Rapid and Sensitive Pathogen Detection by DNA Amplification Using Janus Particle-Enabled Rotational Diffusometry

Ratoon stunting disease (RSD), one of the most important diseases of sugarcane, is caused by the bacterium Leifsonia xyli subsp. xyli (Lxx). Lxx infects sugarcane worldwide and RSD results in high yield losses and varietal degeneration. It is highly challenging to diagnose RSD based on visual symptomatology because this disease does not exhibit distinct external and internal symptoms. In this study, a novel Lxx-specific primer pair Lxx-F1/Lxx-R1 was designed to detect this pathogen using a conventional PCR assay. These primers were then compared with four published Lxx-specific primers and one universal Leifsonia generic primer pair LayF/LayR. Sugarcane leaf samples were collected from Saccharum spp. hybrids in commercial fields (315 samples) and from germplasm clones of five Saccharum species and Erianthus arundinaceus (216 samples). These samples were used for comparative field diagnosis with six conventional PCR assays. Sensitivity tests suggested that the PCR assay with primers Lxx-F1/Lxx-R1 had the same detection limit (1 pg of Lxx genomic DNA) as the primer pairs Cxx1/Cxx2 and CxxITSf#5/CxxITSr#5 and had 10-fold higher sensitivity than the primer pairs Pat1-F2/Pat1-R2, LayF/LayR, and C2F/C2R. Comparison of PCR assays revealed that natural Lxx-infection incidence (6.1%) in field sample evaluation identified by Lxx-F1/Lxx-R1 primers was higher than incidences (0.7 to 3.0%) determined by other primer pairs. Moreover, no nonspecific DNA amplification occurred within these field samples with Lxx-F1/Lxx-R1 primers, unlike with the primer pairs Cxx1/Cxx2 and LayF/LayR. Diverse Leifsonia strains were identified by PCR detection with LayF/LayR primers in the field samples, whereas whether these Leifsonia strains were pathogenic to sugarcane requires further research. Our investigations revealed that the PCR assay with the newly designed primers Lxx-F1/Lxx-R1 could be widely used for RSD diagnosis and Lxx-pathogen detection with satisfactory sensitivity and specificity.

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Instrument-free Lab-on-a-Chip DNA amplification test for pathogen detection

2018 16th Biennial Baltic Electronics Conference (BEC) ◽

10.1109/bec.2018.8600991 ◽

2018 ◽

Author(s):

T. Pardy ◽

T. Rang ◽

C. Kremer ◽

I. Tulp

Keyword(s):

Pathogen Detection ◽

Lab On A Chip ◽

Dna Amplification

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Development of a Sensitive and Rapid Recombinase Polymerase Amplification Assay for Detection of Anaplasma phagocytophilum

Journal of Clinical Microbiology ◽

10.1128/jcm.01777-19 ◽

2020 ◽

Vol 58 (5) ◽

Cited By ~ 1

Author(s):

Le Jiang ◽

Philip Ching ◽

Chien-Chung Chao ◽

J. Stephen Dumler ◽

Wei-Mei Ching

Keyword(s):

