In this study, we chose SYTO-9 as the intercalating dye for the r

In this study, we chose SYTO-9 as the intercalating dye for the real-time PCR platform instead of the commonly used real-time PCR dye SYBR Green I. Based on a previous study [37] comparing the use of these two dyes in real-time PCR, SYTO-9 was found to generate highly reproducible DNA melting curves over a broader range of dye concentrations than SYBR Green I and was far less inhibitory. We also evaluated the use of EvaGreen (Biotium, Hayward, CA) as the intercalating dye on the real-time PCR platform for LAMP, but found it to be inhibitory for LAMP amplifications (data not shown).

The strong linear correlation (r 2 = 0.94-0.99) between the number of V. parahaemolyticus cells in the LAMP reaction and the associated Ct or Tt values over a dynamic range of template concentrations (101 to 106 cells) illustrates the quantitative capability of the toxR-based real-time IWR-1 manufacturer LAMP assays when detecting this organism in both pure culture and spiked oysters. Very few reports have examined the quantitative ability of LAMP. One study monitoring

ammonia-oxidizing bacteria using LAMP also Pifithrin-�� in vivo reported it to possess good quantitative capability between 1 × 104 and 1 × 1010 DNA copies [36]. In spiked oyster samples, we found the detection limit of the toxR-based LAMP assay to be 200 V. parahaemolyticus cells per reaction, which translates to 1.1 × 105 cells per gram of oyster sample. In contrast, the detection limit of the tlh-based LAMP in spike shrimp samples was reported to be 5.3 × 102 CFU/g (2 CFU/reaction) [11]. The U.S. Food and Drug Administration requires that all postharvest-processed oysters have lower than 30 MPN/g of either V. vulnificus or V. parahaemolyticus [38]. This indicates that without enrichment, DNA amplification assays such as LAMP, although potentially

quantitative, lack the needed sensitivity when applied to food samples [23]. Therefore, combining MPN overnight enrichment [19] or pre-enrichment for 6 h [33] with LAMP or other DNA amplification assays is a desirable approach to achieve the needed sensitivity. When testing spiked oyster samples, we observed the time to positive samples (Ct for the real-time PCR platform and Tt for the real-time turbidimeter) was delayed several minutes compared Dapagliflozin to pure culture samples and the detection limit was higher (200 V. parahaemolyticus cells in oyster samples vs. 47 cells in pure culture). Nonetheless, no extensive sample preparation other than homogenization and two simple centrifugation steps was required. This significantly reduced the total assay time. Combined with less than 1 h for the real-time LAMP assay, the complete LAMP detection system was markedly faster than either PCR or conventional methods. Conclusions The toxR-based real-time LAMP assay developed in this study was a highly specific, sensitive, and rapid method for the detection of V. parahaemolyticus in oysters.

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