Agricultural multi-locus genotyping detection methods, devices, apparatuses, and media

Abstract

The application relates to the field of high-throughput sequencing technology, in particular to an agricultural multi-locus typing detection method, device, equipment and medium. The application prepares a to-be-tested agricultural sample by using an automatic sample extraction system, designs primers according to multi-locus typing data of the to-be-tested agricultural sample and a target coding microchip, obtains upstream and downstream primers, carries out product amplification on the to-be-tested agricultural sample according to the upstream and downstream primers, obtains amplification reaction products, carries out mixed hybridization according to the amplification reaction products, the target coding microchip and a streptavidin phycoerythrin conjugate, obtains to-be-tested products, carries out fluorescence detection on the to-be-tested products, and obtains locus typing information of the to-be-tested agricultural sample. The detection method can realize simultaneous typing of multiple SNP loci in one reaction system, and when multiple loci need to be detected, the effective saving of multiple costs such as materials, labor and time can be realized, and the method is convenient and fast.

Description

Technical Field

[0001] This application relates to the field of high-throughput sequencing technology, and in particular to a method, apparatus, equipment and medium for agricultural multi-site genotyping detection. Background Technology

[0002] With the development of high-throughput sequencing technology, various types of high-throughput sequencing technologies have emerged in the agricultural field, such as TaqMan probe method, ARMS-PCR (Amplification Refractory Mutation System PCR) method, and KASP (Kompetitive Allele Specific PCR) method. However, these existing high-throughput sequencing methods cannot simultaneously analyze a large number of samples of a large number of SNP sites. Furthermore, since the agricultural field places greater emphasis on detection cost and site detection efficiency, how to efficiently achieve simultaneous genotyping of multiple SNP sites in a single reaction system has become an urgent technical problem to be solved. Summary of the Invention

[0003] Based on this, this application provides an agricultural multi-site genotyping detection method, apparatus, computer equipment, computer-readable storage medium, and computer program product that can efficiently achieve simultaneous sequencing of multiple SNP sites in a single reaction system.

[0004] This application provides a high-throughput automated detection method for multi-site genotyping in agriculture, the method comprising:

[0005] Fabrication of target-encoded microchips;

[0006] Primers were designed based on multiple genotyping site data of the agricultural samples to be tested and the target coding microchip to obtain upstream and downstream primers;

[0007] Agricultural nucleic acid samples to be tested were prepared using an automated sample extraction system;

[0008] A high-throughput automated sample preparation system was used to mix the reaction system, which included multiple upstream and downstream primers, the agricultural nucleic acid sample to be tested, and the amplification reaction solution.

[0009] The agricultural sample to be tested was amplified using a high-throughput amplification reaction system and the upstream and downstream primers to obtain the amplification reaction product.

[0010] The amplification reaction product, the target-encoded microchip, and the streptavidin-phycoerythrin conjugate were automatically mixed and hybridized to obtain a reaction plate containing the product to be detected; and

[0011] The reaction plate is transferred to the detection module, and the fluorescence detection of the product to be detected is performed to obtain the site typing information of the agricultural sample to be tested.

[0012] The amplification reaction is performed in a PCR reaction plate, and the mixed hybridization reaction is performed in an enzyme-labeled reaction plate.

[0013] In some embodiments, the preparation process of the agricultural nucleic acid sample to be tested using an automated sample extraction system includes:

[0014] The agricultural sample to be tested was ground with liquid, centrifuged, and the supernatant was collected. Then, nucleic acid was extracted using a nucleic acid extraction module to obtain the agricultural nucleic acid sample to be tested.

[0015] In some embodiments, the mixing reaction system utilizing a high-throughput automated sample preparation system includes:

[0016] The high-throughput automated sample preparation system was used to prepare the reaction system for the upstream and downstream primers, the nucleic acid sample, and the amplification reaction solution to obtain the target reaction system.

[0017] The target reaction system is subjected to product amplification reaction according to the preset reaction program of the high-throughput amplification reaction system to obtain the amplification reaction product.

[0018] In some embodiments, the automated hybridization is performed using a high-throughput automated hybridization system preparation system, the preset reaction program of which includes:

[0019] S dilutes the amplification reaction product in a reaction plate to obtain a diluted reaction product;

[0020] S2. Construct a reaction system in a reaction plate based on the diluted reaction product, the target encoded microchip, and the streptavidin-phycoerythrin conjugate to obtain the target hybridization system;

[0021] S3. The reaction plate containing the target hybridization system is shaken and incubated for hybridization to obtain candidate hybridization products;

[0022] S4. The candidate hybridization product is subjected to impurity removal treatment to obtain the product to be tested.

[0023] In some embodiments, the impurity removal process of the candidate hybridization product to obtain the product to be detected includes:

[0024] The candidate hybridization products in the reaction plate are subjected to magnetic adsorption treatment, the supernatant is discarded, and the target encoded microchip hybridization product is retained;

[0025] The hybridization product of the target encoded microchip in the reaction plate is cleaned to obtain the cleaned hybridization product.

