Rapid Detection Method for Salmonella in Agricultural Products and Kit Therefor

Abstract

The present invention relates to an analytical method capable of rapidly detecting Salmonella and a kit for the same. The method of the present invention involves adding a liquid culture medium to an agricultural product sample and performing enrichment culture for a period of one hour or less, capturing pathogens by filtering through a membrane filter, and confirming the presence of Salmonella based on whether colonies are formed by directly contacting the filter with a selective medium such as XLD agar and culturing it. The present invention can effectively detect minute amounts of Salmonella while significantly shortening the enrichment time, and thus can be usefully applied in the field of food safety.

Description

Technology Field

[0001] The present invention relates to a technology for analyzing pathogenic microorganisms, and more specifically, to Salmonella that may be present in food samples such as agricultural products ( Salmonella spp. The present invention relates to an analysis method capable of detecting ) within a short period of time and a kit for performing the same. Background Technology

[0003] Salmonella is a representative pathogen that causes food poisoning. It can enter the human body through various foods, particularly poultry, meat, eggs, and agricultural products, and can cause serious gastrointestinal diseases upon infection. Accordingly, the technology to rapidly and accurately detect the presence of Salmonella in food has been recognized as a critical task in terms of food hygiene and public health.

[0004] Traditionally, the detection of Salmonella has generally involved pre-treating food samples, undergoing a pre-enrichment and enrichment process, and plating them onto selective media for incubation over several days. While this traditional microbial analysis method offers excellent sensitivity and selectivity, the entire analysis process takes more than three days, and there is a possibility of false positives or missed detections when the initial contamination level is low. Furthermore, when samples such as agricultural products have low surface contamination and small bacterial counts, there is a high likelihood of delayed or failed detection using existing methods.

[0005] To address this, molecular biological methods (such as PCR and DNA hybridization) and immunological methods (such as ELISA) have been partially introduced; however, these techniques have limitations, such as requiring expensive equipment and reagents, and still necessitating sample pretreatment and concentration processes. In particular, the technology to effectively concentrate minute amounts of pathogens in samples to ensure sensitivity remains an area requiring improvement. Prior art literature

[0007] Republic of Korea Published Patent Application No. 10-2002-0066091 (August 14, 2002) The problem to be solved

[0008] The present invention aims to solve the problems of the prior art and provides an analysis method and a kit for the same that can detect Salmonella, which may be present in trace amounts in agricultural product samples, with high sensitivity within a short period of time. More specifically, the objective is to provide an analysis method that can effectively capture and detect pathogenic Salmonella with only a short enrichment culture of one hour or less, without the long enrichment culture process required in conventional microbiological analysis.

[0009] In addition, the objective is to provide a pretreatment technology that can rapidly concentrate and culture Salmonella in samples where only a small amount of pathogens may be present on the surface, such as agricultural products, through filtration using a membrane filter and contact culture with a selective medium.

[0010] Furthermore, another objective of the present invention is to provide an integrated kit including enrichment medium, membrane filter, selective medium, etc., so that the above analysis method can be efficiently performed in a field or general experimental environment. means of solving the problem

[0012] To solve the above problem, the present invention provides an analysis method capable of rapidly detecting Salmonella from an agricultural product sample and a kit for the same. The analysis method of the present invention comprises the steps of: adding a liquid culture medium to an agricultural product sample and performing enrichment culture for a period of one hour or less, and then filtering the sample using a membrane filter to capture Salmonella on the surface of the filter; bringing the captured membrane filter into contact with the surface of a selective medium for Salmonella; and selectively culturing the selective medium to form Salmonella colonies and determining their presence.

[0013] Here, the membrane filter is a plate-shaped, flat-structured filtration membrane made of PES, CA, or MCE material, capable of capturing microorganisms in a sample by vacuum filtration or the like. The filter comes into contact with the surface of a Salmonella selective medium, preferably XLD agar, and the contact method can be selected from a method of temporarily applying it to the surface of the medium or a method of placing it on the medium and culturing it. After selective culture, Salmonella present on the surface of the filter receives nutrients from the medium, grows, and forms colonies, thereby allowing confirmation of the presence of Salmonella in the sample.

