Method for detecting and counting low concentrations of Listeria

Abstract

This document relates to a method for detecting and / or counting Listeria in a sample, such as Listeria monocytogenes, the method comprising culturing a sample that may contain Listeria in a medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl.

Description

Technical Field

[0001] This document relates to methods and means for detecting and / or counting Listeria in a sample, such as Listeria monocytogenes, wherein the method comprises culturing the sample that may contain Listeria in a selective growth medium, thereby allowing for a more rapid and sensitive detection of Listeria. Background Art

[0002] Listeria monocytogenes is a foodborne pathogen that causes thousands of cases of listeriosis each year. In Europe alone, approximately 1500 cases are reported each year (EFSA 2018). Although the number of cases of listeriosis is lower than that of salmonellosis and campylobacteriosis, the case fatality rate of listeriosis is higher.

[0003] Listeria monocytogenes is the pathogenic bacterial species that causes the disease listeriosis. It is a Gram-positive facultative anaerobe that can survive in the presence or absence of oxygen. It can grow and multiply inside host cells and is one of the most virulent foodborne pathogens. Listeriosis infections in high-risk individuals can be fatal.

[0004] Listeria monocytogenes (L. monocytogenes) is a ubiquitous bacterium, which means it is present in many different ecological niches such as soil, water, food, food production environments, animals, and humans. It readily forms reservoirs in food production facilities, even in facilities with high hygiene standards and implementing HACCP systems. EFSA has reported the prevalence of Listeria monocytogenes in specific categories of ready-to-eat foods to be 0.5 - 6%. The prevalence is highest in fish and seafood products (EFSA 2018).

[0005] Listeria monocytogenes has the ability to grow at temperatures as low as 0 °C and even as low as -1.5 °C, which allows for proliferation at typical refrigeration temperatures, thereby greatly increasing its ability to evade human food control.

[0006] The people who are most susceptible to becoming infected with Listeria are, in particular, the elderly, immunocompromised individuals, and pregnant women (fetuses). Even a small amount of bacteria, as low as 100 - 1000 colony forming units (cfu) / gram, can cause disease in high-risk populations, and the infection can be fatal and even cause miscarriage in the case of pregnant women. Healthy people are generally not affected by Listeria monocytogenes, but high doses can cause usually harmless mild symptoms. During the past few decades, Norway has recorded 20 - 50 cases of listeriosis each year. When measured per inhabitant, this is comparable to the frequency in other parts of Europe.

[0007] The consequences of listeriosis are very serious and, in severe cases, can lead to death and the social costs are very high. *Listeria monocytogenes* can infect the host's brain, spinal meninges, and / or bloodstream.

[0008] The combination of the following facts has led to strong interest in *Listeria monocytogenes* in legislation and among customers, including buyers of Norwegian fish: listeriosis is a very serious disease for humans; Listeria in food occurs relatively frequently; and it is possible to keep the number of *Listeria monocytogenes* in food below the pathogenic level by relatively simple means.

[0009] Listeria can be analyzed qualitatively or quantitatively. ISO methods 11290-1 (qualitative) and 11290-2 (quantitative) are the standard methods in legislation. However, the detection limits of these methods are 1 cfu / 25 g and 10 cfu / g, respectively, which are not detailed enough for the analysis of process steps where contamination may occur (e.g., during fish slaughter and filleting before the product is sent to the market). People engaged in the fishery trade have described the limitations of the ISO methods in this way: "If the method were used to measure blood pressure, the result would be that there is blood pressure below 1000. This information is not detailed enough to be useful. In the same way, methods are needed to measure the concentration of Listeria in the range between 0.04 cfu / g and 10 cfu / g.

[0010] Listeria is able to grow in food during refrigeration. How high the concentration will be when the consumer eats the food will depend on product conditions, storage conditions, process conditions, and the vulnerability of the consumer who eats it. In some products, such as heat-treated meat intended for use in salads or fish used for sushi or sashimi, if the initial concentration is higher than 2 cfu / g, the concentration can reach a level that poses a food health risk to healthy consumers under reasonably foreseeable conditions, but for vulnerable consumers, the initial concentration needs to be below 0.2 cfu / g. In other products, the concentration can be higher, but in most cases, the concentration acceptable from a food safety perspective is between 0.2 cfu / g and 5 cfu / g. The ISO methods do not distinguish between these concentrations because levels below 10 cfu / g cannot be counted unless many plates, filtered samples, or samples with a higher concentration in the culture medium are obtained in other ways.

[0011] The ISO methods were recently revised (2017). The changes compared to the previous version (1997) are as follows:

[0012] Compared with ISO 11290 2:1998, the main changes are as follows.

[0013] · - The enumeration of Listeria monocytogenes has been modified as listed below.

[0014] · - Primary suspensions in buffered peptone water, semi-Fraser broth with or without supplements, and all applicable diluents mentioned in ISO 6887 (all parts).

[0015] · - The resuscitation step has been removed.

[0016] · - The microscopic aspects, catalase, and CAMP tests for confirmation are optional.

[0017] · - Includes new performance characteristics.

[0018] · - In addition, the enumeration of the genus Listeria has been included in the scope and the title has been changed accordingly.

[0019] In these revisions, although the relevance is known, the enumeration of Listeria monocytogenes concentrations below 10 cfu / g has not been addressed in the revisions.

[0020] The qualitative ISO method 11290-1 includes the steps where Listeria is given optimal growth conditions and plated on a selective agar medium, followed by a confirmation step to qualitatively determine the presence of Listeria in the sample. However, using this qualitative method, the actual number of bacteria in the sample cannot be determined, nor can samples with high or low bacterial numbers be distinguished. This qualitative ISO method takes one week to perform after the laboratory has received the sample.

[0021] In the quantitative ISO method 11290-2, a 10-gram sample is analyzed. One problem with this quantitative determination is that its detection limit is 10 cfu / g, which is insufficient. If the qualitative test is positive but the quantitative test is negative, it can only be concluded that the product has less than 10 cfu / g of Listeria, but it cannot be determined whether there are only 5 cfu or none at all.

[0022] The first step in the quantitative ISO method 11290-2 is to plate serial dilutions of the sample onto the selective growth medium ALOA, and the second step involves confirmation of rhamnose, xylose, Gram, and catalase tests. Lithium chloride is used as the selection pressure in the ALOA medium, and a color indicator is used to indicate presumptive colonies. The confirmation using rhamnose and xylose is based on the fact that only a few bacteria can utilize these sugars. Thus, the quantitative ISO method includes the steps of culturing the sample on a selective agar plate medium for two days, followed by confirmation. This ISO method takes 5 days to perform after the laboratory has received the sample.

[0023] Generally, a combination of the two ISO methods described above is used. However, when it comes to the issue of Listeria in fresh salmon, the combination of these two methods will be insufficient because it is not necessarily required that the fish be completely free of Listeria to be considered safe. There can also be considerable variation within a product batch, and the samples taken may not necessarily be representative of the entire batch.

[0024] One problem with the current method is the relatively low sample volume that can be analyzed, which means that if the bacteria are unevenly distributed in the sample to be tested, bacteria may be missed and the sample is evaluated as Listeria negative.

[0025] European food legislation has set food safety standards for Listeria monocytogenes in ready-to-eat foods. In essence, at any time during the shelf life, the concentration of Listeria monocytogenes should not exceed 100 cfu / g (EU Regulation 2073 / 2005). Some countries have zero tolerance for Listeria monocytogenes, at least in part due to the challenges in low-level Listeria counting, and this option has been discussed in several countries. However, 1) the relatively high prevalence of Listeria monocytogenes in food, 2) the challenge of removing Listeria monocytogenes from production facilities, and 3) the high concentration before the likelihood of listeriosis increases all indicate that zero tolerance is not realistic.

[0026] A concentration of 100 cfu / g is considered a reasonable trade-off between food safety and actual production. It is agreed that this limit is sufficient to avoid listeriosis cases, provided that the analytical method is sensitive and precise enough to detect and count Listeria monocytogenes within the required concentration range, and that the bacteria are distributed evenly enough in the batch so that the samples taken from the batch actually contain a representative concentration of the bacteria.

[0027] Unfortunately, none of these conditions are met. Studies on naturally contaminated food in commercial production have shown that only a few Listeria monocytogenes (usually 1 to 2 per 100 g to per g) are transferred to the product during processing. These bacteria will grow but will remain in the food and form clusters in the food during storage. The sampling plan given in the microbiological criteria of EU Regulation 2073 / 2005 stipulates 5 - 10 samples of 25 g each, but it is doubtful whether this small sample size can cover the variability in concentration. Secondly, food sampling is mainly carried out at the processing level, which means that the concentration of Listeria monocytogenes is much lower than 10 cfu / g. The current detection levels of the reference ISO methods (ISO 11290-1 and 11290-2) are 1 cfu / 25 g in qualitative analysis and 10 cfu / g in enumeration. This means that samples that are positive in qualitative analysis but negative in enumeration are in the range of 0.04 - 9.9 cfu / g. This is a large range, resulting in some food suppliers, customers, and competent authorities having set food recovery limits at the time of detection (0.04 cfu / g = 1 cfu / 25 g), even though this results in food losses, but this represents the minimum risk. Others set the limit at 10 cfu / g, even though these concentrations are too high to ensure that the limit of 100 cfu / g will not be exceeded at the end of the shelf life.

[0028] In summary, when sampling must be carried out at the process level before the product is placed on the market, the implementation of food safety standards for Listeria monocytogenes in ready-to-eat foods is challenging and requires a more precise method in the range of 0.04 - 10 cfu / g.

[0029] Therefore, a faster and even more sensitive analysis is needed, which can also handle larger sample volumes to detect Listeria, such as Listeria monocytogenes, in food samples and swabs used for sampling equipment surfaces.

[0030] Therefore, the object of the present invention is to overcome or at least mitigate one or more of the problems described herein. Summary of the Invention

[0031] This document relates to a method for detecting Listeria rhamnosus in a sample, such as Listeria monocytogenes, the method comprising or consisting of the following steps:

[0032] i) preparing a suspension of the sample that may contain Listeria in a first culture medium, the first culture medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

[0033] ii) incubating the suspension under conditions allowing the growth of Listeria; and

[0034] iii) Identify positive samples.

