• 한국동물위생학회지
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• 제38권3호
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• pp.145-153
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• 2015

In this study, we developed a loop-mediated isothermal amplification (LAMP) with hydroxynaphtol blue dye (HNB) for rapid and direct visual detection of porcine circovirus 2 DNA with high sensitivity and specificity. The LAMP was completed in 40 min at $63^{\circ}C$, and the results of the LAMP can be confirmed by naked eye without any detection process. The sensitivity of the LAMP was 10-fold higher than that of the commercial PCR (cPCR) and real time PCR (rPCR) previously reported. In clinical application, the PCV2 detection rate of the LAMP was the same on porcine tissue samples (75.0%, 36/48) between porcine blood samples (75.0%, 39/52). The PCV2 detection rate (75.0%) of LAMP was higher than those of the cPCR and rPCR (67.3%, 35/52) in blood samples. In conclusion, the LAMP developed in the study could be an useful alternative method for the detection of PCV2 in the swine disease diagnostic laboratories.

• 한국축산식품학회지
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• 제33권5호
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• pp.655-663
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• 2013

본 연구에서는 Salmonella invA 유전자를 마커로 Salmonella 특이 LAMP primer를 제작하였고, inclusivity 및 exclusivity 실험 결과는 각각 100%로 관찰되었다. 순수 배양된 Salmonella 희석액에서의 검출한계는 18.17분에서 $3.2{\times}10^3$ CFU/mL ($R^2$ = 0.9446)으로 관찰되어 신속 간단하고 민감도가 높아 Salmonella 검출을 위한 방법으로 효과적일 뿐만 아니라 간접적인 정량기법으로서도 활용 가능함을 알 수 있었다. 1차 증균 배지인 BPW에 S. Enteritidis 접종 후 0시간 및 12시간 배양 후 LAMP의 검출한계는 각각 $3.2{\times}10^3$ CFU/mL 및 $3.2{\times}10^0$ CFU/mL으로 관찰되었고, 오리도체 시료가 포함된 BPW에 S. Enteritidis 접종 후 0시간 및 12시간 증균액에서의 LAMP 검출한계는 각각 $3.2{\times}10^3$ CFU/mL 및 $3.2{\times}10^0$ CFU/mL으로 관찰되어 PCR과 비교하였을 때 오리도체시료 성분에 의한 영향이 비교적 낮았음을 알 수 있었다. 또한, 실제 도압장에서 채취한 오리도체시료를 BPW에 6시간 배양한 1차 증균액에 개발한 LAMP기법을 적용한 결과, 96%의 민감도 및 84%의 특이도를 보여 오리도체에서의 Salmonella 신속 스크리닝 기법으로서 효과적으로 활용될 수 있음을 알 수 있었다.

• 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.

• 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.

• 한국동물번식학회:학술대회논문집
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• 한국동물번식학회 2004년도 춘계학술발표대회
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• pp.183-183
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• 2004

In the bovine embryo transfer industry, sexing preimplantation embryos is an important management tool. Several methods for bovine embryo sexing utilizing polymerase chain reaction (PCR) have been developed. However, they were not popularized because the methods requiretechnical skills and expensive instruments, and are time consuming. PCR also has the risk of false positives due to DNA contamination during the electrophoresis. (omitted)

• The Plant Pathology Journal
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• 제36권2호
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• pp.170-178
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• 2020

The Lily mottle virus (LMoV) impedes the growth and quality of lily crops in Lanzhou, China. Recently Arabis mosaic virus (ArMV) has been detected in LMoV-infected plants in this region, causing plant stunting as well as severe foliar symptoms, and likely posing a threat to lily production. Consequently, there is a need to develop simple, sensitive, and reliable detection methods for these two viruses to prevent them from spreading. Reverse transcription (RT) loop-mediated isothermal amplification (LAMP) assays have been developed to detect LMoV and ArMV using two primer pairs that match six conserved sequences of LMoV and ArMV coat proteins, respectively. RT-LAMP assay results were visually assessed in reaction tubes using green fluorescence and gel electrophoresis. Our assays successfully detected both LMoV and ArMV in lily plants without the occurrence of viral cross-reactivity from other lily viruses. Optimal conditions for LAMP reactions were 65℃ and 60℃ for 60 min for LMoV and ArMV, respectively. Detection sensitivity for both RT-LAMP assays was a hundredfold greater than that of our comparative RT-polymerase chain reaction assays. We have also found this relatively rapid, target specific and sensitive method can also be used for samples collected in the field and may be especially useful in regions with limited or no laboratory facilities.

• 한국재료학회지
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• 제34권2호
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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.

• Parasites, Hosts and Diseases
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• 제51권3호
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• pp.269-277
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• 2013

Amoebic keratitis (AK) caused by Acanthamoeba is one of the most serious corneal infections. AK is frequently misdiagnosed initially as viral, bacterial, or fungal keratitis, thus ensuring treatment delays. Accordingly, the early detection of Acanthamoeba would contribute significantly to disease management and selection of an appropriate anti-amoebic therapy. Recently, the loop-mediated isothermal amplification (LAMP) method has been applied to the clinical diagnosis of a range of infectious diseases. Here, we describe a rapid and efficient LAMP-based method targeting Acanthamoeba 18S rDNA gene for the detection of Acanthamoeba using clinical ocular specimens in the diagnosis of AK. Acanthamoeba LAMP assays detected 11 different strains including all AK-associated species. The copy number detection limit for a positive signal was 10 DNA copies of 18S rDNA per reaction. No cross-reactivity with the DNA of fungi or other protozoa was observed. The sensitivity of LAMP assay was higher than those of Nelson primer PCR and JDP primer PCR. In the present study, LAMP assay based on directly heat-treated samples was found to be as efficient at detecting Acanthamoeba as DNA extracted using a commercial kit, whereas PCR was only effective when commercial kit-extracted DNA was used. This study showed that the devised Acanthamoeba LAMP assay could be used to diagnose AK in a simple, sensitive, and specific manner.

• 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.

• Journal of Microbiology and Biotechnology
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• 제23권12호
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• pp.1708-1716
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• 2013

Conventional molecular detection methods cannot distinguish between viable and dead Escherichia coli O157 cells. In this study, the loop-mediated isothermal amplification (LAMP) method combined with propidium monoazide (PMA) treatment was developed to selectively detect viable E. coli O157 cells. Four primers, including outer primers and inner primers, were specially designed for the recognition of six distinct sequences on the serogroups (O157) of the specific rfbE gene of the E. coli O157 genome. PMA selectively penetrated through the compromised cell membranes and intercalated into DNA. Amplification of DNA from dead cells was completely inhibited by $3.0{\mu}g/ml$ PMA, whereas the DNA derived from viable cells was amplified remarkably within 1 h by PMA-LAMP. Exhibiting high sensitivity and specificity, PMA-LAMP is a suitable method for evaluating the inactivation efficacy of slightly acidic electrolyzed water in broth. PMA-LAMP can selectively detect viable E. coli O157 cells. This study offers a novel molecular detection method to distinguish between viable and dead E. coli O157 cells.