Integrating Magnetic-Bead-Based Sample Extraction and Molecular Barcoding for the One-Step Pooled RT-qPCR Assay of Viral Pathogens without Retesting

Zhuang, Xinyu; Zhao, Zibin; Feng, Xianzhen; Lui, Grace; Chan, Daniel; Lee, Shui Shan; Hsing, I‐Ming

doi:10.1021/acs.analchem.3c00885

Cited by 3 publications

(1 citation statement)

References 25 publications

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“…The utilization of pooling assays in diagnostic PCR offers significant advantages, including reduced costs, increased testing capacity, and shorter turnaround times, as multiple samples can be simultaneously analyzed in a single assay, providing an efficient and cost-effective approach to detect and monitor pathogens in diagnostics assays (20)(21)(22).…”

Section: Introductionmentioning

confidence: 99%

Ready for new waves: optimizing SARS-CoV-2 variants monitoring in pooled samples with droplet digital PCR

Pacini,

Paredes,

Heckel

et al. 2024

Front. Public Health

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IntroductionThe declaration of the end of the Public Health Emergency for COVID-19 on May 11th, 2023, has shifted the global focus led by WHO and CDC towards monitoring the evolution of SARS-CoV-2. Augmenting these international endeavors with local initiatives becomes crucial to not only track the emergence of new variants but also to understand their spread. We present a cost-effective digital PCR-based pooled sample testing methodology tailored for early variant surveillance.MethodsUsing 1200 retrospective SARS-CoV-2 positive samples, either negative or positive for Delta or Omicron, we assessed the sensitivity and specificity of our detection strategy employing commercial TaqMan variant probes in a 1:9 ratio of variant-positive to variant-negative samples.Results: The study achieved 100% sensitivity and 99% specificity in 10-sample pools, with an Area Under the Curve (AUC) exceeding 0.998 in ROC curves, using distinct commercial TaqMan variant probes.ResultsThematic analysis of the interviews identified three major challenges faced by public health officials in promoting vaccination: dissemination of misinformation in media, political polarization of COVID and its contribution to vaccine acceptance and COVID fatigue, and assessment of the risks associated with disease severity versus vaccine safety considering limited public health resources.DiscussionThe employment of two separate TaqMan probes for both Delta and Omicron establishes dual validation routes, emphasizing the method’s robustness. Although we used known samples to model realistic emergence scenarios of the Delta and Omicron variants, our main objective is to demonstrate the versatility of this strategy to identify future variant appearances. The utilization of two divergent variants and distinct probes for each confirms the method’s independence from specific variants and probes. This flexibility ensures it can be tailored to recognize any subsequent variant emergence, given the availability of its sequence and a specific probe. Consequently, our approach stands as a robust tool for tracking and managing any new variant outbreak, reinforcing our global readiness against possible future SARS-CoV-2 waves.

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Section: Introductionmentioning

confidence: 99%

Ready for new waves: optimizing SARS-CoV-2 variants monitoring in pooled samples with droplet digital PCR

Pacini,

Paredes,

Heckel

et al. 2024

Front. Public Health

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OptoAssay—Light-controlled dynamic bioassay using optogenetic switches

Urban,

Hörner,

Weber

et al. 2024

Sci. Adv.

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Circumventing the limitations of current bioassays, we introduce a light-controlled assay, OptoAssay, toward wash- and pump-free point-of-care diagnostics. Extending the capabilities of standard bioassays with light-dependent and reversible interaction of optogenetic switches, OptoAssays enable a bidirectional movement of assay components, only by changing the wavelength of light. Demonstrating exceptional versatility, the OptoAssay showcases its efficacy on various substrates, delivering a dynamic bioassay format. The applicability of the OptoAssay is successfully demonstrated by the calibration of a competitive model assay, resulting in a superior limit of detection of 8 pg ml −1 , which is beyond those of conventional ELISA tests. In the future, combined with smartphones, OptoAssays could obviate the need for external flow control systems such as pumps or valves and signal readout devices, enabling on-site analysis in resource-limited settings.

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Deciphering dengue: novel RNA barcoding segments for enhanced serotype-specific identification and global surveillance of dengue viruses

Jiang,

Zhao,

Ding

et al. 2024

Front. Microbiol.

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IntroductionDengue viruses (DENVs), the causative agents of dengue hemorrhagic fever and dengue shock syndrome, undergo genetic mutations that result in new strains and lead to ongoing global re-infections.ObjectivesTo address the growing complexity of identifying and tracking biological samples, this study screened RNA barcode segments for the four DENV serotypes, ensuring high specificity and recall rates for DENV identification using segments.ResultsThrough analyzing complete genome sequences of DENVs, we screened eight barcode segments for DENV, DENV-1, DENV-2, DENV-3, and DENV-4 identification. Comparing the screened barcode segments to sequences of known strains and determining the proportion of correctly or incorrectly identified nucleotides, these segments demonstrated an average recall rate at nucleotide level of 91.34% for four DENV serotypes, a specificity of 99.50% at species level within the Flaviviridae family, and a precision rate of 100% for identifying DENVs. For arboviruses, the nucleotide-level specificity was 63.58%. We designed and used the “Barcoding” software to streamline segment design, integrating automated sequence preprocessing, evaluation of barcode segments, and primer design, significantly reducing manual intervention and enhancing overall efficiency. We also established an online database called “Barcodes” for storing and preparing barcode segments.ConclusionThis work established a standard framework for DENV identification and barcode segment selection, promising significant advancements in the real-time management and control of DENVs, thereby enhancing surveillance capabilities and facilitating targeted interventions in dengue outbreak-prone regions.

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Integrating Magnetic-Bead-Based Sample Extraction and Molecular Barcoding for the One-Step Pooled RT-qPCR Assay of Viral Pathogens without Retesting

Cited by 3 publications

References 25 publications

Ready for new waves: optimizing SARS-CoV-2 variants monitoring in pooled samples with droplet digital PCR

Ready for new waves: optimizing SARS-CoV-2 variants monitoring in pooled samples with droplet digital PCR

OptoAssay—Light-controlled dynamic bioassay using optogenetic switches

Deciphering dengue: novel RNA barcoding segments for enhanced serotype-specific identification and global surveillance of dengue viruses

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