• Journal of Microbiology and Biotechnology
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• 제31권4호
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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.

• Genomics & Informatics
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• 제19권3호
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• pp.30.1-30.8
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• 2021

Salmonella species are among the major pathogens that cause foodborne illness outbreaks. In this study, we aimed to develop a loop-mediated isothermal amplification (LAMP) assay for the rapid and sensitive detection of Salmonella species. We designed LAMP primers targeting the hilA gene as a universal marker of Salmonella species. A total of seven Salmonella species strains and 11 non-Salmonella pathogen strains from eight different genera were used in this study. All Salmonella strains showed positive amplification signals with the Salmonella LAMP assay; however, there was no non-specific amplification signal for the non-Salmonella strains. The detection limit was 100 femtograms (20 copies per reaction), which was ~1,000 times more sensitive than the detection limits of the conventional polymerase chain reaction (PCR) assay (100 pg). The reaction time for a positive amplification signal was less than 20 minutes, which was less than one-third the time taken while using conventional PCR. In conclusion, our Salmonella LAMP assay accurately detected Salmonella species with a higher degree of sensitivity and greater rapidity than the conventional PCR assay, and it may be suitable for point-of-care testing in the field.

• The Plant Pathology Journal
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• 제33권2호
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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.

• 한국미생물·생명공학회지
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• 제44권4호
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• pp.493-496
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• 2016

Peach rosette mosaic virus (PRMV)는 1933년 복숭아에서 처음 보고되었으며, 복숭아, 자두, 블루베리, 민들레, 벚나무 등에 감염되는 식물바이러스이다. PRMV는 한국에서 보고된 적이 없으나, 식물검역에서 관리병(control viruses)으로 지정되어 있다. 이번 연구에서는 PRMV를 더욱 신속하고 특이적으로 진단하기 위하여 Loop-mediated isothermal amplification 분석법을 적용한 진단법을 개발하였다. LAMP 방법은 기존의 PCR 방법(RT-PCR 및 nested PCR)과 같은 검출 강도를 가지고 있다. 또한 LAMP 반응을 확인하기 위해 PRMV cDNA을 outer primer sets (Product size 264 bp)로 PCR 한 뒤, Pvu II (CAG/CTG) 제한효소를 처리하였다. 제한효소 처리 결과 2개의 digestion fragments (207 + 57 bp)가 확인되었다. PRMV의 LAMP 진단 방법은 관련 식물로부터 더욱 신속한 모니터링이 가능할 것으로 기대된다.

• 대한화학회지
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• 제55권2호
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• pp.262-267
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• 2011

형광 검출기를 기반으로 한 변성 고성능 액체 크로마토그래피(DHPLC)분석법은 핵산검출에 유용하게 사용되고 있으며 또한 등온증폭 반응법은 병원성 미생물의 효과적인 검출방법으로 알려져 있다. 이 연구에서는 HBV의 조기 분석 방법으로써 사용되는 등온증폭반응법(LAMP)과 변성고성능 액체 크로마토그래피(DHPLC)의 검출한계와 특이성, 그리고 민감도를 평가하였다. 등온증폭 반응법의 검출 시간이 가장 빠른 장점을 보였으나, 변성 고성능 액체 크로마토그래피 분석법이 등온증폭반응법과 real-time PCR분석법과 비교한 결과, 10배 이상의 높은 민감도를 확인 할 수 있었다. 본 결과로서 B형 간염 바이러스의 진단을 위하여, 빠른 검출법으로써 등온증폭 반응법이 유용하게 쓰일 수 있을 것이며 변성 고성능 액체 크로마토그래피 분석법은 좀 더 낮은 병원균의 감염도 검출할 수 있어, 임상에서 유용하게 사용할 수 있을 것이다.

• 한국동물위생학회지
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• 제45권1호
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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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• 제39권4호
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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.

