B-222 Validation of a qPCR Method for Qualitative Detection of Monkeypox Virus

Abstract

Abstract Background The 2022 Monkeypox virus (MPXV) outbreak became a global concern, prompting a multidisciplinary response to increase surveillance and isolate suspected and confirmed cases to reduce disease spread. Quantitative PCR (qPCR) is capable of detecting small amounts of DNA or RNA, and can be used to quickly and accurately confirm MPXV cases, even at early stages of infection, which is crucial for outbreak control and management. Methods qPCR was performed on a 384-well plate with a total reaction volume of 20 μl consisting of 9 μL Acrometrix Monkeypox DNA Positive Control at serial dilutions, 10 μL BactoPure Master Mix, and 1 μL TaqMan Monkeypox Virus Microbe Detection Assay. Positive control dilutions 103, 102, 101, and 100 were diluted with TE Buffer. qPCR was performed on two different Quantstudio 12K Flex instruments. Results The qPCR method validation was performed by calculating precision, accuracy, and linearity on serial dilutions of Acrometrix Monkeypox Virus DNA Positive Control. Precision testing was performed on 2 levels of control: low (101) and high (103) over 5 days with 4 repeats per day. Accuracy and linearity testing were performed on 4 levels with 3 repeats. Coefficient of variation (CV) was less than 5%, specifically 0.9% for the low control and 4.3% for the high control, indicating an acceptable level of precision. Percent recovery fell close to 100%, specifically within 101%-103% for all controls, indicating the results fell close to the expected value. Linearity R2 was close to 1, specifically 0.984, demonstrating the results are proportional to the concentration analyzed. Conclusion Validation of MPXV detection through qPCR was successful. Development and validation of qPCR assays for the detection of positive MPXV cases is essential for managing future outbreaks and combatting future pathogenic strains.