Standardization and evaluation of DNA extraction protocol for Cryptosporidium oocysts from faecal samples in goats

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.