Development of an isothermal recombinase polymerase amplification assay for rapid detection of pseudorabies virus

Yang, Yang; Qin, Xiaodong; Zhang, Wei; Li, Zhiyong; Zhang, Shuaijun; Li, Yanmin; Zhang, Zhidong

doi:10.1016/j.mcp.2017.03.005

Cited by 22 publications

(13 citation statements)

References 8 publications

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“…However, several research groups have studied RPA reaction temperatures that lie outside of the recommended range. 38,44,45,60,[62][63][64][65][66][67][68][69][70][71][72][73][74][75][76][77][78] The largest temperature range was tested between 15°C and 50°C; 62,64,69,70,76 and results indicated the marginal reaction temperature to produce a positive result should be greater than 30°C. [62][63][64]66,67,69,71,74,76,77 However, Sun et al 65 and Poulton and Webster 60 showed that temperature as low as 25°C could still generate a positive signal after RPA amplification and subsequent lateral flow strip detection.…”

Section: Influence Of Temperature and Agitationmentioning

confidence: 99%

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification

Macdonald

Stetten

2019

Analyst

459

365

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Section: Influence Of Temperature and Agitationmentioning

confidence: 99%

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification

Macdonald

Stetten

2019

Analyst

459

365

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“…The displaced template, interacting with the single strand DNA binding (SSB) protein, retains a stable structure, and then extends as the way of PCR by the DNA polymerase when the recombinase is separated from 3’end of the oligonucleotide ( Del Rio et al, 2017 ; Vasileva Wand et al, 2018 ). Since the appearance of the RPA commercial kit from TwistAmp Basic (TWISTDX Ltd, Babraham, UK) in 2014, this rapid amplification technique has been widely used in diagnosis of numerous pathogens, such as human immunodeficiency virus 1 (HIV-1), Ebola virus, Rift Valley fever virus, dengue virus (DENV), pseudorabies virus (PrV), foot-and-mouth disease virus (FMDV), middle east respiratory syndrome coronavirus (MERS-CoV) and so on ( Boyle et al, 2013 ; James et al, 2018 ; Liu et al, 2018 ; Tan et al, 2018 ; Yang et al, 2017 ).…”

Section: Introductionmentioning

confidence: 99%

A novel recombinase polymerase amplification assay for rapid detection of epidemic fowl adenovirus

Liu

et al. 2020

Poultry Science

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Fowl adenovirus (FAdV) has posed a grave threat to the health of poultry and the sudden outbreak highlights the importance of the new rapid diagnostic method for the control and prevention of transmission. Hence, in the present study, a novel recombinase polymerase amplification (RPA) assay, which was suitable for all 12 serotypes (FAdV-1 to 8a and 8b to 11) had been successfully launched to detect FAdV. Also, the entire amplification process could be completed in the isothermal condition when temperature ranged from 26 to 42 °C within no more than 14 min, which was remarkably superior to end-point polymerase chain reaction (PCR, 98 min) with the same detecting sensitivity (as low as 0.1 fg viral DNA), avoiding sophisticated thermal cyclers with simple operation. Additionally, the same primers did not produce positive reactions with other viruses tested, demonstrating that the specificity of the RPA assay was acceptable. Moreover, this developed method could be efficiently used in the diagnosis of FAdV references and epidemic strains from different avian origins, thus making it a rapid, reliable and point-of-care FAdV diagnostics tool, as well as an alternative to end-point PCR.

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“…Traditionally, etiological diagnostic methods for PR include virus isolation [ 14 ], DNA hybridisation [ 15 – 17 ], immunofluorescence [ 17 ], loop-mediated isothermal amplification (LAMP) assay [ 18 ], real-time polymerase chain reaction (PCR) assay [ 19 ], and recombinase polymerase amplification assays (RPA) [ 20 ], none of which are able to distinguish between PRV strains. Several assays, including the LAMP assay [ 21 ], nanoparticle-assisted PCR assay [ 22 ], and two multiplex real-time PCR (qPCR) assays [ 23 ], have been developed to differentiate between wild-type (WT) PRV and gene-deleted PRV vaccines.…”

Section: Introductionmentioning

confidence: 99%

Duplex fluorescence melting curve analysis as a new tool for rapid detection and differentiation of genotype I, II and Bartha-K61 vaccine strains of pseudorabies virus

et al. 2018

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BackgroundRecently, pseudorabies (PR) outbreaks have been reported in a large number of swine herds vaccinated with the Bartha-K61 vaccine in China, the current pseudorabies virus (PRV) belonging to Genotype II is differential genetically from Bartha-K61 vaccine belonging to Genotype I. Furthermore, it has been proved that the Bartha-K61 vaccine cannot provide sufficient protection against the current PRVs in China. Therefore, the accurate and rapid identification of PRVs is essential. The objective of this study is to develop a duplex fluorescence melting curve analysis (FMCA) capable of rapid, simple, high-throughput differentiation of Chinese, European/American and Bartha-K61 vaccine strains of PRV.ResultsPrimers 6F/6R and probes P1/P2, combined with three recombinant plasmids p-B (Bartha-K61), p-N (Genotype I), and p-H (Genotype II), were used to establish the Bicolor FMCA. FAM Tm values (probe P1) and HEX (probe P2) channels of p-B were used as reference values. Tm differences (ΔTm) between detected samples and reference plasmid p-B were calculated in each channel. Bartha-K61 vaccine samples had ΔTm values of ±1 °C in both FAM and HEX channels, Genotype I samples had ΔTm values of ±1 °C in the FAM channel and 4.38 ± 1 °C in the HEX channel, and Genotype II samples had ΔTm values of 6.52 ± 1 °C in the FAM channel and 4.38 ± 1 °C in the HEX channel. The minimum detection limit of the duplex FMCA was approximately 1 × 100 copies per reaction for p-B, p-N, and p-H. The duplex FMCA technique was used to detect and different 198 suspected clinical samples, of which 18 (9%) were positive for Genotype II strains and eight (4%) were positive for Bartha-K61 vaccine strains, and the results were compared with sequencing and phylogenetic analyses, which confirmed that the Bicolor FMCA worked correctly for all samples.ConclusionsIn this study, we developed a duplex FMCA of dual-labeled, self-quenched probes that was performed for rapid detection and differentiation of Genotype I, II and Bartha-K61 vaccine strains of PRV. The duplex FMCA was rapid, simple, and high-throughput, and will likely prove useful for molecular epidemiological investigations and pathogen surveillance of PRV.Electronic supplementary materialThe online version of this article (10.1186/s12917-018-1697-4) contains supplementary material, which is available to authorized users.

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Development of an isothermal recombinase polymerase amplification assay for rapid detection of pseudorabies virus

Cited by 22 publications

References 8 publications

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification

Review: a comprehensive summary of a decade development of the recombinase polymerase amplification

A novel recombinase polymerase amplification assay for rapid detection of epidemic fowl adenovirus

Duplex fluorescence melting curve analysis as a new tool for rapid detection and differentiation of genotype I, II and Bartha-K61 vaccine strains of pseudorabies virus

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