[0026] The cleaned target-encoded microchip hybridization product in the reaction plate is demagnetized to obtain the product to be detected.

[0027] In some embodiments, the step of performing fluorescence detection on the product to be detected to obtain the locus typing information of the agricultural sample to be tested includes:

[0028] Transfer the reaction plate containing the product to be detected to the fluorescence detection system;

[0029] The product to be detected is subjected to optical imaging processing to obtain white light field of view data and fluorescence field of view data;

[0030] The chip location information and encoding information of the agricultural sample to be tested are determined based on the white light field of view data.

[0031] The fluorescence intensity data of each coded microchip is determined based on the fluorescence field of view data;

[0032] When the fluorescence intensity data meets the preset conditions, the locus typing information of the agricultural sample to be tested is determined based on the fluorescence intensity data.

[0033] In some embodiments, primers are designed based on multiple genotyping site data of the agricultural sample to be tested and the target coding microchip to obtain upstream and downstream primers, including:

[0034] Based on the genotyping site data, a first sequence is designed to obtain a first primer sequence that is complementary to and matches the genotyping site data.

[0035] A second primer sequence is designed based on the target encoding microchip to obtain a second primer sequence; wherein the second primer sequence is complementary to a known sequence of the target encoding microchip;

[0036] The first primer sequence and the second primer sequence are connected according to a preset protein to obtain an upstream primer of the upstream and downstream primers.

[0037] In some embodiments, the method further includes:

[0038] Obtain the mother liquor and pre-set primer sequence reagents for the original coded microchip;

[0039] The mother liquor is washed to obtain the target mother liquor;

[0040] The original coding microchip and sequencing reagent are coupled according to the target mother liquor to obtain a candidate coding microchip.

[0041] The candidate encoded microchip is sealed and stored to obtain the target encoded microchip.

[0042] Another aspect of this application provides a high-throughput automated agricultural multi-site typing detection device, the device comprising:

[0043] An automated sample extraction system, wherein the sample extraction system is used to process the supernatant of a ground agricultural sample to be tested and obtain a nucleic acid sample to be tested;

[0044] A high-throughput automated sample preparation system, wherein the sample preparation system includes an amplification system configuration module for amplifying the agricultural sample to be tested according to the upstream and downstream primers and the reaction amplification system to obtain amplification reaction products;

[0045] A high-throughput detection module, wherein the detection system includes a fluorescence detection module, which is used to perform fluorescence detection on the product to be tested to obtain the site typing information of the agricultural sample to be tested;

[0046] In some embodiments, the high-throughput automated sample preparation system further includes an oscillating temperature control module for mixing and hybridizing the amplification reaction products, the target encoded microchip, and a preset mycoagulant-phycoerythrin conjugate to obtain the product to be detected.

[0047] In another aspect, this application provides a computer device including a memory and a processor, wherein the memory stores a computer program, and the processor executes the computer program to implement the steps of the method described above.

[0048] In another aspect, this application provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the steps of the method described above.

[0049] Details of one or more embodiments of this application are set forth in the following description, and other features, objects, and advantages of this application will become apparent from the specification and its claims. Attached Figure Description

[0050] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 This is a flowchart of an agricultural multi-site typing detection method in one embodiment of this application;

[0052] Figure 2 This is a flowchart of a high-throughput agricultural multi-site genotyping automated detection process in one embodiment of this application;

[0053] Figure 3 This is a flowchart illustrating the fabrication steps of the target encoding microchip in one embodiment of this application;

[0054] Figure 4 This is a schematic diagram of a high-throughput agricultural multi-site typing automated detection device template in one embodiment of this application;

[0055] Figure 5 This image shows the typing effect of an agricultural multi-site typing detection method based on an coded microchip on 25 SNP sites in one embodiment of this application.

[0056] Figure 6 This image shows the typing effect of an agricultural multi-site typing detection method based on an coded microchip on 25 SNP sites in one embodiment of this application.

[0057] Figure 7 This image shows the typing effect of an agricultural multi-site typing detection method based on an coded microchip on 25 SNP sites in one embodiment of this application.

[0058] Figure 8 This image shows the typing effect of an agricultural multi-site typing detection method based on an coded microchip on 25 SNP sites in one embodiment of this application.

[0059] Figure 9 This image shows the typing effect of an agricultural multi-site typing detection method based on an coded microchip on 25 SNP sites in one embodiment of this application.

[0060] Figure 10 This image shows the typing effect of an agricultural multi-site typing detection method based on an coded microchip on 25 SNP sites in one embodiment of this application.

[0061] Figure 11 This image shows the typing effect of an agricultural multi-site typing detection method based on an coded microchip on 25 SNP sites in one embodiment of this application.

