The present invention relates to a process for detecting live microbiological contaminants in a food product sample.
Food products are particularly susceptible to contamination with microbiological products, in particular with bacteria, on account of the powerful effect which a contamination has on the health of the persons ingesting the food. In this sphere, live, and therefore active, microorganisms are particularly formidable since they are then able to propagate in the body and transmit severe diseases. There is therefore a definite need to develop a process which enables these live microorganisms to be detected in food products in a manner which is both precise and satisfactory.
A large number of methods exist for detecting bacteria, in particular, in samples. One which may be mentioned, by way of example, is that of culturing the sample in order to increase the number of bacterial cells present until colonies, which can be counted, are observed. Where appropriate, the culture can be followed by an additional step which enables the particular type of bacteria contained in the sample to be identified. These bacteriological methods require a great deal of time and skill on the part of the individuals who carry them out. Thus, it is generally necessary, for example, to incubate for from 24 to 48 hours before being certain of obtaining a positive or negative result.
Other methods have also been envisaged for eliminating the drawbacks of the conventional bacteriological method. Thus, microscopy is frequently used for detecting bacteria in clinical samples. Usually, it is necessary to stain the sample in order to increase the detection limits, a procedure which on the one hand represents a laborious method and, on the other hand, is unsuitable for food samples (1). Some immunological methods have also been successfully developed for detecting certain species which possess surface antigens which can be recognized by specific antibodies. However, such an approach cannot be used for qualitatively determining bacteria in a sample which may contain a large variety of bacterial species which do not possess a common surface antigen.
The European Patent Application EP-A-0 133671 describes a method for determining the presence of bacteria in samples, in particular in media, such as body fluids, which are suitable for diagnostic purposes, which employs nucleic acid hybridization techniques. According to this method, the sample to be tested is first of all subjected to denaturing conditions so as to render the nucleic acids of the bacteria, which are present in the sample, single-stranded. The resulting single-stranded nucleic acids are brought into contact with a polynucleotide probe which possesses a sequence which is homologous with at least one sequence which is common to all the bacterial species which are present in the sample. The probe and the denatured nucleic acids are brought into contact so as to effect a hybridization between the probe and the respective sequences of the bacteria. More particularly, the probe employed comprises at least a part of one of the strands of a gene which codes for the synthesis of a nucleic acid or a protein which is involved in the mechanism by which the proteins are synthesized. Those genes of this type which are cited are, in particular, genes which encode transfer RNAs, ribosomal RNAs, or initiation, elongation or translation termination factors. One of the features of this method is that it is not based on expression but rather on the presence of hybridizable nucleic acids, for example the RNA or the genomic DNA or the extra-chromosomal nucleic acids of the bacteria which are present. The result, which the Applicant views as being an advantage, of this is that the samples do not have that it is not based on expression but rather on the presence of hybridizable nucleic acids, for example the RNA or the genomic DNA or the extra-chromosomal nucleic acids of the bacteria which are present. The result, which the Applicant views as being an advantage, of this is that the samples do not have to be treated so as to guarantee the viability of the bacteria which are present.
Document WO 92/03455 describes compositions and processes for treating and diagnosing infections with Candida, in particular nucleotides which are able to hybridize specifically with a part of the Candida MRNA, in particular the mRNA encoding elongation factors 1 and 2 (TEF1 and TEF2). This document is only directed towards therapeutic or diagnostic applications, either for detecting the presence of Candida in a patient or for inhibiting the activity of this bacterium by blocking the expression of essential proteins.
The document Berg et al., xe2x80x9cMOL CELL. PROBES, Vol. 10, February 1996, pp 7-14xe2x80x9d describes a system for specifically detecting the DNA of microplasmas using the PCR technique. Although it is indicated, on page 12 of this document, that the target sequence for the PCR amplification is the tuf gene, which encodes the elongation factor Tu, this document is only directed towards detecting DNA, and not mRNA on the one hand, and, on the other hand, the method is only a method for detecting bacteria in order to establish a diagnosis in a patient. This document does not envisage any application in the sphere of the invention, in which the specific problem is that of detecting living bacteria.
Thus, there is no known method which makes it possible to detect live microbiological contaminants in a food product and which at the same time discriminates between the live microorganisms and the dead microorganisms and which, moreover, does not pose any problems relating to public health.
The inventors have now discovered that it was possible to detect live microbiological contaminants in a food product sample by detecting, in this sample, the resence of messenger RNA (mRNA) which codes for the synthesis of a protein which is involved in the mechanism by which the proteins of the said contaminants are synthesized.
Different families of microorganisms can be detected in accordance with the invention. Procaryotes, in particular bacteria, unicellular eucaryotes, in particular yeasts, and multicellular eucaryotes, in particular fungi, may be mentioned in a nonlimiting manner. Different species can be identified within these families. Thus, for example, Escherichia, Salmonella and Mycobacterium in the case of bacteria; Saccharomyces and Candida in the case of yeasts; Mucor, Neurospora and Trichoderma in the case of fungi.
The synthesis of proteins by microorganisms comprises steps of transcription and translation. Within the context of translation, nucleic acids and proteins exist which are involved in each of the three basic steps of protein synthesis, i.e. initiation, elongation and termination. invention in order to detect live microbiological contaminants belonging to different species.
Thus, according to one preferred embodiment, the invention relates to a process for non-specifically detecting live contaminants belonging to different species of a family of microorganisms, according to which the MRNA detected is an mRNA which codes for the synthesis of a protein whose primary structure is at least partially conserved between different species.
On the contrary, if a greater specificity of detection between different microorganism species is required, it is possible, according to the invention, to detect different mRNAs which respectively code for the synthesis of a protein whose primary structure is not conserved between different species.
