Cloning and sequencing of 18S rRNA genes and its regions

The partial 18S rRNA gene and the ITS region were amplified from 14 samples that tested positive via RLB using the primer pairs P1/P2 and ITSF/ITS2, respectively (Table ​(Table2).2). PCR amplification of the 18S rRNA gene and ITS sequences was performed in a total volume of 50 μl, with 10 μl of 5 × TransStart FastPfu Buffer, 5 μl of a 2.5 mM dNTP Mixture, 0.1 μM of each primer, 1 μl of TransStart FastPfu DNA Polymerase (Takara Biotechnology, Dalian, China), 2.5 μl of genomic DNA, and double distilled water. The conditions for PCR amplification of the 18S rRNA gene were as follows: an initial denaturation step at 95°C for 2 min; 35 cycles of denaturation for 20 s at 95°C, annealing for 20 s at 55°C, and extension for 45 s at 72°C; and a final extension step of 5 min at 72°C. The PCR amplification conditions for the ITS region were almost the same as for the 18S rRNA gene, except that the annealing temperature used was 54°C in this step. The PCR products were purified using the Easypure Quick Gel Extraction Kit (TransGen Biotech, Beijing, China). The purified amplicons were cloned into the pMD19-T vector (Takara Biotechnology, China), which was then transformed into E. coli JM109 cells (TaKaRa Biotechnology, China) according to the manufacturer's instructions. Three positive colonies of each sample were selected for sequencing (ABI PRISM 377 DNA sequencer).

Primers used to amplify the 18S rRNA gene and ITS region.