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Applied and Environmental Microbiology, March 2005, p. 1135-1141, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1135-1141.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Development of Procedures for Direct Extraction of Cryptosporidium DNA from Water Concentrates and for Relief of PCR Inhibitors

Jianlin Jiang,1 Kerri A. Alderisio,2 Ajaib Singh,3 and Lihua Xiao1*

Division of Parasitic Disease, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia,1 New York City Department of Environmental Protection, Valhalla, New York,2 City of Milwaukee Public Health Laboratories, Milwaukee, Wisconsin3

Received 16 July 2004/ Accepted 7 October 2004


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ABSTRACT
 
Extraction of high-quality DNA is a key step in PCR detection of Cryptosporidium and other pathogens in environmental samples. Currently, Cryptosporidium oocysts in water samples have to be purified from water concentrates before DNA is extracted. This study compared the effectiveness of six DNA extraction methods (DNA extraction with the QIAamp DNA minikit after oocyst purification with immunomagnetic separation and direct DNA extraction methods using the FastDNA SPIN kit for soil, QIAamp DNA stool minikit, UltraClean soil kit, or QIAamp DNA minikit and the traditional phenol-chloroform technique) for the detection of Cryptosporidium with oocyst-seeded samples, DNA-spiked samples, and field water samples. The study also evaluated the effects of different PCR facilitators (nonacetylated bovine serum albumin, the T4 gene 32 protein, and polyvinylpyrrolidone) and treatments (the use of GeneReleaser or ultrafiltration) for the relief from or removal of inhibitors of PCR amplification. The results of seeding and spiking studies showed that PCR inhibitors were presented in all DNA solutions extracted by the six methods. However, the effect of PCR inhibitors could be relieved significantly by the addition of 400 ng of bovine serum albumin/µl or 25 ng of T4 gene 32 protein/µl to the PCR mixture. With the inclusion of bovine serum albumin in the PCR mixture, DNA extracted with the FastDNA SPIN kit for soil without oocyst isolation resulted in PCR performance similar to that produced by the QIAamp DNA minikit after oocysts were purified by immunomagnetic separation.


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INTRODUCTION
 
Waterborne transmission is important in the epidemiology of human cryptosporidiosis. Laboratory diagnosis of Cryptosporidium in water samples has traditionally relied on the identification of oocysts by immunofluorescence assay (IFA) after oocysts in water are concentrated by filtration and floatation or immunomagnetic separation (IMS; such as the ICR method or method 1622 or 1623 of the U.S. Environmental Protection Agency). A disadvantage of immunofluorescence microscopy is that the species nature of Cryptosporidium in environmental samples cannot be assessed, because IFA cannot differentiate Cryptosporidium species or strains from humans and those from various vertebrate animals. PCR has some advantages over immunofluorescence microscopy in detection sensitivity and the ability to differentiate Cryptosporidium species or genotypes. However, PCR is susceptible to the effect of many inhibitors present in samples, which is a major problem in the molecular biological detection of microorganisms in environmental samples (17, 20, 25, 26, 32).

Reduction or removal of PCR inhibitors is an essential component in the molecular detection of microorganisms in environmental samples (34). Three basic approaches have been used in general to overcome the effect of PCR inhibitors, including purification of microorganisms prior to DNA extraction, removal of inhibitors during or after DNA extraction, and relief or suppression of the effect of PCR inhibitors when performing PCR. In the detection of Cryptosporidium, the most commonly used method is the purification of the oocysts by IMS prior to DNA extraction (9, 12, 18, 23, 27, 31, 35), which effectively eliminates or greatly reduces the substances that might be inhibitory to PCR amplification. However, IMS is expensive, and its performance is affected by the type of commercial kits used, pH, and dissociation procedures (7, 16, 30). For the detection of other organisms, modifications of procedures for DNA extraction and purification of DNA after DNA extraction have also been applied to remove PCR inhibitors by chemical flocculation (6) or treatment with Chelex 100 (10) or polyvinylpyrrolidone (8, 10) during DNA extraction and by purification of DNA with anti-inhibitory substances (14) and chromatography (28) after DNA extraction. In addition, several substances, such as bovine serum albumin (BSA) (2, 15, 19), T4 gene 32 protein (15, 21, 24), and polyvinylpyrrolidone (13), have been used to relieve the effects of PCR inhibitors during the PCR step. Some special DNA polymerases that are highly resistant to inhibitors have also been used in PCR (1).

