Rapid detection of Salmonella in milk by combined immunomagnetic separation-polymerase chain reaction assay

Abstract

During the past few years, milk has presented a risk of Salmonella contamination; it has been implicated as the cause in several outbreaks of salmonellosis. Because conventional detection methods require 5 to 7 d for completion and involve several subcultivation stages followed by biochemical and serological tests, rapid and sensitive methods have been sought, mainly at the DNA level. Therefore, a study including milk samples was conducted to evaluate the performance of a combination of 2 techniques--immunomagnetic separation and polymerase chain reaction (PCR)--for the detection of Salmonella. The 16-, 14-, 12-, 10-, and 8-h nonselective pre-enrichment steps before immunomagnetic separation and the high-pure DNA preparation method before PCR were used in a combined assay. Milk samples, which were found to be Salmonella-negative by a reference method, were first inoculated with Salmonella Enteritidis. Next, the shortest pre-enrichment time that is required for detection of 1 or 10 cfu of Salmonella/mL by combined immunomagnetic separation-PCR assay was found by using 16-, 14-, 12-, 10-, and 8-h incubation periods. The detection limit using a 16-, 14-, or 12-h nonselective pre-enrichment was 1 to 10 cfu/mL. However, the sensitivity decreased to 10(1) and 10(2) cfu/mL, respectively, when 10- and 8-h pre-enrichments were used. This assay, in conjunction with a 12-h pre-enrichment, proved to be rapid (overall 16 h) and sensitive (1-10 cfu/mL) for the detection of Salmonella in milk samples and promising for routine use in the detection of Salmonella in milk.

Evaluation of microplate immunocapture method for detection of Vibrio cholerae, Salmonella Typhi and Shigella flexneri from food

“…From there, the pathogen, if present, is isolated on selective agar, and purification and confirmation occur using morphological, biochemical, or physiological tests [3]. Conventional culture methods, however, are often problematic, in that many are time-consuming and require several days to complete, appropriate selective media are not currently available for all bacterial foodborne pathogens, some bacterial pathogens require specific atmospheric or other growth conditions which may be difficult to simulate in the laboratory and some bacterial pathogens may not be culturable by currently available methods [4]. The presence of high background indigenous microflora and complex matrix in food also limits detection of pathogen from food samples.…”

Diagnosis of canine leptospirosis using an immunomagnetic separation-PCR method

“…Recent studies report the immunomagnetic separation (IMS) technique prior to PCR assay as an approach to reduce the effect of inhibitory substances present in biological fluids and food samples (2, 3, 4, 6, 7, 9, 14, 15). For diagnosis of leptospirosis, the IMS-PCR approach was reported to detect Leptospira spp.…”

“…For those reasons, antigen detection tests offer potential advantage over tests based on antibody detection both for early diagnosis and identification of renal carrier status (5). Therefore, research focused on highly sensitive and specific routine tests for leptospira detection in blood and urine samples led to development of several molecular methods for diagnosis of leptospirosis (4, 6, 9, 14). However, the sensitivity and kinetics of PCR diagnostic tests may be dramatically reduced when applied directly to biological samples, such as urine and serum samples (11).…”

Novel Genomic Tools for Specific and Real-Time Detection of Biothreat and Frequently Encountered Foodborne Pathogens

“…immunomagnetic separation and polymerase chain reaction, provided a detection of 1–10 CFU of salmonellae/ml, after a selective pre-enrichment incubation of 12–16 hrs. However, a decreased sensitivity of 10–100 CFU/ml was observed after 8–10 h of pre-enrichment period (Mercanoglu Taban et al, 2009). In this study we have performed a preliminary test to evaluate the use of real-time PCR for detection of S. dysentriae spiked in milk.…”