Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus

Yamazaki, Wataru; Ishibashi, Masanori; Kawahara, Ryuji; Inoue, Kiyoshi

doi:10.1186/1471-2180-8-163

Cited by 136 publications

(112 citation statements)

References 14 publications

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“…The LAMP reaction was performed as described previously [Hill et al, 2008;Yamazaki et al, 2008]. Each reaction mixture (total volume of 25 μL) contained 10× Bst ThermoPol buffer 2.5 μL, MgCl 2 (25 mmol/L) 3 μL, dNTP's (each 2.5 mmol/L) 3 μL, betaine (5 mol/L) 2 μL, inner primers (40 μmol/L) of each 1 μL, outer primers (10 μmol/L) of each 1 μL, loop primer (10 μmol/L) of each 1 μL, Bst DNA polymerase (New England Biolabs, Inc., Ipswich, MA) large fragment 1 μL (8units), DNA template 2 μL, and ddH 2 O×5.5μL.…”

Section: Lamp Primers and Analysis Of Lamp Productsmentioning

confidence: 99%

Rapid and Specific Detection of Escherichia coli O157:H7 in Ground Beef Using Immunomagnetic Separation Combined with Loop-Mediated Isothermal Amplification

Qin¹,

Puthiyakunnon²,

Zhang³

et al. 2018

Pol. J. Food Nutr. Sci.

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Escherichia coli O157:H7 is well known for many foodborne outbreaks that lead to fatal infections in human being worldwide. The objective of this study was to develop a rapid and sensitive method for detection of EHEC O157:H7 from ground beef using a method that combined immunomagnetic separation (IMS) with loop-mediated isothermal amplifi cation (LAMP). The EHEC O157:H7 cells were separated with Dynabeads coated with anti-EHEC O157:H7 after a short enrichment for 4 h. Then, EHEC O157:H7 was identifi ed by LAMP assay for amplifying and detecting the rfbE gene, which is highly conserved in all EHEC O157:H7 strains and exhibits strain-specifi c gene expression. The LAMP method results analyzed with real time turbidity measurements showed a high specifi city and sensitivity, with a positive detection rate of amplifi cation of EHEC O157:H7 DNA diluted to a minimum equivalent concentration of 1.8 × 10 1 CFU/mL, which was 10 times more sensitive than the conventional PCR assay. The IMS followed with LAMP could capture and detect a bacterial concentration as low as 3×10 1 CFU/mL from the meat samples, which was close to the sensitivity of LAMP assay with pure culture. IMS combined with realistic LAMP method is a simple, rapid, highly specifi c gene amplifi cation technology that is suitable for implementing as a screening assay in basic laboratory and fi eld test for detecting food contamination. Unauthenticated Download Date | 5/11/18 5:54 AM

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Section: Lamp Primers and Analysis Of Lamp Productsmentioning

confidence: 99%

Rapid and Specific Detection of Escherichia coli O157:H7 in Ground Beef Using Immunomagnetic Separation Combined with Loop-Mediated Isothermal Amplification

Qin¹,

Puthiyakunnon²,

Zhang³

et al. 2018

Pol. J. Food Nutr. Sci.

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“…The increase in the turbidity of the reaction mixture due to the production of the white precipitate correlates with the amount of DNA synthesized (6, 7, 13). Thus, LAMP assays do not require expensive equipment and are highly precise (3,18,19).Here we describe a rapid and simple LAMP assay for detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. We also determined the sensitivity of this LAMP assay using spiked shrimp samples.…”

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confidence: 99%

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confidence: 99%

Development of a Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of the tdh and trh Genes of Vibrio parahaemolyticus and Related Vibrio Species

Yamazaki

Kumeda

Misawa

et al. 2010

Appl Environ Microbiol

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Thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH) are the major virulence determinants of Vibrio parahaemolyticus. TRH is further differentiated into TRH1 and TRH2 on the basis of genetic and phenotypic differences. We developed a novel and highly specific loop-mediated isothermal amplification (LAMP) assay for sensitive and rapid detection of the tdh, trh1, and trh2 genes of V. parahaemolyticus. The LAMP assay was designed for both combined and individual detection of the tdh, trh1, and trh2 genes and combined detection of the trh1 and trh2 genes. Our results showed that it gave the same results as DNA probes and conventional PCR assays for 125 strains of V. parahaemolyticus, 3 strains of Grimontia hollisae, and 2 strains of Vibrio mimicus carrying the tdh, trh1, and trh2 genes in various combinations. No LAMP products were detected for any of the 20 bacterial strains lacking the tdh, trh1, and trh2 genes. The sensitivities of the LAMP assay for detection of tdh-, trh1-, and trh2-carrying V. parahaemolyticus strains in spiked shrimp samples were 0.8, 21.3, and 5.0 CFU per LAMP reaction tube, respectively. Starting with DNA extraction from a single colony and from spiked shrimp samples, the LAMP assay required only 27 to 60 min and less than 80 min, respectively. This is the first report of a rapid and specific LAMP assay for detection and differentiation of the tdh, trh1, and trh2 genes of V. parahaemolyticus and related Vibrio species.Vibrio parahaemolyticus, which is widely distributed in estuarine, marine, and coastal environments of tropical and temperate zones, causes seafood-borne gastrointestinal disorders in humans (9). Because most clinical isolates of V. parahaemolyticus produce the thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), or both (5,11,14), these products are considered important virulence markers of V. parahaemolyticus (4,5,9,11,14). TDH and TRH are encoded by the tdh and trh genes, respectively. Five sequence variants of the tdh gene (tdh1 to tdh5) can be distinguished, which are Ͼ97% identical (1, 10). The tdh gene has also been detected in Grimontia (Vibrio) hollisae and some strains of Vibrio mimicus isolated from patients with diarrhea (9). The trh gene shares ca. 68% sequence identity with the tdh gene (5). Although trh gene sequences vary somewhat among strains, the trh variants can be clustered into two subgroups represented by two trh genes (trh1 and trh2), which share 84% sequence identity (5).Although most clinical isolates carry the tdh and trh genes, either alone or in combination, approximately 99% of environmental isolates do not possess either gene (9). These genes are therefore considered important virulence and epidemiological markers (5,11,14). Detection of the tdh and trh genes of V. parahaemolyticus using DNA probe methods is time-consuming and laborious. PCR assays, in contrast, although providing rapid detection of both tdh and trh genes (2, 15), require electrophoresis on an agarose gel, which is time-consuming and tedious. A recent ...

