Prepare master mixture using the formula shown in Table 1, which is for one restriction digestion reaction (see Note 4).

Transfer 30 ^L of master mixture to each tube, add 10 ^L of secondary PCR reaction to the tube, and mix well.

Incubate in a 37°C waterbath for 2 h or overnight.

Run electrophoresis on a 2% argrose gel with the entire 40 ^L from the restriction digestion reaction, using procedures standardized in the laboratory.

Identify Cryptosporidium species and genotypes based on RFLP banding patterns (see Note 5).

Table 1

Formula for Master Mixture Preparation

Restriction Water Enzyme enzyme Buffer ( ^L) (^L)

SspI 4 ^L of New England BioLabs Buffer SspI 22 4

Vspl 4 ^L of Promega Buffer D 24 2

Ddel 4 ^L of New England BioLabs Buffer 3 24 2

4. Notes

1. Protocols for EPA methods 1622 and 1623 can be downloaded from the EPA website (http://www.epa.gov/waterscience/methods/1622.pdf and http://www.epa.gov/ waterscience/methods/1623.pdf). Even though filtration of 10-L water samples is recommended, larger volumes of finished water can and should be filtered and used in the analysis. For raw wastewater, filtration with the standard Envirocheck capsule filters can be problematic. We normally process the pellets from 50 mL of grab samples of raw wastewater directly for IMS without filtration, after they are washed twice by centrifuga-tion at 1500g for 10 min.

2. IMS-oocyst pellets can be stored at -20°C before they are used in DNA extraction. No detachment of Dynabeads from oocysts is needed prior to the DNA extraction.

3. The magnesium concentration used in both primary and secondary PCR is 3 mM, which is higher than normal PCR. Even though concentrations lower than 3 mM generally do not work well for the SSU rRNA-based PCR, it is recommended that the magnesium concentration should be optimized in each laboratory prior to sample analysis.

4. We generally do SspI and VspI restriction digestions to differentiate common Cryptosporidium species and genotypes. DdeI digestion is performed to differentiate C. andersoni from C. muris and is done only when results of SspI digestion have shown the RFLP pattern of C. andersoni/C. muris. In most areas, C. andersoni is found much more frequently in water than C. muris.

5. SspI and VspI RFLP patterns for some common Cryptosporidium species and genotypes are shown in Table 2 and Fig. 1, and the DdeI RFLP patterns for C. andersoni and C. muris are shown in Fig. 2. Occasionally, owing to the genetic heterogeneity between parasites of the same genotype and the multicopy nature of the SSU rRNA gene, RFLP banding patterns deviations from the characteristic patterns can occur. New and unusual Cryptosporidium parasites are also sometimes seen in water samples. A few genotypes cannot be differentiated from each other by RFLP analysis. These require DNA sequencing of the secondary PCR product using the secondary forward and reverse primers for confirmation. The most polymorphic region of the SSU rRNA sequences of known Cryptosporidium parasites are shown in Fig. 3. Likewise, isolates within each genotype can differ somewhat in SSU rRNA sequences owing to the presence of heterogeneous copies of the gene and intragenotypic variartions. Thus minor differences (<5 bp) in the SSU rRNA sequences generally do not warrant new genotype designation.

Table 2

Restriction Fragment Length Polymorphism in the SSU rRNA Gene of Common Cryptosporidium spp. and Genotypes

Table 2

Restriction Fragment Length Polymorphism in the SSU rRNA Gene of Common Cryptosporidium spp. and Genotypes


PCR fragment

Ssp I digestion"

Vsp I digestiona

C. muris/C. andersoni

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