This study optimizes the Loop-mediated isothermal amplification (LAMP) assay using activated carbon and bentonite to detect pathogenic Vibrio species in seafood. This pretreatment effectively removes reaction inhibitors, yielding significantly faster, more sensitive, and cost-effective diagnostics compared to conventional methods.
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Research Article
Increased Sensitivity Loop-Amplification Using Activated Carbon and Bentonite for the Detection of Vibrio spp. in Fishery Products
In This Article
Summary
Abstract
The Vibrio genus has more than 100 species, of which 12 are considered pathogenic for humans; species most frequently isolated in food are V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus. The main problem of identifying them through conventional microbiological methods is the high costs and long-term diagnosis. Loop-mediated isothermal Amplification (LAMP) was used for the identification of V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus. Activated carbon with bentonite (ACB) was used to remove inhibitors, improving the sensitivity of the test; pH 6, 7, and 9 were tested, as well as different contact times between the sample and ACB. The LAMP sensitivity method was improved when activated carbon and bentonite were used to remove reaction inhibitors in samples of shrimp, fish, and oyster. LAMP sensitivity, negative predictive value, positive predictive value, and LAMP specificity were equal to or greater than 90% for all samples tested. The LAMP method could be an alternative to identify V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus in fishery products due to faster application, higher sensitivity, and lower costs than the standard method.
Introduction
Species of the Vibrio genus are widely distributed in marine environments and may be associated with fish, mollusks, or crustaceans in a way that can be either pathogenic or commensal. It has been reported that 12 species can be pathogenic for humans, with Vibrio cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus revealing a higher frequency of isolation in fishery products. In the United States, health authorities estimate that Vibrio spp. cause more than 80,000 infections per year1,2,3,4,5. The isolation of Vibrio spp. is achieved through conventional methods that involve pre-enrichment, culture in selective media, and confirmation by biochemical and serological tests6,7. These conventional methods are limited by the prolonged time required to isolate the pathogen (up to 5 days) and their lack of resolving power, which often fails to accurately differentiate closely related species within the Vibrio genus due to similar phenotypic and biochemical profiles6,7,8.
Alternatively, Loop-mediated isothermal amplification (LAMP) has been reported as a rapid, accurate, and inexpensive molecular technique for the identification of pathogens9,10. This method is highly cost-effective and faster than conventional PCR, as it does not require sophisticated equipment like thermocyclers9. The LAMP method allows, in a single step, the detection of the target gene by observing turbidity in the reaction tube caused by the precipitation of magnesium pyrophosphate because it is a stable reaction product at 65 °C, the temperature used in the LAMP technique, so that the increase in turbidity correlates with the amount of DNA synthesized, avoiding the use of agarose gels and transilluminators11,12,13,14,15.
The sensitivity detection of these pathogens can be improved by the elimination of reaction inhibitors like proteases, calcium ions, glycogen, and lipids that can inhibit Bst polymerase or bind to the DNA target, avoiding amplification13,16,17,18,19,20. It has been reported that the combination of activated carbon with bentonite (ACB) has high efficiency in eliminating amplification inhibitors, therefore increasing the sensitivity of Escherichia coli detection in oyster samples, decreasing costs because ACB is cheaper than using commercial kits21.
In this work, we propose to optimize the LAMP method by using activated carbon and bentonite to increase the sensitivity in the identification of V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus directly from fishery products. This optimized method provides practical guidance for routine monitoring and quality control in the food industry, being highly applicable for the rapid diagnosis of pathogens, avoiding the lengthy pre-enrichment stages. Regarding its expected performance, the method demonstrates excellent sensitivity (greater than 98%) and specificity (100%) in matrices such as fish and shrimp. However, a key limitation to consider is that in highly contaminated samples from origin, such as oysters, the ACB system cannot completely eliminate all interfering organic matter, which can slightly decrease the sensitivity of the assay (94%–98%).
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Protocol
This study did not involve human or vertebrate animal subjects; therefore, institutional ethical approval was not required. The reagents and the equipment used are listed in the Table of Materials.
1. Bacterial strains used and extraction of DNA
The Vibrio cholerae no O1 CECT 55, V. parahaemolyticus ATCC 17802, V. vulnificus ATCC 29037, and V. alginolyticus ATCC 17749 strains were grown in brain heart infusion (BHI) broth with 2% NaCl at 37 °C for 24 h. The strains of Escherichia coli ATCC 25922, Cronobacter sakazakii ATCC 12868, Listeria monocytogenes ATCC 49594, and Salmonella Typhimurium ATCC 14028 were cultured in BHI broth without sodium chloride at 37 °C for 24 h to be used as negative controls in the tests. For DNA extraction, an overnight culture was centrifuged at 16,000 x g for 6 min at 25 °C in a centrifuge. The supernatant was carefully decanted to avoid disturbing the cell pellet, and the pellet was thoroughly resuspended by pipetting in 500 μL of TAE buffer solution (Tris-acetic acid-EDTA, pH 8). The suspension was boiled for 6 min to lyse the cells, then immediately cooled on ice for 5 min. A final centrifugation was performed to pellet cellular debris, yielding a clear supernatant containing the extracted DNA, which was subsequently used as a template to amplify by LAMP22,23.
