RPA-CRISPR / Cas12a detection system for genetic detection before blastocyst cavity fluid free DNA single-tube double-gene non-invasive embryo implantation

Abstract

The invention discloses an RPA-CRISPR / Cas12a detection system for genetic detection before blastocyst cavity fluid free DNA single-tube double-gene non-invasive embryo implantation, and belongs to the technical field of genetic detection. The biradical consists of a TBC1D3 gene and a TSPY1 gene; the RPA-CRISPR / Cas12a detection system comprises an RPA (recombinase polymerase amplification) reaction system and a CRISPR / Cas12a reaction system. According to the invention, TSPY1 and TBC1D3 genes are selected as target genes for research, a dual RPA-CRISPR / cas12a detection system is established, embryo preliminary screening of sexual linkage genetic disease patients before implantation can be rapidly completed, a new detection platform is provided for sexual linkage genetic family embryo selection, a foundation is laid for further genetic prenatal breeding, time, manpower and material resource costs are reduced, and the method has a good application prospect. And wounds are reduced.

Description

Technical Field

[0001] The present invention relates to the field of genetic detection technology, and in particular to an RPA-CRISPR / Cas12a detection system for blastocyst cavity fluid free DNA single-tube dual-gene non-invasive pre-implantation genetic detection. Background Art

[0002] Preimplantation genetic testing (PGT) is a detection technology that identifies whether the embryo's genetic material is abnormal during the assisted reproduction process. Currently, it mainly includes polar body biopsy, blastomere biopsy and blastocyst trophoblast cell biopsy. PGT is widely used in the screening of preimplantation aneuploidy (PGT-A), single gene diseases (PGT-M), and chromosome structural rearrangement (PGT-SR), selectively avoiding "problem" embryos and improving clinical pregnancy outcomes. PGT originated in 1990 when Handyside et al. first used PCR technology to detect repetitive sequences on the Y chromosome to screen healthy female embryos, improving the pregnancy outcomes of couples at high risk of sex-linked genetic diseases. Over the past 30 years, advances in whole genome amplification technology have significantly improved the accuracy and reliability of PGT, and more and more high-risk couples are choosing PGT.

[0003] At present, PGT commonly uses invasive biopsy of trophectoderm (TE). Trophoblast cell biopsy relies on expensive equipment and professional and experienced technicians. It is complicated and invasive, and may affect embryo vitality, inconsistent diagnosis of trophoblast cells and inner cell mass, and mosaic embryos. Seeking a minimally invasive or non-invasive sampling method with a short detection cycle and simple operation has practical clinical significance. Studies have shown that both blastocyst cavity fluid (BF) and spent blastocyst culture medium (SECM) contain genetic material. Vera-Rodriguez et al. found that 86% to 94% of free DNA in the culture medium was contaminated by maternal DNA. The research team of Peking University Third Hospital also confirmed the high rate of maternal contamination of free DNA in blastocyst culture medium through DNA methylation sequencing, and there is currently no effective way to solve this contamination.

[0004] Blastocyst cavity fluid is formed by embryonic autocrine during blastocyst development and contains DNA fragments that can be stably amplified. Studies have found that blastocyst cavity fluid can be successfully separated from blastocysts through non-invasive or minimally invasive means, such as intracytoplasmic sperm injection, and genetic analysis can be performed after amplification of free DNA in blastocyst cavity fluid. A research team compared the genetic information of blastocyst cavity fluid and TE cells and found that free DNA in blastocyst cavity fluid can predict the ploidy of embryos. A study conducted second-generation sequencing analysis on free DNA in blastocyst cavity fluid, and high-quality (whole genome amplification) WGA products were obtained. The single gene sequences contained in the blastocyst cavity fluid are highly representative, and the overall amplification efficiency is as high as 84%, further indicating that free DNA in blastocyst cavity fluid is expected to become a source of non-invasive PGT genetic information. Some scholars believe that blastocyst cavity fluid DNA is a safer and simpler PGT diagnostic method to replace TE biopsy, with potential cost advantages and reducing the total cost of PGT. Therefore, the development of trace DNA amplification and detection technology in blastocyst cavity fluid has certain clinical application prospects.

