RU2812147C1 - Method of differentiating listeria monocytogenes from other listeria spp. dot blot method using conjugated antibodies against inlb pathogenicity factor - Google Patents

Abstract

FIELD: microbiology; biotechnology.

SUBSTANCE: method of identifying and differentiating Listeria monocytogenes is described. Microorganisms are grown on an agar nutrient medium containing 0.02% activated carbon. Imprints of colonies are obtained on a nitrocellulose membrane. Colony fingerprints are visualized using polyclonal monospecific antibodies against internalin B conjugated to horseradish peroxidase. The invention can be used for sanitary and epidemiological control of food contamination.

EFFECT: invention makes it possible to differentiate Listeria monocytogenes colonies from other microorganisms and representatives of the genus Listeria.

7 cl, 5 dwg, 1 tbl, 6 ex

Description

Technical field

The invention relates to the field of biotechnology and microbiology and concerns a method for identifying Listeria monocytogenes using the dot blot method. The invention makes it possible to differentiate Listeria monocytogenes colonies from other microorganisms and representatives of the genus Listeria and can be used for sanitary and epidemiological control of product contamination.

State of the art

The topic of microbiological food safety has been actively studied for several decades. In recent years, food safety standards have become significantly stricter in many countries (Van Kreijl et al., 2006). Among the many pathogenic and potentially pathogenic bacteria that cause foodborne illnesses, gram-negative microorganisms Yersiniaspp., Salmonelaspp., Shigellaspp ., and among gram-positive bacteria - Listeria spp., attract special attention.

The genus Listeria consists of seventeen species, of which only strains of the species L. monocytogenes are pathogenic to humans, in particular to people with weakened immune systems, as well as to the elderly and pregnant women (Vázquez-Boland et al., 2001). Clinically, generalized listeriosis most often manifests as bacteremia, meningitis or meningoencephalitis, as well as pregnancy-associated infections manifesting as miscarriage or neonatal sepsis. Invasive listeriosis is life-threatening and is the leading cause of foodborne illness leading to hospitalization in European countries (Koopmans et al., 2022). Listeriosis is often caused by eating contaminated foods, particularly soft cheeses, meats and vegetables.

Since the species Listeria monocytogenes is the only representative of the genus Listeria pathogenic for humans, it is necessary to clearly differentiate this species from closely related ones. A variety of methods are known for detecting L. monocytogenes . Traditional methods for detecting L. monocytogenes involve preconcentration and subsequent isolation of colonies on selective media (Lovette et al., 1987; McClain and Lee, 1988). Individual colonies are examined for their morphology, followed by biochemical or serological identification. Analysis can take up to 6-8 days. Currently, the traditional method for detecting L. monocytogenes in foods is the culture method, which involves: (1) adding a primary and secondary enrichment to the sample; (2) a series of biochemical tests that distinguish between different types of Listeria. However, the numerous similarities between L. monocytogenes and L. innocua and other non-pathogenic Listeria species make the detection of L. monocytogenes in food samples very challenging. Selective media such as PALCAM or Oxford agar are not specific for L. monocytogenes , as they also support the growth of other Listeria species and some other bacterial genera. There is a known method for differentiating L. monocytogenes c (RU 2196827 C1), which consists in determining the hydrolysis of lecithin on a nutrient medium, with and without the addition of activated carbon. To 100 ml of GRM-1 medium (Obolensk) add 5 ml of a 10% suspension of egg yolk in physiological solution and determine the hydrolysis of lecithin without additional additives and in the presence of 0.5% activated carbon. L. monocytogenes shows hydrolytic activity against lecithin only in the presence of activated carbon, unlike other species of Listeria. Despite the relative speed and cheapness, this method requires the isolation of a pure culture, followed by subculture on a specific medium and incubation for 48 hours to obtain the result. A faster method is known (RU 2444567 C1), which is also based on lecithinase activity and takes 16-18 hours, but also requires the preliminary isolation of a pure culture of microorganisms. Since easily perishable food products are often contaminated with L. monocytogenes , there has been a need to develop rapid methods for the detection of L. monocytogenes . Among the existing techniques, PCR and MALDI-TOF based methods are known and provide the best option for differentiation of Listeria spp . However, these methods are expensive and require specialized equipment. There are methods for identifying L. Monocytogenes using oligonucleotide probes, and detection is carried out by direct hybridization of the probes with microbe-specific DNA or RNA (Datta et al., 1987), (RU2730658). The disadvantage of these methods is low sensitivity, since at least 10 ⁵ - 10 ⁶ copies of the target nucleic acid are required. This can be compensated for by a combination with amplification of the target sequence, for example using polymerase chain reaction (PCR). A variety of PCR methods for the detection of L. monocytogenes have been described in the literature (Norton, 2002) (patents US4683195; US4683202 and US4965188). In addition, ligase chain reaction (patent WO 89/09835), self-sustaining sequence replication (EP 329822), transcription-based amplification system (EP 310229). However, all of these methods require specific equipment, and the presence of nucleic acids in samples does not always correspond to the presence of viable pathogens.

