• Journal of Microbiology and Biotechnology
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• 제31권12호
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• pp.1672-1683
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• 2021

Vibrio parahaemolyticus is recognized as one of the most important foodborne pathogens responsible for gastroenteritis in humans. The bla_CARB-17 gene is an intrinsic β-lactamase gene and a novel species-specific genetic marker of V. parahaemolyticus. In this study, a loop-mediated isothermal amplification (LAMP) assay combined with a lateral flow dipstick (LFD) was developed targeting this bla_CARB-17 gene. The specificity of LAMP-LFD was ascertained by detecting V. parahaemolyticus ATCC 17802 and seven other non-V. parahaemolyticus strains. Finally, the practicability of LAMP-LFD was confirmed by detection with V. parahaemolyticus-contaminated samples and natural food samples. The results showed that the optimized reaction parameters of LAMP are as follows: 2.4 mmol/l Mg²⁺, 0.96 mmol/l dNTPs, 4.8 U Bst DNA polymerase, and an 8:1 ratio of inner primer to outer primer, at 63℃ for 40 min. The optimized reaction time of the LFD assay is 60 min. Cross-reactivity analysis with the seven non-V. parahaemolyticus strains showed that LAMP-LFD was exclusively specific for V. parahaemolyticus. The detection limit of LAMP-LFD for V. parahaemolyticus genomic DNA was 2.1 × 10^-4 ng/μl, corresponding to 630 fg/reaction and displaying a sensitivity that is 100-fold higher than that of conventional PCR. LAMP-LFD in a spiking study revealed a detection limit of approximately 6 CFU/ml, which was similar with conventional PCR. The developed LAMP-LFD specifically identified the 10 V. parahaemolyticus isolates from 30 seafood samples, suggesting that this LAMP-LFD may be a suitable diagnostic method for detecting V. parahaemolyticus in aquatic foods.

• Journal of Microbiology and Biotechnology
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• 제25권3호
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• pp.321-327
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• 2015

Nocardia salmonicida is one of the main pathogens of fish nocardiosis. The purpose of this study was to build a loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of N. salmonicida. A set of four primers were designed from the 16S-23S rRNA intergenic spacer region of N. salmonicida, and conditions for LAMP were optimized as incubating all the reagents for 60 min at 64℃. LAMP products were judged with agar gel electrophoresis as well as with the naked eye after the addition of SYBR Green I. Results showed the sensitivity of the LAMP assay was 1.68 × 10³ CFU/ml (16.8 CFU per reaction) and 10-fold higher than that of PCR. The LAMP method was also effectively applied to detect N. salmonicida in diseased fish samples, and it may potentially facilitate the surveillance and early diagnosis of fish nocardiosis.

• 한국어병학회지
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• 제25권3호
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• pp.257-262
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• 2012

본 연구에서는 어체내 IHNV 모니터링에 LAMP법의 사용이 가능 하는지를 검토하기 위해 IHNV를 무지개송어에 인위적으로 감염시킨 후 시간 경과에 따라 LAMP법과 어류세포를 사용한 분리배양법을 이용하여 IHNV 검사를 실시하였다. IHNV를 $10^{6.5}\;TCID_{50}$/fish, $10^{5.5}\;TCID_{50}$/fish, $10^{4.5}\;TCID_{50}$/fish로 복강 주사한 결과, 40%, 0%, 0%의 누적폐사율이 관찰되었다. 폐사어 및 IHNV 접종 후 16일과 28일째에 각 실험구에서 채집한 생존어 5마리를 대상으로 한 IHNV 검사 결과, 폐사어에서 IHNV가 100% (8/8 마리) 분리되었고 (감염가: $10^{4.3}-10^{6.8}\;TCID_{50}/ml$), RT-LAMP법에서도 100% 검출되었다. 16일째 생존한 개체를 대상으로 한 IHNV 검사 결과에서는 $10^{6.5}\;TCID_{50}$/fish, $10^{5.5}\;TCID_{50}$/fish, $10^{4.5}\;TCID_{50}$/fish의 IHNV로 접종한 실험구에서 각각 60% (3/5 마리, 감염가: $10^{2.8}-10^{5.05}\;TCID_{50}/ml$), 20% (1/5 마리, $10^{1.05}\;TCID_{50}/ml$), 60% (3/5 마리, $10^{1.05}-10^{4.8}\;TCID_{50}/ml$) 의 검출율을 보였으나 LAMP법에서는 20% (1/5 마리), 0% (0/5 마리), 20% (1/5 마리) 의 검출율을 나타내었다. 28일째 생존한 개체 및 대조구의 어류에서는 IHNV가 분리 검출되지 않았다. 이상의 연구결과로 LAMP법은 IHNV-생존어에서 바이러스를 모니터링 하는데 한계가 있으나 병어로부터 IHNV를 검출하는데 유용하게 사용될 수 있을 것으로 사료되었다.

• 한국균학회지
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• 제47권4호
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• pp.437-441
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• 2019

국화에 발생하는 반쪽시들음병은 Veriticillium dahliae에 의해 발생하는 진균병으로 국화 재배농가에 상당한 경제적 손실을 야기한다. 일반 식물병원균을 동정하는 방법으로는 병원균을 진단하기까지 상당한 시간이 소요된다. 본 연구에서는V. dahliae를 신속하고 특이적으로 진단하기 위하여 등온증폭기술 (Loop-mediated isothermal amplification, LAMP)을 적용한 검출법을 개발하였다. 이 방법은 반쪽시들음병균의 cellulose-growth-specific protein partial mRNA 유전자 염기서열을 이용하여 4개의 특이 프라이머 세트를 제작하였다. 최적 반응조건 및 시간은 60℃ 내외의 온도조건에서 60분 이내에서 가장 효율이 좋은 것으로 나타났다. 이 등온증폭 검출법은 4종의 토양전염성 병원균과 기주식물의 DNA에는 반응하지 않았다. 따라서 반쪽시들음병균 등온증폭법을 활용한다면 병원균의 감염 유무를 조기에 신속하게 진단할 수 있고, 반쪽시들음병을 효율적으로 모니터링하고 방제할 수 있을 것으로 기대한다.

