Rapid and Specific Detection of the Thermostable Direct Hemolysin Gene in Vibrio parahaemolyticus by Loop-Mediated Isothermal Amplification

Nemoto, Jiro; Sugawara, Chiyo; Akahane, Kenji; Hashimoto, Keiji; Kojima, Tadashi; Ikedo, Masanari; Konuma, Hirotaka; Hara-Kudo, Yukiko

doi:10.4315/0362-028x-72.4.748

Cited by 57 publications

(41 citation statements)

References 23 publications

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“…Out of 19 different bacterial strains we checked, only EHEC O157:H7 (1 standard and 4 wild strains) were positive with LAMP assay to make the specifi city 100%, which is consistent with the previous reports showing very high specifi city of the LAMP assay in detection of EHEC O157:H7 Han & Ge, 2010;Nemoto et al, 2009]. The LAMP sensitivity of 1.8 × 10…”

Section: Discussionsupporting

confidence: 88%

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: Discussionsupporting

confidence: 88%

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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“…However, a sensitivity of 10 1 CFU/ml would be equivalent to 0.05 CFU (0.05 DNA copy) per LAMP reaction tube, theoretically unattainable by molecular detection assays due to the absence of template DNA. The use of overnight culture where a high proportion of cells do not produce CFU was used to partly explain the detection limit of less than one cell (e.g., 0.1 cell) in a LAMP assay designed for virulent V. parahaemolyticus (26). In addition, in that reverse transcriptase LAMP study (42), the big discrepancy of sensitivity (4 logs) between visual observation and gel electrophoresis reported was rather uncommon.…”

Section: Discussionmentioning

confidence: 99%

“…To date, LAMP assays have been developed for Campylobacter spp. (47), Shiga toxinproducing Escherichia coli (14), norovirus (50), Staphylococcus aureus (9), Vibrio parahaemolyticus (5,26), and Vibrio vulnificus (10,12), as well as Salmonella (15,19,20,33,34,45,48). Although LAMP was reported to be rapid, specific, and sensitive, several of the Salmonella LAMP studies (33,34,48) targeted only one specific Salmonella serovar or serogroup, and none of the studies evaluated the quantification of salmonellae in produce samples by LAMP.…”

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confidence: 99%

Rapid Detection of Viable Salmonellae in Produce by Coupling Propidium Monoazide with Loop-Mediated Isothermal Amplification

Chen

Wang

Beaulieu

et al. 2011

Appl Environ Microbiol

147

116

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Recent outbreaks linked to Salmonella-contaminated produce heightened the need to develop simple, rapid, and accurate detection methods, particularly those capable of determining cell viability. In this study, we examined a novel strategy for the rapid detection and quantification of viable salmonellae in produce by coupling a simple propidium monoazide sample treatment with loop-mediated isothermal amplification (PMA-LAMP). We first designed and optimized a LAMP assay targeting Salmonella. Second, the performance of PMA-LAMP for detecting and quantifying viable salmonellae was determined. Finally, the assay was evaluated in experimentally contaminated produce items (cantaloupe, spinach, and tomato). Under the optimized condition, PMA-LAMP consistently gave negative results for heat-killed Salmonella cells with concentrations up to 10 8 CFU/ml (or CFU/g in produce). The detection limits of PMA-LAMP were 3.4 to 34 viable Salmonella cells in pure culture and 6

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“…While our study was in progress, Nemoto and colleagues described a tdh LAMP assay performed with 42 strains of V. parahaemolyticus (8). However, this assay did not include a system to detect the trh1 and trh2 genes, which are also important virulence factors of V. parahaemolyticus.…”

Section: Discussionmentioning

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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Rapid and Specific Detection of the Thermostable Direct Hemolysin Gene in Vibrio parahaemolyticus by Loop-Mediated Isothermal Amplification

Cited by 57 publications

References 23 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

Rapid Detection of Viable Salmonellae in Produce by Coupling Propidium Monoazide 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

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