CN107746882A - A kind of fluorescence quantitative PCR detection system and its application for osteosarcoma gene screening - Google Patents

Abstract

The present invention relates to technical field of biological, especially, is related to a kind of fluorescent quantificationally PCR detecting kit, and further discloses its application for being used to detect osteosarcoma.Fluorescence quantitative PCR detection system of the present invention, including the detection kit to specific site gene, and the score of osteosarcoma risk is calculated to the value-at-risk of osteosarcoma risk according to each site, and carry out crowd's classification is gone according to existing database, the osteosarcoma risk class of individual is obtained by personal score, so as to assess the osteosarcoma risk of person under inspection, guiding excessive risk person makes the life better mode, and avoids risk factor and increase physical examination frequency.At the same time, the present invention is laid a good foundation by establishing Chinese population database to 5 selected gene polymorphism sites to establish the distinctive osteosarcoma assessment of Chinese population.

Description

Fluorescent quantitative PCR detection system for osteosarcoma gene screening and application thereof

Technical Field

The invention relates to the technical field of biological detection, in particular to a fluorescent quantitative PCR detection kit, and further discloses application of the kit in detection of osteosarcoma.

Background

With the continuous development of scientific research, particularly the complete expansion of the human genome project, the application of the latest technology taking gene detection as the leading factor in the field of disease prevention and control is widely concerned. At present, the disease risk prediction by using the theory and method of genetics has already been developed primarily, and the commonly used gene detection method comprises the following steps: direct Sequencing (DS), ligase Detection Reaction (LDR), restriction Fragment Length Polymorphism (RFLP) analysis, denaturing High Performance Liquid Chromatography (DHPLC), and quantitative PCR detection, where direct sequencing with a DNA analyzer is the gold standard, but the method has many problems such as high cost, low accuracy, cumbersome operation, and poor reproducibility.

A Real-time Quantitative PCR detection System (qPCR), also known as a Real-time Quantitative gene amplification (FIA) and a Real-time Quantitative PCR detection technology, is a method for monitoring the whole PCR process in Real time by adding a fluorescent group into a PCR reaction System and utilizing fluorescent signal accumulation, and finally carrying out Quantitative analysis on an unknown template through a standard curve. The qPCR system is a third-generation PCR detection technology, has the outstanding advantages of high detection sensitivity, wide detection linear range, good detection precision and repeatability and the like, is known as the most advanced nucleic acid molecular diagnosis technology for clinical use in the world at present, and can be used for the detection of the motor gene efficiently and accurately.

Single Nucleotide Polymorphisms (SNPs) refer to Single Nucleotide variations in the genome, including transitions, transversions, deletions and insertions, which form genetic markers, which are abundant in number and polymorphism. SNP is a third generation genetic marker, and differences of a plurality of phenotypes of human bodies, susceptibility to drugs or diseases and the like can be related to SNP, so SNP research is an important step of the application of the human genome project. This is mainly because SNP will provide a powerful tool for the discovery of high-risk groups, identification of disease-related genes, drug design and testing, and basic research in biology.

SNPs are quite widely distributed in the human genome, averaging 1 in every 500-1000 base pairs, and estimated to amount to 300 thousands, with even more studies indicating that they occur every 300 base pairs in the human genome. The large number of SNP sites gives people an opportunity to discover various diseases including tumor-associated genomic mutations, and from the viewpoint of experimental operation, discovery of disease-associated genetic mutations by SNPs is easier than that by family studies. Although some SNPs do not directly cause expression of disease genes, they are also important markers because they are adjacent to some disease genes. SNP also plays a great role in basic research, and a series of important achievements are obtained in the fields of human evolution, human population evolution and migration through the analysis of Y chromosome SNP.

More and more documents are available to explain the fluorescent quantitative PCR technology from the application aspect of all aspects, most of the documents disclose specific methods for detecting multiple single fragments by the fluorescent quantitative PCR technology, but in the process of detecting SNP sites by the inventor of fluorescent quantitative PCR, the detection of the single fragment is not enough to meet the detection requirement, and for comprehensive detection with two or more SNP sites, repeated detection is needed, the steps are complicated, and the operation time is long.

