Identifying Molds Recovered from the Unopened Greek Containers by ITS1 Sequence Characterization

Introduction:   Recently, over 200 individuals have reported gastrointestinal illness after consuming the Greek yogurt in several states of United States. The consumers also reported bloating and swelling of some of the product containers.  At first several unopened vials containing yogurt were examined for bacterial and fungal contaminations following conventional microbiological protocols. The recovered molds were genetically typed by ITS1 sequencing. The ITS1 locus has been considered as the “Universal DNA Barcode” to understand the fungal taxonomy.

Purpose:  The major objective of this study was rapid species-identification of molds recovered from unopened vials with contaminated yogurt by ITS1 sequence characterization.

Methods:   In this study, we have used our recently described protocols for DNA extraction, ITS1-specific PCR amplification and bi-directional nucleotide sequencing of PCR amplified ITS1 products for fungi species identification.  The nucleotide sequencing data was analyzed using BioEdit and GENEIOUS programs.

Results:  A total of 14 unopened yogurt vials were analyzed. Molds were recovered from all of these containers and PCR amplified at the ITS1 locus. Using ITS1 amplified products, the bi-directional DNA sequencing resulted high quality bases (> 98% HQ-100% HQ). Analysis of the generated ITS1 sequences confirmed species-identification to all recovered mold samples analyzed. The ITS1 nucleotide sequences obtained in this study matched 100% with the published sequence of Rhizomucor variabilis (GenBank Accession No. JF904893). The intra-specific genetic variation was not noticed.

Significance:  The results suggest that the ITS1 locus is a reliable benchmark for rapid detection and differentiation of human-pathogenic fungi and molds. It will help in achieving the mission of our agency notably analyzing contaminated food with molds of public health importance.