FR2616804A1 - DNA probe intended for detecting Listeria monocytogenes, analytical kit and method which makes it possible to carry out this detection - Google Patents

Abstract

The invention relates to a DNA probe which is useful for detecting Listeria monocytogenes, as well as to a method for carrying out this detection. The said method consists in testing the hybridisation under rigorous conditions of the DNA characteristic of L. monocytogenes with a probe derived from the gene encoding a secreted protein of 60 kd. Application: detection of L. monocytogenes in biological samples, especially foodstuffs.

Description

 The present invention relates to the prevention of contamination and spoilage of food and other products, and medical analysis.

More specifically, it relates to the detection of the presence of organisms belonging to the species Listeria monocytogenes in biological samples.

Listeria monocytogenes is a coccobacillus
Gram-positive which was first identified as a human pathogen in lu29. We now know that L. monocytogenes is very widespread in the environment and can be isolated from human vectors; and, more importantly, that it is a factor responsible for epidemics, sometimes fatal, of food poisoning. For example, it was L. monocytogenes which was responsible for the poisoning epidemics that occurred in Jalisco and Ado Bera from contaminated cheeses, respectively, in California and Arizona.

Infection with the bacteria produces flu-like symptoms that most people can easily cure; however, people who are immunocompromised, such as newborns, alcoholics, cancer patients and pregnant women, can have serious complications, and infection in these individuals is often fatal.

Currently, the detection of contamination by L. monocytogenes requires a culture of the bacteria from a food sample or a biological sample, then conventional methods of identification of the bacteria, a process which, due growth characteristics of the isolate, may take several weeks or months (Albritton, WL, et al., in Manual of
Clinical Microbiology, Lenette, EH, et al., Eds. (1980), American Society for Microbiology, Washington, DC pages 139-142).

 Consequently, it would be useful to have a procedure, which is faster and more sensitive, for the detection and identification of this organism. The use of the gene coding for a protein produced by L. monocytogenes, which is closely linked to the pattern of infection by this bacterial species, appears to constitute such a method.

The protein in question, a secreted protein of 60 kd, is the main protein found in cell cultures of L. monocytogenes. This protein has hemolysin activity. It may be related to similar proteins present in other bacterial strains from which L. monocytogenes must be distinguished, but the discomfort, as indicated below, is characteristic of this species of organism.

The ability of L. monocytogenes to cause disease may, in fact, depend on the presence of hemolysin. Unlike most other invasive infectious agents, this bacterium multiplies in sensitive macrophages and produces foci in parenchyma cells that are resistant to polynuclear leukocytes (PN). The formation of these resistant foci depends on their ingestion by the phagocytes, on the fusion of the phagosome and the lysosome and on the subsequent lysis of the cell. It seems that hemolysin is associated with this process and that its presence is necessary for the latter. It has also been established that partially purified hemolysin can damage isolated lysosomes, causing the release of hydrolytic enzymes and the destruction of
PN.

 Hemolysins belong to a group of sulfhydryl-activated cytolysins, which include streptolysin O from Streptococcus pyrogenes, and cereolysin from Bacillus cereus. A protein with hemolysin activity is the main protein secreted by L. monocytogenes, and has a molecular weight approximately equal to 60 kd.

Other species of the genus Listeria have also been shown to have hemolytic activity but, as described below, the proteins secreted by these species, such as L. ivanoii or L. seeligeri, are not identical to that characteristic of L.

monocytogenes. The 60 kd secreted hemolysin produced by L. monocytogenes is also apparently not homologous to related cytolysins, which can be activated by the sulfhydryl group, produced by B.

thuringiensis or S. pneumoniae.

 It is shown below that the gene coding for the 60 kd protein secreted by L. monocytogenes, having hemolysin activity is useful as a DNA probe for the detection of strains of this species in different samples. The nature of the secreted "protein", whether a single constituent or a mixture, has not been clearly established; however, it is evident that the 60 kd protein fraction present in the supernatant has hemolytic activity.

 The use of DNA probes, in general, for the detection of particular organisms in biological samples is, of course, known.

