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Assay for the diagnosis of dermatophytosis

a technology for dermatophytosis and assays, applied in the field of assays for the diagnosis of dermatophytosis, can solve the problems of long-term and often expensive systemic treatment, false negative results, and low sensitivity of techniques, and achieves the problem of not allowing the rapid and reliable distinction of closely related strains from the genus i>trichophyton

Pending Publication Date: 2021-08-19
EUROIMMUN MEDIZINISCHE LABORDIAGNOSTIKA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention solves the problem of providing a reliable and accurate method for detecting a pathogen associated with a skin, hair, and nail infection in a sample, such as a nail or skin material, using a nucleic acid amplification process. The invention involves a primer pair, a nucleic acid, and a carrier that can be used to detect the pathogen in a sample and identify it as a specific genus or species. The invention can be used in a diagnostic assay or a kit for the diagnosis of a disease associated with a pathogen. The technical effect of the invention is to provide a reliable and accurate method for detecting a pathogen associated with a skin, hair, and nail infection.

Problems solved by technology

Topical therapy is sufficient in most cases, but long term and often expensive systemic treatment is necessary if the infection is caused by specific strains of the genera Microsporum and Trichophyton such as T. verrucosum and T. mentagrophyles.
Although rapid and cheap, this technique has a relatively low sensitivity and shows false negative results in up to 15% of all cases.
In particular, they do not allow the rapid and reliable distinction of closely related strains from the genus Trichophyton and Microsporum, which includes zoophilic and non-zoophilic species.

Method used

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  • Assay for the diagnosis of dermatophytosis
  • Assay for the diagnosis of dermatophytosis
  • Assay for the diagnosis of dermatophytosis

Examples

Experimental program
Comparison scheme
Effect test

example 1

Design of Primers and Probes

[0083]For the identification of T. tonsurans in a sample, the metalloprotease gene was chosen as a useful target region. Based on the comparison of oligonucleotide sequences, a set of primers (forward primer 5′ cy3-GGGAGGGAGACTAGTTG 3′ (SEQ ID NO: 10), reverse primer 5′ cy3-AATTTTTCGCCGCCAAG 3′(SEQ ID NO: 12)) and a species-specific probe (5′ C6-Amino-linker-GATCCTTGGCGTACGGATGCATA 3′(SEQ ID NO: 14)) for the detection of Trichophyton tonsurans were designed. The designed probes were checked for internal repeats, secondary structure, melting temperature and GC content.

DNA Microarray

[0084]The designed probe for T. tonsurans and some controls were spotted with the sciFLEXARRAYER S11 (Scienion AG, Germany) on a solid carrier material as microscopically small spots located at defined positions.

DNA Extraction from Dermatophyte Cultures

[0085]Cultures were performed on dermatophyte test medium agar (SIFIN, Germany, Tenn. 2102). The DNA from cultured T. tonsurans ...

example 2

Material and Methods

[0091]DNA microarrays consist of DNA molecules (probes) that differ from one another by their DNA sequence. When the DNA of an organism contains segments that match to these defined probes at the microarray, the complementary DNA regions bind together (hybridize). Due to fluorescence labeled primers that are used in the polymerase chain reaction (PCR), a positive hybridization between probe and amplified target sequence can be detected via microarray scanner. An evaluated positive signal means that the target sequence could be detected. In this example the detection of the dermatophyte Trichophyton benhamiae (yellow) and Trichophyton concentricum via DNA microarray will be shown. For the verification of probe specificity the most closely related species Trichophyton benhamiae (white) and Trichophyton benhamiae (african) were also included in the analysis. The used method based on the EUROIMMUN DNA microarray platform.

Design of Primers and Probes

[0092]For the iden...

example 3

Material and Methods

[0098]DNA microarrays consist of DNA molecules (probes) that differ from one another by their DNA sequence. When the DNA of an organism contains segments that match to these defined probes at the microarray, the complementary DNA regions bind together (hybridize). Due to fluorescence labeled primers that are used in the polymerase chain reaction (PCR), a positive hybridization between probe and amplified target sequence can be detected via microarray scanner. An evaluated positive signal means that the target sequence could be detected. In this example the detection of the dermatophyte Trichophyton benhamiae (yellow) and Trichophyton benhamiae (white / african) via DNA microarray will be shown. The used method based on the EUROIMMUN DNA microarray platform.

Design of Primers and Probes

[0099]For the identification of Trichophyton benhamiae (yellow) or Trichophyton benhamiae (white / african) in a sample the metalloprotease gene was chosen as a useful target region. Bas...

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Abstract

A primer pair that includes a forward primer and a reverse primer is used to amplify a nucleic acid from a pathogen associated with a skin, hair and nail infection that includes SEQ ID NO: 22. A nucleic acid capable of hybridizing specifically to a nucleic acid sequence from a pathogen associated with a skin, hair and nail infection that includes SEQ ID NO: 22 is provided. A carrier that includes the nucleic acid is provided. A method can be used to detect in a sample a nucleic acid sequence including SEQ ID NO: 22 from a pathogen associated with a skin, hair and nail infection. The primer pair, the nucleic acid, or the carrier may be useful for the diagnosis of a disease. A kit including the primer pair, the nucleic acid, and / or the carrier may be useful for the diagnosis of a disease.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application is a continuation of U.S. application Ser. No. 15 / 938,890, filed on Mar. 28, 2018, which claims the benefit of European Application No. 17000524.3, filed on Mar. 30, 2017. The content of each of these applications is hereby incorporated by reference in its entirety.REFERENCE TO A SEQUENCE LISTING[0002]The present application is accompanied by an ASCII text file as a computer readable form containing the sequence listing entitled, “000911USCONT01_SL_ST25.txt”, created on Mar. 18, 2021, with a file size of 17,190 bytes, the content of which is hereby incorporated by reference in its entirety.BACKGROUND OF THE INVENTIONField of the Invention[0003]The present invention relates to a primer pair comprising a forward primer and a reverse primer for amplifying a nucleic acid from a pathogen associated with a skin, hair and nail infection 20 comprising SEQ ID NO: 22, a nucleic acid capable of hybridizing specifically to a nucleic ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6895C12Q1/04C12Q1/6851C12N15/11
CPCC12Q1/6895C12Q1/6851C12Q1/04C40B40/06
Inventor HARDER, MELANIEGRAESER, YVONNEKUPSCH, CHRISTIANECAVALAR, MARKUS
Owner EUROIMMUN MEDIZINISCHE LABORDIAGNOSTIKA
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