Extraction of DNA

3.1.1. Extraction of Fecal and Environmental DNA

3.1.1.1. ProteinaseK/SDS Method

DNA extracted from feces or water pellets containing Cryptosporidium oocysts is often suitable for PCR detection and genotyping. Since PCR inhibitors may copurify with environmental DNA (16,17), some investigators prefer to purify the oocysts first and then extract DNA from the oocysts. Using purified or partially purified oocysts may also be an advantage when working with a sample containing low concentrations of oocysts. Methods for purifying oocysts are discussed below in Subheading 3.1.2.

1. For the proteinase K/sodium dodecyl sulfate (SDS) extraction method, a 100-500 ^L volume of feces or water pellet is homogenized in STE buffer until a slurry is obtained. The volume of buffer added to the sample depends on the consistency; typically an equal volume of sample and buffer are used.

2. Following homogenization, proteinase K is added at a concentration of 200 ^g/mL and SDS to 0.1%.

3. The samples are then incubated overnight at 45°C and the DNA recovered by absorption to a silica matrix (GeneClean).

3.1.1.2. Beadbeater Method

1. One small volume of feces or water pellet is diluted with 2 vol of 10 mMTris-HCl, pH 8.0, 25 mM EDTA, and 2% SDS in a 2-mL screw-cap microcentrifuge tube.

2. To 1 mL of this slurry, 200 ^L of glass beads (0.5 mm diameter) and an equal volume of phenol/chloroform are added and agitated for 2 min at 5000 rpm in a beadbeater.

3. Samples are spun in a microcentrifuge to separate the phenol and precipitate the beads.

4. The aqueous phase containing the DNA is transferred to a new microcentrifuge leaving behind the pellet containing the beads.

5. NaCl is added to the sample from a 5 M solution to obtain a final concentration of 0.7 M and the solution is extracted with an equal volume of chloroform.

6. Final purification of the DNA is performed with GeneClean.

3.1.1.3. High Pure Extraction Method

1. A portion of 0.5 g of feces is homogenized in 2-5 mL of physiological (0.9%) saline and filtered through gauze.

2. The filtrate is centrifuged at 4000g for 10 min, and the precipitate containing the oocysts is resuspended by vortexing in 200 ^L sterile distilled water.

3. This suspension is then subjected to three cycles of 37/-80°C freeze-thawing.

4. The lysate is then mixed with 200 ^L of binding buffer and 40 ^L proteinase K, both provided with the High Pure PCR template preparation kit, and incubated for 15 min at 72°C.

5. Isopropanol (100 ^L) is then added and the mixture applied to the High Pure filter tube.

6. The tubes are spun at 6000g for 1 min in a microcentrifuge.

7. After centrifugation, 500 ^L of inhibitor removal buffer (from the kit) is added and the centrifugation repeated.

8. The same volume of washing buffer (from the kit) is added to the filter tube and the centrifugation step repeated. This wash cycle is performed twice.

9. One volume of 200 ^L elution buffer (from the kit) heated to 70°C is added to the filter tube, and the tubes are centrifuged at 6000g for 1 min.

10. A 1-10 ^L portion of the eluate containing the DNA is then added directly to the PCR reaction.

3.1.2. Oocyst Purification

3.1.2.1. Oocyst Purification on Density Gradients

Several density media and gradients have been described for floating or sedimenting oocysts from fecal samples. These methods are used individually or in combination with other purification steps until the required level of oocyst purification is achieved.

To our knowledge, no systematic comparison of various oocyst purification methods has been published, and each investigator needs to experiment with different methods before deciding on the one that is most appropriate and meets the requirements of the experiment. Even if a comparative study of different purification protocols were performed, it is likely that the results achieved in one laboratory would not directly apply to another, since the sample matrix has a major impact on oocyst recovery. What may give excellent results with sludge or a diarrheic sample may not be satisfactory with solid fecal samples.

1. In our laboratory, after much experimenting with different gradients, we favor for most fecal samples an initial floatation on a high-density solution followed by sedimentation on an isopycnic gradient or by IMS.

2. We have experimented with two density media for floating oocysts from fecal slurries or sludge samples: 4 M NaCl and 36% w/v Nycodenz. Recoveries with these media are in the 30-50% range. Sodium chloride works well with most samples and is cheap. Nycodenz is more expensive but has the advantage of low osmolarity even when dissolved at high concentration (18), and it appears to float the oocysts better than salt, potentially improving oocyst recovery (Fig. 1).

3. Whichever floatation solution is used, a volume of fecal slurry is mixed with 2 vol of saturated (6.1 M) NaCl and centrifuged at approx 2000g for 15 min.

4. Floatation on Nycodenz is achieved by mixing 1 vol of fecal slurry with 10 vol of 40% Nycodenz, followed by centrifugation under the same conditions.

5. In both methods, but particularly with Nycodenz, the oocysts concentrate in the uppermost layer and can be recovered by aspiration.

6. Before proceeding to the next purification step, the density of the oocyst suspension recovered from the floats is reduced by diluting the oocyst suspension with 2-3 vol of water and precipitation in a microcentrifuge (~3000g).

