CN112226499A - Kit for detecting related susceptibility gene polymorphism of abortion with unknown cause - Google Patents
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Abstract
The invention belongs to the fields of molecular biology and medicine, and relates to a kit for detecting unknown recurrent abortion-related susceptibility gene polymorphism, which covers detection of recurrent abortion-related susceptibility gene polymorphismMTHFRC677T site (rs 1801133) andMTHFRthe a1298C site of the gene (rs 1801131). The invention comprises the gene for detecting susceptibility related to abortion with unknown causeMTHFR2 PCR amplification primer mixed solution of polymorphic sites; a probe mixture solution for the 2 SNP sites; dNTP; 10 × PCR buffer; 25mMMg2+Ions; FastStartTaq enzyme; deionized water, and the like. The inventor screens and combines the Chinese population related susceptibility gene polymorphism with unknown cause recurrent abortion through a large amount of research and practice, and the kit can simultaneously detect 2 polymorphic sites of related susceptibility genes with unknown cause recurrent abortion, is simple and convenient to operate, is quick, has low cost, and has detection flux and detection speedCompared with the prior art, the accuracy of the detection result is greatly improved.
Description
Technical Field
The invention belongs to the fields of molecular biology and medicine, and particularly relates to a kit for detecting related susceptibility gene polymorphism of abortion with unknown cause based on a multiple fluorescence PCR technology, a detection method and application thereof.
Background
Spontaneous abortion is the most common complication in the early pregnancy, and epidemiological research finds that 1% -5% of pregnant women can have Recurrent abortion, and Recurrent abortion (RSA) refers to spontaneous abortion which continuously occurs for 2 or more times, the causes of the Recurrent abortion are extremely complex, the reasons of embryo chromosome abnormality, uterine malformation or intrauterine adhesion and the like are eliminated, while 60% -70% of the Recurrent spontaneous abortion (URSA) with unknown causes cannot be found, so that the Recurrent spontaneous abortion (URSA) brings great physical and mental injuries to the pregnant women, influences family happiness and social stability of the pregnant women, becomes a great public health and social problem influencing reproductive health, and the thought of individualized medicine is adopted, so that personalized treatment is provided after the etiological diagnosis is clear, and the infants born successfully treated by fetus protection treatment are screened for defects and fetus development monitoring in utero, therefore, the basic research and application research of abortion become important contents of priority in the scientific research of population health.
Meanwhile, in recent years, more and more researches show that about 70 percent of sporadic spontaneous abortion has embryo chromosome abnormality which is considered as the most common cause of early spontaneous abortion, but in RSA patients, the probability of the embryo chromosome abnormality only accounts for 25 to 32 percent, only a small part of causes of RSA can be explained, in addition, different factors such as anatomical factors, endocrine factors, infection factors, male specific factors, environmental factors and psychology factors cause spontaneous abortion in different degrees, but classification and action strength of the causes of different researchers do not form consensus yet, in a word, the causes of RSA are more, all the factors are complicated, and the causes of some patients are still unknown, so that great difficulty is brought to clinical diagnosis and treatment, discussion of the causes of RSA is helpful to further know the pathogenesis process of RSA, and diagnosis, treatment and prevention of the disease are improved. With the continuous development of modern medicine, especially the progress of modern molecular cell technology, the research on the etiology of RSA is more intensive, so that the diagnosis and treatment of RSA are further improved, and clinicians should perform corresponding auxiliary examinations on different patients based on detailed knowledge of the related medical history of patients and their partners, so as to quickly and accurately find out the etiology and guide the treatment.
With the development of molecular biotechnology, after traditional cell culture, the chromosome karyotype G-banding technology is the main technical means for examining the cause of spontaneous abortion at present, but the technical requirements are high, time and labor are wasted, the operation process is very easy to be polluted by the external environment, so that the judgment of the result is influenced, chromosome abnormality of less than 3-5 Mb cannot be detected, and 10-40% of samples cannot give detection results due to culture failure, bacterial or fungal infection, maternal blood pollution and the like. Therefore, chromosome abnormalities which are considered to be undetectable by karyotyping can be identified by Fluorescence In Situ Hybridization (FISH), Multiplex ligation-dependent amplification (MLPA), Chromosome Microarray Analysis (CMA), bacterial artificial chromosome labeling-polystyrene microsphere identification/separation (BoBs)TM) Etc. instead of karyotyping. However, FISH and MLPA technologies have the defects of structural abnormality detection and chromosome number and genome detectionLimited coverage, CMA and BoBsTMThe technology is not suitable for being used as first-line detection, has the defects of high detection cost and high detection technology difficulty, and compared with the fluorescence PCR technology based on the melting curve, the fluorescence PCR technology has higher cost performance. The method is generally concerned because of its rapidity, high throughput, low cost of use, accurate results, and the realization of a true tube closure operation.
