Evaluation of different DNA extraction procedures for the detection of Salmonella from chicken products by polymerase chain reaction

Abstract

Polymerase chain reaction (PCR) was used after a short pre-enrichment culture to detect Salmonella subspecies in chicken fillets. A direct PCR assay performed with chicken meat inoculated with Salmonella Typhimurium produced no PCR products. Six different DNA extraction protocols were tested to recover efficiently Salmonella DNA after a short incubation period. Three of them gave results that were reliable, rapid and sensitive. Successful protocols used Proteinase K and/or a centrifugation step to concentrate the samples. For reliable detection of Salmonella subspecies, a few thousand bacterial cells per ml must be present. To obtain this number of bacterial cells with an inoculation of about one cell in 25 g of ionized food products, it was necessary to incubate samples for at least 10 h before PCR. A larger inoculum of approximately 10 cells in 25 g of ionized food products, required 8 h in culture broth to give positive results by PCR-based assay.

Comparison of DNA-extraction methods and Selective Enrichment broths on the detection of Salmonella Typhimurium in swine feces by polymerase chain reaction (PCR)

“…Müller-Kauffmann tetrathionate (MK) was highly inhibitory to PCR when it was performed on pure cultures, which corroborates previous findings (17). However, MK was not inhibitory when PCR was performed using artificially inoculated feces samples (Table 3), probably due to the higher number of salmonella organisms recovered by MK when compared to SC and RV ( Table 2).…”

“…Soumet et al (17) observed that SC was less inhibitory to PCR than MK and RV. However, the results this study indicate that either of three broths wasn't inhibitory to the PCR when the DNA-extraction method used was boiling-centrifugation or salting-out.…”

“…Boiling-centrifugation (M1) as described by Soumet et al (17). One mL aliquots of enrichment broths were centrifuged 13,000 x g, 3 minutes.…”

Prevalence, Detection and Antimicrobial Resistance Pattern of Salmonella in Sudan

“…DNA from each Salmonella serovar and non-Salmonella strain was extracted according to the boiling -centrifugation method (Soumet et al, 1994). A single colony of a pure nutrient agar culture was grown overnight at 37ºC in 1ml Luria -Bertani broth.…”

On-line cell lysis of bacteria and its spores using a microfluidic biochip

“…Standard methods for isolating bacterial DNA rely on cell lysis using combinations of heat, enzymes, chemical lytical agents (detergents) or mechanical forces (sonication, bead milling) [9,10]. However, many such lysis techniques are not amenable for implementation in a microfluidic platform.…”