Method for identifying five species of Trichosporon spp. on basis of high-resolution melting curve

A high-resolution melting and melting curve technology, applied in the field of identification of five species of Trichospora based on high-resolution melting curves, can solve the problem of high PCR amplification fragment yield and purity requirements, and the impact of identification accuracy between Trichosporium species. , the problem of high cost, to avoid cross-contamination, reduce the cost of experimental detection, low cost

Active Publication Date: 2019-09-27
WUHAN UNIV OF SCI & TECH
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  • Abstract
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Problems solved by technology

However, this method has high requirements on the yield and purity of PCR amplified fragments, and needs to be completed with the help of a special gene sequencing analysis system, which takes a long time and costs a lot
In addition, there is also PCR-RFLP technology based on rDNA-ITS fragments, although it can be quickly and accurately identified, but due to the small inter-species differences of Trichosporon rDNA-ITS fragments, the accuracy of inter-species identification of Trichosporum is also limited. greater impact

Method used

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  • Method for identifying five species of Trichosporon spp. on basis of high-resolution melting curve
  • Method for identifying five species of Trichosporon spp. on basis of high-resolution melting curve
  • Method for identifying five species of Trichosporon spp. on basis of high-resolution melting curve

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Embodiment 1

[0045] [Example 1] The method for identifying five kinds of Trichosporum based on high-resolution melting curves

[0046] (1) The method steps for discriminating five kinds of Trichosporum based on high-resolution melting curves are as follows:

[0047] 1. After careful study of Trichosporon asahii (T.asahii), Trichosporon dermatosa (T.inkin), Trichosporon dermatis (T.dermatis), Trichosporon montevideense and Trichosporon dohaense Genome sequence, look for differential target sequences in ITS sequences, and design a pair of specific primers through careful design and screening. The design of the primers cannot be automatically obtained by computer software, and the target sequence fragment and length need to be selected based on rich experience. The primer Tm value was calculated according to the following formula while considering the GC content of the sequence, The target sequence fragment and length were selected to synthesize primers, and a pair of optimal primers capabl...

Embodiment 2

[0080] [Example 2] Application of the method for identifying five kinds of Trichosporum based on high-resolution melting curves

[0081] 1. Select 60 samples of patients with superficial fungal infection from 5 county and city hospitals in Hainan Province, use Potato Dextrose Agar (PDA) containing 0.5% chloramphenicol, isolate and culture at a constant temperature of 28°C as fungal samples to be tested .

[0082] 2. Extract the genomic DNA of the fungal samples to be tested after the above isolation and culture, and use the method of identifying five kinds of Trichosporum based on high-resolution melting curves to identify the samples to be tested, and use QuantStudio TM Real-Time PCRSoftware software was used for result analysis, and by comparing with the standard curve of Example 1, the identification result was determined.

[0083] 3. Identification results: There are 12 positive results, the sample numbers and names of the identification results are shown in Table 3 below...

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Abstract

The invention provides a method for identifying five species of Trichosporon spp. on the basis of a high-resolution melting curve. The five species of Trichosporon spp. comprise Trichosporon asahii, Trichosporon ink in, Trichosporon dermatis, Trichosporon montevideense and Trichosporon dohaense. According to the method, a specific primer pair capable of identifying the five species of Trichosporon spp. simultaneously is utilized, to-be-detected sample DNA is utilized as a template, a high-resolution melting curve reaction system is established, a high-resolution melting curve reaction program is adopted, fluorescence intensity is continuously detected, and a melting curve chart with first-order negative derivatives of fluorescence signals to temperatures as longitudinal coordinates and temperatures as horizontal coordinates is obtained. According to the method, follow-up PCR product electrophoresis detection and sequencing are not needed, the operating process is simplified, the detection efficiency is improved, and sensitivity and specificity are higher.

Description

technical field [0001] The present invention relates to the application of the high-resolution melting curve analysis method, and specifically refers to a method for discriminating five kinds of Trichosporons based on the high-resolution melting curve, and the five kinds of Trichosporons are respectively Trichosporonasahii ), Trichosporon inkin, Trichosporon dermatis, Trichosporon montevideense and Trichosporon dohaense. Background technique [0002] Trichosporon spp., also known as Trichosporon spp., belongs to the kingdom of fungi, Basidiomycota, widely distributed in nature, mainly in tropical and temperate regions, can be found in soil, water and animal feces, and can also live in humans In the human skin, respiratory tract and gastrointestinal tract, it is a common colonization bacteria of skin and mucous membranes. Trichosporum is a kind of opportunistic pathogenic fungus. When the human immune system is functioning normally, it usually does not cause disease. Once th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/04C12Q1/6851C12R1/645
CPCC12Q1/6895C12Q1/6851C12Q2600/16C12Q2527/107C12Q2563/107C12Q2537/143C12Q2561/113C12Q2531/113
Inventor 张玲柳赟昊徐娟李瑞雯付国庆全超曹晓璐
Owner WUHAN UNIV OF SCI & TECH
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