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“…Previous studies have reported the sensitivity of the LAMP method ranges from 10 2 to 10 5 colony forming unit (CFU)/g V. parahaemolyticus in spiked oysters. 17,19) However, seafood with low levels of V. parahaemolyticus has potential risks to human infection because a small number of this pathogen, e.g., ca. 10 2 cells/g seafood, can increase substantially, e.g., >10…”

Section: 7)mentioning

confidence: 99%

“…Therefore, it may be appropriate to target tlh/ldh gene for the detection of V. parahaemolyticus. 11,[15][16][17] The loop-mediated isothermal amplification (LAMP) method described by Notomi et al is more advantageous than the PCR assay because it is more rapid, easier to perform and does not necessarily require expensive thermal cycler machine. 18) In the LAMP method it is possible to amplify DNA fragments within 90 min by using only water bath or heat block.…”

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confidence: 99%

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay Combined with Enrichment Culture for Rapid Detection of Very Low Numbers of Vibrio parahaemolyticus in Seafood Samples

Neogi

et al. 2015

Biol. Pharm. Bull.

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The aim of this study was to develop and evaluate a rapid and effective method to detect Vibrio parahaemolyticus, a leading pathogen causing seafood-borne gastroenteritis. A newly designed loop-mediated isothermal amplification (LAMP) assay including a short enrichment period was optimized. This assay correctly detected all the target strains (n 61) but none of the non-target strains (n 34). Very low numbers of V. parahaemolyticus (2 colony forming unit (CFU) per gram of seafood) could be detected within 3 h and the minimum time of the whole assay was only 5 h. Comparative screening of various seafood samples (n 70) indicated that the LAMP assay is superior to polymerase chain reaction (PCR) and conventional culture methods because it is more rapid and less complex. This highly sensitive LAMP assay can be applicable as the method of choice in large-scale and rapid screening of seafood and environmental samples to detect V. parahaemolyticus strains.Key words Vibrio parahaemolyticus; loop-mediated isothermal amplification; rapid detection; enrichment culture; seafood Vibrio parahaemolyticus is one of the most important seafood-borne pathogen causing gastrointestinal disorders to humans. This halophilic Gram-negative bacterium was first identified as a causative agent of human gastroenteritis in Japan in 1950.1) However, V. parahaemolyticus can occur ubiquitously in the brackish coastal environment of most continents and infections associated with seafood contaminated by this pathogen have occurred throughout the world.2-4) Particularly, the worldwide spread of the V. parahaemolyticus O3 : K6 serotype after its emergence in Asia in 1995, it has become very important to identify this pathogenic species in a rapid and sensitive manner.5) At present, the outbreak of V. parahaemolyticus infections is a significant public health concern in many countries including China, Japan and U.S.A. 6,7)Seafood is very popular in many countries, and simple and specific identification of a pathogen from seafood samples is essential for taking preventive and curative measures. Conventional methods for the diagnosis of V. parahaemolyticus include cultivation of bacteria on selective media followed by biochemical tests. However, the traditional phenotypic identification is problematic, it is very time-consuming requiring 3-7 d and not highly specific because several Vibrio species display similar biochemical characteristics, which make it difficult for rapid identification of V. parahaemolyticus. 8) To circumvent this problem, the identification of V. parahaemolyticus by rapid and specific molecular techniques targeting the genes encoding thermostable direct haemolysin (TDH) and TDH-related haemolysin (TRH) were developed.9-11) However, these genes are only found in pathogenic strains and most V. parahaemolyticus isolates from the environment sources do not produce TDH or TRH. 3,12) Besides, there are reports of toxigenic factors other than TDH or TRH, e.g., type III secretion system 2, associated with clinical V. parahaemolyticus stra...

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Development of a loop-mediated isothermal amplification assay for sensitive and rapid detection of Vibrio parahaemolyticus

Cited by 136 publications

References 14 publications

Rapid and Specific Detection of Escherichia coli O157:H7 in Ground Beef Using Immunomagnetic Separation Combined with Loop-Mediated Isothermal Amplification

Rapid and Specific Detection of Escherichia coli O157:H7 in Ground Beef Using Immunomagnetic Separation Combined with Loop-Mediated Isothermal Amplification

Development of a Loop-Mediated Isothermal Amplification Assay for Sensitive and Rapid Detection of the tdh and trh Genes of Vibrio parahaemolyticus and Related Vibrio Species

Development and Evaluation of a Loop-Mediated Isothermal Amplification Assay Combined with Enrichment Culture for Rapid Detection of Very Low Numbers of Vibrio parahaemolyticus in Seafood Samples

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