With continued development of novel molecular-based technologies for rapid, high-throughput detection of foodborne pathogenic bacteria, the future of conventional microbiological methods such as viable cell enumeration, selective isolation of bacteria on commercial media, and immunoassays seems tenuous. In fact, a number of unique approaches and variations on existing techniques are currently on the market or are being implemented that offer ease of use, reliability, and low cost compared with molecular tools. Approaches that enhance recovery of sublethally injured bacteria, differentiation among species using fluorogenics or chromogenics, dry plate culturing, differentiation among bacteria of interest using biochemical profiling, enumeration using impedence technology, techniques to confirm the presence of target pathogens using immunological methods, and bioluminescence applications for hygiene monitoring are summarized here and discussed in relation to their specific advantages or disadvantages when implemented in a food microbiology setting.

As a means of assessing gene expression and RNA structure/function, nucleic acid sequence‐based amplification (NASBA) involves an isothermic series of reactions using avian myeloblastosis virus reverse transcriptase (RT), RNase H, T7 RNA polymerase, transcript‐specific primers and associated cofactors to amplify large amounts of target RNA. NASBA offers greater speed, sensitivity and versatility compared to other RNA analyses (e.g., RT‐PCR, RNase protection assays and Northern blotting). NASBA allows for target RNA detection by real‐time chemistries, such as SYBR dyes or molecular beacon probes, an advantage in minimizing contamination considering the closed‐tube format of this approach. NASBA has been utilized in diagnostic bacteriology for clinical, environmental and food applications. NASBA kits are currently available for RNA amplification and analyses, a market primarily aimed at the clinical microbiology arena. This article will overview the background and application of NASBA, followed by protocols commonly used for bacterial diagnostics and genomic studies in various experiments. PRACTICAL APPLICATIONS Although not a truly new laboratory method for nucleic acid analysis, NASBA has gained a popular following in recent years in the scientific literature. A diverse array of life science applications for NASBA have developed due to the advent of real‐time detection methods. The field of molecular bacteriology has witnessed a plethora of reports on the use of NASBA in fields ranging from molecular and clinical diagnostics to food microbiology and environmental monitoring. NASBA offers very high speed and sample throughput capabilities, with the sensitivity of real‐time detection chemistries, without necessarily having to confirm or validate signal/amplicon identity. What follows is both a background on the uses of NASBA in bacteriology, as well as a procedural guide on preparing target RNA for subsequent RNA analysis by NASBA. This paper is written from a benchtop pedagogical standpoint and so makes a suitable reference for those learning or teaching NASBA to others.

Although many strains of Bacillaceae are considered nonpathogenic, Bacillus cereus is recognized worldwide as a bacterial pathogen in a variety of foods. The ability of B. cereus to cause gastroenteritis following ingestion of contaminated food is due to the production of enterotoxins. The ubiquity of this genus makes it a persistent problem for quality assurance in food processing environments. The primary objective of this study was to develop and apply a multiplex real-time PCR-based assay for rapid and sensitive detection of enterotoxigenic B. cereus. Template DNA was separately extracted from tryptic soy broth (TSB)-grown and 2.5% Nonfat Dry Milk (NFDM)-grown B. cereus using a commercial system. Three enterotoxin gene fragments (hblC, nheA, and hblA) were simultaneously amplified in real-time followed by melting curve analysis to confirm amplicon identity. Resolution of melting curves (characteristic T(m)) was achieved for each amplicon (hblC = 74.5 °C; nheA = 78 °C; and hblA = 85.5 °C in TSB and 84 °C in NFDM) with an assay sensitivities of 10(1) CFU/ml for both TSB and NFDM-grown B. cereus compared to 10(4) CFU/ml in either matrix using gel electrophoresis. The results demonstrate the potential sensitivity of real-time bacterial detection methods in a heterogenous food matrix using real-time PCR.

Bacillus cereus is traditionally thought to be the only member of its genus accepted as a pathogen in foods like grains, fruits, vegetables, and milk due to the presence of the nonhemolytic (Nhe) operon. However, many other Bacillus spp. may also harbor the Nhe operon and be pathogenic, including not just food-associated gastrointestinal toxicoinfections, but human endophthalmitis as well. Real-time PCR targeted the nheA gene in 37 samples obtained from food, soil, and reference cultures by analyzing the standard deviations of melt peaks. Repetitive element PCR was used to compare the banding patterns of each sample against B. cereus ATCC 14579 and three B. thuringiensis strains to “fingerprint” each isolate. Of the original 43 isolated tested, 37 were Gram-positive rods. The remaining six samples were Gram-positive cocci. Twenty-five of the 37 Gram-positive Bacillus spp. were nheA positive, while twelve were negative. Many of the nheA positive strains were species not previously known to contain Nhe and were capable of causing gastroenteritis in consumers.