2. LAMP procedure
The primers used in LAMP (Table 1) were tested to corroborate the specificity in the identification of V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus. The identification of each Vibrio species using LAMP was performed in reaction tubes by preparing a final volume of 25 μL of the mixture containing: 1.6 μM of each of the primers, 1.4 mM of a mixture of dNTP, 6 mM MgSO4, 800 mM of betaine, 8 U of Bst DNA polymerase, and 5 μL of template DNA. The reaction mixture was gently mixed and incubated in a water bath at 65 °C for 60 min. After the incubation time, the amplification products were separated on a 1.5% agarose gel. The amplification products were then stained using 1 μL of a fluorescent DNA dye diluted 1:100 and revealed using a blue light transilluminator. Another technique to determine if the test was positive in the identification of the pathogen involved adding 4 μL of hydroxynaphthol blue (HNB, 125 μM) to the reaction tubes and observing the color change with the naked eye. A positive test was indicated by the formation of a blue color, and a negative one by a violet color24. Escherichia coli ATCC 25922, Cronobacter sakazakii ATCC 12868, Listeria monocytogenes ATCC 49594, and Salmonella Typhimurium ATCC 14028 were used as negative controls for the LAMP method. The amplifications obtained in LAMP were separated on an agarose gel and observed using a transilluminator. The resulting images were acquired and digitized using the MS 1D Analysis Software.
3. Treatment of samples of oysters, shrimp, and fish for the elimination of Vibrio spp. naturally present in the samples
Ten oyster samples, 10 shrimp samples, and 10 fish samples with an approximate weight of 200 g each were acquired. To remove naturally occurring Vibrio cells, the previously published methodology21 was followed. Fifty grams (50 g) of each sample was weighed and mechanically homogenized in a paddle blender at 260 rpm for 1 min, producing a uniform tissue suspension, which was then stored at -70 °C for 72 h. Subsequently, the samples were completely thawed using a 40 °C water bath, and the samples were washed with 300 mL of saline phosphate buffer solution (0.85% NaCl, pH 9). To ensure that the previous procedure had successfully eliminated Vibrio in the samples, the pre-enrichment methodology indicated in chapter 9 of the Bacteriological Analytical Manual of the Food and Drug Administration7 was performed. The saline solution containing the sample was replaced by 450 mL of alkaline peptone water (APW, pH 9). Serial dilutions were then meticulously prepared in APW up to a 10⁻3 dilution and incubated for 24 h. Finally, an aliquot from each tube was streaked onto a selective medium of thiosulfate-citrate-bile salts-sucrose (TCBS) agar and incubated for 24 h at 37 °C to visually confirm the absence of characteristic Vibrio colonies. All tests were performed in triplicate.
4. Preparation of activated carbon with bentonite (ACB) and evaluation of cellular recovery
To determine the conditions where ACB is most efficient in absorbing inhibitors present in samples of fishery products while maintaining minimal absorption of target cells, the published methodology21 was followed to establish the optimal pH and contact time between ACB and food samples. Activated carbon was thoroughly washed with distilled water and dried at 55 °C. Subsequently, 32.6 g of activated carbon was mixed with 3.04 g of bentonite, stirred continuously at 150 rpm for 24 h to ensure uniform distribution, dried at 55 °C, and sterilized, resulting in a fine, homogeneous dry ACB powder. To evaluate cellular recovery after mixing Vibrio cells with the activated carbon-bentonite (ACB) mixture, strains were grown separately in BHI broth with 2% NaCl until reaching a cell density of 1.5 x 104 CFU/mL. Then, 1 mL of each Vibrio strain was inoculated separately into 100 mL of phosphate buffer (PBS, 0.85% NaCl, adjusted to pH 4, 6, and 9). The bacterial suspension was then combined with 4.6 g of the pre-prepared ACB and left to stir at 150 rpm for 15, 30, and 60 min to facilitate contact. After each designated contact time, a 0.1 mL aliquot was carefully separated, spread-plated onto TCBS agar, and incubated at 37 °C for 24 h before performing the colony count.
