FR2636075A1 - Process for the detection of bacteria, yeasts, parasites and other eurcaryotes especially in foodstuffs - Google Patents

Abstract

For the purpose of detecting, optionally identifying, bacteria and eucaryotes, especially yeasts, fungi and parasites, especially in foodstuffs, a process is applied comprising the steps consisting in: - extracting the ribosomal RNA which may be present in the product, - transcribing the ribosomal RNA into DNA in the presence of reverse transcriptase, - transcribing the DNA strand thus synthesized into a complementary strand in the presence of one or more primers, - amplifying the DNA strand obtained in the presence of at least one pair of primers determining a known detectable sequence, - detecting the amplified sequence. The relevant ribosomal RNA is more particularly 16S rRNA or the corresponding eurcaryotic rRNA. Primers and DNA probes for implementing the said process are also covered.

Description

 Method for detecting bacteria, yeasts, parasites and other eukaryotes, especially in food products.

The invention relates to a method for detecting bacteria in environments liable to be contaminated, in particular in food products, the invention also being able to be applied to the detection of eukaryotes, such as in particular yeasts, fungi, parasites
Methods for detecting and identifying bacteria and eukaryotes have been known for a long time.

However, they rely extensively on the cultivation of cells in a nutrient medium. The transition to culture is a long method which can take from several days to several weeks, - which can be incompatible with the needs of the food industries, especially since the method is not always fast enough and sensitive in the case where the cell concentration is low.

 On the other hand, these cultures intended for the multiplication of bacterial or other cells often require specific or selective culture media, whether for the search for total bacteria or for the search for specific types of bacteria, which multiplies the manipulations. and the risk of error.

 It was therefore interesting to search for a method allowing the detection of bacterial or other cells in food products without passage into cell culture, even in the case of minimal bacterial or cellular concentrations.

 There is a method, described by SAIKI et al., Science, 230, 1530-1534 (1985), which makes it possible to amplify nucleic acid sequences by hybridizing primers and by synthesizing the strand complementary to the sequence from the primer in the presence of nucleotide triphosphates and DNA polymerase or other polymerization enzymes, so that the primer extension product serves as a template for sequence synthesis and so on.

 This method was applied, according to patent application EP-A-0.229.701, to the detection of viruses, including HIV, by amplification of determined sequences characteristic of viral DNA, or possibly of viral 1-RNA present in a humoral or cellular sample. This process includes the use of DNA primers which hybridize in specific locations of the two complementary DNA strands obtained by denaturation of the starting viral double strand. The addition of a polymerization agent such as DNA polymerase induces the replication of the desired sequence. The amplification is ensured by the renewal of the operation.

 This process cannot however be transposed to the field of detection of bacteria or eukaryotes in food products, if only because of minute concentrations of bacterial or nuclear DNA in very specific and variable environments.

 On the other hand, such a method would require a large number of primers and specific detection probes for the identification of bacteria or other cells, therefore a multiplicity of manipulations, which makes it too cumbersome for the present application.

 The sequences of many ribosomal RNAs from a wide variety of bacteria have now been determined. These rRNAs have a great homology so that little attempt has been made to use them for detection purposes. However Ulf B. Gobel et al, in Journal of General Microbiology, 133, 1969-1974 (1987), detected and identified cultures of bacteria, in this case Mycoplasma pneumoniae, after extraction of the RNA, by hybridization of the 16S rRNA with DNA probes complementary to species-specific regions of this ribosomal RNA. This process has been shown to be more sensitive than hybridization of genomic DNA. However, it is unsuitable for detecting bacteria at low concentrations in food products.

 The object of the invention is therefore to provide a method for detecting and / or identifying bacteria or eukaryotes in food products which is highly sensitive and which makes it possible to detect the presence of cells, and if necessary their identification.

 Another object of the invention is to provide such a method which can combine detection and identification with manipulation.

 Yet another objective of the invention is to provide such a method which can be implemented quickly, in particular within a few hours.

The subject of the invention is a method for detecting, and optionally identifying, bacteria, yeasts, fungi, parasites and other eukaryotes, in particular in food products, characterized in that it comprises the steps consisting in:
- extract the RNA.ribosomal possibly present in the product or medium studied,
- transcribe ribosomal RNA into DNA in the presence of reverse transcriptase,
- transcribe the DNA strand thus synthesized into a complementary DNA strand in the presence of one or more primers,
- amplifying the DNA strand obtained, preferably in the presence of at least one pair of primers determining a known detectable sequence,
- detect the amplified sequence.