Anaplasma Phagocytophilum ◽

Pathogenic Bacteria ◽

Pathogen Detection ◽

Limit Of Detection ◽

Dna Amplification ◽

Recombinase Polymerase Amplification ◽

Detection Methods ◽

Content Type ◽

Amplification Assay ◽

Nonspecific Amplification

ABSTRACT Human granulocytic anaplasmosis (HGA) is a tick-borne disease caused by the obligate intracellular Gram-negative bacterium Anaplasma phagocytophilum. The disease often presents with nonspecific symptoms with negative serology during the acute phase. Direct pathogen detection is the best approach for early confirmatory diagnosis. Over the years, PCR-based molecular detection methods have been developed, but optimal sensitivity is not achieved by conventional PCR while real-time PCR requires expensive and sophisticated instruments. To improve the sensitivity and also develop an assay that can be used in resource-limited areas, an isothermal DNA amplification assay based on recombinase polymerase amplification (RPA) was developed. To do this, we identified a 171-bp DNA sequence within multiple paralogous copies of msp2 within the genome of A. phagocytophilum. Our novel RPA assay targeting this sequence has an analytical limit of detection of one genome equivalent copy of A. phagocytophilum and can reliably detect 125 bacteria/ml in human blood. A high level of specificity was demonstrated by the absence of nonspecific amplification using genomic DNA from human or DNA from other closely-related pathogenic bacteria, such as Anaplasma platys, Ehrlichia chaffeensis, Orientia tsutsugamushi, and Rickettsia rickettsii, etc. When applied to patient DNA extracted from whole blood, this new RPA assay was able to detect 100% of previously diagnosed A. phagocytophilum cases. The sensitivity and rapidness of this assay represents a major improvement for early diagnosis of A. phagocytophilum in human patients and suggest a role for better surveillance in its reservoirs or vectors, especially in remote regions where resources are limited.

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Evaluation of Dormant-Stage Inoculum Sanitation as a Component of a Fire Blight Management Program for Fresh-Market Bartlett Pear

Plant Disease ◽

10.1094/pdis-10-14-1082-re ◽

2015 ◽

Vol 99 (8) ◽

pp. 1147-1152 ◽

Cited By ~ 4

Author(s):

Rachel B. Elkins ◽

Todd N. Temple ◽

Carolyn A. Shaffer ◽

Chuck A. Ingels ◽

Steven B. Lindow ◽

...

Keyword(s):

Pathogen Detection ◽

Fire Blight ◽

Lamp Assay ◽

Dna Amplification ◽

Management Program ◽

Fresh Market ◽

Specific Detection ◽

Bloom Period ◽

Four Seasons ◽

Late Stages

From 2010 to 2013, the efficacy of copper-based inoculum sanitation as a component of fire blight management programs was evaluated in commercial pear orchards located in northern California. Forty-one 4-ha sections of orchard were split into two equal-sized plots, with the orchardist applying horticultural oil alone to one plot and horticultural oil plus a fixed copper bactericide to the other plot. These treatments were timed to begin just prior to and finish at the “green tip” phenological stage, which occurs about 5 weeks before full bloom. During bloom, flower samples were collected from the plots and subjected to a loop-mediated isothermal DNA amplification (LAMP) assay for specific detection of Erwinia amylovora. Overall, epiphytic populations of E. amylovora on flowers were detected rarely at midbloom (6% of samples) but commonly at petal fall (44% of samples). In three of four seasons, E. amylovora detection in flower samples at a given bloom stage was significantly suppressed in copper-plus-oil-treated plots compared with oil-only plots. All orchards also received antibiotic treatments during the bloom period and, perhaps as a consequence, the development of fire blight was sporadic and not affected significantly by the copper treatment in any season. The pathogen detection data indicate that copper sanitation may add value to a fire blight management program by delaying the increase of epiphytic populations of E. amylovora in flowers to the late stages of the bloom period, at which time the number of susceptible flowers declines rapidly.

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An Unusual Case ofStreptococcus anginosusGroup Pyomyositis Diagnosed Using Direct 16S Ribosomal DNA Sequencing

Canadian Journal of Infectious Diseases and Medical Microbiology ◽

10.1155/2014/170517 ◽

2014 ◽

Vol 25 (1) ◽

pp. 32-34 ◽

Cited By ~ 5

Author(s):

Andrew Walkty ◽

John M Embil ◽

Kim Nichol ◽

James Karlowsky

Keyword(s):