[0005] There are many types of single gene genetic diseases in my country, and preimplantation genetic testing (PGT-M) can effectively cut off the transmission pathway of single gene diseases. It is mainly suitable for autosomal inheritance, X-linked inheritance, and Y-linked inheritance with clear gene mutation diagnosis or clear gene linkage markers in the family. In 1990, Handyside et al. used PCR technology to detect repetitive sequences on chromosome Y to screen healthy female embryos for the first time, improving the pregnancy outcomes of couples at high risk of sex-linked genetic diseases. In 2013, Palini et al. amplified the TBC1D3 (on chromosome 17) and TSPY1 (on chromosome Y) genes in 29 blastocyst cavity fluid samples by quantitative PCR, confirming that the genetic information contained in the blastocyst cavity fluid has the potential to identify sex-linked genetic diseases, providing the possibility of screening couples carrying sex-linked disease genes.

[0006] PCR is a technology commonly used for gene amplification and can play an important role in preimplantation genetic testing. However, PCR technology has strict requirements for temperature control, is time-consuming and requires specialized operation. In 2006, Piepenburg et al. first used single-stranded DNA binding protein, recombinase (T4 UvsX), and strand displacement DNA polymerase (Bsu) to establish a recombinase polymerase amplification (RPA) amplification system. This system can exponentially amplify target genes at a constant temperature of 37–42°C without relying on large-scale amplification equipment. It has the advantages of simplicity, rapidity, high sensitivity, high specificity, and multi-primer compatibility. It is an experimental technology that is expected to replace PCR.

[0007] With the development and maturity of clustered regularly interspaced short palindromic repeats (CRISPR) technology, after Zhang Feng's team developed the SHERLOCK platform in the field of molecular detection using the cas12 enzyme, other research teams have successively developed the DETECTR rapid detection platform. Cas12 (Cpf1) is a type 2 CRISPR effector protein of the CRISPR system and is a nuclease guided by a single RNA. Under the guidance of crRNA, the protein can specifically bind to the target sequence by recognizing the PAM sequence rich in AT structure, and then specifically cut the target sequence, and then non-specifically cut the surrounding single-stranded DNA. This nuclease activity is called cis- and trans-cutting activity. Based on the characteristics of the above two systems, the advantages of trace nucleic acid templates and high sensitivity of RPA-CRISPR / cas12a technology make up for the shortcomings of existing methods and are widely used in instant visual detection of food safety, microorganisms, parasites, viruses and other aspects. Summary of the invention

[0008] The purpose of the present invention is to provide an RPA-CRISPR / Cas12a detection system for blastocyst cavity fluid free DNA single-tube dual-gene non-invasive preimplantation genetic detection to solve the problems existing in the above-mentioned prior art.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] One of the technical solutions of the present invention is a RPA-CRISPR / Cas12a detection system for non-invasive preimplantation genetic detection of blastocyst cavity fluid free DNA in a single tube with two genes, wherein the two genes are TBC1D3 gene and TSPY1 gene;

[0011] The RPA-CRISPR / Cas12a detection system includes an RPA reaction system and a CRISPR / Cas12a reaction system;

[0012] The RPA reaction system includes TSPY1-F1, TSPY1-R1, TBC1D3-F1 and TBC1D3-R1;

[0013] The CRISPR / Cas12a reaction system includes TBC1D3-crRNA and TSPY1-crRNA;

[0014] The nucleotide sequence of TSPY1-F1 is shown in SEQ ID NO.1, the nucleotide sequence of TSPY1-R1 is shown in SEQ ID NO.2, the nucleotide sequence of TBC1D3-F1 is shown in SEQ ID NO.3, and the nucleotide sequence of TBC1D3-R1 is shown in SEQ ID NO.4;

[0015] The nucleotide sequence of the TBC1D3-crRNA is shown in SEQ ID NO.5, and the nucleotide sequence of the TSPY1-crRNA is shown in SEQ ID NO.6.

[0016] The second technical solution of the present invention is a TBC1D3 and TSPY1 double gene detection method for non-disease diagnosis or treatment purposes, using blastocyst cavity fluid as a detection sample, using DNA in the detection sample as a template, and using the RPA reaction system to perform an amplification reaction;

[0017] The amplified product was added to the CRISPR / Cas12a reaction system, and fluorescence was collected to determine whether TBC1D3 and TSPY1 genes were present in the test sample.

