A blood source marker gene combination for identifying early pregnancy state of cervus nippon, an identification kit and application thereof

Abstract

This invention relates to a blood-derived marker gene combination for identifying early pregnancy status in sika deer, an identification kit, and their applications, belonging to the field of molecular biology detection technology. Addressing the technical problem of the lack of early pregnancy diagnosis methods before embryo implantation in sika deer, and the difficulty in accurately determining pregnancy status, which easily leads to missed opportunities for optimal mating of does, this invention provides a blood-derived marker gene combination for identifying early pregnancy status in sika deer, including… Cbs , Igf2r , xCt and Gclc This invention provides a method for identifying the expression levels of the aforementioned gene transcripts in the peripheral blood of female sika deer 6.5–7.5 days after mating. If the expression levels are significantly upregulated compared to the day of mating, the individual is determined to be pregnant. This invention provides molecular targets for establishing a diagnostic technique for ultra-early pregnancy in sika deer, and the accompanying primers are highly sensitive, specific, and provide stable amplification, enabling timely identification of non-pregnant individuals and subsequent re-mating, thereby improving the reproductive efficiency of sika deer.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology detection technology, and in particular relates to a combination of blood-derived marker genes for identifying the early pregnancy status of sika deer, an identification kit, and their applications. Background Technology

[0002] Sika deer ( Sika deer Sika deer are an important special economic animal in my country. Their antlers and other products have extremely high medicinal and economic value, and their breeding industry plays a vital role in regional economic development. However, sika deer exhibit typical seasonal estrus and single-birth reproductive characteristics, with the estrus period concentrated between August and December each year. The natural twin rate is only about 2%, resulting in low overall reproductive efficiency and slow propagation of improved breeds. This has become a key bottleneck restricting the development of the sika deer breeding industry and genetic improvement.

[0003] To address these issues, the industry has widely adopted artificial insemination of female deer with high-quality male deer semen in recent years to achieve genetic improvement. However, in actual production, due to the difficulty in accurately determining the ovulation time of sika deer, the success rate of artificial insemination is low, easily leading to conception failure in female deer during the breeding season and an increase in the rate of non-pregnant women. This not only increases feeding and management costs but also causes economic losses to breeding enterprises.

[0004] Early pregnancy diagnosis technology can identify non-pregnant does in a timely manner without delaying breeding, allowing for re-mating or rescheduling production plans. This is a key means to improve herd reproductive efficiency and reduce losses due to non-pregnancy. Currently, in sika deer farming, the pregnancy status of does is often determined by observing whether they return to estrus after insemination. However, this method is highly dependent on the subjective experience of technicians, making accurate early diagnosis difficult. Therefore, developing efficient and low-cost early pregnancy diagnosis technology for sika deer is of significant practical importance and application value for improving sika deer reproductive efficiency, accelerating the process of selective breeding, and reducing economic losses in the farming industry.

[0005] The transcriptional dynamics and circulating nucleic acid characteristics of specific functional genes in peripheral blood are gradually becoming important biomarkers for predicting the physiological state of an organism. Traditionally, it was believed that the establishment of pregnancy in ruminants highly depends on the IFNT-ISG pathway, and this was used as the basis for blood pregnancy diagnosis. However, recent studies have shown that IFNT is not a necessary signal for the establishment of pregnancy in ruminants, leading to a re-evaluation of the reliability of using the IFNT-ISG pathway as a core molecular target for pregnancy diagnosis.

[0006] Therefore, systematically discovering and validating novel molecular targets that respond synchronously with the onset of pregnancy will effectively compensate for the limitations of existing detection methods in terms of time window, thus opening up a practical new path for building a diagnostic system for ultra-early pregnancy in sika deer. Summary of the Invention

[0007] This invention addresses the technical problem of the lack of early pregnancy diagnosis methods before embryo implantation in sika deer, making it difficult to accurately determine the pregnancy status and easily missing the optimal re-mating time for the female deer. It provides a blood-derived marker gene combination, identification kit, and its application for identifying the early pregnancy status of sika deer.

[0008] One objective of this invention is to provide a combination of blood-derived marker genes for identifying early pregnancy status in sika deer, wherein the blood-derived marker genes include: Cbs Gene, Igf2r Gene, xCt Genes and Gclc Gene.

