Multifragment melting analysis of yeast species isolated from spoiled fruits.

Abstract

In this study, we developed a novel approach, multifragment melting analysis (MFMA), based on the simultaneous analysis of the melting characteristics of multiple DNA fragments for the differentiation and identification of yeast species isolated from spoiled fruits. In total, 183 yeast isolates recovered from spoiled fruit samples were differentiated and grouped using MFMA. Six different DNA fragments of the 26S rRNA gene showing a high interspecific heterogeneity were amplified, and the PCR products were individually subjected to MFMA. An excellent discrimination and classification were obtained when the melting characteristics of all target DNA fragments for each species were simultaneously assessed. Species-level identification was performed by sequence analysis of representative isolatesfrom each group. In the fruit samples, Hanseniaspora uvarum (Kloeckera apiculata) was found to be the most frequently isolated species followed by members of the Pichia genus, namely P. kluyveri, P. fermentans and P. kudriavzevii (Issatchenkia orientalis). Multifragment melting analysis provided a rapid and reliable approach for discrimination and grouping of a large number of yeast isolates recovered from fruit samples prior to sequence analysis-based identification. The MFMA has great potential for fast andaccurate investigation of yeast communities associated with food spoilage to determine their sources and routes and to prevent contamination.