P-2134. Development and Validation of a Quantitative PCR Assay for the Detection of <i>Mucorales</i> in Human Serum Specimens

Abstract Background Mucormycosis are diseases with high morbidity and mortality caused by fungal species belonging to the order Mucorales. The intended use of a Mucorales qPCR assay is as an adjunct to microbiological, clinical, and radiologic means of diagnosing invasive mucormycosis (IM). Serum collection is a non-invasive alternative to BAL and biopsy. Our design process included sequences from all species within the Mucorales genera: Cunninghamella, Lichtheimia, Absidia, Apophysomyces, Mucor, Rhizomucor, Rhizopus and Saksenaea. The Mucorales qPCR assay is designed to detect and quantify Mucorales DNA extracted from human serum. Methods Primers and probes were designed to target the 18S regions of Mucorales species. Nucleic acid was extracted using the MagMAX™ Cell-Free DNA Isolation Kit and KingFisher™ Flex system. Amplification and detection were performed using TaqMan™ Fast Universal Master Mix and the Applied Biosystems™ QuantStudio 7 Pro. Quantification was performed using Rhizopus oryzae target-bearing linearized plasmid standards with results reported in copies/mL. Analytical validation was performed by spiking negative human serum with four different Mucorales species’ DNA: Rhizopus oryzae, Rhizomucor miehei, Lichtheimia corymbifera, and Cunninghamella bertholletiae. Results The limit of detection was 181 copies/mL, and the lower limit of quantification was determined to be 253 copies/mL as shown in Table 1. Linear regression of dilution data produced an R2=0.997, slope=1.029, and y-intercept=0.212 as show in Figure 1. The Mucorales target detection was 100% (60 of 60) of blinded positive samples with all observed log10 copies/mL results within ±0.5 log10 copies of expected values as shown in Table 2. All (20 of 20) blinded negative samples were negative for Mucorales and positive for Internal Control (IC). Intra-assay precision ranged from 5 – 22 %CV and inter-assay precision ranged from 12 – 17 %CV as Shown in Table 3. Conclusion The validated assay described here demonstrates excellent sensitivity, linearity, precision, and accuracy, providing a reliable means of detecting Mucorales DNA in human serum. This assay represents a non-invasive, rapid, and sensitive method to guide clinical decisions on patient populations at high risk of IM. Disclosures Jerod Davidson, B.A., Eurofins Viracor: Employee James Grantham, B.S., Eurofins Viracor: Employee Clayton Thomas, B.S., Eurofins Viracor: Employee Jamie Nutt, n/a, Eurofins Viracor: Employee Pam Morris, M.S., PMP, Eurofins Viracor: Employee Zachary Kockler, PhD, Eurofins Viracor: Employee Steve Kleiboeker, PhD, Eurofins: employee|Eurofins: Stocks/Bonds (Public Company)