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

Author(s):  
T. Pardy ◽  
T. Rang ◽  
C. Kremer ◽  
I. Tulp
Plant Disease ◽  
2019 ◽  
Vol 103 (12) ◽  
pp. 3251-3258
Author(s):  
Sheng-Ren Sun ◽  
Jun-Lü Chen ◽  
Yao-Yao Duan ◽  
Na Chu ◽  
Mei-Ting Huang ◽  
...  

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.


2020 ◽  
Vol 58 (5) ◽  
Author(s):  
Le Jiang ◽  
Philip Ching ◽  
Chien-Chung Chao ◽  
J. Stephen Dumler ◽  
Wei-Mei Ching

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.


Plant Disease ◽  
2015 ◽  
Vol 99 (8) ◽  
pp. 1147-1152 ◽  
Author(s):  
Rachel B. Elkins ◽  
Todd N. Temple ◽  
Carolyn A. Shaffer ◽  
Chuck A. Ingels ◽  
Steven B. Lindow ◽  
...  

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.


2009 ◽  
Vol 2 (4) ◽  
pp. 199-211 ◽  
Author(s):  
Holger Schulze ◽  
Gerard Giraud ◽  
Jason Crain ◽  
Till T. Bachmann

Author(s):  
Junqing Wu ◽  
Gaurav Soni ◽  
Dazhi Wang ◽  
Carl D. Meinhart

We have developed micropumps for microfluidics that use AC electric fields to drive aqueous fluid motion through micro channels. These pumps operate at relatively low voltages (~5–10Vrms), and high frequencies (~100kHz). They have several distinct advantages over the DC electrokinetic pumps. The low voltages make the pumps well suited for a wide variety of biosensor and “Lab-on-a-Chip” applications (e.g. PCR chip for DNA amplification). The high frequencies minimize electrolysis, so that bubbles do not form on the electrode surfaces, and do not contaminate the working fluid. The pumps can also be used as active valves or precision micro-dispensers.


2015 ◽  
Vol 156 (51) ◽  
pp. 2082-2088
Author(s):  
Kristóf Iván ◽  
Anna Maráz

Detection and identification of food-borne pathogenic bacteria are key points for the assurance of microbiological food safety. Traditional culture-based methods are more and more replaced by or supplemented with nucleic acid based molecular techniques, targeting specific (preferably virulence) genes in the genomes. Internationally validated DNA amplification – most frequently real-time polymerase chain reaction – methods are applied by the food microbiological testing laboratories for routine analysis, which will result not only in shortening the time for results but they also improve the performance characteristics (e.g. sensitivity, specificity) of the methods. Beside numerous advantages of the polymerase chain reaction based techniques for routine microbiological analysis certain drawbacks have to be mentioned, such as the high cost of the equipment and reagents, as well as the risk of contamination of the laboratory environment by the polymerase chain reaction amplicons, which require construction of an isolated laboratory system. Lab-on-a-chip systems can integrate most of these laboratory processes within a miniaturized device that delivers the same specificity and reliability as the standard protocols. The benefits of miniaturized devices are: simple – often automated – use, small overall size, portability, sterility due to single use possibility. These miniaturized rapid diagnostic tests are being researched and developed at the best research centers around the globe implementing various sample preparation and molecular DNA amplification methods on-chip. In parallel, the aim of the authors’ research is to develop microfluidic Lab-on-a-chip devices for the detection and identification of food-borne pathogenic bacteria. Orv. Hetil., 2015, 156(51), 2082–2088.


2014 ◽  
Vol 25 (1) ◽  
pp. 32-34 ◽  
Author(s):  
Andrew Walkty ◽  
John M Embil ◽  
Kim Nichol ◽  
James Karlowsky

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.


2013 ◽  
Author(s):  
Richard Klemm ◽  
Sebastian Schattschneider ◽  
Tobias Jahn ◽  
Nadine Hlawatsch ◽  
Sandra Julich ◽  
...  

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