Identification of parasites
2. Sequencing of Parasite's Cytb
Aim of this step: Sequence part of parasites cytb gene to recognise parasite species/genera in the samples.
To identify the parasites in your sample, first carry out a normal PCR using parasite 7-1 primers. Our PCR reactions contained 1 µM of each primer, 200 nM dNTP's, 0.1 mg/ml BSA, 2.5 mM MgCl2, 0.5 U Biotools DNA polymerase, 1x standard reaction buffer and 1 µl DNA. PCR reaction were done with PCR program of 3 minutes denaturation at 95°C followed by 20 s at 95°C, 20 s at 56°C and finally 20 s at 72°C for 40 cycles. For sequencing, follow the instructions of your sequencing kit and sequencer.
3. Identify parasites species from sequences
Aim of this step: To recognize parasite genera/species cytb sequences from your sample for later use.
To identify parasites species from the sequences it is necessary to make a nucleotide blast query . Once, the parasite species/genera from samples have been identified, you can then check the restrictions sites restriction sites .
