Rapid Identification by Polymerase Chain Reaction of Staphylococcal Exfoliative Toxin Serotype A and B Genes

Abstract

A new system was designed to detect staphylococcal exfoliative toxin A (ETA) and B (ETB) genes by the polymerase chain reaction (PCR). The primer pairs for the ETA gene (eta) were 20 and 20-mer, and its PCR product was a 741-bp eta fragment, while the primer pairs for the ETB gene (etb) were also 20 and 20-mer, and its PCR product was a 629-bp etb fragment. When these primers were simultaneously used in the PCR, the two types of ET were clearly detected as two bands in an ETA and ETB double-producer using only one colony within 3 hr. We examined 66 strains of Staphylococcus aureus isolated from patients with staphylococcal scalded skin syndrome (SSSS) and compared the results obtained by ELISA and PCR. The same results were obtained for 56 of the strains, i.e., 30 strains were ETA producers, 20 strains were ETB producers, and 6 strains were double-producers. However, positive results were obtained for 5 of the 10 non-ET-producing strains. Two of these strains were judged by PCR as ETA producers and three as ETB producers. Thus, PCR is very sensitive and rapid in detecting ETA and ETB gene fragments in colonies isolated from patients with SSSS.

Development and Evaluation of Detection Systems for Staphylococcal Exfoliative Toxin A Responsible for Scalded-Skin Syndrome

“…PCR had the advantage of allowing a clear distinction between ETA and ETB producers as well as double producers (Fig. 2), and this method has been validated in the newborn-mouse model (30). There was a 100% correlation between PCR and the Western blot II assay.…”

“…Since then, a range of different diagnostic tests has been developed for the diagnosis of SSSS (1,10,19,20,22,26,30). However, all of these tests require isolation of the causative staphylococcal strain, which takes time, and the tests are not routinely available in hospital laboratories (7,28).…”

“…PCR was performed according to the method of Sakurai et al (30). Two ETA primers, 5Ј-CTATTTACTGTAGGAGCTAG-3Ј, corresponding to nucleotides 46 to 65, and 5Ј-ATTTATTTGATGCTCTCTAT-3Ј, corresponding to nucleotides 767 to 786 of the eta sequences counted from the initiation codon, and two ETB primers, 5Ј-ATACACACATTACGGATAAT-3Ј, corresponding to nucleotides 167 to 186, and 5Ј-CAAAGTGTCTCCAAAAGTAT-3Ј, corresponding to nucleotides 776 to 795 of the etb sequences counted from the initiation codon, were synthesized (Genoysis; Sigma).…”

“…Isolation of S. aureus from blood cultures or skin lesions of the patient often supports the diagnosis of SSSS (11,18,20,22,23), and production of exfoliative toxin by these strains of S. aureus can be determined using a range of currently available diagnostic tests, including PCR, radioimmunoassay, Ouchterlony immunodiffusion assay, and reverse passive latex agglutination assay (1,10,19,30). However, these tests are not routinely available in hospital laboratories.…”

“…However, these tests are not routinely available in hospital laboratories. Furthermore, they all (even the "gold standard" neonatal-mouse model) require isolation of the causative S. aureus strain from the patient (1,19,20,22,26,30), which occurs only in a small proportion of SSSS patients (6,14). Even when the infective agent is isolated, the time required to obtain and process blood and superficial cultures as well as to isolate and identify the organism (usually 24 to 72 h) makes any further tests to detect the presence of the exfoliative toxins useful only for retrospective confirmation of the diagnosis.…”