Screening and application of pregnant bovine serum aptamers based on magnetism-SELEX (systematic evolution of ligands by exponential enrichment) technology

Abstract

The invention is applicable to the technical field of biology, and provides screening of pregnant bovine serum aptamers based on a magnetic-SELEX technology and application of the pregnant bovine serum aptamers. Based on a magnetic-SELEX (systematic evolution of ligands by exponential enrichment) technology, cow early pregnancy related aptamers are screened by taking pregnant cow serum 21d after fertilization as a target, and homology analysis and secondary structure prediction are performed on the aptamers. The aptamer reacts with negative and positive serum of pregnant cattle, the optimal expression of the L4 aptamer is determined according to the P / N value, and the detection conditions are optimized. The L4 aptamer is used for actual sample detection, the result shows that the L4 aptamer shows high specificity in early pregnancy 14-21 d detection, the OD450 nm value of a positive sample is obviously higher than that of a negative sample, the detection effect is obviously superior to that of a commercial kit, a detection window is advanced to 14 d, and the L4 aptamer has important clinical significance in early pregnancy diagnosis of dairy cows and has good application prospects in early pregnancy diagnosis of dairy cows. A novel tool with low cost and high sensitivity is provided for intensive breeding of dairy cows, and large-scale development of animal husbandry is further promoted.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the screening and application of aptamers for pregnant cow serum based on magnetic-SELEX technology. Background Art

[0002] The early pregnancy diagnosis of cows is of great significance to modern animal husbandry and livestock breeding, which is not only related to the health management of cows, but also directly affects the reproductive efficiency and economic benefits. So far, the main methods for early pregnancy diagnosis of cows include rectal examination, ultrasonic examination, immunological diagnosis (ELISA, RIA, ESA and FLA) and other diagnostic methods (detection of interferon tau-induced genes ISGs and miRNAs in exosomes, etc.). Among them, rectal palpation has become a commonly used early pregnancy detection method in farms due to its relatively simple operation. The pregnancy status can be detected about 40 days after the cow is pregnant. However, its detection accuracy highly depends on the experience of the operator. Ultrasonic examination can determine whether a cow is pregnant about 30 days after pregnancy, but due to its high cost, it is difficult to be widely used in large-scale farming. Immunological detection technology based on antigen-antibody, with high sensitivity, high specificity and wide application range, has become an important tool in modern scientific research and plays an important role in the fields of biomedicine, clinical diagnosis and food safety detection. However, as a macromolecular protein, antibodies are easily affected by factors such as high temperature, pH change and long-term storage, resulting in structural denaturation and inactivation. Moreover, the antibody production process is complex and costly, which is not conducive to large-scale promotion. As for the application research of ISGs and miRNAs in exosomes in the pregnancy diagnosis of dairy cows is still scarce, and their feasibility as pregnancy diagnosis markers still needs to be verified by a large number of clinical trials. With the development of China's dairy farming industry towards large-scale, intensive and standardized directions, these existing early pregnancy detection methods are difficult to meet the growing demand and the development trend of the industry, and there is an urgent need to explore more efficient and accurate early pregnancy diagnosis methods.

[0003] Systematic evolution of ligands by exponential enrichment (SELEX) is a multidisciplinary technique based on the mechanisms of variation, selection, and replication in the process of evolution, involving different fields such as molecular biology, nucleic acid chemistry, materials science, and bioinformatics for affinity ligands against various targets. The aptamers screened by this technique are single-stranded oligonucleotides selected from DNA or RNA molecular libraries, which can form stable secondary or tertiary structures such as hairpins, stem-loops, bulges, and G-quadruplexes, and can specifically bind to target molecules. Aptamers have significant advantages such as high specificity, strong affinity, easy synthesis, good stability, and low production cost. Compared with traditional antibodies, aptamer production does not require animals, and they are small in size and easy to modify, showing great application potential in the fields of biosensing, drug delivery, and targeted therapy, and are also widely used for the detection of biomarkers in serum. Pranav Tripathi et al. combined aptamers with gold nanoenzymes to achieve non-invasive detection of the ovarian cancer biomarker CA125 in serum. The lateral flow chromatography strip method constructed has high specificity and sensitivity, and the detection limit reaches 5.21 U / mL; Munna Lal Yadav et al. screened aptamers of bovine pregnancy-associated glycoprotein (PAG) using non-SELEX technology and successfully detected PAG protein in bovine serum 42 days after artificial insemination.