[0062] Figure 12 This is a visualization of the genotyping results of 25 SNP sites based on the KASP detection method in a comparative example;

[0063] Figure 13 This is a visualization of the genotyping results of 25 SNP sites based on the KASP detection method in a comparative example;

[0064] Figure 14 This is a visualization of the genotyping results of 25 SNP sites based on the KASP detection method in a comparative example;

[0065] Figure 15This is a visualization of the genotyping results of 25 SNP sites based on the KASP detection method in a comparative example;

[0066] Figure 16 This is a visualization of the genotyping results of 25 SNP sites based on the KASP detection method in a comparative example;

[0067] Figure 17 This is a visualization of the genotyping results of 25 SNP sites based on the KASP detection method in a comparative example;

[0068] Figure 18 This is a comparative example of the visualization of genotyping results for 25 SNP sites based on the KASP detection method. Detailed Implementation

[0069] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0070] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0071] the term

[0072] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0073] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These 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 connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: 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, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0074] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", 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.

[0075] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0076] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0077] In this application, terms such as "further," "even further," and "particularly" are used to describe purposes and indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0078] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0079] In this application, the technical features described in an open-ended manner include both closed technical solutions consisting of the listed features and open technical solutions that include the listed features.

[0080] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, 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 to which they are included.

[0081] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0082] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0083] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0084] The term "SNP site" refers to a single nucleotide polymorphism (SNP) that indicates a difference at a specific nucleotide position between individuals at the genomic level. It arises from variations (including transitions, transversions, insertions, or deletions) of a single nucleotide in the DNA sequence.

[0085] This application provides a high-throughput automated detection method for multi-site genotyping in agriculture, such as... Figure 1As shown in the flowchart, the method includes:

[0086] S202, Prepare the target encoding microchip;

[0087] S204. Based on the multiple genotyping site data of the agricultural sample to be tested and the target coding microchip, primers are designed to obtain upstream and downstream primers;

[0088] Agricultural samples to be tested were prepared using an automated sample extraction system;

[0089] S206. A high-throughput automated sample preparation system is used to mix the reaction system, which includes multiple upstream and downstream primers, the agricultural nucleic acid sample to be tested, and the amplification reaction solution.

[0090] The agricultural sample to be tested was amplified using a high-throughput amplification reaction system and the upstream and downstream primers to obtain amplification reaction products, wherein the amplification reaction was carried out in a PCR reaction plate.

[0091] S208. The amplification reaction product, the target encoding microchip and streptavidin phycoerythrin conjugate are automatically mixed and hybridized to obtain the product to be detected, wherein the mixing and hybridization reaction is carried out in an enzyme-labeled reaction plate.

[0092] S210. Transfer the reaction plate to the detection module and perform fluorescence detection on the product to be tested to obtain the site typing information of the agricultural sample to be tested.

[0093] This application utilizes an automated sample extraction system to prepare agricultural samples for testing. Primers are designed based on multiple genotyping site data and a target-encoded microchip in the agricultural samples to obtain upstream and downstream primers. The agricultural samples are then amplified using these primers to obtain amplification reaction products. These amplification reaction products, the target-encoded microchip, and a streptavidin-phycoerythrin conjugate are then mixed and hybridized to obtain the product to be detected. Fluorescence detection of the product to be detected yields the genotyping information of the agricultural samples. This method enables simultaneous genotyping of multiple SNP sites in a single reaction system and effectively saves costs in terms of materials, labor, and time when multiple sites need to be detected, offering convenience and speed.

[0094] In some embodiments, the high-throughput agricultural multi-site genotyping automated detection method of this application employs an automated system combination, specifically including:

[0095] Automated sample extraction system: The agricultural sample to be tested is ground, centrifuged, and the supernatant is collected. Then, nucleic acid is extracted using a nucleic acid extraction module to obtain a nucleic acid sample. The nucleic acid extraction module includes extraction using a magnetic rod and magnetic beads.

[0096] High-throughput automated sample preparation system: The high-throughput automated sample preparation system is used to configure the reaction system of the upstream and downstream primers and the nucleic acid sample to obtain the target reaction system.

[0097] High-throughput amplification reaction system: The target reaction system is subjected to product amplification reaction according to the preset reaction program of the high-throughput amplification reaction system to obtain the amplification reaction product.

[0098] High-throughput automated hybridization system preparation system: according to the preset reaction program of the high-throughput automated hybridization system preparation system.

[0099] Fluorescence detection system: Performs fluorescence detection on the product to be tested to obtain the site typing information of the agricultural sample to be tested.

[0100] In some embodiments, the preparation process using an automated sample extraction system includes:

[0101] The agricultural sample to be tested was ground, centrifuged, and the supernatant was collected. Nucleic acid was extracted using a nucleic acid extraction module to obtain the agricultural nucleic acid sample to be tested.

[0102] In some embodiments, the mixing reaction system utilizing a high-throughput automated sample preparation system includes:

[0103] The high-throughput automated sample preparation system is used to prepare the reaction system for the upstream and downstream primers, the nucleic acid sample, and the amplification reaction solution to obtain the target reaction system.

[0104] The target reaction system is subjected to product amplification reaction according to the preset reaction program of the high-throughput amplification reaction system to obtain the amplification reaction product.

