Nucleic acid combination product and method for detecting short tandem repeat fragments in biological sample DNA

Abstract

The invention belongs to the technical field of assisted reproduction, and relates to a nucleic acid combination product and a method for detecting short tandem repeat fragments in biological sample DNA. The nucleic acid combination product comprises one or more pairs of the following primer pairs: primer pairs with sequences as shown in SEQ ID NO.1 to SEQ ID NO.18, primer pairs with sequences as shown in SEQ ID NO.21 to SEQ ID NO.38, primer pairs with sequences as shown in SEQ ID NO.45 to SEQ ID NO.46, primer pairs with sequences as shown in SEQ ID NO.63 to SEQ ID NO.66, primer pairs with sequences as shown in SEQ ID NO.73 to SEQ ID NO.76, primer pairs with sequences as shown in SEQ ID NO.81 to SEQ ID NO.82, and primer pairs with sequences as shown in SEQ ID NO.85 to SEQ ID NO.102. The nucleic acid combination product can detect one or more of 33 STR sites such as Amelogenin and the like, a scheme is provided for epiploid detection of pre-implantation embryos, and meanwhile, abortion embryos can be traced.

Description

Technical Field

[0001] The present application belongs to the field of assisted reproductive technology, and in particular relates to a nucleic acid combination product and a method for detecting short tandem repeat fragments in biological sample DNA. Background Art

[0002] Assisted reproductive technology is an effective clinical technology to solve infertility. According to statistics from Reproductive Biology and Endocrinology, more than 20% of infertile couples must undergo assisted reproductive technology treatment. Improving the birth rate of assisted reproduction has always been a challenge in the field of assisted reproductive medicine. Selecting excellent embryos for transplantation during assisted reproduction is an effective way to increase the birth rate.

[0003] Currently, pre-implantation DNA methylation detection technology is a new generation of embryo screening method. This method is based on the principle of epigenetics and uses second-generation sequencing technology to evaluate the chromosome copy number variation information and DNA methylation modification information of the embryo, thereby selecting embryos with high developmental potential for transplantation. Clinical data show that this technology can significantly increase the birth rate of assisted reproduction and reduce birth defects. During the assisted reproduction process, some embryos become triploid during in vitro culture, that is, the embryo has 69 chromosomes (normal embryos are diploid, with 46 chromosomes).

[0004] In addition, clinical studies have found that some couples who undergo assisted reproductive treatment experience miscarriage after embryo transplantation. By testing the chromosome copy number variation of the aborted tissue, it was found that the chromosome copy number information obtained from the aborted tissue was inconsistent with the chromosome copy number information obtained from the embryo test before embryo implantation. There are several reasons for this situation: 1. The embryo has a copy number variation during its development after transplantation; 2. The embryo is numbered incorrectly during embryo transplantation, resulting in the wrong embryo being transplanted into the uterus; 3. After the embryo is transplanted, the couple has sex, and the aborted embryo does not come from the transplanted embryo. In view of the above situations, it is necessary to trace the aborted embryo.

[0005] However, the current pre-implantation DNA methylation detection technology cannot detect triploidy. Pre-implantation DNA methylation detection requires biopsy of 6-8 blastocysts' trophoblast cells, and the amount of DNA obtained is about 40pg. Due to the current single-cell amplification technology, the amplified DNA will lose the methylation modification information. Therefore, pre-implantation DNA methylation detection cannot use the existing single-cell amplification technology to amplify the starting DNA to expand the amount of DNA template.

[0006] In view of this, this application is hereby filed. Summary of the invention

[0007] One or more embodiments of the present application provide a nucleic acid combination product and a method for detecting short tandem repeat fragments in biological sample DNA. The technical solutions include the following:

[0008] One or more embodiments of the present application provide a nucleic acid combination product, wherein the nucleic acid combination product includes one or more pairs of the following primer pairs:

[0009] Primer pair M1-1, primer pair M1-2, primer pair M1-3, primer pair M1-4, primer pair M1-5, primer pair M1-6, primer pair M2-1, primer pair M2-3 and primer pair M2-4 having sequences as shown in SEQ ID NO.1 to SEQ ID NO.18,

[0010] Primer pair M3-1, primer pair M3-2, primer pair M3-3, primer pair M3-4, primer pair M3-5, primer pair M3-6, primer pair M4-1, primer pair M4-2 and primer pair M4-4 having sequences as shown in SEQ ID NO.21 to SEQ ID NO.38,

[0011] The primer pair M8-3 whose sequences are shown in SEQ ID NO.45 to SEQ ID NO.46,

[0012] The sequences of primer pair M4-3 and primer pair M2-2 are shown in SEQ ID NO.63 to SEQ ID NO.66,

[0013] The sequences of primer pair M9-4 and primer pair M9-5 are shown in SEQ ID NO.73 to SEQ ID NO.76,

[0014] The primer pair M9-9 whose sequences are shown in SEQ ID NO.81 to SEQ ID NO.82,

[0015] The sequences of primer pair M10-1, primer pair M10-2, primer pair M10-3, primer pair M10-4, primer pair M10-6, primer pair M10-8, primer pair M10-9, primer pair M10-10, and primer pair M10-12 are shown in SEQ ID NO.85 to SEQ ID NO.102.

[0016] In some embodiments of the present application, the nucleic acid combination product further includes one or more pairs of the following primer pairs:

[0017] The primer pair M2-5 whose sequences are shown in SEQ ID NO.19 to SEQ ID NO.20,

[0018] Primer pair M5-1, primer pair M5-2 and primer pair M5-4 having sequences as shown in SEQ ID NO.39 to SEQ ID NO.44,

[0019] Primer pair M6-1, primer pair M6-2, primer pair M6-3, primer pair M7-1, primer pair M7-2, primer pair M7-3, primer pair M7-4 and primer pair M7-5 having sequences as shown in SEQ ID NO.47 to SEQ ID NO.62,

[0020] Primer pair M9-1, primer pair M9-2 and primer pair M9-3 having sequences as shown in SEQ ID NO.67 to SEQ ID NO.72,

[0021] The sequences of primer pair M9-6 and primer pair 9-7 are shown in SEQ ID NO.77 to SEQ ID NO.80,

[0022] The primer pair M9-11 whose sequences are shown in SEQ ID NO.83 to SEQ ID NO.84,

[0023] The sequences of primer pair M10-13 are shown as SEQ ID NO.103 to SEQ ID NO.104.

[0024] In some embodiments of the present application, the number of primer pairs in the nucleic acid combination product is 18 pairs to 52 pairs.

[0025] In some embodiments of the present application, at least one primer in the primer pair is labeled with a fluorescent group;

[0026] Optionally, the fluorescent groups labeled on each primer are independently 6-FAM, ROX, HEX or TAMRA.

[0027] One or more embodiments of the present application also provide a detection kit, which includes the nucleic acid combination product.

[0028] In some embodiments of the present application, the detection kit further includes STR PCR reaction reagents.

[0029] One or more embodiments of the present application further provide a method for detecting short tandem repeat fragments in a biological sample DNA, the detection method comprising the step of using the nucleic acid combination product or the detection kit to detect short tandem repeat fragments in the biological sample DNA to be detected.

[0030] In some embodiments of the present application, the detection method comprises the following steps:

[0031] Extracting DNA from the biological sample to be tested, fragmenting the DNA, performing whole genome amplification, and preparing an amplification product;

[0032] The amplification product is used as a template and the nucleic acid combination product is used to perform STR PCR amplification, and the situation of the short tandem repeat fragments in the DNA of the biological sample to be tested is determined according to the obtained STR PCR amplification result.