[0018] Based on the above technical solution, the present invention has the following technical effects:

[0019] The present invention selects non-invasive sampling without biopsy cells to maintain the integrity of blastocyst cells, which is superior to previous invasive sampling methods. The blastocyst cavity fluid is formed by autosecretion of trophoblast cells and inner cell mass during the development of the blastocyst. The blastocyst cavity fluid is aspirated using a microinjection needle to avoid paternal DNA contamination and maternal DNA contamination of granulosa cells. There are stably amplifiable DNA fragments in the blastocyst cavity fluid, which can be used as a source of genetic information for non-invasive preimplantation genetic testing of embryos. The RPA-CRISPR / Cas12a detection system is expected to become a new preimplantation genetic diagnosis technology. At present, this technology has not been reported in this field.

[0020] The present invention selects TSPY1 and TBC1D3 genes as target genes for research, and establishes a dual RPA-CRISPR / cas12a detection system, which can quickly complete the embryo screening of patients with sex-linked genetic diseases before implantation, providing a new detection platform for embryo selection in sex-linked genetic families, laying the foundation for further genetic eugenics, reducing time, manpower and material costs, and reducing trauma. The molecular detection platform of CRISPR is becoming increasingly mature and perfect, and continuing to explore the application of this detection technology in single-gene diseases before embryo implantation can provide a more efficient and high-quality detection platform for clinical assisted reproduction. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1.Schematic diagram of fluorescence visualization of the Cas12a:crRNA (25nm:25nm) reaction system for detecting the TBC1D3-TSPY1 dual gene in the blastocyst cavity fluid. The TBC1D3 primer pair is F1+R1, and the TSPY1 amplification primer pair is F1+R1.

[0023] Figure 2 .and Figure 1 Corresponding TBC1D3 gene Cas12a fluorescence detection curve.

[0024] Figure 3 .and Figure 1 Corresponding TSPY1 gene Cas12a fluorescence detection curve. DETAILED DESCRIPTION

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0027] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0028] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0029] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0030] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0031] The embodiment of the present invention provides an RPA-CRISPR / Cas12a detection system for non-invasive preimplantation genetic detection of blastocyst cavity fluid free DNA in a single tube with two genes, wherein the two genes are TBC1D3 gene and TSPY1 gene;

[0032] The RPA-CRISPR / Cas12a detection system includes an RPA reaction system and a CRISPR / Cas12a reaction system;

[0033] The RPA reaction system includes TSPY1-F1, TSPY1-R1, TBC1D3-F1 and TBC1D3-R1;

[0034] The CRISPR / Cas12a reaction system includes TBC1D3-crRNA and TSPY1-crRNA;

[0035] The nucleotide sequence of TSPY1-F1 is shown in SEQ ID NO.1, the nucleotide sequence of TSPY1-R1 is shown in SEQ ID NO.2, the nucleotide sequence of TBC1D3-F1 is shown in SEQ ID NO.3, and the nucleotide sequence of TBC1D3-R1 is shown in SEQ ID NO.4;

[0036] The nucleotide sequence of the TBC1D3-crRNA is shown in SEQ ID NO.5, and the nucleotide sequence of the TSPY1-crRNA is shown in SEQ ID NO.6.

[0037] The embodiment of the present invention also provides a TBC1D3 and TSPY1 double gene detection method for non-disease diagnosis or treatment purposes, using blastocyst cavity fluid as a detection sample, using DNA in the detection sample as a template, and using the RPA reaction system to perform an amplification reaction;

[0038] The amplified product was added to the CRISPR / Cas12a reaction system, and fluorescence was collected to determine whether TBC1D3 and TSPY1 genes were present in the test sample.

[0039] In some specific embodiments, the RPA reaction system includes: 29.4 μL Abuffer, 1.5 μL 10 μM forward / reverse primers, 2.5 μL MgOAc and 2 μL DNA template, which are adjusted to 50 μL RPA reaction system with ddH2O;

[0040] The sequences of the forward / reverse primers are shown in SEQ ID NO.1 to SEQ ID NO.4.