[0009] In a preferred embodiment of the present invention, the Cbs The cDNA sequence of the gene is shown in SEQ ID NO.9. Igf2r The cDNA sequence of the gene is shown in SEQ ID NO.10. xCt The cDNA sequence of the gene is shown in SEQ ID NO.11 and Gclc The cDNA sequence of the gene is shown in SEQ ID NO.12.

[0010] The second objective of this invention is to provide a kit for identifying the early pregnancy status of sika deer, the kit comprising: Cbs Gene, Igf2r Gene, xCt Genes and Gclc Specific primer pairs for detecting gene transcript levels.

[0011] In a preferred embodiment of the present invention, the Cbs Specific primers for gene transcript level detection include: an upstream primer with the nucleotide sequence shown in SEQ ID NO.1 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.2.

[0012] In a preferred embodiment of the present invention, the Igf2r Specific primers for gene transcript level detection include: an upstream primer with the nucleotide sequence shown in SEQ ID NO.3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.4.

[0013] In a preferred embodiment of the present invention, the xCt Specific primers for gene transcript level detection include: an upstream primer with the nucleotide sequence shown in SEQ ID NO.5 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.6.

[0014] In a preferred embodiment of the present invention, the GclcSpecific primers for gene transcript level detection include: an upstream primer with the nucleotide sequence shown in SEQ ID NO.7 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.8.

[0015] The third objective of this invention is to provide the application of the above-mentioned blood-derived marker gene combination or identification kit in identifying the early pregnancy status of sika deer.

[0016] The fourth objective of this invention is to provide a method for identifying the pregnancy status of sika deer, the method comprising the following steps: using cDNA extracted from a peripheral blood sample of the female sika deer to be tested as a template, and performing the above-mentioned identification kit... Cbs Gene, Igf2r Gene, xCt Genes and Gclc Transcript level detection of genes; the pregnancy status of sika deer is identified based on the detection results. The peripheral blood of the sika deer was collected on the day of insemination of the female sika deer and on the 6.5-7.5 days after insemination.

[0017] In a preferred embodiment of the present invention, the criterion for identifying the detection result is: if the detection result shows that the peripheral blood sample of the female sika deer being tested contains... (The sentence is incomplete and requires further context to be translated accurately.) Cbs Gene, Igf2r Gene, xCt Genes and Gclc Transcript expression levels of all genes were significantly upregulated compared to day 0 post-fertilization, among which Cbs Gene transcript expression levels were upregulated 46-76 times. Igf2r Gene transcript expression levels were upregulated 46-57 times. xCt Gene transcript expression levels were upregulated 16-25 times. Gclc If the expression level of gene transcripts is upregulated by 8-18 times, the female sika deer being tested is determined to be pregnant.

[0018] Compared with the prior art, the beneficial effects of the present invention are: it provides a combination of blood-derived marker genes for identifying the early pregnancy state of sika deer, including... Cbs , Igf2r , xCt and Gclc Genes provide molecular targets for establishing a diagnostic technique for ultra-early pregnancy in sika deer. RT-qPCR detection confirmed that, compared to day 0 post-insemination, peripheral blood samples from pregnant sika deer on day 7 post-insemination showed significantly higher levels of [genes / targets]. Cbs Gene transcript expression levels were upregulated 46-76 times. P <0.05), Igf2r Gene transcript expression levels were upregulated 46-57 times. P<0.05), xCt Gene transcript expression levels were upregulated 16-25 times. P <0.05), Gclc Gene transcript expression levels were upregulated 8-18 times. P <0.05).

[0019] Based on the aforementioned differences in expression, ROC curves were further used for evaluation. Cbs , Igf2r , xCt and Gclc The efficacy of single-gene and multi-gene combinations in predicting pregnancy outcomes. Results showed that, when using single-gene predictions of pregnancy outcomes, Gclc The predictive efficacy of gene 1 was the best, with an AUC of 0.9167, a sensitivity of 100.00%, and a specificity of 87.50%. The other three genes showed moderate predictive efficacy when used alone. The predictive efficacy of multi-gene combined prediction was significantly improved compared to single-gene prediction, among which… Cbs + Igf2r + xCt , Igf2r + Gclc + xCt and Cbs + Igf2r + Gclc + xCt The AUC values ​​of the ROC curves were all 1.00, achieving 100% sensitivity and 100% specificity. This indicates that these gene combinations can completely and accurately distinguish between pregnant and non-pregnant samples under the detection conditions of this invention, demonstrating good production application value and broad application prospects.

