Rapid Detection of Vibrio parahaemolyticus in Seafood Using Loop-mediated Isothermal Amplification (LAMP) Method
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Vibrio parahaemolyticus has been considered as one of the most important food-borne bacterial pathogens. The loop-mediated isothermal amplification ( LAMP ) that amplifies DNA with high specificity and rapidity under isothermal conditions was applied for rapid detection of V. parahaemolyticus for the first time. A set of four primers, two outer and two inner primers, was designed specifically to recognize the thermolabile hemolysin gene ( tlh ) of V. parahaemolyticus. Genomic DNAs from 28 bacterial strains including 14 V. parahaemolyticus strains were amplified using LAMP, and no amplicon was observed in the other 14 bacterial strains. The detection limits of LAMP assay and pure cultures were 90 fg/LAMP mixture of V. parahaemolyticus genomic DNA and 24 cfu/mL, respectively. And for directly detecting V. parahaemolyticus in artificially contaminated food samples, the detection limit was 89 cfu/g. In addition, 40 seafood samples were tested by LAMP, and 8 of them were V. parahaemolyticus positive. Among the tested positive samples, 6 samples were also detected to be positive by conventional microbiological methods. These results suggest that detection of V. parahaemolyticus by LAMP is an effective and low-cost procedure with high specificity and sensitivity, and no specialized equipment required.Keywords:
Thermolabile
Amplicon
genomic DNA
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Vibrio parahaemolyticus is a leading cause of seafood-related bacterial gastroenteritis and outbreaks worldwide. Sensitive and specific detection methods are needed to better control V. parahaemolyticus infections. This study aimed at developing a highly specific and sensitive loop-mediated isothermal amplification (LAMP) assay for detecting V. parahaemolyticus in oysters. A set of five LAMP primers, two outer, two inner, and one loop were designed based on the published V. parahaemolyticus toxR sequence. Specificity of the assay was evaluated using a panel of 36 V. parahaemolyticus and 39 other strains. The assay sensitivity was determined using serial dilutions of V. parahaemolyticus ATCC 27969 culture ranging from 108 CFU/ml to extinction. The assay was also tested in experimentally inoculated oyster samples. The toxR-based LAMP assay was able to specifically detect all of the 36 V. parahaemolyticus strains without amplification from 39 other strains. The detection limit was 47-470 cells per reaction in pure culture, up to 100-fold more sensitive than that of toxR-PCR. When applied in spiked oysters, the assay was able to detect 1.1 × 105V. parahaemolyticus cells per gram of oyster without enrichment, up to 100-fold more sensitive than that of toxR-PCR. Standard curves generated for detecting V. parahaemolyticus in both pure culture and spiked oyster samples showed good linear relationship between cell numbers and the fluorescence or turbidity signals. The toxR-based LAMP assay developed in this study was sensitive, specific, and quantitative, holding great potential for future field detection of V. parahaemolyticus in raw oysters.
Serial dilution
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genomic DNA
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Objective:To apply the loop-mediated isothermal amplification(LAMP) to test intestinal diarrhea samples and evaluate its feasibility and reliability as a rapid detection method.Methods:145 intestinal diarrhea samples were detected and compared with LAMP and GB/T 4789.7-2008 method.Results:11 positive samples were found by LAMP,same as through GB/T 4789.7-2008 method.Conclusion:LAMP method had the advantages of high specificity,convenience and low cost,which can be used in Vibrio parahaemolyticus detection.
Acute diarrhea
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Primer (cosmetics)
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Abstract V ibrio parahaemolyticus is an important seafood‐borne pathogen associated with gastrointestinal disorders in humans. In this study, the efficacy of a loop‐mediated isothermal amplification ( LAMP ) combined with immunocapture assay was firstly developed for detection of V . parahaemolyticus in pure culture, artificially and naturally contaminated seafood samples. The detection limit was 7.3 × 10 1 cfu/mL for pure culture and artificially contaminated samples by IC‐LAMP , compared with 7.3 × 10 2 cfu/mL by LAMP . Furthermore, among 156 natural seafood samples, 13, 13 and 11 of samples were positive for V . arahaemolyticus by IC‐LAMP , LAMP and culture‐based method, respectively. V . parahaemolyticus was isolated from 11 of the 156 seafood samples by culture‐based method, all of which were positive by IC‐LAMP assay. A comparison between LAMP , IC‐LAMP and culture‐based method indicated that IC‐LAMP was sensitive, specific and reliable for monitoring V . parahaemolyticus contamination in seafood samples. Practical Application Development of rapid, sensitive and specific methods for detection of V . parahaemolyticus is of utmost importance for monitoring and controlling contaminated seafood. In this study, loop‐mediated isothermal amplification combined with the newly developed immunocapture showed higher detection limit than that by LAMP and more specific compared with that by culture‐based method. The IC‐LAMP was fit for monitoring V . parahaemolyticus contamination in seafood samples.
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Immunomagnetic separation
Colony-forming unit
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Primer (cosmetics)
Hemolysin
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Thermolabile
Hemolysin
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The specificity and sensitivity of an improved loop-mediated isothermal amplification (LAMP) method for rapid detection of Vibrio parahaemolyticus strains from various seafood samples had been developed and evaluated in this study. Six primers, including outer primers and inner primers, were specially designed for recognizing six distinct sequences on the target gene of tlh. The optimal reaction condition was found to be 65°C for 45 min, with the detection limit as 10 CFU/ml. Application of LAMP assays was performed on 416 food borne V. parahaemolyticus strains isolated from various seafood samples, and the total detection rate for LAMP and polymerase chain reaction (PCR) assay was found to be 96.2% (400/416) and 85.6% (356/416). This is the first report of an improved and simple LAMP detection assay on V. parahaemolyticus employed procedures of simple template deoxyribonucleic acid (DNA) preparation, equipment for LAMP reaction (water bath) and direct result determination via observation of color change. In addition, this is also the first application of LAMP detection on this considerable amount of V. parahaemolyticus isolates (416 strains for application together with 105 reference strains for establishment) with the total identification rate as 96.2%, as well as its extensive application to marine fish, shrimp, oyster, mussel, jellyfish, cuttlefish and seaweed samples, with detection rate ranging from 89.5 to 100%.
Key words: Loop-mediated isothermal amplification (LAMP), Vibrio parahaemolyticus, seafood samples.
Isothermal process
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Objective To develop an assay of loop-mediated isothermal amplification(LAMP) for detection and identification of Vibrio parahaemolyticus in basic laboratory.Methods The target of Vibrio parahaemolyticus was chosen from gyrB gene,four LAMP primers were designed and reaction conditions were optimized.Results: LAMP method was of high specificity for Vibrio parahaemolyticus.Through the amplification of 14 strains,Vibrio vulnificus was positive,and the other strains were negative.And the detection of LAMP method was finished within 60 minutes,and the detection limit was 25 cfu/ml.Totally 120 seafood samples were detected and positive rate was 62.5%.LAMP method was accord with traditional methods.Conclusion Comparing to traditional method,LAMP method is a good one:saving time,lower requirement of laboratory instruments and of good practicability for Vibrio parahaemolyticus detection.
Vibrio vulnificus
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