[0041] In some specific embodiments, the reaction condition of the amplification reaction is incubation at 39°C for 20 min.

[0042] In some specific embodiments, the CRISPR / Cas12a reaction system includes: 0.25 μL 2 μM cas12a, 0.5 μL 1 μM crRNA, 2 μL 5 μM ssDNA-reporter, 2 μL 10×reaction buffer and 2 μL target DNA, and enzyme-free nucleic acid water is added to adjust the system to 20 μL.

[0043] In some specific embodiments, the method for determining whether TBC1D3 and TSPY1 genes exist in the test sample is: performing fluorescence detection on the TBC1D3 and TSPY1 genes in the test sample respectively;

[0044] When both TBC1D3 and TSPY1 in the same test sample show fluorescence, it indicates the presence of TBC1D3 and TSPY1 genes;

[0045] When only TBC1D3 showed fluorescence in the same test sample, it indicated the presence of TBC1D3;

[0046] When only TSPY1 showed fluorescence in the same test sample, it indicated the presence of TSPY1;

[0047] When neither TBC1D3 nor TSPY1 in the same test sample showed fluorescence, it indicated that the TBC1D3 and TSPY1 genes were absent.

[0048] When both TBC1D3 and TSPY1 genes in the test sample are negative, the result is uninterpretable;

[0049] When any gene in the blastocyst cavity fluid is detected, it is a readable result.

[0050] PCR, RPA (recombinase polymerase amplification) and LAMP (loop-mediated isothermal amplification) are the main amplification methods currently widely used in molecular diagnosis. PCR is the gold standard for nucleic acid detection, but the technology relies on strict temperature control and large-scale amplification instruments. LAMP technology has excellent amplification efficiency and economic benefits, but compared with RPA, it has more complex primer pair design and higher reaction temperature (65°C), which is prone to false positive results from self-primers or primer-primer hybridization, and there is a high risk of contamination. RPA is a simple and applicable molecular diagnostic technology. Compared with PCR, it does not rely on thermal cycling processes and large-scale amplification equipment, and can be rapidly amplified at room temperature. In addition, the technology has significant tolerance to the presence of inhibitors, providing a new technical solution for the development of instant diagnosis platforms.

[0051] CRISPR / cas12a is a widely used detection technology for molecular diagnosis, which can convert molecular information into visual signals, such as lateral flow test strips, visual fluorescence, visual nylon membrane readings, etc., and has the advantages of rapid, sensitive, and specific instant diagnosis. The molecular diagnostic platforms currently developed mainly use single signal detection, but it is still a challenge to achieve CRISPR / Cas multiple biosensors and successfully integrate them into microchips for large-scale detection. To achieve the detection of trace amounts of DNA, it is aimed to continuously improve the detection sensitivity of the CRISPR system and the RPA amplification system. The main methods of the CRISPR system currently include inducing conformational changes in cas12a, photoactivated crRNA, changing the spatial structure of crRNA, 3'-end extension of ssDNA activators, and developing spherical nucleic acid (SNA) reporter genes to increase the trans-cleavage activity of cas12a, and successfully combine electroluminescent biosensors to carry out bedside detection. The present invention utilizes the advantages of the RPA-CRISPR / cas12a fluorescence detection system, such as being non-invasive and simple, to achieve rapid detection of the TSPY1 and TBC1D3 genes in the blastocyst cavity fluid within one hour, thus providing the possibility for fresh cycle transplantation of blastocysts.

[0052] The long arm of the Y chromosome (Yq) contains many amplification and palindromic sequences, which make it easy to self-recombination during spermatogenesis, and therefore easy to chromosomal microdeletion. The key link in screening sex-linked genetic diseases is to detect the Y chromosome, which is also one of the important indications for preimplantation genetic testing. Due to the characteristics of sex-linked genetic diseases, the present invention selects TSPY1 and TBC1D3 genes as target genes for research, and sets up a dual RPA-CRISPR / cas12a detection system, which can quickly complete the embryo screening of patients with sex-linked genetic diseases before implantation, and provides a new detection platform for embryo selection of sex-linked genetic families, laying the foundation for further genetic eugenics, reducing time, manpower and material costs, and reducing trauma. The molecular detection platform of CRISPR is becoming increasingly mature and perfect, and continuing to explore the application of this detection technology in single-gene diseases before embryo implantation can provide a more efficient and high-quality detection platform for clinical assisted reproduction.

