A method for co-detection of bovine mastitis resistance and early embryo gender and its kit

Abstract

This application belongs to the field of nucleic acid detection technology, and particularly relates to a method and kit for co-detecting bovine mastitis resistance and early embryo gender. This application uses a self-made lysis solution and neutralization solution to pretreat the sample, performs whole-genome amplification using Phi29 enzyme, then conducts multiplex PCR amplification based on the developed primer system, and finally combines capillary electrophoresis to achieve co-detection. This application only requires 4-10 embryonic cells to meet the detection requirements, realizing the synchronous detection of bovine early embryo gender and bovine mastitis resistance, and having advantages such as high sensitivity and strong specificity.

Description

Technical Field

[0001] This application belongs to the technical field of nucleic acid detection, and specifically relates to a method and kit for simultaneous detection of bovine mastitis resistance and early embryo sex. Background Art

[0002] In livestock production, many production traits are controlled by gender, and preselection of offspring gender provides opportunities to improve the profitability and sustainability of the livestock industry. As an important economic animal, gender determination and control are of great significance in dairy production. In the dairy embryo transfer industry, embryo sex identification is a powerful tool that can help dairy farmers manage their breeding resources more effectively.

[0003] Bovine mastitis is a pathological disease that causes abnormal milk quality and decreased milk production due to inflammatory reactions in mammary tissue. Its stimulating factors are diverse, including physical damage, chemical irritation, and microbial infection. The universality, high incidence, and intractability of bovine mastitis bring a series of economic losses that cannot be ignored. In addition to directly reducing milk production and deteriorating milk quality, it also causes reduced fertility and increased death and culling rates. Using genetic improvement to enhance mastitis resistance is an important way to improve the udder health of dairy cows. Based on the key single nucleotide polymorphisms (SNPs) of genes related to bovine mastitis resistance, they can be used as molecular markers for bovine mastitis resistance and applied to the genetic breeding of bovine mastitis resistance.

[0004] Simultaneously detecting the sex and mastitis resistance of bovine early embryos can achieve multiple goals such as accelerating genetic progress, reducing breeding costs, and improving the udder health of dairy cows, laying a foundation for the rapid cultivation of high-yield, high-quality, and healthy female cow herds, and is the only way to achieve the rapid propagation of improved dairy cows and the cultivation of characteristic new strains of dairy cows. Combining whole genome amplification with multiplex fluorescence PCR on the CE platform to develop a method for simultaneous detection of sex and key mastitis-resistant SNPs with high detection sensitivity and accuracy is very necessary for screening preferred embryos for bovine early embryo sex and mastitis resistance.

[0005] In view of this, this application is proposed. Summary of the Invention

[0006] The purpose of this application is to provide a method for simultaneously detecting the sex and mastitis resistance of bovine morula early embryos (4 - 10 cells). This method combines whole genome amplification with multiplex fluorescence PCR detection, and through capillary electrophoresis detection, the sex and SNP locus typing of the embryo sample to be detected are obtained. To achieve the above purpose, the following technical solutions are proposed in this application:

[0007] Specifically, this application first proposes a primer composition for simultaneous detection of bovine mastitis resistance and early embryo sex, and the primers are directed against SNP loci and sex identification genes.

[0008] Furthermore, the SNP sites include Chr5:104010752C>T on the CD4 gene, Chr14:2525852T>G on the TRAPPC9 gene, and Chr14:2711615G>A on the TRAPPC9 gene; the sex identification gene includes the Amel gene.

[0009] Even further, the primer sequences of the SNP sites are as follows:

[0010] Primers for the Chr5:104010752C>T site:

[0011] Primer for C typing: 5’-AGGGGGAATCAGAAAAGGA A TC-3’ (SEQ ID NO.1),

[0012] Primer for T typing: (SEQ ID NO.2),

[0013] Common primer: 5’-AGCTCCCAGGCTCAATAATTGT-3’ (SEQ ID NO.3);

[0014] Primers for the Chr14:2525852T>G site:

[0015] Primer for T typing: 5’-GCGCAGTTCTCCTGGAGCTTT C T-3’ (SEQ ID NO.4),

[0016] Primer for G typing: (SEQ ID NO.5),

[0017] Common primer: 5’-ACCATAATATAAAGAAGTAAAACAGCTCCCT-3’ (SEQ ID NO.6);

[0018] Primers for the Chr14:2711615G>A site:

[0019] Primer for G typing: 5’-AGCAGGCTAGGTCTCACGTT G AC-3’ (SEQ ID NO.7),

[0020] Primer for A typing: (SEQ ID NO.8),

[0021] Common primer: 5’-AGAGTGACTACTACCATCCAGTT-3’ (SEQ ID NO.9);

[0022] Wherein: the single underline of "-" represents a mismatched base; the double underline of "=" represents an added base.

