Comparison of a novel semi-nested polymerase chain reaction (PCR) with a uniplex PCR for the detection of Acanthamoeba genome in corneal scrapings

Abstract

A semi-nested polymerase chain reaction (snPCR) was developed to improve the sensitivity of detection of Acanthamoeba sp. genome from corneal scrapings of Acanthamoeba keratitis patients. The snPCR was developed using a laboratory designed inner forward primer targeting the 450-bp product of the 18s rRNA-gene-based PCR. The snPCR was optimized using 11 Acanthamoeba sp. culture isolates and further applied onto 35 corneal scrapings from keratitis patients. The sensitivity and specificity of the snPCR was compared against conventional methods (smear and/or culture-gold standard) and the uniplex PCR described by Schroeder et al. Eleven out of the 35 corneal scrapings were positive by the gold standard and snPCR, whereas only 3 of these 11 were positive by the uniplex PCR. The clinical sensitivity and specificity of the snPCR was 100% when compared with the gold standard. DNA sequencing was performed on first round amplicons of four culture isolates and one specimen, and all of them were identified as genus Acanthamoeba when compared with the GenBank database sequences. Application of snPCR on the 11 culture isolates yielded amplicons ranging 120-160 bp in size, indicating sequence variation among the different culture isolates. The clinical sensitivity of snPCR was higher than the conventional methods and the uniplex PCR reported earlier.