Aims: To demonstrate the DNA sequences of Acanthamoeba castellanii strain using molecular genomic DNA extraction and polymerase chain reactions.
Materials and Methods: Genomic DNA extraction: Four 5 μm thick sequential sections were cut from the formalin-fixed paraffin embedded tissue sample. The micro-sections were placed in a micro-centrifuge tube. Sections were de-paraffinized using 300 μl of mineral oil and incubated at 90°C for 20 minutes to dissolve the wax. The tissue was digested with 50 μg/ ml proteinase K at 48°C and incubated overnight. Genomic DNA (gDNA) was extracted from the solution by adding an equal volume of chloroform/isoamyl alcohol (24:1) to the tube. The gDNA was determined using Nanodrop ND-1000 Spectrophotometer.
Polymerase chain reaction (PCR) assay: PCR assay was carried out on the extracted gDNA to amplify a target sequence of 161bp using Bioneer AccuPower® PCR PreMix in a reaction volume of 20 μl containing 1U of Top DNA polymerase, 250 μM dNTPs, 10 mM Tris‑Hcl (pH 9.0), 30 nM Kcl, 1.5 mM MgCl2, 25 pmol each of the two primers—forward— Aca16Sf1010 (5’-TTATATTGACTTGTACAGGTGCT-3’) and reverse—Aca16Sr1180 (5’-CATAATGATTTGACTTCTTCTCCT- 3’) and the template DNA.
Results: PCR analysis/sequence analysis and alignment: The amplification of the 16S small subunit ribosomal RNA gene produced an expected band, which is 161bp in size. A nucleotide BLAST search was carried out at NCBI to ascertain what the sequence was. The forward and reverse sequences showed great similarity with the same sequence as given by Acanthamoeba castellanii strain sequence ID: gb|AF479520.1| 16S small subunit ribosomal RNA gene.
Conclusion: The high index of suspicion for Acanthamoeba species in this index case presenting histologically as granulomatous amoebic encephalitis was confirmed by molecular polymerase chain reaction as Acanthamoeba castellanii.