CN110747268A

CN110747268A - Application of serum exosome ssc-miR-17-5p as molecular marker for early pregnancy diagnosis of sow

Info

Publication number: CN110747268A
Application number: CN201911011854.9A
Authority: CN
Inventors: 洪林君; 吴珍芳; 蔡更元; 贺艳娟; 周臣; 刘德武; 顾婷; 郑恩琴; 徐铮; 黄思秀
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2019-10-23
Filing date: 2019-10-23
Publication date: 2020-02-04
Anticipated expiration: 2039-10-23
Also published as: CN110747268B

Abstract

The invention relates to the technical field of biology, in particular to application of a serum exosome ssc-miR-17-5p as a molecular marker for early pregnancy diagnosis of sows. The nucleotide sequence of the serum exosome ssc-miR-17-5p is shown in SEQ ID No. 1. The molecular marker has the characteristics of early stage, convenience, quickness, specificity and sensitivity in detecting the pregnancy state, so that the nonpregnant period of the pseudopregnant sow is shortened, the compound is adopted as soon as possible, the production management is facilitated, and the production efficiency is improved. The invention also provides a primer and a kit for detecting the molecular marker, and provides a new way for diagnosing early pregnancy of the sow.

Description

Application of serum exosome ssc-miR-17-5p as molecular marker for early pregnancy diagnosis of sow

Technical Field

The invention relates to the technical field of biology, in particular to application of a serum exosome ssc-miR-17-5p as a molecular marker for early pregnancy diagnosis of sows.

Background

The reproductive performance of the sows is an important index of the production efficiency of the pig industry, one of the reproductive performance indexes is the annual Productivity (PSY) of the sows, which reflects the technical level of pig raising in a pig farm and the core index of the production efficiency, and statistics shows that the PSY of the sows in China is less than 20, and the condition of low production performance seriously restricts the development of the pig industry in China. In order to improve the productivity of sows, the normal breeding cycle of sows is ensured. The early pregnancy diagnosis can confirm whether the bred sows are pregnant or not, timely carry out fetus protection on the sows which are pregnant, timely take compounding measures on the sows which are not pregnant, shorten the non-pregnant period, and early eliminate the sows which are frequently bred to be pregnant. The B-ultrasonic diagnosis is the most popular pregnancy diagnosis method in pig farms at present, but the B-ultrasonic diagnosis needs to be carried out for about 30 days of pregnancy of sows to achieve higher accuracy. In order to accurately judge the pregnancy condition of a sow in the early pregnancy period, exosomes and MicroRNA carried by the exosomes are used as pregnancy markers to quickly and accurately detect the pregnancy condition of the sow.

Exosomes (exosomes) are phospholipid bilayer vesicles 30-150 nm in diameter and are secreted extracellularly by various cells. The exosome can optionally contain various types of nucleic acid molecules, such as DNA, mRNA, microRNA, circRNA, lncRNA and the like, as well as various lipids and proteins. Exosomes have been identified as containing specific protein molecules on their surface (CD9, CD63, CD81, CD82, etc.). Exosomes and their carriers can function as signaling molecules that are transmitted by body fluids to other cellular tissues. There are studies in other species that suggest that blood mirnas may serve as biomarkers of pregnancy. mirnas are endogenous, short 17-25nt non-coding RNAs that disrupt and degrade transcription or inhibit translation by complementary binding to mRNA. miRNA expression is highly tissue specific, with expression levels varying significantly with developmental, physiological, or pathological stages.

There are related literature studies describing the study of exosomes and their carriers as carriers of disease or physiological markers in different body fluids of other species. The invention discloses a method for finding and determining exosome miRNAs with obvious difference in expression in relative nonpregnant/estrus periods during pregnancy and determining the possibility of applying the exosome miRNAs to a molecular marker for early pregnancy diagnosis, wherein the exosome is also present in pig serum and the distribution of the exosome is changed along with the pregnancy state.

