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ABI Prism 7000 SDS platform에서 개발된 Cronobacter sakazakii 검출법을 Cepheid SmartCycler® Ⅱ으로 변환 적용 KCI 등재
Conversion of real-time PCR assays for the detection of Cronobacter sakazakii from the ABI Prism 7700 to the Cepheid SmartCyclerⅡ platform
pp.155-160
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.
Introduction
Materials and Methods
1. Real-time PCR platform
2. Master mix components for ABI Prism 7000 SDS andSmartCycler Ⅱ
3. Optimization cycling temperature and time and MgCl2concentrations for new real-time PCR platform
4. Extraction and preparation of DNA templates for PCR
Results & Disscussion
References
