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

Abstract

This application relates to a nucleic acid combination product and a method for detecting short tandem repeats in biological sample DNA, which belongs to the field of assisted reproductive technology. The nucleic acid combination product includes one or more pairs of the following primer pairs: primer pairs with sequences such as SEQ ID NO.1 to SEQ ID NO.18, SEQ ID NO.21 to SEQ ID NO.38, SEQ ID NO.45 to SEQ ID NO.46, SEQ ID NO.63 to SEQ ID NO.66, SEQ ID NO.73 to SEQ ID NO.76, SEQ ID NO.81 to SEQ ID NO.82, and SEQ ID NO.85 to SEQ ID NO.102. The nucleic acid combination product can detect one or more of 33 STR loci, including Amelogenin, providing a solution for euploidy detection of pre-implantation embryos and tracing the origin of aborted embryos.

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 (ART) is an effective clinical approach to addressing infertility. According to statistics from the journal Reproductive Biology and Endocrinology, over 20% of infertile couples currently require ART treatment. Improving the birth rate through ART has always been a challenge in the field of ART. Selecting high-quality embryos for transfer during ART is an effective method for increasing birth rates.

[0003] Currently, pre-implantation DNA methylation detection technology is a new generation of embryo screening method. This method is based on epigenetic principles and uses second-generation sequencing technology to evaluate the chromosome copy number variation 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 improve 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 undergoing assisted reproductive treatment have experienced miscarriage after embryo transplantation. By testing the chromosome copy number variation of the miscarriage tissue, it was found that the chromosome copy number information obtained from the miscarriage 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 transplantation, resulting in the wrong embryo being transplanted into the uterus; 3. After the embryo is transplanted, the couple has sex, and the miscarried embryo is not from the transplanted embryo. In response to the above situations, it is necessary to trace the miscarriage embryo.

[0005] However, current pre-implantation DNA methylation testing technology cannot detect triploidy. Pre-implantation DNA methylation testing requires biopsy of 6-8 blastocyst trophoblast cells, yielding approximately 40pg of DNA. Because current single-cell amplification technology loses methylation modification information in the amplified DNA, pre-implantation DNA methylation testing cannot use existing single-cell amplification technology to amplify the starting DNA to increase 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 following technical solutions are included:

[0008] One or more embodiments of the present application provide a nucleic acid combination product, comprising one or more 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 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, whose sequences are 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 primer pair M4-3 and the primer pair M2-2 whose sequences are shown in SEQ ID NO.63 to SEQ ID NO.66,

[0013] The primer pair M9-4 and primer pair M9-5 whose sequences 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 shown as 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, whose sequences are 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 shown as 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 primer pair M10-13 has sequences 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 group labeled on each primer is 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 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 tested.

[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 amplified 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 results.

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

[0034] The molar ratio of 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 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 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 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 primer pair M5-1, primer pair M8-3, primer pair M4-3 and primer pair M2-2 is 5±1.0:37.5±1.0:25±1.0:12.5±1.0,

[0037] The molar ratio of 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, primer pair M7-5, primer pair M10-6, primer pair M10-4, primer pair M10-2 and 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 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 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 primer pair M9-6, primer pair M9-1 and 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, primer pair M10-10, primer pair M10-3, primer pair M10-9 and primer pair M10-13 is 10±1.0:10±1.0:10±1.0:5±1.0:15±1.0;

[0042] In each primer pair, 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; alternatively, 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 below, and other features, objects, and advantages of the application will become apparent from the description and from the claims thereof. 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 fully understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present application. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0047] Figures 1 to 8 These are the test results corresponding to Tube1 to Tube8 in Example 1;

[0048] Figures 9 to 17 These are the test results corresponding to Tube1 to Tube8 in Example 2;

[0049] Figures 18 to 25 These are the test results corresponding to Tube1 to Tube8 in Example 3;

[0050] Figure 26 The test results of Example 4 are shown below:

[0051] Figure 27 The test results of Example 5 are shown below:

[0052] Figure 28 The test results of Example 6 are shown below:

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

[0054] Below in conjunction with accompanying drawing, embodiment and example, the application is described in further detail.It should be understood that these embodiment and example are only used to illustrate the application and are not used to limit the scope of the application, and the purpose of providing these embodiment and example is to make the understanding of the disclosure of the application more thorough and comprehensive.It should also be understood that the application can be implemented in many different forms and is not limited to the embodiment and example described herein. Those skilled in the art can make various changes or modifications without violating the connotation of the application, and the equivalent form obtained also falls within the protection scope of the application.In addition, in the description below, a large amount of specific details are given in order to provide a more complete understanding of the application.It should be understood that the 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 pertains. The terms used herein in the specification of this application are for the purpose of describing embodiments and examples only 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 herein include any one of two or more related listed items, and also include any and all combinations of the related listed items, wherein the arbitrary and all combinations include any combination of two related listed items, any more related listed items, or 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 connected by "logical and" and also undoubtedly includes technical solutions 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 of all being 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 four-item combination of A, B, C, and D (that is, the technical solution of all being connected by "logical AND").

