[Real-time PCR in analyzing DNA extraction from Cryptosporidium oocysts].
OBJECTIVE
To compare the quality and quantity of DNA extracted from Cryptosporidium oocysts by different methods.


METHODS
Cryptosporidium oocysts were treated with different kinds of lysis buffers from USA Promega (Promega) and Shanghai Generay (Generay) commercial DNA extraction kits, 2% Triton X-100 and 5% guanidine thiocyanate. The oocysts were then broken down by freeze-thawing, proteinase K and sonication. Genomic DNA was purified using the commercial kits or Chelex-100. Real-time PCR technique was used to determine the copies of Cryptosporidium oocyst wall protein (COWP) gene. The Promega commercial DNA extraction kit was used as control.


RESULTS
The Promega kit resulted in a higher copy number of COWP gene [(6.45-9.86) x 10(6)] than that of Generay commercial DNA kit [(2.38-3.69) x 10(6)], 5% guanidine thiocyanate [(1.27-21.29) x 10(5)] or 2% Triton X-100 [(2.06-866.70) x 10(3)] , respectively. The method of freeze-thawing plus proteinase K plus sonication provided the highest copy number of COWP gene.


CONCLUSIONS
The method of freeze-thawing + proteinase K + sonication is most effective. The effect of DNA extraction by Generay kit and 5% guanidine thiocyanate is similar to that of Promega kit.
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, Key Laboratory of Parasite and Vector Biology, MOH, WHO Collaborating Centre for Malaria, Schistosomiasis and Filariasis, Shanghai 200025, China.
Sheng-Xia
SX
Chen
Liang
L
Wu
Yu-Juan
YJ
Shen
Qiu-Xia
QX
Zhang
Ting-Ting
TT
Li
Xu-Gan
XG
Jiang
Yu-Xin
YX
Xu
Jian-Ping
JP
Cao
D000818:Animals; D003458:Cryptosporidium / Q000737:chemistry* / Q000235:genetics; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification*; D033661:Oocysts / Q000737:chemistry*; D016133:Polymerase Chain Reaction / Q000379:methods*
D016054:DNA, Protozoan