• 농업과학연구
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• 제47권3호
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• pp.673-681
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• 2020

Human enteric Adenovirus 41 (HueAdV-41) is a major waterborne virus that causes human gastroenteritis and is classified as a viral group I double-strand DNA virus, Adenoviridae. HueAdV-41 has been detected with the polymerase chain reaction (PCR) in various samples such as ground water. However, the PCR-based diagnostic method has problems such as reaction time, sensitivity, and specificity. Thus, the loop-mediated isothermal amplification (LAMP) assay has emerged as an excellent method for field applications. In this study, we developed a LAMP system that can rapidly detect HueAdV-41 with high specificity and sensitivity. HueAdV-41 specific LAMP primer sets were tested through a specific, non-specific selection and sensitivity test for three prepared LAMP primer sets, of which only one primer set and optimum reaction temperature were selected. The developed LAMP primer set condition was confirmed as 63℃, and the sensitivity was 1 copy. In addition, to confirm the system, a LAMP positive reaction was developed with the restriction enzyme Taq I (T/GCC). The developed method in this study was more specific, rapid (typically within 2 - 3 hours), and highly sensitive than that of the conventional PCR method. To evaluate and verify the developed LAMP assay, an artificial infection test was done with five cDNAs from groundwater samples, and the results were compared to those of the conventional PCR method. We expect the developed LAMP primer set will be used to diagnose HueAdV-41 from various samples.

• Parasites, Hosts and Diseases
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• 제52권3호
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• pp.305-310
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• 2014

Ascidian soft tunic syndrome (AsSTS) caused by Azumiobodo hoyamushi (A. hoyamushi) is a serious aquaculture problem that results in mass mortality of ascidians. Accordingly, the early and accurate detection of A. hoyamushi would contribute substantially to disease management and prevention of transmission. Recently, the loop-mediated isothermal amplification (LAMP) method was adopted for clinical diagnosis of a range of infectious diseases. Here, the authors describe a rapid and efficient LAMP-based method targeting the 18S rDNA gene for detection of A. hoyamushi using ascidian DNA for the diagnosis of AsSTS. A. hoyamushi LAMP assay amplified the DNA of 0.01 parasites per reaction and detected A. hoyamushi in 10 ng of ascidian DNA. To validate A. hoyamushi 18S rDNA LAMP assays, AsSTS-suspected and non-diseased ascidians were examined by microscopy, PCR, and by using the LAMP assay. When PCR was used as a gold standard, the LAMP assay showed good agreement in terms of sensitivity, positive predictive value (PPV), and negative predictive value (NPV). In the present study, a LAMP assay based on directly heat-treated samples was found to be as efficient as DNA extraction using a commercial kit for detecting A. hoyamushi. Taken together, this study shows the devised A. hoyamushi LAMP assay could be used to diagnose AsSTS in a straightforward, sensitive, and specific manner, that it could be used for forecasting, surveillance, and quarantine of AsSTS.

• 한국동물위생학회지
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• 제40권1호
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• pp.15-20
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• 2017

A two-tube reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was designed for the rapid visual detection of the M gene of all subtypes of avian influenza virus (AIV) and the H5 gene of the H5 subtype of highly pathogenic AIV (HPAIV). The reaction carried out in two tubes in a single step at $58^{\circ}C$ for 40 min, and the assay results could be visually detected by using hydroxynaphthol blue dye. Using M or H5 gene-specific primers, the assay successfully detected all subtypes or H5 subtypes of AIVs, including the Korean representative H5N1 and H5N8 HPAIVs. The detection limit of the assay was approximately $10^{2.0}$ $EID_{50}/reaction$ for the M and H5 genes of H5N1 HPAIV, respectively, and was more sensitive than that of previously reported RT-LAMP and comparable to that of real-time RT-PCR. These results suggest that the present RT-LAMP assay, with its high specificity, sensitivity, and simplicity, will be a useful diagnostic tool for surveillance of currently circulating H5 HPAIVs and other subtypes of AIV in bird population, even in under-equipped laboratories.