[0035] The method may further comprise counting Listeria rhamnosus, wherein the method comprises the step of ia) transferring the suspension prepared in step i) to a multi-well tray, and step iv) performed after step iii) of calculating the concentration of the Listeria in the sample, for example by using the most probable number method.

[0036] Thus, a method for counting Listeria rhamnosus, such as Listeria monocytogenes, comprises or consists of the following steps:

[0037] i) Prepare a suspension of a sample that may contain Listeria in a first culture medium, the first culture medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

[0038] ia) Transfer the suspension obtained in step i) to a multi-well plate;

[0039] ii) Incubate the suspension under conditions that allow Listeria to grow;

[0040] iii) Identify positive samples; and

[0041] iv) Calculate the concentration of the Listeria in the sample, for example by using the most probable number method.

[0042] The method of the present document may further comprise the step of confirming the presence of Listeria monocytogenes by differentiating Listeria monocytogenes from other Listeria species, for example by plating the positive samples identified in step iii) on a second growth medium such as ALOA medium, by using molecular methods, such as by polymerase chain reaction or in situ hybridization ELISA (enzyme-linked immunosorbent assay), VITEC, or API (analytical profile index). This confirmation step is performed after step iii) in the qualitative method and after step iv) in the quantitative method.

[0043] A sample that may contain Listeria rhamnosus can be processed by dividing the sample into small pieces, for example by homogenization, slicing, and / or chopping, before performing step i) and / or in the first culture medium of step i).

[0044] The antibiotic(s) can be one or more of nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide, amphotericin B, preferably a combination of two or more of any of the antibiotics.

[0045] The pH color indicator can be phenol red.

[0046] LiCl may be present in an amount of about 5 - 17 g / l, such as about 7 - 13 g / l, such as about 10 g / l.

[0047] Rhamnose may be present in an amount of about 5 - 17 g / l, such as about 7 - 13 g / l, such as about 10 g / l.

[0048] The sample may be a food sample, an environmental sample, or a sample from an animal such as a human, such as a tissue sample or a fecal sample.

[0049] The food sample may be raw or processed meat, poultry or fish products, vegetables or ready-to-eat food products.

[0050] The environmental sample may be a water sample, a dirt sample or a food industry environmental sample, such as a surface swab sample.

[0051] The method may be performed in a closed system.

[0052] This document also relates to a culture medium for growing and / or detecting Listeria rhamnosus fermentans, such as Listeria monocytogenes, the culture medium comprising:

[0053] a) rhamnose at a concentration of about 5 - 15 g / l, such as about 7 - 13 g / l, such as about 10 g / l;

[0054] b) one or more antibiotics, such as nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide, amphotericin B, preferably at least two of these antibiotics;

[0055] c) a pH color indicator, such as phenol red; and

[0056] d) LiCl at a concentration of about 5 - 17 g / l, such as about 7 - 13 g / l, such as about 10 g / l.

[0057] This document also relates to a kit for detecting and / or counting Listeria rhamnosus fermentans, such as Listeria monocytogenes, in a sample, the kit comprising:

[0058] a) a container containing a culture medium comprising or consisting of rhamnose, one or more antibiotics, a pH color indicator and LiCl;

[0059] b) a container or a porous tray for culturing a sample that may contain Listeria;

[0060] c) an optional colorimetric table for identifying positive samples; and

[0061] d) optional instructions for use.

[0062] The component concentrations of the culture medium and the culture medium of the kit are the same as the component concentrations in the first culture medium described herein.

[0063] This document also discloses a computer-implemented calculator for displaying a prediction of the growth of Listeria, such as Listeria monocytogenes, based on the detection and counting of colony-forming units in a sample by the method according to this document. The calculator includes:

[0064] a) An input device, such as a keyboard or microphone;

[0065] a) An output device, such as a display, a computer or mobile phone screen, or a speaker;

[0066] b) Software in a downloadable or memory, such as an application on a smartphone or a web page;

[0067] Wherein the software accepts an input of any number of colony-forming units in the range of 0.04 cfu / g to 1 cfu / g present in the sample.

[0068] Other features and advantages of the present invention will be apparent from the following detailed description, drawings, examples, and claims.

[0069] Definitions

[0070] The Most Probable Number (MPN) is a method for estimating the concentration of viable microorganisms in a sample by means of growing a liquid culture medium replicated at x-fold dilutions (such as 15x, 10x, or 2x dilutions). The Most Probable Number can be used to estimate microbial populations in soil, water, agricultural products, and especially in samples containing particulate matter that would interfere with plate counting methods.

[0071] Colony-forming unit (cfu) is a unit for estimating the number of viable bacterial or fungal cells in a sample. Viable is defined as the ability to reproduce by binary fission under controlled conditions. Counting using colony-forming units requires culturing the microorganisms and only counting the live cells. The visual appearance of colonies in a cell culture requires significant growth.

[0072] "Cultivating / cultivation", etc. refer to growing or maintaining live cells in a solid or liquid growth or culture medium designed to support the growth of microorganisms or cells. The growth or culture medium contains nutrients essential and relevant for the growth of the expected corresponding microorganisms or cells.

[0073] The ALOA (Listeria agar medium after Ottaviani and Augusti) medium is a selective chromogenic agar medium for the selective and differential isolation of Listeria monocytogenes. The medium is described in the ISO method.

[0074] Polymerase Chain Reaction (PCR) is a method for preparing multiple copies of a DNA sequence, involving repeated reactions using a polymerase.

[0075] In Situ Hybridization ISH is a technique that allows for the precise localization of specific nucleic acid segments within tissue sections. The basic basis of ISH is that if the nucleic acids are sufficiently preserved in a histological specimen, the nucleic acids can be detected by applying a nucleic acid complementary strand attached to a reporter molecule.

[0076] The term "first medium" in the context of this document refers to a medium containing rhamnose, one or more antibiotics, a pH color indicator, and LiCl. An example of the first medium is "SensiList broth", as disclosed elsewhere in this document.

[0077] "Listeria rhamnosus" as used herein refers to Listeria species that are capable of fermenting rhamnose. Examples of such bacteria include Listeria monocytogenes, L. innocua, and L. welshimeri. Description of the Drawings

[0078] Figure 1 Shows an overview of the steps of the method disclosed in this document. The left - hand track is the steps when using the method qualitatively, while the right - hand track shows the steps when using the method quantitatively.

[0079] Figure 2 Shows the stacking pattern used when testing the sensitivity of SensiList broth.

[0080] Figure 3 Shows the results of the sensitivity test of the method of this document.

[0081] Figure 4 Shows the results of the stability test of SensiList broth.

[0082] Figure 5 Shows the results of the tests performed in companies with Listeria monocytogenes in the production facility. Samples 1, 3, 5, and 6 are production environment samples. Samples 3 and 5 turned yellow after incubation. Samples 2 and 4 are 100 g salmon samples.

[0083] Figure 6Shows the results of the analysis in Example 3.

[0084] Figure 7 Shows the results from the presumptive positive samples in Example 3. Detailed implementation

[0085] The present inventors have developed a new method for detecting and / or enumerating Listeria rhamnosus, which has high sensitivity and is at least as accurate as existing methods, which is performed faster and has a detection limit lower than the current ISO method. The method can detect and enumerate very small amounts of Listeria rhamnosus, such as Listeria monocytogenes and Listeria innocua, in large samples or pooled samples (e.g., food samples, such as salmon or chicken), as well as in surface swab samples.

[0086] The principle of the method of this document is to inoculate a sample that may contain Listeria into a culture (growth) medium that is selective for Listeria rhamnosus, such as Listeria monocytogenes and Listeria innocua. Both of these bacteria are able to ferment rhamnose into organic acids, which reduces the pH of the medium. Due to the presence of a pH indicator (e.g., phenol red) in the medium, the color of the culture changes, and the reaction is detected. The change in the color of the culture is sufficient to detect Listeria, limited to Listeria monocytogenes and Listeria innocua. Confirmatory studies can then be performed to distinguish between Listeria monocytogenes and Listeria innocua, for example, by growing the bacteria on ALOA medium that is selective for Listeria monocytogenes. The medium also contains LiCl, which allows for the selection pressure for Listeria and antibiotics depending on the sample type, and it is known that there are bacteria in such samples that are not Listeria.

[0087] Therefore, this document discloses a method for detecting Listeria rhamnosus, such as Listeria monocytogenes, in a sample, the method comprising or consisting of the following steps:

[0088] i) Prepare a suspension of the sample that may contain Listeria in a first medium that contains rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

[0089] ii) Incubate the suspension under conditions that allow Listeria to grow; and

[0090] iii) Identify positive samples (i.e., samples containing Listeria).

[0091] If a quantitative analysis of the number of Listeria monocytogenes in a sample is required, the above method comprises the additional steps of ia) transferring the suspension obtained in step i) to a multi-well tray, and iv) calculating the concentration of said Listeria in said sample, for example by using the most probable number method. Thus, a method for detecting and counting Listeria in a sample comprises or consists of the following steps:

[0092] i) preparing a suspension of the sample that may contain Listeria in a first culture medium, said first culture medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

[0093] ia) transferring the suspension obtained in step i) to a multi-well plate;

[0094] ii) incubating said suspension under conditions that allow the growth of Listeria;

[0095] iii) identifying positive samples (i.e., samples containing Listeria); and

[0096] iv) calculating the concentration of said Listeria in said sample, for example by using the most probable number method.

[0097] To confirm the presence of Listeria monocytogenes in the positive samples identified in the above method (i.e., in samples containing Listeria monocytogenes), the method comprises this confirmation as a further step to be performed after step ii) and after step iv) in the qualitative method. The confirmation step can be carried out, for example, by plating the positive samples on ALOA medium, by using molecular methods such as PCR or in situ hybridization, ELISA, VITEC, and / or API.

[0098] The ISO 11290-1 and ISO 11290-2 methods basically use the same method steps, but in a different order, and the composition of the culture media used in different steps is also different, which allows the integration of more than one step of the ISO method in the same step of the present method, resulting in the selectivity of the method of this document being very similar to that of the ISO method. However, compared with the ISO reference method, the method of this document builds higher selectivity in the first step of the analysis, which in turn results in fewer presumptive positive samples and a faster identification of samples positive for Listeria, such as Listeria monocytogenes.

