P455 Identification of Cryptic species of Aspergillus using Beta-tubulin gene in a tertiary care center in South India

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesTo identify cryptic species of the genus Aspergillus using Beta-tubulin gene by sequence typing.Methods Aspergillus grown from various clinical samples (Ear swab, Bronchial wash, Endotracheal Aspiration, Paranasal sinus, BAL, Sputum) was subcultured on Sabouraud's Dextrose Agar/Oatmeal Agar.Tease mount/Slide culture was done to study the morphological features of the hyphae, size, shape, and arrangement of the conidia.DNA Extraction using phenol-chloroform method: DNA was extracted and purified directly from 4-day-old culture and used as a template for polymerase chain reaction (PCR) amplification.DNA Amplification: A fragment of the target gene were amplified using PCR.The primer pairs used for Beta-tubulin gene was Bt2a (Forward primer) and Bt2b (Reverse primer).Beta-tubulin.Bt2a FGGTAACCAAATCGGTGCTGCTTTCBt2b RACCCTCAGTGTAGTGACCCTTGGCDNA Purification: The PCR product was purified with multi-screen filter plates.Gel electrophoresis: The amplified DNA product was subjected to agarose gel electrophoresis and specific band formation was to be observed for the species of Aspergillus.DNA Sequencing: The purified product will be used as a template for sequencing. An applied biosystem 3730 sequencer will be used to obtain DNA sequences.Phylogenetic Analysis: Parsimony analysis of individual and combined matrices will be conducted using PAUP version 4.0b10 software/free software at phylogeny.fr by the maximum parsimony (MP) and Neighbor-joining methods.The results will be represented using a dendrogram.ResultsA total of 30 Aspergillus isolates were collected and identified using Phenotypic methods.The DNA extraction and PCR amplification were done and sent for Sequencing. The results of Sequence typing are awaited.ConclusionCryptic species are morphologically indistinguishable forms of Aspergillus and their identifications can be confirmed exclusively by using molecular techniques which have led to the description of previously unknown or rare species among different Aspergillus species complex. According to studies, the frequency of such species was found to be 10% in TRANSNET study U.S and 12% in FILPOP study Spain. Most of these species are less susceptible and certain species are multidrug-resistant (azoles and amphotericin B) and the emergence of antifungal-resistance among the Aspergillus species forms the importance in identifying Cryptic species.