Rapid detection of Shigella and enteroinvasive Escherichia coli in produce enrichments by a conventional multiplex PCR assay

Abstract

a b s t r a c tFaster detection of contaminated foods can prevent adulterated foods from being consumed and minimize the risk of an outbreak of foodborne illness. A sensitive molecular detection method is especially important for Shigella because ingestion of as few as 10 of these bacterial pathogens can cause disease. The objectives of this study were to compare the ability of four DNA extraction methods to detect Shigella in six types of produce, post-enrichment, and to evaluate a new and rapid conventional multiplex assay that targets the Shigella ipaH, virB and mxiC virulence genes. This assay can detect less than two Shigella cells in pure culture, even when the pathogen is mixed with background microflora, and it can also differentiate natural Shigella strains from a control strain and eliminate false positive results due to accidental laboratory contamination. The four DNA extraction methods (boiling, PrepMan Ultra [Applied Biosystems], InstaGene Matrix [Bio-Rad], DNeasy Tissue kit [Qiagen]) detected 1.6 Â 10 3 Shigella CFU/ml post-enrichment, requiring w18 doublings to one cell in 25 g of produce pre-enrichment. Lower sensitivity was obtained, depending on produce type and extraction method. The InstaGene Matrix was the most consistent and sensitive and the multiplex assay accurately detected Shigella in less than 90 min, outperforming, to the best of our knowledge, molecular assays currently in place for this pathogen.Published by Elsevier Ltd.

Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols

“…A search of the literature revealed only one DNA extraction from microorganisms from cultured mediums using the Instagene matrix protocol 3 , 10 . This study noted the successful extraction of DNA from this source, but it remains difficult to compare this result with the present study where the extractions were from whole saliva.…”

Development of a lateral flow colloidal gold immunoassay strip for the simultaneous detection of Shigella boydii and Escherichia coli O157:H7 in bread, milk and jelly samples

“…So far, many methods have been developed to replace traditional techniques for food-borne pathogens' detection, for example, ELISA (Feng et al, 2013), chemiluminescence immunoassay (Y. Zhang et al, 2014), real-time PCR (Garrido-Maestu, Chapela, Vieites, & Cabado, 2014, multiplex PCR (Binet, Deer, & Uhlfelder, 2014), loop-mediated isothermal amplification (LAMP) (Kokkinos, Ziros, Bellou, & Vantarakis, 2014), evanescent wave fiber biosensor (Xiao, Rong, Long, & Liu, 2014), electrochemical immunosensors (Chan et al, 2013), surface plasmon resonance immunosensors (Mondani, Roupioz, Delannoy, Fach, & Livache, 2014;Wang, Ye, Si, & Ying, 2013), multiple cycle signal amplification strategy (H. Zhang et al, 2014) and PCR-strip joint detection technique (Chen et al, 2014). These methods have greatly improved sensitivity, specificity, and speed compared to the culture-based methods.…”

Molecular Detection of Shigella spp. Contamination in Ready-to-Eat Salad Samples in West of Tehran

“…Polymerase Chain Reaction PCR method was optimized for amplification of 613 bp segment of ipaH gene using the specific primer pair of RB87: 5´CGGTCAGCCACCCTCTGAG-3′ and RB88: 5′-CTTGACCGCCTTTCCGATACC-3 ′ for detection of Shigella spp., 5 and performed on extracted DNA of all samples (before and after enrichment in Shigella broth).…”