Osteosarcoma is a common bone malignant tumor of non-hematogenous origin in adults and children, accounts for about 20% of primary malignant bone tumor, and is mostly originated from mesenchymal tissue. Osteosarcoma typically occurs well at the metaphysis of long tubular bones and is often poorly prognostic, highly malignant, with a high likelihood of lung metastasis in the early phase, with metastases found in about 10-20% of patients diagnosed, and only about 10% of patients with a prolonged period of asymptomatic phase following treatment. Osteosarcoma is typically characterized by osteoid material that is prone to malignant cell generation, and there have been numerous reports of genetic and epigenetic changes associated with osteosarcoma, but no consensus has been achieved.

With the growing knowledge of stem cell biology and cancer stem cell related knowledge, there is growing evidence that osteosarcoma may be a disease associated with stem cell differentiation, and that the process of osteoblast formation from the progressive differentiation of pluripotent stem cells to continue differentiation into osteocytes is a process that is closely regulated by a series of signaling pathways. Many changes at the molecular level strongly suggest that genetic and epigenetic factors interfere with this differentiation process, which in turn induces osteosarcoma. Although current treatments for osteosarcoma are mainly limited to inhibiting the proliferation of osteosarcoma, promoting differentiation and/or circumventing the differentiation disorder is likely to be an effective adjuvant treatment.

However, the molecular mechanism of osteosarcoma generation is not elucidated, and the abnormal expression of some genes can increase the susceptibility of osteosarcoma, for example, patients with hereditary retinoblastoma and patients with Paget's disease are good groups of osteosarcoma. Cytogenetic studies of osteosarcoma have shown that osteosarcoma has a variety of genetic alterations, resulting in the inactivation of a variety of tumor suppressor genes and overexpression of oncogenes. Among them, the RB gene, P53 gene and Kras gene abnormalities are closely related to the development of osteosarcoma. And the existing data also indicate that the osteosarcoma has a large number of genetic and molecular level changes, including the inactivation of tumor suppressor genes caused by chromosome increase, chromosome decrease or chromosome region recombination, and imbalance of main signal pathways.

The existing osteosarcoma related gene locus is a strong association locus obtained based on the research of large sample volume GWAS of European population, when the existing osteosarcoma related gene locus is applied to Asian population, especially China population, the locus association is weak, and more accurate results are difficult to obtain through detection, so that the establishment of an evaluation system of the China population is very necessary.

Disclosure of Invention

The invention provides a fluorescent quantitative PCR detection kit for osteosarcoma gene screening, and further discloses application of the kit in the field of osteosarcoma gene screening.

In order to realize the purpose, the invention provides the application of the fluorescent quantitative PCR detection system in preparing an osteosarcoma gene screening system.

The invention also discloses a fluorescent quantitative PCR detection system for osteosarcoma gene screening, which comprises a fluorescent quantitative PCR detection kit for detecting the gene to be detected and a detection result contrast evaluation system; the detection result comparison and evaluation system comprises a detection result processing part corresponding to the site and a reference database; the fluorescent quantitative PCR detection kit comprises a fluorescent quantitative PCR detection reagent for detecting at least two osteosarcoma related gene loci.

The relevant sites for the fluorescent quantitative PCR detection comprise sites rs231775, rs1129055, rs7034162, rs1906953 and rs 7591996.

The structure of the sequence of the primer and the probe for detecting the osteosarcoma gene locus is shown as the following table:

the reference database takes the east Asia population genome data in the thousand human genomes as the level of common population, and the detection result is collected into the database in real time.

The detection result contrast evaluation system corresponds the fluorescence quantitative PCR detection result of the gene to be detected to the risk level, wherein the risk value P of the gene to be detected is the product of a plurality of detection gene loci, and the average risk value is P _a And the average variance is S, and the risk level of the gene to be detected is judged according to the average variance:

P _a p is more than or equal to +0.6S and is a high risk level;

P _a +0.2S≤P＜P _a +0.6S is a high risk level;

P _a -0.2S≤P＜P _a +0.2S is the general risk level;

P _a -0.6S≤P＜P _a -0.2S is a lower risk level;

p < Pa-0.6S is a low risk level.

