Detection of Cryptosporidium in stool sample by PCR-RFLP

Extract whole genomic DNA from stool samples.

Positive control DNA for Cryptosporidium parvum may be obtained from ATCC. Alternatively you can extract genomic DNA from wild type Cryptosporidium parvum purchased from Bunchgrass Farms (Deary, Idaho, USA), Waterborne Inc (New Orleans, Louisiana, USA), or Sterling Labs (Tucson, Arizona, USA).

First round of PCR to amplify 18SrRNA

Combine the following in a PCR tube:

5 μl of DNA (from positive control, experimental sample, or water for negative control)

0.1 μM Forward Primer ("out NDIAGF2": CAATTGGAGGGCAAGTCTGGTGCCAGC)

0.1 μM Reverse Primer ("out NDIAGR2": CCTTCCTATGTCTGGACCTGGTGAGT)

1x GoTaq Master Mix (Promega)

Ultrapure water to bring total volume to 25 microliters

Thermocycler program:

95 ºC -5 minutes,

30 cycles:

94 ºC -30 seconds

58 ºC - 1 minute

72 ºC - 30 seconds

Second round of PCR to amplify 18SrRNA

Combine the following in a PCR tube:

1 μl of PCR product from Step 2

0.1 μM Forward Primer ("DIAGF": AAGCTCGTAGTTGGATTTCTG)

0.1 μM Reverse Primer ("DIAGR": TAAGGTGCTGAAGGAGTAAGG)

1x GoTaq Master Mix (Promega)

Ultrapure water to bring total volume to 25 microliters

Thermocycler program:

95 ºC -5 minutes,

45 cycles:

94 ºC -30 seconds

58 ºC - 1 minute

50 ºC - 1 minute

Run 7 μl of PCR product from Step 3 on a 2% agarose gel and visualise by staining with ethidium bromide.

Combine the following and incubate at 37 ºC for 4 hours:

5 μl PCR product from step 4 above

20u Restriction enzyme (Ssp1, Vsp1, Ase1)

1x final concentration Restriction enzyme digestion buffer (corresponds to enzyme used)

1x final concentration BSA

Run digested PCR product from Step 5 on a 2% agarose gel and visualize by staining with ethidium bromide.