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

An early pregnancy and marker technology, 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 improving economic benefits

Active Publication Date: 2020-07-28
XINJIANG ACADEMY OF AGRI & RECLAMATION SCI +1
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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 ewes in the prior art, the present invention aims to provide an exosomal miRNA marker for diagnosis of early pregnancy in ewes. Somatic miRNA markers and their applications

Method used

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

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1: Screening test of exosomal miRNA

[0042] In this example, exosomal miRNAs obtained by separating and purifying peripheral blood were screened for specific exosomal miRNAs related to early pregnancy in ewes using bioinformatics technology. The specific steps are as follows:

[0043] S1. Preparation of serum samples: Collect peripheral blood samples of 17-day mating pregnant and non-pregnant ewes, add coagulant to blood collection tubes (BDvacutainer: Belliver Industrial Estate, Plymouth, BelliverWay, Roborough, PL67BPUK), after collecting peripheral blood , turn the blood collection tube upside down slowly 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. Components; the collected serum can be directly used for subsequent experiments or stored in a -80°C refrigerator.

[0044] S2. Extraction and inspection of exosomal RNA: According to the extraction ...

Embodiment 2

[0064] Example 2: Alignment of exosomal miRNA sequence information

[0065] The information on the two 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 two miRNA sequences.

[0066] Table 2: Two miRNA sequence information

[0067]

[0068] After comparison, it can be seen that the sequence information of the two miRNAs is consistent with the sequencing data, thus proving that the sequences of the two miRNAs oar-miR-22-3p and chi-miR-378-3p obtained in this application are correct.

Embodiment 3

[0069] Example 3: Detection Kit and Application of Exosomal miRNA Markers

[0070] This embodiment provides a detection kit for exosomal miRNA markers, which includes exosomal miRNA markers for the diagnosis of early pregnancy in ewes and reagents commonly used in PCR technology. Selection of exosomal miRNA markers One or two combinations of two miRNA markers oar-miR-22-3p and chi-miR-378-3p.

[0071] 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 oar-miR- 22-3p and chi-miR-378-3p expression.

[0072] Preferably, the specific primers in the kit are specific primers for detecting the expression levels of oar-miR-22-3p and chi-miR-378-3p.

[0073] Preferably, the RNA sequences of the specific primer...

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Abstract

The invention discloses an exosome miRNA marker for early pregnancy diagnosis of ewes and application of the exosome miRNA marker. The exosome miRNA molecular marker comprises oar-miR-22-3p and chi-miR-378-3p. The miRNA molecular marker shows high expression in serum exosomes of ewes in early pregnancy, and when the serum exosome miRNA is independently used for diagnosing and distinguishing the ewes in early pregnancy from the ewes in non-pregnancy, ROC area under the curve (AUC) is 0.891 (0.728, 1.000), 0.922 (0.791, 1.000), respectively; the AUC (95% CI) of the exosome miRNA molecular markeris 0.984 (0.936, 1.000) when early pregnant ewes and non-pregnant ewes are distinguished through combined diagnosis, it can be seen that the exosome miRNA molecular marker has high sensitivity and high specificity in early ewe pregnancy diagnosis, multiple miRNA markers are combined, and superior diagnosis performance is obtained.

Description

technical field [0001] The invention belongs to the field of biotechnology diagnosis. Specifically, the invention relates to an exosome miRNA marker used for ewe 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-chain non-coding RNAs with a length of about 19-2 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 Participate in the embryonic development of animals. 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 highl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/113
CPCC12Q1/6883C12Q2600/158C12Q2600/178C12Q2600/166Y02A50/30
Inventor 黄涛孙义姗孙敬礼杨敏高若男谢苏和军飞孙晓梅陈鑫李清春卢世豪
Owner XINJIANG ACADEMY OF AGRI & RECLAMATION SCI
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