[0020] This invention is the first to construct a criterion for determining very early pregnancy in sika deer based on the aforementioned specific combination of blood-derived marker genes. Furthermore, the primers used with this blood-derived marker gene combination exhibit high sensitivity, good specificity, and excellent linear amplification. It can be used alone or in combination. Cbs , Igf2r , xCt and Gclc Genes can promptly identify non-pregnant individuals and arrange for re-mating, thereby reducing the non-pregnant rate and improving the reproductive efficiency and economic benefits of sika deer populations. Attached Figure Description

[0021] Figure 1 To detect the presence of RT-qPCR in peripheral blood samples from pregnant sika deer on days 0, 7, 15, and 20 post-fertilization. Cbs, Igf2r, Gclc and xCt Trend graph of changes in gene transcript abundance; A represents... Cbs Gene transcript abundance change trend diagram; B is Igf2r Gene transcript abundance change trend diagram; C is GclcGene transcript abundance change trend diagram; D is xCt Gene transcript abundance trend graph; different lowercase letters above the bars indicate significant differences ( P <0.05, with the same lowercase letter indicating no significant difference ( P >0.05); Figure 2 To detect the presence of RT-qPCR in peripheral blood samples from pregnant and non-pregnant sika deer on days 0 and 7 post-insemination. Cbs, Igf2r, Gclc and xCt A comparison of changes in gene transcript abundance; A represents... Cbs Comparison of changes in gene transcript abundance; B represents... Igf2r Comparison of changes in gene transcript abundance; C represents Gclc Comparison of changes in gene transcript abundance; D represents xCt Comparison of changes in gene transcript abundance; different lowercase letters above the bars indicate significant differences. P <0.05); Figure 3 To detect the levels of [unclear - possibly "infectious" or "highly active" substances in the peripheral blood of sika deer on day 7 post-fertilization using RT-qPCR technology] Cbs, Igf2r, Gclc and xCt Minimum amount of cDNA template required for gene transcript; A is Cbs Minimum amount of cDNA template required for gene transcript detection; B is... Igf2r Minimum amount of cDNA template required for gene transcript detection; C is Gclc Minimum amount of cDNA template required for gene transcript detection; D is xCt Minimum amount of cDNA template required for gene transcript detection; Figure 4 To detect the presence of RT-qPCR in peripheral blood samples from 11 sika deer on day 7 post-insemination. Cbs, Igf2r, Gclc and xCt Gel electrophoresis image of gene transcripts; A is Cbs Gene gel electrophoresis image; B is Igf2r Gene gel electrophoresis image; C is Gclc Gene gel electrophoresis image; D is xCt Gene gel electrophoresis image; lanes 1 to 4, lanes 7 to 9 and lane 11 are peripheral blood samples from non-pregnant sika deer; lanes 5, 6 and 10 are peripheral blood samples from pregnant sika deer. Figure 5 ROC curves for predicting pregnancy outcomes using single genes and combinations of multiple genes; A is the ROC curve for predicting pregnancy outcomes using a single gene; B is the ROC curve for predicting pregnancy outcomes using two genes combined; C is the ROC curve for predicting pregnancy outcomes using multiple genes combined. Detailed Implementation

[0022] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously make modifications or appropriate alterations and combinations to the methods and applications described herein without departing from the content and scope of this invention to implement and apply the technology of this invention.

[0023] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the materials, reagents, methods, and instruments used are all conventional materials, reagents, methods, and instruments in the art, and can be obtained commercially by those skilled in the art.

[0024] This application is based on previous laboratory research results, specifically: whole-genome transcriptome sequencing analysis of peripheral blood from sika deer on days 0, 7, 15, and 20 after fertilization, and preliminary observation of the cystine transporter encoding gene based on the whole-genome transcriptome sequencing data. xCt Genes encoding cysteine ​​metabolic enzymes and their downstream derivative synthases Cbs and Gclc and the insulin-like growth factor 2 receptor encoding gene Igf2r Transcript expression was significantly upregulated on day 7 post-fertilization. However, high-throughput transcriptome analysis only provided a large number of candidate genes and failed to directly establish specific marker gene combinations that can be used for precise clinical diagnosis, nor did it establish an effective diagnostic model and specific quantitative thresholds for very early pregnancy.