Numerous direct DNA extraction methods have been used in the preparation of DNA from Cryptosporidium spp., such as the traditional phenol-chloroform extraction method (3) and the use of the commercial FastDNA SPIN kit for soil (5, 8, 22), UltraClean soil kit (11), QIAamp DNA stool minikit (36), and QIAamp DNA minikit (10). However, most of these methods were used for the extraction of DNA from animal or human fecal specimens. Detection of Cryptosporidium oocysts in water samples by these direct DNA extraction methods has rarely been successful (33). High-quality DNA can be obtained from IMS-purified oocysts, but the DNA extracted from them can be used only for the detection of Cryptosporidium spp. Therefore, it is important to directly extract high-quality DNA from water samples without pathogen isolation for the analysis of multiple pathogens.

In this study, we compared the efficiencies of several commonly used DNA extraction methods for the extraction of PCR quality DNA using Cryptosporidium oocyst-seeded samples, DNA-spiked samples, and field wastewater or storm water samples. We also evaluated the effectiveness of several secondary DNA purification treatments and PCR facilitators for the relief from or removal of PCR inhibitors before or during PCR amplification. Based on results of the evaluation, we developed a procedure for Cryptosporidium detection in water samples using a combination of the FastDNA SPIN kit for soil for direct DNA extraction and the addition of high concentrations of nonacetylated BSA to PCR mixtures.


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MATERIALS AND METHODS
 
Cryptosporidium oocysts.
Preparations of 5, 10, 25, or 50 oocysts of the Cryptosporidium parvum Iowa strain, sorted by flow cytometry at the Wisconsin State Health Department Public Health Laboratory, were used for oocyst seeding and the evaluation of detection sensitivity. A preparation of 1,000 oocysts of the same strain in 1 ml of phosphate-buffered saline counted by hemocytometer was used in DNA spiking experiments.

Cryptosporidium-negative water samples.
Storm water samples (used in the oocyst seeding or DNA spiking evaluations) and surface water samples (used in the sensitivity determination) negative for Cryptosporidium oocysts, as determined by microscopy and repeated PCR analysis (at least six times) of DNA extracted with the QIAamp DNA minikit (QIAGEN, Valencia, Calif.) after oocysts were purified with IMS (see method 1 below), were used. All Cryptosporidium-negative samples were collected by the filtration of at least 10 liters of water with the Envirochek HV filter (Pall Gelman, Ann Arbor, Mich.) and washed in a polypropylene tube twice with distilled water by centrifugation at 1,500 x g for 10 min. A 0.5-ml portion of a pellet (the volume used by method 1622 or 1623) per sample was seeded with precounted Cryptosporidium oocysts. Different levels of C. parvum oocysts were used for oocyst seeding to determine the sensitivities of the final direct DNA extraction method (method 5 below) and the standard extraction method (method 1 below). Each method had five replicates (0.5 ml of water pellet/replicate) at each oocyst seeding level for each seeding experiment. The seeding experiment was conducted twice.

DNA extraction.
With the exception of concentrates of samples processed with method 1, which were first subjected to oocyst isolation by IMS, concentrates of all samples were transferred into 2-ml microcentrifuge tubes and centrifuged at full speed for 5 min. One of the following six DNA extraction methods was used to extract the total DNA by following the manufacturer-recommended or a standard protocol. The final volume of DNA preparation was 100 µl per sample. DNA was stored at –20°C before it was used in PCR.

(i) Method 1, DNA extraction with the QIAamp DNA minikit after oocyst isolation by IMS.
Briefly, after adding 1 ml of 10x buffer A, 1 ml of 10x buffer B, and 100 µl of anti-Cryptosporidium Dynabeads from the Dynabead anti-Cryptosporidium kit (Dynal, Lake Success, N.Y.) to a 15-ml tube containing 0.5 ml of water pellets and adjusting the final volume to 10 ml, the tubes were rotated at 20 rpm at room temperature for 1 h. Cryptosporidium oocysts were subsequently captured with a magnetic device (MPC; Dynal) and used directly in DNA extraction without oocyst detachment by adding 180 µl of the ATL buffer from the QIAamp DNA minikit (QIAGEN). The suspension was transferred into a 1.5-ml Eppendorf tube and subjected to five cycles of freeze-thaw (–70°C for 30 min and 56°C for 30 min). DNA was extracted from the purified oocysts with the QIAamp DNA minikit and the manufacturer-recommended procedures.

