Development of conventional and nested PCR assays for the detection of Ophiocordyceps sinensis.
Ophiocordyceps sinensis, endemic to the Tibetan Plateau, is one of the most important medicinal fungi with a huge economic value. In the present study, specific primer pairs were designed based on a comprehensive ITS sequence dataset of O. sinensis and its related fungi, and tested for specificity and sensitivity through PCR experiments using 27 individuals of O. sinensis from different geographical origins and 40 other related fungal species in terms of phylogeny or ecology. A primer pair highly specific to O. sinensis was obtained, yielding a 275 bp PCR product from all the individuals of O. sinensis but no product from the other fungi tested. The detection limit of the primers was demonstrated to be 10 ng of pure O. sinensis DNA for conventional PCR and 10 pg for nested PCR in a 25 µl reaction system. Soil samples collected from the habitat of O. sinensis were also tested using this PCR assay. The results showed that the primer pair and PCR-based assays developed in this study can be applied to the rapid detection of O. sinensis in its natural habitat.
State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, China.
Guo-Sheng
GS
Jin
Xiao-Liang
XL
Wang
Yi
Y
Li
Wen-Jing
WJ
Wang
Rui-Heng
RH
Yang
Shu-Yu
SY
Ren
Yi-Jian
YJ
Yao
D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D021903:DNA, Ribosomal Spacer; D006999:Hypocreales / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D009172:Mycology / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D012988:Soil Microbiology
D017931:DNA Primers; D004271:DNA, Fungal; D021903:DNA, Ribosomal Spacer