[0033] In some embodiments of the present application, in the initial reaction system of STR PCR amplification,

[0034] The molar ratio of the primer pair M1-1, the primer pair M1-2, the primer pair M1-3, the primer pair M1-4, the primer pair M1-5, the primer pair M1-6, the primer pair M4-1, the primer pair M4-2 and the primer pair M4-4 is 10±1.0:2.5±1.0:5±1.0:5±1.0:5±1.0:15±1.0:15±1.0:5±1.0:12.5±1.0,

[0035] The molar ratio of the primer pair M2-1, the primer pair M2-3, the primer pair M2-4, the primer pair M2-5, the primer pair M3-1, the primer pair M3-2, the primer pair M3-3, the primer pair M3-4, the primer pair M3-5 and the primer pair M3-6 is 5±1.0: 15±1.0: 2.5±1.0: 30±1.0: 2.5±1.0: 5±1.0: 10±1.0: 15±1.0: 12.5±1.0: 5±1.0,

[0036] The molar ratio of the primer pair M5-1, the primer pair M8-3, the primer pair M4-3 and the primer pair M2-2 is 5±1.0:37.5±1.0:25±1.0:12.5±1.0,

[0037] The molar ratio of the primer pair M6-1, the primer pair M6-2, the primer pair M6-3, the primer pair M7-1, the primer pair M7-2, the primer pair M7-3, the primer pair M7-4, the primer pair M7-5, the primer pair M10-6, the primer pair M10-4, the primer pair M10-2 and the primer pair M10-1 is 5±1.0:15±1.0:37.5±1.0:5±1.0:7.5±1.0:8.75±1.0:50±1.0:50±1.0:10±1.0:10±1.0:15±1.0:10±1.0,

[0038] The molar ratio of the primer pair M9-11, the primer pair M9-3, the primer pair M9-2, the primer pair M9-7, the primer pair M9-9 and the primer pair M10-12 is 10±1.0:2.5±1.0:10±1.0:5±1.0:25±1.0:37.5±1.0,

[0039] The molar ratio of the primer pair M9-6, the primer pair M9-1 and the primer pair M5-2 is 10±1.0:10±1.0:10±1.0,

[0040] The molar ratio of the primer pair M9-4, the primer pair M9-5 and the primer pair M5-4 is 10±1.0:10±1.0:10±1.0,

[0041] The molar ratio of the primer pair M10-8, the primer pair M10-10, the primer pair M10-3, the primer pair M10-9 and the primer pair M10-13 is 10±1.0:10±1.0:10±1.0:5±1.0:15±1.0;

[0042] In each of the primer pairs, the molar ratio of the forward primer to the reverse primer is 1±0.5:1±0.5.

[0043] In some embodiments of the present application, the biological sample to be tested is a cell sample and a blood sample; optionally, the number of cells in the cell sample is 2-10.

[0044] In some embodiments of the present application, the biological sample to be tested is from a pre-implantation embryo; optionally, the biological sample to be tested is from a trophoblast.

[0045] The details of one or more embodiments of the present application are set forth in the description which follows, and other features, objects, and advantages of the present application will be apparent from the description and its claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0047] Figures 1 to 8 The detection results corresponding to Tube1 to Tube8 in Example 1;

[0048] Figures 9 to 17 The detection results corresponding to Tube1 to Tube8 in Example 2;

[0049] Figures 18 to 25 The detection results corresponding to Tube1 to Tube8 in Example 3;

[0050] Fig.26 It is the test result of Example 4;

[0051] Fig. 27 It is the test result of Example 5;

[0052] Fig.28 It is the test result of Example 6;

[0053] Figure 29 to Figure 32 These are the STR results of the father, the mother, and their normal diploid embryos, triploid embryos, and Sanqin diploid embryos. DETAILED DESCRIPTION

[0054] The present application will be further described in detail below in conjunction with the accompanying drawings, embodiments and examples. It should be understood that these embodiments and examples are only used to illustrate the present application and are not used to limit the scope of the present application. The purpose of providing these embodiments and examples is to make the understanding of the disclosure of the present application more thorough and comprehensive. It should also be understood that the present application can be implemented in many different forms, is not limited to the embodiments and examples described herein, and those skilled in the art can make various changes or modifications without violating the connotation of the present application, and the equivalent form obtained also falls within the protection scope of the present application. In addition, in the description below, a large number of specific details are given in order to provide a more comprehensive understanding of the present application, and it should be understood that the present application can be implemented without one or more of these details.

[0055] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing implementation modes and embodiments and are not intended to limit this application.

[0056] the term

[0057] Unless otherwise specified or incompatible herewith, the terms and phrases used herein shall have the following meanings:

[0058] The terms "and / or", "or / and", and "and / or" used in this article include any one of two or more related listed items, and also include any and all combinations of related listed items, and the arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or", "or / and", and "and / or", it should be understood that in this application, the technical solution undoubtedly includes technical solutions that are all connected by "logical and", and undoubtedly includes technical solutions that are all connected by "logical or". For example, "A and / or B" includes three parallel solutions of A, B and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, the technical solution that is all connected by "logical OR"), and also includes any and all combinations of A, B, C, and D, that is, the combination of any two or any three of A, B, C, and D, and also includes the combination of four of A, B, C, and D (that is, the technical solution that is all connected by "logical AND").

[0059] In the present application, "plurality", "multiple", "multiple times", "multiples", etc., unless otherwise specified, refer to a number greater than 2 or equal to 2. For example, "one or more" means one or greater than or equal to two.

[0060] As used herein, "combination thereof", "any combination thereof", "any combination thereof" etc. include all suitable combinations of any two or more of the listed items.

[0061] Herein, the “suitable” mentioned in “suitable combination”, “suitable method”, “any suitable method”, etc., shall be based on the ability to implement the technical solution of this application, solve the technical problems of this application, and achieve the expected technical effects of this application.

[0062] Herein, “preferred”, “better”, “more preferred” and “suitable” are merely used to describe implementation methods or examples with better effects, and it should be understood that they do not constitute limitations on the scope of protection of this application.

[0063] In the present application, "further", "further", "particularly" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of the present application.

[0064] In this application, "optionally", "optional", and "optional" mean optional or dispensable, that is, any one of the two parallel schemes of "yes" or "no". If multiple "options" appear in a technical solution, unless otherwise specified and there is no contradiction or mutual restriction, each "optional" is independent.

[0065] In the present application, the terms "first", "second", "third", "fourth", etc. in "the first aspect", "the second aspect", "the third aspect", "the fourth aspect", etc. are used only for descriptive purposes and cannot be understood as indicating or implying relative importance or quantity, nor can they be understood as implicitly indicating the importance or quantity of the indicated technical features. Moreover, "first", "second", "third", "fourth", etc. only serve the purpose of non-exhaustive enumeration and description, and it should be understood that they do not constitute a closed limitation on quantity.

[0066] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0067] In the present application, when it comes to numerical intervals (i.e., numerical ranges), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval, and includes the two numerical endpoints (i.e., the minimum and maximum values) of the numerical range, and each numerical value between the two numerical endpoints. Unless otherwise specified, when the numerical interval only refers to the integers within the numerical interval, it includes the two endpoint integers of the numerical range, and each integer between the two endpoints. In this article, it is equivalent to directly listing each integer, such as t is an integer selected from 1 to 10, indicating that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0068] The temperature parameters in this application, unless otherwise specified, are allowed to be either constant temperature treatment or to vary within a certain temperature range. It should be understood that the constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument. Fluctuations within the range of ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are allowed.

