Validation of one-step reverse transcription digital PCR assays for Norovirus GI

• Published in: Analytical biochemistry Vol. 692; p. 115576
• Main Authors: Ko, Bomin, Shin, Taejin, Kim, Boram, Lee, Da-Hye
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2024
• Subjects: Droplet based digital PCR; Nanoplate based digital PCR; Norovirus GI assay; One-step reverse transcription digital PCR; Reference material; Secondary structure; Reference material; Droplet based digital PCR; One-step reverse transcription digital PCR; Secondary structure; Norovirus GI assay; Nanoplate based digital PCR; nanoplate based digital PCR; one-step reverse transcription digital PCR; reference material; droplet based digital PCR
• ISSN: 0003-2697; 1096-0309; 1096-0309
• DOI: 10.1016/j.ab.2024.115576

Regular monitoring of Norovirus presence in environmental and food samples is crucial due to its high transmission rates and outbreak potential. For detecting Norovirus GI, reverse transcription qPCR method is commonly used, but its sensitivity can be affected by assay performance. This study shows significantly reduced assay performance in digital PCR or qPCR when using primers targeting Norovirus GI genome 5291–5319 (NC_001959), located on the hairpin of the predicted RNA structure. It is highly recommended to avoid this region in commercial kit development or diagnosis to minimizing potential risk of false negatives. [Display omitted] •Using primers that target the Norovirus genome 5291-5319 (NC_001959) in commercial kits may lead to reduce assay performace.•Adjusting primer positions improves detection efficiency; avoid placing the forward primer at a haripin in RNA secondary structure prediction.•For the development and evaluation of diagnostic kits, or for quality assessment, use high quality reference materials.