A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions

Özay, Burcu; McCalla, Stephanie E.

doi:10.1016/j.snr.2021.100033

Cited by 78 publications

(56 citation statements)

References 189 publications

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“…3 c, g), six false positive results were obtained from the S. salar system using distilled water, indicating the occurrence of non-specific amplification, which has also been revealed in previous study ( Ye et al, 2018 ). With the aim to differentiate specific amplification from the undesired amplification, two common strategies were available: 1) target-specific detection through the use of target-specific probes or modified primers as biorecognition elements ( Tsugunori et al, 2000 ), 2) avoid to trigger the primer aggregation by adding gold nanoparticle, graphene oxide, and self-avoiding primers ( Özay & McCalla, 2021 ). Generally, the former one is more reliable and cost-effective, and even opens up new application opportunities in the field of multiplex detection.…”

Section: Resultsmentioning

confidence: 99%

Visual detection of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) simultaneously by duplex loop-mediated isothermal amplification

Xue

Fei

et al. 2022

Food Chemistry: Molecular Sciences

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Section: Resultsmentioning

confidence: 99%

Visual detection of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) simultaneously by duplex loop-mediated isothermal amplification

Xue

Fei

et al. 2022

Food Chemistry: Molecular Sciences

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“…concentrations) and reaction environment (e.g. buffer composition), often achieved through iterative experimental validation 3 . Taking the example of LAMP as a comparator, our system can use fewer primers (we only optimised two primer concentrations (1–2 μM) to increase sensitivity and limit false amplification, whilst LAMP requires an additional primer optimisation process, as both FIP/BIP and LP/BP are very important for efficiency).…”

Section: Discussionmentioning

confidence: 99%

“…Currently, the design and implementation of many new isothermal assays is based upon mechanisms involving steric and/or functional interactions between secondary structures of DNA strands 1 , 2 , which can be optimised iteratively to enable rapid nucleic acid analysis 3 . Mechanistically, such secondary structures lead to specific designated products, which are formed in the initial steps and are used as mediators in the subsequent amplification cycling steps.…”

Section: Introductionmentioning

confidence: 99%

Programmable design of isothermal nucleic acid diagnostic assays through abstraction-based models

Reboud

Guo

et al. 2022

Nat Commun

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Accelerating the design of nucleic acid amplification methods remains a critical challenge in the development of molecular tools to identify biomarkers to diagnose both infectious and non-communicable diseases. Many of the principles that underpin these mechanisms are often complex and can require iterative optimisation. Here we focus on creating a generalisable isothermal nucleic acid amplification methodology, describing the systematic implementation of abstraction-based models for the algorithmic design and application of assays. We demonstrate the simplicity, ease and flexibility of our approach using a software tool that provides amplification schemes de novo, based upon a user-input target sequence. The abstraction of reaction network predicts multiple reaction pathways across different strategies, facilitating assay optimisation for specific applications, including the ready design of multiplexed tests for short nucleic acid sequence miRNAs or for difficult pathogenic targets, such as highly mutating viruses.

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“…We selected several common PCR enhancers that are used to alleviate specificity issues in PCR amplification: DMSO, trehalose, Gly-Gly, betaine, 1,2-propanediol, as well as GITC which have been shown to improve amplification efficiency and/or specificity [10][11][12]. Furthermore, we checked whether specificity could be enhanced by increasing assay temperature or by using gRNAs with a shorter spacer sequence as previously observed for CRISPR/Cas9 specificity [13].…”

Section: Assay Optimisation For Efficient Discrimination Of Variantsmentioning

confidence: 99%

VarLOCK - sequencing independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance

Nan

Hoehn

Hardinge

et al. 2022

Preprint

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The COVID-19 pandemic continues to pose a threat to the general population. The ongoing vaccination programs provide protection to individuals and facilitate the opening of society and a return to normality. However, emergent and existing SARS-CoV-2 variants capable of evading the immune system endanger the efficacy of the vaccination strategy. To preserve the efficacy of SARS-CoV-2 vaccination globally, aggressive and effective surveillance for known and emerging SARS-CoV-2 Variants of Concern (VOC) is required. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. In this work, we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to allow rapid and sensitive discrimination of VOCs, that can be used at point of care and/or implemented in the pipelines of small or large testing facilities, and even determine proportion of VOCs in pooled population-level wastewater samples. This technology aims to complement the ongoing sequencing efforts to allow facile and, crucially, rapid identification of individuals infected with VOCs to help break infection chains. Here, we show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants. In addition, we show the rapid adaptability of the technique for new and emerging VOCs such as Omicron.

show abstract

A review of reaction enhancement strategies for isothermal nucleic acid amplification reactions

Cited by 78 publications

References 189 publications

Visual detection of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) simultaneously by duplex loop-mediated isothermal amplification

Visual detection of rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar) simultaneously by duplex loop-mediated isothermal amplification

Programmable design of isothermal nucleic acid diagnostic assays through abstraction-based models

VarLOCK - sequencing independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance

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