[0069] In the present application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass volume percentage.

[0070] All documents mentioned in this application are cited as references in this application, just as each document is cited as reference separately. Unless they conflict with the application purpose and / or technical solution of this application, the cited documents involved in this application are cited with all contents and all purposes. When the cited documents are involved in this application, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the cited documents are involved in this application, the examples and preferred methods of the cited relevant technical features can also be incorporated into this application as references, but are limited to the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, the present application shall prevail or be modified adaptively according to the description of this application.

[0071] In a first aspect of an embodiment of the present application, a nucleic acid combination product is provided, wherein the nucleic acid combination product comprises one or more pairs of the following primer pairs:

[0072] Primer pair M1-1, primer pair M1-2, primer pair M1-3, primer pair M1-4, primer pair M1-5, primer pair M1-6, primer pair M2-1, primer pair M2-3 and primer pair M2-4 having sequences as shown in SEQ ID NO.1 to SEQ ID NO.18,

[0073] Primer pair M3-1, primer pair M3-2, primer pair M3-3, primer pair M3-4, primer pair M3-5, primer pair M3-6, primer pair M4-1, primer pair M4-2 and primer pair M4-4 having sequences as shown in SEQ ID NO.21 to SEQ ID NO.38,

[0074] The primer pair M8-3 whose sequences are shown in SEQ ID NO.45 to SEQ ID NO.46,

[0075] The sequences of primer pair M4-3 and primer pair M2-2 are shown in SEQ ID NO.63 to SEQ ID NO.66,

[0076] The sequences of primer pair M9-4 and primer pair M9-5 are shown in SEQ ID NO.73 to SEQ ID NO.76,

[0077] The primer pair M9-9 whose sequences are shown in SEQ ID NO.81 to SEQ ID NO.82,

[0078] The sequences of primer pair M10-1, primer pair M10-2, primer pair M10-3, primer pair M10-4, primer pair M10-6, primer pair M10-8, primer pair M10-9, primer pair M10-10, and primer pair M10-12 are shown in SEQ ID NO.85 to SEQ ID NO.102.

[0079] The nucleic acid combination product provided in the present application is used to detect one or more of 33 STR loci such as Amelogenin, D3S1358, D1S1656, etc., to provide a solution for euploidy detection of pre-implantation embryos, and at the same time can trace aborted embryos.

[0080] The nucleic acid combination product provided in the present application includes 1 pair, 2 pairs, 3 pairs, 4 pairs...33 pairs of the above-mentioned 33 pairs of primer pairs, optionally includes multiple pairs of the above-mentioned 33 pairs of primer pairs, and further optionally includes the above-mentioned 33 pairs of primer pairs.

[0081] In some examples, the nucleic acid combination product further includes one or more of the following primer pairs:

[0082] The primer pair M2-5 whose sequences are shown in SEQ ID NO.19 to SEQ ID NO.20,

[0083] Primer pair M5-1, primer pair M5-2 and primer pair M5-4 having sequences as shown in SEQ ID NO.39 to SEQ ID NO.44,

[0084] Primer pair M6-1, primer pair M6-2, primer pair M6-3, primer pair M7-1, primer pair M7-2, primer pair M7-3, primer pair M7-4 and primer pair M7-5 having sequences as shown in SEQ ID NO.47 to SEQ ID NO.62,

[0085] Primer pair M9-1, primer pair M9-2 and primer pair M9-3 having sequences as shown in SEQ ID NO.67 to SEQ ID NO.72,

[0086] The sequences of primer pair M9-6 and primer pair 9-7 are shown in SEQ ID NO.77 to SEQ ID NO.80,

[0087] The primer pair M9-11 whose sequences are shown in SEQ ID NO.83 to SEQ ID NO.84,

[0088] The sequences of primer pair M10-13 are shown as SEQ ID NO.103 to SEQ ID NO.104.

[0089] The nucleic acid combination product of the present application is also used to detect one or more of 19 STR loci such as Penta D, D6S477, D22-GATA198B05, and accordingly, includes 1 pair, 2 pairs, 3 pairs, 4 pairs...19 pairs of the above 19 pairs of primer pairs, and optionally includes multiple pairs of the above 19 pairs of primer pairs, and further optionally, includes 19 pairs of the above 19 pairs of primer pairs.

[0090] In some examples, the number of primer pairs in the nucleic acid combination product is 18 pairs to 52 pairs, for example, 18 pairs, 19 pairs, 20 pairs, ... 52 pairs. Accordingly, the nucleic acid combination product can be used to detect 52 STR loci such as Amelogenin, D3S1358, D1S1656, Penta D, D6S477, D22-GATA198B05, etc.

[0091] At present, for euploidy, sample traceability detection mainly uses STR technology, which analyzes the 18 short tandem repeat sequences on the genome (which can easily lead to bias in the results, especially when the amount of DNA input is reduced, the number of effective sites obtained in each test is too low, making it impossible to obtain test results), to determine whether the sample is a uniparent diploid, triploid, tetraploid or other euploid situation, and the embryo can be traced. In conventional STR testing (large amounts of DNA samples), it is required that the number of effective STR sites detected for each sample each time must be greater than or equal to 10, and the test is considered a valid test. However, the current STR technology requires a large amount of starting DNA, and the DNA required for each reaction reaches the microgram level. This makes it impossible to perform STR technology testing when there are only picograms of DNA samples (1 microgram equals 10 6Picograms). Since the biopsy sample before embryo implantation has only 6-8 cells and about 50 picograms of DNA, for the embryonic cells after biopsy, it is necessary to first perform single cell isothermal amplification to expand the template amount of DNA, so that the template amount of DNA reaches the microgram level, and then perform STR detection, and the amplified DNA template can also be used for traditional pre-embryo implantation genetic testing (PGT-A). However, the pre-embryo implantation DNA methylation detection method, as a more effective embryo screening method, will gradually replace the existing pre-embryo implantation genetic testing method. However, if the embryo undergoes pre-implantation DNA methylation detection, STR detection can no longer be performed. This is because, for DNA methylation detection, the DNA after single cell isothermal amplification cannot be used as a template, so the DNA after single cell isothermal amplification will lose the methylation modification information on the original DNA, so DNA methylation detection can only be performed with the original 6-8 cell DNA as a template. The technical challenge currently faced in the clinic is that if the pre-implantation embryo undergoes DNA methylation detection, STR detection can no longer be performed. Therefore, the present application can solve this clinical problem from two directions and provides a stable and feasible clinical solution. Direction 1: Divide the DNA template of 1-10 cells into two parts, and by adjusting the ratio of the two parts, one part can be used for DNA methylation detection and the other part can be used for STR detection, and ensure the stability of the DNA methylation results and the stability of the STR detection results; Direction 2: By increasing the number of sites of the effective segmented tandem repeat sequence, each detection can effectively detect more than or equal to 10 sites. This application takes the nucleic acid combination product for detecting 52 STR sites as an example, which greatly improves the stability of STR, and through sufficient demonstration, when the number of sites of the effective segmented tandem repeat sequence reaches 52, under the condition of a small DNA sample amount (for example, DNA extracted from 5 cells), only 1 / 3 of the DNA sample amount is taken, and the number of effective sites that can be detected each time is more than 10, so it is possible to detect embryos such as triploid, tetraploid, uniparental diploid and other euploid embryos, trace the origin of aborted embryos, and determine the relationship between parents, etc. At the same time, the remaining DNA sample amount is sufficient for DNA methylation detection of pre-implantation embryos, and the DNA methylation detection results can also remain stable.