[0025] The aforementioned preliminary laboratory research results are published in the following literature: Zhang, Y. ; Sun, H. ; Fan, B. ; Liu, L. ; Tang, Y. ; Zhang, Y. ; Zhang, X. ; Chu, X. ; Peng, F. ; Cao, J. ; Xu, B. Transcriptomic Profiling of Peripheral Blood Identifies CandidateGenes for Early Pregnancy Diagnosis in Sika Deer. Animals. 2025, 15, 2960. https: / / doi.org / 10.3390 / ani15202960.

[0026] The sika deer in the following examples came from the Zuojia Sika Deer Breeding Experimental Base, and all animal operations were carried out in accordance with the relevant regulations of the Animal Welfare and Ethics Committee of the Institute of Special Products, Chinese Academy of Agricultural Sciences (NO.ISAPSAEC-2022-081).

[0027] Example 1: Peripheral blood samples from different stages of early pregnancy in sika deer Cbs , Igf2r , Gclc and xCt Gene transcript expression dynamics 1. Collection of peripheral blood samples from sika deer Ten healthy, multiparous female sika deer were selected as experimental animals for a estrus synchronization and insemination experiment. Before the onset of estrus, progesterone vaginal suppositories were placed in the fornix of the cervix of the does. The suppositories were removed 12 days later, and each does was injected intramuscularly with 330 IU of pregnant mare serum gonadotropin (PMS). Estrus was detected using a buck; mounting by the does was considered estrus. Fifty-eight hours after suppository removal, the does in estrus were inseminated, and simultaneously injected intramuscularly with 25 μg of luteinizing hormone-releasing hormone A3 and 1.6 million IU of penicillin. The does were kept isolated from bucks for one month after insemination to prevent conception during this period. On days 0, 7, 15, and 20 after insemination (referred to as D0, D7, D15, and D20, respectively), 10 mL blood samples were collected via the jugular vein, placed in vacuum anticoagulant blood collection tubes, and stored at -80°C for later use. The pregnancy status of the does at the time of peripheral blood sample collection was deduced based on the full-term delivery time of the following year.

[0028] 2. Total RNA extraction and cDNA synthesis Total RNA was extracted from peripheral blood samples of pregnant sika deer at various time points after fertilization using TRIzol reagent according to the manufacturer's instructions. The concentration and purity of total RNA, and OD, were determined using a Nanodrop One spectrophotometer. 260 / 280 The values ​​were all within the range of 1.8-2.0. Following the instructions of the reverse transcription kit (Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR), RNA was reverse transcribed into cDNA and stored at -80℃ for later use.

[0029] 3. RT-qPCR detection of target gene transcript expression levels The Cbs The cDNA sequence of the gene is shown in SEQ ID NO.9. Igf2r The cDNA sequence of the gene is shown in SEQ ID NO. 10. xCt The cDNA sequence of the gene is shown in SEQ ID NO.11 and GclcThe cDNA sequence of the gene is shown in SEQ ID NO. 12.

[0030] Using cDNA extracted from peripheral blood samples of female sika deer as templates, amplification was performed on an AriaMX qPCR instrument using the FastStart Universal SYBR Green Master Rox reaction system. All experimental results were obtained from at least three different does. Actb This is an internal reference gene, and the primer sequence for the internal reference gene is as follows: Actb Upstream primer: 5'-CACCAACTGGGACGACA-3' (SEQ ID NO.13). Actb Downstream primer: 5'-GCATACAGGGACAGCACA-3' (SEQ ID NO.14). Cbs , Igf2r , xCt and Gclc The specific primer pairs for detecting gene transcript expression levels are shown in Table 1.

[0031] RT-qPCR reaction program: 95℃ pre-denaturation for 10 min; 45 cycles of 95℃ denaturation for 30 s and 60℃ annealing extension for 60 s.

[0032] RT-qPCR reaction system: 2 PerfectStart Green qPCR Super Mix 5 μL, 100 μM Forward Primer 0.3 μL, 100 μM Reverse Primer 0.3 μL, 200 ng Template cDNA 1 μL, Nase-Free Water 3.4 μL, the total system is 10 μL.

[0033] The statistical analysis of the data used one-way ANOVA (One-way ANOVA). Multiple comparisons were performed using the Way ANOVA and Tukey test.

