Recombinase polymerase amplification in minimally buffered conditions

Tomar, Saurabh; Lavickova, Barbora; Guiducci, Carlotta

doi:10.1016/j.bios.2021.113802

Cited by 18 publications

(8 citation statements)

References 23 publications

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“…M: DL5000 DNA marker; NC: negative control; 1-8: plasmid concentration ranges from 2.35×10 5 pg/mL-2.35×10 -2 pg/mL. many researchers have conducted studies regarding the optimization of this method, including buffered conditions, specificity enhancement, stirred conditions, and additional methods of detection such as RPA combined with LFD (Moody et al, 2016;Luo et al, 2019;Tomar et al, 2022). It is worth noting that combination of RPA with the latest CRISPR technology has been applied to various bioassay fields (Patchsung et al, 2020;Meng et al, 2021;Jiang H.J.…”

Section: Discussionmentioning

confidence: 99%

Establishment of methods for rapid detection of Prymnesium parvum by recombinase polymerase amplification combined with a lateral flow dipstick

Luo

Huang²,

Jiang³

2022

Front. Mar. Sci.

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Prymnesium parvum is a toxic algal bloom (HAB)-forming species. The toxicity of this alga is a result of a collection of compounds known as prymnesins. Prymnesins exert harmful effects upon fish, shellfish, and mollusks, causing huge economic losses. In the present study, a new method was developed for the detection of P. parvum. The novel method utilizes isothermal amplification, known as recombinase polymerase amplification (RPA), in combination with lateral-flow dipstick (LFD). Herein, a set of primers and probes were designed for internal transcribed spacer (ITS) sequences, and a specific and sensitive RPA-LFD rapid detection method was established for P. parvum. Meanwhile, we verified its feasibility for the detection of environmental samples. It was demonstrated that the optimal amplification temperature and time for RPA were 39°C and 15 min. RPA/RPA-LFD was experimentally verified to be specific, demonstrating no cross-reaction with distinct control microalgae, and furthermore, the total time required for the RPA-LFD experiment was 20 min. Meanwhile, the detection limit for the genomic DNA of P. parvum was 1.5×10-1 pg/μL, and the detection limit for plasmids was 2.35 pg/μL. In addition, the results herein revealed that the RPA-LFD assay was 100 times more sensitive than PCR for detection of P. parvum. In conclusion, we developed an RPA-LFD that does not require precision instruments, and can be utilized for rapid on-site detection of P. parvum. In the future, the RPA-LFD can be considered for practical application for environmental detection of the toxic algal species.

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Section: Discussionmentioning

confidence: 99%

Establishment of methods for rapid detection of Prymnesium parvum by recombinase polymerase amplification combined with a lateral flow dipstick

Luo

Huang²,

Jiang³

2022

Front. Mar. Sci.

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“…A recent study by Tomar et al initially investigated the mechanism of pH changes in RPA assays and provided a valuable guide for the in-depth study of RPA. 15 Additionally, clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems consisting of guide RNA and Cas protein have become increasingly widespread for in vitro diagnostics. Since the sensitivity provided by CRISPR/Cas systems does not meet the detection needs for specific targets, such as the infectious diseases discussed here, the combination of CRISPR/Cas systems and nucleic acid amplification has become an important trend to improve analytical performance further, and has been applied to various detection platforms like microfluidics and biosensors.…”

Section: A Brief Overview Of Detection Methodsmentioning

confidence: 99%

Point-of-care testing of infectious diseases: recent advances

Shang

Guo

2023

Sens. Diagn.

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“…A typical example includes the use of nanomaterial or enzyme-based post-amplication means. [14][15][16] But the preparation of nanoprobes or enzyme-labeling complicates the assay process and also, the large quantities of reagents introduced might easily interfere with the assay reliability. Alternatively, some DNA-based isothermal amplication strategies, for example, rolling circle amplication (RCA), loopmediated isothermal amplication (LAMP), helicase dependent amplication (HDA), recombinase polymerase amplication (RPA), and strand displacement amplication (SDA), are popular for sensitivity improvement.…”

Section: Introductionmentioning

confidence: 99%