(ii) Method 2, direct DNA extraction with the QIAamp DNA minikit without IMS isolation of Cryptosporidium oocysts.
Briefly, 0.5 ml of a water pellet in a 2-ml tube was suspended in 240 µl of the ATL buffer from the QIAamp DNA minikit (QIAGEN). After five cycles of freeze-thaw (–70 and 56°C), the sample was processed further by following the manufacturer-recommended procedures with the exception of tripling the volume of each reagent.

(iii) Method 3, direct DNA extraction with the QIAamp DNA stool minikit.
Briefly, 1.2 ml of buffer ASL from the QIAamp DNA stool minikit (QIAGEN) was added to 0.5 ml of water pellets in a 2-ml tube. After five cycles of freeze-thaw (–70 and 56°C), the sample was processed further by following the manufacturer-recommended procedures.

(iv) Method 4, direct DNA extraction with the UltraClean soil DNA isolation kit.
Briefly, 60 µl of solution S1 and 200 µl of solution IRS from the UltraClean soil DNA isolation kit (Mo Bio, Solana Beach, Calif.) were added to a 2-ml bead solution tube (from the kit) containing 0.5 ml of water concentrates. The tubes were then vortexed at maximum speed for 10 min with a Vortex adapter (Mo Bio) and processed further by following the manufacturer-recommended procedures.

(v) Method 5, direct DNA extraction with the FastDNA SPIN kit for soil.
Briefly, 0.5 ml of water pellets was transferred into a 2-ml tube containing the lysing matrix E from the FastDNA SPIN kit for soil (BIO 101, Carlsbad, Calif.). After 978 µl of sodium phosphate buffer and 122 µl of MT buffer from the kit were added, the tube was vortexed in a FastPrep instrument (BIO 101) for 30 s at the speed setting 5.5. The sample was processed further in accordance with the manufacturer-recommended procedures.

(vi) Method 6, direct DNA extraction by the phenol-chloroform method.
Briefly, after 500 µl of Tris-EDTA buffer was added to 2-ml tubes containing 0.5 ml of water pellets, oocysts were ruptured by five cycles of freeze-thaw (–70 and 56°C). The samples were processed further by the conventional phenol-chloroform DNA extraction method (4).

PCR.
A fragment (~830 bp) of the small subunit (SSU) rRNA gene was amplified by nested PCR as previously described (36) to evaluate the performance of different DNA extraction methods, anti-inhibitory substance treatments, and PCR facilitators. The total volume of the PCR mixture was 100 µl, and it contained 0.5 to 5 µl of DNA, each primer at the concentrations of 0.25 (for primary PCR) and 0.5 µM (for secondary PCR), 0.2 mM deoxynucleoside triphosphate (Applied Biosystems, Foster City, Calif.), 3 mM MgCl2, 1x PCR buffer (GeneAmp 10x PCR buffer; Applied Biosystems), and 2.5 U of Taq DNA polymerase (Promega, Madison, Wis.). PCR amplification consisted of an initial denaturation at 94°C for 4 min; 35 cycles of 94°C for 45 s, 55°C (primary PCR) or 58°C (secondary PCR) for 45 s, and 72°C for 1 min; and a final extension of 72°C for 10 min. PCR products were visualized by 1.5% agarose gel electrophoresis.

DNA spiking experiment.
Five Cryptosporidium-negative storm water samples were used for evaluating the reduction or removal of PCR inhibitors during DNA extraction. The total DNA was extracted by extraction methods 1 to 5 with a set of 0.5-ml portions of water pellets (five replicates per method) as described above, and the final DNA preparation was 100 µl per extraction. DNA was stored at –20°C until it was used in PCR. To determine whether residual PCR inhibitors were present in the extracted DNA, 2 µl of DNA preparation was spiked into a PCR mixture containing 1 µl of a pure C. parvum DNA equivalent of five oocysts as templates. The latter was extracted from 1,000 purified C. parvum oocysts by method 2 and eluted in 200 µl of distilled water.