[0014] The composition of the present invention not only significantly reduces the enrichment culture time of more than 24 hours required in conventional microbiological detection methods to less than 1 hour, but also has the technical feature of improving detection sensitivity by physically and effectively capturing and concentrating pathogens present in trace amounts in a sample. In addition, by providing the elements required for each step of the analysis method of the present invention, namely buffered peptone water, membrane filters, selective media, and biochemical verification means, in the form of a single kit, it enables convenient Salmonella detection in the field or general laboratory environment.

[0015] Accordingly, the present invention provides an analytical means capable of rapidly and effectively detecting Salmonella in agricultural product samples by enabling the entire process, from sample pretreatment, concentration, selective culture, and detection, to be consistently performed within a minimized time. Effects of the invention

[0017] According to the present invention, Salmonella that may be present in agricultural product samples can be rapidly detected in a significantly shorter time than conventional methods. In particular, since pathogens can be effectively concentrated using a membrane filter with only a short enrichment culture of less than one hour and Salmonella colony formation can be induced by selectively culturing on a selective medium, the analysis time can be drastically shortened compared to traditional microbial culture methods.

[0018] In addition, the membrane filter used in the present invention has a plate-like planar structure rather than a hollow fiber form, so the equipment configuration and experimental procedure are simple, and it is easy to apply not only in laboratory environments but also under field inspection conditions. The method of culturing by directly contacting the membrane filter onto a selective medium enables the rapid and selective cultivation of minute amounts of Salmonella captured on the filter, and allows for intuitive confirmation of the presence of infectious agents simply by whether colonies are formed on the selective medium.

[0019] Furthermore, the present invention provides a kit for performing the above analysis method, thereby enabling the inclusion of an enrichment medium, a membrane filter, a selective medium, and a verification means within a single configuration, thereby ensuring consistency and reproducibility of the analysis. Through this configuration, the present invention provides a practical and highly efficient analysis means capable of rapidly and accurately detecting Salmonella even from low-contamination samples such as agricultural products. Brief explanation of the drawing

[0021] Figure 1 shows a microbial pretreatment process through membrane filter filtration. Figure 2 shows the recovery rate of Salmonella after filter filtration. Figure 3 shows the results of Salmonella culture after filtration with a PES membrane filter. Figure 4 shows the results of Salmonella culture after filtration with a CA membrane filter. Figure 5 shows the results of Salmonella culture after filtration with an MCE membrane filter. Figure 6 shows the results of the Salmonella validation for romaine lettuce. Specific details for implementing the invention

[0022] The following detailed description of the invention refers to the accompanying drawings, which illustrate specific embodiments in which the invention may be practiced. These embodiments are described in sufficient detail to enable those skilled in the art to practice the invention. It should be understood that various embodiments of the invention are different but need not be mutually exclusive. For example, specific shapes, structures, and characteristics described herein may be modified from one embodiment to another without departing from the spirit and scope of the invention. It should also be understood that the location or arrangement of individual components within each embodiment may be modified without departing from the spirit and scope of the invention. Accordingly, the following detailed description is not meant to be limiting, and the scope of the invention should be understood to encompass the scope claimed by the claims and all equivalents thereof. Similar reference numerals in the drawings indicate identical or similar components across various aspects.

[0023] One embodiment of the present invention relates to a method for detecting Salmonella from an agricultural product sample, comprising: (a) a step of adding a culture liquid medium to the agricultural product sample and enriching the culture for a period of one hour or less to provide an enriched culture sample; (b) a step of filtering the enriched culture sample through a membrane filter to collect Salmonella on the membrane filter; (c) a step of contacting the membrane filter with the surface of a Salmonella selective medium, wherein the contact includes contacting the filter temporarily with the selective medium or culturing while placed on the selective medium; (d) a step of selectively culturing the selective medium with the membrane filter in contact to form Salmonella colonies; and (e) a step of determining whether Salmonella is present in the sample by confirming whether colonies are formed.