[0105] In some embodiments, the automated hybridization is performed using the high-throughput automated hybridization system preparation system, and its preset reaction program includes:

[0106] S1. The amplification reaction product is diluted in a reaction plate to obtain a diluted reaction product;

[0107] S2. Construct a reaction system in a reaction plate based on the diluted reaction product, the target encoded microchip, and the streptavidin-phycoerythrin conjugate to obtain the target hybridization system;

[0108] S3. The reaction plate containing the target hybridization system is subjected to shaking incubation hybridization to obtain candidate hybridization products;

[0109] S4. The candidate hybridization product is subjected to impurity removal treatment to obtain the product to be tested.

[0110] In some embodiments, the impurity removal process of the candidate hybridization product to obtain the product to be detected includes:

[0111] The candidate hybridization products in the reaction plate are subjected to magnetic adsorption treatment, the supernatant is discarded, and the target encoded microchip hybridization product is retained;

[0112] The hybridization product of the target encoded microchip in the reaction plate is cleaned to obtain the cleaned hybridization product.

[0113] The cleaned target-encoded microchip hybridization product in the reaction plate is demagnetized to obtain the product to be detected.

[0114] In some embodiments, the step of performing fluorescence detection on the product to be detected to obtain the locus typing information of the agricultural sample to be tested includes:

[0115] The reaction plate containing the product to be detected is transferred to the fluorescence detection system;

[0116] The product to be detected is subjected to optical imaging processing to obtain white light field of view data and fluorescence field of view data;

[0117] The chip location information and encoding information of the agricultural sample to be tested are determined based on the white light field of view data.

[0118] The fluorescence intensity data of each coded microchip is determined based on the fluorescence field of view data;

[0119] When the fluorescence intensity data meets the preset conditions, the locus typing information of the agricultural sample to be tested is determined based on the fluorescence intensity data.

[0120] In some embodiments, the step of designing upstream and downstream primers based on multiple genotyping site data of the agricultural sample to be tested and the target coding microchip includes:

[0121] Based on the genotyping site data, a first sequence is designed to obtain a first primer sequence that is complementary to and matches the genotyping site data.

[0122] A second primer sequence is designed based on the target encoding microchip to obtain a second primer sequence; wherein the second primer sequence is complementary to a known sequence of the target encoding microchip;

[0123] The first primer sequence and the second primer sequence are connected according to a preset protein to obtain an upstream primer of the upstream and downstream primers.

[0124] Understandably, two upstream primers need to be designed, with the following differences: the first primer sequence has a 1-base difference at the 3' end, which can match different sites; the second primer sequence needs to be complementary to a known sequence on another target-encoding microchip.

[0125] In some embodiments, the method further includes:

[0126] Obtain the mother liquor and pre-set primer sequence reagents for the original coded microchip;

[0127] The mother liquor is washed to obtain the target mother liquor;

[0128] The original coding microchip and the sequencing reagent are coupled according to the target mother liquor to obtain a candidate coding microchip.

[0129] The candidate encoded microchip is sealed and stored to obtain the target encoded microchip.

[0130] Specifically, the high-throughput agricultural multi-site genotyping automated detection process is as follows: Figure 2 As shown, it includes the following steps:

[0131] I. Providing microchips

[0132] This microchip has two characteristics:

[0133] 1. It contains multiple coding bits, each of which has two possibilities: triangular and planar, corresponding to binary 0 and 1. The coding bit is determined to be 0 or 1 by judging whether it is triangular or planar. Using 12-bit shape encoding, the number of codes reaches 4096.

[0134] 2. It has superparamagnetism and can be gathered using magnetic fields, magnetic frames, etc.

[0135] II. Fabrication of TAG-Microchip

[0136] By utilizing the carboxyl groups on the surface of a microchip and employing an EDC / NHS activation method, nucleic acid probes can be covalently bound to the microchip surface via chemical bonds. These nucleic acid probes are referred to as barcode sequences or TAGs in application. Each encoding microchip can bind a large number of TAG sequences to their 3' ends. At this point, a TAG sequence library is available, allowing for random matching and free combination of encoding microchips with TAG sequences.

[0137] like Figure 3 As shown, the fabrication steps of the target encoding microchip include steps S302 to S308. Wherein:

[0138] Step S302: Obtain the mother liquor of the original encoded microchip and the preset sequencing sequence reagent.

[0139] Among them, the mother liquor of the original coding microchip refers to the mother liquor reagent containing the original coding microchip. The preset sequencing sequence reagent can be the barcode reagent. The original coding microchip has the same characteristics as the target coding microchip. As an unprocessed coding microchip, the original coding microchip can be aggregated using magnetic fields, magnetic racks, etc., to generate target primers.

[0140] It is understood that there is more than one type of encoding microchip and primer sequence reagent; only one type of microchip is coupled with one type of primer sequence reagent. In the current embodiment, 50 different encoding microchips are coupled with 50 different primer sequence reagents, that is, there are 50 target encoding microchips.

[0141] In some embodiments, barcode reagents are obtained from a barcode sequence library as preset sequencing sequence reagents, and a mother liquor containing the original coding microchip is obtained from a coding microchip liquid library. This mother liquor can be used to randomly match and freely combine the original coding microchip with the barcode sequence, thereby designing the target coding microchip.

