skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • Rapid detection of viral, bacterial, fungal, and oomycete pathogens on tomato with microneedles, LAMP on a microfluidic chip, and smartphone device
Title: Rapid detection of viral, bacterial, fungal, and oomycete pathogens on tomato with microneedles, LAMP on a microfluidic chip, and smartphone device
Rapid detection of plant diseases before they escalate can improve disease control. Our team has developed rapid nucleic acid extraction methods with microneedles (MN) and combined these with LAMP assays for pathogen detection in the field. In this work, we developed LAMP assays for early blight (Alternaria linariae, A. alternata, and A. solani) and bacterial spot of tomato (Xanthomonas perforans) and validated these LAMP assays and two previously developed LAMP assays for tomato spotted wilt virus and late blight. Tomato plants were inoculated and disease severity was measured. Extractions were performed using MN and LAMP assays were run in tubes (with hydroxynaphthol blue) on a heat block or on a newly designed microfluidic slide chip on a heat block or a slide heater. Fluorescence on the microfluidic chip slides was visualized using EvaGreen and photographed on a smartphone. Plants inoculated with X. perforans or tomato spotted wilt virus tested positive prior to visible disease symptoms, while P. infestans and A. linariae were detected at the time of visual disease symptoms. LAMP assays were more sensitive than PCR and the limit of detection was 1 pg of DNA for both A. linariae and X. perforans. The LAMP assay designed for early blight detected all three species of Alternaria that infect tomato and is thus an Alternaria spp. assay. This study demonstrates the utility of rapid MN extraction followed by LAMP on a microfluidic chip for rapid diagnosis of four important tomato pathogens.  more » « less
Award ID(s):
2200038
PAR ID:
10527147
Author(s) / Creator(s):
; ; ; ;
Corporate Creator(s):
Editor(s):
NA
Publisher / Repository:
American Phytopathological Society
Date Published:
Journal Name:
Phytopathology®
ISSN:
0031-949X
Subject(s) / Keyword(s):
Detection. Phytophthora infestasn, TSWV, Alternaria sp,Xanthomonas perforans
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; (, Viruses)
    The novel coronavirus SARS-CoV-2 was first isolated in late 2019; it has spread to all continents, infected over 700 million people, and caused over 7 million deaths worldwide to date. The high transmissibility of the virus and the emergence of novel strains with altered pathogenicity and potential resistance to therapeutics and vaccines are major challenges in the study and treatment of the virus. Ongoing screening efforts aim to identify new cases to monitor the spread of the virus and help determine the danger connected to the emergence of new variants. Given its sensitivity and specificity, nucleic acid amplification tests (NAATs) such as RT-qPCR are the gold standard for SARS-CoV-2 detection. However, due to high costs, complexity, and unavailability in low-resource and point-of-care (POC) settings, the available RT-qPCR assays cannot match global testing demands. An alternative NAAT, RT-LAMP-based SARS-CoV-2 detection offers scalable, low-cost, and rapid testing capabilities. We have developed an automated RT-LAMP-based microfluidic chip that combines the RNA isolation, purification, and amplification steps on the same device and enables the visual detection of SARS-CoV-2 within 40 min from saliva and nasopharyngeal samples. The entire assay is executed inside a uniquely designed, inexpensive disposable microfluidic chip, where assay components and reagents have been optimized to provide precise and qualitative results and can be effectively deployed in POC settings. Furthermore, this technology could be easily adapted for other novel emerging viruses. 
    more » « less
  2. ; ; ; (, Biosensors)
    Hepatitis C virus (HCV) infections occur in approximately 3% of the world population. The development of an enhanced and extensive-scale screening is required to accomplish the World Health Organization’s (WHO) goal of eliminating HCV as a public health problem by 2030. However, standard testing methods are time-consuming, expensive, and challenging to deploy in remote and underdeveloped areas. Therefore, a cost-effective, rapid, and accurate point-of-care (POC) diagnostic test is needed to properly manage the disease and reduce the economic burden caused by high case numbers. Herein, we present a fully automated reverse-transcription loop-mediated isothermal amplification (RT-LAMP)-based molecular diagnostic set-up for rapid HCV detection. The set-up consists of an automated disposable microfluidic chip, a small surface heater, and a reusable magnetic actuation platform. The microfluidic chip contains multiple chambers in which the plasma sample is processed. The system utilizes SYBR green dye to detect the amplification product with the naked eye. The efficiency of the microfluidic chip was tested with human plasma samples spiked with HCV virions, and the limit of detection observed was 500 virions/mL within 45 min. The entire virus detection process was executed inside a uniquely designed, inexpensive, disposable, and self-driven microfluidic chip with high sensitivity and specificity. 