[0092] In some examples, at least one primer in the primer pair is labeled with a fluorescent group. In some primer pairs, both primers are labeled with fluorescent groups, such as primer pair M3-5. In some primer pairs, only one primer is labeled with a fluorescent group, such as M1-1.

[0093] The present application does not specifically limit the type of fluorescent group, and the fluorescent group labeled on each primer can independently be but is not limited to 6-FAM, ROX, HEX or TAMRA.

[0094] A second aspect of the embodiments of the present application provides a detection kit, which includes the nucleic acid combination product.

[0095] The present application does not impose any particular limitation on the packaging form of the nucleic acid combination product in the detection kit. For example, each primer pair can be packaged separately or in groups and mixed.

[0096] In addition to the nucleic acid combination product, the detection kit of the present application may also include other reagents for use, such as STR PCR reaction reagents. The STR PCR reaction reagents include, for example, reaction buffer, dNTPs, and DNA polymerase.

[0097] In a third aspect of the embodiments of the present application, a method for detecting short tandem repeat fragments in a biological sample DNA is provided, the detection method comprising the step of using the nucleic acid combination product or the detection kit to detect short tandem repeat fragments in the biological sample DNA to be detected.

[0098] In some examples, the detection method includes the following steps:

[0099] Extracting DNA from the biological sample to be tested, fragmenting the DNA, performing whole genome amplification, and preparing an amplification product;

[0100] The amplification product is used as a template and the nucleic acid combination product is used to perform STR PCR amplification, and the situation of the short tandem repeat fragments in the DNA of the biological sample to be tested is determined according to the obtained STR PCR amplification result.

[0101] In the process of STR PCR amplification, usually, primers of several sites are combined in a PCR tube, and then a DNA template is added for amplification reaction, thereby improving reaction efficiency and reducing experimental costs. However, since the amplification efficiency of each primer is different and the fluorescence carried is also different, it is necessary to combine according to the fluorescent color carried by the primer. More importantly, it is necessary to debug the ratio between the primers when the primers are combined according to the amplification efficiency of the primers in the single reaction, and test the site amplification efficiency when different primers are mixed, so as to avoid the non-characteristic amplification that occurs after the primers are combined. The test results are inaccurate and unstable. Therefore, the present application provides a stable and efficient combination and ratio scheme of primers by combining different primers and repeatedly testing and adjusting the concentrations of different primers under the same combination. For example, the primer pair M1-1, primer pair M1-2, primer pair M1-3, primer pair M1-4, primer pair M1-5, primer pair M1-6, primer pair M4-1, primer pair M4-2 and primer pair M4-4 are divided into one group, the primer pair M2-1, primer pair M2-3, primer pair M2-4, primer pair M2-5, primer pair M3-1, primer pair M3-2, primer pair M3-3, primer pair M3-4, primer pair M3-5 and primer pair M3-6 are divided into one group, the primer pair M5-1, primer pair M8-3, primer pair M4-3 and primer pair M2-2 are divided into one group, the primer pair M6-1, primer pair M6-2, primer pair M6-3, primer pair M7-1, primer pair Primer pair M7-2, primer pair M7-3, primer pair M7-4, primer pair M7-5, primer pair M10-6, primer pair M10-4, primer pair M10-2 and primer pair M10-1 are divided into one group, primer pair M9-11, primer pair M9-3, primer pair M9-2, primer pair M9-7, primer pair M9-9 and primer pair M10-12 are divided into one group, primer pair M9-6, primer pair M9-1 and primer pair M5-2 are divided into one group, primer pair M9-4, primer pair M9-5 and primer pair M5-4 are divided into one group, and primer pair M10-8, primer pair M10-10, primer pair M10-3, primer pair M10-9 and primer pair M10-13 are divided into one group. Multiple primer pairs are grouped for reaction, which improves the detection efficiency.

[0102] In some examples, in the initial reaction system of STR PCR amplification,

[0103] The molar ratio of the primer pair M1-1, the primer pair M1-2, the primer pair M1-3, the primer pair M1-4, the primer pair M1-5, the primer pair M1-6, the primer pair M4-1, the primer pair M4-2 and the primer pair M4-4 is 10±1.0:2.5±1.0:5±1.0:5±1.0:5±1.0:15±1.0:15±1.0:5±1.0:12.5±1.0,

[0104] The molar ratio of the primer pair M2-1, the primer pair M2-3, the primer pair M2-4, the primer pair M2-5, the primer pair M3-1, the primer pair M3-2, the primer pair M3-3, the primer pair M3-4, the primer pair M3-5 and the primer pair M3-6 is 5±1.0: 15±1.0: 2.5±1.0: 30±1.0: 2.5±1.0: 5±1.0: 10±1.0: 15±1.0: 12.5±1.0: 5±1.0,

[0105] The molar ratio of the primer pair M5-1, the primer pair M8-3, the primer pair M4-3 and the primer pair M2-2 is 5±1.0:37.5±1.0:25±1.0:12.5±1.0,

[0106] The molar ratio of the primer pair M6-1, the primer pair M6-2, the primer pair M6-3, the primer pair M7-1, the primer pair M7-2, the primer pair M7-3, the primer pair M7-4, the primer pair M7-5, the primer pair M10-6, the primer pair M10-4, the primer pair M10-2 and the primer pair M10-1 is 5±1.0:15±1.0:37.5±1.0:5±1.0:7.5±1.0:8.75±1.0:50±1.0:50±1.0:10±1.0:10±1.0:15±1.0:10±1.0,

[0107] The molar ratio of the primer pair M9-11, the primer pair M9-3, the primer pair M9-2, the primer pair M9-7, the primer pair M9-9 and the primer pair M10-12 is 10±1.0:2.5±1.0:10±1.0:5±1.0:25±1.0:37.5±1.0,

[0108] The molar ratio of the primer pair M9-6, the primer pair M9-1 and the primer pair M5-2 is 10±1.0:10±1.0:10±1.0,

[0109] The molar ratio of the primer pair M9-4, the primer pair M9-5 and the primer pair M5-4 is 10±1.0:10±1.0:10±1.0,

[0110] The molar ratio of the primer pair M10-8, the primer pair M10-10, the primer pair M10-3, the primer pair M10-9 and the primer pair M10-13 is 10±1.0:10±1.0:10±1.0:5±1.0:15±1.0;

[0111] In each of the primer pairs, the molar ratio of the forward primer to the reverse primer is 1±0.5:1±0.5.

[0112] The present application does not specifically limit the type of biological sample to be tested, including but not limited to cell samples, blood samples (such as peripheral blood samples). In some examples, the biological sample to be tested is a cell sample. The present application does not specifically limit the number of cells contained in the cell sample, for example, 1, 2, 3...10. Optionally, the number of cells in the cell sample is 2-10. The present application does not specifically limit the source of the biological sample to be tested, including but not limited to from a pre-implantation embryo; optionally, the biological sample to be tested comes from the trophoblast.

