• Research in Plant Disease
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• v.21 no.4
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• pp.315-320
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• 2015

Soybean mosaic virus (SMV) is a prevalent pathogen that causes significant yield reduction in soybean production worldwide. SMV belongs to potyvirus and causes typical symptoms such as mild mosaic, mosaic and necrosis. SMV is seed-borne and also transmitted by aphid. Eleven SMV strains, G1 to G7, G5H, G6H, G7H, and G7a were reported in soybean varieties in Korea. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) method allowed one-step detection of gene amplification by simple procedure and needed only a simple incubator for isothermal template. This RT-LAMP method allowed direct detection of RNA from virus-infected plants without thermal cycling and gel electrophoresis. In this study, we designed RT-LAMP primers named SML-F3/B3/FIP/BIP from coat protein gene sequence of SMV. After the reaction of RT-LAMP, products were identified by electrophoresis and with the detective fluorescent dye, SYBR Green I under daylight and UV light. Optimal reaction condition was at $58^{\circ}C$ for 60 min and the primers of RT-LAMP showed the specificity for nine SMV strains tested in this study.

• The Korean Journal of Mycology
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• v.47 no.4
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• pp.437-441
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• 2019

Verticillium wilt disease is caused by a fungal plant pathogen Verticillium dahliae, which attacks commercial crops such as chrysanthemum. The conventional methods so far used to identify this fungal pathogen require high expertise and are time-consuming. Therefore, in this study, we developed an assay for the rapid and specific detection of V. dahliae infection using loop-mediated isothermal amplification (LAMP) method. For this assay, four primers for LAMP were designed for targeting cellulose-growth-specific protein partial mRNA gene in Verticillium dahliae. Under standard condition, the optimum reaction temperature for amplification is around 60 ℃ within 60 minutes. This LAMP assay was designed to amplify only present in V. dahliae. When this LAMP assay applied to the DNAs for four other soil-borne fungi and host plants, no amplification was detected. Therefore, this LAMP assay we developed for V. dahliae is expected to do detection at the early stage of its infection. The fast and reliable detection method will allow us to develop effective management system to monitor and control infection of this pathogen in chrysanthemum plant.

• The Plant Pathology Journal
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• v.32 no.5
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• pp.414-422
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• 2016

Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

• Journal of Microbiology and Biotechnology
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• v.31 no.4
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• pp.501-509
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• 2021

More than half the world's population is thought to be infected with Helicobacter pylori. Although the majority of infected people are asymptomatic, H. pylori infection may cause gastric ulcers and deadly gastric cancer. Owing to the difficulty and invasiveness of current routine culture and diagnostic methods, a highly sensitive and specific noninvasive assay for H. pylori is of interest. This study highlighted the design and performance of a colorimetric magneto loop-mediated isothermal amplification (CM-LAMP) assay to detect H. pylori in spiked saliva samples. LF primers were coated on magnetic nanoparticles by carbodiimide-induced immobilization and functionally used for solid-phase amplification. During the LAMP reaction at 66℃, biotin-tagged FIPs were incorporated into LAMP amplicons. The colorimetric signal developed after the addition of NeutrAvidin horseradish peroxidase conjugate (NA-HRP) and ABTS. None of the tested microorganisms, including closely related bacteria, was shown positive by the CM-LAMP assay except H. pylori isolates. This novel platform was highly specific and 100-fold more sensitive (40 CFU/ml or 0.2 CFU per reaction) than the PCR and conventional LAMP assays for the detection of H. pylori in spiked saliva. Our results demonstrated the feasibility of using this noninvasive molecular diagnostic test to detect H. pylori in saliva samples.

• The Plant Pathology Journal
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• v.34 no.6
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• pp.499-505
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• 2018

Huanglongbing (HLB, Citrus greening disease) is one of the most devastating diseases that threaten citrus production worldwide. Although HLB presents systemically, low titer and uneven distribution of these bacteria within infected plants can make reliable detection difficult. It was known loop-mediated isothermal amplification (LAMP) method has the advantages of being highly specific, rapid, efficient, and laborsaving for detection of plant pathogens. We developed a new LAMP method targeting gene contained tandem repeat for more rapid and sensitive detection of Candidatus Liberibacter asiaticus (CLas), putative causal agent of the citrus huanglongbing. This new LAMP method was 10 folds more sensitive than conventional PCR in detecting the HLB pathogen and similar to that of real-time PCR in visual detection assay by adding SYBR Green I to mixture and 1% agarose gel electrophoresis. Positive reactions were achieved in reaction temperature 57, 60 and $62^{\circ}C$ but not $65^{\circ}C$. Although this LAMP method was not more sensitive than real-time PCR, it does not require a thermocycler for amplification or agarose gel electrophoresis for resolution. Thus, we expect that this LAMP method shows strong promise as a reliable, rapid, and cost-effective method of detecting the CLas in citrus and can be applied for rapid diagnosis is needed.