Further, the mean variance S is calculated as follows:

wherein i is the number of detection sites of the gene to be detected;

mean risk value P _a To pass genomic data of the east Asian population, P _a The calculation formula of (2) is as follows:

wherein the total number of people is N.

The invention also discloses application of the fluorescence quantitative PCR detection system in the osteosarcoma gene screening field.

The invention also discloses a method for screening osteosarcoma genes based on a fluorescence quantitative PCR method, which comprises the step of carrying out fluorescence quantitative PCR detection on osteosarcoma related genes by using the fluorescence quantitative PCR detection system, wherein sites for fluorescence quantitative PCR detection comprise rs231775, rs1129055, rs7034162, rs1906953 and rs7591996 sites.

Preferably, the method for detecting osteosarcoma genes by using a fluorescent quantitative PCR method specifically comprises the following steps:

(1) Amplification of a sample to be detected: according to the structure of the gene locus to be detected, a single fluorescence labeling probe is adopted, and a primer and a probe sequence of a sample to be detected are designed for PCR amplification;

(2) Sample detection: detecting the amplified sites through a melting curve peak pattern graph to obtain a detection result of the corresponding sites;

(3) Processing according to the detection result, counting the risk points of each site to obtain the risk value of the gene to be detected, and corresponding to the risk level in the reference database;

(4) And comprehensively obtaining the gene risk and the related processing result according to the corresponding grade of the detection result.

The fluorescence quantitative PCR detection system comprises a detection kit for a specific locus gene, calculates the osteosarcoma risk score according to the risk value of each locus to the osteosarcoma risk, carries out population classification according to the existing database, and obtains the individual osteosarcoma risk grade through the individual score, so that the osteosarcoma risk of a detected person is evaluated, a high-risk person is guided to improve the life style, and induction factors and increased physical examination frequency are avoided. Meanwhile, the invention establishes a Chinese population database for the selected 5 gene polymorphism sites, and lays a foundation for establishing the evaluation of the osteosarcoma specific to Chinese population.

The fluorescence quantitative PCR detection system can evaluate the osteosarcoma related risk of the examined person. The detection kit provided by the invention has the advantages of strong specificity, high detection rate, high efficiency and low cost, can comprehensively detect and analyze whether the detected population carries osteosarcoma susceptibility genes, evaluates the risk of the detected population suffering from osteosarcoma, screens the susceptible population from common populations, gives personalized guidance suggestions, changes bad living habits and achieves the purpose of prevention.

Drawings

FIG. 1 shows the experimental results of the test sample rs1129055 of the present invention;

FIG. 2 shows the sequencing result of rs1129055 of the test sample according to the present invention;

FIG. 3 shows the experimental results of the test sample rs7034162 of the present invention;

FIG. 4 shows the sequencing result of rs7034162 of the test sample according to the present invention;

FIG. 5 shows the experimental results of the test sample rs1906953 of the present invention;

FIG. 6 shows the sequencing result of rs1906953 of the test sample of the present invention;

FIG. 7 shows the results of the test of rs7591996 in the present invention;

FIG. 8 shows the sequencing result of the detection sample rs7591996 according to the present invention;

fig. 9 is an experimental result of the detection sample rs231775 according to the present invention;

FIG. 10 shows the sequencing result of rs231775 of the test sample according to the present invention;

FIG. 11 is a flow chart of the present invention for evaluation based on the results of the test sample experiment.

Detailed Description

In order to make the aforementioned objects, features and advantages of the present invention comprehensible, embodiments accompanied with figures are described in further detail below.

Example 1

In the embodiment, the osteosarcoma fluorescence quantitative PCR detection kit is used for detecting osteosarcoma related genes, wherein 5 polymorphic sites on five osteosarcoma related genes are detected, including rs231775, rs1129055, rs7034162, rs1906953 and rs7591996 sites, and the total number is 5 sites. And (3) optimizing an amplification system and conditions by using a primer and a probe which are specifically designed, and simultaneously completing the detection of 2 polymorphic sites on two genes in the same system.