[0099] Using the method of this document, it is possible to quantitatively analyze samples up to 125 g with a detection limit of 1 cfu / sample, i.e., 1 cfu / 125 g, and accurately count within a concentration range from 1 cfu / 25 g up to approximately 2000 cfu / g. In the ISO 11290-1 and 11290-2 methods, the sample size is limited to a maximum of 25 g in the qualitative method and to a maximum of 10 g in the quantitative method. Additionally, the detection limit in the ISO methods is 10 cfu / g, unless the method is modified to obtain a more concentrated sample. The selectivity and specificity of the method of this document are the same as those of the reference ISO methods (ISO 11290-1 and 11290-2) currently in use, which means that the number of false negatives and false positives in the method of this document is the same as in the reference methods.

[0100] The method of this document is applicable to the analysis of foods (such as salmon and chicken) and swabs used to collect materials from the surfaces of production facilities. Additionally, the benefit of this method is that sampling and counting can be performed in the production facility, while confirmation and further characterization, such as whole genome sequencing, can be carried out in an external laboratory.

[0101] Another advantage of the method of this document is that sampling and sample preparation can be performed by non-professionals, thus ensuring that food producers or auditors can take responsibility for this part themselves. Negative samples can be evaluated locally, while presumptive positive samples can be sent to the laboratory for confirmation.

[0102] The method disclosed in this document can be used qualitatively and quantitatively (by adding two method steps as described elsewhere in this document), and can be used for pooled samples, such that a single analysis can be used to test compliance with the standards in EU Regulation 2073 / 2005. Due to the excessive growth of false positives, pooled samples are generally more likely to give false negatives, but this is not the case if the method of this document is used quantitatively. In this case, the solution is distributed in many wells, which results in Listeria monocytogenes and false positives being less likely to be in the same chamber as when analyzing the entire solution at once. Therefore, the presence of steps such as steps ia) and iv) described herein, for example, is a significant benefit as it allows for a reduction in the number of false positives.

[0103] Even if the sample has been exposed to abusive temperature conditions during transportation to the laboratory, the method of this document ensures correct counting. This is possible because the start of sample preparation and counting can be carried out together with sampling. This is important because abusive temperature conditions between sampling at the facility and sample preparation in an external laboratory usually lead to an overestimation of the concentration of Listeria in foods and thus to unnecessary waste, recalls, and incorrect decisions.

[0104] Compared to other rapid Listeria detection methods, such as the InSite Listeria test (Hyginia, Watford, UK) and the Path-Chek Hygiene Listeria test (Microgen Bioproducts Ltd., Camberley, UK), the false negative frequency is lower when using the method according to this document. C.T. Schirmer, Solveig Langsrud, Trond Therese Hagtvedt, Even Heir, 2012. Performance of two commercial rapid methods for sampling and detection of Listeria in small-scale cheese-producing and salmon processing environments. Journal of Microbiological Methods, Volume 91, Issue 2, Pages 295 - 300, ISSN 0167-7012, https: / / doi.org / 10.1016 / j.mimet.2012.08.013.)

[0105] ISO methods are given as standards in legislation, and new methods need to be comparable to these methods in terms of specificity and other characteristics related to method validation. For users of analytical methods, the methods need to be cost-effective and deliver results quickly. This is particularly important if the results will influence decisions regarding shipment, recall, additional cleaning, etc. Therefore, an alternative method that meets these criteria and has relevant selectivity and sensitivity is needed.

[0106] The ISO method consists of several steps. The first step is to homogenize the sample in a culture broth and then incubate it in the culture broth (qualitative) or on an agar plate with a specialized growth medium (quantitative method). Over the years, these growth media have been developed and optimized through several steps to obtain

[0107] · Recover stressed cells to avoid false negatives.

[0108] · Improve selectivity by removing bacteria that can also grow on the medium and covering Listeria on the agar plate.

[0109] The growth medium in the ISO method is suitable for counting down to 10 cfu / g and is well-suited for laboratory facilities where open systems such as agar plates can be used. This is not the case in industries where lower concentrations are required and a closed analytical system must be used. It is possible to perform counting in a culture broth using the MPN (Most Probable Number) method, but a way to read the difference between positive and negative wells is needed. The culture broth used for selective enrichment in the ISO method does not allow this. For this reason, if low-level counting using the MPN method should be performed, other growth media are required.

[0110] Although the growth medium in the first step of the ISO method is suitable for detecting and counting Listeria monocytogenes down to 10 cfu / g, presumptive positive colonies still need to be confirmed. In one of the confirmation steps, rhamnose broth is used because Listeria monocytogenes is able to ferment rhamnose and produce acid, which can be detected as a color change of a pH indicator. In the ISO method, there is no selection pressure in the rhamnose broth (which is used later in the ISO method), because only single colonies of isolates that have been adjusted to grow at the incubation temperature and are different from the bacteria in the food can be tested in this way. Therefore, the culture broth does not require stress recovery stimulation.

[0111] In the method of this document ("SensiList" method), a modified version of rhamnose broth is used as the main growth medium and combined with the MPN method to allow counting down to a concentration of 0.2 cfu / g. To achieve this, the rhamnose broth has been designed and adjusted in iterative steps to obtain:

[0112] · Selectivity for Listeria monocytogenes (to avoid false-positive and false-negative bacteria)

[0113] · Detection limit suitable for quantification in the range of 0.2 - 100 cfu / g of food material

[0114] · Stress recovery of bacteria

[0115] · Clear difference between positive and negative results

[0116] · The system needs to be closed to ensure biosafety.

[0117] · The price of the culture medium makes the method cost-effective.

[0118] There are more bacteria that can utilize rhamnose than Listeria. These bacteria should not be able to grow, at least not to a concentration that would give a false-positive signal in the method. Therefore, antibiotics and other selection pressure components such as LiCl used in the initial growth medium in the ISO method have been added to the rhamnose broth used in the initial enrichment step of the method of this document.

[0119] The antibiotic (antibioticum / antibiotics) used in the first culture medium is selected to reduce or avoid false positive results from other rhamnose-fermenting bacteria. Thus, the specific antibiotic used may vary between different sample types. Microorganisms that can give false positive results can be searched for in databases and commercial food samples. Thus, the technician can easily adapt the antibiotic used to the rhamnose-fermenting bacteria that are theoretically present in that specific sample type. Thus, the antibiotic used in the first culture medium is specifically adapted to the sample type and any non-Listeria bacteria that ferment rhamnose that may be present in such sample types.

[0120] The list of antibiotics given in the formulation of the "SensiList" culture medium in this document is specifically adapted to fish samples.

[0121] The recovery of stressed cells involves the cell needing to have sufficient nutrients and good conditions to adapt its metabolism to the growth pattern. Listeria can survive under sub-lethal conditions in a non-growing mode, but this mode cannot be used for detecting bacteria. Thus, the components that cause a selection pressure in the culture medium have been tuned to an optimal state between the conditions for recovery and selectivity. In addition, low concentrations of rich culture medium components, such as meat extract, have been added to the culture medium in order to supply nutrients for stress recovery. This is especially important for samples that contain little organic matter, such as swab samples of production equipment and water.

[0122] The detection principle of the method in this document is the fermentation of rhamnose into acid, which changes the color of the pH indicator. In order to obtain a high enough acid yield, the bacteria need to use rhamnose as a nutrient and energy source, which in turn involves that the amount of other organic substances (including the sample) in the culture medium must be low, otherwise the bacteria will use this as a nutrient and energy source. In addition, the amount of rhamnose must be high enough to allow for the production of sufficient bacteria and acid. The culture medium has been optimized and tested to balance these aspects. It has been found that the amount of rhamnose, the concentration of rhamnose, and the concentration of the acid finally produced are all crucial for the detection of Listeria monocytogenes. The method has been optimized to ensure the correct detection of all samples, even swab samples from water-rich areas.

[0123] The method in this document is generally based on the same principle as the ISO methods (ISO 11290-1 and 11290-2), but the order of the method steps is different and the culture medium has been modified. By implementing these modifications, it has surprisingly been found that the sample volume can be increased, while still maintaining or even increasing the sensitivity of the method, thus allowing for the detection of a smaller number of bacteria in the sample compared to the ISO methods. In addition, the time from sampling to obtaining the result has been greatly reduced.

[0124] The method of this document differs from the ISO method in terms of the steps used, the culture media used in different steps, and the order of the steps. Tables 1 and 2 below list some similarities and differences between the ISO method and the method of this document (Listeria monocytogenes is abbreviated as L. mono).

[0125]

[0126] Table 1

[0127]

[0128] Table 2

[0129] To distinguish between Listeria monocytogenes and Listeria innocua and other false positives that may be caused by other rhamnose-fermenting bacteria, positive samples (i.e., yellow and orange wells if phenol red is used as a pH indicator) can be plated on ALOA medium, and colonies with characteristic regions indicate a positive result for Listeria monocytogenes.

[0130] The method of this document is faster than the ISO method; if classical confirmation tests are used, it takes at most two days to obtain negative samples after going behind, and three days to obtain confirmation results. Using PCR confirmation only takes a few hours. For comparison, the first step in the ISO counting method (i.e., ISO 11290-2) is to plate serial dilutions of the sample onto selective growth medium, and the second step is to confirm rhamnose. This method requires up to 5 ISO detection methods (ISO11290-1, detection level 1 cfu / 25 g), with the following steps: culturing the sample in selective broth for two days, then plating on selective agar, and it takes one week to confirm after the sample is received in the laboratory. In addition, since a larger sample volume can be used, for example, a set of 10 samples can be used in one kit, each sample being 10 grams each. This also results in lower analysis costs for food suppliers.

[0131] Culture Medium

[0132] Three methods are used to make the first culture medium (an example of which is the so-called "SensiList broth" in this document) used in steps i)-iii) of the method of this document selective for Listeria monocytogenes and Listeria innocua.

[0133] Rhamnose is used as a carbon and energy source in the first culture medium. Only a few bacteria can utilize rhamnose, such as Listeria monocytogenes and Listeria innocua. Listeria welshimeri can also ferment rhamnose, but it is a very rare Listeria species compared to Listeria innocua and Listeria monocytogenes. Even if this bacterium grows in a culture medium containing rhamnose, the specificity of the method of the present document is considered to be high enough, at least as high as the currently available methods.