• The Plant Pathology Journal
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• v.35 no.6
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• pp.635-643
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• 2019

To detect Xanthomonas arboricola pv. pruni, a loopmediated isothermal amplification (LAMP) detection method were developed. The LAMP assay was designed to test crude plant tissue without pre-extraction, or heating incubation, and without advanced analysis equipment. The LAMP primers were designed by targeting an ABC transporter ATP-binding protein, this primer set was tested using the genomic DNA of Xanthomonas and non-Xanthomonas strains, and a ladder product was generated from the genomic DNA of X. arboricola pv. pruni strain but not from 12 other Xanthomonas species strains and 6 strains of other genera. The LAMP conditions were checked with the healthy leaves of 31 peach varieties, and no reaction was detected using either the peach leaves or the peach DNA as a template. Furthermore, the high diagnostic accuracy of the LAMP method was confirmed with 13 X. arboricola pv. pruni strains isolated from various regions in Korea, with all samples exhibiting a positive reaction in LAMP assays. In particular, the LAMP method successfully detected the pathogen in diseased peach leaves and fruit in the field, and the LAMP conditions were proven to be a reliable diagnostic method for the specific detection and identification of X. arboricola pv. pruni in peach orchards.

• Korean Journal of Materials Research
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• v.34 no.2
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• pp.63-71
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• 2024

Slipchip offers advantages such as high-throughout, low cost, and simple operation, and therefore, it is one of the technologies with the greatest potential for high-throughput, single-cell, and single-molecule analyses. Slipchip devices have achieved remarkable advances over the past decades, with its simplified molecular diagnostics gaining particular attention, especially during the COVID-19 pandemic and in various infectious diseases scenarios. Medical testing based on nucleic acid amplification in the Slipchip has become a promising alternative simple and rapid diagnostic tool in field situations. Herein, we present a comprehensive review of Slipchip device advances in molecular diagnostics, highlighting its use in digital recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and polymerase chain reaction (PCR). Slipchip technology allows users to conduct reliable droplet transfers with high-throughput potential for single-cell and molecule analyses. This review explores the device's versatility in miniaturized and rapid molecular diagnostics. A complete Slipchip device can be operated without special equipment or skilled handling, and provides high-throughput results in minimum settings. This review focuses on recent developments and Slipchip device challenges that need to be addressed for further advancements in microfluidics technology.

• Korean Journal of Veterinary Service
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• v.38 no.2
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• pp.107-116
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• 2015

In this study, we developed a rapid, sensitive and specific reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) assay for detection of swine influenza viruse (SIV) including major subtypes of swine influenza viruses H1N1, H1N2 and H3N2, and a novel subtype of influenza A virus that accidentally infected in pig population. The RT-LAMP was completed in 40 min at $58^{\circ}C$ and the sensitivity of the RT-LAMP ($1copy/{\mu}L$) was 10-fold higher than conventional reverse transcription-polymerase chain reaction (RT-PCR) ($10copy/{\mu}L$) and the same to real time RT-PCR ($1copy/{\mu}L$). Also, the result of the RT-LAMP can be confirmed without any detection system. Therefore, the RT-LAMP could be a alternative diagnostic method for SIV detection in national SIV monitoring system and clinical diagnostic laboratory in the future.

• The Plant Pathology Journal
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• v.33 no.2
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• pp.184-192
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• 2017

Tylenchulus semipenetrans is an important and widespread plant-parasitic nematode of citrus worldwide and can cause citrus slow decline disease leading to significant reduction in tree growth and yield. Rapid and accurate detection of T. semipenetrans in soil is important for the disease forecasting and management. In this study, a loop-mediated isothermal amplification (LAMP) assay was developed to detect T. semipenetrans using DNA extracted from soil. A set of five primers was designed from the internal transcribed spacer region (ITS1) of rDNA, and was highly specific to T. semipenetrans. The LAMP reaction was performed at $63^{\circ}C$ for 60 min. The LAMP product was visualized directly in one reaction tube by adding SYBR Green I. The detection limit of the LAMP assay was $10^{-2}J2/0.5g$ of soil, which was 10 times more sensitive than conventional PCR ($10^{-1}J2/0.5g$ of soil). Examination of 24 field soil samples revealed that the LAMP assay was applicable to a range of soils infested naturally with T. semipenetrans, and the total assay time was less than 2.5 h. These results indicated that the developed LAMP assay is a simple, rapid, sensitive, specific and accurate technique for detection of T. semipenetrans in field soil, and contributes to the effective management of citrus slow decline disease.

• Parasites, Hosts and Diseases
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• v.54 no.3
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• pp.329-334
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• 2016

Trichomoniasis caused by Trichomonas vaginalis is a common sexually transmitted disease. Its association with several health problems, including preterm birth, pelvic inflammatory disease, cervical cancer, and transmission of human immunodeficiency virus, emphasizes the importance of improved access to early and accurate detection of T. vaginalis. In this study, a rapid and efficient loop-mediated isothermal amplification-based method for the detection of T. vaginalis was developed and validated, using vaginal swab specimens from subjects suspected to have trichomoniasis. The LAMP assay targeting the actin gene was highly sensitive with detection limits of 1 trichomonad and 1 pg of T. vaginalis DNA per reaction, and specifically amplified the target gene only from T. vaginalis. Validation of this assay showed that it had the highest sensitivity and better agreement with PCR (used as the gold standard) compared to microscopy and multiplex PCR. This study showed that the LAMP assay, targeting the actin gene, could be used to diagnose early infections of T. vaginalis. Thus, we have provided an alternative molecular diagnostic tool and a point-of-care test that may help to prevent trichomoniasis transmission and associated complications.