• Korean Journal of Veterinary Service
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• v.45 no.1
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• pp.19-30
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• 2022

In this study, a simple loop-mediated isothermal amplification (LAMP) combined with visual detection method (vLAMP) assay was developed for the rapid and specific detection of African swine fever virus (ASFV), overcoming the shortcomings of previously described LAMP assays that require additional detection steps or pose a cross-contamination risk. The assay results can be directly detected by the naked eye using hydroxynaphthol blue after incubation for 40 min at 62℃. The assay specifically amplified ASFV DNA and no other viral nucleic acids. The limit of detection of the assay was <50 DNA copies/reaction, which was ten times more sensitive than conventional polymerase chain reaction (cPCR) and comparable to real-time PCR (qPCR). For clinical evaluation, the ASFV detection rate of vLAMP was higher than cPCR and comparable to OIE-recommended qPCR, showing 100% concordance, with a κ value (95% confidence interval) of 1 (1.00~1.00). Considering the advantages of high sensitivity and specificity, no possibility for cross-contamination, and being able to be used as low-cost equipment, the developed vLAMP assay will be a valuable tool for detecting ASFV from clinical samples, even in resource-limited laboratories.

• The Plant Pathology Journal
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• v.39 no.4
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• pp.309-318
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• 2023

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.

• Journal of Microbiology and Biotechnology
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• v.33 no.8
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• pp.1101-1110
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• 2023

Streptococcus mutans is the primary causative agent of caries, which is one of the most common human diseases. Thus, rapid and early detection of cariogenic bacteria is critical for its prevention. This study investigated the combination of loop-mediated isothermal amplification (LAMP) and microfluid technology to quantitatively detect S. mutans. A low-cost, rapid microfluidic chip using LAMP technology was developed to amplify and detect bacteria at 2.2-2.2 × 10⁶ colony-forming units (CFU)/ml and its detection limits were compared to those of standard polymerase chain reaction. A visualization system was established to quantitatively determine the experimental results, and a functional relationship between the bacterial concentration and quantitative results was established. The detection limit of S. mutans using this microfluidic chip was 2.2 CFU/ml, which was lower than that of the standard approach. After quantification, the experimental results showed a good linear relationship with the concentration of S. mutans, thereby confirming the effectiveness and accuracy of the custom-made integrated LAMP microfluidic system for the detection of S. mutans. The microfluidic system described herein may represent a promising simple detection method for the specific and rapid testing of individuals at risk of caries.

• Journal of Food Hygiene and Safety
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• v.38 no.5
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• pp.347-355
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• 2023

In this study, polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) were compared in terms of their ability to detect shiga-toxin-producing Escherichia coli (STEC). Various foods were artificially inoculated with STEC to evaluate the limit of detection (LOD), limit of quantification (LOQ), sensitivity, specificity, and efficiency of PCR and LAMP. The LODs were ≤10⁴ and ≤10³ CFU/mL for PCR and LAMP, respectively. The LOQs did not differ between PCR and LAMP. However, of the four considered food types, the sensitivities differed by a maximum of 11.1% for seasoned meat and by a minimum of 8.1% for ground beef. LAMP had higher sensitivity than that of PCR and 100% specificity for all four food types. Therefore, LAMP is a reliable molecular method for detecting STEC as comparable to PCR assay, and its specificity and sensitivity are superior to those of PCR, depending on the food type.

• International Journal of Industrial Entomology and Biomaterials
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• v.48 no.3
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• pp.139-146
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• 2024

For stable silkworm breeding and high-quality sericulture product production, the detection of Pebrine disease in silkworm eggs is critical. Current diagnostic methods can be timeconsuming and complex. This study aimed to develop a simplified and rapid diagnostic method using loop-mediated isothermal amplification (LAMP) technology to detect pebrine infection in silkworm mother moths. Eight primer candidates targeting the ribosomal gene region of microsporidia were designed and evaluated for specificity and detection sensitivity. A simplified nucleic acid extraction method was established, and isothermal amplification was performed using the selected primers. Of these, primers ID30 and ID45 showed no polymerization, while ID5, ID18, and ID76 exhibited nonspecific reactions, making them unsuitable. Primers ID1, ID6, ID45, and ID82 successfully amplified DNA only in the presence of pebrine, with ID82 demonstrating the best reproducibility and sensitivity, detecting as low as 2.5 pg/ul of DNA through electrophoresis and 5 pg/ul via a colorimetric change with phenol red. The entire process, from nucleic acid extraction to detection, was completed within 60 min. The use of the ID82 primer set in LAMP technology offers a promising and efficient approach for the rapid diagnosis of pebrine disease, potentially enhancing quality control in sericulture.

• Journal of the Korean Chemical Society
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• v.52 no.3
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• pp.273-280
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• 2008

Mycobacterium tuberculosis (MTB) remains a major worldwide public health problem. In recent years, the incidence of MTB has been rising. Rapid and reliable diagnosis of Mycobacterium tuberculosis is essential to initiate correct treatment, avoid severe complications, and prevent transmission. LAMP was used to develop a rapid and sensitive laboratory diagnostic system for the MTB. In this research, the loop-mediated isothermal amplification method (LAMP) that amplifies DNA with high specificity and rapidity at an isothermal condition was evaluated for rapid detection of MTB. Undiluted DNA (2.10 × 106 copy/mL), 10-1, 10-2, 10-3, 10-4, 10-5 and 10-6 (copy/mL) of MTB DNA were amplified by PCR and LAMP to determine the sensitivity of the assay. At results, the LAMP assay reported here has the advantages of rapid amplification, high sensitivity, and high specificity and will be useful for rapid and reliable clinical diagnosis of MTB in hospital clinical laboratory.

