Primer combination, MLPA probe, gene chip and kit for detecting microdeletion or/and microduplication of recurrent abortion
阅读说明：本技术 一种检测复发性流产的微缺失或/和微重复的引物组合、mlpa探针、基因芯片及试剂盒 (Primer combination, MLPA probe, gene chip and kit for detecting microdeletion or/and microduplication of recurrent abortion ) 是由 王彬彬 王晶 于 2019-11-19 设计创作，主要内容包括：本发明提供一种检测复发性流产的微缺失或/和微重复的引物组合、MLPA探针、基因芯片及试剂盒,涉及生物技术领域,所述引物组合具有如SEQ ID NO.1-21所示的核苷酸序列,可以被制备成MLPA探针、基因芯片及试剂盒或构建体、重组细胞,还可以作为快速筛选易患复发性流产的生物样品系统的主要部件。本发明提供的引物组合及其应用可以同时检测多个位点的复发性流产微缺失或/和微重复,特异性好,反应灵敏,检测时间短,方法简单、从点突变到大染色体缺失/插入都可以进行检测,成本低廉,临床应用价值高。(The invention provides a primer combination, an MLPA probe, a gene chip and a kit for detecting microdeletion or/and microduplication of recurrent abortion, and relates to the technical field of biology. The primer combination and the application thereof provided by the invention can be used for simultaneously detecting recurrent abortion microdeletion or/and microduplication of a plurality of sites, and have the advantages of good specificity, sensitive reaction, short detection time, simple method, low cost and high clinical application value, and the detection can be carried out from point mutation to large chromosome deletion/insertion.)
1. A primer combination for detecting biological samples of recurrent abortion is characterized in that the primer combination has a nucleotide sequence shown as SEQ ID NO. 1-21.
2. An MLPA probe combination for detecting biological samples of recurrent abortion, which is characterized in that the MLPA probe combination has a nucleotide sequence shown as SEQ ID NO.1-21, wherein each MLPA probe has a fluorescent label and a universal primer, and the universal primer has a nucleotide sequence shown as SEQ ID NO. 22-23.
3. A kit for detecting a biological sample of recurrent abortion, comprising the primer combination of claim 1 or the MLPA probe combination of claim 2.
4. A gene chip for detecting a biological sample of recurrent abortion, comprising a solid support and the primer set of claim 1 or the MLPA probe set of claim 2 attached thereto.
5. A construct for detecting a biological sample of recurrent abortion, which is a vector having the primer combination of claim 1.
6. A recombinant cell for detecting a biological sample of recurrent abortion, which is formed by transforming a vector having the primer combination of claim 1 into a recipient cell.
7. A system for detecting a biological sample of recurrent pregnancy loss, comprising:
a whole genome DNA extraction unit for extracting a whole genome DNA sample from the biological sample;
a whole genome DNA sample processing unit, which is used for denaturing a whole genome DNA sample, hybridizing the whole genome DNA sample with an MLPA probe combination made from the primer combination or the MLPA probe combination in the MLPA probe combination or the kit, and finally amplifying a hybridization product to obtain an analysis sample;
and the sample analysis unit is used for judging whether the analysis sample is a biological sample susceptible to recurrent abortion and giving a judgment result.
8. A method for detecting microdeletion or/and microduplication in recurrent miscarriage, which comprises the steps of detecting a biological sample to be detected by the primer combination of claim 1 or the MLPA probe combination of claim 2 or the kit of claim 3 or the system of claim 4, wherein the method comprises:
extracting whole genome DNA of a biological sample to be detected, and performing denaturation treatment to obtain denatured genome DNA;
carrying out hybridization and ligation reaction on the MLPA probe combination prepared by the primer combination or the MLPA probe combination in the MLPA probe combination or the kit and the deformed genomic DNA to obtain a ligation product;
after the obtained connection product is subjected to PCR reaction, performing capillary electrophoresis on the PCR amplification product to obtain a capillary electrophoresis result;
and (3) analyzing the capillary electrophoresis result, and judging whether the biological sample to be detected is a recurrent abortion microdeletion or/and a micro-repeat sample.
9. Use of the primer combination of claim 1 or the MLPA probe combination of claim 2 or the kit of claim 3 or the system of claim 4 for screening or detecting biological samples with microdeletion/microduplication of the AKT1 gene or/and the PDZD2 gene.
10. Use of the primer combination of claim 1 for constructing a vector or forming a recombinant cell so as to be effective as a model for research relating to recurrent pregnancy loss.
Technical Field
The invention relates to the technical field of biology, in particular to a primer combination for detecting a biological sample susceptible to recurrent abortion and an MLPA probe, a gene chip, a kit, a system and a method prepared from the primer combination.
