Rapid and simple detection of Phytophthora cactorum in strawberry using a coupled recombinase polymerase amplification–lateral flow strip assay

Lu, Xinyu; Xu, Heng; Song, Wen; Yang, Zitong; Yu, Jia; Tian, Yuee; Jiang, Min; Shen, Danyu; Dou, Daolong

doi:10.1186/s42483-021-00089-8

Cited by 21 publications

(13 citation statements)

References 25 publications

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“…faecium and vanA and vanB genes) were 37 °C for 20 min. Several investigations have shown that the RPA-LF can function well at temperatures ranging from 25–45 °C ( Lu et al, 2021 ; Peng et al, 2019 ) and the faint band was visible in 5 min ( Du et al, 2018a ; Peng et al, 2019 ; Srisrattakarn et al, 2020 ; Wu et al, 2020 ). Similarly, our study found that the RPA worked effectively at a wide temperature range (25–42 °C) and incubation times of 5-30 min could generate a visual signal on the test lines.…”

Section: Discussionmentioning

confidence: 99%

Rapid detection ofEnterococcusand vancomycin resistance using recombinase polymerase amplification

Panpru

Srisrattakarn

Panthasri

et al. 2021

PeerJ

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Vancomycin-resistant enterococci (VRE), especially Enterococcus faecium, have been a global concern, often causing serious healthcare-associated infections. We established a rapid approach for detecting E. faecium and vancomycin-resistance genes (vanA and vanB) in clinical samples using isothermal recombinase polymerase amplification (RPA) combined with a lateral-flow (LF) strip. Specific RPA primer sets and probes for ddl (to identify the presence of E. faecium) vanA and vanB genes were designed. The RPA reaction was performed under isothermal condition at 37 °C within 20 min and read using the LF strip within a further 5 min. A total of 141 positive blood-cultures and 136 stool/rectal swab samples were tested using RPA-LF method compared to the conventional PCR method. The RPA-LF method exhibited 100% sensitivity in both blood-culture (60 E. faecium; 35 vanA type and two vanB type) and stool/rectal-swab samples (63 E. faecium and 36 vanA type) without cross-reaction (100% specificity). The lower detection limit of the RPA-LF was approximately 10 times better than that of the conventional PCR method. The RPA-LF method is an alternative rapid method with excellent sensitivity and specificity for detecting E. faecium, vanA, and vanB, and it has the potential to be used as a point-of-care device for VRE therapy and prevention.

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Section: Discussionmentioning

confidence: 99%

Rapid detection ofEnterococcusand vancomycin resistance using recombinase polymerase amplification

Panpru

Srisrattakarn

Panthasri

et al. 2021

PeerJ

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“…First, all these tests were performed with the following commercial RPA kits: TwistDx nfo [27,84,[208][209][210][211], TwistDx Basic [212][213][214][215], TwistDx Exo [51], and Amplifier (Agdia) [27,92,[216][217][218]. The main criterion for the selection of a gene as a target for RPA was maximal specificity for the species of interest.…”

Section: Rpa-based Testsmentioning

confidence: 99%

“…Some tests utilize anti-digoxygenin conjugate with carbon NPs in the test zone [211]. The commercial LFA kits were purchased from Millenia [51,[208][209][210], PCRD nucleic acid detector [84,211,222], BioUSTAR [26], and Agdia [27,92,[216][217][218] or assembled and optimized from different membranes (homemade test strips) [212][213][214]. RPA contains a large amount of DTT that induces aggregation of some GNP conjugates, such as streptavidin-GNP [87].…”

