Immunoquantitative Real-Time PCR for Detection and Quantification of Staphylococcus aureus Enterotoxin B in Foods

Abstract

A real-time immunoquantitative PCR (iqPCR) method for detection of Staphylococcus aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The assay consisted of immunocapture of SEB and real-time PCR amplification of the DNA probe linked to the detection antibody. iqPCR was compared to an in-house enzyme-linked immunosorbent assay (ELISA) using the same couple of capturedetection antibodies and to commercial kits for detection of S. aureus enterotoxins (SE). The iqPCR was approximately 1,000 times more sensitive (<10 pg ml ؊1 ) than the in-house ELISA and had a dynamic range of approximately 10 pg ml ؊1 to approximately 30,000 pg ml ؊1 . iqPCR was not inhibited by any of the foods tested and was able to detect SEB present in these foods. No cross-reactivity with SE other than SEB was observed. Application of iqPCR for detection of SEB in cultures of S. aureus revealed the onset of SEB production after 4 h of incubation at 22, 37, and 42°C, which was in the first half of the exponential growth phase. The total amounts of SEB produced by the two strains tested were larger at 42°C than at 37°C and were strain dependent.The reported detection limits of available commercial systems for detection of Staphylococcus aureus enterotoxins (SE) range from 0.5 to 2 ng enterotoxin per g of food (11,16,18,20,21). The sensitivity of detection methods and their ability to deliver quantitative information concerning the amounts of toxin present may require improvement, as it is possible that the established intoxication dose is underestimated due to constraining detection limits. Immuno-PCR is a detection method that combines the specificity of antibody-antigen recognition and the sensitivity of PCR. Immuno-PCR methods using endpoint detection with classical PCR and electrophoresis of amplicons for detection of Clostridium botulinum neurotoxin type A and E have been described (9,10,22). The immunoquantitative PCR (iqPCR) technology previously described in a patent (24) couples an antibody detection step, similar to an enzyme-linked immunosorbent assay (ELISA), with nucleic acid amplification of a DNA probe linked to the detection antibody by real-time PCR. Use of real-time PCR for quantitative amplification (13,14), unlike endpoint analysis, provides data required for quantification of the target DNA. The results can be expressed in absolute terms by reference to an external quantified standard or in relative terms by reference to another target sequence present in the sample (19).In the present work an iqPCR method for detection of S. aureus enterotoxin B (SEB) was developed and evaluated using both pure cultures and foods. The sensitivity of the iqPCR method was compared to the sensitivities of two commercially available systems (SET-RPLA [Oxoid, Basingstoke, United Kingdom] and VIDAS-SET2 [bioMérieux, Marcy-l'Etoile, France]), as well as to the sensitivity of an in-house ELISA that served as an internal reference for iqPCR. MATERIALS AND METHODSBacterial strains and culture conditions. All st...

Rapid and sensitive method for detection of Staphylococcus aureus enterotoxin genes in milk sample

“…In addition, DNA extracted from the milk sample combined with gene expression by RT-PCR analysis was highly sensitive for detection of enterotoxin Se A and Se B from undiluted and 10-fold diluted milk sample. Similarly, Rajkovic et al [25] detected Se B gene using RT-PCR from food sample, whereas other enterotoxin genes such as Se d, Se e, Se g, Se j, and Se i were also detected in some strains of S. aureus [26]. Likewise, reports showed that nine Se genes were detected with universal primers using SYBR green chemistry [27].…”

“…Furthermore, the RT-PCR method was also proved to be a successfully applicable technique for direct detection of Se from the clinical samples that contain high levels of PCR inhibitors [9]. Rajkovic also compared iqPCR with inhouse ELISA technique for detecting Se b toxin genes in both pure culture and contaminated food sample and found that iqPCR was approximately 1,000 times more sensitive than the in-house ELISA method [25]. Similarly, decade ago, Heller used the different available kit for comparing the sensitivity of the methods for DNA isolation from food samples for detection of Shiga toxin-producing Escherichia coli by RT-PCR [31].…”

Staphylococcal Enterotoxins, Stayphylococcal Enterotoxin B and Bioterrorism

“…In cases of infection with S. aureus, polymerase chain reaction (PCR) assays can determine the presence of the SE gene (Chiang et al, 2008;Rajkovic et al, 2006). Particularly in the case of a toxin that is known to be present in staphylococcal infections, surveillance and monitoring at the clinical level is imperative for differentiating between random outbreaks of the disease and a bioterrorist attack.…”

New generation of electrochemical immunoassay based on polymeric nanoparticles for early detection of breast cancer

“…Alternatively, detection can be achieved through the enhancement of the generated chemiluminescence signal; it was reported that up to 1000-fold signal improvement can be achieved in the presence of certain molecules such as horseradish peroxidase. 4,5 Other signaling mechanisms reported include the 7 DNA reporters, 8,9 and fluorogenic reporters. 10,11 Electrochemical detection is considered as one of the most reliable and efficient techniques to detect biomolecules within biological samples.…”