United States Patent Re-Examined No. B1 4,358,535 in the name of Falkow et al. Generally describes methods of detecting particular organisms in samples using DNA probes. The usual conventional route for the obtaining of DNA from samples for hybridization purposes is, in principle, that of Southern,
E., J. Mol. Biol. (1975) 98: 503-517, cited for reference in this memo. However, other methods of obtaining DNA in appropriate amounts can also be used. For example, amplification of the desired DNA sequences in order to increase the sensitivity of the assay can be carried out using the method de Mullis, K., et al., Science (1985).

The present invention provides a class of probes which are useful for detecting the presence of organisms of the species L. monocytogenes in biological samples, including food samples, using standard DNA extraction and hybridization techniques. The present invention relates more particularly to the gene coding for a secreted protein of 60 kd having a hemolysin activity characteristic of the strains of the species
L. monocytogenes. Probes prepared from this gene hybridize under drastic conditions to DNA fragments of L. monocytogenes, while being unable to hybridize under conditions comparable to DNA encoding putatively for analogous proteins originating from other bacterial species. Due to the specificity of the probes, the present invention provides a method having sufficient specificity, sensitivity and speed to allow food contamination to be determined more effectively or to more effectively verify the infection of patients by this particular organism.

 Accordingly, in one aspect, the present invention relates to probes useful in a method of detecting the presence of Listeria monocytogenes in biological samples, which method comprises detecting the presence or absence of DNA in the sample that s hybrid to the probe under drastic conditions. The DNA probe of the present invention is obtained from the gene coding for a secreted protein of 60 kd from a conventional strain of L. monocytogenes. In other aspects, the present invention relates to particular probes, methods using them and reagent kits containing them.

Other characteristics and advantages of the present invention will emerge from the detailed description which follows, given with reference to the appended drawings in which
Figure 1 shows a map of the fragment
5.2 kb SalI / EcoRI containing the gene coding for the 60 kd protein and
Figure 2 shows the immunoreactivity of cell culture supernatants of the Listeria species with an antiserum prepared against the 60 kd protein secreted by L. monocytogenes.

A. Definitions
As used in the present specification, the expression "drastic conditions" designates hybridization under the conditions described by Southern,
EM, J Mol Biol (1975) (above) as modified by Thomashow, MF, et al,
Cell (1980) 19: 729-739, both cited for reference in this specification, washing being performed using 0.3 x SSC and 0.1 X sodium dodecyl sulfate (DSS) at 68 ° C, or other comparable drastic character protocols. The expression "non-drastic conditions" designates drastic conditions comparable to those obtained using the above method but in which the stains are washed in 3 x SSC, 0.2% of DSS and 5 mM ethylenediaminetetra-acetic acid (EDTA) at 55 "C. It is known that there is a continuum between drastic conditions and non-drastic conditions; however, the present invention has been operationally defined in terms of drastic conditions which are known to allow an adequate distinction between the 60 kd protein secreted by L. monocytogenes and comparable proteins from other species.

 The term "biological sample" designates any material of interest with regard to contamination by L. monocytogenes. In general, these samples fall into two categories - food samples and samples taken from people or animals suspected of harboring the infection. Of course, the method can be applied to samples obtained from any material which can allow the growth of these bacteria.

B. General description
Sample handling
The probes of the present invention are applied to the analysis of samples using procedures which are generally known; for the application of ADE hybridization techniques to the detection of particular DNA present in samples. DNA can be isolated from an appropriate sample of biological material using, for example, the method of Flamm, RK, et al,
Infect Immunol (1984) 44: 157-161. The total DNA is then generally treated with a restriction enzyme such as EcoRI, BamHI, SalI or HindIII in order to obtain fragments of random dimensions which are then subjected to separation according to the dimensions using, for example, l polyacrylamide gel electrophoresis, according to the method described by Southern,
EM (above). The separated DNA fragments are then denatured and hybridized to a suitable probe under conditions suitable for the drastic characters chosen. The methods of hybridizing DNA present in samples to DNA probes are themselves well known in the art.

