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Composition, kit and method for detecting and typing bovine diarrhea pathogens and application of composition, kit and method

A composition and pathogen technology, applied in the field of molecular biology detection, can solve the problems of economic losses in cattle raising, affecting the growth and development of calves and the production performance of adult cattle, etc.

Inactive Publication Date: 2022-07-22
湖南大圣宠医生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The occurrence of this disease seriously affects the growth and development of calves and the production performance of adult cattle, causing huge economic losses to the cattle industry

Method used

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  • Composition, kit and method for detecting and typing bovine diarrhea pathogens and application of composition, kit and method
  • Composition, kit and method for detecting and typing bovine diarrhea pathogens and application of composition, kit and method
  • Composition, kit and method for detecting and typing bovine diarrhea pathogens and application of composition, kit and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1. Primers and probes used in the present invention

[0064] Table 2

[0065]

[0066] Among them, the fluorescent reporter group of Salmonella is FAM; the fluorescent reporter group of Clostridium welchii is HEX; the fluorescent reporter group of Escherichia coli K99 is ROX; the fluorescent reporter group of Shigella dysenteriae is CY5, and the fluorescent reporter group of the probe is CY5. The 3' end also has a BHQ and MGB quenching group.

[0067] The fluorescent reporter group of the upstream primer of Campylobacter jejuni is FAM; the fluorescent reporter group of the upstream primer of the internal standard is HEX; the fluorescent reporter group of the upstream primer of Proteus mirabilis is ROX, and the fluorescent reporter group of the fluorescent primer group is marked in the primer. on one of the T bases.

Embodiment 2

[0068] Embodiment 2, detect the method for bovine diarrhea pathogenic pathogen

[0069] The test samples of the present invention are feces and other samples. DNA nucleic acid is extracted by the company's magnetic bead method, and nucleic acid extraction is performed in the sample processing room as follows:

[0070] The nucleic acid extractor is operated according to the set procedures. For details, please refer to the operating instructions of the nucleic acid extractor.

[0071] Instructions for Pre-aliquoted Extraction Reagents

[0072] According to the number of samples to be tested, take a number of pre-distilled solutions, and add 60 μL of sample to each tube;

[0073] Add 36 μL of pre-dispensing solution 2 to each tube, cover the tube, shake and mix for 30 s, and heat at 60 °C for 4 min;

[0074] Instantaneous centrifugation at low speed, place the centrifuge tube on a 4-hole magnetic separator, and slowly aspirate the waste liquid after 2~3 minutes (*be careful not...

Embodiment 3

[0090] Embodiment 3, the detection result of the composition test sample of the present invention

[0091] The primers and probes shown in Example 1 were used to detect various positive pathogens according to the method of Example 2, and the experimental results were as follows: Figures 1~7 shown. The results show that each channel can be detected normally, and the multiplex PCR system can detect the corresponding targets and type all pathogens.

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Abstract

The invention belongs to the field of molecular biological detection. Specifically, the invention relates to detection of bovine diarrhea pathogens. More specifically, the invention relates to a composition for detecting six bovine diarrhea pathogenic pathogens, namely salmonella, clostridium welchii, escherichia coli K99, shigella dysenteriae, campylobacter jejuni and proteus mirabilis. Meanwhile, the invention further provides a kit containing the composition, application of the composition and a method for detecting and typing bovine diarrhea pathogens. The composition is mainly detected in one tube by using multiple fluorescent PCR and melting curve analysis methods, so that different pathogens can be treated differently, and the treatment and prevention are more effective.

Description

technical field [0001] The invention belongs to the field of molecular biology detection; specifically, it relates to the detection of bovine diarrhea pathogens; Detection of pathogens causing diarrhea in breeding cattle. Background technique [0002] Bovine diarrhea is a symptom of low immunity in cattle, especially calves, caused by pathogenic microorganisms such as bacteria, viruses and parasites in the intestinal tract, nutritional factors, and environmental factors. The clinical manifestations are depression and loss of appetite. Eating, high fever, abdominal distension, abdominal pain, gastroenteritis, diarrhea, foul-smelling stool, darkening, sometimes mixed with blood bubbles, dehydration and acidosis. The etiology of diarrhea in cattle, especially in calves, is complicated. Biological factors, management factors, environmental factors, and nutritional factors can all lead to the occurrence of this disease. Among them, pathogenic microorganism infection is an impor...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/689C12Q1/686C12N15/11C12R1/42C12R1/145C12R1/19C12R1/01
CPCC12Q1/689C12Q1/686C12Q2600/16C12Q2561/113C12Q2563/107C12Q2545/114C12Q2537/143C12Q2527/107
Inventor 毛君杰范文洲王洁汤睿邱先锋罗莎戴立忠
Owner 湖南大圣宠医生物科技有限公司
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