Journal
FOOD CONTROL
Volume 23, Issue 2, Pages 491-498Publisher
ELSEVIER SCI LTD
DOI: 10.1016/j.foodcont.2011.08.019
Keywords
Vibrio; Multiplex PCR; Real-time PCR; Seafood
Categories
Funding
- HUFS-GRRC [2010-B2]
- Seoul RD program
- National Research Foundation of Korea [2009-0092822, 과06A1203] Funding Source: Korea Institute of Science & Technology Information (KISTI), National Science & Technology Information Service (NTIS)
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We developed a multiplex real-time (RTi) PCR method for the simultaneous detection of Vibrio cholerae, V parahaemolyticus, and V. vulnificus using zot, vmrA, and vuuA as the respective target genes. A set of primer pairs specific for those target genes was designed and employed in the SYBR Green-based multiplex RTi-PCR assay. Quantitative analyses with ten-fold serially diluted genomic DNA of each target organism resulted in a linear correlation between C-T values and the amount of each target genome per reaction, with a lower detection level of less than ten genome copies per reaction. Similar sensitivities were observed for Vibrio-spiked seafood samples (oyster, crab meat, and raw fish). After 8 h of enrichment culture of the seafood homogenate in alkaline peptone water, our optimized multiplex RTi-PCR was shown to achieve theoretical maximum sensitivity (ca. 10(0) CFU/gram food homogenate). Our proposed method is simple, robust and readily adaptable in routine laboratories, allowing for high-throughput surveillance of pathogenic Vibrio species in seafood. (C) 2011 Elsevier Ltd. All rights reserved.
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