Oocysts partially purified by floatation can be further purified by sedimentation on a continuous or step gradient. Several rate-zonal or isopycnic gradients have been used, including the popular Sheather's sugar flotation method (19). Cesium chloride, Percoll, and Nycodenz have also been used (20,21) (see Note 3). In our laboratory, we favor sedimentation on a 15/30% w/v Nycodenz step gradient.

1. Depending on the diameter of the centrifugation tube, a 1-5 mL of a 30% Nycodenz solution is used as a cushion.

2. The volume of high and low density fraction will depend on the diameter of the tube. The minimum volume to obtain a good separation of the interphase, where the oocysts will collect, from the pellet at the bottom, and the supernatant at the top, should be used. For instance, for a 13-mm-diameter tube, as used in a SW41 swing bucket rotor (Beckman, Fullerton, CA), 2-3 mL of 30% solution will be sufficient to form a cushion.

3. Onto this cushion, 5-10 mL of 15% solution is slowly layered, taking care not to mix the two phases.

4. Finally, the semipurified oocyst suspension is layered onto the 15% solution.

5. The tubes are then centrifuged in a swing bucket rotor at 10,000-15,000g for 30 min to 1 hour. To avoid the collapse of thin-walled centrifuge tubes during centrifugation, water or mineral oil can be added to increase the sample volume, although in our experience this is not necessary when working at these relative low g-forces.

Fig. 1. Comparison of oocyst floatation on NaCl and Nycodenz. Oocysts (2.75 x 105) were mixed with 3 mL 40% Nycodenz or 3 mL saturated NaCl and spun at 2000g for 15 min approximately. Fractions of 200 ^L were collected starting with the top layer, and the oocysts were counted using a hemocytometer. A mock floatation in water was performed in parallel.

Fig. 1. Comparison of oocyst floatation on NaCl and Nycodenz. Oocysts (2.75 x 105) were mixed with 3 mL 40% Nycodenz or 3 mL saturated NaCl and spun at 2000g for 15 min approximately. Fractions of 200 ^L were collected starting with the top layer, and the oocysts were counted using a hemocytometer. A mock floatation in water was performed in parallel.

6. For most molecular applications, and certainly for PCR analysis, oocysts obtained using a flotation step followed by gradient centrifugation are sufficiently clean, and DNA can be readily extracted using the proteinase K/SDS or the High Pure method described above.

7. Before proceeding with extraction of DNA from oocysts, oocysts are collected from the interphase between the high- and low-density phase using aspiration, diluted with a minimum of 1 vol of water, and precipitated by centrifugation. We have not observed any inhibitory effect to PCR from residual Nycodenz.

3.1.2.2. Oocyst Purification by Immunomagnetic Capture

Immunomagnetic capture of oocysts can be used as a quick alternative to gradient methods, or in combination with floatation or sedimentation. The choice of which method or combination of methods to use will depend on several variables, but primary considerations are sample volume, sample consistency, and the desired purity of the final oocyst preparation. Large sample volumes with low oocyst concentrations will require an initial concentration, and possibly a floatation before the oocysts can be IMS purified. The limitations of IMS are cost and sample volume (maximum 8 mL). Cost of IMS is an important consideration if large numbers of samples need to be processed. We typically use IMS when purifying oocysts from "difficult samples," i.e., samples with large amounts of solids and low concentrations of oocysts, and when high yields and/or high purity is needed. We have used immunomagnetic beads from three different manufacturers (Dynal, ImmuCell, and Aureon Biosystems) and have observed no obvious differences in recovery, although we have not systematically compared the kits. The recovery of non-parvum oocysts with these different products remains to be investigated, and researchers wishing to recover Cryptosporidium oocysts from other species should be aware that these products were developed for the purification of C. parvum. When working with human or bovine fecal samples, fat is often a problem, as it interferes with IMS.

1. To remove fat, fecal samples are first extracted with ether by adding an equal volume of 1% Tween-80, followed by vigorous shaking with a 40% vol of ether.

2. The ether and water phases are separated by centrifugation for 10 min at 4000g and the ether and fatty interphase removed by aspiration (see Note 4).

3. The pellet containing the oocysts can then be washed to remove residual ether and processed directly by IMS.

4. The instructions provided with each IMS kit are similar and sufficiently detailed to perform the procedure.

5. Leighton glass tubes can be purchased from the kit manufacturers, but glass tubes with similar dimension and a flat surface to provide a good contact with the magnets work equally well (see Note 5).

3.2. PCR Analysis of Cryptosporidium Microsatellites

Restriction fragment length polymorphism (RFLP) has been used for identifying Cryptosporidium species (22) and for subspecies identification (9,23). In contrast, microsatellite and DNA sequence polymorphism has revealed the presence of genetic heterogeneity within C. parvum types. These markers are useful for epidemiological studies and source tracking and have recently been applied to the study of genetic recombination (24).