 The invention preferably applies to ribosomal RNA 165 or optionally to ribosomal RNA 235 or 5S or also to the corresponding ribosomal RNA of eukaryotic cells.

 The extraction process is preferably a process of the type described by Kirby K.S. et al., Biochem. J.104, 258-262, 1987. An example of such a method will be described later in detail. It has the double advantage of being fast and ensuring optimal recovery of RNA. By extraction in the sense of the invention means extraction from the bacterial walls, without it being necessary to eliminate the other bacterial constituents or the food constituents.

 Preferably, but not necessarily, the ribosomal RNA is hybridized with that of the two primers which is complementary thereto, but any other means ensuring the start of reverse transcription can be used.

 The RNA strand is deployed, for example by heat or any other means, then transcribed at a temperature compatible with the hybridization of the primer or primers on its specific site or sites. The reverse transcriptase then ensures the synthesis of the DNA strand by extension of the primer so as to include the determined region which will be detected after amplification. Several primers s hybridizing to several locations of the rRNA can also be used.

The synthesis of a DNA strand complementary to the DNA strand synthesized by reverse transcription is then carried out using one or more specific primers.

 Surprisingly, it is found that it is thus possible to transcribe the rRNA into an entire DNA fragment constituted by or comprising the determined region.

 The amplification uses two primers, each of which will hybridize on its specific site located on its complementary DNA strand. The amplification technique can be that described by SAIKI et al., Using in particular a heat-resistant DNA polymerase.

 In accordance with the invention, the primers can frame (by forming part of it or not) variable regions, species specific or homologous regions. Preferably, one of these primers is identical to the primer used in the transcription step. The terms "homologous regions" and "variable regions" are regions of the type described in: Oligonucleotide probes complementary to variable regions of ribosomal RNA discriminate between Mycoplasma species, -Uli B. Gobez et al., J. of General Microbiology ( 1987), 133, 1969-1974.

 The amplified DNA strands may therefore be formed from, or comprise, specific zones or homologous zones, or else comprise homologous zones and specific zones.

 We can therefore either simply detect the presence of bacteria or identify them.

 The DNA primers used in the various stages of the process according to the invention may have a variable length, but, for reasons of selectivity of the hybridization, it is preferred that they contain at least 20 bases. The other factors conditioning the selectivity of the hybridization, such as temperature and salinity, will be adjusted, in known manner, for selective hybridization.

 The actual detection is preferably carried out by hybridization using DNA probes labeled or not covering, preferably in part, the amplified region, the revelation being carried out for example by electrophoresis on polyacrylamide gel 7 bones, or any means usual qualitative or quantitative appropriate.

 The probes can be general or specific, that is to say correspond respectively to homologous regions or to specific variable regions.

 The invention applies to various food products liable to be contaminated with bacteria, in particular milk, meat or vegetable products.

 The invention can also be applied to the detection and identification of bacteria or living eukaryotic cells or microorganisms, in particular yeasts, fungi, parasites, in other media, in particular liquid or tissue samples from animal or human organisms .

 The subject of the invention is also the primers allowing the implementation of the method according to the invention, as well as the new probes allowing the detection of the selected regions. The primers are characterized by a nucleotide sequence derived from a fragment of the rRNA, this sequence preferably comprising at least 15 nucleotides.

Among the general primers, that is to say homologous, appear in particular
(B2) 5 'TAC, CCC1 ACC, TAC, TAG, CTA, AT 3'
(A2) 5 'CTA, CTG, GAA, ACG, GTA, GCT, AA 3'.

Among the primers specific for E. coli appear in particular
(B1) 5 'AAC, TTT, ACT, CCC, TTC, CTC, CC 3'
(B'1) 5 'GTA, ACG, TCA, ATG, AGC, AAA, GG 3'
(These primers can also be used for reverse transcription. Primer B'1 is preferred, primer B1 tends to hybridize also at another site).

 (A1) 5 'AAC, GTC, GCA, AGA, CCA, AAG, AG 3'.

As general probes for the revelation, one can provide in particular
(probe 1) 5 'TAC, CCC, ACC, TAC, TAG, CTA, AT 3'
and / or the complementary sequence
As specific E. coli probes, we can provide in particular
(probe 2) 5 'AAC, GTC, GCA, AGA, CCA, AAG, AG 3'
and / or the complementary sequence.

 Other advantages and characteristics of the invention will appear on reading the following description, given by way of nonlimiting example, from a product containing E. coli, and referring to the single figure representing the DNA transcription. of a partial sequence of the 165 rRNA of E. coli.