[0134] In the method of the present document, the selectivity of rhamnose has been used in the first step (step ii) of enriching bacteria in the method of the present document.

[0135] Rhamnose is present in the first culture medium at a concentration of at least 5 g / l, such as about 5 - 17 g / l, such as about 7 - 13 g / l, such as about 5 g / l, about 6 g / l, about 7 g / l, about 8 g / l, about 9 g / l, about 10 g / l, about 11 g / l, about 12 g / l, about 13 g / l, about 14 g / l, about 15 g / l, about 16 g / l or about 17 g / l, usually about 10 g / l. Analytical grade rhamnose is an expensive component, and since the volume of rhamnose culture solution required in the method of the present document is higher compared to the ISO method, the price of rhamnose is important not only for detection but also for the price of the kit. Therefore, the use of significantly cheaper food grade rhamnose in this method has been tested, and it has been found that rhamnose of this quality gives the same results as the analytical grade, thus reducing the price of performing the method.

[0136] Lithium chloride (LiCl) is also added to the first culture medium. This component has been shown to increase the selection pressure in Listeria selective enrichment media (including in Fraser broth and ALOA plates, which are used as selective enrichment broth and diagnostic agar plate media respectively, in, for example, the ISO method). Lithium chloride is present in an amount of at least about 5 g / l, such as about 5 - 17 g / l, such as about 7 - 13 g / l, such as about 5 g / l, about 6 g / l, about 7 g / l, about 8 g / l, about 9 g / l, about 10 g / l, about 11 g / l, about 12 g / l, about 13 g / l, about 14 g / l, about 15 g / l, about 16 g / l or about 17 g / l, usually about 10 g / l.

[0137] As explained elsewhere in this text, antibiotics are added to minimize the growth of other Gram-positive bacteria. The antibiotics applicable in the methods of this document include one or more of nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide, amphotericin B. Preferably, a combination of two or more of the antibiotics is used from any of the said antibiotics, for example three, four, and even more preferably, all five are used in combination. The antibiotics are generally used at their normal concentrations, for example as specified in the SensiList culture medium formulation below. The need for antibiotics depends on the selection pressure required for the sample to be analyzed. In addition, if the antibiotics used in the first medium are exchanged with other antibiotics having a similar mode of action, the same specificity is expected.

[0138] By using the three methods described above, a selection pressure is achieved that minimizes the growth of bacteria other than Listeria monocytogenes and Listeria innocua (and other rhamnose-fermenting bacteria that may be present in the sample). To be able to distinguish Listeria monocytogenes and Listeria innocua from other rhamnose-fermenting bacteria, further confirmation is required, for example by plating on diagnostic agar (such as ALOA agar), performing polymerase chain reaction or in situ hybridization analysis on specific Listeria monocytogenes genes, or by using methods such as ELISA, VITEC, API and / or performing a MaldiToff test.

[0139] In addition, the first medium may contain an organic carbon source and an energy source for rapid growth and stress recovery in the initial stage. Sodium chloride can be added to obtain preferred bacterial osmotic conditions.

[0140] The first medium also contains a pH indicator. Fermentation is a process that reduces the pH value. Therefore, when bacteria capable of fermenting rhamnose are present in the sample, these bacteria will ferment the rhamnose present in the first medium, which will lower the pH of the medium. This change in pH is detected by including a pH indicator in the first medium. Thus, a change in the color of the culture during incubation of the sample in the first medium indicates the presence of Listeria rhamnose-fermenting species in the sample, such as Listeria monocytogenes and Listeria innocua. A suitable pH indicator for the methods of this document is phenol red, which changes color from red to yellow when Listeria is present in the sample, but any other pH indicator that changes color at a pH similar to phenol red can also be used.

[0141] The first medium can be used as an off-the-shelf product or prepared from single components. The phenol red culture medium and rhamnose can be sterilized by autoclaving at 121 °C. The antibiotics are filter-sterilized and added after autoclaving.

[0142] The first medium can be prepared in different ways, for example:

[0143] - Mix all components into a ready-to-use culture medium (if autoclaving is used to sterilize the medium, this is done before adding antibiotics (which are usually filter-sterilized)).

[0144] - Prepare rhamnose and phenol red broth base as a solution and add antibiotics shortly before use but after autoclaving. Antibiotics are usually less stable than other components in the growth medium, so delaying addition is a way to extend the shelf life of the medium and minimize the probability of false positives.

[0145] - A concentrated medium with rhamnose and phenol red broth can be formulated (e.g., at a 5 - 15x concentration, such as 10x concentrated) and diluted with water before use. Due to the caramelization reaction of the medium, this requires sterile filtration of the medium rather than autoclaving.

[0146] The first culture medium is preferably liquid.

[0147] In all cases, the culture broth can be placed directly into the kit or separately into large-volume flasks or bags.

[0148] The first culture medium comprises or consists of:

[0149] a) Rhamnose at a concentration of about 5 - 15 g / l, such as about 7 - 13 g / l, such as about 10 g / l;

[0150] b) One or more antibiotics, such as nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide, amphotericin B, preferably at least two of these antibiotics;

[0151] c) A pH color indicator, such as phenol red; and

[0152] d) LiCl at a concentration of about 5 g / l to about 15 g / l, such as about 7 g / l to about 13 g / l, such as about 10 g / l.

[0153] An example of the first culture medium that provides good selection pressure is given below. This medium is designated herein as "SensiList Broth":

[0154] SensiList Broth formulation:

[0155] Preparation of 1% rhamnose medium:

[0156] 15 g phenol red broth base

[0157] 10 g rhamnose

[0158] 10 g LiCl

[0159] 1000 ml of distilled water

[0160] Dissolve the components in distilled water. Sterilize at 118 °C for 15 minutes. Cool before adding the antibiotics.

[0161] Prepare the antibiotics:

[0162] 10 mg / ml nalidixic acid

[0163] 200,000 IE / ml polymyxin B

[0164] 2 mg / ml amphotericin B

[0165] 2 mg / ml ceftazidime

[0166] Dissolve the antibiotics in distilled water to the specified concentrations. Sterilize by sterile filtration.

[0167] Complete the SensiList culture medium:

[0168] 1 L of 1% rhamnose medium

[0169] 2 ml of nalidixic acid (10 mg / ml)

[0170] 0.3835 ml of polymyxin B (200,000 IE / ml)

[0171] 5 ml of amphotericin B (2 mg / ml)

[0172] 4 ml of ceftazidime (2 mg / ml)

[0173] Add the antibiotics to the 1% rhamnose diluent. Stir well.

[0174] pH: 7.4 ± 0.2

[0175] The SensiList culture medium is particularly useful for the detection and enumeration of Listeria in fish samples.

[0176] Due to the presence of both rhamnose and LiCl (in combination with the antibiotics), the selection pressure for Listeria obtained in the first culture step ii) in the method of the present document is higher than the selection pressure for Listeria in the quantitative and qualitative ISO methods that use two culture steps to achieve the same result.

[0177] To confirm the presence / absence of Listeria monocytogenes in a sample identified as positive for Listeria rhamnosus in step iii) of the method of this document, the positive sample can be plated on a second growth medium, such as ALOA medium, which differentiates Listeria monocytogenes from Listeria innocua in that the former forms a clear precipitate zone while the latter does not. The ALOA medium contains a specifically purified substrate for phosphatidylinositol-specific phospholipase C, which allows the differentiation of Listeria monocytogenes and Listeria innocua because the former forms an opaque halo around the colonies when metabolizing the substrate. The ALOA medium is preferably a solid medium, such as an agar medium.

[0178] Samples and Sample Preparation

[0179] The sample can be any sample used to check for the presence or absence of Listeria (e.g., Listeria monocytogenes). The sample can be, for example, a food sample, an environmental sample, or a fecal sample.

[0180] The food sample can be, for example, a raw food sample or a sample of processed meat, poultry, or fish products, such as salmon, vegetables, or ready-to-eat food products. The environmental sample can be a water sample (e.g., from a drain tank, a thawing tank, wash water, seawater), a dirt sample, or a food industry environmental sample, such as a surface swab sample or a sample of the surface of equipment.

[0181] Food product samples, usually sized 10 - 125 grams, can be added at a ratio of 1:1 to, for example, buffered peptone water (BPW) or physiological saline and homogenized by hand in a homogenizer bag or in a homogenizer before transfer to the first medium, for example, at a ratio of 1:10 for the homogenized sample to the medium. Alternatively, the sample can be homogenized directly in the first medium.

[0182] Environmental swab samples, such as a cloth (dry or added with buffer) that may contain Listeria, can be added directly to the first medium and left there during incubation. The procedure is otherwise different from that for food samples.

[0183] Water samples, such as process water, including wash water, cooling water, effluent water, etc., can be added to the first medium. As for food product samples, a concentrated first medium can be used to reduce the detection level while limiting the volume in the incubator and thus the space. For example, 11 ml of 10x concentrated first medium can be added to 100 ml of water sample to obtain a detection limit of 1 cfu / 100 ml. The incubation and detection procedures for presumptive positive samples are the same as those for food samples.

[0184] Methods for Detection and / or Counting

[0185] As described above, for all different types of samples, the methods disclosed herein can be performed qualitatively or quantitatively depending on the steps included therein.

[0186] A method for detecting Listeria rhamnosus fermentans, such as Listeria monocytogenes, in a sample (i.e., a qualitative method) comprises or consists of the following steps:

[0187] iv) Prepare a suspension of the sample that may contain Listeria in a first culture medium, the first culture medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

[0188] v) Incubate the suspension under conditions permitting the growth of Listeria; and

[0189] vi) Identify positive samples.

[0190] As described above, the method may further include counting Listeria rhamnosus fermentans, wherein the method includes step ia) transferring the suspension prepared in step i) to a multi-well tray, and step iv) performed after step iii) to calculate the concentration of the Listeria in the sample, for example, by using the most probable number method.

[0191] A method for counting the number of Listeria rhamnosus fermentans, such as Listeria monocytogenes (i.e., a quantitative method) comprises or consists of the following steps:

[0192] i) Prepare a suspension of the sample that may contain Listeria in a first culture medium, the first culture medium comprising rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

[0193] ia) Transfer the suspension obtained in step i) to a multi-well plate;

[0194] ii) Incubate the suspension under conditions permitting the growth of Listeria;

[0195] iii) Identify positive samples; and

[0196] iv) Calculate the concentration of the Listeria in the sample, for example, by using the most probable number method.

