Rapid and visual detection of Meloidogyne hapla using recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay

Plant Disease ◽  
2020 ◽  
Author(s):  
Zhiqiang Song ◽  
Xiai Yang ◽  
Xiaowei Zhang ◽  
Mingbao Luan ◽  
Bing Guo ◽  
...  
Keyword(s):  
Rapid Detection ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Meloidogyne Hapla ◽  
Root Knot Nematode ◽  
Resource Limited ◽  
Lateral Flow Dipstick ◽  
Wide Range ◽  
And Control

The northern root-knot nematode, Meloidogyne hapla, is a biotrophic parasite that infects many crops and causes severe economic losses worldwide. Rapid and accurate detection of M. hapla is crucial for disease forecasting and control. We developed a recombinase polymerase amplification combined with a lateral flow dipstick (RPA-LFD) assay for rapid detection of M. hapla. The primers and a probe were designed based on the effector gene 16D10 sequence and were highly specific to M. hapla. The RPA reaction was performed at a wide range of temperatures from 25 to 45°C within 5 to 25 min, and the amplicon was visualized directly on the LFD within 5 min. The detection limits of the RPA-LFD assay were 10-3 female and 10-2 J2/0.5 g of soil, which was 10 times more sensitive than the conventional PCR assay. In addition, the RPA-LFD assay can detect M. hapla from infested plant roots and soil samples, and the entire detection process can be completed within 1.5 h. These results indicate that the RPA-LFD assay is a simple, rapid, specific, sensitive, and visual method that can be used for rapid detection of M. hapla in the field and in resource-limited conditions.

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

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Xinyu Lu ◽  
Heng Xu ◽  
Wen Song ◽  
Zitong Yang ◽  
Jia Yu ◽  
...  
Keyword(s):  
Crop Production ◽  
Accurate Method ◽  
Fungal Species ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Phytophthora Cactorum ◽  
Lateral Flow Strip ◽  
Alkaline Lysis ◽  
Wide Range

AbstractPhytophthora 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.

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

PeerJ ◽  
2019 ◽  
Vol 7 ◽  
pp. e8083 ◽  
Author(s):  
Tingting Dai ◽  
Tao Hu ◽  
Xiao Yang ◽  
Danyu Shen ◽  
Binbin Jiao ◽  
...  
Keyword(s):  
Reaction System ◽  
Citrus Fruit ◽  
Pcr Amplification ◽  
Brown Rot ◽  
Fungal Species ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Isolation And Identification ◽  
Resource Limited ◽  
Lateral Flow Dipstick

Phytophthora hibernalis, the causal agent of brown rot of citrus fruit, is an important worldwide pathogen and a quarantine pest in China. Current diagnosis of the disease relies on disease symptoms, pathogen isolation and identification by DNA sequencing. However, symptoms caused by P. hibernalis can be confused with those by other Phytophthora and fungal species. Moreover, pathogen isolation, PCR amplification and sequencing are time-consuming. In this study, a rapid assay including 20-min recombinase polymerase amplification targeting the Ypt1 gene and 5-min visualization using lateral flow dipsticks was developed for detecting P. hibernalis. This assay was able to detect 0.2 ng of P. hibernalis genomic DNA in a 50-µL reaction system. It was specific to P. hibernalis without detection of other tested species including P. citrophthora, P. nicotianae, P. palmivora and P. syringae, four other important citrus pathogens. Using this assay, P. hibernalis was also detected from artificially inoculated orange fruits. Results in this study indicated that this assay has the potential application to detect P. hibernalis at diagnostic laboratories and plant quarantine departments of customs, especially under time- and resource-limited conditions.

Development of a reverse-transcription recombinase polymerase amplification assay with a lateral flow assay for rapid detection of avian orthoavulavirus 1

2021 ◽  
Vol 33 (2) ◽  
pp. 308-312
Author(s):  
Jindai Fan ◽  
Wenxian Chen ◽  
Yuanyuan Zhang ◽  
Zhixiang Liu ◽  
Xiaoming Li ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Newcastle Disease ◽  
Cross Reactivity ◽  
Lateral Flow ◽  
Lateral Flow Assay ◽  
Recombinase Polymerase Amplification ◽  
Control Measures ◽  
Economic Losses ◽  
Resource Limited ◽  
Simple Detection

Newcastle disease is an avian infectious disease caused by avian orthoavulavirus 1, also known as Newcastle disease virus (NDV). This disease has caused significant economic losses to the poultry industry worldwide. The rapid and simple detection of NDV infection is crucial to inform the appropriate control measures. We developed a reverse-transcription recombinase polymerase amplification (RT-RPA) assay combined with a lateral flow assay (LFA) for NDV detection. The RPA assay can be completed at 37°C within 20 min, and the RPA result can be visualized by the LFA within 5 min. The NDV RT-RPA-LFA detected NDV specifically with no cross-reactivity with other pathogens. The detection limit of NDV cDNA with our RT-RPA-LFA was 3.34 × 10−3 ng/μL. Consequently, the RT-RPA-LFA showed good potential for the detection of NDV infection in the field, especially in resource-limited settings.

scholarly journals Development and evaluation of recombinase polymerase amplification combined with lateral flow dipstick assays for co-detection of epizootic haemorrhagic disease virus and the Palyam serogroup virus

2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Zhuo-ran Li ◽  
Zhen-xing Yang ◽  
Zhan-hong Li ◽  
Xiang Gao ◽  
Zhong-yan Hu ◽  
...  
Keyword(s):  
Disease Virus ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Economic Losses ◽  
Blood Samples ◽  
Qrt Pcr ◽  
Lateral Flow Dipstick ◽  
Co Detection ◽  
Haemorrhagic Disease ◽  
Analytical Sensitivities

Abstract Background Epizootic haemorrhagic disease virus (EHDV) and the Palyam serogroup viruses (PALV) have led to significant economic losses associated with livestock production globally. A rapid, sensitive and specific method for the detection of EHDV and PALV is critical for virus detection, monitoring, and successful control and elimination of related diseases. Results In the present study, a recombinase polymerase amplification combined with lateral flow dipstick (RPA-LFD) assay for the co-detection of genome segment 1 (Seg-1) of EHDV and PALV was developed and evaluated. The analytical sensitivities of the established RPA-LFD assay in the detection of EHDV and PALV were 7.1 copies/µL and 6.8 copies/µL, respectively. No cross-reaction with other members of the genus Orbivirus, including African horse sickness virus, bluetongue virus, Guangxi orbivirus, Tibet orbivirus and Yunnan orbivirus was observed. The established RPA-LFD assay accurately detected 39 EHDV strains belonging to 5 serotypes and 29 PALV strains belonging to 3 serotypes. The trace back results of quantitative real-time polymerase chain reaction (qRT-PCR) and the established RPA-LFD assay on sentinel cattle were consistent. The coincidence rates of qRT-PCR and the established RPA-LFD assay in 56 blood samples from which EHDV or PALV had been isolated and 96 blood samples collected from cattle farms were more than 94.8 %. The results demonstrated that the established RPR-LFD assay is specific, sensitive and reliable, and could be applied in early clinical diagnosis of EHDV and PALV. Conclusions This study highlights the development and application of the RPA-LFD assay in the co-detection of EHDV and PALV for the first time. The assay could be used as a potential optional rapid, reliable, sensitive and low-cost method for field diagnosis of EHDV and PALV.

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