A microsystem compatible strategy for viable Escherichia coli detection.
This study delineates a microsystem compatible strategy that enables the rapid determination of Escherichia coli viability for the application in food and water monitoring. This approach differentiates the living cells from the dead ones by detecting the presence of a "viability indicator", i.e. mRNAs of a common E. coli GroEL heat shock protein (hsp). Our method starts with a stimulated and controlled transcription of hsp mRNA under an elevated temperature (47 degrees C) for 20min. Following that, the short-life mRNA is rapidly extracted using streptavidin-modified magnetic particles containing biotin-labeled DNA probes complementary to a specific region of the mRNA. The quantification of mRNA by gel electrophoresis and Ag/Au-based electrochemical detection is done after the amplification of mRNAs by reverse transcription-polymerase chain reaction (RT-PCR). Heat shock temperatures and durations that have profound effect to the mRNA transcription were studied and it was found that the mRNA undergoes a rapid minute-by-minute self-degradation after the environment resumes room temperature. Issues such as the DNA contamination that interfere the magnetic particle-based mRNA extraction technique were tackled. A sensitive Ag/Au-based electrochemical analysis method was used to detect the RT-PCR products and a cell concentration as low as 10(2)cfu/ml can be achieved by the electrochemical method, but not by the conventional gel electrophoresis. The strategy demonstrated in this study can be readily implemented in a microsystem and is a step forward for the realization of an integrated bioanalytical microsystem (lab on a chip) for the viable cell detection.
Zhao
Wenting
W
Department of Chemical and Biochemical Engineering, Zhejiang University, 38 Zheda Road, Hangzhou 310027, PR China.
Yao
Shanjing
S
Hsing
I-Ming
IM
D015374:Biosensing Techniques / Q000295:instrumentation* / Q000379:methods; D002470:Cell Survival; D018834:Chaperonin 60 / Q000235:genetics*; D015169:Colony Count, Microbial / Q000295:instrumentation* / Q000379:methods; D004563:Electrochemistry / Q000295:instrumentation / Q000379:methods; D004867:Equipment Design; D019544:Equipment Failure Analysis; D004926:Escherichia coli / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology; D006358:Hot Temperature; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D008904:Miniaturization; D016133:Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D012329:RNA, Bacterial / Q000032:analysis*; D018511:Systems Integration
D018834:Chaperonin 60; D012329:RNA, Bacterial