[0113] In some examples, the DNA fragmentation process includes the steps of chemical lysis and mechanical interruption. Optionally, the initial system of mechanical interruption includes chemical lysis products of DNA, carrier RNA and buffer, and optionally, the buffer includes 0.1×TE buffer. Mechanical interruption uses QSONICA interruption instrument, and the parameter settings include: amplitude of 40%±5%, time of 20s±5s. In addition, since the current single-cell amplification enzyme amplification requires a complete DNA template for effective amplification. However, the total system of cells after embryonic biopsy is only 10μL after cell lysis. If 10μL is directly divided into two parts, there is a risk that the chromosomes cannot be randomly divided, which will cause errors in the results of genetic or epigenetic testing of the embryo. Therefore, the cells after lysis can only be fragmented and fully mixed, and then the lysed samples are divided into two parts. At the same time, in order to ensure the amplification efficiency of the single-cell amplification enzyme, when the cells are fragmented, the fragmentation conditions need to be optimized to ensure that the fragments are at a suitable length, thereby reducing the impact on subsequent DNA amplification. The present application provides a stable fragmentation scheme and conditions through repeated testing and verification, that is, mechanical fragmentation is performed using a QSONICA fragmentation instrument, and the parameter settings include: amplitude of 40%±5%, time of 20s±5s.

[0114] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0115] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0116] Short Tandem Repeat (STR) is composed of short tandem repeat sequences with a length of 2-6 base pairs. These repeat sequences are widely present in the human genome and are a rich source of highly polymorphic markers. The present application is to perform whole genome amplification on a trace amount of the examined embryonic cell sample, and to perform short tandem repeat sequence analysis on it by polymerase chain reaction (PCR). The different number of repetitions of the repeat sequence in the amplified region leads to differences in the allele typing of the STR locus. After separation by capillary electrophoresis, different genotypes can be distinguished by fluorescence detection.

[0117] Example 1

[0118] Taking preimplantation DNA methylation screening as an example, the experimental process is described:

[0119] 1. Sample Preparation

[0120] During the IVF process, the embryo is fertilized and cultured in vitro for 5-6 days, develops to the blastocyst stage, and 5-10 cells are separated from the trophoblast as samples to be tested (containing 5 cells).

[0121] PIMS (a method that uses epigenetic information, i.e. DNA methylation indicators, to select embryos) is used to detect triploidy. Blood samples from the father and mother are required to be collected. The sample volume is 1 mL, and heparin-anticoagulated sterile blood collection tubes are used to ensure strict aseptic collection. DNA is extracted from the father's and mother's peripheral blood samples according to the instructions of the magnetic bead method universal DNA extraction kit.

[0122] The positive controls were genomic DNA extracted from normal B lymphocyte cell lines and trisomy 21 cell lines.

[0123] 2. Reagent Preparation

[0124] (1) Preparation of 0.1× TE buffer: Dilute 1× TE buffer 10 times with nuclease-free water to obtain 0.1× TE buffer.

[0125] (2) Preparation of STR PCR Primer Mix-Tube1 (100 rxns): Take a new 1.5 mL low-absorption EP tube, mark the tube cap, and add the upstream primer F and downstream primer R of each STR locus and the 0.1× TE buffer prepared in step (1) in sequence according to the table below. Vortex thoroughly to mix, and store at -20±5°C in the dark.

[0126] Table 1. Preparation method of STR PCR Primer Mix-Tube1

[0127]

[0128] (3) Preparation of STR PCR Primer Mix-Tube2 (100 ml): Take a new 1.5 mL low-absorption EP tube, mark the tube cap, and add the upstream primer F, downstream primer R of each STR locus and 0.1× TE buffer prepared in step (1) in sequence according to the table below. Vortex thoroughly to mix, and store at -20±5°C in the dark.

[0129] Table 2. Preparation method of STR PCR Primer Mix-Tube2

[0130]

[0131] (4) Preparation of STR PCR Primer Mix-Tube3 (100 ml): Take a new 1.5 mL low-absorption EP tube, mark the tube cap, and add the upstream primer F, downstream primer R of each STR locus and 0.1× TE buffer prepared in step (1) in sequence according to the table below. Vortex thoroughly to mix, and store at -20±5°C in the dark.

[0132] Table 3. Preparation method of STR PCR Primer Mix-Tube3

[0133]

[0134] (5) Preparation of STR PCR Primer Mix-Tube4 (100 ml): Take a new 1.5 mL low-absorption EP tube, mark the tube cap, and add the upstream primer F, downstream primer R of each STR locus and 0.1× TE buffer prepared in step (1) in sequence according to the table below. Vortex thoroughly to mix, and store at -20±5°C in the dark.

[0135] Table 4. Preparation method of STR PCR Primer Mix-Tube4

[0136]

[0137] (6) Preparation of STR PCR Primer Mix-Tube5 (100 ml): Take a new 1.5 mL low-absorption EP tube, mark the tube cap, and add the upstream primer F and downstream primer R of each STR locus and the 0.1× TE buffer prepared in step (1) in sequence according to the table below. Vortex thoroughly to mix, and store at -20±5°C in the dark.

[0138] Table 5. Preparation method of STR PCR Primer Mix-Tube5

[0139]

[0140] (7) Preparation of STR PCR Primer Mix-Tube 6 (100 ml): Take a new 1.5 mL low-absorption EP tube, mark the tube cap, and add the upstream primer F, downstream primer R of each STR locus and 0.1× TE buffer prepared in step (1) in sequence according to the table below. Vortex thoroughly to mix, and store at -20±5°C in the dark.

[0141] Table 6. Preparation method of STR PCR Primer Mix-Tube6

[0142]

[0143] (8) Preparation of STR PCR Primer Mix-Tube7 (100 ml): Take a new 1.5 mL low-absorption EP tube, mark the tube cap, and add the upstream primer F, downstream primer R of each STR locus and 0.1× TE buffer prepared in step (1) in sequence according to the table below. Vortex thoroughly to mix, and store at -20±5°C in the dark.

[0144] Table 7. Preparation method of STR PCR Primer Mix-Tube7

[0145]

[0146] (9) Preparation of STR PCR Primer Mix-Tube8 (100 ml): Take a new 1.5 mL low-absorption EP tube, mark the tube cap, and add the upstream primer F, downstream primer R of each STR locus and 0.1× TE buffer prepared in step (1) in sequence according to the table below. Vortex thoroughly to mix, and store at -20±5°C in the dark.

[0147] Table 8. Preparation method of STR PCR Primer Mix-Tube8

[0148]

[0149]

[0150] Table 9. STR primer sequence information

[0151]

[0152]

[0153]

[0154] 3. Sample Lysis

[0155] Add 7 μL of cell lysis solution and 1 μL of proteinase K to the cell sample tube (20 μL) to a total system of 10 μL. Centrifuge briefly to allow the reagents to accumulate at the bottom of the tube. Vortex mix for 5 seconds. After centrifugation briefly, place the sample in a PCR instrument for lysis.

[0156] 4.DNA fragmentation and sample separation system

[0157] (1) After the lysis is completed, remove the PCR tube from the PCR instrument and centrifuge it briefly (10-15 seconds) to ensure that there are no obvious droplets on the tube wall and lid.

[0158] (2) Add 1 μL of carrier RNA and 9 μL of 0.1×TE buffer to a PCR tube to a total of 20 μL. Vortex to mix and centrifuge briefly to allow the droplets on the tube wall to gather at the bottom of the tube.

[0159] (3) Use QSONICA fragmentation instrument, set the amplitude to 40%, and the time to 20 s to fragment the DNA.

[0160] (4) After disruption, vortex mix and centrifuge briefly to allow the droplets on the tube wall to gather at the bottom of the tube. Use a pipette to remove 1 / 3 of the volume (6.67 μL) and transfer it to a new PCR tube. Add 3.33 μL of 0.1× TE buffer to a total volume of 10 μL and start the next step of whole genome amplification reaction. The remaining 2 / 3 of the sample is used for PIMS library construction.