[0034] Table 1 Cbs , Igf2r , xCt and Gclc Gene-specific primer pair sequences

[0035] like Figure 1 As shown, Cbs , Igf2r , xCt and GclcGene transcript expression levels in peripheral blood of female sika deer on day 7 post-fertilization were significantly upregulated compared to day 0 post-fertilization; among them, Cbs Gene transcript expression levels were upregulated 46-76 times. P <0.05), Igf2r Gene transcript expression levels were upregulated 46-57 times. P <0.05), xCt Gene transcript expression levels were upregulated 16-25 times. P <0.05), Gclc Gene transcript expression levels were upregulated 8-18 times. P <0.05). During the subsequent pregnancy, only xCt The expression level of gene transcripts further increased on day 20 post-fertilization; while Cbs , Igf2r and Gclc The expression level of gene transcripts peaks on day 7 after fertilization and then gradually declines, returning to the baseline level of day 0 after fertilization by day 15 or 20 after fertilization, or even below the baseline level.

[0036] This indicates that on the 7th day after fertilization, female sika deer had [a certain level of blood in their peripheral blood]. Cbs , Igf2r , xCt and Gclc The transcript expression levels of the genes were significantly upregulated compared to day 0 after fertilization, indicating that this gene combination can be used to identify the early pregnancy status of sika deer.

[0037] Example 2: Peripheral blood samples from pregnant and non-pregnant sika deer on day 7 post-insemination Cbs , Igf2r , xCt and Gclc Comparison of gene transcript expression levels 1. Collection of peripheral blood samples from sika deer, extraction of total RNA and synthesis of cDNA Peripheral blood samples of 10 mL each were collected from 3 pregnant sika deer and 3 non-pregnant sika deer on day 0 and day 7 after fertilization in Example 1. The samples were placed in vacuum anticoagulation blood collection tubes and stored at -80℃. The total RNA extraction and cDNA synthesis steps were the same as step 2 in Example 1.

[0038] 2. RT-qPCR detection of target gene transcript expression levels Cbs , Igf2r , xCt and Gclc The RT-qPCR detection procedure for gene transcripts is the same as step 3 in Example 1.

[0039] The difference between the two sets of data was compared using an unpaired method. tUnpaired t Statistical analysis was performed using -test) to... P <0.05 is used as the criterion for determining significance.

[0040] like Figure 2 As shown, compared to the peripheral blood sample on day 0 after fertilization, the peripheral blood of pregnant sika deer on day 7 after fertilization... Cbs , Igf2r , xCt and Gclc The expression levels of gene transcripts were upregulated by 46-76-fold, 46-57-fold, 16-25-fold, and 8-18-fold, respectively (all... P <0.05). In the peripheral blood of non-pregnant sika deer, compared to day 0, on day 7 post-insemination, Cbs Transcripts are only upregulated 3-5 times ( P <0.05), xCt Transcripts are only upregulated 4-5 times ( P <0.05), Igf2r Transcript expression showed no significant change. P >0.05), Gclc Transcript expression was downregulated to 0.1-0.5-fold on day 0. P <0.05). Further intergroup comparisons showed that on day 7 post-insemination, the expression levels of the above four gene transcripts in the peripheral blood of pregnant sika deer were significantly higher than those in non-pregnant sika deer. P <0.05).

[0041] Therefore, the combination of blood-derived marker genes for early pregnancy in sika deer provided by this invention ( Cbs , Igf2r , xCt and Gclc The transcript expression levels of the gene were significantly upregulated in peripheral blood only on day 7 after fertilization in pregnant sika deer, therefore this combination can be used to identify early pregnancy status on days 6.5-7.5 after fertilization in sika deer.

[0042] Example 3: Peripheral blood of sika deer Cbs , Igf2r , [[ID= and ​ Minimum amount of cDNA template required for gene transcript RT-qPCR detection 1. Acquisition and concentration gradient preparation of peripheral blood cDNA from sika deer Peripheral blood samples were collected from three pregnant sika deer on day 7 post-insemination, as described in Example 1. Total RNA extraction and cDNA synthesis were performed following step 2 of Example 1. The prepared RNA extract solutions were then formulated into cDNA solutions with mass concentrations of 200 ng / μL, 20 ng / μL, 2 ng / μL, 0.2 ng / μL, 0.02 ng / μL, 0.002 ng / μL, 0.0002 ng / μL, and 0.00002 ng / μL, respectively.

[0043] 2. Minimum cDNA template amount required for RT-qPCR detection of target gene transcripts in peripheral blood of sika deer Detection of blood-derived marker genes in early pregnancy of sika deer using RT-qPCR ​ , ​ , ​ and ​ The minimum amount of cDNA template required for gene transcripts was determined, and the RT-qPCR detection procedure was the same as step 3 in Example 1. The cDNA solution was diluted with RNase-free water to concentrations of 200 ng / μL, 20 ng / μL, 2 ng / μL, 0.2 ng / μL, 0.02 ng / μL, 0.002 ng / μL, 0.0002 ng / μL, and 0.00002 ng / μL, respectively.

