Bovine serum microRNA molecular marker of milk cow fatty liver disease and milk cow perinatal period-associated metabolic disease

A molecular marker, metabolic disease technology, applied in the field of molecular genetics, to achieve the effect of simple and rapid method, improve accuracy, and avoid economic losses

Active Publication Date: 2016-03-23
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the relationship between serum miRNA and fatty liver in cows

Method used

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  • Bovine serum microRNA molecular marker of milk cow fatty liver disease and milk cow perinatal period-associated metabolic disease
  • Bovine serum microRNA molecular marker of milk cow fatty liver disease and milk cow perinatal period-associated metabolic disease
  • Bovine serum microRNA molecular marker of milk cow fatty liver disease and milk cow perinatal period-associated metabolic disease

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1. Pre-diagnosis of fatty liver disease in dairy cows during the peripartum period and selection of extreme diseased individuals and normal individuals

[0050] 1. Sample Collection

[0051] The experimental animals came from farm A in Taian, Shandong and farm B in Qingdao, Shandong. A total of 131 Chinese Holstein cows were selected in the early lactation period (taking the delivery date as the first day, and the lactation days were 35.5±21.9 days). Free access to food and water. On the day of sample collection, 20ml of blood was collected before feeding in the morning. After standing at room temperature without anticoagulation for 1 hour, the serum was collected by centrifugation at 3000rpm at 4°C, and stored at -80°C after aliquoting.

[0052] 2. Reagents

[0053] Free fatty acid (NEFA) test kit (Nanjing Jiancheng Bioengineering Institute); aspartate aminotransferase IFCC method detection kit (Sichuan Mike Technology Co., Ltd.); blood glucose (GOD-PAP) met...

Embodiment 2

[0066] Example 2. Differential expression of cow serum miRNA in the control group and the test group

[0067] 1. Serum miRNA extraction

[0068] Take 400 μl of the selected dairy cow serum, add 400 μl of lysate MZ, oscillate and mix for 30 sec, place at room temperature for 5 min, add internal reference cel-miR-39-3p, and extract miRNA according to the instructions of the miRcutemiRNA extraction and isolation kit. The extracted miRNA was dissolved in 15 μl RNase-FreeddH 2 O. Take 1μl sample and measure OD 260 / OD 280 and concentration, OD 260 / OD 280 Should be between 1.8-2.0. Store at -80°C for later use.

[0069] 2. miRNA reverse transcription

[0070] cDNA was synthesized by reverse transcription of miRNA using TaKaRa's PrimeScript TM miRNART-PCRKit, operated according to the instruction manual. Reverse transcription system (20μL): 2×miRNAReactionBufferMix10μl, 0.1%BSA2μl, miRNAPrimeScriptRTEnzymeMix2μl, miRNA (1μg) and RNaseFreeddH 2 O total 6μl; reaction cond...

Embodiment 3

[0078] Example 3 Accuracy detection of miR-16-5p and miR-29c-3 as molecular markers for fatty liver diagnosis

[0079] In order to test the accuracy of the diagnosis, the ROCcurves command in the MedCalc11.4.2.0 software was used for analysis, and the ROC (receiver-operating characteristic, ROC) curve was constructed to judge the deviation between the true positive rate and the false positive rate. The specific method is: in the first line of the Date window, name the variable names as diagnosis and test. In diagnosis, fatty liver is 1, non-fatty liver is 0, and test is the corresponding 2. -△△Ct Value, using the Statistics / ROCcurves / ROCcurveanalyze process, with test as a variable and diagnosis as a classification variable, for ROC curve analysis. The area under the ROC curve (AUC) was used to evaluate the sensitivity and specificity of miR-16-5p and miR-29c-3 as molecular markers in the diagnosis of fatty liver in cows. The significance of the ROC curve was tested by the t-...

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Abstract

The invention relates to molecular genetics, and provides a bovine serum microRNA molecular marker of a milk cow fatty liver disease and a milk cow perinatal period-associated metabolic disease. The molecular marker is used for early detection and diagnosis of a diseased cow in production practice of the milk cow, thus greatly improving diagnosis accuracy and greatly avoiding economic loss to the great extent. The marker can be used for variety breeding and idioplasm improvement of the milk cow.

Description

technical field [0001] The invention relates to molecular genetics, and provides bovine serum microRNA molecular markers for dairy cow fatty liver disease and dairy cow perinatal-related metabolic diseases. Background technique [0002] Fatty liver refers to the pathological changes in which the fat content in the liver exceeds 5% (accounting for the wet weight of the liver) due to excessive accumulation of fat in the liver cells due to various reasons. Fatty liver is a major metabolic disease in peripartum dairy cows, and its incidence varies from place to place in the world, ranging from 30% to 70%. The incidence of fatty liver in dairy cows in my country is more than 30%, and in the United States it is 35.0% to 66.0%. Mortality can be as high as 25%. Fatty liver is a major metabolic disease in transition dairy cows, especially in high producing cows. The health and reproductive performance of dairy cows suffering from fatty liver disease will be seriously affected, and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6888C12Q2600/124C12Q2600/158C12Q2600/178
Inventor 王中华师科荣王玉倩林雪彦侯秋玲胡志勇王云
Owner SHANDONG AGRICULTURAL UNIVERSITY
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