1. RNA extraction
The extraction process which will be described is a modification of the phenol method described by Kirby et al. cited above.

Other methods are however possible, although they take longer to implement. These are the processes according to
- Auffray C. and Rougeon F., Eur. J. Biochem, 107, 303-314 (1980),
- Zdzislaw Krawczyk and Carl Wu, Analytical Biochemistry 165, 20-27 (1987),
- Piotr Chomczynski and Nicoletta Sacchi, Analytical
Biochemistry 162, 156-159 (1987).

The process is as follows
- The food product is suspended in a physiological medium.

- the cells (1,108 cells) are recovered by centrifugation of the medium (15 min at 5,000 g) or by vacuum filtration
- add an equal volume (about 5 ml) of the following solution: (1x)
Tri-isopropylnaphthalene sulfonate ... 1% (weight / Vol.)
4-amino salicylate (Na salt) ... 6 \ (W / V)
Tris-HCl (pH = 8.3), final concentration ... 100 mM
Phenolic mixture (see below) ... 6% (V / V)
Phenolic mixture: - highly purified phenol = 500 g
- 8-hydroxyquin-oline = 0.5 g
- saturation of the above with
50 mM Tris-HCl (pH = 8.3).

and 12 to 14 g of glass beads (4.5 to 5 mm in diameter)
- shake with Vortex for 4 min (this step is important because it provides good agitation)
- an equal volume (about 6 ml) of a mixture of phenol, chloroform and iso-amyl alcohol 50/50/1 (V / V) is added, and the mixture is stirred vigorously for 2 min.
- The liquid obtained is introduced into centrifugation tubes and centrifuged at 7,000 g for 5 min, at 5 ° C .;
- the upper aqueous phase is recovered and stirred with
Vortex vigorously for 2 min with an equal volume of a mixture of phenol, chloroform and iso-amyl alcohol 50/50/1 (V / V), then centrifuged at 7,000 g for 5 min at 5C
- the extraction with phenol, chloroform, iso-amyl alcohol is repeated until only a very reduced interface remains
- the volume of the aqueous phase is measured and the nucleic acids are precipitated with 1/9 volume of 3M NaOAc (pH = 6) and with an equal volume of isopropanol
- leave to precipitate for 10 min at 4-C
- centrifuged at 10,000 g for 5 min
- the supernatant is eliminated;
- the sample is washed with 5 ml of absolute ethanol
- the supernatant is removed and the sample is dried; ;
- the sample is dissolved in 10 to 100 Rl of water.

2. Primer hybridization for reverse transcriptase (primer B, see below)
- take from 1 to 8 μl of the RNA solution obtained above
- 20 ng of the oligonucleotide serving as a primer are added
- we bring to a final concentration of
250 mM KCl
2.5 mM Tris-HCl pH = 7
0.25 mM EDTA
in a final volume of 10 'it
- it is heated for 15 minutes at 95 C, then
- 30 minutes at room temperature (or higher)
This temperature is determined by the sequence of the oligonucleotide and by the salt concentration of the medium, in this case 0.25 M. Different methods exist for determining this temperature
- MEINKOTH, J. and WAHLr G. Anal.Biochem. 138, 267-284, 1984,
- WALLACE, RB et al., Nucl. Acids Res., 6, 3543-3656, 1979,
- GOEDDEL, DV et al., Nature, 287, 411-416, 1980,
- SUGGS, SV et al., Proc. Natl. Acad. Sci. USA, 78, 6613-6617, 1981,
- SZOSTAR, JW et al., Methods Enzymol., 68, 419-428, 1979,
- SUGGS, SV et al., ICN-UCLA Symp. Dev. Biol .; 23, 683693, 1981.

3. Synthesis of the DNA Strand by Reverse Transcriptase:
- 20 units of M-MLV reverse transcriptase clone M-MLV (Moloney Murine Leukemia Virus) of reverse transcriptase, from Bethesda Research Laboratories are added
- we adjust to a final concentration of
7 mM Tris-HCl pH = 8.6
7 mM MgCl2
3.5 mM DTT
60 pM dATP, DTTP, DGTP, DCTP
0.6 ng / μl of primer B
75 mM KCl
in a final volume of 33 μl;
- incubate for 30 minutes at 37 ° C.