Dna Sequencing ◽

Ribosomal Dna ◽

Pathogen Detection ◽

Dna Amplification ◽

Viridans Streptococci ◽

Streptococcus Anginosus ◽

16S Ribosomal Dna ◽

Specimen Collection ◽

Focus Of Infection ◽

Single Positive

Bacteria belonging to theStreptococcus anginosusgroup (Streptococcus intermedius,Streptococcus constellatusandStreptococcus anginosus) are capable of causing serious pyogenic infections, with a tendency for abscess formation. The present article reports a case ofS anginosusgroup pyomyositis in a 47-year-old man. The pathogen was recovered from one of two blood cultures obtained from the patient, but speciation was initially not performed because the organism was considered to be a contaminant (viridans streptococci group). The diagnosis was ultimately confirmed using 16S ribosomal DNA sequencing of purulent fluid obtained from a muscle abscess aspirate. The present case serves to emphasize that finding even a single positive blood culture of an organism belonging to theS anginosusgroup should prompt careful evaluation of the patient for a pyogenic focus of infection. It also highlights the potential utility of 16S ribosomal DNA amplification and sequencing in direct pathogen detection from aspirated fluid in cases of pyomyositis in which antimicrobial therapy was initiated before specimen collection.

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Lab-on-Chip platform and protocol for rapid foodborne pathogen detection comprising on-chip cell capture, lysis, DNA amplification and surface-acoustic-wave detection

Sensors and Actuators B Chemical ◽

10.1016/j.snb.2020.128345 ◽

2020 ◽

Vol 320 ◽

pp. 128345

Author(s):

K. Tsougeni ◽

G. Kaprou ◽

C.M. Loukas ◽

G. Papadakis ◽

A. Hamiot ◽

...

Keyword(s):

Acoustic Wave ◽

Surface Acoustic Wave ◽

Pathogen Detection ◽

Foodborne Pathogen ◽

Dna Amplification ◽

Cell Capture ◽

Lab On Chip ◽

Wave Detection ◽

On Chip ◽

Foodborne Pathogen Detection

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Simple and sensitive microbial pathogen detection using a label-free DNA amplification assay

Chemical Communications ◽

10.1039/c6cc02672a ◽

2016 ◽

Vol 52 (47) ◽

pp. 7505-7508 ◽

Cited By ~ 11

Author(s):

Yuhuan Sun ◽

Chuanqi Zhao ◽

Zhengqing Yan ◽

Jinsong Ren ◽

Xiaogang Qu

Keyword(s):

Magnetic Nanoparticles ◽

Pathogen Detection ◽

Dna Amplification ◽

Label Free ◽

Exonuclease Iii ◽

Microbial Pathogen ◽

Free Dna ◽

Amplification Assay

A simple and facile strategy for sensitive pathogen detection has been developed by a combination of quaternized magnetic nanoparticles and a label-free exonuclease III-assisted DNA amplification assay.

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Cleavable hairpin beacon-enhanced fluorescence detection of nucleic acid isothermal amplification and smartphone-based readout

Scientific Reports ◽

10.1038/s41598-020-75795-y ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Xiong Ding ◽

Kun Yin ◽

Ziyue Li ◽

Vikram Pandian ◽

Joan A. Smyth ◽

...

Keyword(s):

Nucleic Acid ◽

Fluorescence Detection ◽

Pathogen Detection ◽

Point Of Care ◽

Lamp Assay ◽

Dna Amplification ◽

Isothermal Amplification ◽

Specific Method ◽

Enhanced Fluorescence ◽

Resource Limited

Abstract Fluorescence detection of nucleic acid isothermal amplification utilizing energy-transfer-tagged oligonucleotide probes provides a highly sensitive and specific method for pathogen detection. However, currently available probes suffer from relatively weak fluorescence signals and are not suitable for simple, affordable smartphone-based detection at the point of care. Here, we present a cleavable hairpin beacon (CHB)-enhanced fluorescence detection for isothermal amplification assay. The CHB probe is a single fluorophore-tagged hairpin oligonucleotide with five continuous ribonucleotides which can be cleaved by the ribonuclease to specifically initiate DNA amplification and generate strong fluorescence signals. By coupling with loop-mediated isothermal amplification (LAMP), the CHB probe could detect Borrelia burgdorferi (B. burgdorferi) recA gene with a sensitivity of 100 copies within 25 min and generated stronger specific fluorescence signals which were easily read and analysed by our programmed smartphone. Also, this CHB-enhanced LAMP (CHB-LAMP) assay was successfully demonstrated to detect B. burgdorferi DNA extracted from tick species, showing comparable results to real-time PCR assay. In addition, our CHB probe was compatible with other isothermal amplifications, such as isothermal multiple-self-matching-initiated amplification (IMSA). Therefore, CHB-enhanced fluorescence detection is anticipated to facilitate the development of simple, sensitive smartphone-based point-of-care pathogen diagnostics in resource-limited settings.