Disclosure of Invention

In order to overcome the defects and shortcomings of the prior art, the invention mainly aims to provide application of a serum exosome ssc-miR-17-5p as a molecular marker for early pregnancy diagnosis of a sow.

The invention also aims to provide a primer for detecting the molecular marker for diagnosing the early pregnancy of the sow.

It is still another object of the present invention to provide a kit comprising the above primer.

The fourth purpose of the invention is to provide the application of the primer and the kit.

The purpose of the invention is realized by the following technical scheme:

an application of a serum exosome ssc-miR-17-5p as a molecular marker for early pregnancy diagnosis of a sow is disclosed, wherein the nucleotide sequence of the serum exosome ssc-miR-17-5p is as follows:

5’-CAAAGUGCUUACAGUGCAGGUAG-3’；

a primer for detecting the molecular marker for diagnosing the early pregnancy of the sow comprises primers ssc-miR-17-5p-F and ssc-miR-17-5p-R, and the nucleotide sequences are shown as follows:

primer ssc-miR-17-5 p-F: 5'-GCCAAAGTGCTTACAGTGC-3', respectively;

primer ssc-miR-17-5 p-R: 5'-GTGCAGGGTCCGAGGT-3', respectively;

the primer for detecting the molecular marker for diagnosing the early pregnancy of the sow preferably further comprises a reverse transcription primer ssc-miR-17-5p _1 of a stem-loop method, and the nucleotide sequence of the primer is as follows:

primer ssc-miR-17-5p _ 1: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTACCT-3', respectively;

the primer for detecting the molecular marker for diagnosing the early pregnancy of the sow preferably further comprises a stem-loop method external reference reverse transcription primer cel-miR-39_1, and the nucleotide sequence of the primer is as follows:

primer cel-miR-39_ 1: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAAGCT-3', respectively;

a kit for detecting the molecular marker for diagnosing the early pregnancy of the sow, which comprises the primer;

the kit for detecting the molecular marker for diagnosing the early pregnancy of the sow preferably further comprises the following components: 2 × TS Reaction Mix, RT/RI Enzyme Mix, gDNA Remover, RNase-free Water, Green qPCRSupermix;

the primer for detecting the molecular marker for diagnosing the early pregnancy of the sow is applied to the preparation of a product for diagnosing the early pregnancy of the sow;

the kit for detecting the molecular marker for diagnosing the early pregnancy of the sow is applied to the preparation of a product for diagnosing the early pregnancy of the sow;

the application comprises the following steps:

extracting exosomes from serum of a sow to be detected, and then extracting RNA from the exosomes; performing fluorescent quantitative PCR after RNA reverse transcription to obtain cDNA;

the reverse transcription system is preferably as follows:

the reverse transcription procedure is preferably as follows:

uniformly mixing the RNA of a sample to be detected, a reverse transcription primer and RNase-free Water, incubating at 65 ℃ for 5min, and carrying out ice bath for 2 min; then adding other reaction components, mixing evenly, and incubating for 15min at 42 ℃; finally heating at 85 ℃ for 5 seconds to obtain cDNA;

the system of the fluorescent quantitative PCR is preferably as follows:

the fluorescent quantitative PCR program is preferably as follows:

pre-denaturation at 94 ℃ for 30 sec; 94 ℃ 5sec, 60 ℃ 30sec, 40 cycles; 94 ℃ for 15sec, 60 ℃ for 1min, 94 ℃ for 15 sec;

and (3) judging the fluorescent quantitative PCR result: if the expression level of ssc-miR-17-5P in the sample to be detected is remarkably increased relative to that of the negative control, and P is less than 0.05, the sow is successfully fertilized; otherwise, the pregnancy is not successful; wherein the negative control is exosome RNA in the serum of the non-pregnant sow;

compared with the prior art, the invention has the following advantages and effects:

(1) according to the invention, through methods such as Illumina sequencing and differential gene screening, the level change of exosome miRNA in serum of 9 th day of pregnancy is firstly determined, the level increase of ssc-miR-17-5p expression in 9 th to 15 th days of pregnancy is further determined, and the miRNA can be used as a new biomarker of early pregnancy of pigs. Compared with the traditional method for diagnosing the pregnancy state by B ultrasonic, the method for diagnosing the pregnancy state by B ultrasonic can achieve higher accuracy only by being carried out for about 30 days of pregnancy of the sow, and the method for detecting the pregnancy state by using the exosome ssc-miR-17-5p has the characteristics of early stage, convenience, rapidness, specificity and sensitivity, so that the non-pregnant period of the non-pregnant sow is shortened, the compounding is carried out as early as possible, the production management is facilitated, and the production efficiency is improved.

(2) In order to identify nonpregnant sows and pregnancy conditions at the early stage of pregnancy, exosomes and miRNA thereof are used as pregnancy markers to quickly and conveniently detect the pregnancy conditions, and primers and a kit for quickly detecting related early pregnancy are further developed, so that a new way is provided for diagnosing early pregnancy of sows.

Drawings

FIG. 1 is a Transmission Electron Microscopy (TEM) identification image of a serum exosome sample prepared in example 1.

FIG. 2 is a band diagram of the serum exosome membrane surface protein molecule shown by Western Blot.

FIG. 3 is a secondary structure diagram of ssc-miR-17-5p _ ssc-miR-17.

Fig. 4 is a diagram of analysis of verification results of high throughput sequencing sample data.

FIG. 5 is a diagram showing the analysis of the validation result of the independent sample differential gene ssc-miR-17-5p in actual production.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1

First, experimental design and sample collection

20 multiparous sows (large white) are selected in a certain breeding farm to carry out estrus synchronization treatment, 15 of the sows are subjected to artificial insemination, and the other 5 sows are subjected to false fertilization treatment. Ear marginal vein blood sampling was performed with a procoagulant tube on day 9 of estrus and day 9, 12, and 15 of pregnancy, respectively. And ultrasound was performed at week 6 after insemination to confirm pregnancy status. Accelerating coagulation is carried out on the collected blood within one hour to separate out serum, and the blood is put into a refrigerator at minus 80 ℃ in time for storage for later use.

Second, separation and identification of serum exosomes

(1) Separation of serum exosomes

Centrifuging the separated serum of the first step at 4 deg.C at 2,000 Xg for 20min to remove floating cells and debris; centrifuging the obtained supernatant at high speed at 11,000 Xg for 30min at 4 deg.C for removing larger membrane vesicles; to further remove particulate matter, the supernatant from the high speed centrifugation was gently squeeze filtered using a 0.22 μm filter (Merck Millipore, Ireland), and the filtered supernatant was ultracentrifuged using an Optima XPN-100 ultracentrifuge (Beckman Coulter, USA) in rotor SW 32Ti at 111,000 Xg for 2h at 4 deg.C; the particles such as exosomes obtained after ultracentrifugation were repeatedly washed and dissolved in PBS and stored in a refrigerator at-80 ℃. Meanwhile, the exosome can be separated by an ultracentrifugation method or an exosome separation and extraction kit.