[0023] Furthermore, the primer sequences of the Amel gene are as follows:

[0024] Forward primer for X typing: 5'-ACAGCTTTGTTCTATCCCAGCC-3' (SEQ ID NO.10),

[0025] Reverse primer for X typing: 5'-AGAGTGTGACTATCTTAGAATTG-3' (SEQ ID NO.11),

[0026] Forward primer for Y typing: 5'-ACAACTTTGCACTGTCCCAGAC-3' (SEQ ID NO.12),

[0027] Reverse primer for Y typing: 5'-AGAGTGTGAAACATCTTAGAATCA-3' (SEQ ID NO.13).

[0028] In some embodiments, the primers are fluorescently labeled;

[0029] Preferably, the fluorescent label is located at the 5' end of the common primer or the 5' end of the reverse primer of the sex identification gene;

[0030] More preferably, the fluorescent label is a FAM fluorescent label.

[0031] This application also provides a kit for co-detection of bovine mastitis resistance and early embryo sex, comprising any one of the foregoing primer compositions;

[0032] Furthermore, the kit further comprises cell lysate, neutralizing solution and Phi29 DNA polymerase.

[0033] Preferably, the alkaline cell lysate and the neutralizing solution are both self-made solutions;

[0034] More preferably, the cell lysate contains 300 - 500 mM of KOH, 75 - 125 mM of DDT and 7.5 - 12.5 mM of EDTA; in some specific embodiments, the alkaline cell lysate contains 400 mM of KOH, 100 mM of DDT and 10 mM of EDTA. The neutralizing solution contains 200 - 400 mM of Tris-Hcl, 150 - 250 mM of Hcl and 200 - 500 mM of Kcl; in some specific embodiments, the neutralizing solution contains 300 mM of Tris-Hcl, 200 mM of Hcl and 300 mM of Kcl.

[0035] Furthermore, the kit also contains 10×Phi29 MAX DNA Polymerase Reaction, MdNTP, Exo-Resistant Random Primer, 2×PCR Master Mix, internal standard, etc.

[0036] Furthermore, the early embryo is a morula embryo.

[0037] The present application also provides an application of the foregoing primer composition in the co-detection of bovine mastitis resistance and early embryo gender.

[0038] The present application also provides an application of the foregoing primer composition in the preparation of a kit for co-detecting bovine mastitis resistance and early embryo gender.

[0039] Furthermore, the co-detection includes the following steps:

[0040] 1) Preparation of the sample: Obtain 4-10 bovine embryo cells, place them in PBS buffer, add alkaline cell lysate, mix well and centrifuge, then add neutralizing solution, mix well and keep on ice for later use;

[0041] 2) Whole genome amplification: Take the product in 1) and add 10×Phi29 MAX DNA Polymerase Reaction Buffer, MdNTP, Phi29 MAX DNA Polymerase, Exo-Resistant Random Primer, nuclease-free pure water; incubate in a PCR instrument for later use;

[0042] 3) PCR amplification and detection: Take the product in 2), add PCR Master Mix, any one of the foregoing primer compositions, nuclease-free pure water, and perform amplification in a PCR instrument; the amplified product is detected by capillary electrophoresis, and the gender and related SNP genotyping results are obtained through analysis of the downloaded data.

[0043] Furthermore, the alkaline cell lysate and the neutralizing solution are both self-made solutions. The cell lysate contains 300-500 mM KOH, 75-125 mM DDT, and 7.5-12.5 mM EDTA; the neutralizing solution contains 200-400 mM Tris-Hcl, 150-250 mM Hcl, and 200-500 mM Kcl.

[0044] In some specific embodiments, the alkaline cell lysate contains 400 mM KOH, 100 mM DDT, and 10 mM EDTA; the neutralizing solution contains 300 mM Tris-Hcl, 200 mM Hcl, and 300 mM Kcl.

[0045] The present application also provides a method for processing and preparing bovine embryo samples, comprising the following steps:

[0046] 1) Sample preparation: Obtain 4-10 isolated bovine embryo cells, place them in PBS buffer, add alkaline cell lysate, mix well, centrifuge, then add neutralizing solution, mix well and keep on ice for later use;

[0047] 2) Whole genome amplification using Phi29: Add the product from 1) to 10×Phi29 MAX DNA Polymerase Reaction Buffer, MdNTP, Phi29 MAX DNA Polymerase, Exo-Resistant Random Primer, and nuclease-free pure water; incubate after amplification with a PCR instrument for later use;

[0048] Further, the cell lysate contains 300-500 mM of KOH, 75-125 mM of DDT, and 7.5-12.5 mM of EDTA; the neutralizing solution contains 200-400 mM of Tris-Hcl, 150-250 mM of Hcl, and 200-500 mM of Kcl.

[0049] The present application also provides a method for co-detection of bovine mastitis resistance and early embryo gender, comprising the following steps:

[0050] 1) Isolate 4-10 embryo cells from the bovine embryo samples to be tested;

[0051] 2) Lyse the isolated cells, then add a whole genome amplification mixture and Phi29 DNA polymerase for an amplification reaction;

[0052] 3) Take a part of the product from 2) and amplify it using a co-detection system for key SNPs of gender and anti-mastitis, and detect it using capillary electrophoresis.