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

[0060] As used herein, "combination thereof", "any combination thereof", "any combination thereof" and the like 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. It should be understood that they do not constitute limitations on the scope of protection of this application.

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

[0064] In this application, the terms "optionally," "optional," and "optional" mean optional or dispensable, i.e., they refer to either option being selected from two parallel options: "with" or "without." If a technical solution contains multiple "optional" clauses, each "optional" clause is independent unless otherwise specified and there are no contradictions or constraints.

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

[0066] In this 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 this application, when referring to a numerical interval (i.e., a numerical range), unless otherwise specified, the optional numerical distribution is considered continuous within the above numerical interval and includes the two numerical endpoints of the numerical range (i.e., the minimum and maximum values), as well as each numerical value between the two numerical endpoints. Unless otherwise specified, when a numerical interval refers only to integers within the numerical interval, it includes the two endpoint integers of the numerical range, as well as 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 merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges included therein.

[0068] Unless otherwise specified, the temperature parameters in this application allow for both constant temperature treatment and temperature fluctuations within a certain temperature range. It should be understood that the constant temperature treatment allows for temperature fluctuations within the accuracy range of instrument control. Fluctuations within ranges such as ±5°C, ±4°C, ±3°C, ±2°C, and ±1°C are permitted.

[0069] In this application, % (w / w) and wt% both refer to 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 in their entirety and for all purposes. When 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 cited documents are involved in this application, the examples and preferred embodiments of the cited relevant technical features may also be incorporated into this application as references, but are limited to the ability to implement 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 adaptively amended according to the description in this application.

[0071] In a first aspect of the embodiments of the present application, a nucleic acid combination product is provided, wherein the nucleic acid combination product includes 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 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, whose sequences are 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 primer pair M4-3 and the primer pair M2-2 whose sequences are shown in SEQ ID NO.63 to SEQ ID NO.66,

[0076] The primer pair M9-4 and primer pair M9-5 whose sequences 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 this application is used to detect one or more of 33 STR sites such as Amelogenin, D3S1358, D1S1656, etc., providing a solution for euploidy detection of pre-implantation embryos and tracing the origin of aborted embryos.

[0080] The nucleic acid combination product provided in this application includes 1 pair, 2 pairs, 3 pairs, 4 pairs...33 pairs of the above 33 pairs of primer pairs, optionally includes multiple pairs of the above 33 pairs of primer pairs, and further optionally includes the above 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 shown as 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, whose sequences are 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 shown as 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 primer pair M10-13 has sequences 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, optionally including multiple pairs of the above 19 pairs of primer pairs, and further optionally including 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 to 52 pairs, for example, 18, 19, 20, ..., 52 pairs. Accordingly, the nucleic acid combination product can be used to detect 52 STR loci, including Amelogenin, D3S1358, D1S1656, Penta D, D6S477, and D22-GATA198B05.

[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 uniparental diploid, triploid, tetraploid or other euploid situation, and the embryo can be traced. In conventional STR testing (a large number 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 only has 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. At the same time, the amplified DNA template can also be used to perform traditional pre-implantation genetic testing (PGT-A). However, the pre-implantation DNA methylation detection method will gradually replace the existing pre-implantation genetic testing method as a more effective embryo screening method. However, if the embryo undergoes pre-implantation DNA methylation detection, STR detection can no longer be performed. This is because, when performing 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, and therefore DNA methylation detection can only be performed using 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 set of stable and feasible clinical solutions. 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 the stability of the DNA methylation results and the stability of the STR detection results can be guaranteed; Direction 2: By increasing the number of effective segmented tandem repeat sequence sites, each test can effectively detect more than or equal to 10 sites. This application uses a nucleic acid combination product for detecting 52 STR sites as an example to greatly improve the stability of STR. And through sufficient demonstration, when the number of effective segmented tandem repeat sequence sites reaches 52, under the condition of a very 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 in each test is more than 10. Therefore, it is possible to detect euploid embryos such as triploid, tetraploid, and uniparental diploid embryos, trace the origin of aborted embryos, and determine parentage, 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 moiety. In some primer pairs, both primers are labeled with fluorescent moieties, such as primer pair M3-5. In some primer pairs, only one primer is labeled with a fluorescent moiety, such as M1-1.

