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Development of reverse transcription loop-mediated isothermal amplification-based assay for rapid and specific
Ankush Kaushik1, Zeeshan Fatima1, Saif Hameed1
1Amity Institute of Biotechnology, Amity University Haryana, Gurugram (Manesar)-122413, Haryana, India.
Background And Purpose:
Due to the ability of Candida auris, a multidrug-resistant human fungal pathogen, to colonize the skin and hospital surfaces, it is pertinent to control its nosocomial outbreaks through rapid diagnosis. Delayed and improper diagnosis of C. auris due to misidentification becomes a major hurdle in the prevention of employment of efficient therapeutics leading to the development of drug resistance. The culture-based methods are slow and less sensitive while PCR-based methods are costly. Loop-mediated amplification (LAMP) is a feasible alternative, but it fails to differentiate between live and dead cells. Therefore, this study aimed to evaluate the diagnostic efficiency of the reverse transcription (RT) LAMP approach and compare it with that of the LAMP assay for the detection of C. auris.
Materials And Methods:
RT-LAMP method was developed for the detection of C. auris and its clinical isolates. The limit of detection (LOD), sensitivity, and specificity were evaluated for the developed method using culture RNA. The RT-LAMP reaction for C. auris detection was standardized using the primers of a specific 869-bp DNA segment (accession no. XM_018317007), encoding a pyruvate: ferredoxin oxidoreductase domain, from the genome of C. auris.
Results:
The LOD for the RT-LAMP method was 1ag contrary to 10fg for LAMP method using DNA. Specificity was 100% as determined using a gram-negative bacteria and several other Candida species. The RT-LAMP method was intraspecific and displayed no cross reaction even with closely related Candida species. The RT-LAMP method was validated on 10 clinical isolates of C. auris and showed 100% concordance with a culture-based method.
Conclusion:
The RT-LAMP-based method in the present study offered a proof of concept that warrants clinical validation on a large number of samples. Therefore, its diagnostic potential for the rapid, sensitive, and specific detection of C. auris could be further exploited in resource-limited regions.
