[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.


CONCLUSIONS
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.
Shen
Yu-juan
YJ
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, WHO Collaborating Center of Malaria, Schistosomiasis and Filariasis, Shanghai 200025, China.
Cao
Jian-ping
JP
Lu
Wei-yuan
WY
Li
Xiao-hong
XH
Liu
Hai-peng
HP
Xu
Yu-xin
YX
Zhou
Xiao-nong
XN
Tang
Lin-hua
LH
Liu
Shu-xian
SX
D000818:Animals; D002648:Child; D016785:Cryptosporidium parvum / Q000235:genetics*; D017931:DNA Primers; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D003967:Diarrhea / Q000469:parasitology; D005243:Feces / Q000469:parasitology; D006801:Humans; D033661:Oocysts / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers; D016054:DNA, Protozoan