[0053] Further, the method for separating cell samples in step 1) of the present application can use any existing separation techniques for common samples such as embryonic cells to separate single-cell samples, such as flow cytometry, laser microdissection technology, etc. As an optimal effect, in some embodiments of the present application, step 1) specifically includes: resuspending the separated embryonic cells in 4 μL of PBS, adding 3 μL of alkaline cell lysate, gently flicking and mixing, and centrifuging. After 10 minutes at 65 °C; adding 3 μL of neutralizing solution to terminate the lysis, mixing and centrifuging to form a cell lysis product. In some ways, the cell lysate contains 300-500 mM of KOH, 75-125 mM of DDT, and 7.5-12.5 mM of EDTA; in some specific ways, the alkaline cell lysate contains 400 mM of KOH, 100 mM of DDT, and 10 mM of EDTA. The neutralizing solution contains 200-400 mM of Tris-Hcl, 150-250 mM of Hcl, and 200-500 mM of Kcl; in some specific ways, the neutralizing solution contains 300 mM of Tris-Hcl, 200 mM of Hcl, and 300 mM of Kcl.

[0054] Further, the cell lysis in step 2) of the present application can be carried out using conventional existing technologies. As an optimal effect, in some specific embodiments of the present application, the whole-genome amplification reaction system in step 2) is 5 μL of 10×Phi29 MAX DNA Polymerase Reaction Buffer (purchased from Novizan), 5 μL of 10 mM dNTP (purchased from Thermo Fisher), 2.5 μL of Phi29 MAX DNA Polymerase (purchased from Novizan), 2.5 μL of 500 uM Exo-Resistant Random Primer (purchased from Thermo Fisher), 25 μL of nuclease-free pure water, and 10 μL of cell lysis product, with a total system of 50 μL.

[0055] Furthermore, the conditions for the amplification reaction in step 2) of the present application are 30 °C for 4-6 hours, and after the reaction, it is preferably inactivated at 65 °C for 10 minutes.

[0056] Further, step 3) of the present application specifically includes taking the product in 2) as a template, adding 2×PCR Master Mix, any one of the foregoing primer compositions, and nuclease-free pure water, and performing amplification in a PCR instrument; the amplified product is detected by capillary electrophoresis, and the off-machine data is analyzed to obtain gender and related SNP genotyping results.

[0057] Compared with the prior art, the present application has at least the following advantages:

[0058] Through the optimization of primer systems, sample processing, etc., and combined with whole genome amplification technology, this application has high detection sensitivity. Only 4-10 embryos are required to meet the detection requirements, and it enables the synchronous detection of the sex of bovine early embryos and key SNPs for anti-mastitis. Compared with current technologies such as LAMP and qPCR, it not only solves the problem of sample volume, but also can detect multiple targets, facilitating embryo screening, providing convenience for excellent animal husbandry, and also making it possible to detect other key sites subsequently.

[0059] In addition, the final detection of this application is based on a capillary electrophoresis platform for product detection, and the detection results have the advantages of high sensitivity, high resolution, simple and easy to operate, and having professional software for result interpretation. Brief Description of the Drawings

[0060] In order to more clearly illustrate the specific embodiments of this application or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of this application. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0061] Figure 1 . Screening and identification of primer combinations at the Chr5:104010752C>T locus on the.CD4 gene (the left is the fragment analysis result, and the right is the corresponding sequencing result);

[0062] Figure 2 . Screening and identification of primer combinations at the Chr14:2525852T>G locus on the TRAPPC9 gene (the left is the fragment analysis result, and the right is the corresponding sequencing result);

[0063] Figure 3 . Screening and identification of primer combinations at the Chr14:2711615G>A locus on the TRAPPC9 gene (the left is the fragment analysis result, and the right is the corresponding sequencing result);

[0064] Figure 4 . Amplification detection results of bovine blood DNA samples;

[0065] Figure 5 . Electrophoresis results of different embryo sample lysis schemes after whole genome amplification;

[0066] Figure 6 . Detection results of the sex and anti-mastitis key SNP locus multiplex amplification detection system after whole gene amplification of the embryo samples lysed by Scheme 1;

[0067] Figure 7. Detection results of the sex and key SNP loci for mastitis resistance in the cracked embryo samples after whole-genome amplification under Scheme II;

[0068] Figure 8 . Detection results of the sex and key SNP loci for mastitis resistance in the cracked embryo samples after whole-genome amplification under Scheme III;

[0069] Figure 9 . Detection results of the sex and key SNP loci for mastitis resistance in the cracked embryo samples after whole-genome amplification under Scheme IV;

[0070] Figure 10 . Detection results of the sex and key SNP loci for mastitis resistance in the cracked embryo samples after whole-genome amplification under Scheme V;

[0071] Figure 11 . Comparison results between Series A and Series B of embryo samples No. 009 and No. 058. Detailed implementation manners

[0072] Those skilled in the art can refer to the content of this application to implement its application. In particular, it should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all regarded as included in this application. The preparation method and application of this application have been described through preferred embodiments, and those related can obviously make changes or appropriate alterations and combinations to the preparation method and application in this application without departing from the content, spirit and scope of this application to implement and apply the technology of this application. Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those of ordinary skill in the art to which this application belongs.

