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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*Comments to Author 
Author affiliations: *PHLS Central Public Health Laboratory, London, UK; †PHLS Communicable Disease Surveillance Centre, London, UK

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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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