title: Development of reverse transcription (RT)-PCR and real-time RT-PCR assays for rapid detection and quantification of viable yeasts and molds contaminating yogurts and pasteurized food products.
abstract: Reverse transcriptase PCR (RT-PCR) and real-time RT-PCR assays have been used to detect and quantify actin mRNA from yeasts and molds. Universal primers were designed based on the available fungal actin sequences, and by RT-PCR they amplified a specific 353-bp fragment from fungal species involved in food spoilage. From experiments on heat-treated cells, actin mRNA was a good indicator of cell viability: viable cells and cells in a nonculturable state were detected, while no signal was observed from dead cells. The optimized RT-PCR assay was able to detect 10 CFU of fungi ml(-1) in pure culture and 10(3) and 10(2) CFU ml(-1) in artificially contaminated yogurts and pasteurized fruit-derived products, respectively. Real-time RT-PCR, performed on a range of spoiled commercial food products, validated the suitability of actin mRNA detection for the quantification of naturally contaminating fungi. The specificity and sensitivity of the procedure, combined with its speed, its reliability, and the potential automation of the technique, offer several advantages to routine analysis programs that assess the presence and viability of fungi in food commodities.
authors.affiliation: Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada le Grazie 15, 37134 Verona, Italy.
authors.forename: Gianluca
authors.initials: G
authors.lastname: Bleve
authors.forename: Lucia
authors.initials: L
authors.lastname: Rizzotti
authors.forename: Franco
authors.initials: F
authors.lastname: Dellaglio
authors.forename: Sandra
authors.initials: S
authors.lastname: Torriani
mesh_terms: D000199:Actins / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001628:Beverages / Q000382:microbiology; D015169:Colony Count, Microbial; D005506:Food Contamination; D005638:Fruit / Q000382:microbiology; D005658:Fungi / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D006358:Hot Temperature; D006801:Humans; D008892:Milk / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013997:Time Factors; D015003:Yeasts / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D015014:Yogurt / Q000382:microbiology*
chemical_list: D000199:Actins; D012333:RNA, Messenger