A multiplex allele specific polymerase chain reaction (MAS-PCR) on the dihydrofolate reductase gene for the detection of Cryptosporidium parvum genotypes 1 and 2


Giles, M; Warhurst, DC; Webster, KA; West, DM; Marshall, JA; (2002) A multiplex allele specific polymerase chain reaction (MAS-PCR) on the dihydrofolate reductase gene for the detection of Cryptosporidium parvum genotypes 1 and 2. Parasitology, 125 (Pt 1). pp. 35-44. ISSN 0031-1820 DOI: https://doi.org/10.1017/S0031182002001786

[img]
Preview
Text - Published Version
License:

Download (308kB) | Preview

Abstract

A multiplex allele specific polymerase chain reaction (MAS-PCR) based on the Cryptosporidium parvum dihydrofolate reductase (dhfr) gene sequence differentiates genotype 1 ('Human') from 2 ('Cattle') in a 1-step reaction. The MAS-PCR was validated on a panel of 34 microscopically positive C. parvum faecal samples of human and animal origin in comparison with 2 published PCR-restriction fragment length polymorphism (RFLP) methods targeting dhfr and the oocyst wall protein (cowp) genes. A validation panel of 37 negative faecal samples of human and animal origin was also tested in comparison with the cowp PCR-RFLP. MAS-PCR was found to be as sensitive for species detection as the most sensitive of the other tests, and detected more mixed genotype infections than the two other tests combined. In addition the MAS-PCR showed equivalent detection sensitivity in comparison with a published nested RFLP targeting the SSU rRNA gene, on a panel of prepared mixed genotype samples. The 1-step reaction is simpler and less expensive to perform than the RFLP methods, while the C. parvum specific amplicons and those for genotypes 1 and 2 (575, 357 and 190 bp respectively) can be easily distinguished on agarose gel.

Item Type: Article
Keywords: *Alleles, Animal, Base Sequence, Cattle, Comparative Study, Cryptosporidium parvum/classification/enzymology/*genetics, DNA, Protozoan/chemistry/genetics, Feces/parasitology, Human, Molecular Sequence Data, Parasite Egg Count, Polymerase Chain Reaction/*methods, Polymorphism, Restriction Fragment Length, Sensitivity and Specificity, Sequence Analysis, DNA, Support, Non-U.S. Gov't, Tetrahydrofolate Dehydrogenase/chemistry/*genetics, Alleles, Animal, Base Sequence, Cattle, Comparative Study, Cryptosporidium parvum, classification, enzymology, genetics, DNA, Protozoan, chemistry, genetics, Feces, parasitology, Human, Molecular Sequence Data, Parasite Egg Count, Polymerase Chain Reaction, methods, Polymorphism, Restriction Fragment Length, Sensitivity and Specificity, Sequence Analysis, DNA, Support, Non-U.S. Gov't, Tetrahydrofolate Dehydrogenase, chemistry, genetics
Faculty and Department: Faculty of Infectious and Tropical Diseases > Dept of Pathogen Molecular Biology
Research Centre: Malaria Centre
PubMed ID: 12166518
Web of Science ID: 177397200004
URI: http://researchonline.lshtm.ac.uk/id/eprint/15747

Statistics


Download activity - last 12 months
Downloads since deposit
219Downloads
351Hits
Accesses by country - last 12 months
Accesses by referrer - last 12 months
Impact and interest
Additional statistics for this record are available via IRStats2

Actions (login required)

Edit Item Edit Item