Modified Multiplex PCR Method for Detection of Pyrogenic Exotoxin Genes in Staphylococcal Isolates

Abstract

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 ...

Comparison of the Prevalence of Genes Coding for Enterotoxins, Exfoliatins, Panton-Valentine Leukocidin and TSST-1 Between Methicillin-Resistant and Methicillin-Susceptible Isolates of Staphylococcus Aureus at the University Hospital in Olomouc

“…Sequences of the primers used as shown in Table 1 have been published by other authors [19][20][21][22][23] . The 25-μl reaction mix contained 5 μl of 10x reaction buffer (100 mM of TrisHCl, pH 8.8, 500 mM of KCl, 1 % Triton X-100, 15 mM of MgCl 2 ) 0.4 μl of dNTPs (10 mM); 50 pmol of primers, 2.5 U of Taq polymerase, 2.…”

Occurrence of methicillin-resistant strains of Staphylococcus aureus at a goat breeding farm

“…The CLSI criteria (2006) were used for the result assessment. In methicillin-resistant S. aureus isolates, multiplex PCR was carried out to detect the presence of genes encoding enterotoxins (sea -sej) (Monday and Bohach, 1999;Lovseth et al, 2004), toxic shock syndrome toxin (tst), exfoliatins A and B (exA, exB) (Mehrotra et al, 2000) and Panton-Valentine leukocidin (lukF -lukS) (Lina et al, 1999). Furthermore, multiplex PCR typing of the staphylococcal cassette chromosome mec (SCCmec) (Milheirico et al, 2007) and macrorestriction analysis using pulsed-field gel electrophoresis (Pantucek et al, 1996) were carried out.…”

Superantigen Profile of Staphylococcus Aureus Isolates from Patients with Atopic Dermatitis in Sri Lanka

“…The presence of genes encoding TSST-1, SEA-E, SEG-I and SElJ-Q were detected for all the isolates by separate PCRs for each superantigen using the primer sets described previously [20,21] (Table 1). PCR products were separated on 1% w/v agarose (Promega) in 1*TAE buffer and visualized under a UV transmitter (Fotodyne TM ).…”