A-073 Validation of a Qualitative PCR for Dengue Virus Detection and Serotyping

Abstract

Dengue is a mosquito-borne viral infection caused by four distinct serotypes: dengue virus 1, 2, 3, and 4 (DENV 1-4). Globally, dengue affects up to 400 million individuals each year and infection can occur multiple times throughout a person’s lifetime. Disease severity varies by the infecting serotype and is often more severe if individuals were previously infected by a different serotype. Analytical validation was performed using contrived serum samples extracted using the MagNA Pure® 96 (MP96) instrument (Roche Molecular Systems) using the DNA and Viral NA Small Volume kit. Real-time PCR using DiaSorin’s Analyte Specific Reagent (ASR) primer probe pairs for each serotype, Dengue 1-4 (MOL9041, MOL9042, MOL9043, MOL9044), was performed using on the QuantStudio™ 7 (QS7) Flex instrument (Applied Biosystems®). The limit of detection (LoD) in serum was established for each serotype using commercial serum (Golden West). The analytical LoD for each serotype was DENV1: 2,540 copies/ml, DENV2: 2,280 copies/ml, DENV3: 832 copies/ml, and DENV4: 496 copies/ml. The assay demonstrated excellent inter-run precision (%CV) for each serotype at 2x LOD: DENV1 2.37%, DENV2 2.83%, DENV3 2.46%, and DENV4 1.84%. Procedural verification was performed by testing 240 contrived serum samples through the assay, 50 for each serotype at varying concentrations, in addition to 40 serum samples expected to be negative for DENV. Categorical detection was shown to be 100% (240/240). Analytical specificity was assessed by testing pathogens that cause similar symptoms including West Nile Virus, Ehrlichia chaffeensis, Anaplasma phagocytophilum, Borrelia burgdorferi, among others and no cross-reactivity was observed with any pathogen tested. Interference studies using a Sun Diagnostics panel containing elevated triglycerides, hemolysate, total protein, conjugated bilirubin, and unconjugated bilirubin revealed that although some interferents affected assay performance at high concentrations, all serotypes remained detectable. Additional titration to clinically relevant interferent levels demonstrated largely mitigated interference. Stability assessments indicated that dengue viral targets in serum remain stable for 48 hours at room temperature, 7 days refrigerated, 30 days frozen, and through two freeze–thaw cycles. The dengue virus serotyping assay using DiaSorin ASR primer pairs demonstrates high accuracy, sensitivity and specificity enabling rapid and reliable differentiation of dengue serotypes. Moreover, clinical samples are stable, allowing for transport to a laboratory without risk of sample degradation. The use of a high throughput molecular assay for dengue virus serotyping allows laboratories to rapidly respond to the potential emergence of dengue virus in the United States.