4. DISCUSSION
Cryptosporidium oocyst wall is highly resistant to physical disruption due to its constituent integral membrane protein and short hydrophobic regions at signal peptide. All COWP subunits are present inside the inner oocyst wall and in wall-forming bodies of mature macrogametes. COWP1 has a striking cysteine periodicity with two cysteine-rich domains, with well conserved motifs. Further, an extensive disulfide-bonded globular structure or intermolecular disulfide bonds adds rigidity to the Cryptosporidium oocyst wall[15,28,29]. The current study explored four different DNA extraction protocols to assist oocyst wall disruption with varying the thermal conditions and reagents concentrations. All the four methods were CTAB-based and attempted on positive Cryptosporidiumspp. oocyst spiked faecal samples till reproduction of consistent results. The conditions used in the Method 4 were effective in reproducing the results comparable to that of commercial faecal DNA extraction kits. The protocol thus developed was simple, less time consuming and economical with no requirement of additional equipment like the Mini Bead-beater or sonicator for oocyst wall disruption.
Molecular techniques demands DNA of high purity and quality for PCR and sequencing analysis that aids in the detection and characterization ofCryptosporidium spp. from faecal samples[30]. The difficulty that arises while extracting DNA from faecal samples is the presence of bile salts and other PCR inhibitors directly influencing the efficiency of PCR despite good recovery rate after disintegrating the thick-walledCryptosporidium oocysts. This protocol could also prove useful in cases of autoinfection with thin walled oocysts or in sub-clinical conditions with minimal faecal oocyst shedding that goes unnoticed by microscopy and conventional DNA extraction based PCR.
Traditionally, the microscopy based detection method is employed for confirmation of Cryptosporidium infection, while it cannot differentiate the species and subtypes of Cryptosporidium species that will be useful in the epidemiological analysis and disease dynamics. On the contrary, molecular techniques could aid species differentiation, possibly further characterization by sub-type grouping and phylogenetic analysis using sequencing methods. Similarly, Real-time PCR can also be an alternate for conventional PCR with its ability for quick detection and non-sequencing based characterization like ‘endpoint genotyping’ or multiplexing [14,31]. Hence, in the current study, we have used 18SSU rRNA gene-based TaqMan® probe real-time PCR to assess the effectiveness of our extracted DNA for such protocols. Based on the machine call, we obtained encouraging results for Method 4, while Method 3, despite failing in conventional PCR could detect the cryptosporidium in 18SSU rRNA gene-based TaqMan® probe real-time PCR.