[0004] Based on the above background, in view of the deficiencies of existing methods for early pregnancy diagnosis in cows and the advantages of aptamers in biomarker detection, the present invention proposes the screening and application of aptamers in pregnant cow serum based on magnetic-SELEX technology. Summary of the Invention

[0005] The purpose of the present invention is to provide the screening and application of aptamers in pregnant cow serum based on magnetic-SELEX technology, aiming to solve the problems proposed in the above background technology.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A specific nucleic acid aptamer for pregnant cow serum, the nucleotide sequence of the aptamer is as shown in SEQ ID NO.4.

[0008] The screening method of the specific nucleic acid aptamer for pregnant cow serum according to the above, includes the following steps:

[0009] Based on magnetic-SELEX technology, post-fertilization 21-day pregnant cow serum was used as the target to screen for aptamers. Through 12 rounds of SELEX screening, aptamers L4, L11, L13, L21, and L40 were enriched; the screened aptamers were subjected to homology analysis and secondary structure prediction, and the screened aptamers were reacted with positive and negative pregnant cow sera respectively. According to the P / N value, the best aptamer was determined to be aptamer L4.

[0010] Further, the nucleotide sequences of the aptamers L11, L13, L21, and L40 are shown in SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 respectively.

[0011] Further, the homology of the aptamer is 63.76%, and the secondary structure contains hairpin and stem-loop structures.

[0012] Application of the specific nucleic acid aptamer of pregnant cow serum described above in the preparation of bovine early pregnancy diagnostic products.

[0013] Further, the optimal detection conditions for the specific nucleic acid aptamer of pregnant cow serum are: ssDNA diluted 100 times, serum diluted 5 times, and substrate acting for 8 min.

[0014] Further, the bovine early pregnancy diagnostic product is used for the early pregnancy diagnosis of pregnant cows 14 - 21 days after fertilization.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] The present invention successfully screened out highly specific aptamers for pregnant cow serum through magnetic-SELEX technology and preliminarily verified their application potential in early pregnancy diagnosis. Aptamer L4 performs excellently in the early pregnancy detection, can advance the detection window to 14 days, and its detection effect is significantly better than that of commercial kits, providing a new tool with low cost and high sensitivity for intensive dairy farming, and thus promoting the large-scale development of the livestock industry. Description of the Drawings

[0017] Figure 1 It is the SELEX screening process.

[0018] Figure 2 It is the determination of the number of SELEX screening rounds.

[0019] Figure 3 It is the homology analysis and structure prediction of the aptamer; where a is the homology analysis of the aptamer sequence; b is the secondary structure prediction of the aptamer sequence.

[0020] Figure 4For aptamer sequence screening and condition optimization; where a is the best aptamer screening; b is the blocking condition optimization; c is the ssDNA dilution factor optimization; d is the serum dilution factor optimization; e is the substrate action time optimization.

[0021] Figure 5 For differential analysis of aptamer detection of positive and negative serum samples; where a is the detection of positive and negative serum samples by L4 aptamer; b is the detection of positive and negative serum samples by commercial kits.

[0022] Figure 6 For median analysis of aptamer detection of positive and negative serum samples; where a is the OD value of serum detected by L4 aptamer 450nm value; b is the serum content value detected by commercial kits; c is the median difference of positive and negative sera detected by L4 aptamer; d is the median difference of positive and negative sera detected by commercial kits. Specific implementation mode

[0023] For a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solutions of the present invention are described in detail below, but it should not be construed as a limitation on the scope of implementation of the present invention.

[0024] The following describes the specific implementation of the present invention in detail in combination with specific embodiments.

