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. 2013 Aug 26;8(8):e72342.
doi: 10.1371/journal.pone.0072342. eCollection 2013.

Development of a real-time resistance measurement for Vibrio parahaemolyticus detection by the lecithin-dependent hemolysin gene

Guiming Xiang  1 , Xiaoyun Pu, Dongneng Jiang, Linlin Liu, Chang Liu, Xiaobo Liu
Affiliations

Development of a real-time resistance measurement for Vibrio parahaemolyticus detection by the lecithin-dependent hemolysin gene

Guiming Xiang et al. PLoS One. .

Abstract

The marine bacterium Vibrio parahaemolyticus (V. parahaemolyticus) causes gastroenteritis in humans via the ingestion of raw or undercooked contaminated seafood, and early diagnosis and prompt treatment are important for the prevention of V. parahaemolyticus-related diseases. In this study, a real-time resistance measurement based on loop-mediated isothermal amplification (LAMP), electrochemical ion bonding (Crystal violet and Mg(2+)), real-time monitoring, and derivative analysis was developed. V. parahaemolyticus DNA was first amplified by LAMP, and the products (DNA and pyrophosphate) represented two types of negative ions that could combine with a positive dye (Crystal violet) and positive ions (Mg(2+)) to increase the resistance of the reaction liquid. This resistance was measured in real-time using a specially designed resistance electrode, thus permitting the quantitative detection of V. parahaemolyticus. The results were obtained in 1-2 hours, with a minimum bacterial density of 10 CFU.mL(-1) and high levels of accuracy (97%), sensitivity (96.08%), and specificity (97.96%) when compared to cultivation methods. Therefore, this simple and rapid method has a potential application in the detection of V. parahaemolyticus on a gene chip or in point-of-care testing.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Scheme of the real-time resistance measurement for V. parahaemolyticus.
Briefly, the lecithin-dependent hemolysin (LDH) gene of V. parahaemolyticus was amplified by LAMP at first. The subsequent two products, DNA and pyrophosphate, both negative ions, were combined with a positive dye (Crystal violet) and positive ions (Mg2+), leading to an increase in the reaction liquid resistance. This resistance was measured in real-time using a electrode, and V. parahaemolyticus concentration was quantitatively detected through a derivative analysis.
Figure 2
Figure 2. Specificity analysis of the real-time resistance measurement.
A. The real-time resistance curve of V. parahaemolyticus and other interfering bacteria. B. End-point resistance of V. parahaemolyticus and other interfering bacteria after a 60-min LAMP assay. C. Derivative analysis of the real-time resistance measurement.
Figure 3
Figure 3. Sensitivity and regression analyses of the real-time resistance measurement.
A. The real-time resistance curve of V. parahaemolyticus in a concentration gradient. B. The derivative analysis of the real-time resistance measurement of V. parahaemolyticus. C. The regression analysis of the real-time resistance measurement of V. parahaemolyticus. Three samples of the same bacterial concentration were measured twice and all values were recorded as mean(n = 6).
See this image and copyright information in PMC

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