Loop-mediated isothermal amplification (LAMP): principle, features, and future prospects

Tsugunori Notomi,Yasuyoshi Mori,Norihiro Tomita,Hidetoshi Kanda 한국미생물학회 2015 The journal of microbiology Vol.53 No.1

Loop-mediated isothermal amplification (LAMP), a newlydeveloped gene amplification method, combines rapidity,simplicity, and high specificity. Several tests have been developedbased on this method, and simplicity is maintainedthroughout all steps, from extraction of nucleic acids to detectionof amplification. In the LAMP reaction, samples areamplified at a fixed temperature through a repetition of twotypes of elongation reactions occurring at the loop regions:self-elongation of templates from the stem loop structureformed at the 3 -terminal and the binding and elongation ofnew primers to the loop region. The LAMP reaction has awide range of possible applications, including point-of-caretesting, genetic testing in resource-poor settings (such as indeveloping countries), and rapid testing of food productsand environmental samples.

Inhibition of Non-specific Amplification in Loop-Mediated Isothermal Amplification via Tetramethylammonium Chloride

장민주,김상효 한국바이오칩학회 2022 BioChip Journal Vol.16 No.3

Loop-mediated isothermal amplification (LAMP) may be used in molecular and point-of-care diagnostics for pathogen detection. The amplification occurs under isothermal conditions using up to six primers. However, non-specific amplification is frequently observed in LAMP. Non-specific amplification has the potential to be triggered by forward and reverse internal primers. And the relatively low reaction temperature (55–65 °C) induces the secondary structure via primer–primer interactions. Primer redesign and probe design have been recommended to solve this problem. LAMP primers have strict conditions, such as Tm, GC contents, primer dimer, and distance between primers compared to conventional PCR primers. Probe design requires specialized knowledge to have high specificity for a target. In polymerase chain reaction (PCR), some chemicals or proteins are used for improving specificity and efficiency. Therefore, we hypothesized that additives can suppress the non-specific amplification. In this study, tetramethylammonium chloride (TMAC), formamide, dimethyl sulfoxide, Tween 20, and bovine serum albumin have been used as LAMP additives. In our study, TMAC was presented as a promising additive for suppressing non-specific amplification in LAMP.

Loop-Mediated Isothermal Amplification Assay Targeting the femA Gene for Rapid Detection of Staphylococcus aureus from Clinical and Food Samples

( Xi Hong Zhao ),( Yan Mei Li ),( Myoung Su Park ),( Jun Wang ),( You Hong Zhang ),( Xiao Wei He ),( Fereidoun ),( Forghani ),( Li Wang ),( Guang Chao Yu ),( Deog Hwan Oh ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.2

In this study, a loop-mediated isothermal amplification (LAMP) method to rapidly detect Staphylococcus aureus strains was developed and evaluated by extensively applying a large number of S. aureus isolates from clinical and food samples. Six primers were specially designed for recognizing eight distinct sequences on the species-specific femA gene of S. aureus. The detection limits were 100 fg DNA/tube and 104 CFU/ml. The LAMP assay was applied to 432 S. aureus strains isolated from 118 clinical and 314 food samples. Total detection rates for the LAMP and polymerase chain reaction assays were 98.4% (306/311) and 89.4% (278/311), respectively.

Rapid, Sensitive, and Specific Detection of Clostridium tetani by Loop-Mediated Isothermal Amplification Assay

( Dong Neng Jiang ),( Xiao Yun Pu ),( Jie Hong Wu ),( Meng Li ),( Ping Liu ) 한국미생물 · 생명공학회 2013 Journal of microbiology and biotechnology Vol.23 No.1

Tetanus is a specific infectious disease, which is often associated with catastrophic events such as earthquakes, traumas, and war wounds. The obligate anaerobe Clostridium tetani is the pathogen that causes tetanus. Once the infection of tetanus progresses to an advanced stage within the wounds of limbs, the rates of amputation and mortality increase manifold. Therefore, it is necessary to devise a rapid and sensitive point-of-care detection method for C. tetani so as to ensure an early diagnosis and clinical treatment of tetanus. In this study, we developed a detection method for C. tetani using loop-mediated isothermal amplification (LAMP) assay, wherein the C. tetani tetanus toxin gene was used as the target gene. The method was highly specific and sensitive, with a detection limit of 10 colony forming units (CFU)/ml, and allowed quantitative analysis. While detecting C. tetani in clinical samples, it was found that the LAMP results completely agreed with those of the traditional API 20A anaerobic bacteria identification test. As compared with the traditional API test and PCR assay, LAMP detection of C. tetani is simple and rapid, and the results can be identified through naked-eye observation. Therefore, it is an ideal and rapid point-of-care testing method for tetanus.