[0025] Example 1: Screening of nucleic acid aptamers in pregnant cow serum;

[0026] I. Materials and reagents;

[0027] Skim milk powder was purchased from Sangon Biotech Co., Ltd.; 96-well ELISA plates were purchased from Guangzhou Jet Bio-Filtration Co., Ltd.; carboxyl magnetic beads were purchased from Wuxi Biomax Biotechnology Co., Ltd.; streptavidin-HRP was purchased from Wuhan Sanying Biotechnology Co., Ltd.; bovine serum was collected from Jiutai Cattle Farm, Changchun, Jilin.

[0028] II. Experimental methods;

[0029] 2.1 Library and primer synthesis;

[0030] The initial library consists of 40 random nucleotides in the center and 19 fixed bases at each end, i.e., 5’-TTGGTTGTAGGTCAGGTGG-N40-CATTCCGCTTACATTCTCG-3’ (the fixed base sequences at both ends are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively). At the same time, three pairs of primers are designed: the upstream primer U26 (5’-TTGGTTGTAGGTCAGGTGG-3’, whose nucleotide sequence is shown in SEQ ID NO.1) and the downstream primer D26 (5’-CGAGAATGTAAGCGGAATG-3’, whose nucleotide sequence is shown in SEQ ID NO.3) for PCR amplification, both of which are oligonucleotide fragments of 19 bases; the upstream primer U26-Bio for ELISA detection of the binding activity with the target, with the 5’ end modified with biotin, i.e., U26-Bio: Bio-TTGGTTGTAGGTCAGGTGG-3’, whose nucleotide sequence is shown in SEQ ID NO.1, and the downstream primer D26; the upstream primer with the 5’ end modified with phosphate for enhancing the stability, binding affinity of the primer and improving the efficiency and accuracy of screening, i.e., U26-Pho: Pho-CGAGAATGTAAGCGGAATG-3’, whose nucleotide sequence is shown in SEQ ID NO.3, and the downstream primer D26. The above library design, primers and the biotinylated aptamers obtained by subsequent screening are all synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0031] 2.2 Preparation and pretreatment of serum;

[0032] The collected pregnant cow positive and negative sera are centrifuged at 4°C and 1500g for 20 min, and the supernatant is taken. Then the supernatant is centrifuged at 4°C and 3000g for 15 min, and the supernatant is collected. Subsequently, the 21-day pregnant cow sera treated as above are mixed in equal amounts (5 positive samples in total of 1200 μL, 5 negative samples in total of 600 μL), and the mixed serum is incubated at 37°C for 15 min until completely mixed. The completely mixed serum is aliquoted into 1.5 mL centrifuge tubes and centrifuged at 15000g and 4°C for 30 min. After centrifugation, all the supernatant is placed in a 1.5 mL centrifuge tube and centrifuged again under the same conditions (15000g, 4°C) for 15 min. All the supernatant is mixed with a vortex mixer for about 20 s, re-aliquoted in 100 μL aliquots per tube, and stored at -20°C.

[0033] 2.3 Activation of carboxyl magnetic beads and coupling with serum;

[0034] Take 100 μL of carboxyl magnetic beads (10 mg / mL) and put them into a 1.5 mL EP tube. Wash the magnetic beads with 500 μL of reaction buffer, remove the supernatant by magnetic separation, and repeat the washing 3 times. Take out 250 μL of 4 mg / mL activator (EDC solution) from the refrigerator and add it to the magnetic beads (dissolved with reaction buffer, prepared fresh). Subsequently, add 2.7 mg of NHS, shake at room temperature for 15 min to prevent the magnetic beads from precipitating and aggregating, and remove the supernatant by magnetic liquid separation.

[0035] Take out 100 μL of the pre - aliquoted pregnant cow serum, melt it at room temperature, add 250 μL of 20% acetonitrile solution (50 μL of acetonitrile + 200 μL of DEPC water) that has been prepared, perform ultrasonic bath for 5 min, and centrifuge at 15000 g and 4 °C for 5 min. Take 300 μL of the serum supernatant after centrifugation, supplement 100 μL of PBS solution, and incubate with the activated magnetic beads for 45 min. Separate the magnetic liquid and discard the serum incubation solution, wash the magnetic beads with PBS solution, and add 1 mL of the prepared blocking solution and continue to incubate for 30 min. Discard the blocking solution, wash the magnetic beads with PBS solution 3 times, and finally resuspend with 100 μL of PBS and place at 4 °C for standby.

