title: Assessment of three commercial DNA extraction kits and a laboratory-developed method for detecting Cryptosporidium and Cyclospora in raspberry wash, basil wash and pesto.
abstract: Polymerase chain reaction (PCR) methods are often used to identify the parasitic protozoa Cryptosporidium parvum and Cyclospora cayetanensis in foods although little has been published regarding the efficacy of available DNA extraction methods. This study reviewed three commonly used commercial DNA extraction kits: FastDNA SPIN Kit for soil, QBiogene (FastDNA), UltraClean™ Soil DNA Isolation Kit, MO BIO Laboratories (MoBio), and QIAamp DNA Mini Stool Kit, Qiagen (QIAamp), as well as a 'homebrew' Universal Nucleic Acid Extraction (UNEX) method. Washes from raspberry and basil as well as commercial pesto samples were seeded with 5000, 500, or 50 C. parvum and C. cayetanensis oocysts. The protocols were assessed for: quantity and quality of the extracted DNA, time to completion, presence of PCR inhibitors and the percentage of samples correctly identified as positive for the two parasites. Real-time and conventional nested PCR assays were used to detect the seeded pathogens. Of the commercial kits, PCR results of samples extracted using FastDNA were statistically similar to QIAamp and both were superior to MoBio. Differences in PCR results among FastDNA, QIAamp and UNEX for detection of Cyclospora were not statistically significant although the UNEX method proved best with Cryptosporidium. Real-time PCR assays targeted the 18S rRNA and the hsp70 genes of C. cayetanensis; overall results were similar to those found using conventional nested PCR targeting the 18S rRNA gene.
authors.affiliation: Division of Virulence Assessment, Office of Applied Research and Safety Assessment, Center for Food Safety and Applied Nutrition, Food & Drug Administration, United States. Joan.Shields@fda.hhs.gov
authors.forename: Joan M
authors.initials: JM
authors.lastname: Shields
authors.forename: Jane
authors.initials: J
authors.lastname: Joo
authors.forename: Richard
authors.initials: R
authors.lastname: Kim
authors.forename: Helen R
authors.initials: HR
authors.lastname: Murphy
mesh_terms: D016785:Cryptosporidium parvum / Q000235:genetics / Q000302:isolation & purification*; D021744:Cyclospora / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000302:isolation & purification*; D005516:Food Microbiology; D006801:Humans; D010274:Parasitology / Q000379:methods*; D016133:Polymerase Chain Reaction; D060888:Real-Time Polymerase Chain Reaction; D013048:Specimen Handling / Q000379:methods*; D013997:Time Factors
chemical_list: D016054:DNA, Protozoan