A simple, rapid, cost-effective and sensitive method for detection of Salmonella in environmental and pecan samples
| Autor: | Shefali Dobhal, M.W. Smith, G. Zhang, Li Ma, C. Rohla |
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| Rok vydání: | 2014 |
| Předmět: |
DNA
Bacterial Salmonella typhimurium Dna template Salmonella medicine.disease_cause Applied Microbiology and Biotechnology Polymerase Chain Reaction Microbiology Preparation method Feces RNA Ribosomal 16S medicine Animals Humans Nuts Bovine serum albumin Soil Microbiology Carya Chromatography biology General Medicine 16S ribosomal RNA DNA extraction RNA Bacterial Specific primers biology.protein Cattle Biotechnology |
| Zdroj: | Journal of applied microbiology. 117(4) |
| ISSN: | 1365-2672 |
| Popis: | Aims PCR is widely used in the routine detection of foodborne human pathogens; however, challenges remain in overcoming PCR inhibitors present in some sample matrices. The objective of this study was to develop a simple, sensitive, cost-effective and rapid method for processing large numbers of environmental and pecan samples for Salmonella detection. This study was also aimed at validation of a new protocol for the detection of Salmonella from in-shell pecans. Methods and Results Different DNA template preparation methods, including direct boiling, prespin, multiple washing and commercial DNA extraction kits, were evaluated with pure cultures of Salmonella Typhimurium and with enriched soil, cattle feces and in-shell pecan each spiked individually with Salmonella Typhimurium. PCR detection of Salmonella was conducted using invA and 16S rRNA gene (internal amplification control) specific primers. The effect of amplification facilitators, including bovine serum albumin (BSA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG) and gelatin on PCR sensitivity, was also evaluated. Conducting a prespin of sample matrices in combination with the addition of 0·4% (w/v) BSA and 1% (w/v) PVP in PCR mix was the simplest, most rapid, cost-effective and sensitive method for PCR detection of Salmonella, with up to 40 CFU Salmonella per reaction detectable in the presence of over 109 CFU ml−1 of background micro-organisms from enriched feces soil or pecan samples. Conclusions The developed method is rapid, cost-effective and sensitive for detection of Salmonella from different matrices. Significance and Impact of the study This study provides a method with broad applicability for PCR detection of Salmonella in complex sample matrices. This method has a potential for its application in different research arenas and diagnostic laboratories. |
| Databáze: | OpenAIRE |
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