Method for identifying five dermatophytes by utilizing high-resolution melting curve

A high-resolution melting, dermatophyte technology, applied in microorganism-based methods, biochemical equipment and methods, and microbial determination/inspection, etc. It can reduce the cost of experimental detection, low cost and simple operation.

Pending Publication Date: 2019-07-26
JINHUA MUNICIPAL CENT HOSPITAL
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

Therefore, by using primer pairs to amplify the CHS gene and then performing sequencing, and performing basic local alignment in NCBI, strain identification can be carried out, but this method is difficult to accurately identify all strains, and there is no amplification phenomenon
In addition, commonly used techniques include polymerase chain reaction-restriction fragment length polymorphism analysis (PCR-RFLP), that is, after PCR amplification of a certain gene fragment, the PCR product is amplified with an appropriate restriction endonuclease. Carry out enzyme digestion, thereby carry out strain identification according to the length polymorphism of enzyme digestion product, but the experimental process of this method is complicated, is not conducive to popularization; Or adopt random amplified polymorphic DNA analysis (RAPD), although has required Small amount of DNA, high sensitivity, simple and rapid operation, convenient large-scale use and low cost, etc., but its repeatability and specificity are often restricted by reaction conditions, and changes in reaction conditions often affect the accuracy of detection results

Method used

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  • Method for identifying five dermatophytes by utilizing high-resolution melting curve
  • Method for identifying five dermatophytes by utilizing high-resolution melting curve
  • Method for identifying five dermatophytes by utilizing high-resolution melting curve

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1 Differentiate the establishment of five kinds of dermatophyte methods and specificity, sensitivity verification

[0042] The present inventors have carefully studied Trichophyton rubrum (T. rubrum), Trichophyton interdigitale (T. interdigitale), Epidermophyton floccosum (E. floccosum), Microsporum canis (M. canis) and Microsporum incurvatum. The genome sequences of five dermatophytes were searched for differential target sequences in the CHS sequence. Through careful design and screening, a pair of specific primers were designed. The nucleotide sequence is shown in SEQ ID NO.1-2. Using this The primers can specifically identify the above five dermatophytes by HRM technique. The design of this primer cannot be automatically obtained by computer software. It is necessary to select the target sequence fragment and length based on rich experience, and calculate the Tm value according to the following formula while considering the GC content of the sequence. Af...

Embodiment 2

[0060] Embodiment 2 Application of the inventive method

[0061] 1. Select 100 samples of patients with dermatophytosis who went to see a doctor in the dermatology department of hospitals in 10 different provinces and cities in the country, and use potato dextrose agar base (PDA) containing 0.5% chloramphenicol to separate under constant temperature conditions of 28°C Culture, and identify culture-positive specimens.

[0062] 2. The genomic DNA of the fungal sample to be tested was extracted by the rough extraction method, and the subsequent identification method was carried out with reference to the operation method of Example 1. ABI software was used to analyze the results, and the CHS differences of different dermatophytes were displayed based on the curve offset and curve shape changes through the standard curve. The identification results are shown in Table 2 below.

[0063] Table 2

[0064]

[0065]

[0066] For the samples for which the corresponding dermatophy...

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Abstract

The invention provides a method for identifying five dermatophytes by utilizing a high-resolution melting curve and belongs to the technical field of high-resolution melting curve analysis. The methodadopts a pair of specific primers and is based on real-time fluorescence PCR melting curve to identify trichophyton rubrum, trichophyton digitorum, epidermophyton floccosum, microsporum caninum and Microsporum incurvatum. The sequence of the pair of specific primers is as shown as SEQ ID NO.1-2. The method has the advantages of high sensitivity, good specificity and high detection speed, can be used for identifying dermatophytes on the aspects of clinical diagnosis, environmental monitoring, food safety and the like, and provides a reliable basis for infection treatment, environmental sanitation and food safety monitoring of dermatophytes.

Description

technical field [0001] The present invention relates to the application of high-resolution melting curve analysis method, in particular to a method for identifying five kinds of dermatophytes using high-resolution melting curves, the five kinds of dermatophytes are respectively Trichophyton rubrum (T.rubrum), Trichophyton interdigitale (T. interdigitale), Epidermophyton floccosum (E. floccosum), Microsporum canis (M. canis) and Microsporum incurvatum. Background technique [0002] Superficial mycosis refers to the disease caused by pathogenic fungi or opportunistic fungi infecting human skin, superficial tissues such as (toe) nails or hair, and is a common and frequently-occurring disease in dermatology clinics. According to reports, the prevalence of the disease in the population can be as high as 20%-25%, and the incidence rate is still increasing year by year. Dermatophytes are the most common pathogenic bacteria in superficial fungal infections. They are a class of kera...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11C12R1/645
CPCC12Q1/6895C12Q1/686C12Q2561/113C12Q2527/107
Inventor 单小云徐娟蔡淑倩周梦诗赵飞龚杰
Owner JINHUA MUNICIPAL CENT HOSPITAL
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