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Whole genome amplification (WGA) for archiving and genotyping of clinical isolates of Cryptosporidium species

Published online by Cambridge University Press:  21 September 2009

MAHA BOUZID ,
DARREN HEAVENS ,
KRISTIN ELWIN ,
RACHEL M. CHALMERS ,
STEPHEN J. HADFIELD ,
PAUL R. HUNTER  and
KEVIN M. TYLER
MAHA BOUZID
Affiliation:
Biomedical Research Centre, School of Medicine, Health Policy and Practice, University of East Anglia, Norwich NR4 7TJ, UK
DARREN HEAVENS
Affiliation:
Genome Lab, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, UK
KRISTIN ELWIN
Affiliation:
UK Cryptosporidium Reference Unit, NPHS Microbiology Swansea, Singleton Hospital, Swansea SA2 8QA, UK
RACHEL M. CHALMERS
Affiliation:
UK Cryptosporidium Reference Unit, NPHS Microbiology Swansea, Singleton Hospital, Swansea SA2 8QA, UK
STEPHEN J. HADFIELD
Affiliation:
UK Cryptosporidium Reference Unit, NPHS Microbiology Swansea, Singleton Hospital, Swansea SA2 8QA, UK
PAUL R. HUNTER
Affiliation:
Biomedical Research Centre, School of Medicine, Health Policy and Practice, University of East Anglia, Norwich NR4 7TJ, UK
KEVIN M. TYLER*
Affiliation:
Biomedical Research Centre, School of Medicine, Health Policy and Practice, University of East Anglia, Norwich NR4 7TJ, UK
*
*Corresponding author: Biomedical Research Centre, School of Medicine, Health Policy and Practice, University of East Anglia, Norwich NR4 7TJ, UK. Tel: +44 (0) 1603 591225. Fax: +44 (0) 1603 591750. E-mail: k.tyler@uea.ac.uk
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Summary

Clinical and environmental isolates of pathogens are often unique and may be unculturable, yielding a very limited amount of DNA for genetic studies. Cryptosporidium in particular are difficult to propagate. Whole genome amplification (WGA) is a valuable technique for amplifying genomic material. In this study, we tested 5 WGA commercial kits using Cryptosporidium clinical isolates. DNA of 5 C. hominis and 5 C. parvum clinical isolates and C. parvum IOWA reference strain were used. The majority of the samples were amplified by all of the kits tested. The integrity and fidelity of the amplified genomic DNA were assessed by sequence analysis of several PCR products of varying length. We found evidence that one kit in particular may be more error prone while another seemed the more suitable kit for Cryptosporidium clinical samples, generating high molecular weight DNA from all the samples with high fidelity. Thus WGA was found to be a useful technique for producing amplified DNA suitable for downstream genotyping techniques and archiving of Cryptosporidium clinical isolates.

Keywords

whole genome amplificationmulti-displacement amplificationCryptosporidium DNAgenotyping
Type
Research Article
Information
Parasitology , Volume 137 , Issue 1 , January 2010 , pp. 27 - 36
Copyright
Copyright © Cambridge University Press 2009

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