Section: Rpa-based Testsmentioning

confidence: 99%

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Иванов

Safenkova

Zherdev

et al. 2021

Plants

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Rapid, sensitive, and timely diagnostics are essential for protecting plants from pathogens. Commonly, PCR techniques are used in laboratories for highly sensitive detection of DNA/RNA from viral, viroid, bacterial, and fungal pathogens of plants. However, using PCR-based methods for in-field diagnostics is a challenge and sometimes nearly impossible. With the advent of isothermal amplification methods, which provide amplification of nucleic acids at a certain temperature and do not require thermocyclic equipment, going beyond the laboratory has become a reality for molecular diagnostics. The amplification stage ceases to be limited by time and instruments. Challenges to solve involve finding suitable approaches for rapid and user-friendly plant preparation and detection of amplicons after amplification. Here, we summarize approaches for in-field diagnostics of phytopathogens based on different types of isothermal amplification and discuss their advantages and disadvantages. In this review, we consider a combination of isothermal amplification methods with extraction and detection methods compatible with in-field phytodiagnostics. Molecular diagnostics in out-of-lab conditions are of particular importance for protecting against viral, bacterial, and fungal phytopathogens in order to quickly prevent and control the spread of disease. We believe that the development of rapid, sensitive, and equipment-free nucleic acid detection methods is the future of phytodiagnostics, and its benefits are already visible.

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“…Based on this technology, a new isothermal DNA amplification technique, recombinase polymerase amplification (RPA), has emerged (Piepenburg et al., 2006). Compared with LAMP, RPA has no strict requirements on template integrity, reaction temperature and reaction time, so it has application potential in portable nucleic acid detection (Lu et al., 2021). The establishment of a rapid, economical and simple technology for the detection of F .…”

Section: Introductionmentioning

confidence: 99%

“…Compared with LAMP, RPA has no strict requirements on template integrity, reaction temperature and reaction time, so it has application potential in portable nucleic acid detection (Lu et al, 2021). The establishment of a rapid, economical and simple technology for the detection of F. fujikuroi in rice seeds and in water samples during seed soaking could provide accurate guidance for the prevention and treatment of bakanae disease.…”

mentioning

confidence: 99%

Rapid detection of Fusarium fujikuroi in rice seeds and soaking water samples based on recombinase polymerase amplification‐lateral flow dipstick

Zhang,

Jin,

et al. 2023

Plant Pathology

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Bakanae disease is a rice seedborne disease caused by the Fusarium (Gibberella) fujikuroi species complex (FFSC), among which F. fujikuroi is the dominant pathogen. Pathogens usually hide inside or on the surface of seeds, and infection occurs mainly at the germination stage. In this study, a method for the detection of F. fujikuroi in rice seeds and seed soaking water samples was established using recombinase polymerase amplification (RPA) technology with lateral flow device (LFD) chromatography test strips. A pair of specific primers and probes based on the cyp51c gene were screened. RPA‐LFD was used to detect 10 F. fujikuroi strains, and the results showed that all of them tested positive and there was no cross‐reaction with other Fusarium or non‐Fusarium species. The target production of the RPA‐LFD assay was obtained at 35–45°C for 8–14 min, and optimal reaction conditions of amplification at 39°C for 8 min is recommended. The sensitivity test showed that the detection limit of the RPA‐LFD test for F. fujikuroi genomic DNA in rice seeds was 100 fg/μL, and the detection limit for F. fujikuroi spores in submerged water samples was 100 spores/mL. In the assay for field samples, it successfully detected F. fujikuroi carried in the seeds of three out of five rice varieties. In addition, the whole RPA‐LFD assay can specifically detect F. fujikuroi within 30 min. This method is expected to become an early field monitoring tool for rice bakanae disease.

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Rapid and simple detection of Phytophthora cactorum in strawberry using a coupled recombinase polymerase amplification–lateral flow strip assay

Cited by 21 publications

References 25 publications

Rapid detection ofEnterococcusand vancomycin resistance using recombinase polymerase amplification

Rapid detection ofEnterococcusand vancomycin resistance using recombinase polymerase amplification

The Potential Use of Isothermal Amplification Assays for In-Field Diagnostics of Plant Pathogens

Rapid detection of Fusarium fujikuroi in rice seeds and soaking water samples based on recombinase polymerase amplification‐lateral flow dipstick

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