Ultrafiltration treatment before PCR amplification.
To remove low-molecular-weight PCR inhibitors from the extracted DNA, 1 µl of DNA was added to 400 µl of distilled water in a Microcon PCR reservoir (Amicon Inc., Beverly, Mass.). The mixture was filtered by centrifugation at 1,000 x g for 15 min to remove the PCR inhibitors. An additional 20 µl of water was added to the reservoir, and the filter was inverted. The ultrafiltered fraction was collected in the reservoir by centrifugation at 1,000 x g for 2 min. All of the eluted DNA solution was used in regular PCR amplification.

GeneReleaser treatment before PCR amplification.
Five microliters of DNA and 15 µl of GeneReleaser reagent (Bioventures Inc., Murfreesboro, Tenn.) were mixed and treated according to the manufacture-suggested program (65°C for 30 s, 8°C for 30 s, 65°C for 90 s, 97°C for 180 s, 8°C for 60 s, 65°C for 180 s, 97°C for 60 s, 65°C for 60 s, and holding at 80°C). The whole mixture was then used directly for regular PCR amplification.

Use of BSA, T4 gene 32 protein, and polyvinylpyrrolidone in PCR.
The effectiveness of PCR facilitators for the relief of PCR inhibitors during PCR amplification was evaluated with the inclusion of nonacetylated BSA (Sigma, St. Louis, Mo.) at different concentrations (50, 200, 400, and 600 ng/µl), 25 ng of T4 gene 32 protein (Roche, Indianapolis, Ind.)/µl, and 1 or 2% (wt/vol) polyvinylpyrrolidone (molecular weight, 360,000; Sigma) in PCR mixtures.

Field evaluation of different DNA extraction methods for the detection of Cryptosporidium.
A total of 55 wastewater samples, each consisting of 250 ml of raw wastewater, were collected from a wastewater treatment plant in Milwaukee from October 2002 to February 2003. A portion (50 ml) of each sample was used for DNA extraction by one of five DNA extraction methods after the sample was centrifuged at 1,500 x g for 10 min. A total of 67 storm water samples from the Malcolm Brook and Ashokan Brook in New York collected from July 2002 to October 2003 were also used in the field evaluation. Storm water samples were collected with preset autosamplers (6700; ISCO, Inc., Lincoln, Neb.) when a predetermined flow rate was reached. Composite samples of 20 liters were filtered through the Envirocheck HV filters (Pall Gelman) by procedures described in reference 29. A portion (0.5 ml) of the eluted pellet of each sample was used for each DNA extraction method. The efficiencies of different DNA extraction methods were determined by amplification of the SSU rRNA gene by nested PCR with the same volume of DNA. Successful PCR amplification and contamination of PCR were monitored by use of one positive DNA control (DNA of Cryptosporidium serpentis, unless specified otherwise) and two negative DNA controls.


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RESULTS
 
Comparison of the efficiencies of six DNA extraction methods with oocyst-seeded samples.
Composite storm water concentrates negative for Cryptosporidium were aliquoted to 30 tubes of 0.5 ml of pellet/tube and were seeded with 50 oocysts of the C. parvum Iowa strain/tube. They were used in DNA extractions with the six methods, with five replicates per method. Regular PCR analysis of the SSU rRNA gene with DNA extracted by these six methods showed that amplification was achieved only with DNA extracted with method 1 (DNA extraction from IMS-purified oocysts with the QIAamp DNA minikit), which produced the expected PCR band for four of five replicates when 1 µl of DNA was used for PCR or three of five replicates when 2 µl of DNA was used for PCR (Table 1 and Fig. 1). All five replicates by method 1 produced PCR amplification in multiple PCR analyses.


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  		TABLE 1. Evaluation of the efficiencies of six DNA extraction methods and the effectiveness of anti-inhibitory treatment of DNA prior to PCR or the use of facilitators in PCR, using Cryptosporidium-negative storm water samples seeded with 50 oocysts of C. parvum



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  		FIG. 1. Impact of PCR inhibitors on PCR amplification. One (top) or two (bottom) microliters of 100 µl of DNA extracted with methods 4 (lanes 1 to 5), 3 (lanes 6 to 10), 2 (lanes 11 to 15), 6 (lanes 16 to 20), 5 (lanes 21 to 25), and 1 (lanes 26 to 30) from storm water samples seeded with 50 C. parvum oocysts was used for PCR analysis of the SSU rRNA gene without secondary purification of the DNA or the inclusion of facilitators in the PCR mixture. Lane 32 (top) was the positive PCR control (DNA of C. serpentis), and lanes 31 to 33 (bottom) were negative PCR controls.

