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Method for identifying Epidermophyton floccosum of rabbit

A technology of Epidermophyton floccosum and rabbits, which is applied in the field of molecular biology and can solve the problems of high cost, difficulty in popularization and application, and high requirements for operators.

Inactive Publication Date: 2011-07-20
林毅 +7
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the diagnosis of rabbit dermatophytosis, there are ITS based on the ribosomal RNA gene region of pathogenic bacteria, primers are designed, PCR technology and sequencing analysis technology are used to identify and diagnose, and random primers are used to amplify the genomic DNA of skin fungi. However, these methods are costly in clinical practice, and the actual operation and analysis methods have high requirements for operators, so it is difficult to popularize and apply them clinically.

Method used

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  • Method for identifying Epidermophyton floccosum of rabbit
  • Method for identifying Epidermophyton floccosum of rabbit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Add 0.05-0.1g of ground mycelium sample to 1mL urea extract (7mol / L Urea, 50mmol / L Tris-HCl pH 8.0, 62.5mmol / LNaCl, 1% SDS), shake well, centrifuge at 12000r / min for 5min, draw The supernatant was centrifuged again at 12000r / min for 5min; the supernatant was transferred to another new tube, and an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) solution was added, and the mixture was shaken vigorously several times. Mix well, centrifuge at 12000r / min for 5min; take the supernatant into a new tube, add an equal volume of isopropanol and 1 / 10 volume of 3mol / L NaAc (pH 5.2), place at -20°C for 30min, centrifuge at 12000r / min for 15min; discard Invert the supernatant to drain the liquid from the tube wall, wash the precipitate with 70% absolute ethanol, let it dry at room temperature for 30 minutes, dissolve it in 50ul double-distilled water, and store it at -20°C for later use.

[0023] Specific primer pair: artificially synthesized forward primer sequence SE...

Embodiment 2

[0031] Inoculate the mycelium of the above-mentioned bacterial strains on fungal selective agar medium (soytone 1%, glucose 4%, agar 1.8%, chloramphenicol 0.005%, cycloheximide 0.05%) in 150ml fungal selective liquid culture base, cultivated at 28°C for 10-15 days, picked mycelium in a mortar, ground it with liquid nitrogen, put 0.05-0.1 g of ground mycelium in a 1.5ml Eppendorf tube, and extracted genomic DNA. The specific operation is as follows:

[0032] 0.05-0.1g ground mycelium sample (100mmol / L Tris-HCl pH9.0, 40mmol / L EDTA pH8.0), shake and mix well, add / add 100μL 10% SDS, 300μL benzyl chloride, shake vigorously, Make the mixture in the tube milky, keep warm at 50°C for 1 hour, oscillate and mix once every few minutes, cool to room temperature, add 300 μL pre-cooled 3mol / L NaAc (pH5.2) solution, mix well, then ice bath for 15 minutes, 11000r / Centrifuge for 15 min; take the supernatant, add an equal volume of chloroform:isoamyl alcohol (24:1), mix well, and centrifuge a...

Embodiment 3

[0042] Trichophyton mentagrophytes standard bacterial strains are inoculated in 150ml mycelia of fungal selective agar medium (soytone 1%, glucose 4%, agar 1.8%, chloramphenicol 0.005%, cycloheximide 0.05%) Fungal selective liquid medium, cultivated at 28°C for 10-15 days, pick mycelia in a mortar, grind with liquid nitrogen, put 0.05-0.1g of ground mycelium in a 1.5ml Eppendorf tube, and use chlorination Genomic DNA was extracted by the benzyl method, and the clinical wool samples were extracted by the benzyl chloride method. The specific operations are as follows:

[0043] 0.05-0.1g ground mycelium sample, 0.01-0.1g rabbit skin hair sample (100mmol / L Tris-HCl pH9.0, 40mmol / L EDTA pH8.0), shake and mix, add 100μL 10% SDS, 300 μL of benzyl chloride, shake vigorously to make the mixture in the tube milky, incubate at 50°C for 1 hour, shake and mix once every few minutes, cool to room temperature, add 300 μL pre-cooled 3mol / L NaAc (pH5.2) solution, mix well , then ice-bathed fo...

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PUM

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Abstract

Epidermophyton floccosum is one of the most common dermatophytosis pathogenic bacteria, and the invention discloses specific primer sequences for identifying the Epidermophyton floccosum. The Epidermophyton floccosum can be specifically identified by the pair of primer sequences through polymerase chain reaction (PCR) amplification, the speed and reliability are high, a theoretical basis is laid for diagnosing dermatophytosis of rabbit and purifying the dermatophytosis in rabbit farms, and technical guidance is provided.

Description

technical field [0001] The invention relates to the field of molecular biology, and mainly relates to the use of primers in polymerase chain reaction tests to detect and identify Epidermophyton flocculus. These primers can be used to detect the dynamic changes of dermatophytosis often occurring in rabbit skin, and lay a theoretical foundation for the diagnosis and treatment of dermatophytosis in rabbits and the purification of dermatophytosis in rabbit farms, and provide technical guidance. Background technique [0002] Dermatomycosis of rabbits is a zoonotic infectious disease with high incidence, strong infectivity and difficult cure. In domestic animals, it mainly infects rabbits, dogs, and cats. It mainly infects the fur of rabbits, and symptoms such as dandruff, scab, hair loss, exudation, folliculitis, and itching appear, which not only affect the growth and development of rabbits, but also reduce the feed rate and resistance. force, and seriously affect the fur quali...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04C12N15/11
Inventor 林毅谢晶曹冶赵素君廖党金李江凌文豪李红
Owner 林毅
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