NDLI: Detection of Cryptosporidium spp. from human faeces by PCR-RFLP, cloning and sequencing

Content Provider	Springer Nature Link
Author	Leone, Annalisa Ripabelli, Giancarlo Sammarco, Michela Lucia Grasso, Guido Maria
Copyright Year	2008
Abstract	In this study, we compared and validated a nested ABC polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) assay to amplify Cryptosporidium parvum oocyst wall protein (COWP) gene fragment with a previous nested PCR-RFLP method. When RFLP of COWP gene did not provide clear results, we further analysed samples by cloning and sequencing for species and genotype identification. ABC-PCR was performed on DNA extracted from human faecal samples collected in England where Cryptosporidium was previously detected by conventional methods. COWP gene amplification was successful for all specimens; RFLP analyses using both RsaI and AluI enzymes recognised 54/55 restriction profiles. Among these, 40 (72.7%) characteristic patterns were generated for C. parvum and six (10.9%) for Cryptosporidium hominis; four (7.3%) showed species mixtures, while the remaining four samples (7.3%) showed atypical genotypes. Cloning and sequencing of mixtures and atypical genotypes identified one C. parvum, one C. hominis and three Cryptosporidium meleagridis. Indeed, molecular tools improved PCR-RFLP results. In conclusion, this study showed that ABC-PCR-RFLP technique was quick, simple and specific. Cloning and sequencing were helpful in characterising mixtures or previously uncharacterised RFLP patterns. All these epidemiological findings are useful information for any preventive intervention to control disease diffusion.
Starting Page	583
Ending Page	587
Page Count	5
File Format	PDF
ISSN	09320113
Journal	Parasitology Research
Volume Number	104
Issue Number	3
e-ISSN	14321955
Language	English
Publisher	Springer-Verlag
Publisher Date	2008-11-01
Publisher Place	Berlin/Heidelberg
Access Restriction	One Nation One Subscription (ONOS)
Subject Keyword	Immunology Microbiology Medical Microbiology
Content Type	Text
Resource Type	Article
Subject	Infectious Diseases Veterinary Parasitology Insect Science

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