Molecular Identification of Isolated Fungi from Unopened Containers of Greek Yogurt by DNA Sequencing of Internal Transcribed Spacer Region

Abstract

In our previous study, we described the development of an internal transcribed spacer (ITS)1 sequencing method, and used this protocol in species-identification of isolated fungi collected from the manufacturing areas of a compounding company known to have caused the multistate fungal meningitis outbreak in the United States. In this follow-up study, we have analyzed the unopened vials of Greek yogurt from the recalled batch to determine the possible cause of microbial contamination in the product. A total of 15 unopened vials of Greek yogurt belonging to the recalled batch were examined for the detection of fungi in these samples known to cause foodborne illness following conventional microbiological protocols. Fungi were isolated from all of the 15 Greek yogurt samples analyzed. The isolated fungi were genetically typed by DNA sequencing of PCR-amplified ITS1 region of rRNA gene. Analysis of data confirmed all of the isolated fungal isolates from the Greek yogurt to be Rhizomucor variabilis. The generated ITS1 sequences matched 100% with the published sequences available in GenBank. In addition, these yogurt samples were also tested for the presence of five types of bacteria (Salmonella, Listeria, Staphylococcus, Bacillus and Escherichia coli) causing foodborne disease in humans, and found negative for all of them.

Internal transcribed spacer (ITS) sequencing reveals considerable fungal diversity in dairy products

“…Although we were able to find one large study that characterized 610 yeast and mold isolates from raw milk and raw milk cheeses (Lavoie et al, 2012), our study represents the largest molecular study characterizing yeast and mold isolates across diverse dairy products. Most other previous studies (Sulaiman et al, 2014;Banjara et al, 2015;Garnier et al, 2017) that used ITS sequencing for characterization of dairy isolates included fewer than 175 isolates. Importantly, our findings are consistent with previous studies that have shown that "ITS barcoding" provides for a standardized ITS AT nomenclature that can transcend taxonomic changes.…”

“…Standardized DNA sequencing methods, sometimes referred to as DNA barcoding, represent robust and rapid methods for fungal identification. Moreover, an accepted universal internal transcribed spacer (ITS) region barcode for fungi has been well documented to allow for reproducible and discriminatory DNA sequencing-based fungal identification (Mayoral et al, 2005;Schoch et al, 2012;Sulaiman et al, 2014).…”

Evcil Hayvanlardan İzole Edilen Mantarların Teşhisinde Kullanılan Konvansiyonel ve ITS Dizi Analizi Metotlarının Karşılaştırılması

“…In order to obtain reliable and faster results, methods based on sequence analysis of ITS regions have been developed and used in routine laboratories [11] . ITS region is accepted and frequently used as a genetic marker for the identification of fungi [12,13] . It is known that ITS regions 1 and 2 are more variable than the other subunit genes among fungi [14,15] .…”

Dual Panel Multiplex PCR Assay for Rapid Detection of Medically Important Fungi and Resistant Species of Candida and Aspergillus

“…The ITS region is a good molecular target for species level identification (Landlinger et al 2009) and is extensively used as a universal DNA barcode in fungal taxonomy studies (Sulaiman et al 2014). Furthermore, it is present at approximately 100 copies per genome (Henry et al 2000) and is not found in prokaryotic and human genomes (Than et al 2012).…”