Effect of anti-inhibitory substance treatments.
Treatment of the extracted DNA with GeneReleaser before PCR amplification resulted in a slight improvement in PCR amplification of DNA extracted by method 1, as all five DNA replicates had consistent PCR amplification. This treatment, however, had no effect on DNA extracted by the other five direct DNA extraction methods (Table 1). Similar results were also obtained with DNA treated with the Ultrafilter, as it improved the quality only of DNA extracted by method 1 (DNA from all five replicates consistently produced PCR amplifications; Table 1).

Effect of PCR facilitators.
The inclusion of polyvinylpyrrolidone in PCR mixtures at the levels of 1% (wt/vol) and 2% (wt/vol) did not improve PCR performance in this study. Only three of five replicates at the 1% level and four of five replicates at the 2% level produced the expected band with DNA extracted by method 1. In contrast, no PCR amplification was found in the analysis of DNA extracted by all of the other five direct DNA extraction methods (Table 1).

The use of BSA at 40 or 60 µg/PCR (at the final PCR concentration of 400 or 600 ng/µl) led to an improvement in the PCR amplification of DNA extracted by method 5 (direct DNA extraction with the FastDNA SPIN kit for soil), which produced amplification with DNA from all five replicates seeded with 50 C. parvum oocysts (Table 1; Fig. 2), whereas inclusion of BSA at 5 or 20 µg/PCR (at the final PCR concentration of 50 or 200 ng/µl) had no significant effect on PCR amplification (Table 1). The addition of 40 or 60 µg of BSA to the PCR mixture also enhanced slightly the amplification of DNA extracted by method 1, as all five replicates at each level had consistent PCR amplification (Table 1; Fig. 2). However, inclusion of BSA in the PCR mixture had no relieving effect on the impact of PCR inhibitors for DNA extracted directly with method 2 (direct DNA extraction with the QIAamp DNA minikit), 3 (direct DNA extraction with the QIAamp DNA stool minikit), 4 (direct DNA extraction with the UltraClean soil kit), or 6 (direct DNA extraction with the phenol-chloroform method) (Table 1).



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  		FIG. 2. Relief of PCR inhibitors with the inclusion of 400 (top) or 600 ng (bottom) of nonacetylated BSA/µl in the PCR mixture. Two microliters of 100 µl of DNA extracted with methods 4 (lanes 1 to 5), 3 (lanes 6 to 10), 2 (lanes 11 to 15), 6 (lanes 16 to 20), 5 (lanes 21 to 25), and 1 (lanes 26 to 30) from storm water samples seeded with 50 C. parvum oocysts was used for PCR analysis of the SSU rRNA gene. Lane 32 was a positive PCR control (DNA of C. serpentis), lane 31 was blank, and lanes 33 to 35 were negative PCR controls.

Inclusion of T4 gene 32 protein at the level of 25 ng/µl also improved PCR amplification of DNA extracted from storm water samples seeded with 50 C. parvum oocysts by methods 1 and 5, as all five DNA replicates derived from each of the two extraction methods had PCR amplification (Table 1). In contrast, no significant improvement was achieved with DNA extracted by methods 2, 3, and 4 (Table 1). Only one concentration of T4 gene 32 protein, which was shown to be effective in other PCR systems (28), was tested in this study because the protein is expensive.

Evaluation of the presence of residual PCR inhibitors in extracted DNA.
Two microliters of DNA extracted by methods 1 to 5 from Cryptosporidium-negative storm water samples was spiked into the PCR mixture for analysis of a pure C. parvum DNA equivalent of five oocysts. Results of the spiking study indicated that method 1 removed more PCR inhibitors than the four direct DNA extraction methods, as four of five replicates from method 1 showed the expected band in PCR when no BSA was used. All DNA extracted by other methods inhibited PCR amplification of the pure C. parvum DNA (Fig. 3). The inclusion of BSA in the PCR mixture reduced the impact of PCR inhibitors in DNA extracted by methods 1 to 5. When BSA was used in the PCR, none of the replicates extracted by method 1 inhibited PCR amplification of the C. parvum DNA. Among direct DNA extraction methods, methods 4 (using the UltraClean soil kit) and 5 (using the FastDNA SPIN kit for soil) had higher efficiency in reducing PCR inhibitors during DNA extraction than other methods because, with the inclusion of 400 ng of BSA/µl in the PCR mixture, none of the five replicates produced by these two methods inhibited PCR amplification of the C. parvum DNA. In contrast, only one of five replicates produced by methods 2 (direct extraction with the QIAamp DNA minikit) and 3 (direct extraction with the QIAamp DNA stool minikit) produced the expected bands when 400 ng of BSA/µl was included in the PCR analysis of the C. parvum DNA (Fig. 3).