(2) Transmission electron microscopy analysis

The negative staining step of the exosome comprises the following steps: firstly, processing a copper mesh, performing glow discharge on the copper mesh to eliminate static electricity, placing a top part with a grid (carbon faces upwards) in a vacuum chamber, and pulling vacuum and the glow discharge for 30-60 seconds. After emptying, it was placed in a petri dish and placed on a clean bench. Then a small piece of Parafilm (sealing film) is cut and tightly attached to the inversely buckled plate vessel; then sucking 10 mu L of the exosome sample prepared in the step (1) by using a pipette and dripping the exosome sample on a sealing film, clamping a copper mesh by using tweezers and slightly sticking the copper mesh on the sample drip upside down, and allowing the copper mesh to be adsorbed (incubated) on the sample drip for 10 min; then a liquid transfer gun is used for sucking 10 mu L of uranium acetate drops on a sealing film beside the sample; after the incubation time is over, slightly clamping the copper mesh on the sample by using forceps, and slightly absorbing the redundant sample buffer solution at the edge of the copper mesh by using Whatman paper; inverting the copper net on 3 wt% uranium acetate drop for adsorption (incubation) for 2 min; after the incubation time is finished, the copper mesh adsorbing the uranium peracetic acid is clamped by using tweezers, excessive liquid is lightly adsorbed by using Whatman paper at the edge of the copper mesh, the negatively dyed copper mesh is placed back into the copper mesh box and naturally dried for observation of a transmission electron microscope, and the coordinate position of each sample placed in the copper mesh box is recorded. Finally using FEI Talos^TMAnd carrying out morphological observation on exosomes by using a field emission transmission electron microscope.

The electron microscope result shows that the serum exosome has good shape, the plasma membrane is obvious and is in an oval concave shape, the size of the exosome is mainly distributed at about 100nm, and most of the diameter of the exosome is distributed at 30-150 nm. The exosome electron microscopy results are shown in figure 1.

(3) Western Blot analysis (Western Blot)

The exosome sample prepared in step (1) was treated with RIPA lysis buffer (CWBIO, PMSF: RIPA ═ 1: 100) to separate total protein, giving exosome protein, and then the protein concentration was measured using a Micro BCA protein assay kit (CWBIO). The isolated exosome proteins were heat denatured and separated by 12% (W/V) SDS-PAGE gel. The exosome proteins were transferred onto polyvinylidene fluoride membranes (Millipore, USA) and blocked with 6% (W/V) skimmed milk powder for 2.5 hours at room temperature. anti-CD 9/CD63(Abcam, UK) and horseradish peroxidase-conjugated goat anti-mouse IgG (Abcam, UK) were then incubated. Blot exposures were performed using the BeyoECL Moon chemiluminescence development kit (Beyotime).

As shown in FIG. 2, Western Blot detected the presence of the exosome surface marker proteins CD9, CD 63.

Thirdly, separation and extraction of exosome RNA

Thawing the exosome sample obtained by ultracentrifugation in the step two (1), and extracting exosome RNA by using miRNeasy Serum/Plasma Kit (Qiagen, Germany) by the specific method as follows:

(1) add 700. mu.L of QIAzol lysine Reagent to each volume of exosome sample, promote dissociation of the nucleoprotein complex by vortexing the tube containing the lysate briefly and incubating at room temperature (15-25 ℃) for 5 min;

(2) to each sample was added exogenous 3.5 μ L cel-miR-39(Qiagen, Germany) as an external reference and mixed well;

(3) adding 90 mu L of chloroform into a test tube containing the lysate, tightly covering the cover of the test tube, violently shaking for 15 seconds, and incubating for 2min at room temperature (15-25 ℃);

(4) centrifuging at 12,000 Xg for 15min at 4 deg.C (after centrifugation, the sample separates into 3 phases: an upper colorless aqueous phase containing RNA, a thin white phase, and a lower red organic phase, the volume of the aqueous phase should be about 400. mu.L);

(5) the upper aqueous phase was transferred to a new collection tube, avoiding any transfer of other liquid phases. Adding 2 times of anhydrous ethanol, and sucking and blowing up and down through a suction pipe for several times to fully mix;

(6) sucking up to 700. mu.L of a sample into an RNeasy MinElute adsorption column, slightly covering the column with a cover, centrifuging the column at room temperature (15-25 ℃) at a speed of not less than 8,000 Xg for 15 seconds, and discarding the filtrate; the step can be repeated for enrichment of the rest sample liquid, and the filtrate is discarded;

