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Abstract
Pathogenic Vibrio spp., Aeromonas spp. and Plesiomonas shigelloides are associated with human gastroenteritis and wound infections, as well as fish diseases. The comprehensive and accurate identification of these pathogens is crucial for the current public health. The present study describes the development of a multiplex assay for the simultaneous identification of ten bacterial pathogens in a single reaction by using a multiplex ligation reaction based on probe melting curve analysis (MLMA). The specificity for target genes was 100%, as assessed with a panel of 67 bacterial pathogens, which indicated no cross-reactions. The detection limit of this assay ranged from 0.8 × 107 CFU/mL to 1.5 × 108 CFU/mL at the pure bacterial culture level and from 0.1 ng to 1.0 ng at the DNA level. The MLMA assay was used to detect ten species of pathogens in 269 clinical and seafood samples, and for further validation, the results were compared with the conventional culture method. The results indicated greater than 90% sensitivity and 100% specificity for each bacterial pathogen tested, and the kappa correlation for all the pathogens ranged from 0.95 to 1.00. Overall, this assay is well suited for public health laboratories for its high throughput, accuracy, and low cost.
Highlights
To diagnose the above pathogens, the conventional method requires bacterial culture in alkaline peptone water, biochemical tests, and slide agglutination assays with specific antisera
To increase the number of target genes and to improve the accuracy of detecting co-infection with two or more bacterial pathogens, we developed a multiplex ligation reaction based on a probe melting curve analysis (MLMA) assay for simultaneous identification of ten species of bacteria
The MLMA assay simultaneously distinguished the O1 and O139 strains from non-O1 and non-O139 strains and was capable of determining whether the detected bacteria had the toxin gene
Summary
To diagnose the above pathogens, the conventional method requires bacterial culture in alkaline peptone water, biochemical tests, and slide agglutination assays with specific antisera. These methods are often time consuming (2 to 7 days) and laborious. To date, no assay system is able to identify all the bacterial pathogens in one reaction In this regard, the development of an efficient, rapid, and accurate molecular method that can identify bacterial pathogens in one assay would be a useful tool for clinical laboratories and for the surveillance and diagnosis of human infections. To increase the number of target genes and to improve the accuracy of detecting co-infection with two or more bacterial pathogens, we developed a multiplex ligation reaction based on a probe melting curve analysis (MLMA) assay for simultaneous identification of ten species of bacteria. Detection can help physicians correctly identify and treat patients affected by these bacterial pathogens and aid epidemiologists in tracking the source of the pathogens as quickly as possible, thereby limiting the economic loss associated with infected seafood
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