3.2.1. Conventional PCR Analysis of C. parvum Microsatellites

1. PCR amplifications are performed in 25-^L volumes containing IX PCR buffer, 2 mM MgCl2, 0.2 mM dNTPs, and 10 pMol of each forward and reverse primer (13). If the amplification products are to be radioactively labeled, 2 pMol of 5' 32[P]-labeled reverse primer is used instead of unlabelled reverse primer.

2. Primers are end-labeled with polynucleotide kinase and [y-32P]ATP.

3. The PCR is run in a conventional thermal cycler using the following temperature parameters: 36 cycles of 15 s at 94°C, 25 s at the respective primer annealing temperature, and 40 s at 72°C. We use a MiniCycler or a PTC-100 thermal cycler from MJ Research.

4. For gel analysis, radiolabeled PCR products (unpurified) are mixed with an equal volume of loading buffer (10 mM NaOH, 95% formamide, 0.05% bromophenol blue, 0.05% xylene cyanol), heat-denatured for 1-2 min at 95-100°C, and fractionated on a 8% polyacryla-mide, 8 M urea sequencing gel at 2000 V/36 W. The electrophoresis buffer is 0.5X TBE in the top chamber and 1X TBE in the bottom chamber.

5. Following electrophoresis, the gel is dried at 80°C under vacuum and the PCR products visualized by autoradiography.

Because of the inconvenience of working with radioactivity, an alternative method to visualize PCR amplicons was developed. In this method, amplicons are electro-phoresed on 10% native polyacrylamide supplemented with 10% Spreadex Polymer NAB and visualized by immersing the gel in an ethidium bromide solution (Fig. 2). Although this method may not resolve bands differing in size by a single base pair, it resolves size differences as small as 2 bp.

3.2.2. Real-Time PCR Analysis

Real-time PCR combined with melting curve analysis (MCA) is currently being evaluated as an alternative method for genotyping C. parvum using microsatellite polymorphisms. Because MCA eliminates the need for electrophoretic analysis, this approach is much quicker than traditional gel analysis of PCR products. In our laboratory we have tested several PCR protocols targeted at microsatellites using a LightCycler real-time PCR system. Although this method is still being evaluated, it appears that repeats containing alternating G/C and A/T are amplified more efficiently than those consisting only AT repeats (see Note 6).

Microsatellite Cp492 (24,25) consists of a variable number of CAC repeats. To date, we have identified 6 Cp492 alleles in C. parvum and a different allele in C. meleagridis. Various primers flanking the repeat were synthesized such that nested amplifications can be performed. Different primer combinations are also useful in case samples fail to amplify owing to the presence of unknown nucleotide polymorphisms in the region flanking the repeat. Because Cp492 is located within an open reading frame, positioning the PCR primers such that their 3' end does not fall on the third codon position reduces the possibility of primer failures.

1. PCR amplification in a LightCycler is performed in 20-^L vol containing 1X LC Fast Start DNA Master Mix for SYBR Green I, 4 mM MgCl2, 10 pMol of each forward and reverse primer, and 1-10 ^L DNA template.

2. The PCR premix and DNA template are added to the LightCycler capillaries, and the capillaries capped and centrifuged using the appropriate centrifuge adapters (Roche Diagnostics).

Fig. 2. Fractionation of Cp492 microsatellite locus on a 10% acrylamide gel supplemented with 10% Spreadex. C. parvum isolates NEMC1 (26), KJHIV, UG565, and YWHIV are type 1; GCH1 (27), MD (28), UG405 (13), and ICP (29) are type 2.

3. A typical amplification is performed using the following temperature conditions: one cycle of 10 min at 95°C, 40 cycles of 0-1 s at 95°C, 4-5 s at the respective annealing temperature, and 7-22 s (depending on the length of template) at 72°C.

4. Temperature transitions of 20°C/s seem to be adequate for most amplifications, but certain sequences seem to amplify better under different conditions. Optimal amplification conditions, including the concentration of MgCl2, need to be determined empirically (see Note 7).

5. PCR products are typed using MCA analysis (Fig. 3) by raising the temperature from 45 to 95°C at a rate of 0.05°C/s.

6. As with traditional PCR methods, a positive control amplification using a standard template and a negative control are included with each reaction.

7. When using a new PCR protocol, it is desirable to confirm by gel analysis that an amplicon of the expected size has been amplified. However, electrophoresis is not needed once the range of melting temperatures for a specific marker has been determined.

4. Notes

1. Several designations for C. parvum types are used in the literature. Here we will adhere to the type 1/type 2 definition adopted in 1999 at a meeting entitled "Cryptosporidium spp. Systematics and Waterborne Challenges in Public Health," sponsored by the EPA Office of Water, Crystal City, Virginia April 29-30.

2. The term isolate is used to describe a population of parasites originating from a host. Isolate may originate from naturally infected animals or humans or may be artificially maintained in the laboratory by serial propagation.

3. Isopycnic gradients of CsCl have also been used by other investigators, but we have experienced difficulty extracting DNA from oocysts purified on CsCl.

4. For extracting the ether supernatant and the fatty interphase, a Pasteur pipet connected to a source of vacuum works well. If clogging of the pipet occurs while one is aspirating the interphase, the opening of the pipet can be enlarged by breaking off the tip of a clean

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