A single-tube nested real-time polymerase chain reaction for sensitive contained detection of Cryptosporidium parvum

Aim: A new real-time polymerase chain reaction (PCR) was developed for sensitive contained detection of Cryptosporidium parvum. Methods and 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. Significance and Impact of the Study: The developed method may be useful for sensitive contained detection of C. parvum in environmental and food samples, after appropriate separation of oocysts.