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  		FIG. 3. The presence of residual PCR inhibitors in DNA extracted by five DNA extraction methods. Two microliters of 100 µl of DNA extracted from a 0.5-ml pellet of Cryptosporidium-negative storm water samples by methods 1 (lanes 1 to 5), 5 (lanes 6 to 10), 3 (lanes 11 to 15), 4 (lanes 16 to 20), and 2 (lanes 21 to 25) was used for PCR amplification of the SSU rRNA gene. One microliter of DNA equivalent to five purified C. parvum oocysts was used as the PCR template without (top) or with (bottom) the inclusion of 400 ng of nonacetylated BSA/µl in the PCR mixture. Lane 27 was a positive PCR control (DNA equivalent to five purified C. parvum oocysts), lane 26 was blank, and lanes 28 to 30 were negative PCR controls.

Detection limits of methods 1 and 5.
To evaluate the efficiency of methods 1 (DNA extraction of IMS-purified oocysts with the QIAamp DNA minikit) and 5 (direct extraction with the FastDNA SPIN kit for soil) with the inclusion of 400 ng of BSA/µl in the PCR mixture, 40 replicates of 0.5 ml of surface water concentrates negative for Cryptosporidium were seeded with 5, 10, 25, or 50 oocysts of C. parvum, with five replicates per method for each seeding level. Method 1 could detect at least five oocysts in seeded samples, because DNA of all five replicates produced PCR amplification (Fig. 4). When method 5 was used for DNA extraction, all five replicates seeded with 50 oocysts were amplified by PCR, but only one of five replicates seeded with 5 oocysts, three of five replicates seeded with 10 oocysts, and four of five replicates seeded with 25 oocysts produced the expected bands (Fig. 4). However, with multiple PCR amplifications of DNA extracted by method 5, two of five replicates seeded with 5 oocysts and all five replicates seeded with 10, 25, and 50 oocysts showed amplification (data not shown).



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  		FIG. 4. Sensitivity of Cryptosporidium PCR detection with DNA extracted by methods 1 and 5. DNA was extracted from a Cryptosporidium-negative surface water sample seeded with 5 (lanes 1 to 5), 10 (lanes 6 to 10), 25 (lanes 11 to 15), and 50 (lanes 16 to 20) oocysts of C. parvum by DNA extraction methods 5 (top) and 1 (bottom). PCR analysis of the SSU rRNA gene was performed with the inclusion of 400 ng of nonacetylated BSA/µl. Lane 21 was a positive PCR control (DNA of C. serpentis), and lane 22 was one of the negative PCR controls.

Efficiencies of five DNA extraction methods for detecting Cryptosporidium in wastewater or storm water samples.
Due to the poor performance of the phenol-chloroform DNA extraction method (method 6) in seeding experiments and our previous study (35), only DNA extraction methods 1 to 5 were evaluated further for efficiency for the detection of Cryptosporidium spp. in 55 wastewater and 67 storm water samples.

When the levels of performance of all five DNA extraction methods were compared by using storm water samples from New York, which was known to have high occurrence of Cryptosporidium oocysts (36), it was found that 82% (55 of 67) of the samples were PCR positive when method 1 was used for DNA extraction, whereas 64% (43 of 67) of samples were positive when DNA was directly extracted from water pellets by method 5 (Table 2). In comparison, only 3% (1 of 33), 2.6% (1 of 39), and 12% (7 of 59) of samples extracted by methods 2, 3, and 4 produced PCR amplification, respectively. When five DNA extraction methods were compared on the basis of the percentage of amplifications of all PCR analyses performed with different volumes (0.5 to 3.0 µl) of DNA of each sample, similar results were obtained; 36% (154 of 426) and 26% (109 of 426) of all PCRs were positive with DNA extracted by methods 1 and 5, compared to 0.5% (1 of 222), 0.4% (1 of 258), and 2% (9 of 378) of PCR amplifications of DNA extracted by methods 2, 3, and 4, respectively (Table 3).