[0197] The method of this document may further include the step of confirming the presence of Listeria monocytogenes by differentiating Listeria monocytogenes from other Listeria species, for example, by plating the positive samples identified in step iii) on a second growth medium such as ALOA medium, by using molecular methods, such as by polymerase chain reaction or in situ hybridization, ELISA, VITEC, or API. This step is performed after step iii) of the qualitative method and step iv) of the quantitative method.

[0198] For both the qualitative and quantitative analysis of the presence of Listeria in a sample, the first step in the method is to prepare a suspension of the sample, which may contain Listeria, in a first culture medium, as described elsewhere herein.

[0199] The sample is typically incubated at a temperature of about 37 °C, although a temperature, for example, between 25 °C and 38 °C, such as about 30 °C, may also be used, and the color of the culture is observed after 1 and 2 days. When phenol red is used as a pH indicator, a change in color, for example, from red to yellow or orange, indicates a presumptive positive sample. The color may be read in this way, or a color code may be found by comparison with a color chart.

[0200] If a qualitative method is to be performed, it is sufficient to incubate the suspension of the sample, which may contain Listeria monocytogenes, to observe whether there is a change in color during the incubation of the sample (i.e., the suspension of the sample, which may contain Listeria monocytogenes, in the first culture medium) in the first culture medium. If a change in color is observed, Listeria monocytogenes, such as Listeria monocytogenes or Listeria innocua, is present in the sample, and thus the sample is positive for Listeria monocytogenes. If no change in color is observed, Listeria monocytogenes is not present in the sample, i.e., the sample is negative for Listeria.

[0201] For a method to be performed to count the concentration of Listeria rhamnosus in a raw sample, a suspension of the sample, which may contain Listeria rhamnosus, in a first culture medium is transferred to a multi-well tray (plate), such as a quantitative tray, such as the Quanti-Tray 2000 from IDEXX. During incubation under conditions such as those described above, the wells containing Listeria rhamnosus change color due to the presence of a pH indicator in the first culture medium. Then, the most likely concentration of Listeria rhamnosus in the raw sample can be estimated using the MPN (Most Probable Number) method. For example, if a quantitative tray is used to estimate the concentration of Listeria rhamnosus in a sample, and the total volume of the suspension of the sample that may contain Listeria rhamnosus is 100 ml, and the sample size is 5 g, the detection limit will be 1 cfu / 5 g of sample, which corresponds to an average concentration of 0.2 cfu / g of sample. If the concentration of Listeria rhamnosus is higher, such as 100 cfu / g, there will be 500 bacteria in a 100 ml solution containing 5 grams of sample. This corresponds to 5 cfu / ml. Then it is very likely that most of the 1 ml wells in the quantitative tray will turn yellow, and at most 50% of the 0.1 ml wells will turn yellow. Using a double concentration of Listeria rhamnosus, at most 100% of the +0.1 ml wells will turn yellow. Above this concentration, all wells will be yellow, and the upper limit of effective counting is reached. The minimum and maximum limits of the counts obtained using 96 1 ml wells and 96 0.1 ml wells will be in the range of 1 cfu / 5 grams (i.e., 0.2 cfu / g) to 200 cfu / gram. If another ratio between the sample and the first culture medium is applied, the detection limit can be adjusted.

[0202] The method of the present document enables the detection and counting of very low levels of Listeria monocytogenes and Listeria rhamnosus in large or pooled samples. The sample size can be up to 100 grams, and the detection level can be as low as 5 cfu / 100 g of food, i.e., 1 cfu / 20 g of food. Due to the large sample size, it is possible to use, for example, a set of 10 samples, each of 10 grams, in one kit. This also results in lower analysis costs for food suppliers. In addition, allowing a larger possible sample size for large or pooled samples is an important factor in overcoming problems caused by uneven distribution of Listeria in food products.

[0203] The analysis performed by the method of this document can be carried out in a production facility because the system can be made closed so that an enriched Listeria-positive sample cannot leak bacteria back into the production facility or the personnel handling the sample. Closing the system means that after the sample is added to the first culture medium, whether the method is quantitative or qualitative, the container (such as a flask, bag, beaker, multi-tray, etc.) is sealed so that the bacteria that may grow in the culture medium do not come into contact with air, the person handling the sample, or the surface in contact with the sample. Sample preparation, such as homogenization and cutting, can be done within the closed system. Gas exchange is not required because Listeria can grow without oxygen. Closing the system can be achieved, for example, by sealing the sample with a lid or plastic film, for example, in a way that only allows the system to be opened with a specific tool. However, the confirmation of the presence of Listeria monocytogenes in a presumptive positive sample must be carried out in a laboratory classified for Listeria analysis because the container containing the culture medium needs to be opened.

[0204] Various samples can be transported to the laboratory for confirmation / testing of the presence of Listeria monocytogenes. If the sample is a presumptive positive sample, i.e., the color has turned yellow after incubating the sample in the first culture medium, a lower temperature should be maintained during transportation to keep the bacteria alive. In the case where the sample is sent out soon after it has been placed in the first culture medium, cooling is not required because the growth during transportation will shorten the detection time after the sample reaches the laboratory.

[0205] There are several methods available for confirming Listeria monocytogenes:

[0206] - Forming zones on ALOA agar. Listeria monocytogenes forms zones while Listeria innocua does not.

[0207] - PCR analysis for detecting Listeria monocytogenes. In fact, this test can be performed before the sample turns yellow because the cell count reached before obtaining a positive PCR reaction is so high that the pH is below the level at which the color turns yellow.

[0208] - In-situ hybridization analysis of specific Listeria monocytogenes genes,

[0209] - Any other identification method, such as ELISA, VITEC, API, MaldiToff test, and whole genome sequencing.

[0210] As can be clearly seen above, the method of the present document allows for obtaining presumptive positive and negative results for both qualitative and quantitative methods within 1 to 2 days, and for confirming positive results after at most one additional day, as opposed to 5 or 7 days for the ISO methods (for quantitative and qualitative methods respectively). Moreover, the advantage of this method is that it does not have to be performed in a laboratory, but can be carried out, for example, at the location where the sample is collected, such as within the industry itself.

[0211] Kit

[0212] The present document also relates to a kit for detecting Listeria in a sample, such as Listeria monocytogenes, the kit comprising:

[0213] a) a container containing a culture medium comprising or consisting of rhamnose, one or more antibiotics, a pH color indicator, and LiCl;

[0214] b) a container or a porous tray for culturing a sample that may contain Listeria;

[0215] c) optionally, a colorimetric chart for identifying positive samples; and

[0216] d) optionally, instructions for use.

[0217] The concentrations of the components of the culture medium in the kit are as disclosed elsewhere herein for the first culture medium. The first culture medium is preferably liquid, but it can also be solid.

[0218] Computer-implemented calculator

[0219] The present document also relates to a computer-implemented calculator for displaying a prediction of the growth of Listeria, such as Listeria monocytogenes, based on the detection and counting of colony-forming units in a sample, the calculator comprising:

[0220] a) an input device, such as a keyboard or a microphone;

[0221] a) an output device, such as a display, a computer or a mobile phone screen, or a speaker;

[0222] b) software that can be downloaded or is in memory, such as an application on a smartphone or a web page;

[0223] Using the input device, the number of colony-forming units is set to any number found in the sample, within the range of 0.04 cfu / g to 10 cfu / g, especially within the range of 0.04 cfu / g to 1 cfu / g. The prior art does not allow for providing such details in the low range.

[0224] The calculator can input parameters, such as the pH value of the sample or the sample type, such as raw salmon, raw fish, sushi, chicken or beef. In addition, there are inputs of parameters such as the temperature and storage time in production, store and household refrigerators, and the number of hours of room temperature and outdoor refrigeration. Based on the inputs, the calculator will calculate according to the stored model, and then display various predictions in the form of charts or numbers, such as the number of days of maximum growth, the most likely growth, the least likely growth, and plot the curves of these against the legal limits, the possible disease limits for exposed consumers, and the possible disease limits for healthy adults.

[0225] The calculator can be implemented as a single application for each food type, or an integrated calculator that provides different options for the user.

[0226] The present invention will be further described in the following embodiments, which do not limit the scope of the present invention described in the claims.

[0227] Experimental section

[0228] Experimental Section

[0229] 1 Example 1: Development and Testing of SensiList Culture Medium

[0230] 1.1 Sensilist culture medium

[0231] The contents in the culture medium will be referred to as the above-mentioned "Sensilist culture medium" hereinafter.

[0232] 1.2 Sensitivity of Sensilist culture medium

[0233] The first study on the selectivity and sensitivity of the SensiList culture medium was performed using salmon samples collected directly from the production company. Salmon fillets were inoculated with 0 cfu / g, 2 cfu / g, 20 cfu / g or 200 cfu / g of Listeria monocytogenes. As the inoculum, a single strain or a mixture of 5 strains was used. The samples were used immediately or stored frozen until use.

[0234] In these studies, the concentrations of LiCl and rhamnose in the SensiList culture medium were tuned.

[0235] Both the SensiList method and the ISO method were used to analyze the samples to compare the results. The ISO method was adjusted to allow the detection level to be reduced in two ways. First, by using five times more plates to plate a larger volume of the sample suspension. This method is not feasible in daily work, but it is applied here to verify the sensitivity of the method herein. Second, as described in the ISO method, a 5x concentration of the sample suspension was used by adding less diluent before inoculation onto the agar medium.

[0236] Analyze using the method of this document: Prepare a salmon sample (product sample) as described below and place it in SensiList culture medium. For qualitative samples (presence / absence), place the sample and the culture medium in a 100 ml or 500 ml flask. To count using the MPN (Most Probable Number) method, place the culture medium containing the sample in a sterile glass tube (1 - 10 ml) or in a Quantitray 2000 from IDEXX with 49 large wells and 24 small wells and a total volume of 100 ml. Incubate the culture medium as described below and observe the color of the culture medium after one and two days. Transfer droplets to an ALOA plate using a sterile needle and further analyze all wells. Blue - green colonies with a typical area are presumed to be positive for Listeria monocytogenes, and blue - green colonies without an area are presumed to be positive for Listeria innocua. Colonies of other colors are negative. Confirm blue - green colonies using Gram staining and catalase according to ISO 11290 - 1.