(7) add 700. mu.L of RWT buffer to RNeasy MinElute adsorption column; slightly covering the cover, centrifuging at a rotating speed of more than or equal to 8,000 Xg for 15 seconds, and discarding the centrifuged filtrate;

(8) add 500. mu.L of RPE buffer to RNeasy MinElute adsorption column; slightly covering the cover, centrifuging at a rotating speed of more than or equal to 8,000 Xg for 15 seconds, and discarding the centrifuged filtrate;

(9) pipette 500 μ L RPE buffer onto RNeasy MinElute adsorption column; covering the cover, centrifuging at a rotating speed of more than or equal to 8,000 Xg for 2min, and discarding the collecting pipe and the filtrate under centrifugation;

(10) put RNeasy MinElute spin column into new 2mL collection tube; opening the cover of the adsorption column, centrifuging at full speed for 5min to dry the adsorption column membrane, and discarding the centrifuged filtrate;

(11) RNeasy MinElute spin column was placed in a new 1.5mL collection tube; 14 μ L RNase free water was added directly to the center of the adsorption column membrane. Slightly covering the cover, allowing the column to stand and incubate for 1min, and then centrifuging at full speed for 1min to elute RNA to obtain a small RNA sample;

fourth, Illumina sequencing and differential gene screening

Use according to manufacturer's recommendations

NEBNext Multiplex of (NEB, USA) prepared a cDNA library for each time period (n-3) of small RNA samples (prepared in step three); library preparation work sequencing was performed on the Illumina Novaseq6000 platform.

Analyzing the sequencing result by utilizing a bioinformatics technology to obtain 6 potential miRNAs markers with significant differential expression, wherein one of the potential miRNAs markers is as follows: the nucleotide sequence of the serum exosome ssc-miR-17-5p is shown as follows:

5’-CAAAGUGCUUACAGUGCAGGUAG-3’；

the precursor ssc-miR-17 sequence of the serum exosome ssc-miR-17-5p is shown as follows, and the secondary structure is shown in FIG. 3:

5’-GAAUAAUGUCAAAGUGCUUACAGUGCAGGUAGUGAUAAUGUGCAUCUACUGCAGUGAAGGCACUUGUAGCAUUAUGG-3’。

fifth, RT-qPCR verifies that the gene ssc-miR-17-5p of differential expression in sequencing data

In a sequencing sample, carrying out fluorescence quantitative differential verification on the differential ssc-miR-17-5p screened from a sequencing result, wherein the specific method comprises the following steps:

(1) synthesis of cDNA by reverse transcription of stem-loop

Uniformly mixing the small RNA sample separated and extracted in the step three with a primer and RNase-free Water, incubating at 65 ℃ for 5min, and carrying out ice bath for 2 min; then adding other reaction components (the reaction system is shown in table 1), mixing evenly, and incubating for 15min at 42 ℃; finally heating at 85 ℃ for 5 seconds to inactivate the RT/RI and the gDNA Remover to obtain cDNA; wherein, exogenous cel-miR-39(Qiagen, Germany) is added as an external reference in the RNA separation process, and the specific stem-loop reverse transcription primer sequence is shown as follows;

TABLE 1 reverse transcription reaction System

(2) Fluorescent quantitative PCR

Diluting the cDNA prepared in the step (1) by 10 times and then using the diluted cDNA as a template, and using ssc-miR-17-5p-F and ssc-miR-17-5p-R as amplification primers to perform fluorescence quantitative PCR, wherein specific reaction systems and procedures are shown in tables 2 and 3, and specific primer sequences are shown as follows:

ssc-miR-17-5p-F：5’-GCCAAAGTGCTTACAGTGC-3’；

ssc-miR-17-5p-R：5’-GTGCAGGGTCCGAGGT-3’；

cel-miR-39-F：5’-GCGCTCACCGGGTGTAAATC-3’；

cel-miR-39-R：5’-GTGCAGGGTCCGAGGT-3’；

TABLE 2 Real time PCR System

TABLE 3 Real time PCR program (two-step procedure)

The results show that: the differences between sows at 9, 12 and 15 days of gestation (P9, P12 and P15) and non-pregnant sows (C9) are obvious and accord with sequencing results, and particularly, the sequencing results are shown in figure 4. And preparing for independent detection in actual production.