Another approach to identify L. monocytogenes from closely related species is the use of immunological methods based on the antigen-antibody reaction. The immunological detection methods described for L. monocytogenes are based primarily on those targets that play a role in the pathogenicity of L. monocytogenes . Some of these genes are known to be located next to each other on the chromosome in pathogenicity islands. Since listeriolysin O (hlyA gene) was first described as essential for pathogenicity, most genotypic detection methods are based on this gene. Antibodies against p60 were also used in different formats of ELISA test systems (Yu et al., 2004) (CN105527441A, CN104099299A), dot blotting (Wicckowska-Szakiel et al., 2002; Yu et al., 2004) and PCR (Patent US5932415). Some antibody detection test kits are already commercially available. However, most of these tests demonstrate low sensitivity and specificity. A review by Banada&Bhunia, 2008 (Banada&Bhunia, 2008) describes some of them. It is known to use antibodies against the InlB and ActA proteins in combination with immunomagnetic beads (CN114574448A) or in immunoblotting (Lathrop et al., 2008). However, when grown in standard media, InlB and ActA are produced at low levels, so kits using antibodies against InlB have low sensitivity. Other immunological methods are often limited to sharing cell surface antigens with other Listeria spp. , making it difficult to differentiate L. monocytogenes from non-pathogenic species commonly found in food samples.

Thus, in the prior art there is a need to develop a new rapid method for identifying L. monocytogenes , which will not have these disadvantages. The new identification method eliminates the steps of accumulation and isolation of pure culture, which take several days, is relatively inexpensive, and also provides sensitivity and specificity in relation to L. Monocytogenes strains. The proposed differentiation method combines traditional microbiological methods, characterized by reliability, with immunological analysis, which provides specificity. In this case, the targets for immunological analysis are surface proteins that are absent in other Listeria spp. Specific surface proteins of L. monocytogenes , absent in other Listeria species, are mainly represented by virulence factors. The surface protein internalin B (InlB), is a virulence factor mediating bacterial invasion of nonprofessional phagocytes, and is absent in other Listeria species. InlB is a secreted protein that can be anchored to the cell surface through noncovalent interactions of its C-terminal GW domains with cell wall teichoic acids (Braun et al., 1997). Like many other virulence factors of L. monocytogenes , the InlB gene is part of a pathogenicity island controlled by the master regulator PrfA (delas Herase et al., 2011). PrfA activity is reduced when L. monocytogenes is cultured on rich medium unless the medium is supplemented with activated carbon or other hydrophobic adsorbent. Hydrophobic adsorbents activate PrfA and induce the expression of PrfA-controlled virulence factors, including InlB (Ermolaeva et al., 2004; Krypotouet al., 2019).

Disclosure of the invention

The technical objective of the claimed invention is an inexpensive and fast method for identifying L. Monocytogenes .

The technical result consists in creating a method for differentiating L. monocytogenes from other species of Listeria spp . the dot blot method, which allows you to obtain results within 12-26 hours from the receipt of food samples to the laboratory and does not require preliminary accumulation and isolation of pure culture and use.

This technical result is achieved by creating a method for differentiating the culture of Listeria monocytogenes from other species of Listeria spp ., which includes growing the test bacteria on a solid nutrient medium containing activated carbon, obtaining an imprint of the colonies on a nitrocellulose membrane, subsequent fixation of the membrane in chloroform, incubation with polyclonal monospecific antibodies against internalin B, conjugated to peroxidase and visualization of activity in the presence of a substrate. At the same time, 0.02% activated carbon was added to the solid nutrient medium of the brain heart extract for growing bacteria. In addition, horseradish peroxidase (HRP) was used as a peroxidase.