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  		TABLE 2. Efficiency of PCR detection of Cryptosporidium spp. in DNA from wastewater samples from Milwaukee and storm water samples from New York extracted by five methodsa


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  		TABLE 3. PCR amplification rates for DNA extracted by five methods from wastewater samples from Milwaukee and storm water samples from New Yorka

When the performance of these five methods was compared with wastewater samples from Milwaukee, it was found that methods 1 to 5 identified Cryptosporidium spp. in 53% (29 of 55), 46% (16 of 35), 38% (21 of 55), 8% (3 of 36), and 55% (30 of 55) of samples, respectively (Table 2). When five DNA extraction methods were compared on the basis of the percentage of amplifications of all PCR analyses performed with different volumes (0.5 to 3.0 µl) of DNA from each sample, it was found that 15% of all PCRs (62 of 402) were positive when DNA extracted by method 1 was used. In contrast, the amplification rates for methods 2 to 5 were 14% (38 of 279), 9% (37 of 402), 4% (10 of 288), and 14% (58 of 402), respectively (Table 3).


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DISCUSSION
 
For environmental samples, the efficiency of DNA extraction methods is determined by the DNA recovery rate and PCR inhibitor reduction during DNA extraction. Because water samples are likely to have only a few Cryptosporidium oocysts in the presence of many other organisms, the recovery of Cryptosporidium DNA during DNA extraction becomes important, and the measurement of total DNA provides no information on the efficiency of Cryptosporidium DNA extraction methods. Thus, in this study, the efficiencies of different DNA extraction methods were evaluated on the basis of the presence or absence of PCR amplification of the SSU rRNA gene of Cryptosporidium.

Environmental samples (water, soil, and food materials) are known to be rich in PCR inhibitors, such as humic acids, which could be coextracted with DNA during the DNA isolation and purification process and which could interfere with PCR amplification (28). Many studies have shown that PCR inhibitors present in water samples reduce or suppress PCR amplification (12, 17, 20, 25, 26, 32). Reduction or elimination of PCR inhibitors prior to, during, or after DNA extraction has become an important step in molecular diagnosis of microbial pathogens in water and other environmental samples. Currently, pathogen isolation by IMS and culture enrichment prior to DNA extraction are standard procedures to eliminate or reduce PCR inhibitors (9, 18, 23, 27, 31, 34, 35). These, however, become impractical for organisms that have no IMS procedures or that cannot be cultured. The use of IMS is also expensive, and this limits the use of samples mostly to single-organism detection. Thus, the development of methods for direct extraction of PCR quality DNA is important for the detection of pathogens in environmental samples.

For PCR detection of Cryptosporidium spp., isolation of oocysts by IMS prior to DNA extraction is currently the standard procedure to remove the PCR inhibitors present in water samples. As shown in this study, without the use of PCR facilitators, only DNA extracted from oocysts purified by IMS from water samples produced PCR amplification. Residual PCR inhibitors were still present in DNA purified by this method, as the efficiency of PCR amplification of DNA could be improved by the secondary purification of extracted DNA or the use of PCR facilitators (Table 1). Therefore, the presence of PCR inhibitors in DNA preparations extracted from water concentrates poses a serious challenge to PCR detection of Cryptosporidium spp.

Several commercial kits for the extraction of DNA from samples rich in PCR inhibitors have been marketed; these include the FastDNA SPIN kit for soil (5, 8), UltraClean soil kit (11), QIAamp DNA stool minikit (37), and QIAamp DNA minikit (10). DNA extracted directly from water samples by these methods in this study, however, failed to produce PCR amplification when PCR facilitators were not used, indicating that residual PCR inhibitors were present in DNA. This was further supported by the results of DNA spiking experiments, which showed that DNA extracted by these methods inhibited PCR amplification of pure C. parvum DNA. Among the direct DNA extraction methods evaluated in this study, DNA prepared by physical lysis methods (bead beating in methods 4 and 5) had less PCR inhibitors than that prepared by chemical methods (methods 2 and 3). The results of a DNA spiking study (Fig. 3) showed that with the addition of PCR facilitators the impact of residual inhibitors in DNA extracted with methods 4 and 5 was relieved. In contrast, PCR amplification was obtained with only one replicate each of DNA extracted by methods 2 and 3. It is likely that more PCR inhibitors, such as humic acids, were extracted from decayed plant materials in water when chemical lysis methods were used for DNA extraction.