• Parasites, Hosts and Diseases
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• 제51권2호
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• pp.237-241
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• 2013

Giardia lamblia is recognized as one of the most prevalent parasites in dogs. The present study aimed to establish a loop-mediated isothermal amplification (LAMP) assay for rapid and specific detection of G. lamblia from dogs. The fecal samples were collected and prepared for microscopic analysis, and then the genomic DNA was extracted directly from purified cysts. The concentration of DNA samples of G. lamblia were diluted by 10-fold serially ranging from $10^{-1}$ to $10^{-5}ng/{\mu}l$ for LAMP and PCR assays. The LAMP assay allows the amplification to be finished within 60 min under isothermal conditions of $63^{\circ}C$ by employing 6 oligonucleotide primers designed based on G. lamblia elongation factor 1 alpha ($EF1{\alpha}$) gene sequence. Our tests showed that the specific amplification products were obtained only with G. lamblia, while no amplification products were detected with DNA of other related protozoans. Sensitivity evaluation indicated that the LAMP assay was sensitive 10 times more than PCR. It is concluded that LAMP is a rapid, highly sensitive and specific DNA amplification technique for detection of G. lamblia, which has implications for effective control and prevention of giardiasis.

• 한국동물위생학회지
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• 제41권1호
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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.

• 식물병연구
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• 제28권2호
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• pp.92-97
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• 2022

Apple stem grooving virus (ASGV) is a high-risk viral pathogen that infects many types of fruit trees, especially pear and apple, and causes serious economic losses across the globe. Thus, rapid and reliable detection assay is needed to identify ASGV infection and prevent its spread. A reliable reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed, optimize, and evaluated for the coding region of coat protein of ASGV in pear leaf. The developed RT-LAMP facilitated the simple screening of ASGV using visible fluorescence and electrophoresis. The optimized reaction conditions for the RT-LAMP were 63℃ for 50 min, and the results showed high specificity and 100-fold greater sensitivity than the reverse transcription polymerase chain reaction. In addition, the reliability of the RT-LAMP was validated using field-collected pear leaves. Furthermore, the potential application of paper-based RNA isolation, combined with RT-LAMP, was also evaluated for detecting ASGV from field-collected samples. These assays could be widely applied to ASGV detection in field conditions and to virus-free certification programs.

• 대한수의학회지
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• 제54권2호
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• pp.113-115
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• 2014

Salmonella are causative agents of gastroenteritis and systemic disease in animals. The invA gene was selected as a target sequence of loop-mediated isothermal amplification (LAMP) assay for diagnosis of Salmonella infection. The detection limits for broth dilution, spiked feces and enrichment were $10^4$, $10^5$ and $10^2$ CFUs/mL, respectively. The LAMP assay developed in the present study may be a reliable method for detection of Salmonella spp. in pig feces.

• 식물병연구
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• 제22권3호
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• pp.178-183
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• 2016

SYMMV는 콩에서 빈번하게 발생하는 바이러스이며, 이 바이러스의 발생률은 계속해서 증가하고 있다. 본 연구에서는 콩에 발생하는 SYMMV를 신속하게 검출하기 위해서 RT-LAMP를 적용하였다. RT-LAMP 방법은 등온에서 단시간에 유전자 증폭이 가능하고, 전기영동 없이도 형광물질을 이용해 바이러스를 검출할 수 있는 이점이 있다. 프라이머는 SYMMV coat protein gene의 염기서열을 기반으로 4개의 프라이머를 설계하였다. 실험결과 SYMMV RT-LAMP는 $65^{\circ}C$에서 50분간 증폭시켰을 때 최적의 효율을 보였다. 또한, RT-LAMP와 RT-PCR과의 민감도를 비교한 결과 RT-LAMP 방법이 10-100배 정도 더 우수한 민감도를 가지는 것으로 밝혀졌다. 본 실험 결과를 토대로 기존의 진단법과 비교하여 높은 민감도와 짧은 소요시간에 이점이 있는 RT-LAMP는 SYMMV의 현장 및 연구실에서의 진단에 적용될 수 있을 것이라 생각된다.

• 생명과학회지
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• 제25권8호
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• pp.903-909
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• 2015

LAMP (Loop-mediated Isothermal Amplification)법은 PCR를 기반으로 등온에서 autocycling 가닥 변위 DNA 합성에 의존하며, Bst polymerase를 사용하여 진단하는 방법이다. 이것은 대상 DNA의 여섯 개의 배열을 인식하는 4개의 특정 primer의 도움을 받아 단시간 안에 병원체를 식별하는 높은 특이성을 지니고 있다. 본 연구에서는 LAMP로 수생에서 위험한 병원체인 Vibrio alginolyticus의 특별한 LAMP primer를 제작하였으며, 신속한 진단을 위해 MgSO₄, dNTP, Betaine, Bst polymerase의 최적 반응 조건의 특이성 및 기존의 PCR보다 10배 정도의 민감하다는 것을 확인하였다. 또한, 디자인 되어진 LAMP primer가 다른 Vibrio 종들 중 오직 V. alginolyticus에서만 반응한 것을 확인 할 수 있었다. 본 논문에서는 병원체 세균인 V. alginolyticus의 빠르고 민감한 효과적인 진단으로 양식 질병들을 조기에 발견할 수 있도록 개발하였다.