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

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

[0095] The present application does not impose any particular limitation on the packaging format of the nucleic acid combination product in the detection kit. For example, each primer pair can be packaged independently 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 the DNA of a biological sample 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 DNA of the biological sample to be tested.

[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 amplified 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 results.

[0101] During STR PCR amplification, primers for several sites are typically combined in a single PCR tube, and then a DNA template is added for the amplification reaction. This improves reaction efficiency and reduces experimental costs. However, because the amplification efficiency of each primer varies and the fluorescence carried is also different, it is necessary to combine primers based on the fluorescent color they carry. More importantly, it is necessary to adjust the ratio of primers when merging based on the amplification efficiency of the primers during individual reactions, as well as to test the site amplification efficiency when different primers are mixed, to avoid the non-specific amplification that occurs after the primers are merged, causing inaccurate and unstable test results. Therefore, this application provides a stable and efficient primer combination and ratio scheme by repeatedly testing and adjusting the concentrations of different primers in 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 grouped together; 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 grouped together; primer pair M9-6, primer pair M9-1, and primer pair M5-2 are grouped together; primer pair M9-4, primer pair M9-5, and primer pair M5-4 are grouped together; and primer pair M10-8, primer pair M10-10, primer pair M10-3, primer pair M10-9, and primer pair M10-13 are grouped together. Multiple primer pairs are grouped for reaction, thereby improving detection efficiency.

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

[0103] The molar ratio of 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 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 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 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 primer pair M5-1, primer pair M8-3, primer pair M4-3 and primer pair M2-2 is 5±1.0:37.5±1.0:25±1.0:12.5±1.0,

[0106] The molar ratio of 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, primer pair M7-5, primer pair M10-6, primer pair M10-4, primer pair M10-2 and 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 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 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 primer pair M9-6, primer pair M9-1 and 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, primer pair M10-10, primer pair M10-3, primer pair M10-9 and primer pair M10-13 is 10±1.0:10±1.0:10±1.0:5±1.0:15±1.0;

[0111] In each primer pair, 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 and 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 pre-implantation embryos; optionally, the biological sample to be tested comes from the trophoblast.

[0113] In some examples, the DNA fragmentation process includes chemical lysis and mechanical fragmentation steps. Optionally, the initial system for mechanical fragmentation includes the chemical lysis product of the DNA, carrier RNA, and a buffer, optionally comprising 0.1× TE buffer. Mechanical fragmentation is performed using a QSONICA fragmentation instrument, with parameter settings including an amplitude of 40% ± 5% and a duration of 20s ± 5s. Furthermore, current single-cell amplification enzymes require a complete DNA template for effective amplification. However, the total volume of cells after cell lysis from an embryonic biopsy is only 10 μL. If this 10 μL is directly divided into two parts, there is a risk that the chromosomes will not be completely randomly divided, which may result in erroneous results in genetic or epigenetic testing of the embryo. Therefore, the cells must be fragmented after lysis and thoroughly mixed before the lysed sample is divided into two parts. Furthermore, to ensure the amplification efficiency of the single-cell amplification enzyme, the fragmentation conditions must be optimized during cell fragmentation to ensure that the fragments are of appropriate length, thereby minimizing the impact on subsequent DNA amplification. This application, through repeated testing and verification, provides a stable fragmentation scheme and conditions, namely, mechanical fragmentation using a QSONICA fragmentation instrument, with parameter settings including: amplitude of 40% ± 5% and time of 20s ± 5s.

[0114] The embodiments of the present application will be described in detail below with reference to the examples. It should be understood that these examples are intended to illustrate the present application only 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 manuals or conventional conditions in this area, or according to the conditions recommended by the manufacturer, or with reference to experimental methods known in the art.

[0115] In the following specific examples, 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 testing accuracy or operational accuracy are allowed.

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

[0117] Example 1

[0118] Taking pre-implantation 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, developing to the blastocyst stage, and 5-10 cells are separated from the trophoblast as a sample to be tested (containing 5 cells).