[0142] Step 304: Wash the mother liquor to obtain the target mother liquor.

[0143] The cleaning process removes impurities and purifies the mother liquor.

[0144] In some embodiments, the mother liquor for encoding microchips is washed with ultrapure water and MEST to obtain a purer target mother liquor for preparing more precise target microchips.

[0145] Step S306: The original coding microchip and sequencing reagent are coupled according to the target mother liquor to obtain the candidate coding microchip.

[0146] In some embodiments, MES and EDC are used to couple the coding microchip and sequencing sequence reagent barcodes. Specifically, utilizing the carboxyl groups on the surface of the original coding microchip, nucleic acid probes are covalently bound to the microchip surface via chemical bonds through an EDC / NHS activation method. These nucleic acid probes are referred to as barcode sequences or TAGs in application. Each coding microchip can bind a large number of barcode sequences to their 3' ends. This application designs a barcode sequence library that allows for random matching and free combination of coding microchips and barcode sequences to obtain candidate coding microchips.

[0147] Step 308: The candidate encoded microchip is sealed and stored to obtain the target encoded microchip.

[0148] In some embodiments, the candidate coding microchip is sealed using TBST after coupling, and then stored in a 4°C refrigerator to obtain the prepared target coding microchip.

[0149] 3. For each SNP site, design two specific upstream primers and one downstream primer.

[0150] In some embodiments, the step of designing upstream and downstream primers based on multiple genotyping site data of the agricultural sample to be tested and the target coding microchip includes:

[0151] Based on the genotyping site data, a first sequence is designed to obtain a first primer sequence that is complementary to and matches the genotyping site data.

[0152] A second primer sequence is designed based on the target encoding microchip to obtain a second primer sequence; wherein the second primer sequence is complementary to the test sequence of the target encoding microchip.

[0153] The first primer sequence and the second primer sequence are connected according to a preset protein to obtain the upstream primer of the upstream and downstream primers.

[0154] Specifically, the upstream primer consists of three parts:

[0155] 1. It is a primer sequence that is complementary to the template and is about 20 bp in length. The two upstream primers differ only by one base at the 3' end in this part, and are used to amplify samples with different genotypes.

[0156] 2. The nucleic acid sequence complementary to the TAG on the coding microchip, also known as the anti-tag, is about 24 bp in length. Two upstream primers use this part of the sequence to complementarily pair onto different coding microchips, thereby distinguishing different samples.

[0157] 3. A spacer modification group is located between the two sequences above, including at least one of C3 Spacer, C6 Spacer, Spacer 9, C12 Spacer, and Spacer 18. It is understood that this application uses the C6 protein to connect the upper and lower sequences and to prevent base addition to the second sequence during downstream primer extension. The downstream primer has a biotin modification at its 5' end.

[0158] 4. Using the primers from step 3 to amplify the template, the two upstream primers competitively amplify the SNP sites. Only the upstream primer whose 3' end bases correctly match the template can extend downwards, and together with the downstream primer, the nucleic acid fragment carrying SNP site information is continuously amplified. The final DNA double-stranded product has one 5' end with an anti-tag nucleic acid sequence complementary to the TAG encoding the microchip, and the other 5' end with a biotin.

[0159] IV. Preparation of the product to be tested

[0160] In some embodiments, the automated hybridization is performed using the high-throughput automated hybridization system preparation system, and its preset reaction program includes:

[0161] S1. The amplification reaction product is diluted in a reaction plate to obtain a diluted reaction product;

[0162] S2. Construct a reaction system in a reaction plate based on the diluted reaction product, the target encoded microchip, and the streptavidin-phycoerythrin conjugate to obtain the target hybridization system;

[0163] S3. The reaction plate containing the target hybridization system is subjected to shaking incubation hybridization to obtain candidate hybridization products;

[0164] S4. The candidate hybridization product is subjected to impurity removal treatment to obtain the product to be tested.

[0165] Specifically, the three DNA products obtained above, two specific encoded microchips, and streptavidin-phycoerythrin (SAPE) are mixed into the same system. The two encoded microchips are each labeled with a different TAG sequence that can complementaryly pair with the upstream primer. The 5' end sequence of one strand of the DNA product can bind complementary to the TAG on one of the encoded microchips, while the 5' biotin on the other strand can bind to streptavidin-phycoerythrin. The final product is a sequence of "encoded microchip + TAG + anti-tag + SNP site amplification product + biotin + streptavidin + phycoerythrin" to be detected.

[0166] The encoded microchip, TAG, Anti-tag, and SNP site amplification products have a one-to-one correspondence; that is, knowing the information of one substance allows you to match the others. After the products to be detected are formed, they aggregate under the influence of a magnetic field, and then the supernatant, especially the avidin-phycoerythrin reagent, is discarded to avoid background fluorescence.

