are foreign body sensation at the back of the pharynx and swallowing infarction sensation. When the tumor is enlarged and the surface is ulcerated, swallowing pain can be caused, and ipsilateral reflex earache is caused, which is often accompanied by progressive dysphagia. If the tumor affects the laryngeal cavity, it causes dyspnea and hoarseness. The incidence of squamous cell carcinoma of larynx is influenced by certain life habits and other external factors, the incidence of male diseases is high, and the male diseases are related to the life habits of smoking, drinking and the like. Therefore, the method can predict the susceptibility of the laryngeal squamous cell carcinoma of the tested population, is favorable for improving the consciousness of the risk of the disease, and the prediction result shows that the population with higher prevalence rate of the laryngeal squamous cell carcinoma can reduce the prevalence rate or discover early treatment by abstaining from the living habits of smoking, drinking and the like.
A large number of studies have shown that genetic polymorphisms of tumor-associated genes play a key role in the development of malignant tumors. However, the development of tumors is a very complex process, and the diagnosis of the disease using changes in a single molecular genetic marker is clearly impossible and not scientific. In the prior art, accurate early warning on tumor susceptibility cannot be performed only through genetic information, and the current early identification and prediction method for tumors needs to be improved. The invention relates to a kit for early warning the susceptibility of laryngeal squamous cell carcinoma by jointly detecting a plurality of STR loci with high relevance to the occurrence of the laryngeal squamous cell carcinoma 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 the laryngeal squamous cell carcinoma by an STR locus fragment analysis method and early warning the susceptibility of the laryngeal squamous cell carcinoma 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 has no significant correlation with the suffering of the laryngeal squamous cell carcinoma of the detected object and the combination of the short tandem sequence repetition times of certain specific STR loci has close relation with the suffering of the laryngeal squamous cell carcinoma of the detected object by analyzing STRs of the genomic DNAs of the laryngeal squamous cell carcinoma detected object and a healthy control detected object and verifying the STRs in a plurality of laryngeal squamous cell carcinoma 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 squamous cell carcinoma of the larynx. 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 sites as references, the susceptibility of squamous cell carcinoma of the larynx 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 inventor surprisingly finds that the short tandem sequence repetition times of each STR locus are obtained by analyzing the cell genome of a detected object, and a statistical analysis method such as discriminant analysis is carried out by taking the repetition times as independent variables, so that early warning can be carried out on the susceptibility of the squamous cell carcinoma of larynx.
On the basis, one of the technical problems solved by the invention is to provide a kit for predicting the susceptibility of the squamous cell carcinoma of larynx, 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 laryngeal squamous cell carcinoma 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 the susceptibility to squamous cell carcinoma of larynx 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 shown in the following table 2, 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 the susceptibility of squamous cell carcinoma of larynx 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 the susceptibility of the squamous cell carcinoma of larynx of the invention, preferably, the LIZ-500 molecular weight internal standard can be preserved at-20 ℃;
in the kit for predicting the susceptibility of the laryngeal squamous cell carcinoma, the deionized formamide can be stored at 2-8 ℃.
Preferably, the kit for predicting the susceptibility to squamous cell carcinoma of larynx of the present invention further comprises instructions for use.
The instruction book describes a method for using the kit for predicting the susceptibility of the laryngeal squamous cell carcinoma, 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:
FLC＝10.750X1-8.272X2+14.088X3+106.284X4-0.418X5+2.056X6+2.288X7+
1.131X8-0.466X9-4.985X10+2.407X11+7.072X12-15.282X13-1550.869
FLN＝11.048X1-8.564X2+14.325X3+108.309X4-0.776X5+1.944X6+2.347X7+
0.795X8-0.807X9-4.929X10+2.523X11+6.583X12-18.780X13-1581.097
wherein, if the subject is female, X is13When the subject is male, X is 013＝1；
(4-4) prediction of laryngeal squamous cell carcinoma susceptibility:
comparison FLCValue sum FLNValue if FLC>FLNPredicting that the probability of the detected object suffering from the laryngeal squamous cell carcinoma is more than or equal to 82.4 percent; if FLC≤FLNAnd predicting that the probability that the detected object does not suffer from the laryngeal squamous cell carcinoma is more than or equal to 83.3 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, 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 the laryngeal squamous cell carcinoma is the sum of the probability of already suffering from the laryngeal squamous cell carcinoma and the probability of suffering from the laryngeal squamous cell carcinoma in the future. Therefore, the method can be used for diagnosing the squamous cell carcinoma of the larynx; the method can also be used for risk early warning of the future suffering from the laryngeal squamous cell carcinoma, can assist a detected object to carry out risk prevention, and reduces the suffering probability of the laryngeal squamous cell carcinoma through the modes of medicine conditioning, change of daily work and rest, diet rule, regular physical examination and the like.