Probes
The probes of the present invention, on which the method of the present invention depends, are obtained from the gene coding for a 60 kd protein secreted by L. monocytogenes; the secreted protein has hemolytic activity. They contain a DNA sequence which is equivalent to the reverse transcript of the mRNA coding for the protein and / or adjacent regions of these sequences as found in the genome, i.e. the native gene . For example, the gene concerned consists of a 5.2 kb EcoRI / Sa1I fragment of pRFlO2 which is deposited (in E. coli Le392) in the American Type Culture
Collection, Rockville, MD, and bears deposit number
ATCC; this 5.2 kb fragment is obtained from the genome of L. monocytogenes 10403. As illustrated below, this 5.2 kb fragment can be used directly as a probe in the method of the present invention or, preferably, a 500 bp HincII / HindIII fragment of this fragment can be used. In general, the probes of the present invention contain at least part of the sequence coding for a 60 kd protein. Thus, in addition to the fragment of total length of 5.2 kb containing the gene, the coding sequence alone comprising the gene can be used, or alternatively a sequence of the latter, comprising at least a sequence of 12 bp. illustrated below, the entire 5.2 kb fragment containing the gene cloned into pBR325 was called pRF102; the 500 bp HincII / Hindili fragment was cloned into pUC8 to obtain pRF106. Thus, pRF102 and / or pRF106 constitute probes of the present invention and contain intervening sequences which contain the genomic DNA sequence coding for the secreted protein of 60 kd present in t. monocytogenes 10403. These probes can be used in the method of the present invention, as can "equivalent" probes.

 The expression "genomic sequence coding for" the secreted protein of 60 kd designates the sequence present in the genome of L. monocytogenes, whether obtained materially from the gene or else synthesized or prepared by another process, as long as the same nucleotide sequence is obtained.

 The expression “equivalent probes” designates a sub-fragment of the intermediate sequences, derived from L. monocytogenes, from pRF102 or pRF106 or else these segments modified only so weakly that they retain their initial specificity by unimportant substitutions of bases. These modified sequences specifically comprise the corresponding portions of the genes associated with the strains of L. monocytogenes differing from those from which the intermediate sequences of pRF102 and pRF106 were isolated.

The availability of the appropriate genomic DNA from pRF102 and pRF106, in particular, provides a means of obtaining these equivalent probes. More specifically, other strains may be used as a source for these equivalents, the DNA originating from these strains, prepared as described herein and subjected to separation by electrophoresis on polyacrylamide or agarose gel , being subject to selection for the appropriate fragments. The gels are explored with the deposited clone or one of these fragments to identify the DNA hybridizing under the drastic conditions required. This DNA is excised from gels and amplified in
E. coli in a manner analogous to that described in this memo.

 As indicated below, the specificity of the subfragment present in pRF106 is greater than that of the larger fragment present in pRF102.

Therefore, in methods for obtaining equivalent probes, it is also preferred to use pRF106. The probability of finding sequences of comparable specificity is thus increased. In another way, pRF102 and pRF106 can be used in combination to obtain suitable fragments.

Whatever the association of initial probes, pRF106 alone, pRF106 / pRF102 associated or pRF102 alone, the gene fragments extracted from other strains of
L. monocytogenes have a high probability of containing the appropriate HincII / HindIII sites to give the fragments corresponding to pRF106, and the specificity of the DNA extracts can be improved by treatment with these enzymes. Thus, a series of probes analogous to pRF106 can be obtained from other strains.

As indicated above, minor changes in the nucleotide sequence can be tolerated, as long as they are not sufficient to suppress the specificity of the probe.

 If desired, the probes can be labeled using standard techniques. (In some cases, it may not be necessary to label probes directly, existing methods for the detection of double-stranded DNA in the presence of single-stranded analogues). However, more conventionally, the probes are labeled directly using isotopes, most often 32 p, by translation of cut or else treatment with a kinase. Alternatively, the DNA may be linked to fluorescent residues, such as a fluorescein or dansyl residue; enzymes, such as horseradish peroxidase or alkaline phosphatase; or chromophoric substances.

C. Kits
The method of the present invention for the detection of L. monocytogenes in biological samples can be conveniently carried out using reagent kits containing the appropriate probes. The probes can be labeled by different techniques, as described above.

The probes are denatured just before hybridization, for example by incubation in a boiling water bath, then by rapid cooling in a mixture of water and ice.

