Evaluation of a loop-mediated isothermal amplification assay based onhrpZgene for rapid detection and identification ofPseudomonas syringaepv.lachrymansin cucumber leaves

Meng, Xianglong; Xie, Xuewen; Shi, Yanxia; Chai, Ali; Ma, Zhanhong; Li, Baoju

doi:10.1111/jam.13356

Cited by 22 publications

(9 citation statements)

References 35 publications

(68 reference statements)

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“…The real-time qPCR LOD is 1.3 times lower, but these values are very similar, in the same order of magnitude. However, the developed biosensor is more sensitive than PCR variation-loop-mediated isothermal amplification (LAMP) [28] and detects 29.7 times less Pseudomonas syringae cells. The last step of sensor evaluation was repeatability/reproducibility and selectivity studies.…”

Section: Psl Bacteria Detection On Developed Electrochemical Immunosementioning

confidence: 99%

Detection of the Plant Pathogen Pseudomonas Syringae pv. Lachrymans on Antibody-Modified Gold Electrodes by Electrochemical Impedance Spectroscopy

Cebula¹,

Żołędowska²,

Dziąbowska³

et al. 2019

Sensors

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The present work describes an impedimetric immunosensor for Pseudomonas syringae pv. lachrymans (Psl) detection. This pathogen infects many crop species causing considerable yield losses, thus fast and cheap detection method is in high demand. In the assay, the gold disc electrode was modified with 4-aminothiophenol (4-ATP), glutaraldehyde (GA), and anti-Psl antibodies, and free-sites were blocked with bovine serum albumin (BSA). Sensor development was characterized by cyclic voltammetry (CV) and antigen detection by electrochemical impedance spectroscopy (EIS) measurements. Seven analyzed strains of Psl were verified as positive by the reference method (PCR) and this immunoassay, proving sensor specificity. Label-free electrochemical detection was in the linear range 1 × 103–1.2 × 105 CFU/mL (colony-forming unit) with an R2 coefficient of 0.992 and a detection limit (LOD) of 337 CFU/mL. The sensor did not interfere with negative probes like buffers and other bacteria. The assay was proven to be fast (10 min detection) and easy in preparation. The advantage was the simplicity and availability of the verified analyte (whole bacteria) as the method does not require sample pretreatment (e.g., DNA isolation). EIS biosensing technique was chosen as one of the simplest and most sensitive with the least destructive influence on the probes compared to other electrochemical methods.

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Section: Psl Bacteria Detection On Developed Electrochemical Immunosementioning

confidence: 99%

Detection of the Plant Pathogen Pseudomonas Syringae pv. Lachrymans on Antibody-Modified Gold Electrodes by Electrochemical Impedance Spectroscopy

Cebula¹,

Żołędowska²,

Dziąbowska³

et al. 2019

Sensors

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show abstract

“…The LAMP assay is a single-step procedure that requires four to six primers that bind laterally to distinct sites using strand-displacement Bst DNA polymerase, permitting extremely specific amplification under isothermal conditions ( Notomi et al, 2000 ; Tomita et al, 2008 ; Li and Macdonald, 2015 ). The amplified products can be detected by gel electrophoresis, turbidimeter, lateral flow dipstick, or the naked eye ( Mori et al, 2004 ; Mao et al, 2012 ; Niu et al, 2012 ; Meng et al, 2017 ); thus, LAMP can be applied in the field using small portable instruments, as well as in laboratories.…”

Section: Introductionmentioning

confidence: 99%

Comparative Evaluation of the LAMP Assay and PCR-Based Assays for the Rapid Detection of Alternaria solani

