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Potential of using real-time PCR-based detection of spoilage yeast in fruit juice—a preliminary study

https://doi.org/10.1016/j.ijfoodmicro.2003.09.002Get rights and content

Abstract

A real-time PCR system was used to differentiate between the common spoilage yeasts, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Candida krusei, Rhodotorula glutinis and Saccharomyces cerevisiae, based on melting peak Tm analysis of the 5.8S rDNA subunit and the adjacent ITS2 region of these yeasts. By using the real-time PCR system and by targeting the citrate synthase (cs 1) gene of C. krusei, it was possible to develop a sensitive detection system to both identify and quantitate the level of C. krusei growth in an artificially contaminated apple juice sample.

Introduction

Improved techniques with increased specificity, discriminatory power and shorter detection times for the identification of spoilage yeasts in foods and drinks are becoming increasingly important in the food sector (Loureiro and Querol, 1999). These techniques allow for the quicker implementation of intervention measures, thus reducing the effects of potential spoilage while also providing a means of monitoring quality control in fermentation processes, such as in the case of wine and beer production (Jespersen et al., 2000). Although significant advances have been made in the more rapid identification of food spoilage yeasts, the discriminatory power of these methods continue to lag behind many of the current molecular-based approaches (Deak, 1995).

A number of PCR-based detection systems have been developed, which are based on the use of either specific (Lopez et al., 2003) or nonspecific (Baleiras Couto et al., 1996) primer sets, offering a high degree of specificity and sensitivity; in a process which is relatively quick and easy to perform and which can be supplemented with additional restriction fragment length polymorphism (RFLP)-based techniques to increase the discriminatory power Esteve-Zarzoso et al., 1999, Yamagishi et al., 1999. In addition, new techniques based on the restriction analysis of yeast mitochondrial DNA have led to improvements involving reductions in both time scale and cost (Lopez et al., 2001). The identification and analysis of yeast isolates by PCR requires around 4–5 h to complete following DNA extraction, which is considered relatively quick, but this can been dramatically improved through the use of real-time PCR.

Real-time PCR combines the discriminatory power of the PCR-based amplification methods with a degree of speed and sensitivity that is superior to that previously achieved. In addition, with the advent of SYBR Green and fluorescent probe technology, we now have the ability to simultaneously detect and quantify DNA from specific targets using real-time PCR Morrison et al., 1998, Rasmussen et al., 1998. In the area of quantitative PCR analysis, the LightCycler™ has greatly simplified the procedure, with the continuous monitoring of samples throughout amplification; allowing for the easy identification of the exponential phase of amplification for the various standard and unknown templates, using either the fluorescent double-strand-specific SYBR Green I dye Morrison et al., 1998, Rasmussen et al., 1998 or a sequence-specific hybridisation probe format Huang et al., 2001, Wellinghausen et al., 2001. Both systems have proven useful in the case of organisms which have been difficult to culture and for which cell numbers may be low within a large background population. These systems coupled to the product analysis software, which differentiates on the basis of melting curve analysis and subsequent Tm determination, can provide differentiation between closely related species which may otherwise be undistinguishable by existing methodologies of culturing and electrophoresis Logan et al., 2001, Mommert et al., 2001.

In this paper we report on the use real-time PCR with the Lightcycler™ to differentiate between the common spoilage yeasts, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Candida krusei, Rhodotorula glutinis and Saccharomyces cerevisiae, based on melting peak Tm analysis of the 5.8S rDNA subunit and the adjacent ITS2 region of these yeasts. In addition, we targeted the citrate synthase (cs 1) gene of C. krusei to allow us to develop a sensitive real-time PCR-based approach to both identify and quantitate the level of growth of this yeast in an artificially contaminated apple juice sample.

Section snippets

Culture, media and culture conditions

Yeast cultures Z. bailii ATCC 2333, Z. rouxii ATCC 52519, C. krusei ATCC 2159 and R. glutinis ATCC 2527 were obtained from the American Type Culture Collection, MD, USA, while the S. cerevisiae culture was obtained from the Microbiology Department, University College Cork, stock collection. Cultures were grown in a 2% (w/v) malt extract broth (MEB) (Difco Laboratories, Detroit, MI, USA) solution at 30 °C. Cultures were maintained at 4 °C on malt extract agar (MEA) plates, comprised of 2% MEB

Results and discussion

The rapid detection and identification of food spoilage yeast contamination of food products is a challenging task particularly from a time standpoint, with both speed and discriminatory power being particularly important. With this in mind, a number of molecular-based methods have been developed which allow for the detection and identification of spoilage yeasts which are routinely associated with a wide range of foods Casey, 2002, Caggia et al., 2001, Cappa and Cocconcelli, 2001, Jespersen et

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    Molecular detection of Candida krusei contamination in fruit juice using the citrate synthase gene cs1 and a potential role for this gene in the adaptive response to acetic acid

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      In this sense, techniques based on DNA amplification have emerged in the last decades with the objective to overcome some of these drawbacks due to their high sensitivity and specificity. Among these methods Polymerase Chain Reaction (PCR) and real-time PCR (qPCR) have been widely used for the detection of spoilage yeasts and moulds in a variety of food products (Casey & Dobson, 2004; Hierro, Esteve-zarzoso, Mas, & Guillamo, 2006; Lozano-Ojalvo et al., 2015; Luque, Córdoba, Rodríguez, Núñez, & Andrade, 2013, 2011; Manonmani, Anand, Chandrashekar, & Rati, 2005; Mayoral, Sanz, Herna, Gonza, & Garcı, 2005; Rawsthorne & Phister, 2006, 2009; Rodríguez et al., 2012; Ros-chumillas, Egea-cortines, Lopez-gomez, & Weiss, 2007; Selma, Elizaquível, Aznar, Elizaquível, & Aznar, 2009; Shimotsu, Asano, Iijima, & Suzuki, 2015; Soares-santos, Pardo, & Ferrer, 2017; Tofalo, Schirone, Perpetuini, Suzzi, & Corsetti, 2012). One of the major drawbacks of most reported PCR/qPCR assays is the lack of an Internal Amplification Control (IAC).

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      Commonly used primers have been based on sequences of the rDNA repeat, such as ITS 1 and 2, or the SSU rRNA gene (Bergman et al., 2007; Khlif et al., 2009; Klingspor and Jalal, 2006; Loeffler et al., 2000; Wellinghausen et al., 2009). This technique is also becoming widely employed in food and beverage analyses and has been used for detection and quantification of spoilage yeasts in orange juice (Casey and Dobson, 2004) as well as in wine fermentations (Cocolin et al., 2001). An advantage of this method is the potential for early detection of contaminating species.

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