Abstract
Acanthamoeba are widely distributed in the environment and are known to cause blinding keratitis and brain infections with greater than 90% mortality rate. Currently, polymerase chain reaction (PCR) is a highly sensitive and promising technique in Acanthamoeba detection. Remarkably, the rate of heating–cooling and convective heat transfer of the PCR tube is limited by low thermal conductivity of the reagents mixture. The addition of nanoparticles to the reaction has been an interesting approach that could augment the thermal conductivity of the mixture and subsequently enhance heat transfer through the PCR tube. Here, we have developed hexagonal boron nitride (hBN) nanoparticle-based PCR assay for the rapid detection of Acanthamoeba to amplify DNA from low amoeba cell density. As low as 1 × 10−4 wt % was determined as the optimum concentration of hBN nanoparticles, which increased Acanthamoeba DNA yield up to ~16%. Further, it was able to reduce PCR temperature that led to a ~2.0-fold increase in Acanthamoeba DNA yield at an improved PCR specificity at 46.2 °C low annealing temperature. hBN nanoparticles further reduced standard PCR step time by 10 min and cycles by eight; thus, enhancing Acanthamoeba detection rapidly. Enhancement of Acanthamoeba PCR DNA yield is possibly due to the high adsorption affinity of hBN nanoparticles to purine (Guanine—G) due to the higher thermal conductivity achieved in the PCR mixture due to the addition of hBN. Although further research is needed to demonstrate these findings in clinical application, we propose that the interfacial layers, Brownian motion, and percolation network contribute to the enhanced thermal conductivity effect.
Highlights
Acanthamoeba is a free-living protozoan widely found in the environment
They can survive as parasites to proliferate within human or animal tissues leading to two major life-threatening infectious diseases known as granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK) that often arise as mild headaches [1,2]
The peak originating from hexagonal boron nitride (hBN) were observed at 2θ angles of 26.8◦, and the nanoparticles display high crystallinity owing to its narrow peak
Summary
Acanthamoeba is a free-living protozoan widely found in the environment. Despite being ubiquitous, they can survive as parasites to proliferate within human or animal tissues leading to two major life-threatening infectious diseases known as granulomatous amoebic encephalitis (GAE) and Acanthamoeba keratitis (AK) that often arise as mild headaches [1,2]. Acanthamoeba detection used hot-start DNA polymerase enzymes to modify the conventional PCR techniques, avoiding non-specific amplification, and increasing the target DNA yield by inactivating the enzyme at lower temperatures [8]. These findings reported 82% specificity, suggesting a possible presence of undesired PCR products as false positives [8]. In this research, the objective is to incorporate hBN nanoparticles of an optimum concentration into PCR assays owing to their relatively high thermal conductivity, chemically stable properties, and low cost Since it can enhance Acanthamoeba DNA yield in PCR and improve the efficiency of PCR parameters (e.g., temperature, step, and cycle), it may aid in the rapid detection of Acanthamoeba
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