et al. 2018

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Early blight (EB), caused by the pathogen Alternaria solani, is a major threat to global potato and tomato production. Early and accurate diagnosis of this disease is therefore important. In this study, we conducted a loop-mediated isothermal amplification (LAMP) assay, as well as conventional polymerase chain reaction (PCR), nested PCR, and quantitative real-time PCR (RT-qPCR) assays to determine which of these techniques was less time consuming, more sensitive, and more accurate. We based our assays on sequence-characterized amplified regions of the histidine kinase gene with an accession number (FJ424058). The LAMP assay provided more rapid and accurate results, amplifying the target pathogen in less than 60 min at 63°C, with 10-fold greater sensitivity than conventional PCR. Nested PCR was 100-fold more sensitive than the LAMP assay and 1000-fold more sensitive than conventional PCR. qPCR was the most sensitive among the assays evaluated, being 10-fold more sensitive than nested PCR for the least detectable genomic DNA concentration (100 fg). The LAMP assay was more sensitive than conventional PCR, but less sensitive than nested PCR and qPCR; however, it was simpler and faster than the other assays evaluated. Despite of the sensitivity, LAMP assay provided higher specificity than qPCR. The LAMP assay amplified A. solani artificially, allowing us to detect naturally infect young potato leaves, which produced early symptoms of EB. The LAMP assay also achieved positive amplification using diluted pure A. solani culture instead of genomic DNA. Hence, this technique has greater potential for developing quick and sensitive visual detection methods than do other conventional PCR strategies for detecting A. solani in infected plants and culture, permitting early prediction of disease and reducing the risk of epidemics.

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“…LAMP isothermal amplification has already been used to detect Pseudomonas syringae pv. lachrymans in cucumber leaves and was found to be a reliable and sensitive method [44]. LAMP assay showed to be a powerful tool for the detection of P. aeruginosa strains, as well [45].…”

Section: Discussionmentioning

confidence: 99%

Loop-mediated isothermal amplification: a rapid molecular technique for early diagnosis of Pseudomonas syringae pv. syringae of stone fruits

Goudarzi

Mortazavi

2020

Journal of Genetic Engineering and Biotechnology

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Background Pathogenic bacteria cause significant economic damages in agriculture. The detection of such bacteria is considered as a continual interest for plant pathologists to prevent disease dissemination. Pseudomonas syringae pv. syringae is one of the most important bacterial pathogens infecting yield and quality of stone fruits throughout the world. Biochemical assays such as a LOPAT and GATTa are common methods to detect this pathogen. Serological tests and culturing on King’s B selective medium also used to isolate this bacterium. Selective media is composed of specific and effective ingredients to inhibit the growth of certain species of microbes in a mixed culture while allowing others to grow. These are used for the growth of only selected microorganisms. King’s B medium can be used as a general medium for the non-selective isolation cultivation and pigment production of Pseudomonas species from foods, cosmetic samples, plants, etc. Nevertheless, the mentioned methods are not enough accurate to differentiate the strains. On the other hand, PCR-based techniques are sensitive and efficient in detecting plant diseases. However, these techniques are not practicable for those researchers who do not have access to a thermal cycler. We have used loop-mediated isothermal amplification to couple with a target. The amplification of syrD gene using loop and bumper primers can be used to prevent disease dissemination. Results The outcome of this investigation indicated more sensitivity of LAMP in comparison to PCR. The direct addition of SYBR Gold in microtube is more sensitive than gel in both LAMP and PCR byproducts so we can eliminate gel electrophoresis, while the LAMP showed high sensitivity and high specificity in comparison to results obtained by cultivation. The described molecular test could detect Pseudomonas syringae pv. syringae type in nearly 1 h, and this is the first time that Lamp molecular detection of Pseudomonas syringae pv. syringae particularly on stone fruits is described and introduced. Conclusions The obtained data confirmed that LAMP is a fast, cheap, and high specific method for the rapid detection of Pseudomonas syringae pv. syringae to the comparison of PCR and culture.

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Evaluation of a loop-mediated isothermal amplification assay based onhrpZgene for rapid detection and identification ofPseudomonas syringaepv.lachrymansin cucumber leaves

Abstract: The method is suitable for direct detection of Psl without strain enrichment and complex DNA extraction from samples in the field, and hence it has the capability to be used for on-site disease diagnosis and field surveys.

Cited by 22 publications

References 35 publications

Detection of the Plant Pathogen Pseudomonas Syringae pv. Lachrymans on Antibody-Modified Gold Electrodes by Electrochemical Impedance Spectroscopy

Detection of the Plant Pathogen Pseudomonas Syringae pv. Lachrymans on Antibody-Modified Gold Electrodes by Electrochemical Impedance Spectroscopy

Comparative Evaluation of the LAMP Assay and PCR-Based Assays for the Rapid Detection of Alternaria solani

Loop-mediated isothermal amplification: a rapid molecular technique for early diagnosis of Pseudomonas syringae pv. syringae of stone fruits

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