[0053] Example 1

[0054] 1Construction of RPA-CRISPR / cas12a detection system

[0055] RPA system construction: Primers were designed based on the gene sequences of TSPY1 gene (Gene ID: 7258) and TBC1D3 (Gene ID: 729873) retrieved from NCBI. Forward primer F1 and reverse primer R1 were designed for TSPY1 gene; forward primer F1 and reverse primer R1 were designed for TBC1D3 gene.

[0056] The single-tube dual-gene RPA system was established, including: 29.4 μL Abuffer, 1.2 μL forward / reverse primers (10 μM), 2.5 μL MgOAc, 2 μL DNA template, and adjusted to 50 μL with ddH2O. After mixing, incubate in a metal bath at 39°C for 20 minutes. The RPA amplification products were purified by a mixture of Tris-saturated phenol / chloroform / isoamyl alcohol (25:24:1), and the primer specificity of the target gene was identified by 2% agarose gel electrophoresis and Sanger sequencing.

[0057] RPA system optimization: optimize the concentration of dual-gene primers, and set the single-gene unidirectional primer concentration gradient to 0.15μM, 0.2μM, 0.24μM, and 0.3μM, respectively. The final amplification results were purified and analyzed by 2% agarose gel and CRISPR / cas12a fluorescence. The results showed that in the single-tube dual-gene RPA detection process, the fluorescence intensity of the TBC1D3 gene primer concentration was 0.24μM higher than that of the primer concentration of 0.15μM, and only the difference between the two was statistically significant (p<0.05). The fluorescence intensity of the TSPY1 gene primer concentration was 0.3μM higher than that of the primer concentration of 0.15μM, and only the difference between the two was statistically significant (p<0.05). The present invention sets the primer concentration to 0.24μM as a condition for subsequent single-tube dual-gene RPA reactions of clinical samples.

[0058] Construction of CRISPR / Cas12a system: 2μL RPA amplification product was added to the cas12a system to establish a system containing 0.25μL cas12a (2μM), 0.5μL crRNA (1μM), 2μL ssDNA-FQ reporter (5μM), 2μL 10x reaction buffer, and 13.25μL ddH2O. The cross-reaction of crRNA of TSPY1 and TBC1D3 genes was verified, and the fluorescence was finally detected by the fluorescence quantitative PCR instrument of the FAM channel. The reaction temperature was 37°C, and the fluorescence was collected every 1min for 40min. After the fluorescence collection was completed, it was placed in the gel imaging system for fluorescence visualization.

[0059] Optimization of CRISPR / Cas12a system: Set Cas12a enzyme (12nM, 25nM, 50nM, 100nM) and crRNA concentration (10nM, 25nM, 50nM, 100nM) to perform orthogonal tests on TSPY1 and TBC1D3 to optimize the optimal ratio of Cas12a and crRNA. The results showed that the Cas12a enzyme concentrations of TSPY1 and TBC1D3 genes obtained better fluorescence effects under the two reaction systems of Cas12a / crRNA (25nm, 25nm) and Cas12a / crRNA (50nm, 50nm), and there was no statistically significant difference in fluorescence intensity between the two systems. In order to improve the cost-effectiveness of detection, the Cas12a / crRNA (25nm, 25nm) reaction system was used to detect TSPY1 and TBC1D3 genes.

[0060] Example 2

[0061] 1. Collection of blastocoel fluid

[0062] In order to explore the genetic information value of blastocyst cavity fluid and further evaluate the clinical detection efficiency of RPA-CRISPR / cas12a technology, this example uses the optimized RPA-CRISPR / cas12a technology to detect abandoned blastocysts. A total of 23 cases of abandoned intracytoplasmic sperm injection-formed blastocysts or fully hatched blastocysts (5CC or 6CC) on day 6 or 7 were collected, and the blastocyst cavity fluid was collected by puncturing the trophoblast cell gap with a microinjection needle, and the remaining blastocysts were placed in sterile, enzyme-free 200μL EP tubes for cryopreservation.