Of all the factors which are involved in protein synthesis, one example which is particularly preferred consists of the elongation factors. Those of these factors which may be mentioned are the EF-1, EF-2, EF-G and EF-TU factors, in the case of bacteria (2), or else the EF-1xcex1 factor (3) in the case of yeasts and fungi. These factors play a fundamental role in protein synthesis in that they determine the length of time during which an aminoacyl tRNA remains associated with the ribosome and with the forming polypeptide chain, which function is referred to by the expression xe2x80x9ckinetic proofreadingxe2x80x9d (4).
There are various reasons why it is particularly advantageous to look for the presence of messenger RNA which encodes an elongation factor. First of all, this gene represents a very suitable marker of the viability of the cells since inactivation of this gene is a lethal event both in procaryotes and eucaryotes (5, 6). Furthermore, the gene which encodes an elongation factor encodes a protein which belongs to those proteins which are most widely expressed in procaryotes and eucaryotes (7, 8), a fact which makes it possible to substantially decrease the cell detection level. Finally, as pointed out above, it is possible to modulate the specificity of the detection insofar as this function is conserved in procaryotes and eucaryotes and the primary structure of this type of gene is very similar (9). It is thus possible to implement means of detection which make it possible to distinguish between procaryotes and eucaryotes or, on the contrary, to non-specifically detect bacteria, yeasts and/or fungi at one and the same time.
Furthermore, mRNA encoding elongation factors has a very short half-life (10, 11). Its presence therefore reveals the presence of cells which were still alive approximately ten minutes before the mRNA was detected.
An mRNA encoding an elongation factor is therefore detected in accordance with a preferred embodiment. In this case, a live microorganism cell within the meaning of the invention is a cell which is able to produce the mRNA corresponding to an elongation factor.
The RT-PCR (polymerase chain reaction combined with reverse transcription) is a method of choice for detecting the presence of messenger RNA according to the invention. This technique consists in carrying out a PCR on an RNA which has previously been transcribed into complementary DNA in the presence of reverse transcriptase and a primer. After the RT stage, the proper PCR stage is carried out under standard conditions in the presence of the DNA to be amplified, two oligonucleotide primers which flank the region to be amplified and four deoxynucleotide triphosphates (DATP, dCTP, dGTP and dTTP), in large molar excess, and the enzyme Taq polymerase.
Naturally, the choice of the primers is a basic requirement, since it makes it possible to target the mRNA which it is desired to detect. The oligonucleotide primers are prepared such that they are specific for the coding region of a gene which encodes an elongation factor. The known elongation factors which are preferably selected are the EF-TU factor in the case of bacteria and the EF-1xcex1 factor in the case of yeasts and fungi. It is for this reason that the primers B1/B2 (5xe2x80x2 CGCTGGAAGGCGACGMRRAG 3xe2x80x2 (SEQ ID NO:1)/5xe2x80x2 CGGAAGTAGAACTGCGGACGGTAG 3xe2x80x2 (SEQ ID NO:2) were, for example, prepared, which primers are specific for a fragment of the bacterial EF-TU elongation factor which is found, in particular, in Salmonella typhimurium, Mycobacterium tuberculosis, Mycobacterium leprae, Escherichia coli, Brevibacterium linens and Streptomyces ramocissimus. 
In the case of yeasts, the primers L1/L2 (5xe2x80x2 TCCATGGTACAAGGGTTGGGAA 3xe2x80x2 (SEQ ID NO:3)/5xe2x80x2 GCGAATCTACCTAATGGTGGGT 3xe2x80x2 (SEQ ID NO:4) were prepared, which primers are specific for a fragment of the yeast EF-1xcex1 elongation factor, which is found both in Saccharomyces cerevisiae and Candida albicans. 
Finally, an example of a nucleotide primer which can be used for detecting messenger RNA which is specific for fungi consists of the pair M1/M2 (5xe2x80x2 GCTGGTATCTCCAAGGATGG 3xe2x80x2 (SEQ ID NO:5)/5xe2x80x2-CGACGGACTTGACTTCRGTGG 3xe2x80x2 (SEQ ID NO:6). These primers are more particularly specific for a fragment of the fungal EF-1xcex1 elongation factor which is found in Mucor racemosus, Neurospora crassa, Trichoderma reesei, Absidia glauca, Aureobasidium pullulans, Histoplasma capsulatum and Puccinia graminis. 
The primers which can be used in accordance with the invention were prepared after comparing the elongation factor-encoding regions of different microorganism species (Lasergene software, Dnastar, Madison, Wis., USA).
In a general manner, the process according to the invention can be characterized by the following steps taken together:
a) a sample of the food product to be tested is withdrawn;
b) the cells are lysed;
c) reverse transcription is carried out;
d) amplification cycles are carried out using oligonucleotide primers which are specific for the coding region of a gene which encodes an elongation factor;
e) the amplification products are separated;
f) the amplification products are visualized.
g) the products as visualized in step f) are compared with the amplification products which are obtained from pure mRNA.
Since the invention is essentially directed towards detecting live microbiological contaminants, it is particularly advantageous to as far as possible remove any additional contamination of the medium with the DNA which is present in the sample. For this reason, according to a preferred embodiment of the invention, an additional step (bxe2x80x2), which is intended to remove the DNA which is present in the sample, is added after step b). For this, DNase I, which does not contain any RNase, can simply be added to the reaction medium. If it is desired to check for the absence of false positives which are linked to the presence of DNA, one possibility consists in carrying out a PCR reaction on the same samples as those used for the RT-PCR.