    more » « less
  3. ; ; ; ; ; ; ; ; ; ; et al (, The Analyst)
    Rapid, simple, inexpensive, accurate, and sensitive point-of-care (POC) detection of viral pathogens in bodily fluids is a vital component of controlling the spread of infectious diseases. The predominant laboratory-based methods for sample processing and nucleic acid detection face limitations that prevent them from gaining wide adoption for POC applications in low-resource settings and self-testing scenarios. Here, we report the design and characterization of an integrated system for rapid sample-to-answer detection of a viral pathogen in a droplet of whole blood comprised of a 2-stage microfluidic cartridge for sample processing and nucleic acid amplification, and a clip-on detection instrument that interfaces with the image sensor of a smartphone. The cartridge is designed to release viral RNA from Zika virus in whole blood using chemical lysis, followed by mixing with the assay buffer for performing reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) reactions in six parallel microfluidic compartments. The battery-powered handheld detection instrument uniformly heats the compartments from below, and an array of LEDs illuminates from above, while the generation of fluorescent reporters in the compartments is kinetically monitored by collecting a series of smartphone images. We characterize the assay time and detection limits for detecting Zika RNA and gamma ray-deactivated Zika virus spiked into buffer and whole blood and compare the performance of the same assay when conducted in conventional PCR tubes. Our approach for kinetic monitoring of the fluorescence-generating process in the microfluidic compartments enables spatial analysis of early fluorescent “bloom” events for positive samples, in an approach called “Spatial LAMP” (S-LAMP). We show that S-LAMP image analysis reduces the time required to designate an assay as a positive test, compared to conventional analysis of the average fluorescent intensity of the entire compartment. S-LAMP enables the RT-LAMP process to be as short as 22 minutes, resulting in a total sample-to-answer time in the range of 17–32 minutes to distinguish positive from negative samples, while demonstrating a viral RNA detection as low as 2.70 × 10 2 copies per μl, and a gamma-irradiated virus of 10 3 virus particles in a single 12.5 μl droplet blood sample. 
    more » « less
  4. ; ; ; ; ; ; ; (, Frontiers in Bioengineering and Biotechnology)
    The bacterial wilt pathogenRalstonia pseudosolanacearum (Rps)colonizes plant xylem vessels and blocks the flow of xylem sap by its biofilm (comprising of bacterial cells and extracellular material), resulting in devastating wilt disease across many economically important host plants including tomatoes. The technical challenges of imaging the xylem environment, along with the use of artificial cell culture plates and media in existingin vitrosystems, limit the understanding ofRpsbiofilm formation and its infection dynamics. In this study, we designed and built a microfluidic system that mimicked the physical and chemical conditions of the tomato xylem vessels, and allowed us to dissectRpsresponses to different xylem-like conditions. The system, incorporating functional surface coatings of carboxymethyl cellulose-dopamine, provided a bioactive environment that significantly enhancedRpsattachment and biofilm formation in the presence of tomato xylem sap. Using computational approaches, we confirmed thatRpsexperienced linear increasing drag forces in xylem-mimicking channels at higher flow rates. Consistently, attachment and biofilm assays conducted in our microfluidic system revealed that both seeding time and flow rates were critical for bacterial adhesion to surface and biofilm formation inside the channels. These findings provided insights into theRpsattachment and biofilm formation processes, contributing to a better understanding of plant-pathogen interactions during wilt disease development. 
    more » « less
  5. ; ; ; ; ; ; (, Small)
    Abstract The ability to detect pathogens specifically and sensitively is critical to combat infectious diseases outbreaks and pandemics. Colorimetric assays involving loop‐mediated isothermal amplification (LAMP) provide simple readouts yet suffer from the intrinsic non‐template amplification. Herein, a highly specific and sensitive assay relying on plasmonic sensing of LAMP amplicons via DNA hybridization, termed as plasmonic LAMP, is developed for the severe acute respiratory syndrome‐related coronavirus 2 (SARS‐CoV‐2) RNA detection. This work has two important advances. First, gold and silver (Au–Ag) alloy nanoshells are developed as plasmonic sensors that have 4‐times stronger extinction in the visible wavelengths and give a 20‐times lower detection limit for oligonucleotides over Au counterparts. Second, the integrated method allows cutting the complex LAMP amplicons into short repeats that are amendable for hybridization with oligonucleotide‐functionalized Au–Ag nanoshells. In the SARS‐CoV‐2 RNA detection, plasmonic LAMP takes ≈75 min assay time, achieves a detection limit of 10 copies per reaction, and eliminates the contamination from non‐template amplification. It also shows better detection specificity and sensitivity over commercially available LAMP kits due to the additional sequence identification. This work opens a new route for LAMP amplicon detection and provides a method for virus testing at its early representation. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email