[0036] 2.4 SELEX screening;

[0037] The SELEX screening procedure is as Figure 1 shown: Coupling the processed pregnant cow positive and negative sera with magnetic beads respectively for standby. Incubate the initial ssDNA library with the magnetic bead - coupled protein, wash to remove the unbound sequences, and after eluting the bound sequences by magnetic separation, perform PCR amplification. Prepare the PCR product into single - stranded DNA as the secondary library for the next round of screening. After 12 rounds of amplification, ligate, transform, and clone - sequence the aptamers to obtain the nucleic acid sequences that specifically bind to the target. The specific operation steps are as follows:

[0038] Positive and negative screening: When pre - treating the library, take 2 OD (2500 pmol) of the aptamer library, heat at 95 °C for 5 min, and place on ice for 10 min for standby. Positive screening: Wash the magnetic bead - coupled positive serum 3 times with 100 μL of PBS and resuspend, mix with ssDNA (2 OD aptamer library), and incubate at 37 °C for 1 h. Retain the supernatant for screening other targets. Wash the magnetic beads 2 times with 100 μL of 1xPBS, and finally resuspend with 100 μL of Wahaha water, heat at 95 °C for 5 min, and aspirate the supernatant as the PCR template for preparing the secondary library. This experiment was carried out for 12 rounds of screening. Negative screening was carried out in the 3rd, 6th, 10th, and 12th rounds. The treatment and experimental process of negative screening and the secondary library are roughly the same as those of positive screening. The difference is that the secondary library is first incubated with the magnetic bead - coupled negative serum, and then the aptamers in the unbound supernatant are collected and incubated with the magnetic bead - coupled positive serum to enrich the aptamers that can specifically bind to the positive serum.

[0039] Library amplification: Using the screened ssDNA as a template, exponential enrichment in the magnetic-SELEX screening process was achieved by PCR amplification technology. The PCR amplification system is shown in Table 1, and the reaction conditions are: pre-denaturation at 94°C for 30 s, denaturation at 94°C for 6 s, annealing at 57°C for 4 s, extension at 72°C for 4 s, additional extension at 72°C for 30 s, with 25 reaction cycles.

[0040] Table 1. PCR system

[0041]

[0042] Digestion and purification of amplification products: Lambda Exonuclease digestion was used to prepare the ssDNA of the secondary library. The digestion conditions for the 50 μL reaction system are shown in Table 2:

[0043] Table 2. Digestion system

[0044]

[0045] The PCR digestion products were purified to remove small molecules, doped magnetic bead particles, proteins and other interfering substances in the solution to obtain high-concentration ssDNA. The specific operation is as follows: Add 1 / 10 volume of 3M CH3COONa solution to the PCR digestion products and mix well; add 2 volumes of ice-cold ethanol, gently mix evenly and place at 4°C for 2 h, centrifuge at 4°C and 12,000 rpm for 30 min, carefully remove the supernatant, avoiding touching the tube wall, and let the remaining liquid evaporate automatically. Add half volume of pre-cooled 70% ethanol, invert several times up and down, centrifuge at 4°C and 12,000 rpm for 10 min, carefully remove the supernatant; evaporate the remaining liquid completely at room temperature (do not dry), and make up the volume to the working volume with the screening buffer. After measuring the concentration, store at -20°C for later use.

[0046] Ligation, transformation and cloning sequencing of enriched aptamers: Using the aptamers obtained from the last round of screening as a template, primers without biotin and phosphate modifications were used to enrich dsDNA by symmetric PCR amplification (the PCR reaction system, conditions and purification steps are the same as above). The recovered DNA products were ligated to the pMD19-T vector, transformed into competent cells, and then spread on an LB solid medium plate containing X-Gal, IPTG, and Amp. Single colonies were picked and inoculated into 5 mL of LB liquid medium containing Amp resistance, and cultured with shaking at 37°C for 18 h. Then, the bacterial liquid was taken for PCR verification, and the bacterial liquid with correct verification was sent to Sangon for sequencing.