Disclosure of Invention
Aiming at the defects of the prior art, the invention mainly aims to provide a simple, medium-flux, high-efficiency and low-cost kit for detecting the susceptibility gene polymorphism related to the abortion with unknown cause, which is suitable for Chinese people. The kit is used for simultaneously detecting 2 polymorphic sites of susceptibility genes related to abortion with unknown cause and is also suitable for a common fluorescent PCR instrument, so that the genotyping of the 2 polymorphic sites of the susceptibility genes related to abortion with unknown cause of Chinese people is accurately carried out. It includes:
1) used for detecting susceptibility genes related to recurrent abortion with unknown causeMTHFRC677T site of gene andMTHFRa mixture of PCR amplification primers at position a1298C of the gene;
2) a probe mixture solution aiming at the 2 SNP sites, whereinMTHFRThe fluorescent group at the 5 'end of the probe at the C677T site of the gene is FAM, and the quenching group at the 3' end is BHQ 1; the above-mentionedMTHFRThe fluorescent group at the 5 'end of the probe at the A1298C site of the gene is ROX, and the quenching group at the 3' end is BHQ 2;
3）dNTP；
4) 10 × PCR buffer;
5）25mM Mg2+ions;
6) fast StartTaq enzyme;
7) deionized water.
The detectionMTHFRThe PCR amplification primer sequences of the C677T locus of the gene are consistent, and the specific sequences are shown in SEQ ID No.1 to SEQ ID No. 2.
The detectionMTHFRThe PCR amplification primer sequences of A1298C sites of the gene are consistent, and the specific sequences are shown as SEQ ID No.3 to SEQ ID No. 4.
The detectionMTHFRThe PCR probe sequences of the C677T site of the gene are consistent, and the specific sequence is shown in SEQ ID No. 5.
The detectionMTHFRThe PCR probe sequences of A1298C sites of the genes are consistent, and the specific sequences are shown as SEQ ID No. 6.
Further, to aim at theMTHFRC677T site of gene andMTHFRthe final concentrations of the probes designed for the A1298C locus of the gene were 0.1. mu.M and 0.16. mu.M, respectively.
Still another object of the present invention is to provide a method for detecting a susceptibility gene polymorphism associated with abortion of unknown cause, which comprises:
(1) extracting the genome DNA of a sample to be detected;
(2) adding the DNA obtained in step (1) toMTHFRC677T site of gene andMTHFRand carrying out PCR amplification in the PCR reaction solution of the A1298C site of the gene, carrying out melting analysis on an amplification product, and carrying out data acquisition and analysis to obtain a genotyping result.
The invention also aims to provide application of the susceptibility gene polymorphism detection kit related to the recurrent spontaneous abortion with unknown reasons of any Chinese population in detection of the recurrent spontaneous abortion population.
It is a further object of the present invention to provide a detection system comprising the kit of the present invention.
The invention has the beneficial effects that:
1. through a large amount of research and practice, the inventor screens and combines susceptibility gene polymorphism sites related to recurrent abortion with unknown reasons of Chinese people through the search of a Google database, a PubMed database and an HGMD database, constructs the kit, can simultaneously perform fusion analysis on SNP sites of 2 different genes by using the kit, obtains different genotypes at one time, overcomes the defects of the existing mutation detection method, is simple and convenient to operate and low in cost, greatly improves the detection flux, the accuracy of the detection result and the like, and has high large-scale clinical application value.
2. Based on the improved molecular beacon probe used for the melting curve method, the improved molecular beacon probe is successfully applied to the detection of the polymorphic sites of C677T and A1298C of MTHFR gene, only one improved molecular beacon probe is used respectively, and the detection kit for the folate metabolism gene polymorphism, which has the advantages of low cost, simple operation, clear result interpretation and easy clinical popularization, is established. The invention adopts the improved molecular beacon probe result multiple fluorescence melting curve technology, establishes a high-efficiency amplification system, has no uncapping pollution, and is low in price, high-efficiency and accurate.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following detailed description is given with reference to the preferred embodiments of the present invention and the accompanying drawings. The detailed description of the present invention is given in detail by the following examples and the accompanying drawings.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this application, illustrate embodiment(s) of the invention and together with the description serve to explain the invention without limiting the invention. In the drawings:
FIG. 1 is a process flow chart of genetic testing using the detection kit for susceptibility gene polymorphism related to abortion caused by unknown recurrence of Chinese people according to an exemplary embodiment of the present invention;
FIG. 2 is a graph of melting peak curves obtained in an exemplary embodiment of the present invention;
FIG. 3 is a graph showing the results of an amplification curve obtained in an exemplary embodiment of the present invention.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.
