Development of nanoparticle-assisted PCR assay in the rapid detection of brain-eating amoebae

Abstract

Brain-eating amoebae (Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri) have gained increasing attention owing to their capacity to produce severe human and animal infections involving the brain. Early detection is a pre-requisite in successful prognosis. Here, we developed a nanoPCR assay for the rapid detection of brain-eating amoebae using various nanoparticles. Graphene oxide, copper and alumina nanoparticles used in this study were characterized using Raman spectroscopy measurements through excitation with a He-Ne laser, while powder X-ray diffraction patterns were taken on a PANanalytical, X'Pert HighScore diffractometer and the morphology of the materials was confirmed using high-resolution transmission electron microscopy (HRTEM). Using nanoparticle-assisted PCR, the results revealed that graphene oxide, copper oxide and alumina nanoparticles significantly enhanced PCR efficiency in the detection of pathogenic free-living amoebae using genus-specific probes. The optimal concentration of graphene oxide, copper oxide and alumina nanoparticles for Acanthamoeba spp. was determined at 0.4, 0.04 and 0.4 μg per mL respectively. For B. mandrillaris, the optimal concentration was determined at 0.4 μg per mL for graphene oxide, copper oxide and alumina nanoparticles, and for Naegleria, the optimal concentration was 0.04, 4.0 and 0.04 μg per mL respectively. Moreover, combinations of these nanoparticles proved to further enhance PCR efficiency. The addition of metal oxide nanoparticles leads to excellent surface effect, while thermal conductivity property of the nanoparticles enhances PCR productivity. These findings suggest that nanoPCR assay has tremendous potential in the clinical diagnosis of parasitic infections as well as for studying epidemiology and pathology and environmental monitoring of other microbes.

Simultaneous Detection of Bovine Rotavirus, Bovine Parvovirus, and Bovine Viral Diarrhea Virus Using a Gold Nanoparticle-Assisted PCR Assay With a Dual-Priming Oligonucleotide System

“…Thus, thermal conductivity is enhanced in nanoparticle-containing PCR systems that enables attaining the target temperature in a shorter time. Efficient heat transfer generates a larger number of amplicons, thereby improving the sensitivity of the reaction (Gabriel et al, 2018). We developed a multiplex DPO-nanoPCR assay for detecting BRV, BPV, and BVDV that is 100-1000 times more sensitive than conventional multiplex PCR.…”

Development of a Conventional RT-PCR Assay for Rapid Detection of Porcine Deltacoronavirus with the Same Detection Limit as a SYBR Green-Based Real-Time RT-PCR Assay

“…This indicates that RT-PCR can detect PDCoV with a detection limit of 1000 copies. Recently, nano-particle-assisted PCR assays were used to increase the sensitivity of conventional PCR [22][23][24]. In the detection of duck Tembusu virus, the detection limit of nano-particle-assisted PCR was 1.8 × 10 2 copies [23].…”

hBN Nanoparticle-Assisted Rapid Thermal Cycling for the Detection of Acanthamoeba

“…DNA band, as described previously [ 27 ]. The third DNA extraction method was also performed to the above-stated cell number counts, using the GeNet Bio Kit method based on the manufacturer’s protocol [ 28 ]. For Chelex, Proteinase K, and GeNet Bio Kit DNA extraction methods, nano dropping of the supernatant containing the A. castellanii DNA was measured for DNA concentrations.…”

“…DNA, 1 μL of 2 mM dNTPs mixture, 8.5 μL of nuclease-free water, 1 μL of 25 μM MgCl 2, and 1 μL of each 10 μM forward and reverse Acanthamoeba genus-specific primer sequences ( Table 1 ) [ 31 ]. PCR thermocycler reaction involved initial denaturation at 94 °C for 3 min followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 50 °C for 30 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 10 min with an expected PCR amplified product size of 950 bp for A. castellanii [ 28 ]. PCR assay was repeated 3 times to observe the consistency of visible band strength.…”

“…For nano-PCR, hBN nanoparticles were added into PCR reactions to enhance the amplification of T4 genotype DNA using various hBN concentrations (0.4–1 × 10 − 8 wt % hBN). To determine optimum hBN concentration, nano-PCR involved PCR protocol parameters, as described previously [ 28 ]. Next, to improve PCR efficiency in the rapid detection of Acanthamoeba , nano-PCR used hBN optimum concentration to test the effects of PCR parameters (e.g., temperature, step, and cycle).…”