[0073] The following terms or definitions are provided only to assist in understanding this application. These definitions should not be construed as having a scope less than that understood by those skilled in the art.

[0074] Unless otherwise defined hereinafter, the meanings of all technical terms and scientific terms used in the specific implementation manners of this application are intended to be the same as those commonly understood by those skilled in the art. Although the following terms are believed to be well understood by those skilled in the art, the following definitions are still provided to better explain this application.

[0075] As used in this application, the terms "comprise", "include", "have", "contain" or "involve" are inclusive or open-ended and do not exclude other unlisted elements or method steps. The term "consisting of" is considered a preferred embodiment of the term "comprising". If a certain group is defined hereinafter as including at least a certain number of embodiments, this should also be understood as disclosing a group preferably consisting only of these embodiments.

[0076] The indefinite or definite article used when referring to a singular noun, such as "a" or "an", "the", includes the plural form of the noun.

[0077] The terms "about" and "substantially" in this application indicate the accuracy range that those skilled in the art can understand and still ensure the technical effect of the feature being discussed. This term generally represents ±10% deviation from the indicated value, preferably ±5%.

[0078] In addition, the terms first, second, third, (a), (b), (c), and the like in the specification and claims are used to distinguish similar elements and are not necessary for describing the order or time sequence. It should be understood that the terms applied in this way can be interchanged in appropriate circumstances, and the embodiments described in this application can be implemented in an order different from that described or illustrated in this application.

[0079] The term "nucleic acid" or "nucleic acid sequence" in this application refers to any molecule, preferably a polymeric molecule, containing ribonucleic acid, deoxyribonucleic acid, or analog units thereof. The nucleic acid can be single-stranded or double-stranded. The single-stranded nucleic acid can be a nucleic acid of one strand of denatured double-stranded DNA. Alternatively, the single-stranded nucleic acid can be a single-stranded nucleic acid not derived from any double-stranded DNA.

[0080] The technical solutions of this application will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are part of the embodiments of this application, rather than all of them. All other embodiments obtained by those of ordinary skill in the art based on the embodiments in this application without creative efforts belong to the scope of protection of this application.

[0081] Example 1 Screening of Key SNP Loci for Bovine Sex and Anti-Mastitis and Optimization of Primer Design

[0082] This application selects the bovine AMEL gene for sex identification. The anti-mastitis loci were finally selected as Chr5:104010752C>T on the CD4 gene, Chr14:2525852T>G and Chr14:2711615G>A on the TRAPPC9 gene through preliminary analysis and evaluation.

[0083] For the above loci, this application first conducts preliminary primer design. According to the principle of allele-specific PCR, each specific primer can only bind to the DNA template of the corresponding genotype and amplify. The preliminary primer sequences are as follows:

[0084] The locus Chr5:104010752C>T on the CD4 gene:

[0085] C-genotyping primer: 5’-AGGGGGAATCAGAAAAGGATTC-3’

[0086] T-genotyping primer: 5’-TGCCAGGGGGAATCAGAAAAGGATTT-3’

[0087] Common primer: 5’-AGCTCCCAGGCTCAATAATTGT-3’

[0088] Chr14:2525852T>G locus on the TRAPPC9 gene

[0089] T-genotyping primer: 5’-GCGCAGTTCTCCTGGAGCTTTTT-3’

[0090] G-genotyping primer: 5’-CTTAGCGCAGTTCTCCTGGAGCTTTTG-3’

[0091] Common primer: 5’-ACCATAATATAAAGAAGTAAAACAGCTCCCT-3’

[0092] Chr14:2711615G>A locus on the TRAPPC9 gene

[0093] G-genotyping primer: 5’-AGCAGGCTAGGTCTCACGTTAAC-3’

[0094] A-genotyping primer: 5’-CTCACTGCAGGCTAGGTCTCACGTTAAT-3’

[0095] Common primer: 5’-AGAGTGACTACTACCATCCAGTT-3’

[0096] In order to coordinate the amplification efficiency, improve the product peak shape, and facilitate capillary electrophoresis detection, the present application further makes specific base modifications or processing to the primers.

[0097] Exemplarily, taking the primer screening of the Chr5:104010752C>T locus on the CD4 gene as an example, the applicant introduces different intensities of base mismatches and modifications to the 5’-end and 3’-end of the C-genotyping primer and the T-genotyping primer respectively for the primers at this locus. For the specific screening and evaluation result data, see Figure 1 , using the samples of C-genotyping, T-genotyping, and heterozygous CT-genotyping determined by the gold standard as templates for amplification detection respectively. The results show that after adjustment and modification, they are all correct and there are no miscellaneous peaks in the detection.