Background
Recurrent Spontaneous Abortion (RSA) is a common pathological pregnancy in gynecology, and refers to a person who has suffered 2 or more Spontaneous abortions. Studies have shown that recurrent abortion is caused by a variety of causes, among which genetic factors are closely related to the occurrence of recurrent abortion, and chromosomal microdeletions and microduplications are also common types of genetic factors. Chromosomal microdeletions/microreplications refer to deletions or duplications on the chromosome that are 1.5kb to 10Mb in length. Although the incidence of each microdeletion or/and microduplication syndrome is low, due to the limitation of clinical detection technology, a large number of patients with recurrent abortion caused by microdeletion or/and microduplication cannot be detected in the examination and diagnosis before pregnancy, so that the mind, body and even family of the patients suffer from serious trauma and huge economic burden. Therefore, if the chromosome microdeletion/microreplication can be detected during pregnancy preparation or before embryo transplantation, it will be of great significance to the patients with recurrent abortion.
Since pregnancy is a complex process with multiple links, more studies are being made on recurrent abortion at present, chinese patent document CN 107177696a for prevention and treatment of recurrent abortion by detecting three genetic susceptibility genes TNF- α, CTLA4, MTHFR, chinese patent document CN 103221421a for detection of predisposition to recurrent abortion by examining the human annexin a5(ANXA5) promoter in a non-maternal sample, in particular a sample obtained from the (prospective) biological father, or by examining the human annexin a5(ANXA5) promoter in a chorionic biopsy or amniocentesis sample obtained from said female, or by examining certain nucleotide point mutations in the human annexin a5(ANXA5) promoter in a sample obtained before or during the morula stage of an in vitro fertilized embryo obtained from said in vitro fertilized embryo, chinese patent document CN108486241A for diagnosis of recurrent abortion by detecting mRNA biomarkers CN106987593A and ESR genetic susceptibility gene samples of chinese
Because of the many reasons for recurrent abortion, the current detection methods do not necessarily detect all recurrent abortions. Moreover, the existing microdeletion/microreplication diagnostic technology still has the problems of low detectable rate, only target diagnosis and the like, for example, the high resolution karyotype analysis method mainly used in the microdeletion/microreplication diagnostic technology can only find microdeletion abnormality of a small part of larger fragments, the fluorescence in situ hybridization method has good specificity, but only target diagnosis can be performed.
Disclosure of Invention
In order to solve the problem of low detection rate of recurrent abortion caused by microdeletion and microreplication in the prior art and make the detection whether the biological sample to be detected is a biological sample of recurrent abortion more complete and accurate, the invention provides a MLPA probe combination, a kit, a gene chip and a system which can rapidly screen and detect whether the biological sample is a biological sample susceptible to recurrent abortion.
To achieve the technical object of the present invention, the first aspect of the present invention provides a primer set for detecting a biological sample of recurrent abortion, which has a nucleotide sequence as shown in SEQ ID No. 1-21.
The primer combination can simultaneously detect microdeletion/microduplication in AKT1 gene and PDZD2 gene in a biological sample, thereby screening whether the biological sample to be detected is a biological sample susceptible to recurrent abortion.
In order to achieve the technical object of the present invention, another aspect of the present invention provides an MLPA probe set for detecting a biological sample of recurrent abortion, which has the nucleotide sequences shown in SEQ ID nos. 1 to 21, respectively:
wherein each MLPA probe is provided with a fluorescent label and a universal primer.
Wherein, the universal primer pair has the nucleotide sequence shown as SEQ ID NO.22-23 in sequence.
The probe combination can simultaneously detect microdeletion/microduplication of AKT1 gene and PDZD2 gene in a biological sample, thereby screening whether the biological sample to be detected is a biological sample susceptible to recurrent abortion.
In still another aspect, the present invention provides a kit for detecting a biological sample of recurrent abortion, which has the above-mentioned MLPA probe combination.
Wherein, the kit also comprises a reaction solution.
Wherein the reaction solution comprises a conventional buffer for MLPA hybridization ligation reaction, ligase, polymerase, and a buffer for PCR reaction, ligase, polymerase or other related reagents.
Specifically, the hybridization reagent is:
wherein the amplification reaction reagent is:
among them, the above reagents are all commercially available.
In order to achieve the technical purpose of the invention, the invention also provides a gene chip for detecting the biological sample of recurrent abortion, which comprises a solid phase carrier and a gene probe attached to the solid phase carrier.
Wherein the gene probe has a nucleotide sequence shown as SEQ ID NO. 1-21.
Wherein, the solid phase carrier is any one solid phase carrier suitable for manufacturing gene chips.
To achieve the technical object of the present invention, the present invention further provides a system for detecting a biological sample of recurrent abortion, comprising:
a whole genome DNA extraction unit for extracting a whole genome DNA sample from the biological sample;
a whole genome DNA sample processing unit, which is used for hybridizing and amplifying the denatured whole genome DNA sample and the MLPA probe combination made from the primer combination or the MLPA probe combination in the MLPA probe combination or the kit to obtain an analysis sample;
and the sample analysis unit is used for judging whether the analysis sample is a biological sample susceptible to recurrent abortion and giving a judgment result.
