P423 Recombinase polymerase amplification for Mucormycosis

Abstract

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PMObjectives(1) To develop a Recombinase polymerase amplification (RPA)-based assay to diagnose mucormycosis. (2) To determine the analytical sensitivity and cross-reactivity of the developed assay. (3) To validate the assay in clinical samples. (4) To determine the turnaround time for the developed assay.MethodsMultiple alignments of the ITS region of Rhizopus arrhizus were used to identify conserved sequences and design 30-32 bp long primers and 45 bp long Exo probes for RPA-based assay as per TwistDX guidelines (Cambridge, UK) using the PrimedRPA program (Higgins et al. 2019) and subject to in silico specificity check using NCBI primer blast. Genomic DNA and plasmid clone of the target region were spiked into human DNA and were used to determine analytical sensitivity. DNA from 35 clinically relevant fungi and bacteria were used to determine the cross-reactivity. A total of 40 sinus tissue samples from patients with microscopy and culture-confirmed mucormycosis and 40 sinus tissue samples from patients without mucormycosis were used to determine the diagnostic sensitivity and specificity of the assay. DNA from relevant fungi, bacteria and molds were extracted using the MPbio FastDNA kit as per the manufacturer’s instructions with an elution volume of 100 μl. All extracted DNA samples were subjected to GAPDH PCR before RPA testing to rule out the presence of inhibitors as per established protocols. A 50 μl RPA reaction using Twistamp Exo kit as per manufacturer's instruction but using 10 μl template instead of 1 μl. The reactions were set up and inserted into a portable Qiagen ESE quant TS2.4 fluorometer incubated at 39°C and fluorescence was acquired every 30 sec over 20 min. The tubes were removed from the fluorometer mixed well and reinserted after 4 min as per kit instructions. Positive control and no template control were set up with each reaction. All relevant data were entered in Microsoft excel and diagnostic sensitivity and specificity were calculated using the Medcalc free online statistical software.ResultsA total of 1246 primer-probe combinations were designed and checked for in silico specificity using NCBI primer blast. Of these 1246 primer probes, 8 primers and 1 probe was finalized for in vitro testing to determine the best primer and probe set. The finalized primers had analytical sensitivity of 10pg and 10 copies and showed no cross-reactivity with any of the 35 clinically relevant fungi and bacteria. All extracted DNA showed amplified GAPDH gene demonstrating the absence of PCR inhibitors in extracted DNA. The sensitivity and specificity of the assay were 97.5% (95% CI: 86.8-99.9) and 97.6% (95% CI: 87.1-99.9). The turnaround time for RPA was 6 h.ConclusionReal-time RPA can be used to rapidly and reliably diagnose mucormycosis.