[0098] Similarly, for the primers at the Chr14:2525852T>G locus of the TRAPPC9 gene, different intensities of base mismatches and modifications were introduced at the 5' and 3' ends of the T-genotyping primers and G-genotyping primers respectively. The screening and evaluation result data are shown in Figure 2 , using the samples of T-genotyping, G-genotyping and heterozygous TG-genotyping determined by the gold standard as templates for amplification detection respectively. After adjustment, no heterozygous peaks were detected and the effect was better.

[0099] For the primers at the Chr14:2711615G>A locus of the TRAPPC9 gene, different intensities of base mismatches and modifications were introduced at the 5' and 3' ends of the G-genotyping primers and A-genotyping primers respectively. The specific screening and evaluation result data are shown in Figure 3 , using the samples of G-genotyping, A-genotyping and heterozygous GA-genotyping determined by the gold standard as templates for amplification detection respectively. The results showed that after adjustment and modification, they were all correct and there were no heterozygous peaks, and the effect was significantly better.

[0100] Based on the above methods, the primers for each locus were redesigned and optimized, and finally the sequences of the primer combinations of this application were determined as follows:

[0101] Primers for the Chr5:104010752C>T locus:

[0102] C-genotyping primer: 5’-AGGGGGAATCAGAAAAGGA A TC-3’(SEQ ID NO.1),

[0103] T-genotyping primer: (SEQ ID NO.2),

[0104] Common primer: 5’-AGCTCCCAGGCTCAATAATTGT-3’(SEQ ID NO.3);

[0105] Primers for the Chr14:2525852T>G locus:

[0106] T-genotyping primer: 5’-GCGCAGTTCTCCTGGAGCTTT C T-3’(SEQ ID NO.4),

[0107] G-genotyping primer: (SEQ ID NO.5),

[0108] Common primer: 5’-ACCATAATATAAAGAAGTAAAACAGCTCCCT-3’(SEQ ID NO.6);

[0109] Primers for the Chr14:2711615G>A locus:

[0110] G-genotyping primer: 5’-AGCAGGCTAGGTCTCACGTT G AC-3’ (SEQ ID NO.7),

[0111] A-genotyping primer: (SEQ ID NO.8),

[0112] Common primer: 5’-AGAGTGACTACTACCATCCAGTT-3’ (SEQ ID NO.9);

[0113] Wherein: the single underline of “-” represents a mismatched base; the double underline of “=” represents an added base.

[0114] Furthermore, the primer sequences of the Amel gene are as follows:

[0115] Forward primer for X-genotyping: 5’-ACAGCTTTGTTCTATCCCAGCC-3’ (SEQ ID NO.10),

[0116] Reverse primer for X-genotyping: 5’-AGAGTGTGACTATCTTAGAATTG-3’ (SEQ ID NO.11),

[0117] Forward primer for Y-genotyping: 5’-ACAACTTTGCACTGTCCCAGAC-3’ (SEQ ID NO.12),

[0118] Reverse primer for Y-genotyping: 5’-AGAGTGTGAAACATCTTAGAATCA-3’ (SEQ ID NO.13).

[0119] Wherein, 1. The single underline of “-” represents a mismatched base; 2. The double underline of “=” represents an added base, and all primers for the detection sites are labeled with FAM fluorescence at the 5’ end, and the reverse primers for the sex sites are labeled with FAM fluorescence at the 5’ end.

[0120] Example 2 Performance evaluation: Comparison between the multiplex amplification detection system for bovine sex and key SNP sites related to mastitis resistance and the sequencing results

[0121] The multiplex amplification detection system in this example includes PCR Master Mix, internal standard, etc. The main components of PCR Master Mix include hot-start Taq enzyme, amplification buffer, and primers for each site, etc. The primers are the primer parts determined in Example 1, and all primers are mixed according to the ratio explored in the experiment to prepare primer Mix.

[0122] In this embodiment, 60 blood DNA samples of dairy cows with known genders were amplified and detected. At the same time, the amplification results were verified by sequencing to confirm the effectiveness, specificity, and accuracy of the detection system in this application. The specific steps are as follows:

[0123] I. Detection of multiplex amplification system

[0124] 1) Sample preparation

[0125] Take fresh dairy cow blood with known gender, extract genomic DNA using a blood extraction kit, measure the concentration and purity of DNA using NanoDrop2000 (Thermo), dilute the DNA to the corresponding concentration, and store it at 4°C or -20°C for later use;

[0126] 2) Preparation of amplification system

[0127] Prepare the PCR amplification system according to the components in Table 1. After mixing well by oscillation, aliquot according to the number of samples.

