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Food-borne illnesses caused by pathogenic bacteria still occur at unacceptably high frequencies in industrialized nations and developing countries. Recently, a report from the Centers for Disease Control and Prevention presented estimates and causes of food-borne outbreaks for known illnesses in the United States from 1983 to 1992 and for passive and active surveillance for 1992 to 1997 and 1996 to 1997, respectively (13). The estimates ranged from 6 to 81 million cases of food-borne illnesses per year, with 5,000 deaths occurring annually. Although food safety initiatives have since been introduced to reduce contamination by food handlers and to improve sanitary conditions at the sites where foods are grown, harvested, and processed, food-borne illnesses attributed to these potential sources of contamination continue. This is illustrated by periodic reports of major outbreaks, such as those recently linked to contaminated sprouts in which Salmonella spp. or Escherichia coli O157:H7 was identified as the causative pathogen (18).

Increased public awareness of the health-related and economic impact of food-borne contamination and illness has resulted in greater efforts to develop more sensitive methods of pathogen detection and identification. Advances in molecular biology technology, particularly the PCR, have allowed for more reliable microbial identification and surveillance. PCR has also become a valuable tool for investigating food-borne outbreaks and identifying the responsible etiological agents. PCR techniques have provided increased sensitivity, allowed for more rapid processing times, and enhanced the likelihood of detecting bacterial pathogens. In addition to the analysis of foods, PCR has also been successfully applied to the detection and identification of pathogenic organisms in clinical and environmental samples (16, 21).

The reliability of PCR detection methods depends, in part, on the purity of the target template and the presence of sufficient numbers of target molecules. With such complex matrices as foods, steps must be taken to limit the effects of any potentially inhibitory compounds present that may limit PCR amplification of the intended target (4, 11, 17). This is in addition to enrichment steps that are frequently required to enhance PCR detection sensitivities and overcome problems of low pathogen numbers. Other means must be employed, however, when selective enrichment methods are not possible or do not exist. These other means include immunomagnetic separation (2, 7) and filter (19) formats, both of which are designed to concentrate microorganisms and remove potential PCR inhibitors.

In this study, we developed a protocol that uses FTA filters to prepare bacterial DNA templates derived from pure cultures and from artificially contaminated foods without arduous processing, preenrichment, and purification steps. The FTA filter is a fibrous matrix impregnated with chelators and denaturants that effectively traps and lyses microorganisms on contact (3). Released DNA is sequestered and preserved intact within the membrane. Following a series of brief washes to remove cell debris and other nonbinding contaminants, filters can be used directly in PCR assays or as a solid medium to store samples for later use. With this filter-based technology, enhanced detection sensitivities have been observed (14) compared to the detection sensitivities obtained with conventional template preparations. Templates from low numbers of target cells can be efficiently and rapidly prepared with minimal handling and sample loss. Previously, FTA filters have been used as a blood storage medium (3, 5), for bacterial ribotyping (15), and for preparation of plant genomic DNA (12). The uses of these filters have also been extended to include preparation of PCR templates from parasitic organisms isolated from foods and clinical specimens (14). In the present study, gram-negative and gram-positive bacteria were tested to determine the applicability of FTA filters in PCR detection. Furthermore, we examined the limits of detection of Shigella flexneri cells when they were seeded onto different foods (bean sprouts, alfalfa sprouts, cilantro, lettuce, tomatoes, beef, and apple cider). In addition, a comparison was made between PCR amplification from templates prepared directly from washed produce and PCR amplification from filters. Whereas several investigators have described different methods for preparing template DNA for PCR (20), including the use of other filter types (1), the protocol described here is a more efficient, sensitive, and uniform method for PCR template preparation.

