Method for detecting endotoxin poisoning of livestock by mutation of host liver mitochondrial gene

A mitochondrial gene and endotoxin technology, applied in biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of time-consuming and laborious, high detection cost, inaccurate detection results of different types of livestock, etc., to achieve high detection cost, Good reproducibility, the effect of reducing the risk of death from endotoxin poisoning in livestock

Inactive Publication Date: 2014-08-20
YUNNAN AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Claims
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Problems solved by technology

The above two methods are time-consuming and labor-intensive, the detection cost i

Method used

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  • Method for detecting endotoxin poisoning of livestock by mutation of host liver mitochondrial gene
  • Method for detecting endotoxin poisoning of livestock by mutation of host liver mitochondrial gene
  • Method for detecting endotoxin poisoning of livestock by mutation of host liver mitochondrial gene

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Embodiment 1

[0043] Step 1. Prepare the reagents in the mtDNA extraction step, the formula is as follows:

[0044] (1) SE solution (pH: 7.8): 0.25M sucrose, 2.5mM CaCl2, 30mM Tris-HCl, 10mM EDTANa2;

[0045] (2) Solution Ⅰ (TEN, pH8.0): 0.15M NaCl, 10mM EDTANa2, 10mM Tris-HCl;

[0046] (3) Solution Ⅱ: 1% SDS, containing 0.2N NaOH, ready to use, mix well, and use immediately;

[0047] (4) Solution Ⅲ: 3M Kac, which contains 3M KAc and 5M glacial acetic acid, mix well, autoclave, store at room temperature for later use.

[0048] (5) TE solution (pH: 8.0): 10mM Tris-HCl, 0.1mM EDTANa2, 20ug / ml DNase-free RNase.

[0049] Step 2. Liver mtDNA extraction:

[0050] (1) Take 0.5g ~ 1.0g of liver tissue as a sample, wash away blood stains in ice-precooled SE solution, then transfer to 6ml ice-precooled SE solution, cut into pieces, then transfer to a 10ml centrifuge tube, and use a high-speed disperser at 1500rpm Homogenize 3 times with an interval of 20 sec each time, and homogenize for 10 sec e...

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Abstract

The invention relates to a method for detecting endotoxin poisoning of livestock by mutation of host liver mitochondrial gene, which saves time and effort and has credible results. According to the method, host liver mitochondrial DNA (deoxyribonucleic acid) is extracted, a gene specific primer of mitochondria cytochrome C oxidase (COI) is designed, and a liver COI gene segment is analyzed and sequenced by a PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) technology; and if a sample PCR stripe of a livestock poisoned by endotoxin swims abnormally, and the sequenced COI gene segment is subjected to T-G replacement point mutation simultaneously at 5322 and 5331, the host body is poisoned by the endotoxin and has toxic effect. The method has good repeatability, specificity and sensibility, and has good application value.

Description

technical field [0001] The invention relates to a method for detecting endotoxin poisoning in livestock, in particular to a method for detecting endotoxin poisoning in livestock by utilizing point mutations in the mitochondrial COI gene of the host liver. Background technique [0002] Bacterial infection and abuse of antibiotics in the process of large-scale raising of livestock can easily lead to endotoxin poisoning in livestock, and the detection of endotoxin poisoning is an important means of diagnosing livestock diseases. The traditional method is to separate and prepare the toxin, give it to mice, and judge it by clinical symptoms, or collect blood, and use the expensive limulus test for diagnosis. The above two methods are time-consuming and labor-intensive, the detection cost is high, and the detection results are inaccurate for different types of livestock. Contents of the invention [0003] The purpose of the present invention is to provide an accurate, rapid, si...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6858C12Q1/6869C12Q2531/113C12Q2565/131
Inventor 高洪严玉霖彭辂周铭涛陈利平陈玲赵汝
Owner YUNNAN AGRICULTURAL UNIVERSITY
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