Detection and Resolution of Cryptosporidium Species and Species Mixtures by Genus-Specific Nested PCR-Restriction Fragment Length Polymorphism Analysis, Direct Sequencing, and Cloning

Author:

Ruecker Norma J.12,Hoffman Rebecca M.3,Chalmers Rachel M.4,Neumann Norman F.25

Affiliation:

1. Department of Microbiology and Infectious Diseases, University of Calgary, 2500 University Dr. NW, Calgary, Alberta T2N 1N4, Canada

2. Alberta Provincial Laboratory for Public Health, 3030 Hospital Drive NW, Calgary, Alberta T2N 4W4, Canada

3. Wisconsin State Laboratory of Hygiene, 465 Henry Mall, Madison, Wisconsin 53706

4. UK Cryptosporidium Reference Unit, Public Health Wales, Microbiology ABM, Singleton Hospital, Swansea SA2 8QA, Wales

5. School of Public Health, University of Alberta, Rm. 3-50 University Terrace, Edmonton, Alberta T6G 2T4, Canada

Abstract

ABSTRACT Molecular methods incorporating nested PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene of Cryptosporidium species were validated to assess performance based on limit of detection (LoD) and for detecting and resolving mixtures of species and genotypes within a single sample. The 95% LoD was determined for seven species ( Cryptosporidium hominis , C. parvum , C. felis , C. meleagridis , C. ubiquitum , C. muris , and C. andersoni ) and ranged from 7 to 11 plasmid template copies with overlapping 95% confidence limits. The LoD values for genomic DNA from oocysts on microscope slides were 7 and 10 template copies for C. andersoni and C. parvum , respectively. The repetitive nested PCR-RFLP slide protocol had an LoD of 4 oocysts per slide. When templates of two species were mixed in equal ratios in the nested PCR-RFLP reaction mixture, there was no amplification bias toward one species over another. At high ratios of template mixtures (>1:10), there was a reduction or loss of detection of the less abundant species by RFLP analysis, most likely due to heteroduplex formation in the later cycles of the PCR. Replicate nested PCR was successful at resolving many mixtures of Cryptosporidium at template concentrations near or below the LoD. The cloning of nested PCR products resulted in 17% of the cloned sequences being recombinants of the two original templates. Limiting-dilution nested PCR followed by the sequencing of PCR products resulted in no sequence anomalies, suggesting that this method is an effective and accurate way to study the species diversity of Cryptosporidium , particularly for environmental water samples, in which mixtures of parasites are common.

Publisher

American Society for Microbiology

Subject

Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology

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