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.

Rapid Identification and Enumeration of Saccharomyces cerevisiae Cells in Wine by Real-Time PCR

“…Numerous real-time PCR systems have been developed to detect and enumerate bacteria, especially pathogens. Due to the advantages afforded by the technique, it was later extended to yeasts in the clinical setting (1,22,27,29) and in food technology (2,3). Currently, the detection of wine spoil-age yeast using this method is limited to the species Dekkera bruxellensis (21).…”

Application of PCR-Based Methods to Dairy Products and to Non-Dairy Probiotic Products

“…Direct comparisons of Cq values with a standardised amount of RNA have also been used to investigate the effect of cell separation from the cheese matrix before RNA extraction . Bleve et al (2003) observed a correlation between standard plate counts of yeasts and moulds present in spoiled commercial food products and the Cq values obtained by reverse transcription qPCR analysis with primers targeting the fungal actin gene. To follow gene expression of P. freudenreichii and Lb.…”

“…RNA extraction methods involving prior separation of the cells from cheeses and other dairy products have been used in several studies (Randazzo et al, 2002;Bleve et al, 2003;Sanchez et al, 2006;Bogovic Matijasic et al, 2007;Smeianov et al, 2007;Makhzami et al, 2008;Rantsiou et al, 2008a;Rantsiou et al, 2008b;Ulvé et al, 2008;Duquenne et al, 2010;Falentin et al, 2010;Cretenet et al, 2011;La Gioia et al, 2011;Masoud et al, 2011;Taïbi et al, 2011). The recovery of microbial cells is done following similar protocols than for DNA extraction methods (see above).…”

A New Concept for Efficient Sensitivity Amplification of a QCM Based Immunosensor for TNF-α by Using Modified Magnetic Particles under Applied Magnetic Field

“…Various detection methods have been employed for the monitoring of TNF-α such as ELISA, reverse transcription polymerase chain reaction (RT-PCR), flow cytometric assay, surface plasma resonance (SPR) and quartz crystal microbalance (QCM). [10][11][12][13][14][15][16] However, the high cost and slow turnaround times of conventional enzyme linked photometric or PCR based methods indicate a need for more sensitive but simpler analytical techniques. To meet this need, a sensor-based system which is simple to use, inexpensive, disposable and highly sensitive, is becoming increasingly important in TNF-α analysis.…”