[0128] Table 1. PCR amplification system

[0129] Component Name Addition Amount in 20 μL System (μL) 2×PCR Master Mix 10 Primer Mix 2 Nuclease-Free Water 7 Template 1 Total 20

[0130] 3) Add template

[0131] Add 1 μL of each prepared DNA sample to the corresponding PCR reaction tube. At the same time, set a negative control: 1 μL of nuclease-free water.

[0132] 4) PCR amplification

[0133] Place each reaction tube in the reaction slot of the PCR amplifier, and set the reaction system to 20 μL. Perform PCR amplification according to the program in Table 2.

[0134] Table 2. PCR reaction program

[0135]

[0136]

[0137] 5) Capillary electrophoresis detection of amplification products

[0138] Prepare a loading mixture containing molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) × number of test samples, vortex for 10 - 15 seconds; aliquot 9 μL of the formamide and internal standard mixture into each test well using a pipette; add 1 μL of the amplification product to the formamide and internal standard mixture, and cover with a sealing plate cover. Perform detection according to the steps in the user manual of the genetic analyzer.

[0139] 6) Data analysis

[0140] Import relevant files into the GeneMapper software, input the original data (.fsa file) from the detector, and analyze the data.

[0141] 7) Result genotyping determination

[0142] As shown in the figure, Figure 4 It is the amplification detection result diagram of bovine blood DNA samples, where SNPs are shown as corresponding genotypes; for the AMEL locus, female samples are shown as "XX" and male samples are shown as "XY".

[0143] II. Sanger sequencing:

[0144] According to the determined SNP locus information, search for the gene sequences of each locus, and design sequencing primers in the regions at least 100 bp upstream and downstream of the corresponding SNP loci (synthesized by Sangon Biotech Co., Ltd.). Use the designed sequencing primers for each locus to amplify the fragments where each locus of the target samples is located. After electrophoresis detection, when it is determined that there are amplification products and the size of the target fragment is correct, send the samples to Sangon Biotech Co., Ltd. for Sanger sequencing.

[0145] III. Comparison between CE results and sequencing results

[0146] Based on the capillary electrophoresis detection diagram, analyze and obtain the result genotypes of each sample. Analyze the data obtained by the sequencing company for sequencing, and obtain the result analysis of the sequencing primers for each SNP locus of 60 samples. The comparison results are shown in Table 3:

[0147] Table 3. Sequencing results of 60 samples and comparison results with capillary electrophoresis (CE)

[0148]

[0149]

[0150]

[0151] According to the statistical results, it can be seen that the detection results of this detection system are completely consistent with the detection results of the gold standard (Sanger sequencing), indicating that this detection system can accurately detect the gene genotypes of samples.

[0152] Example 3 Detection system optimization: Exploration and establishment of embryo sample processing methods

[0153] It is necessary to identify the sex of bovine embryos and at the same time detect the SNP of gene loci related to bovine mastitis resistance. The difficulty lies in that on the premise of minimizing damage to the embryos, it is difficult for the obtained embryonic cells to achieve the sensitivity for co-detection of sex and SNP of genes related to mastitis resistance. Therefore, a series of treatments need to be carried out on the embryo samples so that after the detection template amount reaches a certain level, the sex and SNP can be detected. Combining whole genome amplification with multiplex fluorescence PCR detection can achieve this goal.

[0154] In this example, embryo samples at the morula stage are selected for treatment. The embryo cell treatment solution mainly includes alkaline cell lysate and neutralizing solution, etc. The components in the cell treatment solution are mixed and tested according to gradient ratios to determine the optimal concentration range. The specific operations are as follows:

[0155] 1) Preparation and preservation of samples

[0156] 4 - 10 embryonic cells are isolated from each bovine embryo sample to be detected, stored in 4 μL of PBS buffer, placed in a 0.5 mL PCR tube, 3 μL of alkaline cell lysate is added, gently flicked and mixed, and centrifuged. After 10 min at 65 °C; 3 μL of neutralizing solution is added to terminate the lysis, and after mixing and centrifuging, it is placed on ice for standby. Among them, the treatment of embryonic cells is compared and tested according to the following scheme:

[0157] Scheme 1: The component concentrations of the alkaline cell lysate are as follows: 200 mM of KOH, 50 mM of DDT, 5 mM of EDTA; the component concentrations of the neutralizing solution are as follows: 100 mM of Tris-Hcl, 100 mM of Hcl, and 100 mM of Kcl

[0158] Scheme 2: The component concentrations of the alkaline cell lysate are as follows: 300 mM of KOH, 75 mM of DDT, 7.5 mM of EDTA; the component concentrations of the neutralizing solution are as follows: 200 mM of Tris-Hcl, 150 mM of Hcl, and 200 mM of Kcl.

[0159] Scheme 3: The component concentrations of the alkaline cell lysate are as follows: 400 mM of KOH, 100 mM of DDT, 10 mM of EDTA; the component concentrations of the neutralizing solution are as follows: 300 mM of Tris-Hcl, 200 mM of Hcl, and 300 mM of Kcl.

