Review articleThe use of NASBA for the detection of microbial pathogens in food and environmental samples
Introduction
For many microbial pathogens, the environmental route of transmission is important, with water and food being particularly significant. Often, the numbers of pathogens are not high, which can complicate their detection in samples taken from these environments. The polymerase chain reaction, with its potential for exquisite sensitivity, can mediate detection of low numbers of microorganisms rapidly and with a high degree of specificity, and its use for this has been propounded in various published studies. So far, there have been few published methods for detection of foodborne or waterborne pathogens using the alternative technique, nucleic acid sequence-based amplification (NASBA) (Chan and Fox, 1999) and those which are available concern very few pathogenic types. This review will detail the publications that have appeared to date. The advantages and limitations of NASBA will be overviewed, with regard to its use in analysis of food and environmental samples. In addition, the issue of detection of viable target will be discussed, as will other relevant issues such as sample pretreatment. Finally, a view of the future of NASBA-based methods, in relation to the current position of PCR-based method for the detection of food and environmental pathogens, will be provided.
Section snippets
NASBA
NASBA is specifically designed to detect RNA and employs three enzymes: a reverse transcriptase, RNaseH and T7 RNA polymerase, which act in concert to amplify sequences from an original single-stranded RNA template. Oligonucleotide primers, complementary to sequences in the target RNA, are incorporated in the reaction. One primer also contains a recognition sequence for T7 RNA polymerase. The reaction contains both dNTPs and NTPs. The first primer binds to the RNA, allowing the reverse
NASBA-based detection methods for pathogens in food and water
The following sections will focus on the particular microorganisms for which NASBA-based methods have been reported, describing salient aspects within each study. The studies are summarised in Table 1.
The issue of viability
The detection of messenger RNA has been proposed as an indicator of cell viability Bej et al., 1991, Bej et al., 1996, Klein and Kuneja, 1997, del Mar Lleò et al., 2000, as defined by capability of cell division, metabolism or gene transcription. Messenger RNA can have a short half-life within viable cells, and is rapidly degraded by specific enzymes (RNases) which are themselves very stable even in environments outside the cell itself (Sela et al., 1957). Simpkins et al. (2000) showed that
Sample pretreatment
Generally, NASBA's requirements for sample pretreatment are similar to those of PCR, in that nucleic acids must be extracted from the target organism in the food or environmental matrix and delivered to the reaction in a reasonable pure solution, i.e. one that does not contain any inhibitors of the reaction. The reaction itself is more complex than PCR in that it requires three enzymes and the effect of potential inhibitors has not been studied in detail, as for PCR (Rossen et al., 1992), nor
Future directions
Nucleic acid-based diagnostics for pathogenic microorganisms, in its main current incarnation as PCR, is very young compared to traditional detection methods. However, it is considered that PCR can be established as a routine reference method, alongside traditional techniques, within the next decade (Hoorfar and Cook, 2002). Currently, there are international collaborative efforts to produce PCR-based methods for foodborne pathogens which are suitable for standardization, and thereafter
Acknowledgments
The unpublished work from the author's laboratory was funded by the United Kingdom Food Standards Agency.
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