[0063] 2. Blastocoel fluid detection

[0064] The RPA amplification primers and crRNA sequences of TBC1D3 and TSPY1 genes are shown in Table 1. Before RPA isothermal amplification, the blastocyst cells were lysed at 95°C for 10 minutes and centrifuged at 3000r / min for 3 minutes. The RPA system was then added to the EP tubes containing the blastocyst cavity fluid and blastocyst cells, respectively. The metal bath was incubated at 39°C for 20 minutes, and 2μLRPA amplification products were added to the CRISPR / cas12a detection system and placed in the ABI 7500 fluorescence quantitative PCR instrument to collect fluorescence. The collection time at 37°C was 40 minutes, and the collection was performed once every 1 minute. After that, the gel imaging system (iBright 1500) was used for photography under green light, and the green light setting condition was Filter Se: Green (Ex: 488, Em: 528).

[0065] Fluorescence detection was performed on the TBC1D3 and TSPY1 genes in the test samples respectively;

[0066] When both TBC1D3 and TSPY1 in the same test sample show fluorescence, it indicates the presence of TBC1D3 and TSPY1 genes;

[0067] When only TBC1D3 showed fluorescence in the same test sample, it indicated the presence of TBC1D3;

[0068] When only TSPY1 showed fluorescence in the same test sample, it indicated the presence of TSPY1;

[0069] When neither TBC1D3 nor TSPY1 in the same test sample showed fluorescence, it indicated that the TBC1D3 and TSPY1 genes were absent.

[0070] When both TBC1D3 and TSPY1 genes in the test sample are negative, the result is uninterpretable;

[0071] When any gene in the blastocyst cavity fluid is detected, it is a readable result.

[0072] Table 1 RPA amplification primers and crRNA sequences of TBC1D3 and TSPY1 genes

[0073]

[0074]

[0075] Single-tube dual-gene RPA reaction system

[0076] The amplification kit for RPA reaction was purchased from Amp Future (Changzhou, China). The reaction components included 29.4 μL Abuffer, 1.5 μL forward / reverse primers (10 μM), 2.5 μL MgOAc, and 2 μL DNA template. The mixture was adjusted to 50 μL RPA reaction system with ddH2O, mixed well, and incubated in a constant temperature device at 39°C for 20 min.

[0077] CRISPR / Cas12a reaction system

[0078] Lba Cas12a (Cpf1) was purchased from Meige Biotechnology Co., Ltd. (Guangzhou, China). The components of the cas12a reaction system included: 0.25 μL cas12a (2 μM), 0.5 μL crRNA (1 μM), 2 μL ssDNA-reporter (5 μM), 2 μL 10xreaction buffer, 2 μL target DNA, and enzyme-free nucleic acid water was added to adjust the system to 20 μL.

[0079] 3. Test results

[0080] The present invention collected 23 discarded fresh blastocysts, and simultaneously collected the blastocyst cavity fluid and the remaining blastocyst cells to verify the reliability of the technology and evaluate the clinical application value of the single-tube dual-gene CRISPR / Cas12a reaction system.

[0081] The detection efficiency of the single-tube dual-gene system of cas12a / crRNA (25nm, 25nm) for TSPY1 and TBC1D3 genes was evaluated. A total of 23 pairs of blastocyst samples were tested using the single-tube dual-gene RPA system. The results are shown in Figures 1 to 3 . In the 23 pairs of blastocysts tested, the TBC1D3 gene detection rate was 87% (20 / 23), and the proportion of negative uninterpretable results in the blastocyst cavity fluid double gene test was only 8.6%. Among the interpretable results, the accuracy of TSPY1 gene detection was 90.5% (19 / 21). It is suggested that the cas12a detection system cas12: crRNA (25nm: 25nm) can be used for the detection of free DNA in the blastocyst cavity fluid.