[0237] Repeat the study approximately 10 times in triplicate using salmon inoculated with 2 cfu / g, 20 cfu / g, or 200 cfu / g of Listeria monocytogenes. In some experiments, place one Listeria - positive sample (cube, approximately 2×2×2 cm) together with 9 negative samples of the same size and stack them in different forms, as Figure 2 shown. The aim is to study whether the positive sample was detected in the following three sampling procedures: i) analyze all samples in the stack, ii) analyze the culture medium after shaking the sample with the culture medium and then incubating the culture medium, and iii) analyze the water released from the fish.

[0238] 1.3 Selectivity of SensiList Culture Medium

[0239] Search for false - positive and false - negative bacteria in two ways:

[0240] 1. Select strains used as positive and negative control strains in Listeria diagnosis and kit tests.

[0241] 2. Salmon from different suppliers were purchased from stores in the Oslo area and cultured in SensiList culture medium without antibiotics and LiCl to allow the growth of all bacteria capable of utilizing rhamnose. Plate tubes in which the color of the culture medium changed from red to yellow during incubation on blood agar and ALOA agar and isolate the bacteria. Use the isolates in subsequent experiments to 1) formulate an antibiotic composition in SensiList culture medium and 2) test the stability of the culture medium.

[0242] 1.4 Confirm Presumed Positive Samples Using PCR

[0243] The following methods were used to test the confirmation using PCR:

[0244] Using A blood and tissue kit (Qiagen) was used to extract DNA from Listeria monocytogenes (strain VI 58361, serotype 1 / 2a) cultured in growth medium broth. The following growth media were used: SensiList broth, SensiList broth without LiCl and antibiotics, SensiList broth without antibiotics. In addition, half Fraser medium and buffered peptone water were included as controls. Listeria monocytogenes colonies were transferred from agar plates to different broths. DNA was extracted using the kit according to the procedure from the supplier, and the DNA concentration was measured using a Nanodrop (a spectrophotometer from ThermoFischer for measuring DNA concentration).

[0245] Using the BioRad iQ- for DNA extraction and PCR Listeria monocytogenes II kit, and the isolated DNA was analyzed using qPCR (quantitative PCR) according to the following genetic markers: ORF2819, ORF2110, lmo1118, lmo0737, plcA, and prs.

[0246] 1.5 Stability of SensiList broth

[0247] The stability of different formulations of SensiList broth was examined as follows:

[0248] Four formulations of SensiList broth were prepared in a large enough quantity to perform multiple analyses during a one-year storage period. The formulations used were SensiList broth without LiCl and antibiotics, broth containing LiCl but no antibiotics, broth with antibiotics added immediately before use, and full broth. The experiment was repeated with three independent batches of each formulation.

[0249] Experiments were performed using individual strains from a group of Listeria monocytogenes (8 strains), other Listeria species (2 strains), and other bacteria that may give false positive results. The latter strains were selected based on the author's own experiments on salmon (see above), strains used to test the ISO method, and strains that gave false positive results in other Listeria monocytogenes kits. The strains are described in more detail below.

[0250] A. Listeria monocytogenes strains:

[0251] a. 2 strains were used to test the ALOA medium in the ISO method.

[0252] i. Listeria monocytogenes serotype 4b VI 60847 (WDCM00021 / ATCC 13932)

[0253] ii. Listeria monocytogenes serotype 1 / 2a, VI 51285 (WDCM00109 / CCUG 15527)

[0254] B. Listeria monocytogenes strains used in experiments with SensiList broth:

[0255] a. VI 51503

[0256] b. VI 58363 (00EB250LM)

[0257] c. VI 58365 (00EB254LM)

[0258] d. VI 59994

[0259] e. VI 59998

[0260] f. VI 58366

[0261] C. Listeria innocua: Used to test the ALOA medium in the ISO method.

[0262] a. VI 51284 (WDCM00017 / CCUG 15531)

[0263] D. Listeria ivanovii: Used to test the ALOA medium in the ISO method.

[0264] a. VI 51040 (ATCC 19119)

[0265] E. Other strains:

[0266] a. Used to test ALOA:

[0267] i. Enterococcus faecalis (VI 52179).

[0268] ii. Escherichia coli VI 51656 (WDCM00013 / CCUG 17620)

[0269] b. Candida albicans VI06652 (ATCC10231)

[0270] F. In earlier versions of SensiList, 18 strains from salmon produced false positive results when no antibiotics were applied

[0271] a. Five Hafnia alvei strains: VI 54910, VI 54914, VI 54918, VI 54924, VI 54927

[0272] b. Five Citrobacter strains: VI 54915, VI 54916, VI 54928, VI 54931, VI 54940

[0273] c. Two Aeromonas strains: VI 54922, VI 54926

[0274] d. Two Enterobacter cloacae strains: VI 54935, VI 54941

[0275] e. One Alcaligenes faecalis strain: VI 54929

[0276] f. One Enterococcus faecalis strain: VI 54946

[0277] g. One Bacillus cereus strain: VI 54948

[0278] h. One uncertain identification, possibly Macrococcus caseolyticus: VI 54952

[0279] G. Two strains that give false positives in other kits

[0280] a. Carnobacterium maltaromaticum: VI 61406

[0281] b. Enterococcus casseliflavus: VI 61407

[0282] H. Strains capable of fermenting rhamnose:

[0283] a. Lactobacillus rhamnosus: VI 60846

[0284] Culture Listeria monocytogenes strains and cold-adapt them at 7°C for 7 days to adapt them to the representative stress conditions of food samples. Culture other strains that may give false positive samples overnight at 37°C and transfer them to BHI medium. Apply different temperatures to prepare for the worst-case scenario: The Listeria monocytogenes strains should be given the actual stress to detect false negative results, while the false positive strains should be given the best option for growth in the culture broth during incubation.

[0285] Inoculate (0.1 ml) the preparation of SensiList culture broth (0.9 ml) into a 96-well plate (type). Dilute the mixture ten-fold in eight steps in a tray, resulting in one column for each strain, as shown in Tray 1 below. Three trays are required for each culture medium preparation to test all strains. Cover the plate before incubation at 37°C and observe the color change after 1 and 2 days of incubation.

[0286]

[0287]

[0288] Table 3: Distribution of strains used in the stability test in 96-well plates (12 columns and 8 rows per plate). Each column contains 1 strain diluted 10-fold. Row A contains the first 10-fold diluted strain, row B contains the second 10-fold diluted strain, and so on. The top 5 alphanumerics are the identification of each strain.

[0289] 1.6 Treating samples for analysis by the methods of this document

[0290] 1.6.1 Food product samples

[0291] Add 10 - 125 g of the sample to buffered peptone water (BPW) at a ratio of 1:1 and homogenize by hand or in a bag or in a homogenizer. Add 10 ml of the homogenized solution to 90 ml of SensiList culture medium. Incubate the whole sample at 37 °C and observe the color after 1 day and 2 days. A change in the solution from red to yellow indicates a presumptive positive sample. The color can be read in this way, or the color code can be found by comparison with a colorimetric table. For example, a colorimetric table from www.gioco.no Colorimetric table of color Y50R indicates a positive result.

[0292] If prepared as described above, the test solution contains 5 g of the product. Including the whole solution, this results in a detection level of 1 cfu / 5 g. If another detection level is required, this detection level can be obtained by applying another ratio between the sample and the culture medium. Some examples are given below. If a large sample is needed, for example, a pooled sample of 5 * 25 g samples, to meet the criterion of no microorganisms present in 25 g measured in 5 samples, the total volume can be restricted by adding the culture medium as a concentrated solution. For example, 6-fold concentrated 125 g sample + 125 ml BPW + 50 ml SensiList culture medium restricts the total volume to 300 ml. This is a high enough liquid fraction to observe the color change, but some foods will require a higher dilution factor in quantitative analysis.

[0293]

[0294]

[0295] Table 4. Examples of different culture medium to sample ratios considering the required detection level.

[0296] 1.6.2 Swabs from production environment samples

[0297] The swabs are various types of cloth, which can be either dried or buffered and can be added directly to the SensiList culture medium and left in the container during incubation. The procedure is otherwise different for food products.

[0298] 1.6.3 Water samples

[0299] Process water, including wash water, cooling water, leachate, etc., can be added to the SensiList culture medium. For product samples, concentrated culture medium can be used to reduce the detection level while limiting the volume in the incubator and thus the space. For example, 11 ml of 10x SensiList culture medium can be added to 100 ml of water sample to obtain a detection limit of 1 cfu / 100 ml. The incubation and detection procedures for presumptive positive samples are the same as those for food samples.

[0300] 1.6.4 Counting method - Quantitative analysis

[0301] All categories of samples can be performed qualitatively or quantitatively. To count the concentration of Listeria in the sample, the liquid fraction of the above SensiList culture medium solution is transferred to, for example, a quantitative plate, where the culture medium is separated in 96 small and large wells. If the entire volume (100 ml) contains 1 Listeria, one of the wells will turn yellow while the others will remain red. The most probable concentration can be estimated using the MPN method. In the case of a 100 ml solution containing 5 g of sample, the detection limit will be 1 cfu / 5 g of sample, which corresponds to an average concentration of 0.2 cfu / g of sample. If the concentration of Listeria is high, for example 100 cfu / g, there will be 500 bacteria in a 100 ml solution containing 5 g of sample. This corresponds to 5 cfu / ml. Then it is very likely that all 1 ml wells will turn yellow, while at most 50% of the 0.1 ml wells will turn yellow. Using double the concentration of Listeria, up to 100% of the +0.1 ml wells will turn yellow. Above this concentration, all wells will be yellow and the upper limit of effective counting is reached. The minimum and maximum limits of counting obtained using 96 1 ml wells and 96 0.1 ml wells will be in the range of 1 cfu / 5 g to 200 cfu / g.

[0302] The detection limit can be tuned by using another ratio between the sample and the SensiList culture medium.