In this example, a single fluorescently labeled probe, roche, was used480 platform, finally detecting rs231775, rs1129055, rs7034162, rs1906953 and rs7591996 sites through melting curve peak pattern.

Specifically, in the dual fluorescence quantitative PCR detection method for the sites rs231775, rs1129055, rs7034162, rs1906953 and rs7591996, the design of primers and probes for the sites rs231775, rs1129055, rs7034162, rs1906953 and rs7591996 is shown in the following table 1.

TABLE 1 primer and Probe design

For the sites rs231775, rs1129055, rs7034162, rs1906953 and rs7591996, the reagent reaction systems shown in the following tables 2 to 4 were designed respectively:

TABLE 2 Dual reagent System for sites rs1129055 and rs7034162

TABLE 3 Dual reagent System for sites rs1906953 and rs7591996

TABLE 4 reagent system at site rs231775

The sites rs231775, rs1129055, rs7034162, rs1906953 and rs7591996 were subjected to PCR amplification, and PCR reaction program (cycle) setting parameters were designed as shown in table 5 below.

TABLE 5 PCR reaction parameter settings

According to the designed primers and the PCR parameters of the kit, the locus rs231775, rs1129055, rs7034162, rs1906953 and rs7591996 is subjected to PCR amplification, and the amplification result is detected, wherein the detection results are respectively shown in the figures 1-10.

In the test results shown in FIGS. 1-10:

detecting the result of the rs1129055 experiment as AG, and determining the result AG;

detecting the result of the rs7034162 experiment as AT and the sequencing result AT;

the test result of the rs1906953 sample is TT, and the sequencing result is TT;

detecting whether the experimental result of the sample rs7591996 is AC, the reverse chain is GT, and the sequencing result is GT;

the test result of the detection sample rs231775 is GG, the reverse chain is CC, and the sequencing result is CC.

And the retest is carried out through sequencing, so that the experimental method has accuracy and reliability.

Example 2

The 5 sites in example 1 were obtained from Bezzina et al and from the articles published by doctor Ar-king, respectively, and the two susceptible sites directly associated with osteosarcoma were obtained from GWAS studies, and the risk value of risk allelic type, OR value, was calculated for the two sites.

GWAS studies have found that common genetic variations in the NFIB, GRM4 genes and 2p25.2 genes may be associated with osteosarcoma development. Mirabello L et al found that the SNP site (rs 7034162) in the NFIB gene is a risk factor for osteosarcoma cell migration, proliferation and colony formation. The rs7034162 mutation (genetic variation of germ cells) is a susceptibility gene for tumor cell metastasis.

Osteosarcoma is the most common primary bone malignancy in adolescents and young adults. To better understand the genetic cause of osteosarcoma, savage S a et al performed a multi-stage genome-wide association study, including 941 osteosarcoma patients and 3291 european adult control group without cancer. The GRM4 gene is involved in the inhibition of intracellular signal transduction and cyclic adenosine monophosphate (cAMP) signaling cascades. In animal model studies, cAMP-dependent protein kinase (prkar 1. Alpha.) is an oncogene of osteosarcoma and is involved in tumorigenesis. These all suggest that the cAMP pathway plays a significant role in osteosarcoma. Variation of the SNP site (rs 1906953) on the GRM4 gene alters the cAMP pathway, thereby affecting the development of osteosarcoma.

The 5 sites were evaluated separately, and the genotype and risk value (OR) were calculated as:

the OR values of AA-AG-GG genotypes of the rs231775 locus of the CTLA-4 gene are 1.9881-1.41-1 respectively;

the OR values of AA-AG-GG genotypes at the rs1129055 loci of the CD86 gene are 2.0449-1.43-1 respectively;

the OR values of AA-AT-TT genotypes AT the rs7034162 loci of the NFIB genes are 5.9049-2.43-1 respectively;

the OR values of TT-TC-CC genotypes at the site of 1906953 of the GRM4 gene rs 5363 are 2.4649-1.57-1 respectively;

the OR values of CC-CA-AA genotypes of the rs7591996 sites on 2p25.2 are 1.9321-1.39-1 respectively.