[0121] PIMS (a method that uses epigenetic information, namely DNA methylation markers, to optimize embryo selection) for triploidy testing requires blood samples from both the father and the mother. The sample volume is 1 mL, collected using sterile, heparinized blood collection tubes, and strict aseptic procedures are ensured. DNA is extracted from the blood samples according to the instructions for a universal magnetic bead 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-fold with nuclease-free water to obtain 0.1× TE buffer.

[0125] (2) Preparation of STR PCR Primer Mix-Tube 1 (100 rxns): Take a new 1.5 mL low-adsorption 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 of STR PCR Primer Mix-Tube1

[0127]

[0128] (3) Preparation of STR PCR Primer Mix-Tube 2 (100 ml): Take a new 1.5 mL low-adsorption 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.

[0129] Table 2. Preparation 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-adsorption 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.

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

[0133]

[0134] (5) Preparation of STR PCR Primer Mix-Tube 4 (100 ml): Take a new 1.5 mL low-adsorption 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.

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

[0136]

[0137] (6) Preparation of STR PCR Primer Mix-Tube 5 (100 ml): Take a new 1.5 mL low-adsorption 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 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-adsorption 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.

[0141] Table 6. Preparation 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-adsorption 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.

[0144] Table 7. Preparation 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-adsorption 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.

[0147] Table 8. Preparation 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 buffer 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, centrifuge briefly, and 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 volume of 20 μL. Vortex to mix thoroughly and centrifuge briefly to allow the droplets on the tube wall to aggregate 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 9.4 μL Pre-Amplification Enzyme 0.6μL Sample (interruption product) 10 μL Total reaction volume 20 μL

[0165] (2) Place the PCR tube on a PCR instrument and perform pre-amplification according to the following procedure (heated 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 1.3 μL Nuclease-free water 2.5 μL Pre-amplification products 20 μL Total reaction volume 50μL

[0173] (2) Place the PCR tube on a PCR instrument and perform pre-amplification according to the following procedure (heated 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 briefly to allow the liquid to pool. 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 removing 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 outwards. 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 and discard the residual liquid 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) The PCR tube was centrifuged lightly and placed on a magnetic rack. After the solution was clarified, the supernatant was transferred 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 self-prepared STR PCR Primer Mix-Tubes 1 to 8 in Tables 1 to 8, and take out the NEB Prepare High-Fidelity DNA Polymerase (Cat. No. M0491) by adding 5×Q5 Reaction Buffer, 10mM dNTPs, and nuclease-free water. After the reagents are dissolved, mix thoroughly and centrifuge briefly. Mix the Q5 High-Fidelity DNA Polymerase by inversion and centrifuge briefly. Place on ice until ready to 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 Mix 1

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

[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 2.5 μL Q5 High-Fidelity DNA Polymerase 0.25 μL Template DNA (10 ng / μL) 2μL Nuclease-free water 14.75μL Total reaction volume 25 μL

[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 2.5 μL Q5 High-Fidelity DNA Polymerase 0.25 μL Template DNA (10 ng / μL) 2μL Nuclease-free water 14.75μL Total reaction volume 25 μL

[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 2.5 μL Q5 High-Fidelity DNA Polymerase 0.25 μL Template DNA (10 ng / μL) 2μL Nuclease-free water 14.75μL Total reaction volume 25 μL

[0202] Table 18. STR PCR Amplification Mix 5

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

[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 2.5 μL Q5 High-Fidelity DNA Polymerase 0.25 μL Template DNA (10 ng / μL) 2μL Nuclease-free water 14.75μL Total reaction volume 25 μL

[0206] Table 20, STR PCR Amplification Mix 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 2.5 μL Q5 High-Fidelity DNA Polymerase 0.25 μL Template DNA (10 ng / μL) 2μL Nuclease-free water 14.75μL Total reaction volume 25 μL

[0211] (5) Vortex the PCR amplification mixture for 5-10 seconds, and then divide 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 to the reaction tube containing the PCR amplification solution.

[0214] (8) Cover the reaction plate or tube tightly and centrifuge gently to centrifuge 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 cycling program, and the reaction time is approximately 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, this application performs STR testing on the micro-cell sample under Item 1. The test results show the consistency of the test results of each STR locus in Tubes 1-8 with the test results of the positive control. In the sample testing of this example, a total of 15 valid STR loci (the STRs marked with boxes) were detected. Therefore, using the method of this example to perform STR testing on the sample under Item 1 can effectively determine the polyploidy detection results.

