The Use of a DNA-Intercalating Dye for Quantitative Detection of Viable Arcobacter spp. Cells (v-qPCR) in Shellfish
Salas Massó, Nuria
Linh, Quyen Than
Chin, Wai Hoe
Andree, Karl B.
Furones, M. Dolors
Figueras, María José
Bang, Dang Duong
The genus Arcobacter (Vandamme et al., 1991), comprised of Campylobacter-related species, are considered zoonotic emergent pathogens. The presence of Arcobacter in food products like shellfish, has an elevated incidence worldwide. In this study, we developed a specific viable quantitative PCR (v-qPCR), using the dye propidium monoazide (PMA), for quantification of the viable Arcobacter spp. cells in raw oysters and mussels. The high selectivity of primers was demonstrated by using purified DNA from 38 different species, 20 of them from the genus Arcobacter. The optimization of PMA concentration showed that 20 μM was considered as an optimal concentration that inhibits the signal from dead cells at different concentrations (OD550 from 0.2 to 0.8) and at different ratios of live: dead cells (50:50 and 90:10). The v-qPCR results from shellfish samples were compared with those obtained in parallel using several culture isolation approaches (i.e., direct plating on marine and blood agar and by post-enrichment culturing in both media). The enrichment was performed in parallel in Arcobacter-CAT broth with and without adding NaCl. Additionally, the v-qPCR results were compared to those obtained with traditional quantitative (qPCR). The v-qPCR and the qPCR resulted in c.a. 94% of positive detection of Arcobacter vs. 41% obtained by culture approaches. When examining the reduction effect resulting from the use of v-qPCR, samples pre-enriched in Arcobacter-CAT broth supplemented with 2.5% NaCl showed a higher reduction (3.27 log copies) than that of samples obtained directly and those pre-enriched in Arcobacter-CAT broth isolation (1.05 and 1.04). When the v-qPCR was applied to detect arcobacter from real shellfish samples, 15/17 samples tested positive for viable Arcobacter with 3.41 to 8.70 log copies 1g-1. This study offers a new tool for Arcobacter surveillance in seafood.
63 - Agricultura. Silvicultura. Zootècnia. Caça. Pesca
Is part of
Frontiers in Microbiology
Salas-Massó, Nuria, Quyen Than Linh, Wai Hoe Chin, Anders Wolff, Karl B. Andree, M. Dolors Furones, María José Figueras, and Dang Duong Bang. 2019. "The Use Of A DNA-Intercalating Dye For Quantitative Detection Of Viable Arcobacter Spp. Cells (V-Qpcr) In Shellfish". Frontiers In Microbiology 10. Frontiers Media SA. doi:10.3389/fmicb.2019.00368.
Grant agreement number
MICINN/Programa Nacional de Proyectos de Investigación Fundamental/AGL2011-30461-C02-02/ES/Evaluación del impacto de la presencia de las bacterias potencialmente patogenas aeromonas y arcobacter en aguas regeneradas y en la seguridad alimentaria/BACTRERISK
EU/FP7/311846/EU/Protecting the health of Europeans by improving methods for the detection of pathogens in drinking water and water used in food preparation/AQUAVALENS
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