[0024] In one embodiment of the present invention, the membrane filter may be made of PES (polyethersulfone), CA (cellulose acetate), or MCE (mixed cellulose esters).

[0025] In one embodiment of the present invention, the membrane filter may not be a hollow fiber membrane.

[0026] In one embodiment of the present invention, the membrane filter may have a flat-sheet membrane structure.

[0027] In one embodiment of the present invention, the liquid medium may include buffered peptone water.

[0028] In one embodiment of the present invention, the enrichment culture step may be performed at a temperature of 36±1℃ for 1 hour.

[0029] In one embodiment of the present invention, the selective medium may include XLD agar.

[0030] In one embodiment of the present invention, the agricultural product sample may be at least one of lettuce, perilla leaves, spinach, or napa cabbage.

[0031] In one embodiment of the present invention, the culture step may be performed on a selective medium for 24 hours.

[0032] In one embodiment of the present invention, the step of contacting a membrane filter with a selective medium may include culturing while maintaining the filter placed on the selective medium.

[0033] In one embodiment of the present invention, detection may be possible even when the concentration of Salmonella present in the sample is 1 CFU / 25 g or less.

[0034] Another embodiment of the present invention relates to a kit for detecting Salmonella from an agricultural product sample, comprising: (a) buffered peptone water for enriching the sample; (b) a membrane filter made of PES, CA, or MCE material for filtering the enrichment culture solution; and (c) XLD agar for selectively culturing Salmonella captured in the membrane filter.

[0035] The liquid culture medium is a solution for enriching the culture of Salmonella that may be present in agricultural product samples, and has a composition designed to enable the pathogenic microorganism to survive and recover even when exposed to stressful environments (drying, acidity, refrigeration, etc.). In one embodiment, the liquid culture medium may be composed of Buffered Peptone Water (BPW). BPW contains defatted peptone as a main component and is adjusted to a pH of 7.0 ± 0.2, providing conditions suitable for the growth of Salmonella in various food sample environments. Enrichment culture is generally performed at a temperature of 36 ± 1°C, and limiting the culture time to one hour or less can contribute to shortening the overall analysis time.

[0036] A membrane filter is a filtration membrane designed to effectively capture Salmonella from a sample that has been enriched and subsequently cultured in direct contact with a selective medium. The membrane filter used in the present invention has micropores smaller than the cell size of Salmonella and is configured to selectively capture pathogenic microorganisms in the sample. In one embodiment, the membrane filter is composed of polyethersulfone (PES), cellulose acetate (CA), or mixed cellulose esters (MCE). These materials have excellent microbial adhesion properties, minimal physical damage during filtration, and the ability to maintain structural stability even when cultured in contact with a selective medium.

[0037] The pore size of the filter is generally set to a range of 0.22 μm to 0.45 μm, which is a range capable of providing appropriate capture performance considering the size of bacterial pathogens such as Salmonella (approx. 0.5 to 1.5 μm). The membrane filter has a flat-sheet structure and is configured with a diameter of 35 to 60 mm or 47 mm, allowing it to be easily applied to the surface of a filtration device and a selective medium. Hollow fiber filters are not used, and since the present invention is based on a configuration in which the filter is in direct contact with the surface of the selective medium, a membrane with a flat structure is suitable. This configuration provides structural conditions that allow microorganisms captured on the filter to absorb nutrients from the selective medium and grow, while simultaneously facilitating subsequent colony observation.

[0038] The step of contacting a membrane filter with the surface of a selective Salmonella medium is a process for selectively culturing Salmonella captured on the filter surface through filtration, and is performed by placing the membrane filter in close contact with the upper surface of the selective medium. The selective medium used at this time is preferably a solid medium, such as XLD agar, composed to allow only Salmonella to grow selectively, and selective culture can be achieved by temporarily pressing the filter onto the surface of the medium or by placing it flat on the medium and introducing it directly into an incubator. This contact method allows the bacterial cells attached to the filter to grow by absorbing moisture and nutrients from the selective medium, while simultaneously limiting the growth of non-target microorganisms by the inhibitory components of the selective medium, thereby providing a culture environment in which the presence of Salmonella can be clearly confirmed.

