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Traditional methods of assaying water samples for the presence of pathogens rely on the culture of enteric bacteria (enterococci or coliforms) in conjunction with biochemical tests. These methods have several advantages: they are inexpensive, easy to perform, reproducible, and acceptable by those government agencies responsible for setting water quality standards. Molecular biology-based techniques such as PCR may offer distinct advantages in terms of sensitivity and specificity; however, one of the more critical factors in the use of PCR is the ability to provide a quality nucleic acid template free of any inhibitory substances (11). Investigators have evaluated inexpensive sample preparation methods for a variety of pathogens. Orlandi and Lampel (9) used the FTA filter paper-based method to rapidly extract PCR-ready DNAs from Cyclospora spp., Cryptosporidium parvum, and microsporidia, while Lampel et al. (8) used FTA paper to extract DNAs from bacteria (Shigella, Salmonella, and Listeria spp.). Carnevale et al. (2) used Whatman filter paper to obtain DNA from stool samples containing the microsporidian Enterocytozoon bieneusi. Another type of rapid sample preparation matrix is Chelex resin by Bio-Rad (Hercules, Calif.); Hallier-Soulier and Guillot (5) used this to extract DNA from Cryptosporidium parvum oocysts for PCR. A newer rapid extraction reagent is the Xtra Bind matrix which Kozwich et al. (7) used to detect C. parvum viral symbiont RNA via a reverse transcriptase PCR assay.

Here, we report on our use of Instagene matrix (Bio-Rad), Prepman matrix (PE Biosystems, Foster City, Calif.), Xtra Amp tubes (Ansys Diagnostics, Lake Forest, Calif.), and Isocode paper (Schleicher and Schuell, Keene, N.H.) to extract PCR-ready DNAs from pure cultures of E. coli O157:H7 and C. parvum oocysts.

Sample collection and DNA extraction. Overnight cultures of bacterial stocks were done in Luria-Bertani broth, or minimal lactose broth, at 37°C on a rocking platform. The tubes containing the cultures were centrifuged at a medium speed (1,000 × g) on a tabletop centrifuge to pellet the cells. All but 100 µl of the supernatant was discarded, and the pellet was resuspended. Aliquots were removed and processed as described below. Colonies of E. coli O157:H7 on blood agar plates were removed with a sterile plastic loop and vortexed in 100-µl volumes of sterile water in a microcentrifuge tube to dislodge the bacteria. The sample was then centrifuged, the supernatant was discarded, and the pellet was resuspended in 30 µl of sterile water and processed as described below.

DNA amplification protocols. The E. coli lacZ TaqMan probe was 5' (6-carboxyfluorescein) CGC CTT ACT GCC TGT TTT GAC 6-carboxy-N,N',N,N'-tetramethylrhodamine, and the primers were lacZ forward (5' GTC CCG CAG CGC AGA C; nucleotides 5889 to 5904) and lacZ reverse (5' GCA GCG TTG TTG CAG TGC; nucleotides 6236 to 6253), which amplify a 364-bp region of the E. coli lacZ gene (GenBank accession no. AE000141.1 [1]). A variety of C. parvum genes were used as targets for PCR, namely, Cp11 (3), 18S rRNA (12), and Cp41 (GenBank accession no. AF144621 [6]). Primers for this gene were outer forward primer Cp41OF, 5' GAG GAG ATG GAC TAT TCT AGG; outer reverse primer Cp41OR, 5' GCA ACA GTA GTA AGA GTG GTA; inner forward primer Cp41 IF, 5' TGT ATG AAT TGG ATA TAT TAT TA; and inner reverse primer Cp41 IR, 5' GTA AAA GCA ACA CCA TTA CTA. To confirm that the extracted DNA was of sufficient quality for use in PCR, 50 ng of an internal positive control consisting of a cloned fragment of a 15-kDa C. parvum gene was spiked into PCR mixtures and amplified with the Cp11 primer set.

lacZ amplification from E. coli cultures. Results from 10-ml overnight cultures of E. coli O157:H7 of one of three replicate experiments evaluating three different DNA extraction methods are shown in Fig. 1. Based on the fluorescence intensities of the lacZ TaqMan probe reactions, the strongest positive reaction was obtained with 20 µl of the Isocode-extracted DNA. Results with 5 µl of PrepMan-extracted DNA and with 5 µl of Isocode-extracted DNA were very similar, with greater fluorescence emissions than those observed for Xtra Amp-extracted DNA and 1 µl of PrepMan-extracted DNA. This pattern was consistent for all three replicates.