The second technical problem solved by the invention is to provide a method for predicting the susceptibility of the squamous cell carcinoma of larynx, 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 laryngeal squamous cell carcinoma in preparing a laryngeal squamous cell carcinoma diagnosis product.
The fourth technical problem solved by the present invention is to provide a laryngeal squamous cell carcinoma 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 squamous cell carcinoma of the larynx and outputting 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 FLC＝10.750X1-8.272X2+14.088X3+106.284X4-0.418X5+2.056X6+2.288X7+1.131X8-0.466X9-4.985X10+2.407X11+7.072X12-15.282X13-1550.869
Second discrimination function FLN＝11.048X1-8.564X2+14.325X3+108.309X4-0.776X5+1.944X6+2.347X7+0.795X8-0.807X9-4.929X10+2.523X11+6.583X12-18.780X13-1581.097
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 FLCAnd a second discrimination function FLNIf F is the result of the calculation ofLC>FLNOutputting a prediction result that the probability of the detected object suffering from the laryngeal squamous cell carcinoma is more than or equal to 82.4%; if FLC≤FLNThen, a prediction result that "the probability that the subject does not suffer from laryngeal squamous cell carcinoma is not less than 83.3%" is output.
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 laryngeal squamous cell carcinoma susceptibility prediction system in preparing a laryngeal squamous cell carcinoma prediction product, a laryngeal squamous cell carcinoma diagnosis product and an oral cavity and throat health auxiliary product.
The sixth technical problem to be solved by the invention is to provide a laryngeal squamous cell carcinoma prediction product, a laryngeal squamous cell carcinoma diagnosis product, or an oral cavity and throat health auxiliary product, which comprises the laryngeal squamous cell carcinoma 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 laryngeal squamous cell carcinoma susceptibility.
Drawings
FIG. 1 is a schematic diagram of modules included in a data processing and determining device in the prediction system for the susceptibility of squamous cell carcinoma of larynx 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 laryngeal squamous cell carcinoma susceptibility prediction system
A system for predicting susceptibility to squamous cell carcinoma of the larynx 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 squamous cell carcinoma of the larynx and outputting 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 FLC＝10.750X1-8.272X2+14.088X3+106.284X4-0.418X5+2.056X6+2.288X7+1.131X8-0.466X9-4.985X10+2.407X11+7.072X12-15.282X13-1550.869
Second discrimination function FLN＝11.048X1-8.564X2+14.325X3+108.309X4-0.776X5+1.944X6+2.347X7+0.795X8-0.807X9-4.929X10+2.523X11+6.583X12-18.780X13-1581.097
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;
X10of short tandem sequences of the larger of the two alleles of STR-5The number of repetitions;
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 FLCAnd a second discrimination function FLNIf F is the result of the calculation ofLC>FLNOutputting a prediction result that the probability of the detected object suffering from the laryngeal squamous cell carcinoma is more than or equal to 82.4%; if FLC≤FLNThen, a prediction result that "the probability that the subject does not suffer from laryngeal squamous cell carcinoma is not less than 83.3%" is output.
Example 2 kit for predicting susceptibility to squamous cell carcinoma of larynx
A kit for predicting susceptibility to squamous cell carcinoma of larynx, 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 developing squamous cell carcinoma of the larynx of the subject
The detected object is: the male, 59 years old, visits the otorhinolaryngology department of the Mirabilis-friendship Hospital of Jilin university, signs an informed consent on the premise of fully informing the examination purpose and application, and collects 1mL of anticoagulation blood through the peripheral vein.