4. Amplification
- the solution is brought to a concentration of
50 mM KCl
10 mM Tris-HCl pH = 8.6
2.5 mM MgCl2
200 pg / ml Gelatin
200 pM dNTP
10 ng / μl of primer A
10 ng / μl of primer B
in a final volume of 100'il
- 4 TAQ units are added (Thermus Aquaticus DNA Polymerase from New England Biolabs, ref .: KALEDIN, AS et al.,
Biokhimiia, 45 (4), 644-651, 1980)
- 90 seconds heating at 93-C
- it is brought to room temperature for 90 seconds or at a higher temperature, and / or for a longer time as appropriate
- incubate 120 seconds at 46-C
- this temperature cycle is repeated 12 times
- we add 4 units of TAQ
- and repeat this cycle 12 times (or more).

 The amplification step can advantageously be carried out in an apparatus such as that described in European patent application EP-A-O 236 069.

5. Revelation
The amplification can be visualized by different methods and in particular by electrophoresis on 7% polyacrylamide gel or hybridization using probes partially covering the amplified area.

 The above method was applied to two cases, with primers and probes according to the invention, reported in the figure.

 The figure represents a DNA sequence corresponding to a partial sequence of the E. coli 16S rRNA. The DNA strand complementary to the first is not shown. Probes and primers are located there, it being understood that some of them have their specific site on the strand shown (hereinafter B1, B'1, B2, probe 1), others on the complementary strand not shown (ci -after Al, A2, probe 2).

Case 1: specific primer, general probe
- primer A
5'AAC, GTC, GCA, AGA, CCA, AAG, AG 3 = Al primer
- primer B
5'AAC, TTT, ACT, CCC, TTC, CTC, CC 3 '= primer B1
5'GTA, ACG, TCA, ATG, AGC, AAA, GG 3 '= primer B'1
(these primers B are also used for reverse transcriptase)
- probe = 5'TAC, CCC, ACC, TAC, TAG, CTA, AT 3 '= probe 1,
we can also use the complementary probe, that is to say 5'ATT, AGC, TAG, TAG, GTG, GGG, TA 3 ', or the gods together.

Case 2: general primer, specific probe
- primer A
5 'CTA, CTG, GAA, ACG, GTA, GCT, AA 3' = primer A2
- primer B
5'TAC, CCC, ACC, TAC, TAG, CTA, AT 3 '= primer B2
- probe: 5'AAC, GTC, GCA, AGA, CCA, AAG, AG 3 '= probe 2
or the complementary, or both together.

 The results demonstrated the effectiveness of the specific primers Al and B'1 as well as of the general probe 1 and of the general primers h2 and B2 as well as of the specific probe 2. By story, the primer B1 was found to be little efficient because it hybridizes at another location indicated by X in the figure.

 These two cases are of course not limiting. We can consider all possible cases, in particular the following case: general primer, general probe.

 It is understood that the example described makes it possible, using a commercial machine, and with fairly unskilled personnel, to detect, or even identify the presence of bacteria in a sample of food product, for example milk or a can, in four to six hours, most of the operations can be automated.

The process is sensitive to the presence of a few bacteria to a few hundred bacteria per ml.

Claims (10)

 1. Method for detecting, and possibly identifying, bacteria and eukaryotes, in particular yeasts, fungi and parasites, especially in food products, characterized in that it comprises the steps consisting in  - extract the ribosomal RNA possibly present in the product,  - transcribe ribosomal RNA into DNA in the presence of reverse transcriptase,  transcribe the DNA strand thus synthesized into a complementary strand in the presence of one or more primers,  - amplifying the DNA strand obtained in the presence of at least one pair of primers determining a known detectable sequence,  - detect the amplified sequence.  2. Method according to claim 1, characterized in that the said steps are applied to the rRNA 165 or the corresponding eukaryotic rRNA.  3. Method according to one of claims 1 and 2, characterized in that one proceeds to an extraction with phenol.  4. Method according to any one of claims 1 to 3, characterized in that the sequence determined by the primers corresponds to a homologous sequence of rRNA.  5. Method according to any one of claims 1 to 3, characterized in that the sequence determined by the primers corresponds to a specific sequence of rRNA species.  6. Method according to any one of claims 1 to 5, characterized in that the DNA is amplified by succession of thermal cycles with denaturation of DNA at high temperature in the presence of heat-resistant polymerase.  7. Method according to any one of claims 1 to 6, characterized in that the amplified sequence is detected using a nucleotide probe.  8. Primers for implementing the method according to any one of claims 1 to 7, characterized in that they are derived from a bacterial rRNA sequence.  9. Primers according to claim 8, characterized in that they comprise one of the sequences described under the references A1, A2, B1, B'1, B2, or the complementary sequences.  10. DNA probes for implementing the method according to any one of claims 1 to 7, characterized in that they comprise one of the sequences described under the references (probe 1), (probe 2), or complementary sequences.