A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, <i>Phytophthora hibernalis</i>

Dai, Tingting; Hu, Tao; Yang, Xiao; Shen, Danyu; Jiao, Binbin; Xu, Yue

doi:10.7717/peerj.8083

Cited by 19 publications

(19 citation statements)

References 26 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The design of species-specific primers is critical for P. cactorum detection. The nucleotide sequence of the Ypt1 gene varies sufficiently among Phytophthora species, and therefore has been widely used as a target for molecular detection of Phytophthora pathogens (Konig et al 2015 ; Dai et al 2019b ; Yu et al 2019 ; Lu et al 2020 ). In this study, we also designed specific LF-RPA primers based on the Ypt1 gene for P. cactorum detection.…”

Section: Discussionmentioning

confidence: 99%

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

Song

et al. 2021

Phytopathol Res

Self Cite

View full text Add to dashboard Cite

Phytophthora cactorum is a devastating pathogen that infects a wide range of plants and causes Phytophthora rot disease, which has resulted in great economic losses in crop production. Therefore, the rapid and practicable detection of P. cactorum is important for disease monitoring and forecasting. In this study, we developed a lateral flow recombinase polymerase amplification (LF-RPA) assay for the sensitive visual detection of P. cactorum. Specific primers for P. cactorum were designed based on the ras-related protein gene Ypt1; all 10 P. cactorum isolates yielded positive detection results, whereas no cross-reaction occurred in related oomycete or fungal species. The detection limit for the LF-RPA assay was 100 fg of genomic DNA under optimized conditions. Combined with a simplified alkaline lysis method for plant DNA extraction, the LF-RPA assay successfully detected P. cactorum in naturally diseased strawberry samples without specialized equipment within 40 min. Thus, the LF-RPA assay developed in this study is a rapid, simple, and accurate method for the detection of P. cactorum, with the potential for further application in resource-limited laboratories.

show abstract

Section: Discussionmentioning

confidence: 99%

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

Song

et al. 2021

Phytopathol Res

Self Cite

View full text Add to dashboard Cite

show abstract

“…www.nature.com/scientificreports/ be performed within a broad range of temperatures (37-42 °C) 42 . The reaction needed a single set of primers, whereas LAMP needs as many as four to six primers 39 . The quick lateral flow immunochromatographic assay optimized using the Twist Amp nfo probe which saved an additional 60 to 80 min compared to RPA where products have to be visualized by agarose gel electrophoresis.…”

Section: Discussionmentioning

confidence: 99%

“…The RPA approach is an emerging isothermal, low cost, rapid, and point-of-care diagnostic tool 23 . It is a highly sensitive, reliable nucleic acid based method and has become a rapid detection tool for many pathogens including viruses 34 , bacteria 19 , 27 , and Phytophthora species 39 . It is also used for detection of RNA viruses without the need for a separate step to synthesize cDNA by RT-RPA 21 , 36 , 37 , 40 .…”

Section: Discussionmentioning

confidence: 99%

Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus

Ghosh

Kokane

Gowda

2020

Sci Rep

View full text Add to dashboard Cite

Tristeza is a highly destructive disease of citrus caused by the phloem-limited, flexuous filamentous Citrus tristeza virus (CTV) in the genus Closterovirus and the family Closteroviridae. It has been a major constraint for higher productivity and has destroyed millions of citrus trees globally. CTV is graft transmissible and spread through use of virus infected nursery plants. Therefore, virus detection by using specific and reliable diagnostic tools is very important to mitigate disease outbreaks. Currently, the standard molecular techniques for CTV detection include RT-PCR and RT-qPCR. These diagnostic methods are highly sensitive but time consuming, labor intensive and require sophisticated expensive instruments, thus not suitable for point-of-care use. In the present study, we report the development of a rapid, sensitive, robust, reliable, and highly specific reverse transcription-RPA technique coupled with a lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA). RT-RPA technique was standardized to amplify the coat protein gene of CTV (CTV-p25) and detect double labeled amplicons on a sandwich immunoassay by designing specific labeled primer pair and probe combinations. The optimally performing primer set (CTRPA-F1/CTRPA-R9-Btn) and the corresponding TwistAmp nfo probe (CTRPA-Probe) was optimized for temperature and reaction time using purified cDNA and viral RNA as template. The sensitivity of the developed assay was compared with other detection techniques using in vitro-transcribed RNA. The efficacy and specificity of the assay was evaluated using CTV positive controls, healthy samples, field grown citrus plants of unknown status, and other virus and bacterial pathogens that infect citrus plants. The RT-RPA-LFICA was able to detect ≤ 141 fg of RNA when cDNA used as a template. The assay detected ≤ 0.23 ng/µl of CTV RNA when directly used as template without cross-reactivity with other citrus pathogens. Best results were achieved at the isothermal temperature of 40 °C within 15–20 min. The study demonstrated that RT-RPA-LFICA has potential to become an improved detection technique for end users in bud-wood certification and quarantine programs and a promising platform for rapid point-of-care diagnostics for citrus farmers and small nurseries in low resource settings.

show abstract

“…Recently, Phytophthora species-specific RPA assays targeting the single copy gene Ypt1 have been developed Fig. 3 Amplification curves of RPA assay for concentrations ranging from 1 ng to 10 fg purified genomic DNA of P. fragariae (Dai et al 2019a;Dai et al 2019b;Yu et al 2019). In contrast, the mitochondrial atp9-nad9 marker is a multicopy locus and exhibits enough sequence divergence for designing assay on species-level identification of Phytophthora.…”

Section: Discussionmentioning

confidence: 99%

Development and evaluation of a recombinase polymerase amplification assay for rapid detection of strawberry red stele pathogen

et al. 2020

View full text Add to dashboard Cite

Phytophthora fragariae causes drastic damage in strawberry crops. P. fragariae infects strawberry roots and causes red stele root rot. Although P. fragariae is a quarantine organism, its spread in Finland continues as more and more fields contract the disease. The spread can be halted through developing rapid and reliable detection assays. We have developed a rapid recombinase polymerase amplification (RPA) assay for P. fragariae targeting the Phytophthora mitochondrial DNA intergenic atp9-nad9 marker. The assay is DNA-extraction free and capable of detecting as low as 10 fg of P. fragariae genomic DNA. We found the assay reliable for diagnosing field plants when samples are adequately collected. We also applied the RPA assay to the detection of the pathogen in the soil through coupling the assay with baiting with the host plant. The results suggest that if only a small number of samples are analysed, the baiting results will not be reliable.

show abstract

A recombinase polymerase amplification-lateral flow dipstick assay for rapid detection of the quarantine citrus pathogen in China, Phytophthora hibernalis

Cited by 19 publications

References 26 publications

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

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

Development of a reverse transcription recombinase polymerase based isothermal amplification coupled with lateral flow immunochromatographic assay (CTV-RT-RPA-LFICA) for rapid detection of Citrus tristeza virus

Development and evaluation of a recombinase polymerase amplification assay for rapid detection of strawberry red stele pathogen

Contact Info

Product

Resources

About