A single-tube nested real-time polymerase chain reaction for sensitive contained detection of Cryptosporidium parvum.
OBJECTIVE
A new real-time polymerase chain reaction (PCR) was developed for sensitive contained detection of Cryptosporidium parvum.


RESULTS
The method is a nested PCR targeting a specific region of rDNA of C. parvum, which takes place in one tube, using different annealing temperatures to control the first and the second rounds of PCR, with real-time fluorogenic probe-based detection of the second round of PCR. The DNA-based detection limit of the method was 2 fg, which corresponds to approx. one genome per reaction. The detection level determined using diluted samples of C. parvum oocysts was ten oocysts per millilitre.


CONCLUSIONS
The method facilitates sensitive detection of C. parvum thanks to the nested format, while reducing the risk of laboratory contamination thanks to the single-tube, real-time fluorimetric format.


CONCLUSIONS
The developed method may be useful for sensitive contained detection of C. parvum in environmental and food samples, after appropriate separation of oocysts.
Minarovicová
J
J
Department of Microbiology and Molecular Biology, Food Research Institute, SK-82475 Bratislava 26, Slovakia.
Kaclíková
E
E
Krascsenicsová
K
K
Siekel
P
P
Kuchta
T
T
D016785:Cryptosporidium parvum / Q000235:genetics / Q000302:isolation & purification*; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
D016054:DNA, Protozoan