Since the incidence of osteosarcoma is less than 10%, the OR value approximates the RR value, i.e. the risk value. The risk value of each locus is calculated according to the genotype, namely the risk value of the homozygous risk allele is the square of the OR value, the risk value of the heterozygous risk allele is the OR value, the homozygous non-risk allele is 1, each SNP locus has a susceptibility value according to the detected genotype for one subject, the score is defined as S, then the susceptibility value of the individual osteosarcoma of the subject is P, and the P value is the product of the OR values of the five genotypes, namely:

according to the gene detection result, the individual osteosarcoma risk is evaluated, and an osteosarcoma susceptibility database of Chinese population is established.

The genome data of east Asia population in thousands of genomes is taken as the common population level, and the total population is defined as N. The genotypes of the east Asia population loci in the thousand human genomes are divided equally:

wherein the total number of people is N.

And the average variance is calculated out,

classifying the osteosarcoma risk values of east Asia population according to Pa and S, and corresponding the individual osteosarcoma risk value P of the examined person to the population classification, wherein the population classification has five levels, namely five risk levels of high-slightly high-general-slightly low-low, specifically:

p is more than or equal to Pa +0.6S and is a high risk level;

the higher risk level is that P is more than or equal to Pa +0.2S and less than Pa + 0.6S;

the general risk level is that P is more than or equal to Pa-0.2S and less than Pa + 0.2S;

the lower risk level is that P is more than or equal to Pa-0.6S and less than Pa-0.2S;

p < Pa-0.6S is a low risk level.

Based on osteosarcoma risk level divided by east Asia population, P value is more than or equal to 20.35 is high risk level, P value is more than or equal to 315.54 and less than 20.35 is higher risk, P value is more than or equal to 10.74 and less than 15.54 is general risk, P value is more than or equal to 5.93 and less than 10.74 is lower risk, and P value is less than or equal to 5.93 is lower risk.

As shown in FIG. 11, the evaluation process is performed according to the experimental results of the test samples, and the value of the OR of 5 items is multiplied according to the gene test results to obtain the susceptibility value, i.e., the risk value, of the osteosarcoma of the individual. Obtaining individual osteosarcoma risk level by a grading method correspondingly, and comprehensively obtaining the osteosarcoma risk of the examinee according to the level corresponding to the P value of the examinee, thereby providing an individual health management scheme.

Example 3

The method comprises the steps of collecting a gene sample of a detected person, extracting DNA, and detecting by using a Roche LightCycler480 real-time quantitative instrument to obtain a result as shown in a graph 1,3,5,7,9.

The results of the test for the following subjects are shown in table 6 below.

The gene rs231775 of CTLA-4 gene of a certain detected person is GG, and the corresponding OR value is 1; the rs1129055 genotype of the CD86 gene is AG, and the corresponding OR value is 1.43; the rs7034162 genotype of NFIB gene is AT, and the corresponding OR value is 2.43; the rs1906953 genotype of GRM4 gene is CC, and the corresponding OR value is 1; the rs7591996 genotype at 2p25.2 is CC, corresponding to an OR value of 1.9321. The above OR values are derived from literature studies at NCBI.

The subject's individual osteosarcoma susceptibility value P =1 × 1.43 × 2.43 × 1 × 1.9321=6.71 was calculated.

TABLE 6 Gene test results of the subjects

	rs231775	rs1129055	rs7034162	rs1906953	rs7591996
						Subject of the test	GG	AG	AT	CC	CC
HG00403	AG	AA	AT	CC	AC
						HG00404	GG	GA	AT	CC	CC
HG00406	AG	GA	TT	CC	CC

There are 504 east asians in the thousand people genome database, for example, the person P =1.41 × 2.0449 × 2.43 × 1.39=9.74 of HG00403, and the rest can be calculated as well, so that the genotype of the east asian population locus is divided into P average _a =13.14, mean variance =12.01.