[0167] V. Fluorescence Detection

[0168] The coding and fluorescence are detected using an optical imaging system. In white light, the instrument's algorithm identifies coded microchips of different shapes and determines their location. In fluorescence, laser excitation causes phycoerythrin bound to specific coded microchips to fluoresce. The genotype is determined by reading the fluorescence intensity of the corresponding coded microchip, and the SNP information of the template can be obtained from the upstream primer corresponding to the coded microchip with the strongest fluorescence intensity.

[0169] Based on the same inventive concept, this application also provides an agricultural multi-site typing detection device for implementing the above-mentioned agricultural multi-site typing detection method. The solution provided by this device is similar to the solution described in the above method; therefore, the specific limitations of one or more embodiments of the agricultural multi-site typing detection device provided below can be found in the limitations of the agricultural multi-site typing detection method described above, and will not be repeated here.

[0170] Another aspect of this application provides a high-throughput automated agricultural multi-site typing detection device 2000, such as... Figure 4 As shown, the device includes:

[0171] An automated sample extraction module 2001, wherein the sample extraction module is used to grind the agricultural sample to be tested and obtain the nucleic acid sample to be tested;

[0172] High-throughput automated sample preparation module 2002, wherein the sample preparation system includes an amplification system configuration module for amplifying the agricultural sample to be tested according to the upstream and downstream primers to obtain amplification reaction products;

[0173] Optionally, the module is used to perform hybridization of the amplification reaction product, the target encoded microchip, and the streptavidin phycoerythrin conjugate to obtain the product to be detected.

[0174] High-throughput detection module 2003: The detection system includes a fluorescence detection module for performing fluorescence detection on the product to be detected to obtain the site typing information of the agricultural sample to be tested.

[0175] This application provides a computer device, including a memory and a processor, wherein the memory stores a computer program, and the processor executes the computer program to implement the steps of the method described in any of the above claims.

[0176] In another aspect, this application provides a computer-readable storage medium having a computer program stored thereon, which, when executed by a processor, implements the steps of the method described above.

[0177] It is understood that the coded microchip provided in this application includes one or more of the following classification functions and applications:

[0178] (1) It can accurately detect SNP and Indel sites in a single well and a single sample. (2) It can perform high-throughput sample genotyping using the matching instrument. (3) Application in genotyping of crops such as rice and maize. (4) Application in variety identification of crops such as rice and maize. (5) Application in authenticity and / or purity identification of crops such as rice and maize. (6) Application in detection of breeding materials of crops such as rice and maize. (7) Application in quality monitoring of seed production or propagation of crops such as rice and maize. (8) Application in the preparation of breeding chips for crops such as rice and maize. (9) Application in molecular marker-assisted breeding of crops such as rice and maize. (10) Application in molecular fingerprint analysis of germplasm resources of crops such as rice and maize.

[0179] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0180] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0181] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0182] Example 1

[0183] This embodiment takes rice as an example and uses the high-throughput agricultural multi-site typing automated detection method of this application for detection.

[0184] In this embodiment, 25 representative loci were selected from the "SNP Marker Method for Identifying the Authenticity of Rice Varieties" issued by the Ministry of Agriculture and Rural Affairs of the People's Republic of China on December 15, 2021, to detect 92 rice varieties used in production practice.

[0185] I. Fabrication of Encoded Microchips

[0186] 1. Fabrication of TAG-encoded microchips

[0187] (1) Prepare the coded microchip mother liquor and the corresponding TAG reagent.

[0188] (2) The mother liquor of the coded microchip is cleaned with ultrapure water and MEST.

[0189] (3) Use MES and EDC to couple the encoding microchip and TAG.

[0190] (4) After coupling, TBST is used to close it.

[0191] (5) After sealing, store in a refrigerator at 4°C.

[0192] 2. PCR product amplification

[0193] (1) Synthesize upstream and downstream primers based on the genotyping site.

[0194] (2) Extract DNA from the plant to be tested.

[0195] (3) Prepare a 10 μL reaction system.

[0196] (4) Set up the reaction program.

[0197] (5) Perform PCR reaction.

[0198] 3. Hybridization detection

[0199] (1) Dilute the reaction product by 10 times.

[0200] (2) Take 5 μL of the diluted product and add the corresponding two TAG-encoded microchips and SAPE. Mix the three components into a system.

[0201] (3) Hybridize by shaking at 55℃ for about 20 minutes.

[0202] (4) Under the action of magnetic force, magnetic beads are adsorbed, the supernatant is removed, and the reaction system is washed 3 times.

[0203] (5) Demagnetize and resuspend the hybridization product.

[0204] (6) Take photos under white light and fluorescence respectively.

[0205] (7) Output the fluorescence values ​​of different encoded microchips.

[0206] II. Testing Process

[0207] 1. Based on the SNP marker method for identifying the authenticity of rice varieties, 25 upstream and downstream primers were selected and synthesized. The 5' ends of the two upstream primers were connected to specific anti-tag sequences with C6 protein. The 5' ends of the downstream primers were all modified with biotin.

[0208] 2. Select 50 types of coded microchips, and add a specific TAG sequence to each type of coded microchip using the EDC / NHS activation method.

