Volume 7, Number 1—February 2001
Research
Nested Polymerase Chain Reaction for Amplification of the Cryptosporidium Oocyst Wall Protein Gene
Susana Pedraza-Díaz*, Corinne Amar*, Gordon L. Nichols†, and Jim McLauchlin*
Table 1
Sensitivities of different polymerase chain reaction procedures for Cryptosporidium gene sequences
| PCR procedurea | |||||||
|---|---|---|---|---|---|---|---|
| Dilutions of DNA sample | N-COWP | E-COWP | COWP | TRAP-C1 | TRAP-C2 | 18S rDNA 1 | 18s rDNA 2 |
| Human, genotype 1 | |||||||
| Undiluted | +++b | ++ | ++ | ++ | ++ | ++ | ++ |
| 10-1 | +++ | ++ | ++ | ++ | ++ | ++ | ++ |
| 10-2 | +++ | + | ++ | -- | + | + | ++ |
| 10-3 | +++ | -- | -- | -- | -- | + | ++ |
| 10-4 | -- | -- | -- | -- | -- | -- | -- |
| Human, genotype 2 | |||||||
| Undiluted | +++ | ++ | ++ | ++ | ++ | ++ | ++ |
| 10-1 | +++ | + | ++ | + | -- | ++ | ++ |
| 10-2 | +++ | -- | + | + | -- | + | ++ |
| 10-3 | +++ | -- | -- | -- | -- | ± | ± |
| 10-4 | -- | -- | -- | -- | -- | -- | -- |
| Human, genotype 3 (Cryptosporidium meleagridis) | |||||||
| Undiluted | +++ | + | + | + | -- | + | ++ |
| 10-1 | +++ | ± | -- | -- | -- | + | ± |
| 10-2 | +++ | -- | -- | -- | -- | -- | -- |
| 10-3 | -- | -- | -- | -- | -- | -- | -- |
| 10-4 | -- | -- | -- | -- | -- | -- | -- |
| Ovine, genotype 2 (Moredun strain) | |||||||
| Undiluted | +++ | ++ | ++ | ++ | ++ | ++ | ++ |
| 10-1 | +++ | + | ++ | + | + | ++ | ++ |
| 10-2 | +++ | ± | ± | -- | -- | + | ++ |
| 10-3 | +++ | -- | -- | -- | -- | -- | ± |
| 10-4 | -- | -- | -- | -- | -- | -- | -- |
aPolymerase chain reaction (PCR) procedures used for gene fragments: N-COWP = nested Cryptosporidium oocyst wall protein gene (553 bp, this study); E-COWP = extended COWP (769 bp, this study); COWP = unnested procedure (553 bp, 5); TRAP-C1 = thrombospondin-related adhesive protein Cryptosporidium 1 (506 bp, 6); TRAP-C2 = thrombospondin-related adhesive protein Cryptosporidium 2 (369 bp, 4); 18S rDNA 1 (422 bp, 8,17); 18S rDNA 2 (435 bp, 18).
bSemiquantitative results on the basis of strength of ethidium bromide staining in electrophoresis gels: strong (+++), moderate (++), weak (+), very weak (±), and amplification not detected (-).
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