If the probes are to be marked with
32 means of P, they are generally supplied in the unlabeled state due to the short half-life of the marker. However, DNA, labeled with an enzyme or with a fluorescent body can be provided in labeled form.

The kit may additionally contain additional reagents in suitable containers for extracting DNA from the cells present in the sample, digesting the DNA with a suitable restriction enzyme, and separating the fractions on gels. serving as supports. Typical carrier gels include polyacrylamide and agarose.

The nature and quantity of the reagents and supports depend on the convenience and the design of the particular labeling method used, on the type of sample and on the quantity of samples analyzed. These variants are within the skill of the art, and are provided for convenience. The kit also contains instructions for carrying out the analysis.

D. Example of separation process
Preparation of the protein called hemolysin and antisera
The gene encoding L. hemolysin

monocytogenes 10403 was investigated using the immunological reactivity of the encoded protein vis-à-vis the antisera produced against the secreted soluble protein. (The frequent presence of soluble hemolysin in L. monocytogenes has been demonstrated by Njokubi
Obi et al., J Bacteriol (1963) 86: 1-8, who tested the hemolytic activity of 112 strains of
L. monocytogenes on sheep blood agar plates. 91% of the strains exhibited such activity. Hemolysin has been shown to be generally part of sulfhydryl-activated cytolysins (Smyth, CJ, et al., In Bacterial
Toxins and Cell Membranes (1978), Jeljaszewicz et al., Eds., Academic Press Inc., NY, pages 129183)).

The 60 kd protein exhibiting hemolysin activity, originating from this particular strain, was purified from L. monocytogenes 10403 cultivated overnight in 2 liters of EHI broth which were dialyzed, starting from broth
BHI concentrated (4x) overnight at 4 "C to remove large proteins (pore diameter 2.4 Nananeters). The culture supernatant was collected by centrifugation at 10,000 g and the supernatant was adjusted using of 10 mM EDTA and 0.02 X of sodium azide. Crystalline sodium sulphate was added at 70% saturation with slow stirring, then precipitated overnight at 4 C. The precipitate was collected by centrifugation at 10,000 g for 10 10 minutes at pH 7.2, and resuspended in distilled water The suspension was dialyzed against 100 mM tris.

The resuspended protein has been shown to have hemolysin activity using the method of Kingdon, GC, et al., Infect
Immunol (1970) 1: 356-362 using washed red cells from sheep in 1% suspension.

 The resuspended protein containing hemolysin was then subjected to gel electrophoresis by suspension in lysis buffer (tris HCl 125 mM, pH 6.8, DSS 2%, glycerol 10%, sea captoethanol 0, 7 M and bromophenol blue 0.003%) and treated with steam for 10 minutes before submitting the samples to electrophoresis according to the conventional Laemmli technique. The separated proteins were isolated from the gel, if necessary, or else transferred to nitrocellulose by electrophoresis at 500 mA for 3 hours.

 Only one main protein band was observed in the supernatant; it exhibited electrophoretic mobility corresponding to a peptide of 60 kd. This 60 kd peptide exhibited hemolytic activity in addition, red cells lysed with the crude supernatant were associated with a 60 60 kd peptide, and antisera produced against the purified 60 kd protein inhibited the hemolytic activity of the crude supernatant.

 Antisera were produced against the crude supernatant and against the purified 60 kd peptide. The preparation was carried out by conventional methods using doses of injection emulsified in the complete Freund's adjuvant, with subsequent doses of booster injection in the incomplete Freund's adjuvant. Rabbits were injected subcutaneously in several places with approximately 500 mg of crude supernatant protein or 200 mg of purified hemolysin at intervals of two weeks, and were bled one week after the third injection.

Gene preparation
L. monocytogenes 10403 obtained from ML Gray was used as a source of genomic library. Total DNA was prepared as described by Flamm et al, Infect Immunol (1984) (above), digested with Sau3AI and the fragments were separated by agarose gel electrophoresis. The 15 to 23 kb fragments were separated from the gels and ligated to EMBL3A vector DNA (Frischauf, A. et al., J Mol Biol (1983) which was digested with BamHI and EcoRI. ligation was introduced into a phage using the in vitro method of Hohn, B., Methods Enzymol (1979) 68: 209-218) and amplified in E. coli NM535 (Frischauf,
A., et al. (Above)).

