PCR-SSCP-based reconstruction of the original fungal flora of heat-processed meat products Samart Dorn-In a, ⁎, Christina S. Hölzel a , Tobias Janke a , Karin Schwaiger a , Joachim Balsliemke b , Johann Bauer a a Chair of Animal Hygiene, WZW, TUM, Weihenstephaner Berg 3, 85354 Freising, Germany b Bavarian Health and Food Safety Authority, Eggenreuther Weg 43, 91058 Erlangen, Germany abstract article info Article history: Received 21 August 2012 Received in revised form 17 December 2012 Accepted 27 December 2012 Available online 4 January 2013 Keywords: Fungi Primer PCR SSCP Heat processed meat Plant Food processing of spoiled meat is prohibited by law, since it is a deception and does not comply with food safety aspects. In general, spoilage of meat is mostly caused by bacteria. However, a high contamination level of fungi could be also found in some meat or meat products with certain preserving conditions. In case that unhygienic meat is used to produce heat processed products, the microorganisms will be deactivated by heat, so that they cannot be detected by a standard cultivation method. Therefore, this study aimed to develop and apply a molec- ular biological method—polymerase chain reaction and single strand conformation polymorphism (PCR-SSCP)— to reconstruct the original fungal flora of heat processed meat. Twenty primer pairs were tested for their specificity for fungal DNA. Since none of them fully complied with all study criteria (such as high specificity and sensitivity for fungal DNA; suitability of the products for PCR-SSCP) in the matrix “meat”, we designed a new reverse primer, ITS5.8R. The primer pair ITS1/ITS5.8R amplified DNA from all tested fungal species, but not DNA from meat-producing animals or from ingredients of plant origin (spices). For the final test, 32 DNA bands in acrylamide gel from 15 meat products and 1 soy sauce were sequenced—all originating from fungal spe- cies, which were, in other studies, reported to contaminate meat e.g. Alternaria alternata, Aureobasidium pullulans, Candida rugosa, C. tropicalis, C. zeylanoides, Eurotium amstelodami and Pichia membranifaciens, and/or spices such as Botrytis aclada, Guignardia mangiferae, Itersonilia perplexans, Lasiodiplodia theobromae, Lewia infectoria, Neofusicoccum parvum and Pleospora herbarum. This confirms the suitability of PCR-SSCP to specifically detect fungal DNA in heat processed meat products, and thus provides an overview of fungal species contaminat- ing raw material such as meat and spices. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Meat consumption is globally increasing every year: the growth rate of livestock and meat production has increased annually by 2.2% during 2002 to 2010 (FAO, 2010). Parallel to this trend, public interest is in- creasing in food quality and food safety due to concerns about contam- ination by microorganisms such as bacteria and fungi. To detect and quantify contaminating microorganisms, the conventional cultivation is always applied as a standard method. This method is not applicable for the reconstruction of original flora in raw material of heat processed meat products such as sausage and canned meat, since most of the veg- etative bacteria, fungal hyphae and fungal spores are sensitive to heat (Samson et al., 2000; Wijnands et al., 2009). The other alternative methods such as the detection of endotoxin from bacteria or mycotoxin from fungi can be used, since toxins are relatively heat resistant (Magalhães et al., 2007; Samson et al., 2000). However, not all bacteria and fungi can produce toxin. For the quantification of fungal biomass, ergosterol detection and chitin assay are also applied, but they do not allow any species identification. Moreover, ergosterol should be used cautiously as a biomarker for fungi, since they will be degraded severely if the sample is exposed to sunlight (Mille-Lindblom et al., 2004). Although the deactivated microorganism are not harmful to human health, their high level presence in food may indicate a hygienic prob- lem of raw material, especially in case of spoiled meat which was used to produce heat processed food. Therefore, in order to evaluate the hy- gienic status of raw material in these products, a molecular biological method was applied. Polymerase Chain Reaction (PCR) is the basic method for this purpose in order to produce PCR-amplicons for further application such as Single Strand Conformation Polymorphism (SSCP), Denaturing Gradient Gel Electrophoresis (DGGE), Temperature Gradi- ent Gel Electrophoresis (TGGE) and Terminal-Restriction Fragment Length Polymorphism (T-RFLP) analysis. These molecular fingerprinting methods are suitable for the investigation of microbial communities in complex samples such as soil, environmental dust and fresh water (Ege et al., 2011; Lee et al., 1996; Okubo and Sugiyama, 2009; Peters et al., 2000; Robinson et al., 2009). However, the T-RFLP method, which uses an automatic sequencer, does not allow sequencing of the DNA of International Journal of Food Microbiology 162 (2013) 71–81 ⁎ Corresponding author. Tel.: +49 8161 71 5503; fax: +49 8161 71 4516. E-mail address: Samart.Dorn-In@wzw.tum.de (S. Dorn-In). 0168-1605/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijfoodmicro.2012.12.022 Contents lists available at SciVerse ScienceDirect International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro