Direct colony PCR of several medically important fungi using Ampdirect plus.
Direct colony polymerase chain reaction (DCPCR) is a useful molecular biological technique for application in the field of mycology. In this study, all of the 63 fungal strains examined, including those of the genera Candida and Aspergillus, were amenable to DNA amplification using an Ampdirect(R) Plus kit, which allows direct PCR amplification with no requirement for DNA extraction, following 1 h of rapid fungal lysis. Moreover, we compared DCPCR of 35 strains, representing 20 species, using Ampdirect PCR and standard PCR with no lysis buffer. Thirty-four of these strains (97.14%) yielded positive results on Ampdirect PCR, while only 11 (including Aspergillus fumigatus TIMM1776) of the 35 strains (31.43%) showed PCR products when standard PCR reagents were used. Ampdirect DCPCR was also applicable to DNA amplification for spore and hyphal cells. This approach reduces DNA template preparation time before PCR from fungal colonies, and also reduces the cost of PCR.
Alshahni
Mohamed M
MM
Teikyo University Institute of Medical Mycology, Tokyo 192-0395, Japan.
Makimura
Koichi
K
Yamada
Tsuyoshi
T
Satoh
Kazuo
K
Ishihara
Yumiko
Y
Takatori
Kosuke
K
Sawada
Takuo
T
D005658:Fungi / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D025301:Hyphae / Q000235:genetics; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009181:Mycoses / Q000175:diagnosis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D011933:Reagent Kits, Diagnostic; D012680:Sensitivity and Specificity; D013172:Spores, Fungal / Q000235:genetics
D011933:Reagent Kits, Diagnostic