The biological sample can be any sample of whole genome DNA which can be extracted, including but not limited to blood, amniotic fluid cells, skin tissue and the like.
Wherein, the whole genome DNA sample processing unit comprises a hybridization reaction agent and an amplification reaction agent.
Wherein, the hybridization reagent is:
wherein the amplification reaction reagent is:
wherein the whole genome DNA sample is denatured in the whole genome DNA sample treatment unit under such a condition that the whole genome DNA is treated at 98 ℃ for 5 minutes.
Wherein the whole genome DNA sample processing unit hybridizes the denatured whole genome DNA sample with the primer combination or the MLPA probe combination under the operating conditions that the denatured whole genome DNA sample reacts with the primer combination or the MLPA probe combination at 95 ℃ for 1min and the hybridization reaction is carried out at 60 ℃ for 3 hours.
Wherein the operating conditions for amplifying the hybridization product are: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 10s, annealing at 60 deg.C for 20s, and polymerization at 72 deg.C for 20s, and after 35 cycles, prolonged polymerization at 72 deg.C for 10min, and storing at 4 deg.C.
The sample analysis unit adopts a capillary electrophoresis device, and judges whether the analysis sample is a biological sample susceptible to recurrent abortion or not according to an electrophoresis detection result.
In order to achieve the technical object of the present invention, the present invention further provides a method for detecting recurrent abortion microdeletion or/and microduplication, which uses the above primer combination or MLPA probe combination or kit or system to detect a biological sample to be detected, comprising:
extracting a whole genome DNA solution of a biological sample to be detected, and performing denaturation treatment to obtain denatured genome DNA;
carrying out hybridization and ligation reaction on the MLPA probe combination prepared by the primer combination or the MLPA probe combination in the MLPA probe combination or the kit and the deformed genomic DNA to obtain a ligation product;
after the obtained connection product is subjected to PCR reaction, performing capillary electrophoresis on the PCR amplification product to obtain a capillary electrophoresis result;
and (3) analyzing the capillary electrophoresis result, and judging whether the biological sample to be detected is a recurrent abortion microdeletion or/and a micro-repeat sample.
Wherein the denaturation treatment is carried out by treating the DNA solution at 98 ℃ for 5 minutes.
Wherein the reaction solution of the hybridization ligation reaction is as follows:
wherein the reaction conditions of the hybridization and ligation reaction are that the reaction is carried out for 1min at 95 ℃, and the hybridization reaction is carried out for 3 hours at 60 ℃.
Wherein the reaction solution of the PCR reaction is as follows:
wherein the reaction conditions of the PCR reaction are as follows: pre-denaturation at 95 deg.C for 5min, denaturation at 95 deg.C for 10s, annealing at 60 deg.C for 20s, and polymerization at 72 deg.C for 20s, and after 35 cycles, prolonged polymerization at 72 deg.C for 10min, and storing at 4 deg.C.
Advantageous effects
1. The method can simultaneously detect the microdeletion or/and the microduplication of a plurality of sites in a biological sample in one tube by utilizing a primer combination or an MLPA probe or a gene chip or a kit or a system with the nucleotide sequence shown in SEQ ID NO.1-23, thereby judging whether the biological sample is a recurrent abortion sample of the microdeletion or/and the microduplication, and the method has the advantages of good specificity, sensitive reaction, short detection time, simple method, capability of detecting deletion/insertion of large chromosomes from point mutation, low cost and high clinical application value.
2. The primer combination provided by the invention further enriches the pathogenic gene map of recurrent abortion and provides scientific basis for early pathogenic gene screening research and intervention treatment research of recurrent abortion.
Drawings
FIG. 1 is a graph showing the results of capillary electrophoresis conducted in example 2 of the present invention;
FIG. 2 is a system for rapid screening of biological samples susceptible to recurrent abortion, provided in example 3 of the present invention.
Detailed Description
The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Unless otherwise indicated, the techniques used in the examples are conventional and well known to those skilled in the art, and may be performed according to the third edition of the molecular cloning, laboratory Manual, or related products, and the reagents and products used are also commercially available. Various procedures and methods not described in detail are conventional methods well known in the art, and the sources, trade names, and components of the reagents used are indicated at the time of first appearance, and the same reagents used thereafter are the same as those indicated at the first appearance, unless otherwise specified.
In addition, it should be noted that the primer combination, the MLPA probe, the kit, the gene chip, the system and the method for screening a biological sample prone to recurrent abortion provided by the present invention are all completed by the inventors of the present application after hard creative work and optimization work.
The features and advantages described in the methods section herein before for screening biological samples susceptible to recurrent pregnancy loss are equally applicable to a system or kit for screening biological samples susceptible to recurrent pregnancy loss and will not be described in further detail herein.