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Development of a loop-mediated isothermal amplification assay for rapid detection of Trichosporon asahii in
Jianfeng Zhou1, Yong Liao2, Haitao Li2
1Department of Dermatology, General Hospital of Beijing Military Command of PLA, No.5, Nanmencang, Dongcheng District, Beijing 100010, China ; The Clinical Medical College in the Beijing Military Region of Second Military Medical University of PLA, Beijing 100700, China.
Abstract:
Invasive trichosporonosis is a deep mycosis found mainly in immunocompromised hosts, and the major pathogen is Trichosporon asahii. We detected the species-specific intergenic spacers (IGS) of rRNA gene of T. asahii using a loop-mediated isothermal amplification (LAMP) assay in 15 isolates with 3 different visualization methods, including SYBR green detection, gel electrophoresis, and turbidimetric methods. The LAMP assay displayed superior rapidity to other traditional methods in the detection time; that is, only 1 h was needed for detection and identification of the pathogen DNA. Furthermore, the detection limit of the LAMP assay was more sensitive than the PCR assay. We also successfully detect the presence of T. asahii in samples from experimentally infected mice and samples from patients with invasive trichosporonosis caused by T. asahii, suggesting that this method may become useful in clinical applications in the near future.
Insights
A new loop-mediated isothermal amplification (LAMP) assay rapidly detects Trichosporon asahii, the main cause of invasive trichosporonosis. This sensitive method identifies the fungal pathogen DNA in just one hour, showing promise for clinical use.
Area of Science:
- Mycology
- Medical Microbiology
- Molecular Diagnostics
Background:
- Invasive trichosporonosis is a serious fungal infection primarily affecting immunocompromised individuals.
- Trichosporon asahii is identified as the predominant pathogen responsible for invasive trichosporonosis.
- Accurate and rapid diagnostic methods are crucial for managing this deep mycosis.
Purpose of the Study:
- To develop and evaluate a species-specific loop-mediated isothermal amplification (LAMP) assay for detecting Trichosporon asahii.
- To compare the performance of the LAMP assay with traditional methods and PCR for pathogen identification.
- To assess the potential clinical applicability of the LAMP assay in diagnosing invasive trichosporonosis.
Main Methods:
- A loop-mediated isothermal amplification (LAMP) assay was designed to target species-specific intergenic spacers (IGS) of the rRNA gene of Trichosporon asahii.
- The assay was tested on 15 isolates of T. asahii using three visualization techniques: SYBR green, gel electrophoresis, and turbidimetry.
- Sensitivity was compared against the polymerase chain reaction (PCR) assay, and the method was validated using experimentally infected mouse samples and clinical patient samples.
Main Results:
- The LAMP assay achieved species-specific detection of T. asahii DNA within one hour, demonstrating superior rapidity compared to conventional methods.
- The detection limit of the LAMP assay was found to be more sensitive than that of the PCR assay.
- Successful detection of T. asahii was confirmed in both experimentally infected mice and clinical samples from patients with invasive trichosporonosis.
Conclusions:
- The developed LAMP assay offers a rapid, sensitive, and potentially valuable tool for the identification of Trichosporon asahii.
- This molecular diagnostic method shows significant promise for timely clinical application in the diagnosis of invasive trichosporonosis.
- The assay's efficiency and sensitivity suggest it could improve patient management and outcomes for invasive fungal infections.