[0303] 1.7 Testing the method of this document with artificially and naturally contaminated samples

[0304] 1.7.1 Case 1: Artificially and Naturally Contaminated Salmon

[0305] Analyze the thawed water released from the inoculated samples using the SensiList method and the improved ISO method. The procedure is as described above, and the research objectives are in the results section.

[0306] 1.7.2 Case 2: Heat-Treated Contaminated Chicken

[0307] Inoculate the samples with a low concentration of Listeria monocytogenes and analyze them according to the food sample protocol described above.

[0308] 1.7.3 Case 3: Naturally Contaminated Meat and Dairy Products, Processed Products from Several Animal Species

[0309] Analyze the samples according to the food sample protocol described above.

[0310] 1.7.4 Case 4: Swabs for testing the production surface after cleaning

[0311] Inoculate and analyze the samples according to the protocol for the environmental samples described above.

[0312] 1.7.5 Case 5: Testing in Production Companies

[0313] Send the SensiList culture medium in the flask to the salmon slaughter and filleting facility. A brief note of caution and a color map are also provided. The company collects samples according to the normal procedure, adds the samples to the SensiList culture medium, and incubates the culture medium at 37°C. After incubation, the company takes photos and sends the photos to us for discussion of the results. They also send the samples to a local laboratory for confirmation.

[0314] 1.8 Results

[0315] 1.9 Development of the method

[0316] 1.9.1 Accuracy and sensitivity of the method - Detection level

[0317] For concentrations of 200 cfu / g and 20 cfu / g, there is a good correlation between the inoculation concentration, the concentration counted by the method in this document, and the concentration counted by the ISO method. For salmon at 2 cfu / g, Listeria monocytogenes was detected only using the method in this document. This is as expected because the detection level of the ISO method is 10 cfu / g when performed according to the standard procedure. For all concentrations, the presumptive positive concentrations were confirmed.

[0318] The sensitivity of the method was further tested to investigate whether it was necessary to homogenize the whole salmon fillet or whether it was sufficient to shake the sample in SensiList broth. One positive sample was mixed with nine negative samples to obtain an accurate inoculation level of Listeria monocytogenes at a very low concentration (0.2 - 20 cfu / g). The theoretical concentration in the broth was 10 times lower because the sample and the broth were mixed at a 1:2.5 ratio. The results are shown in Figure 3 as follows.

[0319] All inoculated samples were detected by the method of this document, even those with the lowest concentration of Listeria monocytogenes. The ISO method only detected the highest concentration (20 cfu / g). After incubating the broth overnight, the concentration of all samples detected by the ISO method was 2 cfu / g or higher, but only one of the three samples had a concentration of 0.2 cfu / g, even though Listeria monocytogenes was detected immediately by the method of this document after shaking the salmon fillet with the broth. The most likely explanation for this observation is that only a small volume (0.1 - 1.0 ml solution) was plated using the ISO method, while the whole broth was analyzed using the method of this document.

[0320] 1.9.2 False positive and false negative bacteria

[0321] Similar experiments as described above were performed using different mixtures of Listeria strains and salmon samples, but with a modified version of the ISO method to obtain higher sensitivity of the method (see Materials and Methods). In all cases, there was a good correlation between the methods, but here too, the method of this document was found to be the most sensitive. However, in samples containing Listeria innocua, either alone or in mixture with Listeria monocytogenes, the method of this document gave a higher concentration of presumptive positives than of confirmed positives compared to the ISO method. This is because Listeria innocua is also able to ferment rhamnose and has a similar tolerance to antibiotics and LiCl as Listeria monocytogenes. Therefore, a confirmation step is needed to separate Listeria monocytogenes from Listeria innocua.

[0322] Strains 1 - 18 were false positives obtained from salmon purchased from a fish delicatessen. The strains were identified as Hafnia alvei, Citrobacter spp., Aeromonas spp., Enterobacter cloacae, Alcaligenes faecalis, Enterococcus faecalis, Bacillus cereus, and Macrococcus sp. (the latter is uncertain) using the Malditoff technique. Strains 19 - 24 were Listeria monocytogenes. The conclusion was that the antibiotics tested were sufficient to avoid false positive results.

[0323] 1.9.3 Stability of SensiList broth

[0324] The test strains were cultured in different SensiList culture broth preparations for a period of up to one year. The results after 48-hour incubation of all strains in SensiList culture broth in fresh culture broth and culture broth stored for approximately one year before use are as follows. As expected, all strains of Listeria monocytogenes (columns 1 - 7 of tray A and column 8 of tray C) and Listeria innocua (column 8 of tray A) gave positive results on all preparations. However, other strains gave false positive results only in preparations without LiCl and / or without antibiotics. For all preparations containing antibiotics, whether the antibiotics were added immediately before use or had been present in the culture broth for one year, all three batches obtained correct results, indicating that the full culture broth was stable and accurate. The results are as Figure 4 shown.

[0325] 1.9.4 Confirmation of presumptive positive results

[0326] In all experiments using SensiList culture broth, the confirmation tests described on ALOA and in the ISO method gave correct results when plated.

[0327] Confirmation using PCR technology also indeed gave correct results. Commercially available kits for DNA isolation provided sufficient amounts and quality of DNA in all test cases to perform PCR analysis. The qPCR method applied was the same as the method used to determine the genetic serogroup, also known as the molecular serogroup. The results for one of the following strains are listed in the table below. For all preparations of SensiList culture broth, the same gene gave the correct signal. The negative signal (marked as 0) was negative because the test strain was serogroup IIa.

[0328] Any other PCR method for detecting Listeria monocytogenes can be used, but this method was chosen because it can identify both Listeria monocytogenes and the serogroup. Testing for serogroups is usually performed in a separate analysis. Here, by using it as a confirmation step, confirmation and sufficient characterization have been obtained in the preliminary analysis of the sample to check for links to outbreaks or similarities to other isolates.

[0329]

[0330]

[0331] Table 5

[0332] 2 Example 2: Demonstration of the availability of the method of this document for detecting Listeria in food samples

[0333] 2.1 Case 1: Artificially and Naturally Contaminated Salmon

[0334] The above research was carried out using salmon. The results were good in all cases.

[0335] In addition, research was also carried out on frozen and thawed salmon fillets. The thawed water released was sampled using the ISO (ALOA) method and the method of this document. Two different mixtures of Listeria monocytogenes were applied and inoculated at concentrations of 2 cfu / g and 20 cfu / g. For incubation, two temperatures were tested. The results are shown in Table 5 below. In all the samples analyzed, the ISO method gave 6 negative results, while the method of this document gave only 1 negative result in the inoculated samples. All the differences were observed for the lowest inoculation level. The decrease in the count with the increase in the frozen storage time of the fish seems to be due to the reduction of live bacteria during frozen storage.

[0336] The conclusion of this experiment is that the method of this document is suitable for detecting and quantifying low concentrations of Listeria in fresh and frozen salmon.

[0337]

[0338]

[0339]

[0340]

[0341] Table 6

[0342] 2.2 Case 2: Contaminated heat-treated chicken

[0343] Heat-treated chicken was inoculated with Listeria monocytogenes and analyzed using the method of this document and the modified ISO (ALOA) with samples of a lower dilution factor, resulting in a detection level of 1 cfu / g. The aim was to first investigate whether the chicken interfered with the SensiList culture medium and gave false positive results, and second, whether the concentration was correctly estimated. The results are given below.

[0344] The method of this document produced correct detections in all cases, while the modified ISO method gave false negative results. This is most likely due to the higher sensitivity of the method of this document.

[0345] The conclusion is that heat-treated chicken does not interfere with the method of this document.

[0346] In addition, compared with the modified ISO method, the method of this document provides accurate counting, accurately counting as low as 0.2 cfu / g using a quantitative tray and accurately detecting as low as 1 - 3 cfu / total sample, see Table 6.

[0347]

[0348]

[0349] Table 7

[0350] 2.3 Case 3: Naturally contaminated meat and dairy products, products processed from several animal species

[0351] Naturally contaminated food samples were obtained from the reference laboratory for Listeria monocytogenes in European food. The samples were frozen, and the Listeria might have died during storage. However, the samples were first used to test the interference of the food matrix on the method of this document, second to test the interference on the normal background flora, and third to test the counting level.

[0352] Also in this case, a modified ISO method was used to obtain a sufficiently low detection level for comparison with the method of this document.

[0353] The results are shown in the table below.

[0354] Generally, there is a good correlation between the modified ISO methods. The method of this document detected Listeria monocytogenes in one more sample (noodles with chicken) than the ISO method, which might be due to higher sensitivity. The measured concentration was 0.4 cfu / g.

[0355] No interference from the tested food matrix was observed to cause false positive or false negative results when using the method of this document.

[0356] One of the said samples contained Listeria welshimeri. This gave false positive results when using SensiList and on ALOA. The results show that false positive results can be obtained in both the inventive method and the prior art methods used for the detection and counting of Listeria. However, compared with Listeria innocua and Listeria monocytogenes, Listeria welshimeri is a very rare Listeria species, and the specificity of the method of this document is also considered to be high enough, at least as high as the currently available methods.

[0357] The conclusion from this study is that the method of this document produced true results for naturally contaminated samples. No significant negative interference from complex food matrices was observed. See Table 7.

[0358]

[0359]

[0360] Table 8.

[0361] 2.4 Case 4: Swabs for testing production surfaces after cleaning

[0362] Swabs from the surface of production equipment for testing were obtained from three facilities. Such swabs were supplied with neutralizing solution. The test was performed to study whether the material, the neutralizing solution, or residues from the cleaning solution would interfere with the method of this document, for example by introducing growth-inhibiting compounds or increasing buffer capacity, both of which could result in possible false-negative results.

[0363] Samples obtained from these companies were inoculated with 0 cfu / swab, 15 cfu / swab, or 1500 cfu / swab of Listeria monocytogenes. The swabs were placed in SensiList culture medium, and the solution was transferred to a counting plate and incubated as described above.

[0364] None of the uninoculated swabs gave presumptive positive results. For swabs inoculated with 15 cfu of Listeria monocytogenes, 4 - 9 out of 96 wells turned yellow and were confirmed positive for Listeria monocytogenes. For swabs inoculated with 1500 cfu, 58 - 96 wells turned yellow. In a few cases, some wells changed from red to orange but not to yellow. The color change was below the color change qualified for presumptive positive results.

