2.11. Conventional PCR amplification of 18SSU rRNA gene
The PCR product for 18SSU rRNA gene of about 1325bp was amplified by primary PCR from each sample using the oligonucleotide primers Cryp_18ssuP_F (5´-TTCTAGAGCTAATACATGCG-3´) and Cryp_18ssuP_R (5´-CCCTAATCCTTCGAAACAGGA-3´) in the primary PCR[27]. The PCR was carried out in 20μl volume containing 2.0μl (10ng/μl) of genomic DNA, 10μl 2X Master Mix (Takara, Prime Star Max 2X PCR Master Mix), 2μl Forward Primer, 2μl Reverse Primer, and 4μl Nuclease Free Water up to 20μl. The reaction mixture was initially incubated at 94°C for 5 min for initial denaturation and then a total 35 cycles of reaction were performed with denaturation at 94°C for 1 min, primer annealing at 56°C for 1 min, strand extension at 72°C for 1 min. The final extension was done at 72°C for 10 min.
For the secondary/nested PCR product of 834bp was amplified, 2μl of the primary product was used as the template and primers viz., Cryp_18ssuS_F (5´ -GGAAGGGTTGTATTTATTAGATAAAG-3´) and Cryp_18ssuS_R (5´-AAGGAGTAAGGAACAACCTCCA-3´) were used [27]. The PCR reaction and the cycling conditions were the same as for the primary PCR was used in the nested PCR and the annealing temperature was 57°C. The amplification of specific PCR products was checked by gel electrophoresis in 1.8% agarose.