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Detection of Salmonella in 24 hours
February 09, 2007The food and drink we consume have to pass strict quality controls. Nevertheless, these measures are not always sufficient, given that sometimes certain foodstuffs can still give rise to food poisoning, most often caused by micro-organisms. The Salmonella bacterium is undoubtedly one of the best known of these. At the University of the Basque Country (UPV/EHU) they are developing a new, rapid-detection system (within 24 hours) for Salmonella.
It is currently a laborious process to detect Salmonella in food. An analytical study is carried out in the laboratory by means of conventional microbiological techniques and the results take a week, a delay which creates problems for the food industry
In 2002 the Department of Immunology, Microbiology and Parasitology at the UPV/EHU together with the company, Laboratorios Bromatológicos Araba, and the Leioa Technological Centre, decided to carry out collaborative work in order to try to develop new, faster methods for Salmonella detection.
Genetic methods
A requisite for such genetic methods is to know the genome of this bacterium well. Fortunately there are several strains of Salmonella which have been totally sequenced. It is also known that there are certain genes that are specific to Salmonella that are not found in any other bacteria nor, for that matter, in any other living being. Thus, if we detect these genes, it means the presence of Salmonella. Although we may not detect the entire micro-organism, we can find the DNA of this bacteria.
The study of this DNA has given rise to technical developments which enable the detection of the presence or absence of Salmonella within 24 hours in food. Nevertheless, these methods based on the detection of DNA have a drawback. DNA is a very stable molecule that enables its study in persons who have died many years before. The same can happen in bacteria, i.e. it may be that we are identifying the DNA but that the bacteria have been destroyed by pasteurisation or sterilisation. The researchers have shown that the detection of the DNA in itself is not sufficient to identify the Salmonella given that, using this technique, it is not possible to know if the bacterium is dead or alive.
So the UPV/EHU found another, more specific marker for the viability of the bacteria - messenger RNA; an unstable and easily degradable molecule which is only produced when the bacteria is in the multiplication phase (and thus capable of producing infection), and is subsequently destroyed. Armed with this knowledge, the UPV/EHU research team designed a procedure to extract this RNA from foodstuffs, with subsequent transformation of this RNA into DNA and the detection of the latter.
Working with RNA means working with great precision and speed, because it can give us false negative results, i.e. indicate that there is no salmonella when, in fact, there is, the molecule having degraded. The extraction procedure is a fundamental one: once the messenger RNA is extracted, it is transformed into DNA by means of inverse transcription; a process whereby a DNA copy is synthesised. This DNA copy is detected by certain probes previously developed by the research team. In fact, the probes are DNA chains that are complementary to Salmonella genes marked with a fluorescent compound. If the DNA copy and the complementary DNA unite, the fluorescent compound emits a signal detectable in real time. This device, moreover, enables the quantification of the reaction, i.e. it tells us the number of Salmonella cells present in the food sample.
What the UPV/EHU researchers are proposing, in fact, is a combination of techniques: extraction on the one hand; the design of probes for and detection of DNA and RNA molecules on the other. They are techniques complementary to the traditional cell cultures and that enable the analysis of more samples in less time, thus enhancing food safety globally.
Elhuyar Fundazioa
Genetic methods
A requisite for such genetic methods is to know the genome of this bacterium well. Fortunately there are several strains of Salmonella which have been totally sequenced. It is also known that there are certain genes that are specific to Salmonella that are not found in any other bacteria nor, for that matter, in any other living being. Thus, if we detect these genes, it means the presence of Salmonella. Although we may not detect the entire micro-organism, we can find the DNA of this bacteria.
The study of this DNA has given rise to technical developments which enable the detection of the presence or absence of Salmonella within 24 hours in food. Nevertheless, these methods based on the detection of DNA have a drawback. DNA is a very stable molecule that enables its study in persons who have died many years before. The same can happen in bacteria, i.e. it may be that we are identifying the DNA but that the bacteria have been destroyed by pasteurisation or sterilisation. The researchers have shown that the detection of the DNA in itself is not sufficient to identify the Salmonella given that, using this technique, it is not possible to know if the bacterium is dead or alive.
So the UPV/EHU found another, more specific marker for the viability of the bacteria - messenger RNA; an unstable and easily degradable molecule which is only produced when the bacteria is in the multiplication phase (and thus capable of producing infection), and is subsequently destroyed. Armed with this knowledge, the UPV/EHU research team designed a procedure to extract this RNA from foodstuffs, with subsequent transformation of this RNA into DNA and the detection of the latter.
Working with RNA means working with great precision and speed, because it can give us false negative results, i.e. indicate that there is no salmonella when, in fact, there is, the molecule having degraded. The extraction procedure is a fundamental one: once the messenger RNA is extracted, it is transformed into DNA by means of inverse transcription; a process whereby a DNA copy is synthesised. This DNA copy is detected by certain probes previously developed by the research team. In fact, the probes are DNA chains that are complementary to Salmonella genes marked with a fluorescent compound. If the DNA copy and the complementary DNA unite, the fluorescent compound emits a signal detectable in real time. This device, moreover, enables the quantification of the reaction, i.e. it tells us the number of Salmonella cells present in the food sample.
What the UPV/EHU researchers are proposing, in fact, is a combination of techniques: extraction on the one hand; the design of probes for and detection of DNA and RNA molecules on the other. They are techniques complementary to the traditional cell cultures and that enable the analysis of more samples in less time, thus enhancing food safety globally.
Elhuyar Fundazioa
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