Application of serum ssc-miR-194b as molecule marker for detection of piglet intestinal tract stress injury
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A ssc-mir-194b, intestinal technology, applied in the field of livestock genetic engineering, can solve problems such as no diagnostic value
Active Publication Date: 2015-10-14
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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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
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Embodiment 1
[0022] 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:
[0023] 1. Extraction of total RNA from intestinal samples of piglets
[0024] 24 piglets (6 piglets per litter) were selected from 4 litters of 25-day-old piglets with the same parity and birth date as the research objects, and were divided into lactation group and weaning group (weaning on the same day) on average. 1d, 4d and 7d after weaning piglets in the weaning group, respectively, 4 piglets were slaughtered in each group (1 piglet per litter). The jejunal tissue samples were collected, and the total RNA samples of piglet intestinal tissues were extracted by Trizol method.
[0025] 2. Small RNA library construction
[0026] The total RNA samples of 4 biological repetitions of each treatment group were converted according to the concentration and mixed in equal amounts to form RNA samp...
Embodiment 2
[0050] Example 2: In this example, the ssc-miR-194b obtained in Example 1 was used to mark intestinal stress injury in weaned piglets.
[0051] (1) Randomly select 24 weaned piglets and mark them as Nos. 1-24 respectively, collect whole blood of weaned piglets, and prepare serum samples No. 1-24;
[0052] (2) Total RNA in serum samples was extracted by Plasma / Serum Exosome Kit (Norgen, 49200) reagent;
[0053] (3) Using miRNeasy Mini Kit (Qiagen 217004) kit to extract serum microRNA in Total RNA;
[0054] (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), 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, place the uniformly mixed reaction system at 42°C for 60min, then place at 7...
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Abstract
The present invention belongs to the field of biology, and relates to applications of serum ssc-miR-194b as a molecule marker for detection of piglet intestinal tract stress injury. According to the present invention, the nucleotide sequence of the serum ssc-miR-194b is represented by SEQ ID NO.1, the stem-loop primer nucleotide sequence required by the reverse transcription of the ssc-miR-194b is represented by SEQ IDNO.2, the forward primer nucleotide sequence required by the amplification of the ssc-miR-194b is represented by SEQ ID NO.3, and with the primers, the diagnosis on the weaned piglet intestinal tract stress injury can be achieved, and the diagnosis method comprises serum total RNA extraction, micromolecule RNA separation, cDNA synthesis, and PCR amplification and identification; and the guide is provided for the prevention, the diagnosis and the treatment of the weaned piglet intestinal tract stress injury, and the new molecule marker is provided for the marker-assisted selection of the pig anti-stress strain.
Description
technical field [0001] The invention belongs to the technical field of livestock genetic engineering, and in particular 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 worldwide, 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 main target of weaning stress is the piglet's gastrointestinal tract, where damage to the small intestine is particularly severe. Weaning stress-induced tissue damage and remodeling of the piglet small intestine seriously affects the development and maturation of the piglet gut, thereby restricting piglet health and growth. Similarly, intestinal damage caused by weaning stress, resulting in a decline in the synthesis and absorption of gastrointestinal enzymes, is considered t...
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