[0365] The conclusion from this study was that the method of this document gave correct test results for Listeria monocytogenes in swabs taken from the production environment, and the counting was adequate.

[0366] 2.5 Case 5. Testing of production samples in a salmon production company

[0367] A salmon processing company took samples of process water and cotton plugs from "sluk" and inserted the said samples into SensiList culture medium. The samples were incubated in the company and sent to a local laboratory for confirmation. The results after the first culture were as Figure 4 shown.

[0368] According to the company, sampling and reading of the color change were easy. Confirmation of the samples showed that the yellow flasks contained Listeria innocua, while the red flasks did not contain Listeria spp.

[0369] Water and "sluk" / drainage samples were collected with absorbents (packing). None of these caused any interference.

[0370] Salmon samples were 100 g each.

[0371] As Figure 5 shown, the conclusion of this study was that SensiList was user-friendly enough for the company. The presumptive positive results were consistent with the results of the laboratory.

[0372] Example 3: Comparison of food-grade rhamnose and analytical-grade rhamnose

[0373] Rhamnose is a rare carbohydrate and is relatively expensive. Since this component is used in the first culture medium according to this document and has a large volume, this component will have a great impact on the price of the method / kit. To check whether food-grade rhamnose can be used instead of analytical-grade rhamnose, two versions of SensiList culture medium were prepared and compared. The comparison was carried out in two large-scale tests:

[0374] 1. Stability tests of the culture medium were carried out using single-cell cultures with the same strain. Qualitative methods were applied.

[0375] 2. Natural and uncontaminated samples from two fish slaughter and processing companies were used. The samples were water, wet swabs, dry swabs, and fish meat. Both qualitative and quantitative methods were applied. After culturing in SensiList culture medium, the following three agar media were used for confirmation: blood agar, Rapid L′mono, and ALOA for confirmation.

[0376] Results

[0377] The tests with single strains correctly identified analytical-grade rhamnose and food-grade rhamnose in all cases.

[0378] For samples from fish production facilities, there was a good correlation between the samples, which means that the positive and negative results were positive for both food-grade rhamnose and analytical-grade rhamnose. However, there were some deviations. This was probably due to the very low concentration of Listeria monocytogenes, i.e., never exceeding 6 cfu / total sample. At such a low concentration, if the transfer of Listeria to either of the two culture media was random, this in turn meant that only the culture medium containing Listeria had the possibility of giving a positive result.

[0379] The following gives an overview of the results from one company, and the samples were collected over time. Crosses indicate negative samples, while Pos indicates detected. All concentrations were very low. The previous revised version of the ISO method used less diluent, resulting in a counting detection level of 2 cfu / g, which was not sufficient to detect Listeria in these samples.

[0380] Positive samples from the company: no more than 3 positive wells: indicating <6 cfu / sample

[0381]

[0382]

[0383] Table 9 This table is an example used in the industry. The Norwegian text in the leftmost column shows the positions in the production line. The top row shows the dates of the samples, where "Pos" means positive sample. The rightmost column has notes on the tests, such as "swab of 10 fish fillets", "meat sample", "water".

[0384] The results of three of the samples in the sample are given in Figure 6 Shown. The left box represents the qualitative method, in which 1 Listeria monocytogenes in the sample can be detected, and the sample in this case is 20 ml of water. Only the left sample is positive. The same sample is analyzed using the quantitative method (right small figure). In this case, 100 m1 of the sample (10 ml of water and 90 ml of SensiList culture medium) is transferred to the quantitative plate. Only one well changes color from red to yellow, which means that the most probable concentration of Listeria monocytogenes in the water is 1 cfu / 10 ml. The results are obtained after incubation for 24 hours.

[0385] The SensiList method (i.e., the method of this document) is applicable to all samples, but the dilution ratio needs to be considered, especially for very wet samples and for fish meat. On the one hand, this is due to the dilution of the SensiList culture medium, because the pH is related to the amount of acid produced, and thus related to the concentration of rhamnose. On the other hand, a high relative amount of other carbon sources and energy sources, such as fish meat, may cause bacteria to grow on these rather than on rhamnose, and will not produce a sufficient amount of acid. If a high enough proportion of the culture medium is used, both of these challenges can be easily solved in the SensiList culture medium.

[0386] Figure 7 The results of presumptive positive samples are given in. Two wells are yellow, some are orange, and most wells are red. The material from each well is transferred to three different growth media to test specificity. Only the yellow wells hemolyze on blood agar, and typical areas of Listeria monocytogenes appear on ALOA agar. The orange and red wells either do not grow or grow bacteria that are not Listeria monocytogenes. Therefore, the sensitivity and selectivity of SensiList using rhamnose culture medium are as good as those found when using analytical grade rhamnose before.

[0387] It should be understood that although the present invention has been described in conjunction with its detailed description, the foregoing description is intended to illustrate rather than limit the scope of the present invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

[0388] Unless there is a clear description to the contrary, each of the preferred features described herein can be used in combination with any and all of the other preferred features described herein.

[0389] References

[0390] Vitullo et al., Real-time PCRs assay for serogrouping Listeria monocytogenes and differentiation from other Listeria spp., Molecular and Cellular Probes, Volume 27, Issue 1, February 2013, Pages 68-70.

[0391] C.T. Schirmer, Solveig Langsrud, Trond Therese Hagtvedt, Even Heir, 2012. Performance of two commercial rapid methods for sampling and detection of Listeria in small-scale cheese producing and salmon processing environments. Journal of Microbiological Methods, Volume 91, Issue 2, Pages 295-300, ISSN 0167-7012, https: / / doi.org / 10.1016 / j.mimet.2012.08.013

Claims

1. A method for detecting Listeria rhamnosus in a sample, the method comprising or consisting of the following steps: i) preparing a suspension of the sample that may contain Listeria in a first culture medium, wherein the first culture medium is selective for Listeria rhamnosus and the first culture medium comprises: rhamnose at a concentration of 5 - 17 g / l; antibiotics, wherein the antibiotics are three, four or all five of nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide and amphotericin B; a pH color indicator; and LiCl at a concentration of 5 - 17 g / l; ii) incubating the suspension under conditions allowing the growth of Listeria; and iii) identifying positive samples by detecting a color change of the suspension; The method is not used for diagnosing diseases.

2. The method according to claim 1, the method further comprising counting Listeria rhamnosus, the method comprising step ia) transferring the suspension prepared in step i) to a multi-well tray, and step iv) calculating the concentration of the Listeria in the sample, which is performed after step iii).

3. The method according to claim 2, wherein step iv) of calculating the concentration of the Listeria in the sample, which is performed after step iii), is by using the most probable number method.

4. The method according to claim 1, the method further comprising a step of confirming the presence of Listeria monocytogenes, which is performed after step iii) in claim 1.

5. The method according to claim 2, the method further comprising a step of confirming the presence of Listeria monocytogenes, which is performed after step iv) in claim 2.

6. The method according to claim 4, wherein the step of confirming the presence of Listeria monocytogenes is by plating the positive sample identified in step iii) on a second growth medium.

7. The method according to claim 4, wherein the step of confirming the presence of Listeria monocytogenes is by using a molecular method, wherein the molecular method is polymerase chain reaction, in situ hybridization, enzyme-linked immunosorbent assay, whole genome sequencing or analysis of characteristic indices.

8. The method according to claim 1, wherein the sample that may contain Listeria rhamnosus is obtained by dividing the sample into small pieces before performing step i) and / or in the first culture medium of step i).

9. The method according to claim 1, wherein the antibiotics are four or all five of nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide and amphotericin B.

10. The method according to claim 1, wherein the pH color indicator is phenol red.

11. The method according to claim 1, wherein the sample is a food sample, an environmental sample, or a sample from an animal.

12. The method according to claim 11, wherein the food sample is raw or processed meat, poultry or fish products, vegetables or ready-to-eat food products.

13. The method according to claim 11, wherein the environmental sample is a water sample, a soil sample or a food industry environmental sample.

14. The method according to claim 1, wherein the method is performed in a closed system.

15. A culture medium for growing and / or detecting Listeria rhamnosus, wherein the culture medium is liquid and selective for Listeria rhamnosus, and the culture medium comprises: a) Rhamnose at a concentration of 5 - 15 g / l; b) Antibiotics, wherein the antibiotics are three, four or all five of nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide and amphotericin B; c) A pH color indicator; and d) LiCl at a concentration of 5 g / l to 15 g / l.

16. A kit for detecting and / or counting Listeria rhamnosus in a sample, the kit comprising: a) A container containing a culture medium, wherein the culture medium is liquid and selective for Listeria rhamnosus, and the culture medium comprises or consists of: Rhamnose at a concentration of 5 - 15 g / l; Antibiotics, wherein the antibiotics are three, four or all five of nalidixic acid, ceftazidime, polymyxin B sulfate, cycloheximide and amphotericin B; A pH color indicator; and LiCl at a concentration of 5 - 15 g / l; b) A container or a porous tray for culturing a sample that may contain Listeria; and c) Optionally, a colorimetric table for identifying positive samples; and d) Optionally, instructions for use.

17. The method according to claim 1, the culture medium according to claim 15 or the kit according to claim 16, wherein the rhamnose is present at a concentration of 7 - 13 g / l.

18. The method according to claim 1, the culture medium according to claim 15 or the kit according to claim 16, wherein the rhamnose is present at a concentration of 10 g / l.

19. The method according to claim 1, the culture medium according to claim 15 or the kit according to claim 16, wherein the LiCl is present at a concentration of 7 - 13 g / l.

20. The method according to claim 1, the culture medium according to claim 15 or the kit according to claim 16, wherein the LiCl is present at a concentration of 10 g / l.

21. A computer-implemented calculator for displaying a prediction of the growth of Listeria based on the detection and counting of colony-forming units in a sample by the method according to claim 1, the calculator comprising: a) An input device; b) An output device; c) Software in a downloadable or memory; wherein the software accepts an input of any number of colony-forming units in the range of 0.04 cfu / g to 1 cfu / g present in the sample.

22. The method according to claim 1, the culture medium according to claim 15, the kit according to claim 16 or the computer-implemented calculator according to claim 21, wherein the Listeria is Listeria monocytogenes.

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