[0039] A selective medium is a medium for selectively culturing only specific pathogens, preferably Salmonella spp., among the various microorganisms that may be present in a sample. In the present invention, it serves to efficiently proliferate Salmonella captured on a membrane filter and induce the formation of colonies in a visually distinguishable form. The selective medium used in one embodiment is XLD agar (Xylose Lysine Deoxycholate agar), which contains components such as sucrose, xylose, lysine, and iron sulfate, thereby causing Salmonella to undergo specific metabolic reactions (e.g., It is designed to form black-centered colonies depending on the generation, while simultaneously possessing both selectivity and indicator properties to inhibit the growth of other intestinal bacteria or non-target bacteria. This selective medium provides an environment in which bacteria captured on the filter surface can grow, and at the same time allows for intuitive determination of culture results, thereby improving the speed and accuracy of the entire detection process.

[0040] Example 1. Measurement of recovery rate by membrane filter filtration method

[0041] Salmonella typhimurium used in this study ( Salmonella typhimurium, NCTC 12023 was supplied by BioMerieux Korea (bioMerieux, France) and used. Standard strains were diluted in physiological saline (0.85% saline) to a concentration of 120 CFU per 10 mL and then homogenized. The prepared homogenates (10 mL) were filtered under reduced pressure using membrane filters (PES, CA, MCE). After filtration, the filters were placed directly onto the selective media for each strain and incubated for 24 hours at the appropriate culture temperature for each strain; the results were expressed in CFU / mL. Figure 1 illustrates the microbial pretreatment process through membrane filter filtration.

[0042] In order to compare the detection limits of the general analysis method and the filter filtration pretreatment method, the number of bacteria in the same homogenate was confirmed by plating a diluted homogenate onto a selective medium in accordance with Article 8. General Test Methods 4. Microbiological Test Methods of the "Standards and Specifications for Food" (Notification of the Ministry of Food and Drug Safety).

[0043] Figure 2 shows the recovery rate of Salmonella after filtration. Figure 2 shows the results of directly culturing the filter on a selective medium after filtering 10 mL of a homogenate prepared with a standard Salmonella typhimurium strain in physiological saline (0.85% saline) to a level of 120 CFU. The recovery rate is calculated by dividing the number of detected bacteria by the number of inoculated bacteria and multiplying the result by 100. With the conventional analysis method, countable colonies were detected at 2 CFU / mL; however, after filtration using filters (PES, CA, MCE), colonies were detected at 100, 60, and 50 CFU / mL, respectively, resulting in recovery rates of 83.33%, 50.83%, and 46.67%, respectively. The highest recovery rate was confirmed when using the PES filter.

[0044] Example 2. Application of membrane filter filtration method to pathogenic microorganisms

[0045] One colony of the standard strain was inoculated into 25 mL of the strain's enrichment medium (BPW) and homogenized for 30 seconds using a pulsifier (Macrogen, Seoul, Korea). The homogenate was cultured for 24 hours at an appropriate culture temperature for each strain, and samples were taken at 1, 2, 3, 4, 8, 12, and 24 hours of culture to homogenize the enrichment culture and prepare 10 mL of diluted solution. The prepared homogenate (10 mL) was filtered under reduced pressure using a PES (Polyethersulfone) membrane (47 mm, 0.22 μm) (CVS, Sanford, USA), an MCE (Mixed cellulose ether) membrane (47 mm, 0.22 μm) (Millipore, Ireland), and a CA (Cellulose acetate) membrane (47 mm, 0.22 μm) (CVS, Sanford, USA) filter. After filtration, the filter was placed directly onto the selective medium (XLD agar) of each strain and cultured at an appropriate temperature for 24 to 48 hours, after which the culture results were observed.

