Comparison Between Specific DNA-amplifications using Recombinase Polymerase Amplification (RPA) and using Polymerase Chain Reaction (PCR)

Min, Kim Jeng; 임수진,; truongatai,; 왕지희,; 이칠우,; Yoon, Byoung-Su

doi:10.17519/apiculture.2017.04.32.1.41

Cited by 2 publications

(2 citation statements)

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“…However, because of the method itself, it still has some defects. Such as RPA reactions produce more non-specific amplifications relative to PCR, a difficult phenomenon to avoid [28,51]. Therefore, it is necessary to screen primers in order to minimize the interference of non-specific amplification for subsequent experiments.…”

Section: Discussionmentioning

confidence: 99%

Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay for the Rapid and Sensitive Detection of Pseudo-nitzschia multiseries

Yao,

Luo,

Zong

et al. 2024

IJMS

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The harmful algal bloom (HAB) species Pseudo-nitzschia multiseries is widely distributed worldwide and is known to produce the neurotoxin domoic acid, which harms marine wildlife and humans. Early detection and preventative measures are more critical than late management. However, the major challenge related to early detection is the accurate and sensitive detection of microalgae present in low abundance. Therefore, developing a sensitive and specific method that can rapidly detect P. multiseries is critical for expediting the monitoring and prediction of HABs. In this study, a novel assay method, recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD), is first developed for the detection of P. multiseries. To obtain the best test results, several important factors that affected the amplification effect were optimized. The internal transcribed spacer sequence of the nuclear ribosomal DNA from P. multiseries was selected as the target region. The results showed that the optimal amplification temperature and time for the recombinase polymerase amplification (RPA) of P. multiseries were 37 °C and 15 min. The RPA products could be visualized directly using the lateral flow dipstick after only 3 min. The RPA-LFD assay sensitivity for detection of recombinant plasmid DNA (1.9 × 100 pg/μL) was 100 times more sensitive than that of RPA, and the RPA-LFD assay sensitivity for detection of genomic DNA (2.0 × 102 pg/μL) was 10 times more sensitive than that of RPA. Its feasibility in the detection of environmental samples was also verified. In conclusion, these results indicated that the RPA-LFD detection of P. multiseries that was established in this study has high efficiency, sensitivity, specificity, and practicability. Management measures made based on information gained from early detection methods may be able to prevent certain blooms. The use of a highly sensitive approach for early warning detection of P. multiseries is essential to alleviate the harmful impacts of HABs on the environment, aquaculture, and human health.

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Section: Discussionmentioning

confidence: 99%

Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay for the Rapid and Sensitive Detection of Pseudo-nitzschia multiseries

Yao,

Luo,

Zong

et al. 2024

IJMS

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show abstract

“…Unfortunately, RPA reactions produce more non-specific amplifications relative to PCR, a difficult phenomenon to avoid (Kim et al, 2017). Therefore, it is necessary to screen primers to minimize the interference of non-specific amplification for subsequent experiments.…”

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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Comparison Between Specific DNA-amplifications using Recombinase Polymerase Amplification (RPA) and using Polymerase Chain Reaction (PCR)

Cited by 2 publications

References 0 publications

Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay for the Rapid and Sensitive Detection of Pseudo-nitzschia multiseries

Recombinase Polymerase Amplification Combined with Lateral Flow Dipstick Assay for the Rapid and Sensitive Detection of Pseudo-nitzschia multiseries

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

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