In addition, the specified technical result is achieved by the fact that the use of a method for differentiating a culture of Listeria monocytogenes from other species of Listeria spp has been developed. for assessing the contamination of food and clinical samples.

Brief description of drawings

In fig. 1 scheme for the production of polyclonal monospecific antibodies against InlB conjugated with horseradish peroxidase.

In fig. Figure 2 shows a scheme for the determination of Listeria monocytogenes in food samples by dot blotting.

Figure 3 shows a dot immunoassay with a-InlB-IgG-HRP conjugate (purified InlB-specific IgG conjugated with HRP).

A - imprints of membranes of L. monocytogenes EGDe colonies grown on BHI agar (with the addition of 0.2% carbon);

B - dot immunoassay of the membrane with the a-InlB-IgG-HRP conjugate.

In fig. Figure 4 shows the verification of the dot blot results.

L. monocytogenes EGDe and L. innocua SLCC 3379 were taken in a 1:1 ratio and grown on BHIC agar for 24 hours. Imprinted colonies were selected before washing, then the membrane was treated with a-InlB-IgG-HRP conjugate as described above. To identify L. monocytogenes, PCR was performed using primers specific for the inlB gene.

A - prints of colonies grown on BHIC agar;

B - results of dot immunoassay before imprinting on the membrane;

B - PCR products were separated on a 1% agarose gel; the inlB gene fragment has a size of 872 bp. The numbers indicate individual colonies.

In fig. Figure 5 shows the detection of L. monocytogenes in artificially inoculated milk samples.

Raw milk samples were inoculated with L. Monocytogenes EGDe to the indicated concentrations. Samples of 100 μl were applied to selective PALCAM medium and non-selective BHIC agar.

Membership of the L. monocytogenes species was confirmed by PCR for bacteria grown on PALCAM agar and by a developed dot blot immunoassay for bacteria grown on BHIC agar.

Implementation of the invention

The method proposed by the authors for differentiating L. monocytogenes in food products is as follows:

1. Inoculate samples of food products onto a solid nutrient medium of brain heart extract containing 0.02% activated carbon, which makes it possible to increase the expression of InlB in the medium and thus distinguish the pathogenic species of Listeria L.monocytogenes not only from bacteria of another genus, but also from non-pathogenic listeria, such as Listeriainnocua and Listeriaivanovii .

2. Next, an imprint of the colonies is made from the solid nutrient medium onto a nitrocellulose membrane, followed by fixation in chloroform.

3. The nitrocellulose membrane is then incubated with polyclonal monospecific anti-InlB antibodies conjugated to horseradish peroxidase (HRP).

4. Next, a series of washes are performed to remove excess antibodies and visualization using horseradish peroxide substrate (TMB).

Dot blot, on the one hand, combines the immunoblotting technique (the use of a membrane as an antigen carrier and the ability to evaluate all colonies on a solid nutrient medium), on the other hand, the simplicity of enzyme immunoassay, since there is no need for special sample preparation, protein electrophoresis and subsequent blotting of the analyzed proteins mixtures. Thus, the method used will simplify both sample preparation, since it is possible to analyze all colonies at once on a solid nutrient medium, and subsequent screening procedures.

The proposed differentiation method can be used as an independent method for the identification and differentiation of Listeria monocytogenes, or in combination with other identification methods, for example in combination with PCR methods or biochemical identification.

The invention can be made in the form of a test system containing specific anti-InlB antibodies conjugated to HRP.

The implementation of the invention is confirmed by examples

Example 1. Preparation of hyperimmune sera.

To obtain hyperimmune sera, recombinant idInlB (amino acid residues 36-321) was used as an antigen. Three rabbits of the Soviet Chinchilla breed weighing about 3-4 kg were immunized. The first immunization was carried out intravenously (0.25 mg of antigen), subcutaneously (0.375 mg + 0.5 ml of complete Freund's adjuvant (PAF), intramuscularly in the paws (0.375 mg + 0.5 ml of incomplete Freund's adjuvant (NAF)). A month later and The immunization cycle was repeated for two weeks, but without intramuscular injection. After two months, intravenous and intramuscular injections were carried out. The serum titer was assessed in an enzyme-linked immunosorbent assay. The immunization scheme for rabbits to obtain hyperimmune sera, indicating the amount of antigen, is presented in Table 1.

