Exosome miRNA marker for early pregnancy diagnosis of sows and application of exosome miRNA marker

A technology of early pregnancy and markers, applied in the field of biotechnology diagnosis, can solve the problems of low diagnostic value, low specificity and sensitivity, and achieve the effect of reducing the number of non-productive days, reducing the number of non-productive days, and shortening the parity interval

Active Publication Date: 2020-08-07
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Aiming at the technical problems of low specificity, sensitivity and low diagnostic value of exosomal miRNA markers used in the diagnosis of early pregnancy in so

Method used

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  • Exosome miRNA marker for early pregnancy diagnosis of sows and application of exosome miRNA marker
  • Exosome miRNA marker for early pregnancy diagnosis of sows and application of exosome miRNA marker
  • Exosome miRNA marker for early pregnancy diagnosis of sows and application of exosome miRNA marker

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1: Screening test of exosomal miRNA

[0049] In this example, exosomal miRNAs obtained by separating and purifying peripheral blood were screened for specific exosomal miRNAs related to early pregnancy in sows using bioinformatics technology. The specific steps are as follows:

[0050] S1, preparation of serum samples: collect peripheral blood samples of 14-day mating pregnant and non-pregnant sows, add coagulant in blood collection tubes (coagulation-promoting tubes, Nanchang Ganda Medical Instrument Co., Ltd.), after collecting peripheral blood, Slowly invert the blood collection tube and mix well. The mixed whole blood is centrifuged at 4000×g at 4°C for 15 minutes. The upper layer of yellow translucent liquid is the serum to be collected. ; The collected serum can be directly used for subsequent experiments or stored in a -80°C refrigerator.

[0051] S2. Extraction and inspection of exosomal RNA: According to the extraction steps and requirements of the QIA...

Embodiment 2

[0071] Example 2: Alignment of exosomal miRNA sequence information

[0072] The information on the three miRNA sequences obtained in this application was sorted out by searching the information disclosed in the miRBase database. See Table 2 for the information on the three miRNA sequences.

[0073] Table 2: Three miRNA sequence information

[0074]

[0075] After comparison, it can be seen that the sequence information of the three miRNAs is consistent with the sequencing data, thus proving that the sequences of the three miRNAs obtained in this application are correct.

Embodiment 3

[0076] Example 3: Detection Kit and Application of Exosomal miRNA Markers

[0077] This embodiment provides a detection kit for exosomal miRNA markers, which includes exosomal miRNA markers for the diagnosis of early pregnancy in sows and reagents commonly used in PCR technology. Selection of exosomal miRNA markers Combination of ssc-miR-192, ssc-miR-146a-5p, ssc-miR-149 three miRNA markers.

[0078] Preferably, the detection reagents in the kit include 2×miRNA RT Reaction Buffer, miRNA RTEnzyme Mix, RNase-Free ddH 2 O, 2×miRcute Plus miRNAPreMix, Reverse Primer, miRNA first-strand cDNA, 50×ROX Reference Dye, 75% ethanol, serum exosome lysate, chloroform and absolute ethanol, the above detection reagents are used to detect ssc-miR- 192. Expression levels of ssc-miR-146a-5p and ssc-miR-149.

[0079] Preferably, the specific primers in the kit are specific primers for detecting the expression levels of ssc-miR-192, ssc-miR-146a-5p, and ssc-miR-149.

[0080] Preferably, the mi...

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Abstract

The invention discloses an exosome miRNA marker for early pregnancy diagnosis of sows and application of the exosome miRNA marker. The exosome miRNA molecular marker is prepared from ssc-miR-192, ssc-miR-146a-5p and ssc-miR-149; when the serum exosome miRNA is independently used for diagnosing and distinguishing early pregnant sows and non-pregnant sows, the areas under the ROC curve (AUC) are respectively 0.863, 0.885 and 0.857; the combined AUC of the ssc-miR-192 and the ssc-miR-146a-5p is 0.921; the combined AUC of the ssc-miR-192 and the ssc-miR-149 is 0.902; the combined AUC of the ssc-miR-146a-5p and the ssc-miR-149 is 0.910; when the three miRNAs, namely ssc-miR-192, ssc-miR-146a-5p and ssc-miR-149, are combined, the combined AUC is 0.959; The exosome miRNA molecular marker providedby the invention has high sensitivity and high specificity in early sow pregnancy diagnosis, and has excellent diagnostic performance by combining a plurality of miRNA markers.

Description

technical field [0001] The invention belongs to the field of biotechnology diagnosis. Specifically, the invention relates to an exosome miRNA marker used for sow pregnancy diagnosis and application thereof. Background technique [0002] Exosomes are vesicles with a size of 30-100nm that are produced by cells and released into the extracellular environment, and are commonly found in fluids such as saliva, blood, urine, and milk of animals. microRNAs (miRNA) are short non-coding RNAs with a length of about 19-22 nucleotides, which play a regulatory role by complementing part or all of the sequences in the 3'-UTR region of mRNA, leading to transcriptional repression of target genes, thereby participating in Animal embryonic development. The unique form of miRNAs present in exosomes makes them ideal biomarkers. Studies have shown that exosomes, as a delivery form of extracellular miRNA, can protect the internal miRNA from ribonuclease degradation, making miRNA highly stable ex...

Claims

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Application Information

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IPC IPC(8): C12Q1/6888C12N15/113
CPCC12Q1/6888C12Q2600/178C12Q2600/158C12Q2600/124Y02A50/30
Inventor 黄涛高若男李涛邱梅玉谢苏孙义姗和军飞公红斌陈鑫李清春卢世豪
Owner SHIHEZI UNIVERSITY
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