[0209] 3. DNA was extracted from 92 rice leaf tissue samples, including 2 samples of Nipponbare and 9311 with known genotypes. The DNA was extracted using the standard procedure of the MGI Easy Plant Genomic DNA Extraction Kit (GT) independently developed by BGI Genomics.

[0210] 4. DNA sample quality testing: The levels of protein and organic matter contamination in genomic DNA were measured using a Nanodrop spectrophotometer. The A260 / 280 ratio of genomic DNA should be between 1.8 and 2.0, and the A260 / 230 ratio should be between 1.8 and 2.2. The concentration of DNA working solution should be above 10 ng / μL.

[0211] 5. Genotype detection: The samples were tested according to the experimental steps in the technical solution of this application. To ensure the authenticity of the results, the two samples, Nipponbare and 9311, were repeated twice and two water controls were added. Finally, the original fluorescence value data of the test samples were generated, and the genotype of the samples was obtained through the prescribed interpretation method.

[0212] 6. Data Analysis: Using the analysis software built into the instrument, which is independently developed by BGI Genomics, and referring to the custom-constructed fractal intervals, based on... Figure 5 The detection results of molecular markers are interpreted and categorized as follows: (a) homozygous genotype GG is shown as a black square; (b) homozygous genotype AA is shown as a white square; (c) homozygous genotype CC is shown as a black circle; (d) homozygous genotype TT is shown as a white circle; (e) heterozygous genotypes A / G or C / T are shown as black triangles; (f) control results are shown using... Display; (g) Use with mixed or no amplified signal show.

[0213] II. The reagents and equipment used in the examples are shown in Table 1 below:

[0214] Table 1

[0215]

[0216] III. The specific primer sequences in this embodiment are shown in Table 2 below:

[0217] Table 2. 25 SNP sites and corresponding TAG information

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224] IV. The genotyping results of the 90 rice samples coded by the microchip are shown in Table 3 below. The genotyping effect is as follows: Figures 5 to 11 As shown in Table 3. The alleles in Table 3 represent:

[0225] GG: This indicates that the individual is homozygous at this gene locus, and both alleles are "G".

[0226] TT: This indicates that the individual is homozygous at this gene locus, and both alleles are "T".

[0227] AA: This indicates that the individual is homozygous at this gene locus, and both alleles are "A".

[0228] CC: This indicates that the individual is homozygous at this gene locus, and both alleles are "C".

[0229] G / A: This indicates that the individual is heterozygous at this gene locus, with one allele being "G" and the other being "A". G / T: This indicates that the individual is heterozygous at this gene locus, with one allele being "G" and the other being "T".

[0230] Table 3. Microchip typing results of 25 coding sites in 90 rice samples

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243] Comparative Example 1

[0244] In this comparative example, KASP genotyping was performed on 25 loci in 90 rice samples using the KASP technique. The results are shown in Table 4, and the genotyping results are illustrated in the figure below. Figures 12-18 As shown.

[0245] The classification results in Table 4 indicate that:

[0246] FF: This usually represents a fluorescent signal indicating that a sample is homozygous for an allele. For example, if a sample's genotype at a certain SNP locus is FF, it may mean that the sample is homozygous at that locus, and both alleles are of a specific base (such as AA or TT).

[0247] HH: This indicates a different fluorescent signal, representing a homozygous allele. For example, if the genotyping result is HH at the same SNP locus, it may indicate that the sample is a different homozygote at this locus, and both alleles are different specific bases (such as GG or CC).

[0248] F:H: indicates a mixed signal, meaning the individual is heterozygous at this site.

[0249] Missing: This indicates that no signal was detected at this site, and the detection failed.

[0250] Table 4. KASP typing results at 25 loci in 90 rice samples

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259] Result verification:

[0260] 1. Result Comparison: Comparison of genotyping results with KASP test results. The genotyping results obtained following the above steps will be compared with the results obtained using KASP technology.

[0261] 2. Conclusion: The results showed that the two methods achieved a 99.8% concordance rate for the genotypes of the tested materials. Inconsistencies occurred in the absence of some KASP markers (e.g., ChSeed-11, ChSeed-38, and ChSeed-88). These results demonstrate that the high-throughput automated multi-site genotyping method for agriculture provided in this application can reliably genotype 90 rice materials, providing reliable authenticity determination results, and exhibits higher sensitivity compared to KASP technology.

[0262] The same operation described above can be used to achieve purity detection when testing multiple individual samples.

[0263] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A high-throughput automated detection method for multi-site genotyping in agriculture, characterized in that, The method includes: Fabrication of target-encoded microchips; Primers were designed based on multiple genotyping site data of the agricultural samples to be tested and the target coding microchip to obtain upstream and downstream primers; Agricultural nucleic acid samples to be tested were prepared using an automated sample extraction system; A high-throughput automated sample preparation system was used to mix the reaction system, which included multiple upstream and downstream primers, the agricultural nucleic acid sample to be tested, and the amplification reaction solution. The agricultural sample to be tested was amplified using a high-throughput amplification reaction system and the upstream and downstream primers to obtain the amplification reaction product. The amplification reaction product, the target-encoded microchip, and the streptavidin-phycoerythrin conjugate were automatically mixed and hybridized to obtain a reaction plate containing the product to be detected; and The reaction plate is transferred to the detection module, and the fluorescence detection of the product to be detected is performed to obtain the site typing information of the agricultural sample to be tested. The amplification reaction is performed in a PCR reaction plate, and the mixed hybridization reaction is performed in an enzyme-labeled reaction plate.