The following procedure was carried out using the kit of example 2 according to the method described in the kit instructions:
(1) extracting sample DNA: extracting blood genome DNA by using a DNA extraction kit;
(2) PCR reaction
(2-1) taking out the STR-1 primer, the STR-2 primer, the STR-3 primer, the STR-4 primer, the STR-5 primer, the STR-6 primer and the PCR amplification reaction solution from a refrigerator, balancing to room temperature, fully dissolving each component, and respectively and rapidly centrifuging for 10 seconds;
(2-2) adding 100ng of sample DNA into 60 mu L of PCR amplification reaction solution, adding deionized water to supplement to 115.2 mu L, fully and uniformly mixing, quickly centrifuging for 10 seconds, and subpackaging the mixed solution into 6 PCR reaction tubes according to 19.2 mu L/hole;
(2-3) respectively adding an STR-1 primer, an STR-2 primer, an STR-3 primer, an STR-4 primer, an STR-5 primer and an STR-6 primer into the 6 PCR reaction tubes in the step (2-2) according to 0.8 mu L/hole; covering a PCR reaction tube cover, recording the sample adding condition, quickly centrifuging for 10 seconds, then transferring the PCR reaction tube to a corresponding position of a sample groove of a PCR amplification instrument, recording the placing sequence, and starting the PCR amplification reaction; the amplification reaction conditions are as follows: 3 minutes at 95 ℃; 30 seconds at 95 ℃, 30 seconds at 60 ℃ and 30 seconds at 72 ℃ for 10 cycles; 30 seconds at 95 ℃, 30 seconds at 55 ℃, 30 seconds at 72 ℃ and 20 cycles; 6 groups of PCR amplification products are obtained at 72 ℃ for 6 minutes;
(3) STR fragment analysis
(3-1) adding 990 mu L of deionized formamide into 10 mu L of LIZ-500 molecular weight internal standard, fully and uniformly mixing, quickly centrifuging for 10 seconds, respectively adding into a sequencing reaction tube according to 10 mu L/hole, and quickly centrifuging for 10 seconds;
(3-2) adding the 6 groups of PCR amplification products into 6 sequencing reaction tubes according to 1 mu L/hole respectively, and quickly centrifuging for 10 seconds; then transferring the sequencing reaction tube to a corresponding position of a sample tank of a PCR (polymerase chain reaction) amplification instrument, heating at 98 ℃ for 5 minutes, immediately placing the sequencing reaction tube on an ice-water mixture after the program is finished, rapidly cooling to 0 ℃, and rapidly centrifuging for 10 seconds; then transferring the sequencing reaction tube to a corresponding position of a sample groove of an STR locus fragment analyzer, recording the placement sequence, and performing fragment analysis detection;
(4) analysis and determination of results
(4-1) respectively recording the fragment lengths of two alleles at each site of STR-1, STR-2, STR-3, STR-4, STR-5 and STR-6 according to the fragment analysis result: the length of the smaller of the two STR-1 alleles is recorded as L1And the length of the larger fragment of the two STR-1 alleles is designated as L2(ii) a The length of the smaller fragment of the two STR-2 alleles is recorded as L3And the length of the larger fragment of the two STR-2 alleles is designated as L4(ii) a The length of the smaller fragment of the two STR-3 alleles is recorded as L5And the length of the larger fragment of the two STR-3 alleles is marked as L6(ii) a The length of the smaller fragment of the two STR-4 alleles is recorded as L7And the length of the larger fragment of the two STR-4 alleles is marked as L8(ii) a The length of the smaller of the two STR-5 alleles is recorded as L9And the length of the larger fragment of the two STR-5 alleles is designated as L10(ii) a The length of the smaller fragment of the two STR-6 alleles was designated L11And the length of the larger fragment of the two STR-6 alleles is marked as L12(ii) a The results show that: l is1＝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。
