[Detection of Cryptosporidium parvum in fecal samples using polymerase chain reaction combined with non-radioactive labeled DNA probe hybridization].

AIM

To develop a non-radioactive labeled probe hybridization technique for the detection of the PCR products of Cryptosporidium parvum DNA in fecal samples and evaluate its specificity and sensitivity.

METHODS

DNA of C. parvum was prepared directly from C. parvum infected patients' fecal samples by the direct lysis method, and was used as the template for PCR. A pair of oligonucleotide primers was synthesized and used to prime the amplification of a 452 bp target fragment specific for C. parvum. The PCR products were directly spotted or transferred on to NC membrane. Then, the PCR products were hybridized with the biotin-labeled oligonucleotide probe and coloured by a substrate BCIP (5-bromo-4-chloro-3-indolyl-phosphate).

RESULTS

Positive hybridized signals were only showed in the PCR products of DNA extracted from the fecal samples of C. parvum infected patients, but not in the other DNA of P. yoelii, L. donovani, G. lamblia, C. albicans, E. coli and human leucocytes. The lowest detectable amount of both dot or Southern blotting hybridization was 0.1 pg of C. parvum DNA.

CONCLUSION

A combination of PCR and non-radioactive biotin-labeled probe hybridization assay is highly sensitive, specific, and relatively simple and safe for the detection of the PCR products of C. parvum in fecal samples.