Development of an Automated, Non-Enzymatic Nucleic Acid Amplification Test

Zimmers, Zackary; Boyd, Alexander D.; Stepp, Hannah E.; Adams, Nicholas M.; Haselton, Frederick R.

doi:10.3390/mi12101204

Cited by 3 publications

(4 citation statements)

References 34 publications

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“…The assay requires manual processing to transfer products through the sequential enzymatic reactions, which can be burdensome on the operator and increase the risk of laboratory contamination from opening tubes containing RT‐PCR amplicons. Future automation by strategies such as magnetic processing or robotic liquid handling through a self‐contained platform would minimize contamination and technical burden 44–46 . Optimizing the workflow to enable testing of unextracted samples would further improve the processing burden 39 .…”

Section: Discussionmentioning

confidence: 99%

“…Future automation by strategies such as magnetic processing or robotic liquid handling through a self‐contained platform would minimize contamination and technical burden. 44 , 45 , 46 Optimizing the workflow to enable testing of unextracted samples would further improve the processing burden. 39 The ligation‐based assay may also benefit from adopting alternative amplification and detection strategies that do not require a real‐time PCR instrument.…”

Section: Discussionmentioning

confidence: 99%

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Ligation‐based assay for variant typing without sequencing: Application to SARS‐CoV‐2 variants of concern

Nelson

Shilts

Pakala

et al. 2022

Influenza Resp Viruses

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Background COVID‐19 prevalence has remained high throughout the pandemic with intermittent surges, due largely to the emergence of genetic variants, demonstrating the need for more accessible sequencing technologies for strain typing. Methods A ligation‐based typing assay was developed to detect known variants of severe acute respiratory syndrome virus 2 (SARS‐CoV‐2) by identifying the presence of characteristic single‐nucleotide polymorphisms (SNPs). General principles for extending the strategy to new variants and alternate diseases with SNPs of interest are described. Of note, this strategy leverages commercially available reagents for assay preparation, as well as standard real‐time polymerase chain reaction (PCR) instrumentation for assay performance. Results The assay demonstrated a combined sensitivity and specificity of 96.6% and 99.5%, respectively, for the classification of 88 clinical samples of the Alpha, Delta, and Omicron variants relative to the gold standard of viral genome sequencing. It achieved an average limit of detection of 7.4 × 104 genome copies/mL in contrived nasopharyngeal samples. The ligation‐based strategy performed robustly in the presence of additional polymorphisms in the targeted regions of interest as shown by the sequence alignment of clinical samples. Conclusions The assay demonstrates the potential for robust variant typing with performance comparable with next‐generation sequencing without the need for the time delays and resources required for sequencing. The reduced resource dependency and generalizability could expand access to variant classification information for pandemic surveillance.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Ligation‐based assay for variant typing without sequencing: Application to SARS‐CoV‐2 variants of concern

Nelson

Shilts

Pakala

et al. 2022

Influenza Resp Viruses

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“…The three sequential reactions are initially optimized for reaction product transfer by direct manual pipetting, and then a magnetic bead transfer is incorporated into this assay to overcome buffer incompatibility and increase product transfer. Combined with our prior work demonstrating magnetic bead transfer in a closed system without pipetting, − the improvement in analytical sensitivity provides a pathway for the incorporation of SNP detection into resource-limited settings with access to standard PCR instrumentation.…”

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confidence: 99%

Magnetic Bead Processing Enables Sensitive Ligation-Based Detection of HIV Drug Resistance Mutations

et al. 2022

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HIV develops single nucleotide polymorphisms (SNPs), some of which lead to drug resistance mutations (DRMs) that prevent therapeutic viral suppression. Genomic sequencing enables healthcare professionals to select effective combination antiretroviral therapy (ART) to achieve and maintain viral suppression. However, sequencing technologies, which are resource-intensive, are limited in their availability. This report describes the first step toward a highly specific ligation-based SNP discrimination method with endpoint PCR detection, which is more suitable for resource-limited clinics. The approach is based on magnetic bead processing to maximize reaction product transfer and minimize the carryover of incompatible buffer for three consecutive enzymatic reactionsreverse transcription (RT), oligonucleotide ligation assay (OLA), and PCR. The method improved PCR detection following RT → OLA by 8.06 cycles (∼250-fold) compared to direct pipette processing and detected between 103 and 104 RNA copies per reaction. In studies with synthesized nucleic acids based on the well-studied HIV mutation, K103N, the assay successfully differentiated between wild-type and mutant for RNA targets with high specificity. With further development, this design provides a pathway for SNP detection with more accessible PCR instrumentation and is a step toward a self-contained processing approach that incorporates the SNP specificity of the ligation reaction for more effective clinical management of DRMs in resource-constrained settings.

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“…Since the simulation results were promising, a laboratory sample of the biosensor was designed and manufactured, but further experiments are needed. In a similar line of research, Zimmers and co-workers [ 12 ] conceived a novel diagnostic platform for the detection of target DNA. In brief, the target DNA is captured by magnetic beads and then loaded into a microfluidic reaction tape with the other reaction solutions.…”

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confidence: 99%

Editorial for the Special Issue on Micro/Nanofluidic and Lab-on-a-Chip Devices for Biomedical Applications

2022

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Development of an Automated, Non-Enzymatic Nucleic Acid Amplification Test

Cited by 3 publications

References 34 publications

Ligation‐based assay for variant typing without sequencing: Application to SARS‐CoV‐2 variants of concern

Ligation‐based assay for variant typing without sequencing: Application to SARS‐CoV‐2 variants of concern

Magnetic Bead Processing Enables Sensitive Ligation-Based Detection of HIV Drug Resistance Mutations

Editorial for the Special Issue on Micro/Nanofluidic and Lab-on-a-Chip Devices for Biomedical Applications

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