Method for detecting rabbit dermatophyte and trichophyton mentagrophytes through dual quantitative PCR detection

A technology of Trichophyton mentagrophytes and dermatophytes, applied in the field of detection of Trichophyton mentagrophytes, can solve the problems of only qualitative judgment, unable to quantify, low accuracy, etc., avoiding false positive results, time-consuming Short, cost-saving effect

Inactive Publication Date: 2015-09-02
HEBEI UNIV OF ENG
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Problems solved by technology

The common characteristics of these methods are specificity and sensitivity. Because conventional PCR needs electrophoresis analysis, it is necessary to determine whether there is amplification and the approximate length of the amplification product, so it takes a long time, the accuracy is low, non-specific amplification is prone to occur, and it can only be qualitative. judgment, not quantifiable

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  • Method for detecting rabbit dermatophyte and trichophyton mentagrophytes through dual quantitative PCR detection
  • Method for detecting rabbit dermatophyte and trichophyton mentagrophytes through dual quantitative PCR detection
  • Method for detecting rabbit dermatophyte and trichophyton mentagrophytes through dual quantitative PCR detection

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Embodiment Construction

[0031] 1. Primer synthesis:

[0032] a. Specific primers for skin fungi:

[0033] Upstream primer Dm1: 5'-CTGCGGAAGGATCATTAACCCTGGA-3',

[0034] Downstream primer Dm25'-AAGAGATCCGTCCTGGATGTTGAAAG-3';

[0035] b. Specific primers for Trichophyton mentagrophytes:

[0036] Upstream primer F: 5'-GCAAA GAAG CCTGG AAGAAG-3',

[0037] Downstream primer R: 5'-GGAGACCATCTGTGAGAGTTG-3;

[0038] 2. Genomic DNA extraction:

[0039] Inoculate Trichophyton mentagrophytes, Microsporum canis, Epidermophyton flocculus, Trichophyton rubrum, Candida albicans, and Aspergillus fumigatus in potato dextrose liquid culture medium, culture on a shaker at 27°C for 72 hours, and wait until spherical mycelia appear , filter the mycelium with eight layers of gauze, wash it twice with PBS, squeeze out the water of the mycelium, put the collected mycelium into a mortar, add an appropriate amount of liquid nitrogen, and grind the mycelium quickly when the liquid nitrogen is about to evaporate completely. ...

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Abstract

The invention provides a method for detecting rabbit dermatophyte and trichophyton mentagrophytes through dual quantitative PCR detection. The method comprises the steps that 1 primer is compounded; 2 genomic DNA is extracted; 3 the SYBR Green I fluorogenic quantitative PCR is conducted; 4 a SYBR Green I fluorogenic quantitative PCR melting curve is drawn; 5 result determination is conducted. The method for detecting rabbit dermatophyte and trichophyton mentagrophytes through dual quantitative PCR detection has the advantages that detecting on amplified product can be conducted in a close state, the post processing of a common PCR is leaved out, a false positive result caused by the pollution of the amplified product is avoided, the sensitivity is high, accurate quantification on beginning target gene of the reaction can be achieved further, the liner relation is good, and only 2 hours are needed to make a regular laboratory examination and send out a report.

Description

technical field [0001] The invention relates to a method for detecting Trichophyton mentagrophytes in rabbits, in particular to a method for detecting rabbit skin fungi and Trichophyton mentagrophytes by double quantitative PCR. Background technique [0002] Rabbit dermatophytosis is a highly contagious zoonotic contact skin disease caused by filamentous fungi invading the cuticle and its appendages. With the intensive development of the rabbit industry, the occurrence of rabbit skin mycosis is becoming more and more common. Dermatomycosis in rabbits not only affects the fur of rabbits, but also seriously affects their health, fur quality and economic benefits. The more important thing is that the disease can be transmitted to humans, which has a greater negative impact on public health. Therefore, the disease has been increasingly paid attention to by the medical and veterinary circles at home and abroad. [0003] Dermatophytes mainly involve Trichophyton (such as Trichop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/04
CPCC12Q1/6851C12Q2531/113C12Q2537/143C12Q2563/107
Inventor 刘彦威
Owner HEBEI UNIV OF ENG
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