351 Loop-mediated isothermal amplification for the detection of cytomegalovirus infection and drug resistance at the point of care

Abstract

OBJECTIVES/GOALS: To develop a loop-mediated isothermal amplification (LAMP) assay for the detection of cytomegalovirus (CMV) infection and drug resistance. METHODS/STUDY POPULATION: We designed core and loop primers sets utilizing the NEB LAMP Primer Design Tool. Each set contained four or six primers targeting major-immediate early genes – essential for viral entry/replication – or regions known to confer resistance to the antiviral drug ganciclovir. Optimization of reactions conditions was achieved employing DNA reference materials. Reactions were visualized through a change in color as amplification reactions accumulated. Successful reaction conditions were selected based on specific amplification products in less than 60 minutes. Limits of detection were evaluated as the main performance outcome. RESULTS/ANTICIPATED RESULTS: Genomic data were extracted and used to design a series of LAMP primers (48 total) that aimed to detect specific genomic regions of CMV. Using this strategy, we successfully designed and identified eight primer sets that showed high 100% sensitivity and 100 % specificity, when detecting > 1.00 x 10^5 copies/mL of CMV gDNA. We are in the process of characterizing a new set of primers to determine the diagnostic utility of a LAMP assay in detecting selected single-nucleotide mutations at the UL97 loci. The expected outcomes at completion include: (1) the identification of LAMP primers to detect drug-resistance mutants, (2) defining optimal conditions for successful reactions, and (3) determining limits of detection for subsequent validation with clinical specimens. DISCUSSION/SIGNIFICANCE: CMV infection remains one of the most dangerous infectious agents for immunocompromised hosts, newborns, and unborn children. This study will describe a proof-of-concept LAMP assay for the genotypic detection of drug resistance in CMV-infected individuals and hence, create new avenues for selection of effective therapies to treat CMV disease.