The sensitivity of PCR detection of Cryptosporidium oocysts in fecal samples using two DNA extraction methods.
BACKGROUND
The implementation of cost-effective intervention strategies for zoonotic protozoa relies on the development of sensitive and accurate diagnostic methods. We carried out a study to evaluate the accuracy of a PCR method for the detection of Cryptosporidium spp. oocysts in fecal samples from cattle.


METHODS
Fecal samples were spiked with different numbers of oocysts and the limit of detection of the method was determined. Two methods of DNA extraction were assessed: glass beads and freeze-thawing using liquid nitrogen. A nested PCR approach was developed targeting the Cryptosporidium SSU rRNA and TRAP-C2 genes. Agreement between the diagnosis of Cryptosporidium spp. at the SSU rRNA and TRAP-C2 loci was quantified using the kappa-coefficient.


RESULTS
Compared with the freeze-thawing method, the glass beads method was found to be a better way of extracting DNA from Cryptosporidium oocysts (sensitivities were 83 and 100%, respectively). The limits of detection for glass beads and freeze-thaw were low, 1 and 10 oocyst/g fecal samples, respectively. Forty-six percent of the field samples previously classified as negative for Cryptosporidium parvum by the flotation-concentration and enzyme-linked immunosorbent assay methods showed DNA with the PCR protocol.


CONCLUSIONS
Primers for SSU rRNA are more successful in producing an amplification than primers for the TRAP-C2 gene which makes the former PCR protocol the approach of choice for detecting Cryptosporidium parvum oocysts in field samples.
Lindergard
Gabriella
G
Section of Epidemiology, Department of Population Medicine and Diagnostic Science, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA.
Nydam
Daryl V
DV
Wade
Susan E
SE
Schaaf
Stephanie L
SL
Mohammed
Hussni O
HO
D000818:Animals; D001483:Base Sequence; D003457:Cryptosporidiosis / Q000175:diagnosis; D003458:Cryptosporidium / Q000235:genetics* / Q000302:isolation & purification; D017931:DNA Primers; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000469:parasitology*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D017931:DNA Primers; D016054:DNA, Protozoan