• Food Science of Animal Resources
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• v.33 no.5
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• pp.610-616
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• 2013

Loop-mediated isothermal amplification (LAMP) is an emerging detection technology for the amplification of DNA under isothermal conditions. The aim of this study was to develop a rapid and reliable LAMP technique for the detection of Cronobacter sakazakii in milk powder. In order to enhance the sensitivity and specificity, LAMP primers targeting outer membrane protein A (ompA) gene of C. sakazakii were designed using Explorer V4 software. Thirty seven C. sakazakii strains and 13 pathogenic microorganisms were used for comparative detection of C. sakazakii using polymerase chain reaction (PCR), real-time PCR, and LAMP. LAMP developed in this study could specifically detect C. sakazakii strains without cross-reactivity with other foodborne pathogens. LAMP products amplified from ompA gene of C. sakazakii were digested with with HhaI and NruI enzyme. The specificity of LAMP was confirmed by restriction fragment length polymorphism (RFLP) analysis. LAMP could detect C. sakazakii within 1 h without bacterial culture and its detection limit was as low as 1 CFU/mL C. sakazakii in milk. In the comparison of the sensitivity, LAMP showed 10,000- and 100-times higher detection limit than PCR or real-time PCR, respectively. Therefore, this study can conclude that LAMP is a rapid and reliable detection technique for C. sakazakii contaminated in powdered milk.

• Korean Journal of Veterinary Service
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• v.41 no.1
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• pp.29-39
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• 2018

In this study, we developed a sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid visual detection of foot-and-mouth disease virus (FMDV) circulated in Korea. The RT-LAMP was completed in 40 min at $62^{\circ}C$ and the results of the assay were directly detected by naked eye without any detection process. The assay specifically amplified all 7 serotypes of FMDV RNAs but not amplified other viral and cellular nucleic acids. The sensitivity of the RT-LAMP was $10^2$, $10^3$ and $10^3TCID_{50}/mL$ for serotype O, A and Asia 1 FMDV, respectively, which was comparable to conventional reverse transcription polymerase chain reaction (RT-PCR) and relatively lower than that of real time quantitative RT-PCR (qRT-PCR). Clinical evaluation of the RT-LAMP using different serotypes of Korean and foreign FMDV strains showed a 100% (35/35) agreement with the results of the RT-PCR and qRT-PCR. These results indicated that RT-LAMP assay developed in this study could be a valuable diagnostic method for FMDV monitoring and surveillance.

• The Plant Pathology Journal
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• v.36 no.1
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• pp.76-86
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• 2020

Cucumber mosaic virus (CMV) is damaging to the growth and quality of lettuce crops in Lanzhou, China. Recently, however, for the first time an isolate of lettuce necrotic yellows virus (LNYV) has been detected in lettuce crops in China, and there is concern that this virus may also pose a threat to lettuce production in China. Consequently, there is a need to develop a rapid and efficient detection method to accurately identify LNYV and CMV infections and help limit their spread. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed to detect the nucleoprotein (N) and coat protein (CP) genes of LNYV and CMV, respectively. RT-LAMP amplification products were visually assessed in reaction tubes separately using green fluorescence and gel electrophoresis. The assays successfully detected both viruses in infected plants without cross reactivity recorded from either CMV or LNYV or four other related plant viruses. Optimum LAMP reactions were conducted in betaine-free media with 6 mM Mg²⁺ at 65℃ for LNYV and 60℃ for 60 min for CMV, respectively. The detection limit was 3.5 pg/ml and 20 fg/ml using RT-LAMP for LNYV and CMV plasmids, respectively. Detection sensitivity for both RT-LAMP assays was greater by a factor of 100 compared to the conventional reverse transcription polymerase chain reaction assays. This rapid, specific, and sensitive technique should be more widely applied due to its low cost and minimal equipment requirements.

• Bulletin of the Korean Chemical Society
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• v.34 no.4
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• pp.1035-1038
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• 2013

Rolling circle amplification (RCA) of DNA on an aligned-carbon nanotube (a-CNT) surface was electrically interfaced by the a-CNT based filed effect transistor (FET). Since the electric conductance of the a-CNT will be dependent upon its local electric environment, the electric conductance of the FET is expected to give a very distinctive signature of the surface reaction along with this isothermal DNA amplification of the RCA. The a-CNT was initially grown on the quartz wafer with the patterned catalyst by chemical vapor deposition and transferred onto a flexible substrate after the formation of electrodes. After immobilization of a primer DNA, the rolling circle amplification was induced on chip with the a-CNT based FET device. The electric conductance showed a quite rapid increase at the early stage of the surface reaction and then the rate of increase was attenuated to reach a saturated stage of conductance change. It took about an hour to get the conductance saturation from the start of the conductance change. Atomic force microscopy was used as a complementary tool to support the successful amplification of DNA on the device surface. We hope that our results contribute to the efforts in the realization of a reliable nanodevice-based measurement of biologically or clinically important molecules.