[0047] III. Experimental results:

[0048] 3.1 SELEX screening conditions;

[0049] During the SELEX screening process, in order to screen out nucleic acid aptamers with high specificity and affinity, it is necessary to ensure sufficient library volume and target substance input. In the first round of screening, 2 OD (2500 pmol) of the random library is input to ensure rich nucleic acid structures in the library. A large amount of the target is input to increase the chance of binding between the random library and the target substance and avoid missed screening. To obtain aptamers with strong affinity and high specificity as soon as possible, the number of washing times is gradually increased in each round, and as the number of screening rounds increases, the library usage and target substance usage are gradually reduced round by round to more quickly screen out the ligands that bind to the target substance. The screening conditions for each round are shown in Table 3:

[0050] Table 3. SELEX Screening Conditions

[0051]

[0052] 3.2 Verification of the screening products;

[0053] The screening products were verified by indirect ELISA. The positive serum and negative serum of pregnant cows at 21 d were diluted 10-fold and coated on the ELISA plate, 100 μL per well, and incubated overnight at 4 °C. The liquid in the plate was discarded, and the plate was washed 3 times with a plate washer. Then, 100 μL of 5% skim milk powder was added and blocked at 37 °C for 1 h. The biotinylated screening products of each round above were heated and denatured at 95 °C for 5 min, then immediately ice-bathed for 10 min. After dilution to an appropriate concentration, 100 μL per well was added to the ELISA plate and incubated at 37 °C for 1 h. The plate was washed 3 times, 1:4000-fold diluted HRP-labeled streptavidin was added, and incubated at 37 °C for 1 h. After washing, TMB chromogenic solution was added. The substrate reaction was terminated after 15 min, and the OD 450nm value was measured using an ELISA reader. The results are as Figure 2 shown. The OD 450nm value of the positive sample in the first round was 0.5. After 12 rounds of screening, the OD 450nm value gradually increased to 1.4. The OD 450nm value increased significantly with the number of screening rounds, indicating that the specific binding ability of the target specific sequences in the aptamer library gradually increased. The effect of the fifth round was better than that of the first four rounds, and the difference between the negative and positive sera was large, indicating that the screening effect of this round was good. After the sixth round of reverse screening, the effect decreased slightly, presumably related to the decrease in the input amount of ssDNA, but the overall trend was still enrichment. After the tenth round of reverse screening, the OD 450nm value of the negative and positive sera decreased significantly to 0.4, presumably because the number of washing times was increased and the input amount of ssDNA was reduced in this round. However, the OD 450nm value increased in the eleventh round, indicating that the dominant aptamers were still retained during the screening process. Until the twelfth round of screening, the OD 450nm value of the negative serum decreased, and the OD 450nm value of the positive serum increased, and the gap between the two widened significantly, fully proving that the target specific sequences in the aptamer library were successfully enriched.

[0054] 3.3 High-throughput sequencing results of nucleic acid aptamers specific to pregnant cow serum;

[0055] After 12 rounds of screening, the aptamers specifically binding to pregnant cow serum were amplified and purified, cloned into the pMD19-T vector, 50 monoclonal colonies were randomly selected, and 21 bacterial solutions were selected for sequencing after being verified correct by PCR. Finally, 5 different nucleic acid aptamer sequences were obtained (as shown in Table 4), successfully realizing the screening and identification of nucleic acid aptamers specific to pregnant cow serum.

[0056] Table 4. SELEX-screened aptamer sequences

[0057]

[0058]

[0059] Example 2: Homology analysis and secondary structure analysis of nucleic acid aptamers specific to pregnant cow serum;

[0060] The 5 aptamer sequences obtained by sequencing were subjected to multiple sequence alignment and homology analysis using DNAMAN software. The secondary structures of the 5 aptamers were predicted through the online analysis website http: / / www.unafold.org / .

[0061] Experimental results: Through sequence alignment and homology analysis, the overall homology among the 5 aptamer sequences was 63.76%, indicating that they have a certain degree of conservation but significant diversity, and may correspond to different target binding sites or structural features ( Figure 3 a) in the figure). The predicted results of the secondary structures of the aptamers showed that all 5 aptamers exhibited different hairpin structures, stem-loop structures, and bulge structures, which could stack planar groups and form hydrogen bonds, participating in the formation of tertiary binding sites between nucleic acid aptamers and targets, suggesting their high structural integrity and functional potential ( Figure 3 b) in the figure).

