Standardization and evaluation of DNA extraction protocol for
Cryptosporidium oocysts from faecal samples in goats
Abstract
DNA extraction from stools is the major hurdle in the detection of
Cryptosporidium infection due to complex oocyst wall and presence
of PCR inhibitors. There is no conventional full-proof DNA extraction
method which efficiently recovers DNA from Cryptosporidium.
Alternatively, commercial DNA kits costs dearer and unaffordable in
smaller laboratories with limited funding. To address this, the current
study explored for an efficient DNA extraction method from
Cryptosporidium oocysts. Four different DNA-extraction
methodologies were developed at different Cetyltrimethylammonium
Bromideconcentrations and temperature-cycles and analyzed for
quality-quantity parameters. Four different types of recipes were used,
in brief which includes 1. Sonication+3 cycles of chill-thaw+ (24:1)
Chloroform:Isoamyl alcohol, 2. Liquid Nitrogen+ thaw (@70ºC)+ (24:1)
Chloroform: Isoamyl alcohol, 3. Sonication + 3 cycles of freeze that +
(24:1) Chloroform: Isoamyl alcohol and 4. Three cycles of ‘snap-chill
(@-80ºC) and boil (@95ºC) +(24:1) Chloroform: Isoamyl alcohol’. Among
these, a protocol involving three cycles of ‘snap-chill and boil’
(Method-4) could successfully recover Cryptosporidium DNA with
better quality-quantity parameters with consistency and repeatability
and lack of PCR inhibitors as evidenced by the workability of this
method confirmed by conventional-PCR and real-time PCR for 18
small-subunit-ribosomal RNA ( 18SSU rRNA). The repeated deep
freezer and boil cycles successfully disrupted the thick chitin-rich
oocyst wall of Cryptosporidium leading to precipitation of
nucleic acids by chloroform-isoamyl alcohol. The current study aims to
introduce a cost-effective method that overcomes the bottlenecks faced
with the conventional DNA extraction techniques for
Cryptosporidium directly from faecal samples.