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 Rapid, Equipment-Free Method for Detecting Phytophthora infestans in the Field Using a Lateral Flow Strip-Based Recombinase Polymerase Amplification Assay

Plant Disease ◽  
2020 ◽  
Vol 104 (11) ◽  
pp. 2774-2778
Author(s):  
Xinyu Lu ◽  
Ying Zheng ◽  
Fan Zhang ◽  
Jia Yu ◽  
Tingting Dai ◽  
...  
Keyword(s):  
Late Blight ◽  
Phytophthora Infestans ◽  
Cost Effective ◽  
Fungal Species ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Lateral Flow Strip ◽  
Global Food Security ◽  
Wide Range ◽  
Field Samples

Late blight, caused by the oomycete Phytophthora infestans, is a major constraint on the production of potatoes and tomatoes as well as a constant threat to global food security. An early diagnostic tool is important for the effective management of late blight in the field. Here, in combination with a simplified DNA extraction method, we developed a lateral flow strip-based recombinase polymerase amplification (LF-RPA) assay for the rapid, equipment-free detection of P. infestans. This assay targets the Ras-related protein (Ypt1) gene and can be performed over a wide range of temperatures (25 to 45°C). All 12 P. infestans isolates yielded positive detection results using the LF-RPA assay, and no cross-reaction occurred with related oomycetes or fungal species. With this assay, the detection limit was 500 fg of genomic DNA in optimized conditions. Furthermore, by combining a simplified polyethylene glycol-NaOH method for extracting DNA from plant samples, the entire LF-RPA assay enabled the detection of P. infestans within 30 min with no specialized equipment. When applied to field samples, it successfully detected P. infestans in naturally diseased potato plants from eight different fields in China. Therefore, the LF-RPA assay is simple, rapid, and cost-effective and has potential for further development as a kit for diagnosing late blight in resource-limited settings or even on-site.

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.

Fungi Associated with Decay of Some Philippine Dipterocarps and Its Ecological Functions and Significance at Mt. Makiling, Laguna, Philippines

2012 ◽  
Vol 3 (1) ◽  
Author(s):  
Edwin R. Tadiosa ◽  
Ernesto P. Militante ◽  
Nelson M. Pampolina
Keyword(s):  
Wood Decay ◽  
Fungal Species ◽  
Economic Losses ◽  
Frequency Diversity ◽  
Ecological Functions ◽  
Transect Line ◽  
Wide Range ◽  
Integral Role ◽  
Harmful Organisms ◽  
Fungal Association

This study endeavored to document some of the fungal species affecting Philippine dipterocarps in Mt. Makiling. Collection of macroscopic fungi was conducted in five selected sites. A 1-2 km transect line was laid out per site, and four 10 x 10 m2 quadrat sampling plots were established to determine existing fungi. These fungi were quantified using ecological parameters such as density, frequency, diversity and evenness value. Twenty-seven species of macroscopic fungi belonging to fifteen genera were found associated with dipterocarps. These were characterized morphologically and anatomically prior to identification. The Ganoderma, Polystictus, Auricularia, Fomes and Polyporus species dominated the study areas. Among the dipterocarp host species, white lauan (Shorea contorta) has a wide range of fungal association. Decay capacity tests on heartwood and sapwood using inoculants were conducted in the laboratory on four dipterocarp species. Weight loss in percent, and absorbability of water in grams after 20 weeks of incubation were measured and compared. These fungi are commonly considered harmful organisms that cause great economic losses in wood. Nevertheless, they could be beneficial to the ecosystem. This permits the growth of young, vigorous trees and thereby, play an integral role in maintaining the dynamic and ever-changing nature of the forests. Keywords - Micology, Fungi, Wood Decay, Philippine Dipterocarps, Ecological Functions, Mt. Makiling

scholarly journals One-Step Reverse-Transcription Recombinase Polymerase Amplification Using Lateral Flow Strips for the Detection of Coxsackievirus A6

2021 ◽  
Vol 12 ◽  
Author(s):  
Jia Xie ◽  
Xiaohan Yang ◽  
Lei Duan ◽  
Keyi Chen ◽  
Pan Liu ◽  
...  
Keyword(s):  
Reverse Transcription ◽  
Clinical Performance ◽  
Quantitative Polymerase Chain Reaction ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Clinical Samples ◽  
Effective Vaccine ◽  
Lateral Flow Strip ◽  
Coxsackievirus A6 ◽  
One Step

Hand, foot, and mouth disease (HFMD) is a common infectious disease affecting mainly children under 5 years of age. Coxsackievirus A6 (CVA-6), a major causative pathogen of HFMD, has caused outbreaks in recent years. Currently, no effective vaccine or antiviral treatments are available. In this study, one-step reverse-transcription recombinase polymerase amplification (RT-RPA), combined with a disposable lateral flow strip (LFS) assay, was developed to detect CVA-6. This assay can be performed in less than 35 min at 37°C without expensive instruments, and the result can be observed directly with the naked eye. The sensitivity of the RT-RPA-LFS was 10 copies per reaction, which was comparable to that of the conventional real-time quantitative polymerase chain reaction (qPCR) assays. Moreover, the assay specificity was 100%. The clinical performance of the RT-RPA-LFS assay was evaluated using 142 clinical samples, and the coincidence rate between RT-RPA-LFS and qPCR was 100%. Therefore, our RT-RPA-LFS assay provides a simple and rapid approach for point-of-care CVA-6 diagnosis.

scholarly journals Detection of Helminth Ova in Wastewater Using Recombinase Polymerase Amplification Coupled to Lateral Flow Strips

Water ◽  
2020 ◽  
Vol 12 (3) ◽  
pp. 691
Author(s):  
Vivek B. Ravindran ◽  
Basma Khallaf ◽  
Aravind Surapaneni ◽  
Nicholas D. Crosbie ◽  
Sarvesh K. Soni ◽  
...  
Keyword(s):  
Wastewater Treatment ◽  
Wastewater Treatment Plants ◽  
Cost Effective ◽  
Lateral Flow ◽  
Recombinase Polymerase Amplification ◽  
Detection Methods ◽  
Water Current ◽  
Recycled Water ◽  
Lateral Flow Strip ◽  
Skilled Personnel

Ascaris lumbricoides is a major soil-transmitted helminth that is highly infective to humans. The ova of A. lumbricoides are able to survive wastewater treatment, thus making it an indicator organism for effective water treatment and sanitation. Hence, Ascaris ova must be removed from wastewater matrices for the safe use of recycled water. Current microscopic techniques for identification and enumeration of Ascaris ova are laborious and cumbersome. Polymerase chain reaction (PCR)-based techniques are sensitive and specific, however, major constraints lie in having to transport samples to a centralised laboratory, the requirement for sophisticated instrumentation and skilled personnel. To address this issue, a rapid, highly specific, sensitive, and affordable method for the detection of helminth ova was developed utilising recombinase polymerase amplification (RPA) coupled with lateral flow (LF) strips. In this study, Ascaris suum ova were used to demonstrate the potential use of the RPA-LF assay. The method was faster (< 30 min) with optimal temperature at 37 °C and greater sensitivity than PCR-based approaches with detection as low as 2 femtograms of DNA. Furthermore, ova from two different helminth genera were able to be detected as a multiplex assay using a single lateral flow strip, which could significantly reduce the time and the cost of helminth identification. The RPA-LF system represents an accurate, rapid, and cost-effective technology that could replace the existing detection methods, which are technically challenged and not ideal for on-site detection in wastewater treatment plants.

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.

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