Detection of Escherichia coli in Meat with an Electrochemical Biochip

Abstract

Detection of foodborne pathogenic and spoilage bacteria by RNA-DNA hybridization is an alternative to traditional microbiological procedures. To achieve high sensitivity with RNA-DNA-based methods, efficient bacterial lysis and release of nucleic acids from bacteria are needed. Here we report the specific detection of the hygiene indicator microorganism Escherichia coli in meat by use of electrochemical biochips. We improved RNA isolation from bacteria in meat juice from pork and beef. Samples, either naturally or artificially contaminated by E. coli, were enriched by incubation in full or minimal medium. A combined treatment of the samples with lysozyme, proteinase K, and sonication resulted in efficient cell disruption and high total RNA yields. Together with optimization of enrichment time, this ensures high sensitivity of electrochemical measurements on biochips. A short enrichment period and the triple-lysis regimen in combination with electrochemical biochip measurement were tested with 25 meat samples. The lower limit of detection of the biochip was approximately 2,000 CFU of E. coli per ml. The entire analysis procedure (5 h of enrichment, triple lysis, and biochip detection) has a lower limit of detection of 1 CFU of E. coli per ml within a total time needed for analysis of 7 h.

Biosensors for the analysis of microbiological and chemical contaminants in food

“…The widely accepted gold standards for monitoring pathogens in food are microbiological methods [51][52][53][54]. Microbiological methods generally consist of an enrichment stage (preenrichment and/or selective enrichment), culturing in selective or differential agar plates to isolate cultures, followed by phenotype analysis or metabolic fingerprint analysis to confirm the result [49,53].…”

Visual detection of microRNA with lateral flow nucleic acid biosensor

“…MicroRNAs (miRNAs) are a class of small (∼18-25 nucleotides) noncoding RNAs that serve as post-transcriptional regulators of gene expression in a broad range of plants, viruses and mammals (Heidenreich et al 2010; Wightman et al 1993). Mature miRNAs are considered to regulate gene expression by catalyzing the cleavage of messenger RNA and incorporation into RNA-induced silencing complex (RISC) where they interact with complementary sites on messenger RNA as well as downstream regulation of the expression of target messenger RNA molecules (Engels and Hutvagner 2006; Meister and Tuschl 2004).…”

Electrochemical Detection of RNA

“…The method was validated using contaminated meat juice samples. The detection limit of the biochip applying this complex sample matrix was approximately 2,000 cfu mL À1 E. coli (Heidenreich et al 2010). Walter et al (2011) described also an electrochemical biosensor for detection of E. coli 16S rRNA using pAP redox cycling by nicotinamide adenine dinucleotide (NAD).…”