Example 2 RT-qPCR independently verifying early pregnancy differential gene ssc-miR-17-5p in actual production

(1) In a group of sows from an independent expanded sample (n-8), which were subjected to the above-described estrus synchronization treatment and artificial insemination, sow sera were collected for 0 day (day of insemination), 9 days of estrus, 9, 12 and 15 days of gestation, respectively.

(2) Referring to example 1, an exosome sample was isolated using ultracentrifugation and exosome RNA was extracted using RNA extraction Kit miRNeasy Serum/Plasma Kit (Qiagen, Germany), exogenous cel-miR-39(Qiagen, Germany) was added as an external reference during extraction, and all candidate genes and target gene primers were diluted according to Oligo synthesis manufacturer (BGI) protocol. A cDNA library of miRNA was created by a stem loop primer method using a reverse transcription kit (transcgen, CHINA) using all samples.

(3) The synthesized cDNA library was diluted 10-fold, and three miRNA cdnas were initially detected by real-time PCR on an ABI 7300 real-time PCR instrument (life, USA), the specific reaction system and procedure are shown in the fluorescent quantitative PCR section of example 1.

(4) Gene expression raw data were processed using Microsoft Excel, using 2^-ΔΔCtCalculating the relative expression level of each miRNA, analyzing by using a paired t test method and an analysis of variance method, and obtaining a P value<0.05 was a significant difference and was analyzed using GraphPad Prism 6 software for graphical statistics.

Independent quantification results of 6 specifically different miRNAs showed that the expression level of ssc-miR-17-5P was very significantly increased in maternal serum-derived exosomes collected as early as day 9 of pregnancy, with P < 0.01. See figure 5 for details.

By RNA sequencing and independent qPCR analysis of porcine serum exosome miRNAs, the present invention first determined the level changes of exosome miRNAs in serum earliest on day 9 of pregnancy. The results indicate that circulating serum exosome mirnas may play an important role in early pregnancy in pigs. In particular, we determined that during days 9 to 15 of gestation, the levels of ssc-miR-17-5p expression are increased, and that these mirnas can all serve as novel biomarkers for early pregnancy in pigs.

EXAMPLE 3 tailing reverse transcription method

The same results can be obtained by the tailing method in addition to the stem-loop reverse transcription method.

(1) Synthesis of cDNA by tailing transcription

Referring to example 1, an exosome sample was isolated using ultracentrifugation and exosome RNA was extracted using RNA extraction Kit miRNeasy Serum/Plasma Kit (Qiagen, Germany), exogenous cel-miR-39(Qiagen, Germany) was added as an external reference during extraction, and all candidate genes and target gene primers were diluted according to Oligo synthesis manufacturer (BGI) protocol. Carrying out general reverse transcription by a tailing method by using a full-type gold kit TransScript miRNA First-Strand cDNAsSynthesis Super Mix to create a cDNA library of miRNA, wherein a reverse transcription primer is provided by the TransScript miRNA First-Strand cDNA Synthesis Super Mix kit. Preparing a reaction system according to the table 4, mixing the reaction system evenly and gently, and incubating the mixture for 1 hour at 37 ℃; the RT Enzyme Mix was inactivated by heating at 85 ℃ for 5 seconds.