[0062] Example 3: Preliminary application and applicability analysis of nucleic acid aptamers specific to pregnant cow serum;

[0063] I. Preliminary application of aptamers;

[0064] To screen aptamers that can distinguish between positive and negative sera of pregnant cows and explore the application potential of aptamers in the early pregnancy diagnosis of pregnant cows. After condition optimization, the following standard operating procedures were adopted:

[0065] Dilute pregnant cow's positive and negative sera by 5 times their volume, and fix 100 μL per well in CBS (0.05 mol / L, pH 9.6) on a transparent 96-well microplate, and coat overnight at 4 °C; after washing 3 times with PBST, dilute the biotin-labeled aptamer to 0.1 μM with 0.01 M PBS, incubate in a metal bath at 95 °C for 5 min, and then incubate on ice for 10 min. Then add 100 μL per well to the microplate and incubate at 37 °C for 1 h; after washing with PBST, add streptavidin-HRP (1:4000) at 100 μL per well and incubate at 37 °C for 1 h; after washing with PBST, add 100 μL of TMB substrate to each well, incubate at 37 °C for 8 min, and immediately add 50 μL of stop solution per well, and read the OD 450nm absorbance value at this position.

[0066] Experimental results: Use the 5 selected aptamers for the detection of pregnant cow's positive and negative sera respectively. According to the principle that the larger the P / N value, the more significant the signal of positive samples is higher than that of negative samples and the less non-specific binding, Figure 4 as shown in a of, the P / N value of L4 shows the best performance among them. Therefore, L4 is selected for subsequent experiments. Through the exploration of the blocking treatment link, Figure 4 as shown in b of, whether there is blocking treatment has no significant impact on the detection results. Considering the time cost comprehensively, blocking treatment will not be carried out in subsequent experiments. To obtain the best detection conditions, optimize the ssDNA dilution factor (12.5 times - 0.8 μM, 100 times - 0.1 μM, 200 times - 0.05 μM, 1000 times - 0.01 μM, 2000 times - 0.005 μM), serum dilution factor (undiluted, 5 times, 10 times, 20 times, 40 times) and substrate action time (5 min, 8 min, 12 min, 15 min, 20 min). Finally, it is determined that ssDNA is diluted 100 times ( Figure 4 as shown in c of), serum is diluted 5 times ( Figure 4 as shown in d of), and the substrate acts for 8 min ( Figure 4 as shown in e of) are the best experimental conditions, providing a reliable basis for the subsequent application of aptamers in the detection of pregnant cow sera.

[0067] II. Aptamer applicability analysis;

[0068] To evaluate the effectiveness of the selected aptamers in practical applications, conduct an aptamer applicability analysis experiment and compare their detection performance with that of commercial kits.

[0069] Twenty cows in the Changchun Jiutai cattle farm were monitored after fertilization, and serum samples were collected at 14 days, 18 days, 21 days, and 28 days after fertilization respectively. After the later monitoring of the cattle farm, 8 cows were positive and 12 cows were negative. The serum samples collected from these 20 cows at the four time points were uniformly tested, and each sample was tested in parallel 3 times. At the same time, a commercial kit was used for parallel determination to evaluate the applicability of the screened aptamer.