[0161] 5. Whole genome amplification-preamplification

[0162] (1) Place the sample tube on ice, add 9.4 μL Pre-Amplification Buffer and 0.6 μL Pre-Amplification Enzyme to the sample tube, mix gently, and centrifuge to the bottom of the tube.

[0163] Table 10

[0164] Components Volume added per sample Pre-Amplification Buffer 9.4μL Pre-Amplification Enzyme 0.6μL Sample (interruption product) 10μL Total reaction volume 20μL

[0165] (2) Place the PCR tube on a thermal cycler and perform pre-amplification according to the following procedure (heat cover: 101-105°C):

[0166] Table 11

[0167]

[0168] Note: After the pre-amplification reaction, the sample should be placed at 4°C and it is recommended to proceed to the next amplification reaction immediately; if a pause is required, the pre-amplification product can be stored at 4°C overnight or at -20°C for 2 weeks.

[0169] 6. Whole genome amplification-amplification

[0170] (1) Place the sample tube on ice, add 26.2 μL Amplification Buffer, 1.3 μL Amplification Enzyme, and 2.5 μL nuclease-free water to the sample tube, mix gently, and centrifuge to the bottom of the tube.

[0171] Table 12

[0172] Components Volume added per sample Amplification Buffer 26.2μL Amplification Enzyme 1.3μL Nuclease-free water 2.5μL Pre-amplification products 20μL Total reaction volume 50μL

[0173] (2) Place the PCR tube on a thermal cycler and perform pre-amplification according to the following procedure (heat cover: 101-105°C):

[0174] Table 13

[0175]

[0176] 7. Purification of Whole Genome Amplification Products

[0177] (1) Vortex and briefly centrifuge each PCR tube containing whole genome amplification products, then add 50 μL of resuspended DNA purification magnetic beads.

[0178] (2) Vortex to mix thoroughly and incubate at room temperature for 5 min.

[0179] (3) Centrifuge the sample tube momentarily to collect the liquid, and place the PCR tube on a magnetic rack to separate the magnetic beads. After the solution is clarified, carefully discard the supernatant (leave 3-5 μL at the bottom to avoid taking away the magnetic beads).

[0180] (4) Add 200 μL of freshly prepared 80% ethanol to the PCR tube on the magnetic stand, incubate for 30 seconds, and discard the supernatant.

[0181] (5) Repeat step (4), then place the sample tube in a centrifuge with the magnetic beads facing outward. Centrifuge briefly to allow the droplets on the tube wall to gather at the bottom. Place the tube back on the magnetic rack and use a 10 μL pipette to aspirate the remaining beads at the bottom.

[0182] (6) Open the tube cap and allow the magnetic beads to dry in air for about 2-3 minutes until there is no reflection or excess liquid on the surface of the magnetic beads.

[0183] (7) Remove the sample tube from the magnetic stand and add 20.5 μL of 0.1× TE buffer on top of the magnetic beads.

[0184] (8) Vortex mix using an oscillator and incubate at room temperature for 5 min.

[0185] (9) Centrifuge the PCR tube slightly and place it on a magnetic rack. After the solution is clarified, transfer the supernatant to a new 1.5 mL centrifuge tube to complete the purification.

[0186] 8. Quantification of Whole Genome Amplification Products

[0187] The purified whole genome amplification product was quantified using a fluorometer.

[0188] 9. STR-PCR

[0189] STR-PCR amplification setup:

[0190] (1) Thoroughly melt the prepared STR PCR Primer Mix-Tubes 1 to 8 in Tables 1 to 8, and take out the NEB 5×Q5 Reaction Buffer, 10mM dNTPs, and nuclease-free water in High-Fidelity DNA Polymerase (Cat. No. M0491). After the reagents are dissolved, mix thoroughly and centrifuge briefly. Invert and mix the Q5 High-Fidelity DNA Polymerase, centrifuge briefly, and place on ice for later use.

[0191] (2) Determine the number of amplification reactions including gDNA extracted from peripheral blood of embryos and their parents.

[0192] (3) Use clean 0.2 mL PCR tubes and label them correctly for batch amplification.

[0193] (4) Add the final volume of each reaction component to a 1.5 ml sterile centrifuge tube.

[0194] Table 14. STR PCR Amplification Mixture 1

[0195] PCR reaction mixture components Volume added per sample 5×Q5 Reaction Buffer 5μL 10mM dNTPs 0.5μL STR PCR Primer Mix-Tube1 2.5μL Q5 High-Fidelity DNA Polymerase 0.25μL Template DNA (10 ng / μL) 2μL Nuclease-free water 14.75μL Total reaction volume 25μL

[0196] Table 15. STR PCR Amplification Mix 2

[0197] PCR reaction mixture components Volume added per sample 5×Q5 Reaction Buffer 5μL 10mM dNTPs 0.5μL STR PCR Primer Mix-Tube2 2.5μL Q5 High-Fidelity DNA Polymerase 0.25μL Template DNA (10 ng / μL) 2μL Nuclease-free water 14.75μL Total reaction volume 25μL

[0198] Table 16. STR PCR Amplification Mix 3

[0199] PCR reaction mixture components Volume added per sample 5×Q5 Reaction Buffer 5μL 10mM dNTPs 0.5μL STR PCR Primer Mix-Tube3 2.5μL Q5 High-Fidelity DNA Polymerase 0.25μL Template DNA (10 ng / μL) 2μL Nuclease-free water 14.75μL Total reaction volume 25μL

[0200] Table 17. STR PCR Amplification Mix 4

[0201] PCR reaction mixture components Volume added per sample 5×Q5 Reaction Buffer 5μL 10mM dNTPs 0.5μL STR PCR Primer Mix-Tube4 2.5μL Q5 High-Fidelity DNA Polymerase 0.25μL Template DNA (10 ng / μL) 2μL Nuclease-free water 14.75μL Total reaction volume 25μL

[0202] Table 18. STR PCR Amplification Mixture 5

[0203] PCR reaction mixture components Volume added per sample 5×Q5 Reaction Buffer 5μL 10mM dNTPs 0.5μL STR PCR Primer Mix-Tube5 2.5μL Q5 High-Fidelity DNA Polymerase 0.25μL Template DNA (10 ng / μL) 2μL Nuclease-free water 14.75μL Total reaction volume 25μL

[0204] Table 19. STR PCR Amplification Mix 6

[0205] PCR reaction mixture components Volume added per sample 5×Q5 Reaction Buffer 5μL 10mM dNTPs 0.5μL STR PCR Primer Mix-Tube6 2.5μL Q5 High-Fidelity DNA Polymerase 0.25μL Template DNA (10 ng / μL) 2μL Nuclease-free water 14.75μL Total reaction volume 25μL

[0206] Table 20, STR PCR Amplification Mixture 7

[0207]

[0208]

[0209] Table 21. STR PCR Amplification Mix 8

[0210] PCR reaction mixture components Volume added per sample 5×Q5 Reaction Buffer 5μL 10mM dNTPs 0.5μL STR PCR Primer Mix-Tube8 2.5μL Q5 High-Fidelity DNA Polymerase 0.25μL Template DNA (10 ng / μL) 2μL Nuclease-free water 14.75μL Total reaction volume 25μL

[0211] (5) Vortex the PCR amplification mixture for 5-10 seconds, and then dispense the PCR mixture into each reaction tube.

[0212] (6) Add the template DNA (20 ng) of the corresponding sample to the reaction tube containing the PCR amplification mixture.

