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Application of serum ssc-mir-194b as a molecular marker for detecting intestinal stress injury in piglets

A technology of ssc-mir-194b and serum, applied in the field of livestock genetic engineering, can solve problems such as lack of diagnostic value

Active Publication Date: 2018-04-20
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the detection of the changes of the above indicators are all relative values, and the corresponding control samples are required as a reference and the conclusion can be drawn by analyzing the significant difference of the data, so it has no diagnostic value

Method used

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  • Application of serum ssc-mir-194b as a molecular marker for detecting intestinal stress injury in piglets
  • Application of serum ssc-mir-194b as a molecular marker for detecting intestinal stress injury in piglets
  • Application of serum ssc-mir-194b as a molecular marker for detecting intestinal stress injury in piglets

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Example 1: In this example, ssc-miR-194b was obtained by analyzing the correlation between serum microRNA and intestinal microRNA of weaned piglets, including the following steps:

[0024] 1. Total RNA extraction from piglet intestinal samples

[0025] Twenty-four piglets (6 piglets per litter) were selected from 4 litters and 25-day-old piglets with the same parity and date of birth, and were divided into lactation group and weaning group (weaning on the same day). The piglets in the weaning group were slaughtered 1d, 4d and 7d after weaning, and 4 piglets (1 per litter) were slaughtered in each group. Jejunal tissue samples were collected, and total RNA samples of piglet intestinal tissue were extracted by Trizol method.

[0026] 2. Small molecule RNA library construction

[0027] The total RNA samples of 4 biological replicates in each treatment group were converted by concentration and mixed in equal amounts to form RNA sample pools, and 15% denaturing polyacrylam...

Embodiment 2

[0051] Example 2: In this example, the ssc-miR-194b obtained in Example 1 was used to mark intestinal stress injury in weaned piglets.

[0052] (1) Randomly select 24 weaned piglets and mark them as Nos. 1 to 24, collect whole blood of weaned piglets, and prepare serum samples of Nos. 1 to 24;

[0053] (2) Using Plasma / Serum Exosome Kit (Norgen, 49200) reagent to extract total RNA in serum samples;

[0054] (3) using miRNeasy Mini Kit (Qiagen 217004) kit to extract serum microRNA in Total RNA;

[0055] (4) Use the ssc-miR-194b reverse transcription primer shown in SEQ ID NO.2 to reverse transcribe the microRNA to obtain cDNA: the reverse transcription system is: microRNA sample 2.5ul (40ng / ul), ssc-miR-194b reverse transcription primer 0.5ul (2uM), dNTP 0.25ul (10mM each),

[0056] 5×RT buffer 1.0ul, RNase inhibitor 0.25ul (40U / ul), M-MLV 0.5ul (200U / ul), the total volume of the reaction system is 5.0ul, put the uniformly mixed reaction system at 42°C for 60min, and then pla...

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Abstract

The invention belongs to the field of biology, and relates to the application of serum ssc-miR-194b as a molecular marker for detecting intestinal stress damage in piglets. The nucleotide sequence of serum ssc-miR-194b is shown in SEQ ID NO.1, reversed The nucleotide sequence of the stem-loop primer required to record ssc-miR-194b is shown in SEQ ID NO.2, and the nucleotide sequence of the forward primer required to amplify ssc-miR-194b is shown in SEQ ID NO. As shown in 3, the above primers can be used to diagnose the intestinal stress injury of weaned piglets. The diagnostic method includes the extraction of serum total RNA, the isolation of small molecule RNA, cDNA synthesis, PCR amplification and identification. It provides guidance for the prevention, diagnosis and treatment of tract stress injury, and provides a new molecular marker for marker-assisted selection of pig stress-resistant strains.

Description

technical field [0001] The invention belongs to the technical field of livestock genetic engineering, and particularly relates to the application of serum ssc-miR-194b as a molecular marker for detecting intestinal stress injury in piglets. Background technique [0002] Piglet weaning stress is a major problem in pig farming all over the world, causing huge economic losses to the pig industry every year. Piglet diarrhea caused by stress is the most common and most harmful stress disease in pig production. The primary target of weaning stress is the gastrointestinal tract of piglets, where damage to the small intestine is particularly severe. Small intestinal tissue damage and remodeling induced by weaning stress severely affects the development and maturation of the piglet's intestine, thus restricting the health and growth of piglets. Similarly, the decrease of gastrointestinal enzyme synthesis and absorption capacity caused by intestinal damage caused by weaning stress i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883
CPCC12Q1/6883C12Q2600/158C12Q2600/178
Inventor 陶新徐子伟
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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