Multiplex PCR for detection of Escherichia coli O157:H7 in foods

Abstract

Escherichia coli O157:H7 is well known enterohemorrhagic pathogen responsible for infections among animals including a man. The main source of this bacterium is cattle, that is mostly asymptomatic and through that E. coli O157:H7 can simple transfer to food products. Therefore, there is a need for rapid, sensitive and specific detection method. The present work is focused on its detection by a heptaplex polymerase chain reaction, which targets genes from known virulent regions of E. coli O157:H7. According to obtained results this approach is able to reach the detection sensitivity of 4 colony-forming units (CFU) from a culture and 6 and 8 CFU from milk and meat samples, respectively, independently of tested sample volume.

Development of a Multiplex PCR Assay for Detection of Shiga Toxin-Producing Escherichia coli, Enterohemorrhagic E. coli, and Enteropathogenic E. coli Strains

“…Evaluation of the specificity revealed no significant cross-reactivity of the primers with other E. coli pathotypes. The detection threshold of the assay was determined to be comparable to other PCR-based methods for detection of E. coli isolates (Aranda et al, 2007 ; Antikainen et al, 2009 ; Vidová et al, 2011 ). Of particular significance is the observation that defined combinations of lpfA subtypes permitted differentiation of EHEC and EPEC clonal groups.…”

Improved Multiplex Polymerase Chain Reaction for Rapid Staphylococcus Aureus Detection in Meat and Milk Matrices

“…The supernatant (5 µl) was added directly to the PCR mixture. In the case of NaClO samples, prepared according to our previous study (VIDOVÁ et al, 2011), the 1 ml of the S. aureus overnight culture was treated with NaClO solution in resulting concentration of 5 × 10 −4 % for 10 min. In both cases, the viability of the cells was verified by counting the number of CFU on LB-agar plate after overnight incubation at 37°C.…”

“…PCR assay was carried out directly from S. aureus cells prepared according to procedure described previously by VIDOVÁ et al (2011). The amplification programme consisted of denaturation at 95°C for 5 min, 30 cycles of denaturation at 95°C for 60s, annealing at 55°C for 90s and extension at 72°C for 2 min, followed by a final extension at 72°C for 8 min.…”

“…Therefore, in this work was DNA extraction step excluded and replaced by direct use of washed bacterial cells into polymerase chain reactions. According to our previous experience (CHOTÁR et al, 2006;VIDOVÁ et al, 2011), bacterial cells prepared by washing method with NaClO pretreatment were used. Briefly, two ways to prepare cell suspensions from overnight culture before washing steps were used; and samples (5 µL) after washing with sterile water were used directly to PCR.…”

Establishment and Application of a Visual DNA Microarray for the Detection of Food-borne Pathogens

“…The former has been widely used to simultaneously detect multiple pathogens. [3][4][5][6][7][8] For the mPCR assay, the capacity of primers for specific amplification and amplicons with different size length fragments must be taken into consideration for subsequent detection by gel electrophoresis. However, gel electrophoresis is not accurate for pathogens identification, other techniques have to be used, such as sequencing, microarray hybridization, 2 capillary electrophoresis, 9 etc.…”