Separate colonies of infected E. coli were tested using the plaque uplift technique (Young, RA et al, Proc Natl
Acad Sci USA (1983) 80: 1194-1198) to demonstrate the presence of the 60 kd polypeptide, using the two anti-hemolysin antisera described above.

Several plates were found to react with the antisera and phage from one of the plates,
L5, has been selected.

 Restriction analysis showed that the dimensions of the insert sequence in L5 corresponded to 15.7 kb, and the insert sequence was separated and digested with EcoRI / Sa1I, then cloned into pBR325 digested with EcoRI / Sa1I and transformed into E .

coli LE392 obtained from Anquist, L. Digestion of L5 with SA1I / EcoRI gave three fragments of 5.2 kb (SA1I / EcoRI); 3.5 kb (EcoRI / EcoRI); and 7.0 kb (EcoRI / SalI). The immunological analysis of the spots carried out for the 60 kd polypeptide in the transformed bacteria has shown that the sequence coding for the 60 kd protein is located in the 5.2 kb SalI / EcoRI fragment.

 The plasmid pBR325 containing the 5.2 kb interlayer sequence was called pRF7o2 and the orientation of the interlayer sequence was studied using the TnphoA transposon. This transposon is inserted randomly into the DNA present in other plasmids and carries a part of the alkaline phosphatase gene, which allows the detection of the product obtained by translation. Consequently, colonies containing the appropriate interlayer sequences in the direction of transcription and translation of the invaded gene sequences are detectable by a staining test. The use of this method and the analysis of the DNA of colonies which are are found to detectably detect alkaline phosphatase and to show hemolysin activity has resulted in the map shown in Figure 1.

 The HincII / HindIII portion of the approximately 500 bp coding sequence shown in Figure 1 was cloned into pUC8 digested with HincII / Hindili to obtain the additional plasmid pRF106.

Use of probes
pRF102 and pRF106 are useful as probes for detecting the presence of L. monocytogenes in the presence of other Gram-positive bacteria. Seventeen strains of L. monocytogenes were examined for the presence of a DNA fragment exhibiting homology with pRF106 after extraction of the DNA, digestion with EcoRI and separation by gel electrophoresis.

Under drastic hybridization conditions, as defined above, all of the strains possessed an EcoRI fragment of approximately 13 kb which hybridized with the probe. There were no other hybridized fragments, even under mildly drastic conditions.

The specificity of pRF106, probe used for -L. monocytogenes as opposed to other Gram-positive bacteria, was tested in the manner described in the previous paragraph, the results being presented in FIG. 2. The results concerning the following strains are presented in lanes 1 to 11 of FIGS. 2a and 2b , respectively, for drastic and non-drastic conditions
channel 1, L. murrayi, ATCC 25401
track 2, L. denitrificans, ATCC 14870
lane 3, L. grayi, ATCC 19120
lane 4, L. innocua, ATCC 33090
lane 5, L. seeligeri, ATCC 35967
track 6, L. ivanovii, ATCC 19119
lane 7, L. monocytogenes, 10403
lane 8, L. monocytogenes, D3A
lane 9, L. monocytogenes, Scott A
lane 10, L. monocytogenes, EGD
lane 11, L. monocytogenes, ATCC 19111.

 As shown in FIG. 2a, under drastic conditions corresponding to the definition above, only pathways 7 to 11, containing the DNA digested by EcoRI originating from the strains of L. monocytogenes, 32 showed hybridization with the probe labeled with P.

For use in this analysis, pRFîO6 was marked by the cut-off translation method of
Maniatis, T. et al., Proc Natl Acad Sci
USA (1975) 72: 1184-1188, modified as described by Thomashow, MF, et al., Cell (1980) 19: 729-739.

 As shown in FIG. 1b, under slightly drastic conditions meeting the above definition, the strains of channels 4 to 6 also present hybridization with the pRF106 probe by a small fragment; even under these conditions, the strains tested in channels 1 to 3 did not give hybridization fragments.