Sensitivity of the PCR assay with pure cultures. S. flexneri 2457T and Salmonella enterica serotype Typhimurium phage type DT-104 were grown overnight in Luria broth at 37°C in a shaking water bath. Cells were diluted 1:100 in 5 ml of Luria-Bertani broth and incubated as described above for approximately 4 h to obtain a cell count of 10⁹ to 10¹⁰ CFU/ml. Listeria monocytogenes cells were grown overnight at 37°C either in brain heart infusion broth or on agar (1.5%) medium. Bacterial cells from cultures grown for either 4 h or overnight (Listeria cultures) were serially diluted with Butterfield's phosphate buffer. PCR detection sensitivities were then compared by using templates prepared either from boiled lysates or by FTA filter application. Aliquots (10 µl) were transferred to thin-wall PCR tubes (Perkin-Elmer), covered with a few drops of mineral oil (Sigma), boiled for 5 min, and then placed on ice until needed. From the same dilutions, 10-µl aliquots were also applied to FTA filters (Fitzco, Inc.). After application of the bacterial cells, the filters were air dried on a heating block at 56°C (15 to 20 min). The FTA filters were washed twice with FTA purification buffer (GIBCO/BRL) for 2 min and twice in 10 mM Tris (pH 8.0) containing 0.1 mM EDTA for 2 min. The filters were air dried as described above, and either the filters were stored at 20°C or the spotted areas were removed with a 6-mm-diameter hole puncher and directly used as templates for PCR. For PCR templates prepared by boiling, the reaction mixtures (total volume, 25 µl) contained the following: 1× PCR buffer (Qiagen), 200 µM (each) dATP, dCTP, dGTP, and dTTP, and each PCR primer at a concentration of 0.2 µM. The mixtures were overlaid with mineral oil. Enzyme (Qiagen Taq DNA polymerase; 1.5 U) was added after the reaction vessels reached 80°C. For reaction mixtures applied onto FTA filters, the conditions were the same, except that the total reaction volume was 100 to 200 µl and the buffer and DNA polymerase (2.5 U/100 µl of reaction mixture) were purchased from Promega. The total number of cycles was 30, and the annealing temperature was 60°C; the denaturing and extension temperatures were 94 and 72°C, respectively. Each step of the cycle was 1 min long. The PCR primers used for amplifying the ipaH gene of Shigella (10) were 5'-GTTCCTTGACCGCCTTTCCGATACCGTC-3' and 5'-GCCGGTCAGCCACCCTCTGAGAGTAC-3'; the primers used for amplifying the invA gene of salmonellae (9) were 5'-ACCACGCTCTTTCGTCTGG3' and 5'-GAACTGACTACGTAGACGCTC-3'; and the primers used for amplifying the hemolysin gene of L. monocytogenes were 5'-CGGAGGTTCCGCAAAAGATG-3' and 5'-CCTCCAGAGTGATCGATGTT-3' (8). The expected size of the ipaH PCR product is 600 bp; the expected size of the invA PCR product is 941 bp; and a 234-bp product results from targeting the hemolysin gene of L. monocytogenes. PCR-amplified products were visualized in 1% agarose gels in 0.5× Tris-acetate-EDTA (pH 7.8) buffer with ethidium bromide (0.2 µg/ml) by using a Run-one gel system (Embi-Tec).

View larger version (52K): [in this window] [in a new window]   FIG. 1.   Comparison of detection limits of PCR-based assays with lysates and FTA filters as templates. (I) PCR products generated from 10-fold serially diluted S. flexneri cells (lane A, 5 × 104 CFU; lane B, 5 × 103 CFU; lane C, 5 × 102 CFU; lane D, 50 CFU) when boiled lysates and FTA filters were used as PCR templates. (II and III) Same comparison performed with S. enterica serotype Typhimurium and L. monocytogenes, respectively. The arrows indicate the expected size of each amplicon.

PCR data from unseeded and seeded foods. Table 1 shows the background microbial population that was enumerated for each food tested. Foods with moderate to large microbial floras were selected in order to assess their impact on detecting low levels of the targeted bacteria (Shigella) when they were applied to FTA filters. Untreated food samples were also tested by using the PCR-based assay for Shigella detection to determine if the microbial content yielded any nonspecific amplicons. The samples chosen for analysis included samples of bean sprouts, alfalfa sprouts, lettuce, tomato, cilantro, ground beef, and apple cider. Solid samples (10 g) were placed in 250-ml beakers and washed once with 10 ml of 1× phosphate-buffered saline with gently agitation for 15 to 20 min at room temperature. The wash buffer was decanted from each food sample, and serial dilutions were then made to 10³; 10 µl from the 10-ml wash and 10 µl of each dilution were used as PCR templates. Apple cider was assayed directly and by using serial dilutions as described above. Each sample was evaluated directly with the PCR assay or spotted onto FTA filters. No amplicons were observed with either template preparation method in the absence of the appropriate target (data not shown).