[0044] Statistical analysis of the data employed linear regression, using the logarithm of cDNA concentration as the criterion. 10 Using cDNA (ng / μL) as the independent variable and the corresponding Cq value detected by qPCR as the dependent variable, a standard curve was fitted, and the coefficient of determination R was used to determine the curve. 2 Evaluate the linear relationship.

[0045] As shown in Tables 2 to 5, the lowest cDNA concentration that can stably detect specific amplification and effective Cq value in an RT-qPCR reaction is used as the criterion for determining the detection rate. ​ , ​ , ​ and ​ The minimum amount of cDNA template required for gene transcripts. Among them, [the following was detected]. ​ The minimum amount of cDNA template required for gene transcripts is 0.0002 ng / μL; detection ​ The minimum amount of cDNA template required for gene transcripts is 20 ng / μL; detection ​ The minimum amount of cDNA template required for gene transcripts is 2 ng / μL; detection ​ The minimum amount of cDNA template required for gene transcripts is 0.2 ng / μL.

[0046] like ​ As shown, it has been verified that it is used for detection. ​ and ​ The specific primers for gene transcripts exhibited ideal amplification efficiency within their linear amplification range, and the coefficients of determination of the standard curves were all greater than 0.92. The Cq value increased systematically with decreasing template concentration, verifying the reliability and high sensitivity of this RT-qPCR detection method.

[0047] Table 2 ​ Primer pair amplification results

[0048] Table 3 ​ Primer pair amplification results

[0049] Table 4 ​ Primer pair amplification results

[0050] Table 5 ​ Primer pair amplification results

[0051] Example 4: Based on ​ , ​ , ​ and ​ Validation of the accuracy of gene transcription level changes in identifying early pregnancy status in sika deer 1. Assessment of the accuracy of early pregnancy diagnosis in sika deer based on peripheral blood biomarkers using a double-blind design. Eleven healthy, multiparous female sika deer were selected and subjected to estrus synchronization and insemination according to the method described in step 1 of Example 1. Peripheral blood samples were collected on the 7th day after insemination, and the deer were kept alone for one month without contact with male deer to prevent pregnancy during this period. The 11 samples were randomly numbered and subjected to double-blind testing. The pregnancy status on the 7th day after insemination was deduced based on the full-term delivery time of the female deer the following year to determine the accuracy of the detection method of this invention.

[0052] 2 、 RT-qPCR detection of target gene transcript expression levels The total RNA extraction and cDNA synthesis steps are the same as step 2 in Example 1.

[0053] Detection ​ RT-qPCR reaction program for gene transcripts: 95℃ pre-denaturation for 10 min; 20 cycles of 95℃ denaturation for 30 s and 60℃ annealing extension for 60 s.

[0054] Detection ​RT-qPCR reaction program for gene transcripts: 95℃ pre-denaturation for 10 min; 35 cycles of 95℃ denaturation for 30 s and 60℃ annealing extension for 60 s.

[0055] Detection ​ RT-qPCR reaction program for gene transcripts: 95℃ pre-denaturation for 10 min; 35 cycles of 95℃ denaturation for 30 s and 60℃ annealing extension for 60 s.

[0056] Detection ​ RT-qPCR reaction program for gene transcripts: 95℃ pre-denaturation for 10 min; 33 cycles of 95℃ denaturation for 30 s and 60℃ annealing extension for 60 s.

[0057] 3. Agarose gel electrophoresis analysis of target gene RT-qPCR amplification products right ​ , ​ , ​ and ​ The RT-qPCR amplification products of the gene were subjected to agarose gel electrophoresis. Electrophoresis conditions: 2% agarose gel, 105 V, 130 mA, 40 min. Loading system: 2 μL of 6× Loading Buffer, 10 μL of RT-qPCR product, and a total system volume of 12 μL.

[0058] like ​ As shown, ​ , ​ , ​ and ​ The agarose gel electrophoresis results of the gene transcripts after RT-qPCR detection showed that specific target bands of the expected size were detected in lanes 5, 6, and 10, indicating pregnant sika deer; no specific bands were observed in the remaining lanes, indicating non-pregnant sika deer.