[0160] Scheme 4: The component concentrations of the alkaline cell lysate are as follows: 500 mM of KOH, 125 mM of DDT, 12.5 mM of EDTA; the component concentrations of the neutralizing solution are as follows: 400 mM of Tris-Hcl, 250 mM of Hcl, and 500 mM of Kcl.

[0161] Solution 5: The component concentrations of the alkaline cell lysate are as follows: 600 mM KOH, 150 mM DDT, and 15 mM EDTA; the component concentrations of the neutralizing solution are as follows: 500 mM Tris-Hcl, 300 mM Hcl, and 500 mM Kcl.

[0162] 2) Whole genome amplification

[0163] Add 5 μL of 10×Phi29 MAX DNA Polymerase Reaction Buffer (purchased from Novizan), 5 μL of 10 mM dNTP (purchased from Thermo Fisher), 2.5 μL of Phi29 MAX DNA Polymerase (purchased from Novizan), 2.5 μL of 500 μM Exo-Resistant Random Primer (purchased from Thermo Fisher), and 25 μL of nuclease-free pure water to the sample in 1) above, for a total system of 50 μL; incubate at 30 °C in a PCR instrument for 4 h, and then take it out of the machine after 10 min at 65 °C. Take 2 μL of the product taken out of the machine and detect whether there is amplification by agarose gel electrophoresis. The results are shown in Figure 5 .

[0164] The results of the electrophoresis experiment show that after lysing cells using Solutions 2, 3, and 4 and then performing whole genome amplification, the amplification effect is good, with clear and bright bands, which is in line with the results of whole genome amplification. Among them, the electrophoresis band of Solution 3 is relatively brighter, superior to other solutions.

[0165] 3) Sample detection

[0166] Take the undiluted product or appropriately diluted product in 2) and perform amplification detection using the steps of the multiplex amplification detection system in Example 2. The detailed capillary detection results are shown in Figures 6 - 10 . According to the detection results, the peak height and locus detection rate of the final detection results of different embryo sample treatment solutions are statistically analyzed, as shown in Table 4.

[0167] Table 4 Statistics of the final CE detection results of different embryo sample treatment solutions

[0168]

[0169] The detection results show that the average detection peak height of Solution 3 > Solution 1 > Solution 4 > Solution 5 > Solution 2, and the amplification efficiency of Solution 3 is significantly higher than that of other solutions; in the CE results, the smaller the ratio of the low peak to the high peak, the greater the difference in the peak height of the locus detection in the system, indicating a greater bias in whole genome amplification. The ratio of Solution 3 is greater than that of others, indicating that the result of whole genome amplification under this condition is better than that of other solutions; for the locus detection situation, locus loss occurs in both Solution 1 and Solution 5, while Solutions 2, 3, and 4 can all detect all loci. Considering the comprehensive efficiency and locus detection situation, the result of Solution 3 is the best.

[0170] In this example, it is determined by comparing different cell lysis protocols that the detection effect is optimal when the cell lysate is 300 - 500 mM KOH, 75 - 125 mM DDT, and 7.5 - 12.5 mM EDTA, and the neutralizing solution is 200 - 400 mM Tris-Hcl, 150 - 250 mM Hcl, and 200 - 500 mM Kcl. When the component concentrations of the alkaline cell lysate are 400 mM KOH, 100 mM DDT, and 10 mM EDTA; and the component concentrations of the neutralizing solution are 300 mM Tris-Hcl, 200 mM Hcl, and 300 mM Kcl, the lysis effect and subsequent detection effect are the best. Moreover, whole-genome amplification using Phi29 enzyme can simultaneously detect the gender and key SNPs for anti-mastitis in 4 - 10 embryonic cells.

[0171] Example 4 Detection of real samples: Detection of the gender of bovine early embryos and key SNP sites for anti-mastitis

[0172] In this example, the method for co-detection of the gender of bovine early embryos and key SNPs for anti-mastitis determined above is used to detect real embryo samples. The samples of morula embryos are divided into two parts. One part is sampled according to the real sampling operation (about 4 - 10 cells, named series B); the other part is all the remaining embryonic cells (about 130 cells, named series A), which are used as a control. The two samples of the same embryo are detected according to the following steps:

[0173] 1) Sample preparation

[0174] The two samples of the same embryo are resuspended in 4 μL of PBS buffer, placed in a 0.5 mL PCR tube, 3 μL of alkaline cell lysate is added, gently flicked and mixed, and centrifuged. After 10 min at 65 °C, 3 μl of neutralizing solution is added to terminate the lysis. After mixing and centrifuging, it is placed on ice for standby. Among them, the component concentrations of the alkaline cell lysate are as follows: 400 mM KOH, 100 mM DDT, 10 mM EDTA; the component concentrations of the neutralizing solution are as follows: 300 mM Tris-Hcl, 200 mM Hcl, and 300 mM Kcl.

