A simple PCR condition for detection of a single cyst of Acanthamoeba species

Abstract

Acanthamoeba is a free-living protozoan with a worldwide distribution in a variety of natural and artificial habitats. It has even been found in contact lens solution. Acanthamoeba spp. can cause infections such as granulomatous amoebic encephalitis and amoebic keratitis. Specific and sensitive diagnosis of Acanthamoeba infections can prevent clinical symptoms from worsening. Recently, PCR technique has been used for Acanthamoeba diagnosis. Unfortunately the dormant cyst of Acanthamoeba is resistant to chemical reagents; thus, most extraction of DNA uses a commercial DNA extraction kit for obtaining DNA for further use in polymerase chain reaction (PCR). Therefore in the present study, we improved the ability to diagnose Acanthamoeba using a simplified PCR technique. Interestingly, heating at 94°C for 10 min could release DNA which is amplified with specific primers designed from 16S rRNA. The PCR product is about 180 bp. This technique is a simple and efficient method for detection of Acanthamoeba-even a single cyst-and does not require high-cost reagents or complicated procedures to extract DNA.

Comparative analyses of different genetic markers for the detection of Acanthamoeba spp. isolates

“…During the last few years, molecular techniques (PCR) are increasingly being used in the identification of the genotype (Booton et al 2005, Caumo et al 2009, Edagawa et al 2009, Goldschmidt et al 2012, Howe et al 1997, Itahashi et al 2011, Jain and Tilak 2011, Laummaunwai et al 2012, Le Calvez et al 2012. Our results confirmed the usefulness of PCR for the early, rapid and sensitive diagnosis of pathogenic Acanthamoeba spp.…”

Advances in the Diagnosis and Treatment of Acanthamoeba Keratitis

“…Le Calvez et al [27] recently described a multiplexed quantitative PCR method which is able to distinguish individual Acanthamoeba species in free-living mixed flora samples. Laummaunwai et al [28] developed an algorithm for DNA extraction which is able to produce viable DNA from a single Acanthamoeba cyst; previously cystic DNA extraction has been the Achilles heel of the technique. These advances mean that real-time PCR is becoming more and more relevant in diagnostics, with particular benefits being that it is a much faster technique than growing Acanthamoeba cultures and does not require adjuvant biopsy, so it can be implemented earlier, on cases of lower clinical suspicion.…”

Rapid and sensitive diagnosis of Acanthamoeba keratitis by loop-mediated isothermal amplification

“…Therefore, development of a sensitive, convenient and rapid method for laboratory detection of Acanthamoeba to assist AK diagnosis is urgently required. PCR has been widely used in the detection of various pathogens, including Acanthamoeba [13,14,17,18], because of its relatively high sensitivity and effectiveness, but PCR requires precise equipment, such as a thermal cycler for the amplification of DNA, which limits its application in the clinic. However, the LAMP assay is a simple diagnostic tool that can be completed in a regular laboratory water bath within 70 min, and the final amplified product of the LAMP assay can be detected through unaided visual examination by adding Gene-Finder to the reaction tube.…”