[0046] Assuming that agricultural products are contaminated with a minimum amount of pathogenic microorganisms (1 colony / 25 g), 1 colony / 25 g of Salmonella was actually inoculated. Then, based on the existing culture method according to Article 8, General Test Methods, 4, Microbiological Test Methods of the "Standards and Specifications for Food" (Notification of the Ministry of Food and Drug Safety), the enrichment culture time was set to 1, 2, 3, 4, 8, 12, and 24 hours. The enrichment culture solution for each culture time was filtered through a membrane filter and cultured to confirm the detection results of Salmonella. The results are shown in Tables 1 to 3 and Figures 3 to 5.

[0047] Table 1 shows the quantitative values ​​of Salmonella according to the analysis method (PES filter filtration, conventional analysis method). Table 2 shows the quantitative values ​​of Salmonella according to the analysis method (CA filter filtration, conventional analysis method). Table 3 shows the quantitative values ​​of Salmonella according to the analysis method (MCE filter filtration, conventional analysis method).

[0048] Figure 3 shows the results of Salmonella culture after filtration with a PES membrane filter. Figure 4 shows the results of Salmonella culture after filtration with a CA membrane filter. Figure 5 shows the results of Salmonella culture after filtration with an MCE membrane filter.

[0049]

[0050]

[0051]

[0052] Analysis results of Salmonella after 24 hours of enrichment culture according to the conventional culture method were 3.8 × 10⁻⁶ 8 It was found to be... On the other hand, as a result of culturing the enrichment culture after filtering it, 9.3×10⁻⁶ were obtained for each filter (PES, CA, MCE) after 1 hour of enrichment culture. 5 , 1.2 x 10 6 , 1.3 x 10 6 It was detected at the CFU / mL level.

[0053] The method of filtering the enrichment culture solution and culturing it in a medium allows for the detection of pathogenic microorganisms even when a short enrichment culture (1 hour) is performed, as Salmonella adheres to the filter and the bacteria are concentrated when the culture solution is filtered.

[0054] When using a method that allows the culture solution of pathogenic microorganisms to be concentrated using a filter, it can be used as a primary rapid screening method when Salmonella is detected in agricultural products.

[0055] Example 3. Verification of the validity of a microbiological analysis method for agricultural products applying an efficient pretreatment step

[0056] Analysis results of Salmonella after 24 hours of enrichment culture according to the conventional culture method were 3.8 × 10⁶ 8 It was found to be... On the other hand, as a result of culturing the enrichment culture after filtering it, 9.3×10 after 1 hour of enrichment culture, for each filter (PES, CA, MCE), respectively. 5 , 1.2 x 10 6 , 1.3 x 10 6It was detected at the CFU / mL level.

[0057] The method of filtering the enrichment culture solution and culturing it in a medium allows for the detection of pathogenic microorganisms even when a short enrichment culture (1 hour) is performed, as Salmonella adheres to the filter and the bacteria are concentrated when the culture solution is filtered.

[0058] When using a method that allows the culture solution of pathogenic microorganisms to be concentrated using a filter, it can be used as a primary rapid screening method when Salmonella is detected in agricultural products.

[0059] Example 3. Verification of the validity of a microbiological analysis method for agricultural products applying an efficient pretreatment step

[0060] To validate the membrane filter filtration pretreatment analysis method, Salmonella by target sample (lettuce) ( Salmonella typhimurium Validation was performed for ). 25g of the target sample was aseptically divided into sterile bags (Whirl-pak, 10 x 30 cm, Nasco, Fort Atkinson, Wi, USA) and Salmonella ( Salmonella typhimurium ), Listeria ( Listeria monocytogenes ) 1 colony was artificially inoculated. 225 mL of peptone water buffer was added to the inoculated sample, and the mixture was homogenized using a pulsifier for 30 seconds. The sample homogenate was filtered under reduced pressure using a membrane filter (PES, CA, MCE), and the filtered filter was placed directly onto the selective medium (XLD agar) of each strain and incubated at an appropriate temperature for 24 hours, after which the culture results were observed.