Table 1. Immunization scheme for rabbits to obtain hyperimmune serum to InlB. PAF - Freund's complete adjuvant; NAF - incomplete Freund's adjuvant; IV - intravenously; IM - intramuscularly into the paw, subcutaneously along the lateral surfaces of the body at 5 points on one side. time, weeks No. Volume, ml protein mass, mg Adjuvant method of administration 0 1st immunization 1 0.25 - IV 1 0.375 0.5 ml PAF PC 1 0.375 0.5 ml NAF i/m 4 2nd immunization 1 0.25 - IV 1 0.75 0.5 ml NAF PC 6 3rd immunization 1 0.25 - IV 1 0.75 0.5 ml NAF PC 8 4th immunization 1 0.25 - IV 1 0.375 0.5 ml NAF i/m

Example 2. Preparation of polyclonal monospecific antibodies against InlB.

To obtain a specific immunosorbent, the purified protein corresponding to the region of the internalin domain of internalin B (idInlB) was immobilized on BrCN-Sepharose ® 4B. Hyperimmune rabbit serum was applied to the column with a simmunosorbent. The column was washed with phosphate-buffered saline containing 0.3 M NaCl to remove unbound impurities. InlB-specific antibodies were eluted with 4.5 M MgCl ₂ and dialyzed in phosphate-buffered saline, pH 7.4 (PBS). Purified InlB-specific IgG antibodies were dissolved to a concentration of 5 mg/ml in PBS and stored in 50% glycerol at -20°C. The production scheme is shown in Fig. 1.

Example 3. Preparation of anti-InlB IgGHRP conjugate.

Anti-InlB IgG was subsequently conjugated with horseradish peroxidase using a two-step method and glutaraldehyde as a cross-linker. Stage 1. Binding of horseradish peroxidase to glutaraldehyde. 10 mg of horseradish peroxidase was dissolved in 0.2 ml of 0.1 M phosphate buffer containing 0.15 M NaCl and 1.25% glutaraldehyde, pH 6.8, by stirring overnight at room temperature. Excess glutaraldehyde was removed by dialysis against 0.15 M NaCl. Stage 2. Binding of horseradish peroxidase-glutaraldehyde conjugate to IgG. The resulting horseradish peroxidase solution was diluted to 1 ml using 0.15 M NaCl. Then 1 ml of IgG solution (5 mg/ml) containing 0.15 M NaCl and 0.1 ml of 1 M carbonate buffer, pH 9.5, was added. The mixture was incubated at 4 °C for 24 hours, then 0.1 ml of 0.2 M lysine solution was added to stop the reaction. The mixture was incubated for 2 hours at room temperature and dialyzed overnight against PBS. The a-InlB-IgG-horseradish peroxidase conjugate (a-InlB-IgG-HRP) was precipitated with an equivalent volume of a saturated NH ₄ (SO ₄ ) ₂ solution and dissolved in 1 ml of phosphate buffer. The solution was dialyzed against phosphate buffered saline (PBS). The dialyzed solution was centrifuged at 10,000 g for 30 min, the precipitate was removed, BSA was added to a concentration of 1% and filtered through a filter with a pore diameter of 0.2 μm. The resulting a-InlB-IgG - HRP conjugate is stored at -20 °C.

Example 4. Determination of Listeria monocytogenes by dot blot.