These results are consistent with those obtained using immunoreactivity as a criterion. When culture supernatants of the same species of
Listeria than that tested in FIG. 2 were fractionated on a polyacrylamide gel (PAGE) containing 10% DSS, transferred to nitrocellulose and treated with an antiserum produced against the hemolysin of 60 kd purified on gel, the results presented on the figure 3 were obtained
The L. monocytogenes strains presented on channels 7 to 11 showed a clear immunoreactivity with the antisera against the 60 kd protein. There was a cross-reactivity with the antiserants by the supernatants of the strains (lanes 4 to 6) which exhibited hybridization with the probe under slightly drastic conditions. Only L. innocua showed the presence of the only 60 kd protein. L. seeligeri showed the presence of a single protein of 60 ka, while L. ivanovii gave two compounds of 60 kd and 67 kd.

 Negative results, under all drastic conditions, were also obtained when DNA digestion elements from different other Gram-positive bacteria were tested using the pRF106 probe. No DNA hybridization was detectable in the supernatants of B. cereus, B thuringiensis, S. pyrogenes and S. pneumoniae.

 The pRF102 probe has slightly lower specificity, as it recognizes DNA fragments from L. seeligeri, L. innocua et t. ivanovii, in addition to L. monocytogenes, under weakly and strongly drastic conditions. Thus, the pRF106 probe is clearly preferred.

 On January 6, 1988, the plasmid pRF102 transformed into E. coli LE392 was deposited in the American Type Culture Collection, in Rockville, MD, and was admitted in accordance with the Budapest Treaty. The deposit number for this culture is 67599. All restrictions on obtaining this deposit will be lifted irrevocably by the grant of United States patents, based on this application. This filing is made for convenience of the practitioner, and should not be construed as an admission that a written description may be inadequate. On the contrary, the Applicant considers that the written description appearing in this memo fully enables those skilled in the art to practice the present invention. Furthermore, the accessibility of this deposit does not constitute a license to put the present invention into practice in contravention of the rights attributed to the Applicant due to any law on national patents.

 It goes without saying that the present invention has only been described for explanatory purposes, but is in no way limitative, and that numerous modifications can be made without departing from its scope.

Claims (11)

 1. DNA probe useful for detecting the presence of Listeria monocytogenes, characterized in that it comprises the genomic DNA sequence coding for the 60 kd protein secreted by L. monocytogenes 10403 or, at least, a 12 bp fragment thereof, or an equivalent probe.  2. Probe according to claim 1, characterized in that it comprises the DNA interlayer sequence present in pRF102 or its equivalent, or at least a 12 bp fragment thereof.  3. Probe according to claim 1, characterized in that it comprises the DNA interlayer sequence of pRF106 or its equivalent, or even at least a 12 bp fragment thereof.  4. Probe according to claim 1, characterized in that the equivalent probe is the sequence. of genomic DNA coding for the 60 kd protein secreted by a strain of L. monocytogenes other than the strain 10403, or at least a 30 bp fragment thereof.  5. A probe according to claim 1, characterized in that it is conjugated to a marker.  6. Probe according to claim 5, charac  32 terized in that the marker is P.  7. A probe according to claim 1, characterized in that it is pRF106.  8. Method for detecting the presence of Listeria monocytogenes in a biological sample, characterized in that it consists  detecting in the sample the presence or absence of DNA which hybridizes under drastic conditions with a DNA probe comprising the genomic DNA sequence coding for the 60 kd protein secreted by L. monocytogenes 10403, or at least a 30 bp fragment thereof, or an equivalent probe.  9. Method according to claim 8, characterized in that it comprises the steps consisting in carrying out the digestion of the DNA of the sample with a restriction enzyme, in separating the fragments on gel, and in testing the fragments separated in as regards hybridization under drastic conditions with the DNA probe.  10. Method according to claim 9, characterized in that the restriction enzyme is EcoRI.  11. Reagent kit useful for detecting the presence of L. monocytogenes in a sample, characterized in that it comprises the DNA probe according to claim 1, with additional reagents for the preparation of DNA from the sample and the instructions for carrying out the analysis.