Improved Template Preparation for PCR-Based Assays for Detection of Food-Borne Bacterial Pathogens

Abstract

Shigella flexneri, Salmonella enterica serotype Typhimurium, and Listeria monocytogenes were applied to FTA filters, and the filters were used directly as templates to demonstrate their sensitivity and applicability in PCR-based detection assays. With pure cultures, the sensitivities of detection by FTA filter-based PCR were 30 to 50 and 200 CFU for the gram-negative enterics and Listeria, respectively. Different numbers of S. flexneri cells were used in controlled contamination experiments with several different foods (produce, beef, and apple cider). Aliquots from concentrated food washes subsequently spotted onto FTA filters and assayed by PCR gave consistently positive results and detection limits similar to those observed with pure-culture dilutions. This universal method for PCR template preparation from bacterial cells is rapid and highly sensitive and reduces interference from food-associated inhibitors of PCR. In addition, its broad applicability eliminates the need for multiple methods for analysis of food matrices.

Targeting Single-Nucleotide Polymorphisms in the 18S rRNA Gene To Differentiate Cyclospora Species from Eimeria Species by Multiplex PCR

“…A direct comparison of the utility of these methods and that presented in the present study has not been done; however, the present protocol provides several distinct advan- tages for the examination of such diverse and complex matrices as foods, water sources, environmental samples, and clinical 5qolates. When coupled with the advances reported on the utility of FTA filters for the detection of human pathogens by PCR (14,21), the use of SNP primers in a multiplex PCR protocol provides timeliness, sensitivity, and selectivity, all of which are paramount considerations in the detection of human pathogens. In a single assay, this approach also provides a means to detect and differentiate among mixed populations of similar but distinct species of microorganisms such as the closely related protozoan parasites presented here.…”

“…DNA templates from parasite isolates (C. cayetanensis and Eimeria spp.) were prepared by applying 100 to 1,000 oocysts onto FTA filters (Fitzco, Inc., Maple Plain, Minn.) in volumes ranging from 2 to 10 l. FTA filters were then processed as previously described and used directly as the source of DNA template for PCR (14,21). DNA templates for C. cercopitheci, C. colobi, and C. papionis were prepared according to the protocol of Da Silva et al (4).…”

Comparative Analysis of the Schleicher and Schuell IsoCode Stix DNA Isolation Device and the Qiagen QIAamp DNA Mini Kit

“…In addition to the reduced time for purifying DNA, the IsoCode Stix procedure included only one reagent (MBG water), reducing the number of manipulations needed to obtain pure nucleic acid, improving the ease of sample handling, and minimizing the risk of cross-contamination. A unique feature of the IsoCode Stix is their impregnation with chelators and denaturants reported to retard bacterial and viral growth; inhibit nuclease activity, thus minimizing nucleic acid degradation; and release template DNA from organisms during processing (7,8,9,13,18,21). Impregnated membrane-based technology has provided enhanced detection sensitivities compared to those with conventional preparations (18).…”

“…The low concentration of DNA from pathogenic agents present in typical samples makes such applications necessary (3,18,22). In addition, a method with a flexible protocol applicable to numerous matrix types that is efficient at removing inhibitory substances found in clinical material that interfere with PCR amplification of the intended target is imperative (3,12,13,15,18,20,25,26). Further, the proposed sample processing method should facilitate reproducibility, production of DNA for long-term storage, and minimal cross-contamination (10,18,19).…”

Detection of Salmonella in Food Samples by Culture and Polymerase Chain Reaction Methods

“…The removal of the inhibitory substances is a major step in the preparation of the samples for PCR based detection of food pathogens. Although these inhibitory substances limit the application of PCR directly to food samples, the application of PCR based assays to enrichment broths has been more successful [9]. This study was carried out to evaluate a rapid (12 hour) method for detection of Salmonella in food samples and compare it with the conventional method.…”