A kind ofThe kit, the method and the application can aim at the existing Chinese population recurrent abortion related susceptible gene polymorphism detection kit and method for the Chinese population recurrent abortionMTHFRC677T site (rs 1801133) andMTHFRthe A1298C locus (rs 1801131) of the gene is used as a detection target, and the method for detecting the susceptibility gene polymorphism related to the abortion with unknown reasons, which has the advantages of simple and convenient operation, rapidness, low cost, greatly improved detection flux, accuracy of detection results and the like compared with the prior art, is provided.
The invention relates to a susceptibility gene polymorphism detection kit related to abortion with unknown cause, which comprises: primer mixture, PCR probe mixture, dNTP, 10 XPCR buffer solution and 25mM Mg2+Ions, deionized water, FastTaq enzyme.
From the above description, the beneficial effects of the present invention are: based on the improved molecular beacon probe used for the melting curve method, the improved molecular beacon probe is successfully applied to the detection of the polymorphic sites of C677T and A1298C of MTHFR gene, only one improved molecular beacon probe is used respectively, and the detection kit for the folate metabolism gene polymorphism, which has the advantages of low cost, simple operation, clear result interpretation and easy clinical popularization, is established. The invention adopts the improved molecular beacon probe result multiple fluorescence melting curve technology, establishes a high-efficiency amplification system, has no uncapping pollution, and is low in price, high-efficiency and accurate.
TABLE 1MTFHRPrimer and probe sequence of gene C677T and A1298C site
Further, the concentration of PCR primers and the concentration of probes are optimally configured, so that the yield of each site can be considered during the melting reaction, the final concentrations of the primers at the C677T site and the A1298C site of the MTHFR gene are respectively 0.4 mu M and 0.5 mu M, and the final concentrations of the primers at the C677T site and the A1298C site of the MTHFR gene are respectively 0.4 mu M and 0.5 muMTHFRC677T site of gene andMTHFRthe final concentrations of the probes designed for the A1298C locus of the gene were 0.1. mu.M and 0.16. mu.M, respectively.
Further, the C677T probe of the MTHFR gene is marked with FAM fluorescent group and BHQ1 quenching group, and the A1298C probe of the MTHFR gene is marked with ROX fluorescent group and BHQ2 quenching group.
The lengths of the amplified fragments at positions C677T and A1298C of the MTHFR gene of the present invention, specific primer sequences and probe sequences can be referred to Table 1.
The detection method based on the kit comprises the following steps: amplifying and enriching 2 target fragments of a detected sample by multiplex PCR amplification, wherein the PCR amplification and enrichment conditions are set as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 deg.C for 30s, annealing at 50-55 deg.C for 90s, extension at 72 deg.C for 5min, and 40 cycles. Then, a melting curve analysis is carried out, and the melting program is as follows: 30s at 95 ℃; 5min at 40 ℃; FAM and ROX channel fluorescence signals are collected at 40-80 ℃.
And (5) judging a result: and judging the result according to the Tm value corresponding to the melting peak, and detecting 3 peak types of wild type, heterozygous mutation and homozygous mutation by each probe. Finally, the site C677T of MTHFR gene is preferred: FAM channel DTm65 ℃. + -. 0.6 ℃, site A1298C: the ROX channel DTm72 ℃. + -. 0.5 ℃.
The specific implementation mode has the advantages of low cost, simple operation, clear result interpretation and easy clinical popularization, and is suitable for detecting the polymorphism of the susceptibility genes related to the recurrent abortion with unknown reasons. An improved molecular beacon probe is combined with a multiple fluorescence melting curve technology to establish a high-efficiency amplification system, and the method has the advantages of no uncapping pollution, low price, high efficiency and accuracy.
The technical solution of the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.
Referring to fig. 1 to fig. 3, in this example, a kit (96 persons) for detecting polymorphisms at positions C677T and a1298C of MTFHR, a susceptibility gene related to abortion with unknown cause, is provided, which can complete in vitro screening of polymorphisms of susceptibility genes related to abortion with unknown cause in chinese.
1. Test sample
The sample is from DNA library of recurrent abortion with unknown reasons, such as reproduction and genetic center of Suzhou city hospital, obstetrics and gynecology department, and women's health department, and all patient samples in the library are signed with informed consent before collection, and committed to cooperate with subsequent diagnosis and treatment and follow-up visit.