[0213] (7) Set up a blank control for amplification. Add nuclease-free water instead of template DNA into the reaction tube containing the PCR amplification solution.

[0214] (8) Cover the reaction plate or tube tightly and centrifuge gently to move the reaction solution to the bottom of the reaction tube and eliminate bubbles.

[0215] (9) Amplification cycle parameter settings:

[0216] 1) Place the reaction tube in a PCR instrument, select and run the following cycle program, and the reaction time is about 1.5 hours.

[0217] Table 22. STR PCR cycle program

[0218]

[0219] 2) After the reaction is completed, store the amplified product at -20°C in a dark environment.

[0220] 10. Capillary electrophoresis sequencing

[0221] 11. Results Analysis

[0222] like Figures 1 to 8 As shown, the present application performs STR detection on the trace cell sample under item 1. The test results show the consistency of the test results of each STR locus in Tube1-Tube8 and the test results of the positive control. In the sample test of this embodiment, a total of 15 valid STR loci (STR marked with boxes) were detected. Therefore, the STR test of the sample under item 1 using the method of this embodiment can effectively determine the polyploidy test results.

[0223] Example 2

[0224] Another sample preparation was performed according to Example 1 and STR detection was performed on the sample. The results are as follows Figures 9 to 17 As shown, the results show that in the detection of this embodiment, a total of 20 valid STR loci (STR marked with boxes) were detected, and the consistency of the detection result of each STR locus and the detection result of the positive control was consistent.

[0225] Example 3

[0226] Another sample preparation was performed with reference to Example 1 and STR detection was performed on the sample. Figures 18 to 25 As shown, the results show that in the detection of this embodiment, a total of 14 valid STR loci (STR marked with boxes) were detected, and the consistency of the detection result of each STR locus and the detection result of the positive control was consistent.

[0227] Combining Embodiment 1 to Embodiment 3 Figures 1 to 25 It can be seen that, by using the method provided in the present application, the number of effective STR loci that can be detected for each sample is greater than 10, and effective result judgment can be performed.

[0228] Example 4

[0229] This embodiment is a variation of embodiment 1, and the only difference from embodiment 1 is that multiplex PCR is performed in a combined tube. Fig.26 As shown, when D6S477, D22-GATA198B05, D15S659, and D19S253 are combined in a multiplex PCR, D22-GATA198B05 and D15S659 cannot be amplified; when the primer pairs in Tube3 and Tube4 are combined in one tube for PCR amplification, D19S253 will have low amplification efficiency, resulting in low fluorescence intensity at this site and cannot be used for judgment.

[0230] Example 5

[0231] This embodiment is a variation of embodiment 1. The only difference from embodiment 1 is that the primer pair in Tube3 and the primer pair in Tube8 are combined in one tube for PCR amplification. Fig. 27 As shown, when the primer pair in Tube3 and the primer pair in Tube8 are combined in one tube for PCR amplification, non-specific amplification of D19S433 will occur.

[0232] Example 6

[0233] This embodiment is a variation of embodiment 1, and the only variation from embodiment 1 is the comparison of the fragmentation condition test. As shown in Figure 28: Figure 1 is the capillary electrophoresis result of a large number of DNA samples (5 cells) as a positive control STR PCR amplification; Figure 2 is the capillary electrophoresis result of STR PCR after 5 cells were interrupted for 20 seconds and then subjected to WGA. All 6 STR sites were amplified and the peak height was basically consistent with the positive control; Figure 3 is the capillary electrophoresis result of STR PCR after 5 cells were interrupted for 3 minutes and then subjected to WGA. Only 2 of the 6 STR sites were correctly amplified; Figure 4 is the capillary electrophoresis result of 1 / 3 of the 5 cells that were interrupted to 8 minutes according to the previous PIMS library construction process. None of the 6 STR sites were successfully amplified.

[0234] Regarding the analysis of the embodiment of the best detection of 52 STR loci in this application. STR PCR amplification is performed after single-cell amplification enzyme amplification, and single-cell amplification will introduce random errors in STR loci. Using the method of Example 1 of the present application, STR detection was performed on the two trace cell samples prepared under Item 1 of Reference Example 1, and the detection results are as follows: Sample 1 called 18 STR loci, and sample 2 called 14 STR loci. The loci marked in yellow in the table are valid STR loci detected in both tests, a total of 11, and meet ≧10 loci to meet the judgment standard. If the STR loci are reduced, the number of call pairs may be less than 10, resulting in a decrease in the number of correct STR loci and a risk of not meeting the judgment requirements.

[0235] Table 23

[0236]

[0237]

[0238] Schematic diagram of the use of STR in this application to determine uniparental diploidy, triploidy, etc.:

[0239] like Fig.29At a certain STR locus, the mother has a diallele peak of 6-fold and 8-fold 1:1, and the father has a diallele peak of 7-fold and 10-fold 1:1.

[0240] like Fig.30 As shown, the normal diploid embryos born to the couple may have the following two allele peaks at this STR locus: embryo 1: 6-fold and 7-fold, embryo 2: 6-fold and 10-fold, embryo 3: 7-fold and 8-fold, or embryo 4: 8-fold and 10-fold, a 1:1 biallelic peak.

[0241] like Fig.31 , triploidy means that each cell has three complete sets of chromosomes, namely 3n, which is a chromosomal aberration with abnormal chromosome number. PIMS detection of triploidy is based on paternity testing as the judgment standard. When the STR loci of the embryo have ≥10 tri-allelic genotypes or unbalanced di-allelic peaks with a peak height ratio of 2:1, the probability that the embryo is triploid is ≥99.99%, which can support the judgment that the detected embryo is triploid. The triploid embryos born by the couple may appear in the following situations at the STR loci: embryo 1: 6-fold, 7-fold and 10-fold tri-allelic peaks; embryo 2: 6-fold, 7-fold and 8-fold tri-allelic peaks; embryo 3: 7-fold, 8-fold and 10-fold tri-allelic peaks; embryo 4: 6-fold, 8-fold and 10-fold tri-allelic peaks of 1:1:1. If all 52 STR loci are combined to detect ≥10 such loci, the embryo is triploid.

[0242] like Fig.32 As shown in the figure below, the ectopic gamete and the normal gamete are fertilized to form a monomeric zygote. During the cleavage process, the monomer is replicated to become a disomy. As shown in the figure below, the possible situation of the uniparental diploid embryo born by this couple at this STR locus should be: embryo 1: 7 weight or embryo 2: 10 weight, and the embryo is maternal uniparental homodisomy.

[0243] During meiosis, the non-disjunction of homologous chromosomes or sister chromatids causes the fertilization of disomic gametes with normal monosomic gametes to form a trisomic zygote. Due to self-rescue, one of the chromosomes is randomly lost during the cleavage process, forming a uniparental diploid. As shown in the figure below, the possible situation of the uniparental diploid embryos born by the couple at this STR locus should be: Embryo 3: 6-fold and 8-fold 1:1 diallele peaks (heterozygous), the father has a 7-fold monoallele peak (homozygous), and embryo 3 detects the presence of 6-fold and 8-fold 1:1 diallele peaks at this STR locus, which is consistent with the mother's result. Both homologous chromosomes are inherited from the mother. If such STR loci are ≥10, the embryo is a maternal uniparental heterodisome.

