Although PCR technology has obvious limitations, the potentially high degree of sensitivity and specificity explains why it has been the first choice of most analytical laboratories interested in detection of genetically modified (GM) organisms (GMOs) and derived materials. Because the products that laboratories receive for analysis are often processed and refined, the quality and quantity of target analyte (e.g. protein or DNA) frequently challenges the sensitivity of any detection method. Among the currently available methods, PCR methods are generally accepted as the most sensitive and reliable methods for detection of GM-derived material in routine applications. The choice of target sequence motif is the single most important factor controlling the specificity of the PCR method. The target sequence is normally a part of the modified gene construct, for example a promoter, a terminator, a gene, or a junction between two of these elements. However, the elements may originate from wildtype organisms, they may be present in more than one GMO, and their copy number may also vary from one GMO to another. They may even be combined in a similar way in more than one GMO. Thus, the choice of method should fit the purpose. Recent developments include event-specific methods, particularly useful for identification and quantification of GM content. Thresholds for labelling are now in place in many countries including those in the European Union. The success of the labelling schemes is dependent upon the efficiency with which GM-derived material can be detected. We will present an overview of currently available PCR methods for screening and quantification of GM-derived DNA, and discuss their applicability and limitations. In addition, we will discuss some of the major challenges related to determination of the limits of detection (LOD) and quantification (LOQ), and to validation of methods.

A modified multiplex PCR method for detection of nine Staphylococcus aureus enterotoxin genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) and one form of immunoreactive toxic shock syndrome toxin based on a previously published method (S. R. Monday and G. A. Bohach, J. Clin. Microbiol. 37:3411-3414, 1999) has been developed. The modified PCR protocol seems robust and gives reliable results.Staphylococcal enterotoxins (SE), toxic shock syndrome toxin (TSST), and exfoliative toxins A and B belong to a family of related pyrogenic toxins produced by Staphylococcus aureus (4, 10). By virtue of its variety of enterotoxins S. aureus is an important food-borne pathogen. In addition to the well-characterized SEA, SEB, SEC, SED, and SEE, new serological types of SEs (SEG, SEH, SEI, SEJ, SEK, SEL, SEM, SEN, SEO, SEP, SEQ, SER, and SEU) have been identified in recent years (5,6,7,11,13,15,16,18). Five of the SE genes (seg, sei, sem, sen, and seo) belong to the same enterotoxin gene cluster (egc), and detection of one of these genes usually indicates the presence of all five enterotoxin genes (5, 11). For routine detection of SEs, commercially produced kits, such as reverse passive latex agglutination assays and enzyme-linked immunosorbent assays, are most commonly used. However, these methods are, to date, designed only to detect SEA, SEB, SEC, SED, and SEE. As an alternative to these more traditional methods, the PCR approach can provide detection of toxin genes and is presently designed to detect at least nine SE genes (sea, seb, sec, sed, see, seg, seh, sei, and sej) (1,9,12,14).The objective of this study was initially to establish the multiplex PCR method for detection of the toxin genes in staphylococcal isolates published by Monday and Bohach (12). According to their article, any of the nine SE genes (sea, seb, sec, sed, see, seg, seh, sei, and sej), the TSST gene, and the 16S rRNA gene can be amplified in a single multiplex reaction (results were not shown). However, while establishing the described method on two different types of thermocyclers, no detection of seb, sec, and tsst could be observed, and the amplification of sec and sei gave various results. Some modifications and adaptations were therefore considered necessary.The following modifications were made to Monday and Bohach's method (12): (i) using another DNA isolation method; (ii) redesigning four primers (seb-sec forward, seb reverse, sei forward, and tsst reverse); (iii) splitting the multiplex into two PCRs; (iv) significantly decreasing the amount of 16S rRNA primers; and (v) adding four cycles to the PCR program.Bacterial isolates and DNA isolation. The S. aureus isolates used in this study are listed in Table 1. One colony of each isolate was incubated overnight in 3 ml of brain heart infusion broth medium at 37°C with agitation. Overnight culture (1.5 ml) was transferred to an Eppendorf tube and was centrifuged for 2 min at 17,500 ϫ g. The pellet was resuspended in 1ϫ Tris-EDTA buffer, and the cells were then lysed with 10 l of 10-mg/ml ...

Mashed potato made with raw bovine milk was suspected to have been the source of a food poisoning outbreak. Almost 8 x 10(8)Staphylococcus aureus CFU g(-1) were detected in the mashed potato. S. aureus was also found in bulk milk from the farm that had supplied milk for the mashed potato. Isolates from mashed potato and bulk milk were positive for the gene encoding staphylococcal enterotoxin H (seh), and the corresponding protein toxin, SEH, was detected by ELISA in the mashed potato. Production of SEH by S. aureus isolates from mashed potato (n = 4) and bulk milk (n = 4) was also demonstrated by ELISA. Sequencing of seh from one mashed potato isolate and one bulk milk isolate confirmed that the gene was a variant seh, and that the genes in both isolates were identical. Macrorestriction of chromosomal DNA with Sma1 followed by pulsed-field gel electrophoresis of seh-positive S. aureus from mashed potato and bulk milk revealed indistinguishable banding patterns between isolates from both sources. It seems likely that raw bovine milk used in the preparation of mashed potato contained S. aureus that subsequently produced sufficient SEH in the mashed potato to cause food poisoning.

The genetical relatedness between epidemiologically linked fecal VRE strains from poultry farmers (n = 5) and their broilers (n = 7) at five avoparcin-exposed Norwegian farms was examined. Pulsed-field gel electrophoresis (PFGE) of bacterial chromosomal digests and structural analysis of vanA resistance elements was performed. Animal and human Enterococcus faecium strains at one farm were genetically closely related with indistinguishable vanA elements and a single band position difference in PFGE analysis. Examination of the vanA elements in genetically unrelated strains by restriction enzyme digestion of Tn1546 long-PCR amplicons and ORF2-vanR intergenic sequencing revealed a pool of at least two distinct vanA gene cluster groups in the two reservoirs. The results indicate that transmission of VanA glycopeptide resistance in enterococci between human and animal at avoparcin-exposed farms can occur by direct transfer of VRE strains as well as horizontal spread of resistance genes between strains.

Aim: To find out if testing of up to 10 Staphylococcus aureus isolates from each sample from raw milk and raw milk products for staphylococcal enterotoxin (SE) might increase the chances of identifying potential sources of food intoxication. Methods and Results: Altogether 386 S. aureus isolates were tested for the presence of SE by reversed passive latex agglutination (SET-RPLA), and SE genes (se) by a multiplex polymerase chain reaction (PCR). In 18 of 34 (53%) S. aureus positive samples a mixture of SE and/or se positive and negative isolates were identified. Multiplex PCR increased the number of potential SE producing strains, i.e. isolates that harboured se, with 51% among the product and 48% among the raw bovine milk isolates. Examination by pulsed-field gel electrophoresis mostly confirmed clonal similarity among isolates sharing SE/se profile, but did not further differentiate between them. Conclusions: Isolates of S. aureus collected from one sample may show great diversity in SE production and different plating media seem to suppress or favour different strains of S. aureus. Significance and Impact of the Study: Several isolates of S. aureus from each sample should be tested for enterotoxin production in cases with typical SE intoxication symptoms with methods that are able to reveal new SE/se.