TABLE 4 reverse transcription reaction System

(2) Fluorescent quantitative PCR

Diluting cDNA obtained by reverse transcription by 10 times and using the diluted cDNA as a template, wherein an upstream Primer is a miRNA specific Primer, a downstream Primer is a Universal Primer Universal miRNA qPCR Primer (10 mu M) provided by a TransScript miRNA First-Strand cDNA Synthesis Super Mix kit, a specific reaction system and a program are shown in tables 5 and 6, and a Primer sequence is designed according to target miRNA as shown in the following table:

ssc-miR-17-5p_2：5’-GCCAAAGTGCTTACAGTGC-3’；

TABLE 5 qPCR System

TABLE 6 qPCR procedure (two-step procedure)

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

SEQUENCE LISTING

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Claims

1. An application of a serum exosome ssc-miR-17-5p as a molecular marker for diagnosing early pregnancy of a sow is characterized in that the nucleotide sequence of the serum exosome ssc-miR-17-5p is as follows:

5’-CAAAGUGCUUACAGUGCAGGUAG-3’。

2. a primer for detecting a molecular marker for diagnosing early pregnancy of a sow is characterized by comprising primers ssc-miR-17-5p-F and ssc-miR-17-5p-R, wherein the nucleotide sequences of the primers are as follows:

ssc-miR-17-5p-F：5’-GCCAAAGTGCTTACAGTGC-3’；

ssc-miR-17-5p-R：5’-GTGCAGGGTCCGAGGT-3’。

3. the primer for detecting the molecular marker for diagnosing the early pregnancy of the sow as claimed in claim 2, which further comprises a reverse transcription primer ssc-miR-17-5p _1 of stem-loop method, and the nucleotide sequence of the reverse transcription primer ssc-miR-17-5p _1 is shown as follows:

primer ssc-miR-17-5p _ 1: 5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTACCT-3' are provided.

4. A kit for detecting a molecular marker for diagnosing early pregnancy of a sow, which is characterized by comprising the primer of claim 2 or 3.

5. The kit for detecting the molecular marker for diagnosing the early pregnancy of the sow as claimed in claim 4, which is characterized by further comprising the following components: 2 XTS Reaction Mix, RT/RI Enzyme Mix, gDNA Remover, RNase-freeWater, Green qPCR Supermix.

6. Use of the primer for detecting the molecular marker for diagnosing early pregnancy of a sow as claimed in claim 2 or 3 in the preparation of a product for diagnosing early pregnancy of a sow.

7. Use of the kit for detecting a molecular marker for diagnosing early pregnancy of a sow as claimed in claim 4 or 5 in preparing a product for diagnosing early pregnancy of a sow.

8. The use of the kit for detecting the molecular marker for diagnosing early pregnancy of a sow as claimed in claim 7 in the preparation of a product for diagnosing early pregnancy of a sow, which is characterized by comprising the following steps:

extracting exosomes from serum of a sow to be detected, and then extracting RNA from the exosomes; and performing fluorescent quantitative PCR after RNA reverse transcription to obtain cDNA.

9. The use of the kit for detecting the molecular marker for diagnosing early pregnancy of a sow as claimed in claim/8 in the preparation of a product for diagnosing early pregnancy of a sow, wherein the kit comprises:

the reverse transcription system is as follows:

the reverse transcription procedure is as follows:

uniformly mixing the RNA of a sample to be detected, a reverse transcription primer and RNase-free Water, incubating at 65 ℃ for 5min, and carrying out ice bath for 2 min; then adding other reaction components, mixing evenly, and incubating for 15min at 42 ℃; finally, the cDNA was obtained by heating at 85 ℃ for 5 seconds.

10. The use of the kit for detecting the molecular marker for diagnosing early pregnancy of a sow as claimed in claim/8 in the preparation of a product for diagnosing early pregnancy of a sow, wherein the kit comprises:

the system of the fluorescence quantitative PCR comprises:

the fluorescent quantitative PCR program comprises:

pre-denaturation at 94 ℃ for 30 sec; 94 ℃ 5sec, 60 ℃ 30sec, 40 cycles; 94 ℃ for 15sec, 60 ℃ for 1min and 94 ℃ for 15 sec.