[0223] Example 2

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

[0225] Example 3

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

[0227] Combined with Example 1 to Example 3 Figures 1 to 25 It can be seen that, using the method provided in this 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. The only difference from embodiment 1 is that multiplex PCR is performed in a combined tube. Figure 26 As shown, when D6S477, D22-GATA198B05, D15S659, and D19S253 are combined in a multiplex PCR tube, 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. Figure 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 example is a variation of Example 1, differing only in the comparison of fragmentation conditions tested. As shown in Figure 28: Figure 1 shows the capillary electrophoresis results of a large DNA sample (5 cells) used as a positive control for STR PCR amplification; Figure 2 shows the capillary electrophoresis results of 5 cells fragmented for 20 seconds followed by WGA and then STR PCR. All six STR loci were amplified, with peak heights essentially consistent with the positive control; Figure 3 shows the capillary electrophoresis results of 5 cells fragmented for 3 minutes followed by WGA and then STR PCR. Only two of the six STR loci were correctly amplified; Figure 4 shows the capillary electrophoresis results of a one-third sample of 5 cells fragmented for 8 minutes according to the previous PIMS library construction process. None of the six STR loci were successfully amplified.

[0234] Regarding the analysis of the embodiment of the optimal detection of 52 STR loci in this application. STR PCR amplification is performed after amplification by single-cell amplification enzyme. 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 two trace cell samples prepared under Item 1 of Reference Example 1. The test 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, which meet the judgment criteria of ≥10 loci. 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 the risk of not meeting the judgment requirements.

[0235] Table 23

[0236]

[0237]

[0238] Schematic diagram of the use of STRs for uniparental diploidy, triploidy, etc. in this application:

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

[0240] like Figure 30 As shown, the normal diploid embryos born to this 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 diallele peak.

[0241] like Figure 31 Triploidy, or 3n, is a chromosomal aberration characterized by the presence of three complete sets of chromosomes in each cell. PIMS testing for triploidy uses paternity testing as a criterion. If an embryo's STR loci contain ≥10 triallelic peaks or an unbalanced diallelic peak height ratio of 2:1, the probability of the embryo being triploid is ≥99.99%, supporting the diagnosis of triploidy. The possible patterns of triploid embryos at these STR loci for this couple are: embryo 1: triallelic peaks of 6-, 7-, and 10-fold; embryo 2: triallelic peaks of 6-, 7-, and 8-fold; embryo 3: triallelic peaks of 7-, 8-, and 10-fold; and embryo 4: triallelic peaks of 6-, 8-, and 10-fold in a 1:1:1 ratio. If ≥10 such peaks are detected across all 52 STR loci, the embryo is triploid.

[0242] like Figure 32 As shown, the missing gamete fertilizes with a normal gamete to form a monosomic zygote. During cleavage, the monosomic zygote replicates and becomes disomic. As shown in the figure below, the possible outcomes of a uniparental diploid embryo born to this couple at this STR locus are: embryo 1:7 weights or embryo 2:10 weights, indicating that the embryo is maternally uniparental homodisomic.

[0243] During meiosis, nondisjunction of homologous chromosomes or sister chromatids results in the fertilization of a disomic gamete with a normal haploid gamete, forming a trisomic zygote. Due to self-rescue, one of the chromosomes is randomly lost during cleavage, resulting in uniparental disomy. As shown in the figure below, the possible patterns of uniparental disomy at this STR locus in this couple are: embryo 3: a 1:1 ratio of 6-fold and 8-fold biallelic peaks (heterozygous), the father's 7-fold monoallelic peak (homozygous), and embryo 3's detection of a 1:1 ratio of 6-fold and 8-fold biallelic peaks at this STR locus, consistent with the mother's results. Both homologous chromosomes are inherited from the mother. If there are ≥10 such STR loci, the embryo is a maternally uniparental heterodisomic.

[0244] The various technical features of the above-mentioned implementation modes and examples can be combined in any appropriate manner. In order to make the description concise, not all possible combinations of the various 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 the description in this specification.

[0245] The above-described embodiments only express several implementation methods of the present application, which facilitate a specific and detailed understanding of the technical solutions of the present application, but cannot be understood as limiting 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 variations and improvements can be made, which all fall within the scope of protection of the present application. In addition, it should be understood that after reading the above-mentioned teaching content of the present application, those skilled in the art can make various changes or modifications to the present application, and the equivalent forms obtained also fall within the scope of protection of the present application. It should also be understood that the technical solutions obtained by those skilled in the art through logical analysis, reasoning or limited experiments on the basis of the technical solutions provided in the present application are all within the scope of protection of the claims attached to the present application. Therefore, the scope of protection of the patent application of the present application shall be based on the content of the attached claims, and the description and drawings can be used to interpret the content of the claims.

