Combination of reverse transcription and multienzyme restriction fragment length polymorphism analysis for rapid detection of Escherichia coli

Abstract

Cultivation methods are used to monitor pathogenic microorganisms in foods. However, the current methods require a few days to produce results, and products are often released for sale before the results of microbiological analysis become available. We developed an RNA extraction and microorganism detection system using model food samples inoculated with Escherichia coli K-12 and O157:H7 (GTC 14536) (0 CFU/g and 1×101-104 CFU/g). Before RNA extraction, live or dead cells were inoculated into the food samples, the samples were homogenized, and the extracted RNAs were used to synthesize cDNAs using random 6-mer. PCR was used to analyze the target genes, and the PCR products were digested with two restriction enzymes (HhaI and HaeIII) to analyze restriction fragment length polymorphism (RFLP). PCR confirmed the RNA extraction and cDNA synthesis of up to 1×101 CFU/g samples of live cells. Multienzyme RFLP (MeRFLP) showed that the sizes of the DNA fragments obtained were consistent with the theoretical fragment sizes, suggesting that reverse transcription-MeRFLP (RT-MeRFLP) could identify the target bacteria. These results suggest that RT-MeRFLP, which does not require culture and can be completed within 6.5 h, is a promising approach for a low-cost, rapid, and reliable system for identifying bacteria in food. © 2013 Hosoda A, et al.