2.12. TaqMan® probe based Real Time PCR for 18SSU rRNA
For 18SSU rRNA TaqMan® probe Real-time PCR, we have used the oligonucleotide primers and probes designed in-house[14] in a previous study. The real-time PCR master mix was prepared in duplicates for each sample in a final reaction volume of 25µl in 8 strip PCR tubes with optically compatible caps assayed in the real-time thermal cycler (CFX96 Real-time PCR system®, Bio-Rad) with the reagent preparation and thermal conditions as mentioned in the table (Table 2). The 109bp amplicon post-conventional PCR was confirmed by gel electrophoresis.
(Table 2 here)
In the present study, CTAB method was used for the DNA extraction fromCryptosporidium oocysts. The major problems ofCryptosporidium DNA extraction were associated with the presence of thick wall of Cryptosporidium oocysts and other organic and bile salts available in faecal material that inhibits PCR enzymes.In the current study, extractions weredone with CTAB buffer that effectively removes the faecal organic compounds and act as a detergent which solubilizes the cell wall, lipid membrane and denature the protein ofCryptosporidium oocysts along with thermal manipulation steps.
In the present study, DNA extraction from Cryptosporidium oocysts was standardized by the CTAB method. The main bottlenecks ofCryptosporidium DNA extraction are due to the thickCryptosporidium oocyst wall and other organic and bile salts available in faecal material that inhibits PCR enzymes. In the current study, CTAB based extractions has effectively managed to remove the faecal organic compounds and with its detergent action, it solubilized the cell wall, lipid membrane and denature the protein ofCryptosporidium oocystsalong with thermal manipulation steps.