5. Evaluation of the detection limit of V. cholerae, V. parahaemolyticus, V. vulnificus, and V. alginolyticus in oysters, shrimp, and fish samples treated with activated carbon and bentonite
Each of the Vibrio species used in this study was grown in BHI broth, and their cell densities were precisely adjusted to 1.5 x 104, 7.5 x 103, 3.8 x 103, 1.5 x 103, 7.5 x 102, 3 x 102, 1.5 x 102, and 76 CFU/mL (A600 nm ~0.25) as measured using a spectrophotometer. Subsequently, 1 mL of each Vibrio culture was used to inoculate 50 g of each sample (oysters, fish, shrimp) that were previously treated to eliminate any Vibrio presence. The inoculated samples were homogenized in a paddle blender at 360 rpm for 30 s and diluted with 450 mL of APW (pH 6). The mixture was then centrifuged at 260 x g for 5 min at 4 °C to separate large debris. The resulting supernatant was decanted into flasks containing 23 g of ACB, stirred at 150 rpm for 15 min to absorb inhibitors, and filtered using filter paper, yielding a clear eluate devoid of large particulates. This eluate was then centrifuged at 16,000 x g for 10 min at 4 °C, resulting in a visible cell pellet at the bottom of the tube, which was subsequently used to extract DNA. Each Vibrio species was identified by the LAMP method. Negative controls consisted of samples inoculated with strains of Escherichia coli ATCC 25922, Cronobacter sakazakii ATCC 12868, Listeria monocytogenes ATCC 49594, and Salmonella Typhimurium ATCC 14028. The reaction mixture was incubated in a water bath at 65 °C for 60 min. After the incubation time, the amplification products were separated on a 1.5% agarose gel and stained with a fluorescent DNA dye. Gels were revealed in a blue light transilluminator. Finally, 4 μL of hydroxynaphthol blue (125 μM) was added to each tube for naked-eye detection24.
6. Determination of positive predictive value, negative predictive value, specificity, and sensitivity of the LAMP method
The sensitivity, specificity, and predictive values of the LAMP method were determined using 30 samples (10 of shrimp, 10 of oyster, and 10 of fish). Each sample was inoculated with the minimum detectable concentration established for each species of Vibrio. To determine the percentage of sensitivity and specificity as well as negative predictive value (NPV) and positive predictive value25, a contingency table was used to summarize the true positives (TP), true negatives (TN), false positives (FP), and false negatives (FN) obtained during the experiments. (Table 2). From these data, the following formulas were applied:
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Results
Bacterial strains used and the extraction of DNA
DNA was successfully extracted from all pathogenic and negative control strains to be used as templates for the downstream LAMP assays.
LAMP procedure
Primers used in this work were specific for the amplification of the molecular target in each Vibrio species. The amplification products obtained by LAMP when Vibrio strains were used demonstrated that each primer was specific in the targ...
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Discussion
This study establishes optimized conditions to enhance the sensitivity of loop-mediated isothermal amplification (LAMP) for detecting V. cholerae, V. parahaemolyticus, V. alginolyticus, and V. vulnificus in contaminated seafood samples. By circumventing the traditional pre-enrichment stage, diagnostic time is significantly reduced, offering a highly efficient and rapid approach for directly identifying pathogens from fishery products.
A key methodological im...
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Disclosures
The authors declare no competing interests.
Materials
| Name | Company | Catalog Number | Comments |
|---|---|---|---|
| Activated carbon | Sigma-Aldrich | C2764 | |
| Bentonite | Sigma-Aldrich | 682659 | |
| Betaine | Sigma-Aldrich | B0300 | |
| Bio-Image systems transilluminator | Bio-Image systems | MBE-CP-01 | |
| Bst DNA polymerase | New England Biolabs | M0374 | |
| Centrifuge (model J2-HS) | Beckman Coulter | https://www.americaninstrument.com/products/beckman-j2-hs-high-speed-1273g-centri | |
| Cronobacter sakazakii | ATCC | 12868 | |
| dNTP mixture | Thermo Fisher Scientific | R0241 | |
| Escherichia coli | ATCC | 25922 | |
| Hydroxynaphthol blue | Sigma-Aldrich | 219916 | |
| Listeria monocytogenes | ATCC | 49594 | |
| Midori Green Advanced | Nippon Genetics | MG01 | |
| MS 1D Analysis Software | Major Science | MBE-IMG-SW | |
| Salmonella Typhimurium | ATCC | 14028 | |
| Spectrophotometer (model 6300) | Jenway | 630531 | |
| Stomacher Circulator (model 400) | Seward | 0400/000/AJ | |
| Vibrio alginolyticus | ATCC | 17749 | |
| Vibrio cholerae no O1 | CECT | 55 | |
| Vibrio parahaemolyticus | ATCC | 17802 | |
| Vibrio vulnificus | ATCC | 29037 | |
| Whatman paper | Whatman | 1001-055 |
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