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A Disposable and Multi-Chamber Film-Based PCR Chip for Detection of Foodborne Pathogen

Sensors ◽

10.3390/s18093158 ◽

2018 ◽

Vol 18 (9) ◽

pp. 3158 ◽

Cited By ~ 8

Author(s):

Nam Bae ◽

Sun Lim ◽

Younseong Song ◽

Soon Jeong ◽

Seol Shin ◽

...

Keyword(s):

Foodborne Pathogens ◽

Pathogen Detection ◽

Detection System ◽

Foodborne Pathogen ◽

Pcr Amplification ◽

Dna Amplification ◽

Adhesive Film ◽

Thermal Transfer ◽

Polymerase Chain ◽

Structurally Stable

Since the increment of the threat to public health caused by foodborne pathogens, researches have been widely studied on developing the miniaturized detection system for the on-site pathogen detection. In the study, we focused on the development of portable, robust, and disposable film-based polymerase chain reaction (PCR) chip containing a multiplex chamber for simultaneous gene amplification. In order to simply fabricate and operate a film-based PCR chip, different kinds of PCR chambers were designed and fabricated using polyethylene terephthalate (PET) and polyvinyl chloride (PVC) adhesive film, in comparison with commercial PCR, which employs a stereotyped system at a bench-top scale. No reagent leakage was confirmed during the PCR thermal cycling using the film PCR chip, which indicates that the film PCR chip is structurally stable for rapid heat cycling for DNA amplification. Owing to use of the thin film to fabricate the PCR chip, we are able to realize fast thermal transfer from the heat block that leads to short PCR amplification time. Moreover, using the film PCR chip, we could even amplify the target pathogen with 10 CFU mL−1. The artificially infected milk with various concentration of Bacillus cereus was successfully amplified on a single film PCR chip. On the basis of the reliable results, the developed film PCR chip could be a useful tool as a POCT device to detect foodborne pathogens via genetic analysis.

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2-LED-µSpectrophotometer for Rapid On-Site Detection of Pathogens Using Noble-Metal Nanoparticle-Based Colorimetric Assays

Applied Sciences ◽

10.3390/app10082658 ◽

2020 ◽

Vol 10 (8) ◽

pp. 2658 ◽

Cited By ~ 1

Author(s):

Cornelia Reuter ◽

Matthias Urban ◽

Manuel Arnold ◽

Ondrej Stranik ◽

Andrea Csáki ◽

...

Keyword(s):

Legionella Pneumophila ◽

Pathogen Detection ◽

Color Change ◽

Colorimetric Assay ◽

Point Of Care ◽

Colorimetric Detection ◽

Dna Amplification ◽

Metal Nanoparticle ◽

Colorimetric Assays ◽

Induced Aggregation

Novel point-of-care compatible methods such as colorimetric assays have become increasingly important in the field of early pathogen detection. A simple and hand-held prototype device for carrying out DNA-amplification assay based on plasmonic nanoparticles in the colorimetric detection is presented. The low-cost device with two channels (sample and reference) consists of two spectrally different light emitting diodes (LEDs) for detection of the plasmon shift. The color change of the gold-nanoparticle-DNA conjugates caused by a salt-induced aggregation test is examined in particular. A specific and sensitive detection of the waterborne human pathogen Legionella pneumophila is demonstrated. This colorimetric assay, with a simple assay design and simple readout device requirements, can be monitored in real-time on-site.

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