Rapid and specific detection of the thermostable direct hemolysin gene in Vibrio parahaemolyticus by loop-mediated isothermal amplification.

Several investigators have reported that thermostable direct hemolysin (TDH) and TDH-related hemolysin are important virulence factors of Vibrio parahaemolyticus, but it has been difficult to detect these factors rapidly in seafood and other environmental samples. A novel nucleic acid amplification method, termed the loop-mediated isothermal amplification (LAMP), which amplifies DNA with high specificity and rapidity under isothermal conditions, was applied. In this study, we designed tdh gene-specific LAMP primers for detection of TDH-producing V. parahaemolyticus. The specificity of this assay was evaluated with 32 strains of TDH-producing V. parahaemolyticus, one strain of TDH-producing Grimontia hollisae, 10 strains of TDH-nonproducing V. parahaemolyticus, and 94 strains of TDH-nonproducing bacteria, and the sensitivity was high enough to detect one cell per test. Moreover, to investigate the detection of TDH-producing V. parahaemolyticus in oysters, the LAMP assay was performed with enrichment culture in alkaline peptone water of oyster samples inoculated with TDH-producing V. parahaemolyticus and TDH-nonproducing V. parahaemolyticus and V. alginolyticus after enrichment in alkaline peptone water. These results suggest that the LAMP assay targeting tdh gene has high sensitivity and specificity and is useful to detect TDH-producing V. parahaemolyticus in oyster after enrichment.

[Rapid detection of Vibrio vulnificus using a loop-mediated isothermal amplification method].

Vibrio vulnificus is found in marine waters near the coast around the world. Infection with this gram-negative rod, via ingestion of raw seafood or via a skin wound following contact with contaminated estuarine or marine water, can cause necrotizing fasciitis and sepsis. Most of patients with Vibrio vulnificus infection have underlying liver dysfunction or diabetes mellitus. Due to the high mortality and short latent periods, control of this infection depends on early identification of the bacterial species and prompt initiation of intensive care. Accordingly, the development of a technique that can identify this microbe quickly and accurately is of great importance. Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method to detect specific genes with rapidity and high sensitivity. In this study, we developed LAMP for the detection of Vibrio vulnificus. Using 28 Vibrio vulnificus strains and 53 other bacterial strains, we confirmed the high specificity of this method. Moreover, our LAMP method also showed high sensitivity, with a minimum detection level of one colony-forming unit per test. Furthermore, we developed simplified and conventional pretreatments for the method using experimental animal models. All of these attempts have lod to our non being able to detect Vibrio vulnificus within 1 hour.

Sensitive and rapid detection of Vero toxin-producing Escherichia coli using loop-mediated isothermal amplification.

A loop-mediated isothermal amplification (LAMP) assay was developed to detect Vero toxin (VT)-producing Escherichia coli rapidly (within 60 min). The 24 strains of VT-producing E. coli were successfully amplified, but 6 strains of non-VT-producing E. coli and 46 bacterial species other than E. coli were not. The sensitivity of the LAMP assay was found to be >0.7 c.f.u. per test using serogroups O157, O26 and O111 of VT-producing E. coli; this sensitivity is greater than that obtained by PCR assay. Furthermore, the LAMP assay was examined for its ability to detect VT-producing E. coli in food because of the difficulty of detection in food samples. The recovery of VT-producing E. coli by LAMP assay from beef and radish sprouts inoculated with the pathogen was high, similar to that obtained using culture methods with direct plating and/or plating after immunomagnetic separation. Although PCR assay was unable to recover VT-producing E. coli from half of the radish samples, LAMP assay was successful in most samples. In addition, VT-producing E. coli was successfully detected in cultures of the beef samples by LAMP assay, but not by the culture method. The LAMP products in naturally contaminated beef samples were analysed to confirm the specific amplification of the VT-encoding gene, and were found to show a specific ladder band pattern on agarose gel after electrophoresis. Additionally the sequences of the LAMP products coincided well with the expected sequences of the VT-encoding gene. These results indicate that the proposed LAMP assay is a rapid, specific and sensitive method of detecting the VT-producing E. coli.

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

Loop-mediated isothermal amplification (LAMP) assays targeting the rpoD and toxR genes were developed to detect Vibrio parahaemolyticus. All 78 tested V. parahaemolyticus strains yielded positive results within 40 min, while negative results were obtained for 69 strains of other organisms even at 60 min. For V. parahaemolyticus ATCC 17802 in pure culture, the detection limits of LAMP assays targeting rpoD and toxR were 3.7 and 450 CFU per test, respectively. Due to the higher sensitivity of rpoD-LAMP, it was further evaluated for the ability to detect V. parahaemolyticus in seafood samples. V. parahaemolyticus populations spiked in short-necked clams were enumerated by the most-probable-number (MPN) method combined with the rpoD-LAMP assay and the MPN method with a culture method using agar medium. The MPN-rpoD-LAMP method had better sensitivity and was more rapid than the conventional method. These results indicate that the MPN-LAMP assay targeting the rpoD gene is a specific, sensitive, and rapid method to enumerate V. parahaemolyticus organisms.