Anti-inhibitory substance treatments, such as GeneReleaser or ultrafiltration treatment before PCR, and the inclusion of PCR facilitators during PCR were also used for the removal of or relief from PCR inhibitors in DNA preparation. GeneReleaser or ultrafiltration treatment before PCR only slightly improved the sensitivity of method 1 in this study (Table 1). Many studies have shown that the inclusion of BSA (2, 15, 19), T4 gene 32 protein (15, 21, 24), and polyvinylpyrrolidone (13) in PCR could improve the performance of PCR. In both oocyst seeding and DNA spiking experiments in this study, the inclusion of 400 ng of BSA/µl or 25 ng of T4 gene 32 protein/µl in the PCR mixture overcame the impact of inhibitors on PCR amplification when DNA was extracted with the FastDNA SPIN kit for soil (Table 1; Fig. 2 and 3). Despite the usefulness of the T4 gene 32 protein for PCR, its expense, which increases the cost of PCR by $1.50 to $2.00 per reaction, has limited its use in PCR. Because of the low number of Cryptosporidium oocysts present in environmental samples, each water sample is usually analyzed by PCR at least three times, which would cost $4.50 to $6.00 more per sample if the T4 gene 32 protein is used for PCR. The inclusion of 400 ng of BSA/µl in the PCR mixture economically relieves the impact of residual PCR inhibitors present in DNA extracted by method 5, and the effective concentration was the same as that previously determined to be the optimum to eliminate the inhibitory effects of 1 ng of humic acids/µl, 100 µM FeCl3, manure, marine water, and freshwater (15). Nevertheless, the use of BSA or T4 gene 32 protein was not able to overcome the effect of PCR inhibitors present in DNA extracted directly by other direct-extraction methods. Contrary to the results of a previous study (13), the use of 1 or 2% polyvinylpyrrolidone failed to reduce the effect of PCR inhibitors in this study. The polyvinylpyrrolidone used in this study, however, had a molecular weight (360,000) different from those (25,000 to 30,000) of the polyvinylpyrrolidone used in the previous study.

The combination of direct DNA extraction with the FastDNA SPIN kit for soil and inclusion of BSA as a PCR facilitator produced good PCR detection of Cryptosporidium oocysts in field water samples. When storm water samples were analyzed, method 5 led to the detection of more positive samples or a higher percentage of PCR amplification than the other three direct DNA extraction methods and had a performance approaching that of method 1. However, when wastewater samples were analyzed, methods 1, 2, 3, and 5 had similar performance levels, with detection of Cryptosporidium spp. in 53% (29 of 55), 46% (16 of 35), 38% (21 of 55), and 55% (30 of 55) of samples, respectively. The better performance by the other direct DNA extraction methods in the analysis of wastewater samples was probably the result of less PCR inhibitors in urban wastewater than in storm water. The low rate of positive detection in wastewater samples for method 4, with 8% (3 of 36) positive samples (Table 2), indicated that the poor performance of method 4 was due more to low DNA recovery than to PCR inhibition, a finding supported by DNA spiking experiments, which showed that DNA extracted by this method had less PCR inhibitors than DNA extracted by methods 2 and 3 (Fig. 3).

In summary, results of the study suggest that direct DNA extraction with the FastDNA SPIN kit for soil in combination with the use of a high concentration of BSA represents an effective tool for PCR detection of Cryptosporidium oocysts in water samples. This reduces the cost of current PCR detection of Cryptosporidium oocysts in water samples significantly and enables the use of extracted DNA for the analysis of other pathogens. It may also facilitate the development of detection methods for pathogens that have no IMS isolation or culture enrichment techniques, especially in the era of bioterrorism.


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ACKNOWLEDGMENTS
 
This study was supported in part by funds from the AWWA Research Foundation.

We thank staff at the New York City Department of Environmental Protection, the Milwaukee Metropolitan Sewerage District, and the City of Milwaukee Public Health Laboratories for assistance in sample collection.


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FOOTNOTES
 
* Corresponding author. Mailing address: Division of Parasitic Disease, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Mail Stop F-12, 4770 Buford Hwy., Atlanta, GA 30341-3717. Phone: (770) 488-4840. Fax: (770) 488-4454. E-mail: lax0{at}cdc.gov. Back


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Applied and Environmental Microbiology, March 2005, p. 1135-1141, Vol. 71, No. 3
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.3.1135-1141.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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