Cryptosporidium parvum mixed genotypes detected by PCR-restriction fragment length polymorphism analysis

Abstract

Combinations of 10 Cryptosporidium parvum oocysts, with various ratios of genotype I to genotype II, were isolated and subjected to PCR-restriction fragment length polymorphism analysis. Amplification of both genotypes in these samples ranged from 31 to 74% and yielded no information about the genotype proportions. In addition, since both genotypes were not always detected, amplification of a single genotype is not conclusive evidence that the sample contains only a single genotype.

FIG. 1.

Two percent ethidium bromide-stained agarose gel illustrating nested-PCR amplification of an 18S rRNA segment within C. parvum and genotype determination with VspI RFLP analysis. Approximately 200 ng of nested-PCR amplicon from singly or mixed genotypes was subjected to VspI digestion for 8 h at 37°C. Lane 1, genotype I amplicon; lane 2, genotype II amplicon; lane 3, digested genotype I amplicon illustrating 503- and 90-bp fragments; lane 4, digested genotype II; lane 5, digestion of both genotypes within a single reaction tube; lane M, 50-bp molecular weight marker (Gibco BRL, Grand Island, N.Y.).