2. The method according to claim 1, characterized in that, The preparation process of the agricultural nucleic acid sample to be tested using the automated sample extraction system includes: The agricultural sample to be tested was ground with liquid, centrifuged, and the supernatant was collected. Then, nucleic acid was extracted using a nucleic acid extraction module to obtain the agricultural nucleic acid sample to be tested. Optionally, the mixing reaction system utilizing the high-throughput automated sample preparation system includes: The high-throughput automated sample preparation system was used to prepare the reaction system for the upstream and downstream primers, the nucleic acid sample, and the amplification reaction solution to obtain the target reaction system. The target reaction system is subjected to product amplification reaction according to the preset reaction program of the high-throughput amplification reaction system to obtain the amplification reaction product.

3. The method according to claim 2, characterized in that, The automated mixing and hybridization is performed using a high-throughput automated hybridization system, the preset reaction program of which includes: S1. The amplification reaction product is diluted in a reaction plate to obtain a diluted reaction product; S2. Construct a reaction system in a reaction plate based on the diluted reaction product, the target encoded microchip, and the streptavidin-phycoerythrin conjugate to obtain the target hybridization system; S3. The reaction plate containing the target hybridization system is shaken and incubated for hybridization to obtain candidate hybridization products; S4. The candidate hybridization product is subjected to impurity removal treatment to obtain the product to be tested.

4. The method according to claim 3, characterized in that, The impurity removal process performed on the candidate hybridization products to obtain the product to be detected includes: The candidate hybridization products in the reaction plate are subjected to magnetic adsorption treatment, the supernatant is discarded, and the target encoded microchip hybridization product is retained; The hybridization product of the target encoded microchip in the reaction plate is cleaned to obtain the cleaned hybridization product. The cleaned target-encoded microchip hybridization product in the reaction plate is demagnetized to obtain the product to be detected.

5. The method according to any one of claims 1 to 4, characterized in that, The step of performing fluorescence detection on the product to be detected to obtain the locus typing information of the agricultural sample to be tested includes: Transfer the reaction plate containing the product to be detected to the fluorescence detection system; The product to be detected is subjected to optical imaging processing to obtain white light field of view data and fluorescence field of view data; The chip location information and encoding information of the agricultural sample to be tested are determined based on the white light field of view data. The fluorescence intensity data of each coded microchip is determined based on the fluorescence field of view data; When the fluorescence intensity data meets the preset conditions, the locus typing information of the agricultural sample to be tested is determined based on the fluorescence intensity data.

6. The method according to any one of claims 1 to 4, characterized in that, Primers were designed based on multiple genotyping site data of the agricultural sample to be tested and the target coding microchip to obtain upstream and downstream primers, including: Based on the genotyping site data, a first sequence is designed to obtain a first primer sequence that is complementary to and matches the genotyping site data. A second primer sequence is designed based on the target encoding microchip to obtain a second primer sequence; wherein the second primer sequence is complementary to a known sequence of the target encoding microchip; The first primer sequence and the second primer sequence are connected according to a preset protein to obtain an upstream primer of the upstream and downstream primers.

7. The method according to any one of claims 1 to 4, characterized in that, The method further includes: Obtain the mother liquor and pre-set primer sequence reagents for the original coded microchip; The mother liquor is washed to obtain the target mother liquor; The original coding microchip and sequencing reagent are coupled according to the target mother liquor to obtain a candidate coding microchip. The candidate encoded microchip is sealed and stored to obtain the target encoded microchip.

8. A high-throughput automated detection device for multi-site genotyping in agriculture, characterized in that, The device includes: An automated sample extraction system, wherein the sample extraction system is used to process the supernatant of a ground agricultural sample to be tested and obtain a nucleic acid sample to be tested; A high-throughput automated sample preparation system, wherein the sample preparation system includes an amplification system configuration module for amplifying the agricultural sample to be tested according to upstream and downstream primers and a reaction amplification system to obtain amplification reaction products; A high-throughput detection module, wherein the detection system includes a fluorescence detection module, which is used to perform fluorescence detection on the product to be tested to obtain the site typing information of the agricultural sample to be tested; Optionally, the high-throughput automated sample preparation system further includes a oscillation temperature control module for mixing and hybridizing the amplification reaction product, the target encoded microchip, and the preset myvidin-phycoerythrin conjugate to obtain the product to be detected.

9. A computer device comprising a memory and a processor, wherein the memory stores a computer program, characterized in that, When the processor executes the computer program, it implements the steps of the method according to any one of claims 1 to 7.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that, When the computer program is executed by a processor, it implements the steps of the method according to any one of claims 1 to 7.

Images