[0175] 2) Whole-genome amplification

[0176] Add 5 μL of 10×Phi29 MAX DNA Polymerase Reaction Buffer (purchased from Novizan), 5 μL of 10 mM dNTP (purchased from Thermo Fisher Scientific), 2.5 μL of Phi29 MAX DNA Polymerase (purchased from Novizan), 2.5 μL of 500 μM Exo-Resistant Random Primer (purchased from Thermo Fisher Scientific), and 25 μL of nuclease-free water to the sample in step 1) above, for a total system volume of 50 μL. Incubate at 30 °C for 4 h in a PCR instrument, and then take it out of the instrument after incubating at 65 °C for 10 min for standby use.

[0177] 3) Sample detection

[0178] Prepare the PCR amplification system according to the components in the following table. After mixing well by oscillation, aliquot according to the number of samples.

[0179] Component Name Addition Amount in 20 μL System (μL) 2×PCR Master Mix 10 Primer Mix 2 Nuclease-Free Water 7 Template 1 Total 20

[0180] Add 1 μL of the original product solution or dilution prepared in step 2) above to the corresponding PCR reaction tube. At the same time, set up a positive control (1 μL of bovine blood DNA sample) and a negative control (1 μL of nuclease-free water).

[0181] Place each reaction tube into the reaction slot of the PCR amplifier, and perform the PCR reaction according to the reaction program in the following table. Set the reaction system volume to 20 μL.

[0182]

[0183]

[0184] Prepare the loading mixture mixed with molecular weight internal standard and formamide: (0.5 μL of molecular weight internal standard + 8.5 μL of formamide) × the number of test samples, vortex and mix well for 10 - 15 seconds; aliquot 9 μL of the formamide and internal standard mixture into each test well using a pipette; add 1 μL of the amplified product to the formamide and internal standard mixture, and cover with a sealing plate cover. Perform the detection according to the steps in the user manual of the genetic analyzer.

[0185] Import the relevant files into the GeneMapper software, input the original data (.fsa file) taken out of the detector, and analyze the data. Taking embryo No. 009 and embryo No. 058 as examples, the detection maps are shown in Figure 11 . The detection results of all test samples are sorted out in the following table:

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193] In this embodiment, a total of 96 embryo samples were detected. The results of Series A and Series B were 100% consistent. This result shows that the detection method of the present application has high sensitivity and accuracy and can be used for the sex identification of early bovine embryos and the detection of key SNP typing for anti-mastitis.

[0194] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, not to limit them; although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A kit for bovine mastitis resistance and early embryo sex co-detection, characterized in that, It includes a primer composition; it also includes a cell lysate, a neutralization solution, and Phi29 DNA polymerase; the cell lysate contains 300 - 500 mM of KOH, 75 - 125 mM of DDT, and 7.5 - 12.5 mM of EDTA; the neutralization solution contains 200 - 400 mM of Tris-Hcl, 150 - 250 mM of Hcl, and 200 - 500 mM of Kcl; the kit also includes: 10×Phi29MAX DNA Polymerase Reaction, MdNTP, Exo-Resistant Random Primer, and 2×PCR Master Mix; The primers are for SNP sites and sex identification genes; the SNP sites are Chr5:104010752C>T of the CD4 gene, Chr14:2525852T>G of the TRAPPC9 gene, and Chr14:2711615G>A of the TRAPPC9 gene; the sex identification gene is the Amel gene; The primer sequences of the SNP sites are as follows: Primers for the Chr5:104010752C>T site: C-genotyping primer: 5’-AGGGGGAATCAGAAAAGGA A TC-3’, T-type primer: Common primer: 5’-AGCTCCCAGGCTCAATAATTGT-3’; Primers for the Chr14:2525852T>G site: T-typing primer: 5’-GCGCAGTTCTCCTGGAGCTTT C T-3’, G genotyping primer: Common primer: 5’-ACCATAATATAAAGAAGTAAAACAGCTCCCT-3’; Primers for the Chr14:2711615G>A site: G genotyping primer: 5’-AGCAGGCTAGGTCTCACGTT G AC-3’, A genotyping primer: Common primer: 5’-AGAGTGACTACTACCATCCAGTT-3’; Among them, the single underline of the "-" represents the mismatched base, and the double underline of the "=" represents the added base; The primer sequences of the Amel gene are as follows: Forward primer for X typing: 5’-ACAGCTTTGTTCTATCCCAGCC-3’, Reverse primer for X typing: 5’-AGAGTGTGACTATCTTAGAATTG-3’, Forward primer for Y typing: 5’-ACAACTTTGCACTGTCCCAGAC-3’, Reverse primer for Y typing: 5’-AGAGTGTGAAACATCTTAGAATCA-3’; The primers are fluorescently labeled; the fluorescent label is located at the 5’ end of the common primer and the 5’ end of the reverse primer of the sex identification gene; the fluorescent label is the FAM fluorescent label.

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