[0082] In summary, the present invention selects non-invasive sampling without biopsy cells to maintain the integrity of blastocyst cells, which is superior to previous invasive sampling methods. Blastocyst cavity fluid is formed by autosecretion of trophoblast cells and inner cell mass during blastocyst development. The blastocyst cavity fluid is aspirated with a microinjection needle to avoid paternal DNA contamination and maternal DNA contamination of granulosa cells. There are DNA fragments that can be stably amplified in the blastocyst cavity fluid, which can be used as a source of genetic information for non-invasive preimplantation genetic testing of embryos. The RPA-CRISPR / Cas12a detection system is expected to become a new preimplantation genetic diagnosis technology. At present, this technology has not been reported in this field. The present invention combines RPA isothermal amplification technology with CRISPR / Cas12a detection technology. The technology is simple and efficient, and preliminary test results can be obtained within half an hour to one hour, which is conducive to the transplantation of fresh blastocysts, improves clinical efficiency, and can provide an instant detection platform for the diagnosis of sex-linked genetic diseases.

[0083] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A RPA-CRISPR / Cas12a detection system for non-invasive preimplantation genetic testing of blastocyst cavity fluid free DNA in a single tube with two genes, characterized in that: The double genes are TBC1D3 gene and TSPY1 gene; The RPA-CRISPR / Cas12a detection system includes an RPA reaction system and a CRISPR / Cas12a reaction system; The RPA reaction system includes TSPY1-F1, TSPY1-R1, TBC1D3-F1 and TBC1D3-R1; The CRISPR / Cas12a reaction system includes TBC1D3-crRNA and TSPY1-crRNA; The nucleotide sequence of TSPY1-F1 is shown in SEQ ID NO.1, the nucleotide sequence of TSPY1-R1 is shown in SEQ ID NO.2, the nucleotide sequence of TBC1D3-F1 is shown in SEQ ID NO.3, and the nucleotide sequence of TBC1D3-R1 is shown in SEQ ID NO.4; The nucleotide sequence of the TBC1D3-crRNA is shown in SEQ ID NO.5, and the nucleotide sequence of the TSPY1-crRNA is shown in SEQ ID NO.

6.

2. A method for detecting two genes of TBC1D3 and TSPY1 for non-disease diagnosis or treatment purposes, characterized in that: Using the blastocyst cavity fluid as a test sample and the DNA in the test sample as a template, and using the RPA reaction system of claim 1 to perform an amplification reaction; The amplified product is added to the CRISPR / Cas12a reaction system of claim 1, and fluorescence is collected to determine whether TBC1D3 and TSPY1 genes are present in the test sample.

3. The TBC1D3 and TSPY1 double gene detection method according to claim 2, characterized in that: The RPA reaction system includes: 29.4 μL Abuffer, 1.5 μL 10 μM forward / reverse primers, 2.5 μL MgOAc and 2 μL DNA template, which are adjusted to 50 μL RPA reaction system with ddH2O; The sequences of the forward / reverse primers are shown in SEQ ID NO.1 to SEQ ID NO.

4.

4. The TBC1D3 and TSPY1 double gene detection method according to claim 2, characterized in that: The reaction condition of the amplification reaction is incubation at 39° C. for 20 min.

5. The TBC1D3 and TSPY1 double gene detection method according to claim 2, characterized in that: The CRISPR / Cas12a reaction system includes: 0.25 μL 2 μM cas12a, 0.5 μL 1 μM crRNA, 2 μL 5 μM ssDNA-reporter, 2 μL 10×reaction buffer and 2 μL target DNA, and enzyme-free nucleic acid water is added to adjust the system to 20 μL.

6. The TBC1D3 and TSPY1 double gene detection method according to claim 2, characterized in that: The method for determining whether the TBC1D3 and TSPY1 genes are present in the test sample is as follows: performing fluorescence detection on the TBC1D3 and TSPY1 genes in the test sample respectively; When both TBC1D3 and TSPY1 in the same test sample show fluorescence, it indicates the presence of TBC1D3 and TSPY1 genes; When only TBC1D3 showed fluorescence in the same test sample, it indicated the presence of TBC1D3; When only TSPY1 showed fluorescence in the same test sample, it indicated the presence of TSPY1; When neither TBC1D3 nor TSPY1 in the same test sample showed fluorescence, it indicated that the TBC1D3 and TSPY1 genes were absent. When both TBC1D3 and TSPY1 genes in the test sample are negative, the result is uninterpretable; When any gene in the blastocyst cavity fluid is detected, it is a readable result.

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