[0061] In order to compare the detection limits of the existing analysis method and the filter filtration pretreatment method, the number of bacteria in the same homogenate was confirmed by plating a diluted homogenate onto a selective medium in accordance with Article 8. General Test Methods 4. Microbiological Test Methods of the "Standards and Specifications for Food" (Notification of the Ministry of Food and Drug Safety).

[0062] 25 g of lettuce was weighed, inoculated with one Salmonella colony, and 225 mL of buffered peptone water was added and incubated at 36±1℃ for 1 hour. The enrichment culture was filtered, placed on a selective medium, and incubated at 36±1℃ for 24 hours, and suspected colonies were biochemically confirmed. As a reference method, incubation for 24 hours was performed according to the existing analysis method.

[0063] Figure 6 shows the results of the Salmonella validation for romaine lettuce. After enriching the Salmonella-contaminated samples for 1 hour, the Salmonella concentration in the culture medium was 6.4 × 10⁶ as shown in Table 4. 7 ~ 9.1×10 7 It was found to be at the CFU level, and the result obtained using the existing analysis method was 6.4×10 8 It was found to be at a level similar to CFU. Compared to existing analytical methods, the filter filtration method shortened the enrichment time and enabled rapid confirmation of the presence or absence of pathogenic microorganisms through the effect of concentrating a large amount of pathogenic microorganisms.

[0064] Explanation of the symbols delete

Claims

Claim 1 A method for detecting Salmonella (Salmonella spp.) from an agricultural product sample, comprising: (a) a step of adding a culture liquid medium to the agricultural product sample and enriching the culture for a period of one hour or less to provide an enriched culture sample; (b) a step of filtering the enriched culture sample through a membrane filter to collect Salmonella on the membrane filter; (c) a step of contacting the membrane filter with the surface of a Salmonella selective medium, wherein the contact includes contacting the filter temporarily with the selective medium or culture while placed on the selective medium; (d) a step of selectively culturing the selective medium contacted with the membrane filter to form Salmonella colonies; and (e) a step of determining whether Salmonella is present in the sample by checking whether colonies are formed, wherein the liquid medium comprises buffered peptone water, the selective medium comprises XLD agar, the agricultural product sample comprises at least one of lettuce, perilla leaves, spinach, or Chinese cabbage, and the step of contacting the membrane filter with the selective medium comprises placing the membrane filter so as to be in close contact with the upper surface of the selective medium, wherein detection is possible even when the concentration of Salmonella present in the sample is 1 CFU / 25 g or less, and wherein the sample is not cultured for enrichment after the collection step. Claim 2 A method for detecting Salmonella according to claim 1, characterized in that the membrane filter is composed of PES (polyethersulfone), CA (cellulose acetate), or MCE (mixed cellulose esters). Claim 3 A method for detecting Salmonella according to claim 2, characterized in that the membrane filter is not a hollow fiber membrane. Claim 4 A method for detecting Salmonella according to claim 2, characterized in that the membrane filter has a flat-sheet membrane structure. Claim 5 delete Claim 6 A method for detecting Salmonella according to claim 1, characterized in that the enrichment culture step is performed for 1 hour at a temperature of 36℃ ±1℃. Claim 7 delete Claim 8 delete Claim 9 A method for detecting Salmonella according to claim 1, characterized in that the culture step is performed on a selective medium for 24 to 48 hours. Claim 10 delete Claim 11 delete Claim 12 A kit for detecting Salmonella from an agricultural product sample comprises: (a) buffered peptone water for enriching the sample; (b) a membrane filter made of PES, CA, or MCE material for filtering the enrichment culture solution; and (c) XLD agar for selectively culturing Salmonella captured on the membrane filter, wherein the enrichment culture is carried out for a period of 1 hour or less, the agricultural product sample is at least one of lettuce, perilla leaves, spinach, or Chinese cabbage, the membrane filter is placed so as to be in close contact with the upper surface of the XLD agar, detection is possible even when the concentration of Salmonella present in the sample is 1 CFU / 25 g or less, and the sample is not enriched after capturing Salmonella on the membrane filter.

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