Preliminary Studies on the Detection of Bacillus cereus and Its Toxins: Comparing Conventional and Immunological Assays with a Direct Polymerase Chain Reaction Method

Abstract

Aims: Experiments were conducted to evaluate the specificity and rapidity of the application of conventional, immunoassay and direct Polymerase Chain Reaction (dPCR) techniques in the detection of Bacillus cereus and its toxins in contaminated rice.
 Place and Duration of Study: Department of Life Sciences, Glasgow Caledonian University, UK, between May 2015 and December 2015.
 Methodology: Conventional testing for the presence of B. cereus and associated toxins was achieved using Polymixin Egg-yolk Mannitol Agar (PEMBA) culture plates while immunoassays were conducted using a commercially available Reverse Passive Latex Agglutination (TD0950 BCET-RPLA, Oxoid, UK) kit. Direct PCR was used to detect the HBL-E gene in the food samples and the PCR amplicons were visualised after separation by gel electrophoresis.
 Results: Total Mean B. cereus count was recorded as 5.3 ×107 cfu / g of the rice sample from the PEMBA plate culture. The PEMBA method was the least in terms of rapidity of assay completion. Further assays such as the RPLA and dPCR assays were tested for potential in overcoming this limitation. Results from the immunological study showed that sample agglutination with sensitised latex beads was only positive up to the 10-2 dilution which is an indication of the presence of the Haemolysin BL enterotoxin (HBL-E). The PCR assay had the lowest limit of detection (5.3 ×106 cfu / g) thereby suggesting that the method has very low sensitivity for the bacteria. However, the PEMBA method was more sensitive but had highest detection limit (100 cfu / g) than the RPLA assay (5.3 ×105 cfu / g).
 Conclusion: Although the dPCR had the advantage of producing results within a short time, it was less sensitive to B. cereus than RPLA and PEMBA assays. A development of improved highly sensitive assay for the toxin has the potential to enhance food safety.

Modernization of Control of Pathogenic Micro-Organisms in the Food-Chain Requires a Durable Role for Immunoaffinity-Based Detection Methodology—A Review

“…Another variation, known as reversed passive latex agglutination (RPLA), is the immobilization of probing immunoglobulins on latex particles which are incubated with food extracts to detect bacterial toxins (the target antigen) to form lattice structures. Examples are the detection of staphylococcal enterotoxins SEA, SEB, SEC, and SED [ 124 ] or Bacillus cereus enterotoxin [ 125 ]. Although RPLA is more sensitive than PCR methods, this method can be less sensitive and less specific than the less easy-to-use cell cytotoxicity tests applied for the same goal [ 126 , 127 ].…”