Novel multiplex PCR for the detection of the Staphylococcus aureus superantigen and its application to raw meat isolates in Korea Sun Young Hwang a , So Hyun Kim a , Eun Joo Jang c , Nam Hoon Kwon a , Young Kyung Park a , Hye Cheong Koo a,b , Woo Kyung Jung a , Jun Man Kim a , Yong Ho Park a,b, ⁎ a Department of Microbiology, College of Veterinary Medicine and BK21 Program for Veterinary Science, Seoul National University, Sillim-dong, Gwanak-gu, Seoul, 151-742, Republic of Korea b KRF Zoonotic Disease Priority Research Institute, College of Veterinary Medicine, Seoul National University, Sillim-dong, Gwanak-gu, Seoul, 151-742, Republic of Korea c Seegene, Inc., Teawon Bldg., Bangyi-dong, Songpa-gu, Seoul, 138-050, Republic of Korea Received 23 November 2006; received in revised form 9 February 2007; accepted 27 February 2007 Abstract A multiplex PCR assay that allows for the rapid screening of the 19 genes that encode staphylococcal enterotoxins (SEs) (sea to see, and seg to sei), SE-like (SEl) toxins (sej to ser, and seu), and toxic shock syndrome toxin-1 (TSST-1) (tst) was developed in this study. These toxins are included in the pyrogenic toxin superantigen (PTSAg) family and are responsible for many diseases such as staphylococcal food poisoning (SFP) and TSS. The primers were designed based on dual priming oligonucleotide (DPO) technology to detect all of the 19 SAg genes in three sets of PCR. The developed multiplex PCR was applied to 143 Staphylococcus aureus strains isolated from pork and chicken meat in Korea. Almost 50% of the strains possessed at least one of the 19 SAg genes. The most frequently found genes were seg, sei, sem, and sen (53 isolates, 37%), which were often found simultaneously in the same isolate. In those isolates, the seo (39 isolates, 27%) or seu (6 isolates, 4%) genes were frequently found together and this combination (seg, sei, sem, sen, and seo or seu) was considered to be a part of the enterotoxin gene cluster (egc). The sea gene (10 isolates, 7%) was the gene most frequently detected out of all the classical SE genes (sea to see). Although these classical SEs are considered to be major etiological factors in SFP, newly described SE or SEl genes (seg to ser, and seu) were more frequently detected than the classical SE genes in this study. There was no isolate detected containing the seb, sec, sek, sel, or seq genes. S. aureus possessing mobile genetic elements known to encode these SAg genes, such as egc, were presumed to be widely distributed among pork and chicken meats in Korea. The multiplex PCR developed in this study could be applied to the investigation of SAg genes in S. aureus strains isolated from various sources. © 2007 Published by Elsevier B.V. Keywords: Staphylococcal enterotoxin; Superantigen; Multiplex PCR; DPO; egc 1. Introduction Staphylococcus aureus produces staphylococcal enterotox- ins (SEs) which are responsible for staphylococcal food poisoning (SFP), a major type of food-borne illness (Balaban and Rasooly, 2000) and the toxic shock syndrome toxin-1 (TSST-1), which is known to cause TSS in humans (Dinges et al., 2000). Both the SEs and TSST-1 are included in the pyrogenic toxin superantigen (PTSAg) family. SAgs, unlike conventional antigens, bind to the outside of the major histocompatibility complex (MHC) class II molecules and form a complex with the Vβ chain of a T-cell receptor (TCR). It leads to the stimulation of T-cell proliferation in a nonspecific manner causing the host immune system to be finally suppressed (Dinges et al., 2000; Chang et al., 2005). SEs are named according to their emetic activities as the etiological factor of SFP, but the exact mechanism is not yet fully understood. Several SEs are described as SE-like (SEl) toxins because they lack the emetic properties or their emetic effects have not been tested in a primate model (Lina et al., 2004). Thus, a International Journal of Food Microbiology 117 (2007) 99 – 105 www.elsevier.com/locate/ijfoodmicro ⁎ Corresponding author. Department of Microbiology, College of Veterinary Medicine, Seoul National University, Sillim-dong, Gwanak-gu, Seoul, 151-742, Republic of Korea. Tel.: +82 2 880 1257; fax: +82 2 871 7524. E-mail address: yhp@snu.ac.kr (Y.H. Park). 0168-1605/$ - see front matter © 2007 Published by Elsevier B.V. doi:10.1016/j.ijfoodmicro.2007.02.013