• Animal Bioscience
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• 제36권4호
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• pp.654-670
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• 2023

Objective: This study aimed to develop loop-mediated isothermal amplification (LAMP) combined with lateral flow dipstick (LFD) and compare it with LAMP-AGE, polymerase chain reaction (PCR), and standard Salmonella culture as reference methods for detecting Salmonella contamination in animal products and animal production environmental samples. Methods: The SalInvA01 primer, derived from the InvA gene and designed as a new probe for LFD detection, was used in developing this study. Adjusting for optimal conditions by temperature, time, and reagent concentration includes evaluating the specificity and limit of detection. The sampling of 120 animal product samples and 350 animal production environmental samples was determined by LAMP-LFD, comparing LAMP-AGE, PCR, and the culture method. Results: Salmonella was amplified using optimal conditions for the LAMP reaction and a DNA probe for LFD at 63℃ for 60 minutes. The specificity test revealed no cross-reactivity with other microorganisms. The limit of detection of LAMP-LFD in pure culture was 3×10² CFU/mL (6 CFU/reaction) and 9.01 pg/μL in genomic DNA. The limit of detection of the LAMP-LFD using artificially inoculated in minced chicken samples with 5 hours of pre-enrichment was 3.4×10⁴ CFU/mL (680 CFU/reaction). For 120 animal product samples, Salmonella was detected by the culture method, LAMP-LFD, LAMP-AGE, and PCR in 10/120 (8.3%). In three hundred fifty animal production environmental samples, Salmonella was detected in 91/350 (26%) by the culture method, equivalent to the detection rates of LAMP-LFD and LAMP-AGE, while PCR achieved 86/350 (24.6%). When comparing sensitivity, specificity, positive predictive value, and accuracy, LAMP-LFD showed the best results at 100%, 95.7%, 86.3%, and 96.6%, respectively. For Kappa index of LAMP-LFD, indicated nearly perfect agreement with culture method. Conclusion: The LAMP-LFD Salmonella detection, which used InvA gene, was highly specific, sensitive, and convenient for identifying Salmonella. Furthermore, this method could be used for Salmonella monitoring and primary screening in animal products and animal production environmental samples.

• Journal of Microbiology and Biotechnology
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• 제29권10호
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• pp.1543-1552
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• 2019

Salmonella is a common zoonotic and foodborne pathogen that causes high morbidity and mortality in developing countries. In this study, we established and validated a polymerase spiral reaction (PSR) assay which targeted the conserved invasion gene (invA) of Salmonella by SYBR Green I indicator methods. Subsequently, assays for determination of the optimal conditions for optimal specificity and sensitivity of PSR were performed. We performed comprehensive evaluations using loop-mediated isothermal amplification (LAMP) and real-time PCR. A total number of 532 samples of daily food were analyzed by PSR. Twenty-seven bacterial strains were tested in the specificity assay, from which positive results were obtained only for 14-Salmonella strains. However, none of the 13 non-Salmonella strains was amplified. Similarly with LAMP and real-time PCR, the detection limit of the PSR assay was 50 CFU/ml. The PSR method was also successfully applied to evaluate the contamination with Salmonella in 532 samples of daily food, corroborating traditional culture method data. The novel PSR method is simple, sensitive, and rapid and provides new insights into the prevention and detection of foodborne diseases.

• The Plant Pathology Journal
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• 제34권3호
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• pp.191-198
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• 2018

Fast and accurate diagnosis is needed to eradicate and manage economically important and invasive diseases like fire blight. Loop-mediated isothermal amplification (LAMP) is known as the best on-site diagnostic, because it is fast, highly specific to a target, and less sensitive to inhibitors in samples. In this study, LAMP assay that gives more consistent results for on-site diagnosis of fire blight than the previous developed LAMP assays was developed. Primers for new LAMP assay (named as DS-LAMP) were designed from a histidine-tRNA ligase gene (EAMY_RS32025) of E. amylovora CFBP1430 genome. The DS-LAMP amplified DNA (positive detection) only from genomic DNA of E. amylovora strains, not from either E. pyrifoliae (causing black shoot blight) or from Pseudomonas syringae pv. syringae (causing shoot blight on apple trees). The detection limit of DS-LAMP was 10 cells per LAMP reaction, equivalent to $10^4$ cells per ml of the sample extract. DS-LAMP successfully diagnosed the pathogens on four fire-blight infected apple and pear orchards. In addition, it could distinguish black shoot blight from fire blight. The $B{\ddot{u}}hlmann$-LAMP, developed previously for on-site diagnosis of fire blight, did not give consistent results for specificity to E. amylovora and on-site diagnosis; it gave positive reactions to three strains of E. pyrifoliae and two strains of P. syringae pv. syringae. It also, gave positive reactions to some healthy sample extracts. DS-LAMP, developed in this study, would give more accurate on-site diagnosis of fire blight, especially in the Republic of Korea, where fire blight and black shoot blight coexist.

• 한국동물위생학회지
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• 제36권2호
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• pp.79-86
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• 2013

Mycobacterium (M.) bovis, a member of the M. tuberculosis complex (MTC), is a re-emerging, zoonotic agent of bovine tuberculosis whose prevalence probably depends on variations in direct exposure to cattle and ingestion of raw milk. Accurate species differentiation of M. bovis and M. tuberculosis is needed to distinguish between human and zoonotic tuberculosis. This study successfully developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection and differentiation of M. bovis and M. tuberculosis, however showed negative reactions in eight non-tuberculous mycobacteria (NTM) samples and ten other bacterial species. Sensitivity of this assay for detection of genomic M. bovis DNA was 10 $fg/{\mu}l$. And this assay successfully detected M. bovis in bovine clinical specimens. In conclusion, the LAMP assay is a simple and powerful tool for rapid detection of M. bovis in both pure bacterial culture and in clinical samples.

• Journal of Microbiology and Biotechnology
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• 제22권7호
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• pp.1021-1028
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• 2012

In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid, sensitive, and inexpensive detection of nervous necrosis virus (NNV) in olive flounder, Paralichthys olivaceus, in Korea. A set of six specific primers was designed to target the RNA 2 gene encoding the coat protein of Korean NNV strains. The RT-LAMP reaction successfully detected NNV after 30 min at $65^{\circ}C$. When the sensitivities among RT-LAMP, RT-PCR, and nested RTPCR were compared, the RT-LAMP was shown to be able to detect the RNA template at $2.58{\times}10^{-2}\;TCID_{50}/ml$, whereas the RT-PCR and nested RT-PCR were only able to detect the RNA template at $2.58{\times}10^2\;TCID_{50}/ml$ and $2.58TCID_{50}/ml$, respectively. Thus, the sensitivity of the RT-LAMP assay was higher than those of the RT-PCR assays. In the specificity test of the RT-LAMP, 2 genotypes of NNVs (SJNNV and RGNNV) were positive; however, no other fish viruses were positive with the primers, indicating that the RT-LAMP assay is only specific to NNV. A total of 102 olive flounder were collected from hatcheries between 2009 and 2011. The occurrence of NNV in olive flounder was determined to be 53.9% (55/102) by the RT-LAMP. On the other hand, the prevalence based on the nested RT-PCR and RT-PCR results was 33.8% (34/102) and 20.6% (21/102), respectively. This result indicates that the RT-LAMP assay developed in this study is suitable for early field diagnosis of NNV with high sensitivity.

• 대한의생명과학회지
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• 제25권1호
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• pp.75-82
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• 2019

We developed the multiplex LAMP assay using 16S rRNA, femA and mecA genes for direct detection of the methicillin resistance in Staphylococci from positive blood culture. To simultaneously recognize Staphylococci genus, S. aureus and methicillin resistance, three sets of six primers for 16S rRNA, femA and mecA were designed, respectively. The performance of LAMP assay was affirmed using VITEK system for the phenotypic methods of identification and for oxacillin and cefoxitin antimicrobial susceptibility. The optimal condition for LAMP assay was obtained under $64^{\circ}C$ for 50 min. The detection limit was determined to be of 20 copies and CFU/reaction ($10^4CFU/mL$). For clinical application of comparison with phenotypic methods, the sensitivity and specificity of the LAMP with femA gene for detecting S. aureus was 95.31% and 100%, respectively. The sensitivity and specificity of the LAMP with mecA gene for detecting methicillin resistance was 98.46% and 100%, respectively. The multiplex LAMP assay with femA and mecA gene successfully detected all of MRSA (38 isolates) isolates from 103 Staphylococci in blood cultures. The LAMP assay developed in this study is sensitive, specific, and of excellent agreement with the phenotypic methods.

• 대한의생명과학회지
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• 제28권4호
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• pp.334-339
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• 2022

African swine fever virus (ASFV) is a highly contagious and lethal pathogen that poses a threat to the global pork industry. The World Organization for Animal Health (WOAH) has placed strict surveillance measures for ASFV. The possibility of long-term survival of ASFV in raw meat or undercooked pork has been reported. Accordingly, the problem of secondary infection in food waste from households or waste disposal facilities has emerged, raising the need for ASFV monitoring of food waste. However, most of the previously reported ASFV gene detection methods are focused on clinical monitoring of pigs. There are very few cases in which their application in waste has been verified. Since ASFV diagnosis requires rapid monitoring and immediate action, loop-mediated isothermal amplification (LAMP) may be suitable, but this requires conformity assessment for LAMP to be used as a diagnostic technique. In this study, six LAMP methods were evaluated, and two methods (kit and manual) were recommended for use in diagnosing ASFV in food waste.

• 한국군사과학기술학회지
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• 제27권4호
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• pp.516-527
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• 2024

Rapid, early, accurate detection and identification of the various pathogenic agents associated with the development of biological weapons is critical in preventing loss of life and limiting the impact of these organisms when used against civilian or military targets. The aim of this study was to produce a system for the simple, rapid, accurate and simultaneous detection and identification of Ricin, Botulinum toxin B and Staphylococcal enterotoxin B as a proof of principle for developing field appropriate reverse transcription loop-mediated isothermal amplification systems for the accurate identification of potential biological threats. These systems were designed to facilitate the identification of potential threats even in remote or resource-limited locations.

• Asian Pacific Journal of Cancer Prevention
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• 제16권17호
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• pp.7409-7414
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• 2015

Loop-mediated isothermal amplification (LAMP) developed by Notomi et al. (2000) has made it possible to amplify DNA with high specificity, efficiency and rapidity under isothermal conditions. The ultimate products of LAMP are stem-loop structures with several inverted repeats of the target sequence and cauliflower-like patterns with multiple loops shaped by annealing between every other inverted repeats of the amplified target in the similar strand. Because the amplification process in LAMP is achieved by using four to six distinct primers, it is expected to amplify the target region with high selectivity. However, evaluation of reaction accuracy or quantitative inspection make it necessary to append other procedures to scrutinize the amplified products. Hitherto, various techniques such as turbidity assessment in the reaction vessel, post-reaction agarose gel electrophoresis, use of intercalating fluorescent dyes, real-time turbidimetry, addition of cationic polymers to the reaction mixture, polyacrylamide gel-based microchambers, lateral flow dipsticks, fluorescence resonance energy transfer (FRET), enzyme-linked immunosorbent assays and nanoparticle-based colorimetric tests have been utilized for this purpose. In this paper, we reviewed the best-known techniques for evaluation of LAMP amplicons and their applications in molecular biology beside their advantages and deficiencies. Regarding the properties of each technique, the development of innovative prompt, cost-effective and precise molecular detection methods for application in the broad field of cancer research may be feasible.

• Journal of Microbiology and Biotechnology
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• 제28권11호
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• pp.1928-1936
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• 2018

Recently, human infections caused by severe fever with thrombocytopenia syndrome virus (SFTSV), which can lead to fatality, have dramatically increased in East Asia. With the unavailability of vaccines or antiviral drugs to prevent and/or treat SFTSV infection, early rapid diagnosis is critical for prevention and control of the disease. Here, we report the development of a simple, rapid and sensitive portable detection method for SFTSV infection applying reverse transcription-loop mediated isothermal amplification (RT-LAMP) combined with one-pot colorimetric visualization and electro-free reaction platform. This method utilizes a pocket warmer to facilitate diagnosis in a resource-limited setting. Specific primers were designed to target the highly-conserved region of L gene of SFTSV. The detection limit of the RT-LAMP assay was approximately $10^0$ viral genome copies from three different SFTSV strains. This assay exhibited comparable sensitivity to qRT-PCR and 10-fold more sensitivity than conventional RT-PCR, with a rapid detection time of 30 to 60 minutes. The RT-LAMP assay using SFTSV clinical specimens has demonstrated a similar detection rate to qRT-PCR and a higher detection rate compared to conventional RT-PCR. Moreover, there was no observed cross-reactive amplification of other human infectious viruses including Japanese Encephalitis Virus (JEV), Dengue, Enterovirus, Zika, Influenza and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). This highly sensitive, electro- and equipment-free rapid colorimetric visualization method is feasible for resource-limited SFTSV field diagnosis.