Association for Molecular Pathology (AMP) Annual Meeting & Expo

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Why use an exogenous RNA VLP control?

An exogenous reference control is crucial in molecular assays as it eliminates false negatives and false positives, ensures accurate detection, validates extraction and amplification, enables quantification and normalization, and promotes standardization in inter-laboratory comparisons. This is particularly important in workflows involving RNA due to the presence of ubiquitous ribonucleases (RNases). Unreliable RNA controls create data interpretation challenges and impose significant time, effort, and expense during validation and execution. Therefore, utilizing an exogenous RNA VLP control is crucial to mitigate these issues by protecting the RNA from nucleases.

A computational design and encapsulation to create VLPs

We used an algorithm to create a non-repetitive 1kb sequence and encapsulated synthetic DNA to make VLPs. The sequence was assembled from short, partially overlapping k-mer subsequences that are not found in the human genome or in over 1000 human-pathogen genomes. The algorithm ensures nonhomology by densely incorporating nonhomologous subsequence building blocks. Furthermore, we conducted laboratory spike-in experiments using diverse raw samples (e.g., wastewater, plasma, saliva) and conducted rigorous stability testing to validate its performance.

Our VLP exogenous RNA control demonstrates exceptional accuracy and stability throughout the RNA processing workflow; it is an optimal choice for various molecular applications.

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Why a syndromic panel assay design pipeline?

The rise of respiratory infectious diseases, particularly highlighted by the SARS-CoV-2 pandemic, has prompted a surge in qPCR-based testing for the fast and accurate identification of multiple respiratory pathogens. One challenge has been to design sensitive and specific multiplex assays within a large syndromic panel that exhibit no cross-reactivity. To address this obstacle, we utilized Takara Bio’s SmartChip Real-Time PCR system, which is able to perform massively parallel single-plex qPCR reactions at the nanoliter scale across multiple samples and reactions without the need for complex degenerate or multiplex designs.

A new system method for efficient design of syndromic panels

To complement the SmartChip system, we have developed a pipeline to streamline the process of designing large syndromic assay panels. Leveraging this pipeline, we successfully generated a comprehensive respiratory qPCR assay panel comprising six viral targets commonly associated with respiratory infections and several bacteria targets associated with co-infections and pneumonia. Notably, these targets include: SARS-CoV-2, Influenza A H1N1/H3N2, Influenza B, and Respiratory Syncytial Virus A/B. The pipeline employs computational tools to optimize primer and probe selection and consists of several steps: (1) the retrieval of relevant strain information from databases, (2) strain filters based on clade and date of collection, and (3) alignment-based assay filters encompassing strain-inclusivity, exclusivity, and other qPCR design criteria.

Our study showcases the successful implementation of an assay design and characterization pipeline, combining in-silico analyses and experimental evaluation.