From a plate with a pure culture of L. monocytogenes, a single colony is loop-cultured into liquid BHI medium. Incubate at 37 °C in a thermostatic shaker at 180 rpm for 18 hours. Ten-fold dilutions are made from the resulting overnight cultures. Sow 100 μl of the sixth, seventh and eighth dilutions on BHI agar plates containing 0.2% activated carbon (BHIC) for 24 hours. The grown colonies are imprinted on a nitrocellulose membrane (Bio-Rad, USA). The membrane is dried and then immersed in chloroform for 15 minutes to fix. The membrane is then dried to remove residual chloroform and incubated for 30 minutes in blocking buffer containing 2% bovine serum albumin in phosphate-buffered saline, pH 7.4 (PBS) at room temperature. The developed a-InlB-IgG-HRP conjugate is diluted 1:4000 in Tris-buffered saline solution with the addition of 0.05% nonionic surfactant Tween-20 (TTBS) and the membrane is incubated in it at room temperature for 1 hour, using a shaker with speed 150 rpm. The membrane is washed 3 times for 5 minutes each with TTBS. Colonies are detected using TMB substrate. The reaction is stopped by washing the membranes with distilled water. The general definition scheme is presented in Fig. 2. A color change from light to dark purple indicates a positive reaction and the presence of colonies of the species L. monocytogenes (Fig. 3).

Example 5. Evaluation of the effectiveness of the proposed method in mixed crops.

The species closest to L. monocytogenes is L. innocua , which is also commonly found in dairy products. To evaluate the potential of InlB as a marker for differentiating L. monocytogenes from L. innocua in a mixed culture, we took overnight cultures of L. monocytogenes EGDe and L. innocua SLCC3379 in ratios of 1:1, 1:5, 1:10 and applied the mixed cultures on BHI agar containing 0.02% charcoal. The colonies of the two species were morphologically indistinguishable, but dot immunoassay with a-InlB-IgG-HRP conjugate applied to the colony fingerprints allowed the colonies to be unambiguously identified as positive. The proportion of positive colonies corresponded to the percentage of L. monocytogenes in the mixed culture. To confirm the results, positive and negative colonies were tested using PCR with primers InlBF (5'-aca-agc-gga-tcc-tat-cac-tgt-gcc-3') and InlBR (5'-tgt-aaa-gct- ttt-tca-gtg-gtt-ggg-3'). Only colonies that were positive in the dot immunoassay gave a signal in the PCR assay, confirming the specificity of the assay (Fig. 4).

Example 6. Evaluation of the effectiveness of the proposed approach using artificially inoculated milk.

An overnight culture of L. monocytogenes strain EGDe is concentrated by centrifugation, washed and resuspended in phosphate-buffered saline (PBS). The suspension, taken at a concentration of 10 ⁹ CFU/ml in accordance with the McFarland standard, was diluted to 10 ³ and 10 ⁴ CFU/ml in PBS. Bacterial suspensions were mixed with raw milk samples in a ratio of 1:9 (bacterial suspension: milk). Samples were subcultured onto BHI agar containing 0.2% charcoal. Dot immunoassay is carried out as described in example 4. Uninoculated milk samples are used as a control. The absence of L. monocytogenes in milk is confirmed by plating on PALCAM agar medium (Fig. 5).

Industrial applicability

All the examples provided confirm the effectiveness of the proposed method for differentiating L. monocytogenes and its industrial applicability.

Literature:

1. Egor V. Kalinin, Yaroslava M. Chalenko, Parfait Kezimana, Yaroslav M. Stanishevskyi, Svetlana A. Ermolaeva. Combination of growth conditions and InlB-specific dot-immunoassay for rapid detection of Listeria monocytogenes in raw milk. Journal of Dairy Science, 2023, https://doi.org/10.3168/jds.2022-21997.

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Claims (7)

1. A method for differentiating a culture of Listeria monocytogenes from other species of Listeria spp ., including growing test bacteria on a solid nutrient medium containing activated carbon, obtaining an imprint of colonies on a membrane, subsequent fixation of the membrane in chloroform, incubation with polyclonal monospecific antibodies and visualization of HRP activity in the presence of a substrate. 2. The differentiation method according to claim 1, characterized in that it includes the growth of Listeria monocytogenes on a solid nutrient medium of brain heart extract with the addition of 0.02% activated carbon. 3. The differentiation method according to claim 2, characterized in that to identify Listeria monocytogenes, prints are made on a nitrocellulose membrane. 4. The differentiation method according to claim 3, characterized in that when incubated with polyclonal monospecific antibodies, an antigen-antibody reaction occurs. 5. The differentiation method according to claim 4, characterized in that it is performed using antibodies against internalin B. 6. The differentiation method according to claim 5, characterized in that it is performed using HRP-conjugated polyclonal monospecific antibodies against internalin B. 7. Application of the differentiation method according to claim 1 to assess the contamination of food products.