[0244] The technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. To make the description concise, not all possible combinations of the technical features in the above-mentioned implementation modes and examples are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0245] The above-described embodiments only express several implementation methods of the present application, which is convenient for understanding the technical solution of the present application in detail, but it cannot be understood as a limitation on the scope of protection of the patent application. It should be pointed out that for ordinary technicians in this field, without departing from the concept of the present application, several deformations and improvements can be made, which all belong to the protection scope of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, the technicians in this field can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the protection scope of the present application. It should also be understood that the technical solutions obtained by the technicians in this field through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the protection scope of the claims attached to the present application. Therefore, the protection scope of the patent of the present application shall be based on the content of the attached claims, and the description and drawings can be used to explain the content of the claims.

Claims

1. Nucleic acid combination products, It is characterized in that The nucleic acid combination product includes one or more pairs of the following primer pairs: Primer pair M1-1, primer pair M1-2, primer pair M1-3, primer pair M1-4, primer pair M1-5, primer pair M1-6, primer pair M2-1, primer pair M2-3 and primer pair M2-4 having sequences as shown in SEQ ID NO.1 to SEQ ID NO.18, Primer pair M3-1, primer pair M3-2, primer pair M3-3, primer pair M3-4, primer pair M3-5, primer pair M3-6, primer pair M4-1, primer pair M4-2 and primer pair M4-4 having sequences as shown in SEQ ID NO.21 to SEQ ID NO.38, The primer pair M8-3 whose sequences are shown in SEQ ID NO.45 to SEQ ID NO.46, The sequences of primer pair M4-3 and primer pair M2-2 are shown in SEQ ID NO.63 to SEQ ID NO.66, The sequences of primer pair M9-4 and primer pair M9-5 are shown in SEQ ID NO.73 to SEQ ID NO.76, The primer pair M9-9 whose sequences are shown in SEQ ID NO.81 to SEQ ID NO.82, The sequences of primer pair M10-1, primer pair M10-2, primer pair M10-3, primer pair M10-4, primer pair M10-6, primer pair M10-8, primer pair M10-9, primer pair M10-10 and primer pair M10-12 are shown in SEQ ID NO.85 to SEQ ID NO.

102.

2. The nucleic acid combination product according to claim 1, It is characterized in that The nucleic acid combination product also includes one or more pairs of the following primer pairs: The primer pair M2-5 whose sequences are shown in SEQ ID NO.19 to SEQ ID NO.20, Primer pair M5-1, primer pair M5-2 and primer pair M5-4 having sequences as shown in SEQ ID NO.39 to SEQ ID NO.44, Primer pair M6-1, primer pair M6-2, primer pair M6-3, primer pair M7-1, primer pair M7-2, primer pair M7-3, primer pair M7-4 and primer pair M7-5 having sequences as shown in SEQ ID NO.47 to SEQ ID NO.62, Primer pair M9-1, primer pair M9-2 and primer pair M9-3 having sequences as shown in SEQ ID NO.67 to SEQ ID NO.72, The sequences of primer pair M9-6 and primer pair 9-7 are shown in SEQ ID NO.77 to SEQ ID NO.80, The primer pair M9-11 whose sequences are shown in SEQ ID NO.83 to SEQ ID NO.84, The sequences of primer pair M10-13 are shown as SEQ ID NO.103 to SEQ ID NO.

104.

3. The nucleic acid combination product according to any one of claims 1 to 2, It is characterized in that The number of primer pairs in the nucleic acid combination product is 18 pairs to 52 pairs.

4. The nucleic acid combination product according to any one of claims 1 to 2, It is characterized in that At least one primer in the primer pair is labeled with a fluorescent group; Optionally, the fluorescent groups labeled on each primer are independently 6-FAM, ROX, HEX or TAMRA.

5. A detection kit, It is characterized in that The detection kit comprises the nucleic acid combination product according to any one of claims 1 to 4; Optionally, the detection kit further comprises STR PCR reaction reagents.

6. A method for detecting short tandem repeat fragments in biological sample DNA, It is characterized in that The detection method comprises the step of using the nucleic acid combination product according to any one of claims 1 to 4 or the detection kit according to claim 5 to detect short tandem repeat fragments in the DNA of the biological sample to be detected.

7. The method for detecting short tandem repeat fragments in biological sample DNA according to claim 6, It is characterized in that The detection method comprises the following steps: Extracting DNA from the biological sample to be tested, fragmenting the DNA, performing whole genome amplification, and preparing an amplification product; The amplification product is used as a template and the nucleic acid combination product is used to perform STR PCR amplification, and the situation of the short tandem repeat fragments in the DNA of the biological sample to be tested is determined according to the obtained STR PCR amplification result.

8. The method for detecting short tandem repeat fragments in biological sample DNA according to claim 7, It is characterized in that In the initial reaction system of STRPCR amplification, The molar ratio of the primer pair M1-1, the primer pair M1-2, the primer pair M1-3, the primer pair M1-4, the primer pair M1-5, the primer pair M1-6, the primer pair M4-1, the primer pair M4-2 and the primer pair M4-4 is 10±1.0:2.5±1.0:5±1.0:5±1.0:5±1.0:15±1.0:15±1.0:5±1.0:12.5±1.0, The molar ratio of the primer pair M2-1, the primer pair M2-3, the primer pair M2-4, the primer pair M2-5, the primer pair M3-1, the primer pair M3-2, the primer pair M3-3, the primer pair M3-4, the primer pair M3-5 and the primer pair M3-6 is 5±1.0: 15±1.0: 2.5±1.0: 30±1.0: 2.5±1.0: 5±1.0: 10±1.0: 15±1.0: 12.5±1.0: 5±1.0, The molar ratio of the primer pair M5-1, the primer pair M8-3, the primer pair M4-3 and the primer pair M2-2 is 5±1.0:37.5±1.0:25±1.0:12.5±1.0, The molar ratio of the primer pair M6-1, the primer pair M6-2, the primer pair M6-3, the primer pair M7-1, the primer pair M7-2, the primer pair M7-3, the primer pair M7-4, the primer pair M7-5, the primer pair M10-6, the primer pair M10-4, the primer pair M10-2 and the primer pair M10-1 is 5±1.0:15±1.0:37.5±1.0:5±1.0:7.5±1.0:8.75±1.0:50±1.0:50±1.0:10±1.0:10±1.0:15±1.0:10±1.0, The molar ratio of the primer pair M9-11, the primer pair M9-3, the primer pair M9-2, the primer pair M9-7, the primer pair M9-9 and the primer pair M10-12 is 10±1.0:2.5±1.0:10±1.0:5±1.0:25±1.0:37.5±1.0, The molar ratio of the primer pair M9-6, the primer pair M9-1 and the primer pair M5-2 is 10±1.0:10±1.0:10±1.0, The molar ratio of the primer pair M9-4, the primer pair M9-5 and the primer pair M5-4 is 10±1.0:10±1.0:10±1.0, The molar ratio of the primer pair M10-8, the primer pair M10-10, the primer pair M10-3, the primer pair M10-9 and the primer pair M10-13 is 10±1.0:10±1.0:10±1.0:5±1.0:15±1.0; In each of the primer pairs, the molar ratio of the forward primer to the reverse primer is 1±0.5:1±0.

5.

9. The method for detecting short tandem repeat fragments in biological sample DNA according to any one of claims 6 to 8, It is characterized in that The biological samples to be tested are cell samples and blood samples; Optionally, the number of cells in the cell sample is 2-10.

10. The method for detecting short tandem repeat fragments in biological sample DNA according to any one of claims 6 to 8, It is characterized in that The biological sample to be tested is from a pre-implantation embryo; Optionally, the biological sample to be tested comes from the trophoblast.

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