[0059] 4. Validation of the accuracy of early pregnancy diagnosis based on electrophoresis results By retrospectively estimating the gestation status of the does on the 7th day after insemination based on their full-term calving time the following year, the actual calving records for the following year showed that sika deer numbers 5, 6, and 10 were pregnant, while the rest were not. This result was completely consistent with the electrophoresis results. Therefore, it is evident that the invention designed… ​ , ​ , ​ and ​ The gene primer pairs have good specificity, and the established method for early pregnancy identification in sika deer has an accuracy rate of 100%.

[0060] Example 5: Based on ROC curves ​ , ​ , ​and ​ The efficacy of single-gene and multi-gene combinations in predicting pregnancy outcomes 1. Collection of peripheral blood samples from sika deer and detection of target gene transcripts Peripheral blood samples were collected from 11 female sika deer on day 7 after fertilization in Example 4. The total RNA extraction and cDNA synthesis steps were the same as step 2 in Example 1. ​ , ​ , ​ and ​ The RT-qPCR detection procedure for gene transcripts is the same as step 3 in Example 1.

[0061] 2. Based on ROC curves ​ , ​ , ​ and ​ The efficacy of single-gene and multi-gene combinations in predicting pregnancy outcomes Based on the time of full-term calving of the does the following year, the pregnancy status on the 7th day after insemination was deduced, resulting in 8 non-pregnant samples and 3 pregnant samples. The above samples were then analyzed. ​ , ​ , ​ and ​ The Cq values ​​obtained from gene RT-qPCR were used, and ROC curves were applied to evaluate the efficacy of single-gene and multi-gene combinations in predicting pregnancy outcomes.

[0062] based on ​ , ​ , ​ and ​ ROC curves were plotted using Cq values ​​obtained from gene transcript analysis to evaluate the efficacy of single-gene prediction of pregnancy outcomes. The ROC curve was plotted with sensitivity on the ordinate, and 1 - specificity (1 / 2)^2. The x-axis represents the specificity of the data; the Cq value of each gene corresponding to the maximum Youden's index is used as the cut-off value; the area under the curve (AUC) is used to judge the prediction accuracy, and the closer the AUC value is to 1, the higher the prediction value.

[0063] As shown in Table 6 ​ The gene exhibited the best predictive efficacy, with an AUC of 0.9167, a sensitivity of 100.00%, a specificity of 87.50%, and a Youden index of 0.8750, corresponding to a Cut The value of off is Cq < 33.07. ​ The gene ROC curve had an AUC of 0.7917, a sensitivity of 66.67%, a specificity of 75.00%, and a Youden index of 0.4167, corresponding to a Cut... The value of off is Cq < 34.48. ​ Genes and ​ The predictive efficacy of the genes was quite similar, with both having an AUC of 0.7083, sensitivity and specificity of 66.67% and 75.00% respectively, and a Youden index of 0.4167. ​ Gene Cut The off value is Cq < 19.97, indicating a cut in the xCt gene. The value of off is Cq < 34.67.

[0064] like ​ As shown in Figure A, when using a single gene to predict pregnancy outcomes... ​ The predictive efficacy of the gene is the best, with the highest AUC value of 0.9167 on its ROC curve, indicating high diagnostic accuracy. The other three genes have moderate predictive efficacy when used alone.

[0065] Table 6. ROC curve analysis of single-gene prediction of pregnancy outcomes

[0066] based on ​ , ​ , ​ and ​ ROC curves were plotted using binary logistic regression analysis of Cq values ​​obtained from gene transcript detection to evaluate the efficacy of two-gene and multi-gene combinations in predicting pregnancy outcomes. The ROC curves were plotted with sensitivity on the ordinate and 1-specificity on the abscissa, and AUC was used to determine predictive accuracy; a higher AUC value indicates a higher predictive value.

[0067] As shown in Table 7, in the two-gene combination group, ​ + ​ , ​ + ​ , ​ + ​ and ​ + ​ The AUC of the ROC curves were all 0.9167, among which... ​ + ​ and ​ + ​ The sensitivity and specificity reached 100.00% and 87.50%, respectively; ​ + ​ and ​ + ​ The sensitivity of all samples was 100.00%, and the specificity was 75.00%. ​ + ​ and​ + ​ The AUC of the ROC curve was further improved to 0.9583, with sensitivity and specificity reaching 100.00% and 87.50%, respectively. In the three-gene combination group, ​ + ​ + ​ The ROC curve had an AUC of 0.9583, a sensitivity of 100.00%, and a specificity of 87.50%; while ​ + ​ + ​ and ​ + ​ + ​ The AUC of the ROC curves all reached 1.0000, with both sensitivity and specificity at 100.00%. Even after combining all four genes, the AUC of the ROC curve remained at 1.0000, with both sensitivity and specificity at 100.00%. This indicates that under the detection conditions of this invention, ​ + ​ + ​ and ​ + ​ + xCt The combination of three genes and the combination of four genes can achieve ideal predictions with no errors.