(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]＝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。
(4-3) prediction of susceptibility to laryngeal squamous cell carcinoma in a subject using a computer running the system for predicting susceptibility to laryngeal squamous cell carcinoma 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 FLC＝10.750X1-8.272X2+14.088X3+106.284X4-0.418X5+2.056X6+2.288X7+1.131X8-0.466X9-4.985X10+2.407X11+7.072X12-15.282X13-1550.869＝1386.718
Second discrimination function FLN＝11.048X1-8.564X2+14.325X3+108.309X4-0.776X5+1.944X6+2.347X7+0.795X8-0.807X9-4.929X10+2.523X11+6.583X12-18.780X13-1581.097＝1381.062
Analyzed, determined and result output module compared FLCValue sum FLNValue, FLC>FLNAnd outputting a prediction result that the probability of the detected object to suffer from the laryngeal squamous cell carcinoma is more than or equal to 82.4 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 developing squamous cell carcinoma of the larynx of the subject
The detected object is: female, 72 years old, visit the otorhinolaryngology department of the Mirabilis-friendship Hospital, Jilin university, with sufficient informed examination purpose and use, under the voluntary premise, sign informed consent, and collect 1mL of anticoagulation blood via peripheral vein.
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＝268.17，L2＝268.17，L3＝404.17，L4＝404.17，L5＝228.76，L6＝228.76，L7＝386.85，L8＝390.67，L9＝311.75，L10＝311.75，L11＝260.20，L12＝263.85。
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]＝14；X8＝round[(L8-359)/2]＝16；
X9＝round[(L9-278)/2]＝17；X10＝round[(L10-278)/2]＝17；
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 laryngeal squamous cell carcinoma described in example 1, a prediction of susceptibility of a subject to suffer from laryngeal squamous cell carcinoma is made:
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 FLC＝10.750X1-8.272X2+14.088X3+106.284X4-0.418X5+2.056X6+2.288X7+1.131X8-0.466X9-4.985X10+2.407X11+7.072X12-15.282X13-1550.869＝1650.871
Second discrimination function FLN＝11.048X1-8.564X2+14.325X3+108.309X4-0.776X5+1.944X6+2.347X7+0.795X8-0.807X9-4.929X10+2.523X11+6.583X12-18.780X13-1581.097＝1655.76
Analyzed, determined and result output module compared FLCValue sum FLNValue, FLC≤FLNThe result of prediction that "the subject does not suffer from laryngeal squamous cell carcinoma at a probability of 83.3%" is output.
The subject is diagnosed as vocal cord polyp after treatment, and the clinical diagnosis result of the subject 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
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Claims (7)
1. A kit for predicting susceptibility to squamous cell carcinoma of larynx, 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 instruction book describes a using method of the reagent kit for predicting the susceptibility of the laryngeal squamous cell carcinoma, 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:
FLC＝10.750X1-8.272X2+14.088X3+106.284X4-0.418X5+2.056X6+2.288X7+1.131X8-0.466X9-4.985X10+2.407X11+7.072X12-15.282X13-1550.869
FLN＝11.048X1-8.564X2+14.325X3+108.309X4-0.776X5+1.944X6+2.347X7+0.795X8-0.807X9-4.929X10+2.523X11+6.583X12-18.780X13-1581.097
wherein, if the subject is female, X is13When the subject is male, X is 013＝1；
(4-4) prediction of laryngeal squamous cell carcinoma susceptibility:
comparison FLCValue sum FLNValue if FLC>FLNPredicting that the probability of the detected object suffering from the laryngeal squamous cell carcinoma is more than or equal to 82.4 percent; if FLC≤FLNAnd predicting that the probability that the detected object does not suffer from the laryngeal squamous cell carcinoma is more than or equal to 83.3 percent.
2. The laryngeal squamous cell carcinoma susceptibility prediction kit of claim 1, wherein: the using concentration of the STR-1 primer, the STR-2 primer, the STR-3 primer, the STR-4 primer, the STR-5 primer and the STR-6 primer is 10 mu M.