2. Detection scheme
The scheme adopts a case control research method, and detects the single nucleotide polymorphisms of the sites C67 677T and A1298C of MTHFR genes by using a PCR fluorescent probe technology for 170 cases of patients (case groups) suffering from recurrent abortion with unknown reasons in the south Suzhou area and 170 cases of normal women (control groups).
3. The result of the detection
The frequency of occurrence of TT genotype at MTHFR C677T site in case group (34.1%) was significantly higher than that in control group (17%), the frequency of T allele (58.8%) was also higher than that in control group (45.3%), the difference was statistically significant (P < 0.05), while the frequency of CC genotype and the frequency of C allele at MTHFR A1298C site in case group were not statistically significant (P > 0.05) compared to that in control group, see tables 2, 3.
The risk of the patients with CC genotype in the group population developing unexplained recurrent abortion was set as a control (set value of 1.0) for the C677T site of the MTHFR gene. The risk of abortion of patients carrying the CT genotype is 1.266 times that of the control, the 95% CI value is (0.76-2.10), but the difference is not statistically significant (P is 0.36); the risk of abortion of patients carrying TT genotype is 2.895 times of that of the control group, 95% of CI values are (1.58-5.32), and the difference is statistically significant (P is 0.001).
TABLE 2 MTHFR genotype frequency distribution comparison [ n (%) ]
TABLE 3MTHFRComparison of allele frequency distributions [ n (%) ]
In conclusion, the research suggests that the polymorphism at the C677T locus of the MTHFR gene may be a genetic risk factor for the onset of recurrent abortion with unknown reasons for Han people in the southern Suzhou region, so that the polymorphism at the C677T locus of the MTHFR gene is detected before pregnancy, and the supplementation of folic acid is increased in a targeted manner according to the genotype, so that the blindness of treatment can be effectively avoided, and the method has important guiding significance for couples suffering from recurrent abortion with unknown reasons.
The screening method and the kit have the advantages of convenient use, simple operation, low cost and direct and reliable detection result, and are suitable for large-scale screening of genetic polymorphism detection related to recurrent abortion with unknown reasons of Chinese people.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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cgtggaggag ctgaccagtg aagaaagtgt ccacg 35
Claims (6)
1. A kit for detecting polymorphism of susceptibility genes related to abortion with unknown cause, which is characterized by comprising:
1) used for detecting susceptibility genes related to recurrent abortion with unknown causeMTHFRC677T site of gene andMTHFRPCR amplification primer mixture of A1298C locus of gene;
2) a probe mixture solution for the C677T site and the A1298C site;
3）dNTP；
4) 10 × PCR buffer;
5）25mM Mg2+ions;
6) fast StartTaq enzyme;
7) deionized water;
wherein the PCR amplification primer sequence of the C677T site is shown as SEQ ID No.1 to SEQ ID No. 2;
the PCR amplification primer sequence of the A1298C site is shown as SEQ ID No.3 to SEQ ID No. 4;
the PCR probe sequence of the C677T site is shown in SEQ ID No. 5;
the PCR probe sequence of the A1298C site is shown as SEQ ID No. 6.
2. The kit for detecting the polymorphism of the susceptibility gene related to abortion with unknown cause of relapse according to claim 1, wherein the kit comprises: to the saidMTHFRC677T site of gene andMTHFRthe final concentrations of the probes designed for the A1298C locus of the gene were 0.1. mu.M and 0.16. mu.M, respectively.
3. The method for detecting susceptibility genes associated with abortion with unknown cause of recurrent abortion in claim 1, comprising the steps of:
1) extracting a DNA sample to be detected, and adjusting the concentration of the DNA sample to be detected to 20 ng/mu L;
2) using the PCR amplification primer pairMTHFRC677T site of gene andMTHFRPCR amplification is carried out on the A1298C locus of the gene and other different gene loci;
3) and (3) carrying out melting curve analysis on the polymorphic sites of the target gene of the PCR product, and obtaining the screening results of 2 sites at one time.
4. The method for detecting susceptibility genes associated with abortion with unknown cause of recurrent, as claimed in claim 3, wherein the PCR amplification conditions are set as follows: pre-denaturation at 95 ℃ for 10 min; denaturation at 95 deg.C for 30s, annealing at 50-55 deg.C for 90s, extension at 72 deg.C for 5min, and 40 cycles.
5. The method for detecting susceptibility genes associated with abortion with unknown cause of relapse according to claim 3, wherein the step 5(3) comprises the following steps: 30s at 95 ℃; 5min at 40 ℃; FAM and ROX channel fluorescence signals are collected at 40-80 ℃.
6. Use of a kit according to any one of claims 1 to 5 for the detection of a susceptibility gene associated with abortion of unknown cause.
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Application publication date: 20210115 |