title: A rapid method for extracting oocyst DNA from Cryptosporidium-positive human faeces for outbreak investigations.
abstract: We describe a rapid method for extracting and concentrating Cryptosporidium oocysts from human faecal samples with subsequent DNA preparation for mainstream PCR applications. This method consists of extracting faecal lipids using a modified water-ether treatment and releasing DNA from semi-purified oocysts by freeze thawing in lysis buffer. Following immunomagnetisable separation (IMS), recovery rates of 29.5%, 43.2% and 49.8% were obtained from oocyst-negative solid, semi-solid and liquid faeces, respectively, seeded with 100 +/- 2 C. parvum oocysts, which were enumerated by flow cytometry. A retrospective analysis was conducted on 92 positive human faecal samples including 78 oocyst-positive cases from 2 UK cryptosporidiosis outbreaks (outbreak A = 34 samples, outbreak B = 44 samples) and 14 oocyst-positive, sporadic cases. We used primers targeting the Cryptosporidium oocyst wall protein gene (COWP; STN-COWP), the 18S rRNA (direct PCR) and the dihydrofolate reductase gene (dhfr, MAS-PCR) fragments to evaluate extracted DNA by PCR. PCR inhibitors were present in 20 samples when template was co-amplified with the 18S rRNA gene primers and an internal control. Template dilution (1/5) in polyvinylpyrrolidone (10 mg ml(-1), pH 8.0) transformed four PCR-negative samples to PCR-positive and increased amplicon intensity in previously positive samples. Eighteen of 20 PCR-negative samples produced visible amplicons when Taq polymerase concentration in the STN-COWP PCR was increased from 2.5 to 5 U. The STN-COWP PCR assay amplified 90 of 92 samples (97.8%) and the MAS-PCR assay amplified 70 of 92 samples (76.1%) tested. In the absence of inhibitors, DNA equivalent to 3 C. parvum oocysts was amplified.
authors.affiliation: Scottish Parasite Diagnostic Laboratory, Stobhill Hospital, Glasgow, UK.
authors.forename: Rosely A B
authors.initials: RA
authors.lastname: Nichols
authors.forename: John E
authors.initials: JE
authors.lastname: Moore
authors.forename: Huw V
authors.initials: HV
authors.lastname: Smith
mesh_terms: D000818:Animals; D003457:Cryptosporidiosis / Q000453:epidemiology* / Q000469:parasitology; D003458:Cryptosporidium / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000032:analysis / Q000302:isolation & purification*; D004196:Disease Outbreaks; D005243:Feces / Q000469:parasitology*; D006801:Humans; D033661:Oocysts / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D016054:DNA, Protozoan