3. The laryngeal squamous cell carcinoma susceptibility prediction kit of claim 1, wherein: the PCR amplification reaction solution is a mixed solution of the following reagents: TaqDNA polymerase 5U/. mu. L, Tris-HCl 100mM, KCl 500mM, ethylphenylpolyethylene glycol 0.8 vol%, MgCl225mM, dNTP 10mM and deionized water; wherein Tris-HCl has a pH of 8.8 at 25 ℃.
4. The laryngeal squamous cell carcinoma susceptibility prediction kit of claim 1, wherein: the sample is whole blood, buccal swab or buccal tissue of a subject.
5. Use of the laryngeal squamous cell carcinoma susceptibility pre-test kit of any one of claims 1 to 4 for preparing a laryngeal squamous cell carcinoma diagnostic product.
6. A system for predicting susceptibility to squamous cell carcinoma of the larynx, 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 make a prediction of the susceptibility of the squamous cell carcinoma of larynx and outputting the result,
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 FLC＝10.750X1-8.272X2+14.088X3+106.284X4-0.418X5+2.056X6+2.288X7+1.131X8-0.466X9-4.985X10+2.407X11+7.072X12-15.282X13-1550.869
Second discrimination function FLN＝11.048X1-8.564X2+14.325X3+108.309X4-0.776X5+1.944X6+2.347X7+0.795X8-0.807X9-4.929X10+2.523X11+6.583X12-18.780X13-1581.097
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;
X7short tandem of the smaller of the two alleles of STR-4The number of repetitions of the sequence;
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 of the two alleles of STR-2,L4is 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 FLCAnd a second discrimination function FLNIf F is the result of the calculation ofLC>FLNOutputting a prediction result that the probability of the detected object suffering from the laryngeal squamous cell carcinoma is more than or equal to 82.4%; if FLC≤FLNThen, a prediction result that "the probability that the subject does not suffer from laryngeal squamous cell carcinoma is not less than 83.3%" is output.
7. Use of the laryngeal squamous cell carcinoma susceptibility prediction system of claim 6 for the preparation of a laryngeal squamous cell carcinoma prediction product or a laryngeal squamous cell carcinoma diagnostic product.
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Citations (1)
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Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1313130A (en) * | 2001-03-23 | 2001-09-19 | 黄石 | Application of RIZ gene in detecting and treating tumor showing MSI positive |
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| US20170107577A1 (en) * | 2014-03-11 | 2017-04-20 | The Council Of The Queensland Institute Of Medical Research | Determining Cancer Aggressiveness, Prognosis and Responsiveness to Treatment |
| WO2015149034A2 (en) * | 2014-03-27 | 2015-10-01 | Life Technologies Corporation | Gene fusions and gene variants associated with cancer |
| CN106011067A (en) * | 2016-07-22 | 2016-10-12 | 浙江省肿瘤医院 | Esophageal cancer cell line and application thereof |
-
2018
- 2018-07-12 CN CN201810765727.7A patent/CN108866189B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW201243331A (en) * | 2011-03-18 | 2012-11-01 | Baylor Res Inst | Line-1 hypomethylation as a biomarker for early-onset colorectal cancer |
Non-Patent Citations (4)
| Title |
|---|
| Androgen Receptor in Laryngeal Carcinoma: Could There Be an Androgen-Refractory Tumor?;Anastasios K Goulioumis等;《ISRN Oncol》;20111129;第2011卷;第1-5页 * |
| Fisher linear discriminant analysis for classification and prediction of genomic susceptibility to stomach and colorectal cancers based on six STR loci in a northern Chinese Han population;Shuhong Hao等;《PeerJ》;20190528;第7卷;e7004 * |
| Microsatellite and chromosome instability in squamous cell laryngeal carcinoma;M Sasiadek等;《Int J Oncol》;20010831;第19卷(第2期);第401-405页 * |
| 喉鳞状细胞癌细胞系的建立及其生物学特性研究;王茹等;《中华耳鼻咽喉头颈外科杂志》;20170131;第52卷(第1期);第44-48页 * |
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