[0070] Experimental results: Using the optimized conditions, the L4 aptamer was used to identify the negative and positive serum samples of pregnant cows at 14 days, 18 days, 21 days, and 28 days after fertilization respectively. The results are as Figure 5 shown in a below. The OD 450nm values of the positive samples at each time point were significantly higher than those of the negative samples. When detecting the serum samples at 14 days and 21 days, the difference in OD 450nm values between the positive and negative samples was extremely significant, which was significantly better than the detection effects at 18 days and 28 days. This result fully indicates that the L4 aptamer has great potential in the detection of negative and positive pregnant cows. Further analysis found that the OD 450nm value of the 28-day positive sample was lower than that at 14 days, 18 days, and 21 days. It is speculated that the L4 aptamer has a higher affinity for the target in the early pregnancy stage, and then the binding efficiency decreases due to the decrease in target abundance or structural changes. In addition, OD 450nm values were also detected in the negative serum, which may be due to the fact that while the aptamer preferentially binds to the target molecule in the positive sample, there is still a certain degree of non-specific binding, such as cross-reaction or background adsorption, resulting in signals in the negative samples. The results of parallel detection of negative and positive serum samples with a commercial kit are as Figure 5 shown in b below. In the detection of pregnancy from 14 to 28 days, there was no significant difference in the content or signal intensity of the negative and positive serum samples. Although this kit can detect the content of EPF, it has obvious deficiencies in the differentiation of early pregnancy.

[0071] Considering that in the detection of actual samples such as serum, a small number of samples may have extremely high or low values due to individual differences or pathological conditions, using the median as an indicator of central tendency can effectively reduce the interference of extreme values and more robustly reflect the actual performance of the detection method. The median of the L4 aptamer in detecting the negative and positive serum of pregnant cows indicates that the OD 450nm value of the positive serum is higher than that of the negative ( Figure 6 shown in a below); the median of the commercial kit in detecting the negative and positive serum of pregnant cows is between 0.9 - 1.4 ng / mL, and there is basically no difference between the negative and positive ( Figure 6 shown in b below). Analyzing the median difference between the negative and positive sera, the results show that the median difference curve of the L4 aptamer in detecting the negative and positive serum of pregnant cows is flat, and the difference at 21 days is the largest, indicating that the detection effect at this time point is the best ( Figure 6in c); while the difference in the median values of positive and negative sera of pregnant cows detected by the commercial kit is close to 0, indicating that although its detection stability is good, it cannot effectively distinguish whether a cow is pregnant ( Figure 6 in d). Generally speaking, aptamer L4 is significantly superior to the commercial kit in terms of the specificity and effectiveness of early pregnancy detection in pregnant cows.

[0072] Summary:

[0073] Magnetic-SELEX technology couples the target with magnetic beads, significantly improving the screening efficiency. Compared with traditional SELEX, the advantages of this study are rapid separation of binding sequences, reduction of non-specific adsorption, and effective removal of non-target aptamers through a counter-selection strategy (rounds 3, 6, 10, and 12). At the initial stage of the study, a high-concentration library (2500 pmol) and target were used to ensure the comprehensiveness of the screening. Subsequently, the ssDNA dosage was gradually reduced and the washing intensity was increased round by round to gradually enrich high-affinity aptamers. The screening results showed that the OD 450nm value increased from 0.5 in the first round to 1.4 in the 12th round, and the difference between positive and negative samples in the 12th round was obvious, verifying the effectiveness of this strategy. However, the OD 450nm value decreased briefly after counter-selection in the 6th and 10th rounds, which may be related to the reduction of the library input amount or the loss of some high-affinity sequences due to excessive washing intensity. In the future, the screening process can be further optimized by dynamically adjusting the counter-selection intensity, such as gradually reducing the amount of target coupling.

[0074] The function of aptamers is closely related to their secondary structures. In this invention, although the homology of the 5 aptamers is only 63.76%, they all form stable hairpin and stem-loop structures, and such conformations can bind to target molecules through hydrogen bonds and base stacking forces. For example, the continuous GGG region in the L4 aptamer sequence may form a G-quadruplex structure, enhancing the binding stability with pregnancy-related proteins. However, the diversity of aptamers may also lead to differences in target binding sites. For example, the L40 aptamer may target different epitopes due to an additional stem-loop structure, but its P / N value is low, indicating that structural complexity may introduce non-specific binding. In subsequent studies, the interaction mode between aptamers and targets can be clarified through molecular docking or surface plasmon resonance (SPR), and the core binding domain can be screened by combining truncation experiments to simplify the structure and improve specificity.

