[Preparation of DNA from Cryptosporidium parvum oocysts for PCR detection].

OBJECTIVE

To establish three methods of DNA extraction from Cryptosporidium parvum oocysts and test by PCR.

METHODS

After three freeze-thaw cycles, three kinds of templates were extracted from the oocysts by Chelex-100, phenol/chloroform or genomic DNA purification system kit, and used for PCR detection. According to the sequence of a C. parrum gene (L.16996), a pair of primers was designed and synthesized, and used for PCR. The sensitivity of the template by Chelex 100 method was also tested by PCR.

RESULTS

One 446 bp PCR product was observed by agarose gel electrophoresis for all three kinds of templates. The PCR sensitivity by Chelex-100 extracted DNA reached for detection of a specimen containing only 1/2 oocyst.

CONCLUSION

The three kinds of extraction can all be served as templates for PCR detection of C. parvum oocysts, while Chelex 100 method is simpler, quicker and more reliable for DNA extraction of the parasite.