The subject P<P _a -0.2s =13.14-0.2 x 12.01, so osteosarcoma risk level is low, so the osteosarcoma risk level of the subject is lower than or equal to the general risk level, but does not represent that the subject must not suffer from such diseases, thus suggesting that the subject continues to maintain optimistic mood, and a healthy lifestyle, a routine physical examination per year.

The above embodiments of the present invention are described in detail, and the principle and the implementation of the present invention are explained by applying specific embodiments, and the above description of the embodiments is only used to help understanding the method of the present invention and the core idea thereof; meanwhile, for a person skilled in the art, according to the idea of the present invention, there may be variations in the specific embodiments and the application scope, and in summary, the content of the present specification should not be construed as a limitation to the present invention.

Sequence listing

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Claims (10)

1. The fluorescent quantitative PCR detection system is used for preparing an osteosarcoma gene screening system.

2. A fluorescence quantitative PCR detection system for osteosarcoma gene screening is characterized by comprising a fluorescence quantitative PCR detection kit for detecting a gene to be detected and a detection result contrast evaluation system; the detection result comparison and evaluation system comprises a detection result processing part corresponding to the site and a reference database;

the fluorescent quantitative PCR detection kit comprises a fluorescent quantitative PCR detection reagent for detecting at least two osteosarcoma related gene loci.

3. The fluorescent quantitative PCR detection system for osteosarcoma gene screening of claim 2, wherein the relevant sites for fluorescent quantitative PCR detection include rs231775, rs1129055, rs7034162, rs1906953, and rs7591996 sites.

4. The fluorescent quantitative PCR detection system for osteosarcoma gene screening of claim 3, wherein the primer and probe sequence structure for detecting said osteosarcoma gene locus is shown in the following table:

5. the fluorescent quantitative PCR detection system for osteosarcoma gene screening of any one of claims 2 to 4, wherein the reference database is the genome data of east Asian population in thousand human genomes as the common population level, and the detection result is collected into the database in real time.

6. The fluorescence quantitative PCR detection system for screening osteosarcoma genes of any one of claims 2-5, wherein the detection result control evaluation system is used for mapping the fluorescence quantitative PCR detection result of the gene to be detected into a risk grade, wherein the risk value P of the gene to be detected is the product of multiple detection gene sites, and the average risk value is P _a And the average variance is S, and the risk level of the gene to be detected is judged according to the average variance:

P _a p is more than or equal to +0.6S and is a high risk level;

P _a +0.2S≤P＜P _a +0.6S is a high risk level;

P _a -0.2S≤P＜P _a +0.2S is the general risk level;

P _a -0.6S≤P＜P _a -0.2S is a lower risk level;

p < Pa-0.6S is a low risk level.

7. The fluorescent quantitative PCR detection system for osteosarcoma gene screening of claim 6, wherein:

the mean variance S is calculated as follows:

wherein i is the number of detection sites of the gene to be detected;

mean risk value P _a To pass genomic data of the east Asian population, P _a The calculation formula of (2) is as follows:

wherein the total number of people is N.

8. Use of the fluorescent quantitative PCR detection system of any one of claims 2-7 in the field of osteosarcoma gene screening.

9. A method for screening osteosarcoma genes based on a fluorescent quantitative PCR method, which is characterized by comprising the step of carrying out fluorescent quantitative PCR detection on osteosarcoma related genes by using the fluorescent quantitative PCR detection system of any one of claims 2-7, wherein sites for fluorescent quantitative PCR detection comprise rs231775, rs1129055, rs7034162, rs1906953 and rs7591996 sites.

10. The method for detecting osteosarcoma gene based on fluorescent quantitative PCR method according to claim 9, which comprises the following steps:

(1) Amplification of a sample to be detected: according to the structure of the gene locus to be detected, a single fluorescence labeling probe is adopted, and a primer and a probe sequence of a sample to be detected are designed for PCR amplification;

(2) Sample detection: detecting the amplified sites through a melting curve peak pattern graph to obtain a detection result of the corresponding sites;

(3) Processing according to the detection result, obtaining the risk value of the gene to be detected after counting the risk points of each site, and corresponding to the risk level in the reference database;

(4) And comprehensively obtaining the gene risk and the related processing result according to the corresponding grade of the detection result.