[0075] The present invention selects time windows at 14 days, 18 days, 21 days, and 28 days after fertilization for aptamer applicability detection. Its scientific basis stems from the stage characteristics of embryonic development and the actual requirements of pregnancy diagnosis. Around 14 days after fertilization, the mother begins to recognize pregnancy and secrete pregnancy-related proteins, and the pregnancy status can be preliminarily judged through blood tests. By 18 days, it is the critical period of maternal pregnancy recognition. The embryo inhibits luteal regression by secreting signaling molecules such as interferon tau and EPF, and their concentrations increase significantly. Detection at this time can reduce the risk of false negatives. At the same time, the maternal progesterone level increases significantly, further supporting pregnancy maintenance. When the embryo completes implantation and establishes stable maternal-fetal signal transmission, detection at this time can reduce the risk of false negatives. At the same time, the maternal progesterone level increases significantly, further supporting pregnancy maintenance. At 21 days, uterine caruncles and cotyledons appear, supporting placental development and embryonic nutrient supply. It is a critical period of embryonic development, and this time point is connected to the estrous cycle of cows (about 21 days). If not pregnant, reproductive management can be restarted in time, thereby optimizing the reproductive efficiency of the cattle herd and reducing breeding costs. 28 days is the traditional pregnancy confirmation time, such as rectal palpation. Selecting this time point aims to compare with conventional methods and significantly improve the detection accuracy. L4 aptamer is superior to commercial early pregnancy diagnostic kits in the detection of negative and positive sera in the early pregnancy stage of 14 - 21 days. The OD 450nm value of positive samples is significantly higher than that of negative samples, and the statistical difference is significant. Compared with the aptamer detection of samples at 42 days of pregnancy based on PAG protein by Munna Lal Yadav et al., this result advances the detection window to 14 days, which has crucial clinical significance for the early pregnancy diagnosis of dairy cows. It can help breeders master the pregnancy status of dairy cows earlier, reasonably arrange breeding plans, and improve breeding efficiency. However, the OD 450nm values of negative and positive samples at 28 days both decrease, indicating that the aptamer may be more suitable for earlier pregnancy diagnosis. This phenomenon may be related to the concentration changes of stage-specific target molecules such as hormones or exosomal miRNAs, or it may be that the aptamer conformation cannot adapt to the dynamic modification of the target, or it may be because when screening the aptamer considering the estrous cycle of cows, it targets the negative and positive sera of pregnant cows at 21 days. At 21 days, the embryo is usually in the early development stage, has been implanted and begins to develop. Therefore, the OD 450nm values of negative and positive samples both decrease during the detection at 28 days.

[0076] The above is only the preferred implementation mode of the present invention. It should be noted that for those skilled in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these should also be regarded as the protection scope of the present invention, and these will not affect the implementation effect of the present invention and the practicality of the patent.

Claims

1. Specific nucleic acid aptamer for pregnant cow serum, characterized in that, The nucleotide sequence of the aptamer is shown as SEQ ID NO.

4.

2. The screening method of the specific nucleic acid aptamer for pregnant cow serum according to claim 1, characterized in that It includes the following steps: Based on magnetic-SELEX technology, using the serum of pregnant cows at 21 days after fertilization as the target to screen for aptamers. Through 12 rounds of SELEX screening, aptamers L4, L11, L13, L21, and L40 are enriched; perform homology analysis and secondary structure prediction on the screened aptamers, and react the screened aptamers with the positive and negative sera of pregnant cows respectively. According to the P / N value, the best aptamer is determined to be aptamer L4.

3. The screening method according to claim 2, wherein The nucleotide sequences of the aptamers L11, L13, L21, and L40 are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8 respectively.

4. The screening method according to claim 2, wherein The homology of the aptamer is 63.76%, and the secondary structure contains hairpin and stem-loop structures.

5. The application of the specific nucleic acid aptamer of pregnant cow serum according to claim 1 in the preparation of bovine early pregnancy diagnostic products.

6. The application according to claim 5, characterized in that, The optimal detection conditions for the specific nucleic acid aptamer of pregnant cow serum are: ssDNA is diluted 100 times, serum is diluted 5 times, and the substrate acts for 8 minutes.

7. The application according to claim 5, wherein The bovine early pregnancy diagnostic product is used for the early pregnancy diagnosis of pregnant cows at 14 - 21 days after fertilization.

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