Claims

1. A nucleic acid combination product, characterized in that: The nucleic acid combination product includes 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 shown in SEQ ID NO.1 to SEQ ID NO.18, The primer pair M2-5 whose sequences are shown in SEQ ID NO.19 to SEQ ID NO.20, 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, whose sequences are shown in SEQ ID NO.21 to SEQ ID NO.38, 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, The primer pair M8-3 whose sequences are shown in SEQ ID NO.45 to SEQ ID NO.46, 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, whose sequences are shown in SEQ ID NO.47 to SEQ ID NO.62, The primer pair M4-3 and the primer pair M2-2 whose sequences are shown in SEQ ID NO.63 to SEQ ID NO.66, Primer pair M9-1, primer pair M9-2 and primer pair M9-3 having sequences shown in SEQ ID NO. 67 to SEQ ID NO. 72 The primer pair M9-4 and primer pair M9-5 whose sequences are shown in SEQ ID NO.73 to SEQ ID NO.76, 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 M9-9 whose sequences are shown in SEQ ID NO.81 to SEQ ID NO.82, The primer pair M9-11 whose sequences are shown in SEQ ID NO.83 to SEQ ID NO.84, 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, whose sequences are shown in SEQ ID NO.85 to SEQ ID NO.102, Primer pair M10-13 whose sequences are shown in SEQ ID NO. 103 to SEQ ID NO. 104; 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 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, The 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, The primer pair M9-6, primer pair M9-1 and primer pair M5-2 are divided into one group, The primer pair M9-4, primer pair M9-5 and primer pair M5-4 are divided into one group, The 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.

2. The nucleic acid combination product according to claim 1, characterized in that At least one primer in the primer pair is labeled with a fluorescent group.

3. The nucleic acid combination product according to claim 2, characterized in that The fluorescent groups labeled on each primer are independently 6-FAM, ROX, HEX or TAMRA.

4. A detection kit, characterized in that The detection kit comprises the nucleic acid combination product according to any one of claims 1 to 3.

5. The detection kit according to claim 4, characterized in that The detection kit also includes STR PCR reaction reagents.

6. A method for detecting short tandem repeats in DNA of a biological sample for non-diagnostic purposes, characterized in that: The detection method comprises the step of using the nucleic acid combination product according to any one of claims 1 to 3 or the detection kit according to any one of claims 4 to 5 to detect short tandem repeat fragments in the DNA of a biological sample to be tested.

7. The method for detecting short tandem repeats in non-diagnostic biological sample DNA according to claim 6, 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 amplified product is used as a template and the nucleic acid combination product is used to perform STR PCR amplification, and the situation of short tandem repeat fragments in the DNA of the biological sample to be tested is determined according to the obtained STR PCR amplification results.

8. The method for detecting short tandem repeats in DNA of a biological sample for non-diagnostic purposes according to claim 7, characterized in that: In the initial reaction system of STR PCR amplification, The molar ratio of 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 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 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 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 primer pair M5-1, primer pair M8-3, primer pair M4-3 and primer pair M2-2 is 5±1.0:37.5±1.0:25±1.0:12.5±1.0, The molar ratio of 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, primer pair M7-5, primer pair M10-6, primer pair M10-4, primer pair M10-2 and 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 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 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 primer pair M9-6, primer pair M9-1 and 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, primer pair M10-10, primer pair M10-3, primer pair M10-9 and primer pair M10-13 is 10±1.0:10±1.0:10±1.0:5±1.0:15±1.0; In each primer pair, 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 repeats in DNA of a biological sample for non-diagnostic purposes according to any one of claims 6 to 8, characterized in that: The biological samples to be tested are cell samples and blood samples.

10. The method for detecting short tandem repeats in non-diagnostic biological sample DNA according to claim 9, characterized in that: The number of cells in the cell sample is 2-10.

11. The method for detecting short tandem repeats in non-diagnostic biological sample DNA according to any one of claims 6 to 8, characterized in that: The biological sample to be tested comes from a pre-implantation embryo.

12. The method for detecting short tandem repeats in DNA of a biological sample for non-diagnostic purposes according to claim 11, characterized in that: The biological sample to be tested comes from the trophoblast.

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