View larger version (31K): [in this window] [in a new window]   FIG. 1.   Results of E. coli lacZ gene TaqMan assay performed on DNAs extracted from broth cultures of the E. coli O157:H7 Odwalla strain using the Xtra Amp tube, Isocode paper, and PrepMan matrix reagents. Thirty microliters (out of 100 µl) of an overnight culture pellet was extracted with the Xtra Amp and PrepMan reagents, and 10 µl was extracted with the Isocode paper. Fifty microliters of the PCR master mix plus 5 µl of Amp Enhance solution were used in the Xtra Amp tube. For the other two methods, 5- and 1-µl aliquots of the PrepMan-extracted DNA and 20- and 5-µl aliquots of Isocode paper-extracted DNA were used as the templates. Fluorescence readings for the 6-carboxyfluorescein reporter dye were obtained after 43 cycles of PCR, were corrected for background, and are plotted on the y axis.  Fluorescence, change in fluorescence.

PCR on DNA extracted from oocysts. Results from over 30 replicate samples indicated that larger numbers of oocysts (>200) provided positive PCR results with one round of amplification but that obtaining strong positives from 100 or fewer oocysts required a nested PCR. As an example, results from Cp11, Cp41, and 18S rRNA PCR assays done on Instagene-extracted DNAs are shown in Fig. 2. Figure 2A shows positive reactions for aliquots of 2,000 and 348 oocysts, but not 16 oocysts, amplified by one round of PCR with either Cp11 or 18S rRNA. When Cp41 gene PCR was performed on another dilution series of 1,673, 206, and 80 oocysts, samples with 1,673 and 206 oocysts were positive by primary PCR and samples with all three amounts were positive by nested PCR (Fig. 2B). In another experiment, nested Cp11 gene PCR was done on very small quantities of oocysts (n = 3) and positive reactions were observed for three replicate samples (Fig. 2C). If we assume uniform distribution of oocyst DNA in the Instagene matrix and we use 20 µl (of the total 200 µl) of Instagene-derived DNA, the theoretical number of oocysts detected by the nested Cp11 gene PCR under these idealized conditions is less than 1. However, in contrast to a report describing the use of a Chelex-based DNA extraction procedure in conjunction with one round of 18S rRNA PCR to detect 1 oocyst in 20 liters of source water (5), we found that nested PCR was necessary to detect even 80 or fewer oocysts in a 50-µl PCR mixture. A similar requirement for nested 18S rRNA PCR to detect small numbers of oocysts enumerated by the use of micromanipulators was reported by Sturbaum et al. (10).

View larger version (39K): [in this window] [in a new window]   FIG. 2.   Results of PCR performed on DNA extracted from purified C. parvum oocysts using the Instagene matrix. The quantity of oocysts assayed is indicated above each lane. Lanes L contain the DNA ladder (rung sizes are labeled), lanes Xc contain the extraction control, and lanes ntc, ntc1, and ntc2 contain the no-template controls. (A) 18S rRNA gene (left side of the DNA ladder) and Cp11 gene (right side of the DNA ladder) PCR results with DNAs from 2,000, 348, and 16 oocysts. (B) Results of nested Cp41 gene PCR on 1,673, 206, and 80 oocysts. (The band located between the primary (1°) and secondary (2°) PCR products is an artifact of the gel electrophoresis.) (C) Results of nested Cp11 gene PCR on three replicate samples containing three oocysts.

View larger version (35K): [in this window] [in a new window]   FIG. 3.   Results of nested Cp41 gene PCR performed on two replicate samples of DNA extracted from 650 and 65 oocysts of C. parvum using Xtra Amp tubes. Lane L, DNA ladder with rung sizes indicated; lanes Xc, extraction control; lanes 1° ntc and 2° ntc, primary and secondary no-template controls, respectively.