Conversion of real-time PCR assays for the detection of Cronobacter sakazakii from the ABI Prism 7700 to the Cepheid Smart Cycler Ⅱ platform
2011
Chon, J.W., Konkuk University, Seoul, Republic of Korea | Song, K.Y., Konkuk University, Seoul, Republic of Korea | Kim, D.H., Konkuk University, Seoul, Republic of Korea | Park, J.H., Konkuk University, Seoul, Republic of Korea | Hyeon, J.Y., Konkuk University, Seoul, Republic of Korea | Lee, J.I., Konkuk University, Seoul, Republic of Korea | Seo, K.H., Konkuk University, Seoul, Republic of Korea
Real-time PCR could help to provide answers to urgent questions about the incidence, prevalence, and epidemiology of currently emerging food-borne bacteria and diseases as identification and detection tools. The objective of this study was carried out to examine several critical parameters that must be optimized when converting from the ABI Prism 7000 SDS platform to the Cepheid SmartCycler Ⅱ so as to directly use the same primer and probe sequences. A lyophilized master mix-OmniMix HS bead, MgCl₂ concentration, and PCR cycling conditions were evaluated so as to convert to a new platform, Smartcycler Ⅱ. The best optimal cycling conditions to detect Cronobacter sakazakii on SmartCycler Ⅱ were as follow: initial denaturation at 95℃ for 2 min followed by 45 cycles of 95℃ for 15 s, and 60℃ for 60 s using OmniMix HS bead contained 6 mM MgCl₂ concentration. And the Ct value was 16.97 compared to 23.84 of Ct value in ABI Prism 7000 SDS. This result showed that when the several analytical parameters were taken the consideration for optimization, it could be performed assays between real-time PCR platforms. Also it is need of further study to develop the new single multiplex real-time PCR method for determining various Cronobacter spp. including three subspecies, too.
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