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

Abstract

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...

A cross-priming amplification assay coupled with vertical flow visualization for detection of Vibrio parahaemolyticus

“…The other bacterial strains were obtained from the culture collection of the Huzhou Center for Disease Control and Prevention. DNA was extracted by boiling samples, as previously described [12].…”

“…The sensitivity of the assay for pure cultures and spiked oysters was determined by bacterial colony counts as previously described [7,12]. Oysters were obtained from a local retail market and confirmed to be V. parahaemolyticus-negative following previously described methods Table 1 Specificity of the CPA-VF and real-time PCR assays for V. parahaemolyticus.…”

A highly sensitive and flexible magnetic nanoprobe labeled immunochromatographic assay platform for pathogen Vibrio parahaemolyticus

“…For example, a toxR-based LAMP assay was able to detect 1.1 × 10 5 V. parahaemolyticus cells per gram in spiked oyster without enrichment (Chen and Ge, 2010). In contrast, LAMP based on tlh/tdh gene was capable to detect 2 CFU/g V. parahaemolyticus in seafood within 5 h (Di et al, 2015). However, LAMP assay is more timeconsuming than MNP/ICTS.…”

Virulence gene expression of Vibrio parahaemolyticus in the viable but nonculturable state

“…These methods were used to determine the total number of V. parahaemolyticus, which were then used to estimate the numbers of the pathogenic subtype (Di et al, 2015;Malcolm et al, 2015). Molecular techniques such as gene-specific probe and PCR are increasingly used to detect pathogenic V. parahaemolyticus defined by the presence of certain virulence markers (Malcolm et al, 2015).…”

“…Molecular techniques such as gene-specific probe and PCR are increasingly used to detect pathogenic V. parahaemolyticus defined by the presence of certain virulence markers (Malcolm et al, 2015). A loop-mediated isothermal amplification method has been recently developed and has since been coupled with other molecular techniques (Di et al, 2015;Malcolm et al, 2015;Notomi et al, 2015). These newer DNA-based methods, in conjunction with the conventional biochemical tests often require the bacterial cells to be cultured on typical growth media first.…”