[0068] like Figure 5 As shown in B, Igf2r + xCt and Gclc + xCt The ROC curve has the highest AUC value and the best predictive efficacy in the two-gene combination group.

[0069] like Figure 5 As shown in C, CBS + Igf2r + xCt , Igf2r + Gclc + xCt and CBS + Igf2r + Gclc + xCt The curves all passed through the point (0, 1), which means that the sensitivity and specificity reached 100% and the AUC value was 1.0000. This indicates that these gene combinations can completely and accurately distinguish between pregnant and non-pregnant samples under the detection conditions of this invention.

[0070] Table 7. ROC curve analysis of two-gene and multi-gene combined prediction of pregnancy outcomes

[0071] The specific embodiments of the present invention disclosed above are merely illustrative of the invention. These embodiments do not exhaustively describe all details, nor do they limit the invention to the specific embodiments described. Many modifications and variations can be made based on the content of this specification. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention.

Claims

1. A combination of blood-derived marker genes for identifying early pregnancy status in sika deer, characterized in that, The blood-derived marker genes include: CBS Gene, Igf2r Gene, xCt Genes and Gclc Gene.

2. The blood-derived marker gene according to claim 1, characterized in that, The CBS The cDNA sequence of the gene is shown in SEQ ID NO.

9. Igf2r The cDNA sequence of the gene is shown in SEQ ID NO.

10. xCt The cDNA sequence of the gene is shown in SEQ ID NO. 11 and Gclc The cDNA sequence of the gene is shown in SEQ ID NO.

12.

3. A kit for identifying early pregnancy status in sika deer, characterized in that, The identification kit includes CBS Gene, Igf2r Gene, xCt Genes and Gclc Specific primer pairs for detecting gene transcript levels.

4. The identification kit according to claim 3, characterized in that, The CBS Specific primers for gene transcript level detection include: an upstream primer with the nucleotide sequence shown in SEQ ID NO.1 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.

2.

5. The identification kit according to claim 3, characterized in that, The Igf2r Specific primers for gene transcript level detection include: an upstream primer with the nucleotide sequence shown in SEQ ID NO.3 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.

4.

6. The identification kit according to claim 3, characterized in that, The xCt Specific primers for detecting gene transcript levels include: an upstream primer with the nucleotide sequence shown in SEQ ID NO.5 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.

6.

7. The identification kit according to claim 3, characterized in that, The Gclc Specific primers for detecting gene transcript levels include: an upstream primer with the nucleotide sequence shown in SEQ ID NO.7 and a downstream primer with the nucleotide sequence shown in SEQ ID NO.

8.

8. The use of the blood-derived marker gene combination according to any one of claims 1 to 2 or the identification kit according to any one of claims 3 to 7 in identifying the early pregnancy status of sika deer.

9. A method for identifying the pregnancy status of a sika deer, characterized in that, The method includes the following steps: using cDNA extracted from the peripheral blood sample of the female sika deer to be tested as a template, and performing tests using the identification kit described in any one of claims 3 to 7. CBS Gene, Igf2r Gene, xCt Genes and Gclc Transcript level detection of genes, and identification of sika deer pregnancy status based on the detection results; The peripheral blood samples from the sika deer were collected on the day of insemination and 6.5-7.5 days after insemination.

10. The method according to claim 9, characterized in that, The criteria for identifying the test results are as follows: if the test results show that the peripheral blood sample from the female sika deer tested was positive for COVID-19 on days 6.5-7.5 after fertilization... CBS Gene, Igf2r Gene, xCt Genes and Gclc Transcript expression levels of all genes were significantly upregulated compared to day 0 post-fertilization, among which CBS Gene transcript expression levels were upregulated 46-76 times. Igf2r Gene transcript expression levels were upregulated 46-57 times. xCt Gene transcript expression levels were upregulated 16-25 times. Gclc If the expression level of gene transcripts is upregulated by 8-18 times, the female sika deer being tested is determined to be pregnant.

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