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.