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Nocardia salmonicida, the Causative Agent of Nocardiosis in Fish

( Qun Xia Li ),( Hong Lian Zhang ),( Yi Shan Lu ),( Jia Cai ),( Bei Wang ),( Ji Chang Jian ) 한국미생물 · 생명공학회 2015 Journal of microbiology and biotechnology Vol.25 No.3

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°C. 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.

Development of a Loop-mediated Isothermal Amplification Assay for Detecting Listeria monocytogenes prfA in Milk

조애리,동희진,서건호,조성범 한국식품과학회 2014 Food Science and Biotechnology Vol.23 No.2

A rapid and sensitive loop-mediated isothermalamplification (LAMP) assay was developed for detectingListeria monocytogenes prfA in milk. The inclusivity of 23L. monocytogenes and the exclusivity of 16 non-L. monocytogenes strains were both 100% in the assay. Thelimit of detection (LoD) of the LAMP assay in Listeriaenrichment broth (LEB) was 2.22 CFU/mL after 12 h and24 h of incubation. The LoDs of the LAMP assay in LEBwith artificially contaminated milk (LEB-M) incubated for12 h (2.22×101 CFU/mL) and 24 h (2.22 CFU/mL) werelower than those of the PCR and real-time PCR assays. Comparison of the LoDs in LEB with those in LEB-Mshowed that the LAMP assay was less influenced by themilk compounds than the real-time PCR assay. Our resultsindicate that the LAMP assay can be utilized as a potentialscreening tool for L. monocytogenes in milk.

Loop-mediated isothermal amplification (LAMP)법을 이용한 Vibrio alginolyticus의 신속 진단법 개발

홍승현(Seung-hyun Hong),허문수(Moon-Soo Heo) 한국생명과학회 2015 생명과학회지 Vol.25 No.8

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. Loop-mediated isothermal amplification (LAMP), a PCR-based diagnostic method, is based on autocycling strand displacement DNA synthesis in the presence of exonuclease-negative Bst DNA polymerase under isothermal conditions. With the help of four specific primers that recognize six different sequences of a target DNA, LAMP has high specificity in pathogenic identification in a short time. Hence, in the present study, LAMP is used as a diagnostic tool in the identification of the most dreadful aquatic pathogenic species, Vibrio alginolyticus, and to develop species-specific LAMP primers and optimization of LAMP reaction conditions such as annealing temperature, elongation time, and other PCR chemical concentrations, including MgSO₄, dNTPs, Betaine, and Bst polymerase. The optimized LAMP primers were also checked for specificity with other Vibrio species, which showed that the designed primers were very specific to V. alginolyticus After the first introduction of a species name like this one, the first part (“Vibrio” in this case) should be abbreviated to only the first letter.only. These are usually the most harmful pathogens of the Vibrio species that appear in shrimp and crabs. The results also revealed that the LAMP assay could be 10-fold more sensitive than conventional PCR in detecting V. alginolyticus. This could be the first report on using a rapid and highly sensitive technique, the LAMP assay, in the effective diagnosis of the pathogenic bacteria V. alginolyticus, which could help in the early detection of diseases, particularly in aquaculture.

Loop-mediated isothermal amplification (LAMP)법을 이용한 Streptococcus parauberis의 신속 진단

문경미(Kyung-Mi Moon),김동휘(Dong-Hwi Kim),허문수(Moon-Soo Heo) 한국생명과학회 2014 생명과학회지 Vol.24 No.4

Loop-mediated isothermal amplification (LAMP)법은 등온에서 DNA 주형을 변성시키지 않고 실시하기 때문에, autocycling 가닥 변위 DNA 합성에 의존한다. 그래서 고가의 PCR 장비를 필요로 하지 않고 등온 유지가 가능한 저가의 장비인 항온 수조, 오븐, 온장고 등에서 증폭이 가능하다. 본 연구진은 Streptococcus parauberis의 random primer중에서 5개를 선정하여, 신장도가 높은 2개의 primer를 이용하여 최적 반응온도 및 최적 반응시간, 최적 반응 조건들을 확립하였다. 그리고 기존의 PCR과 LAMP의 민감도의 비교 분석을 측정한 결과, LAMP의 높은 검출 한계를 확인할 수 있었다. 본 논문에서는 non-target DNA의 영향을 받지 않고 등온 조건 하에서 DNA를 증폭시킬 수 있는 LAMP법과 SYBR-green I를 이용하여 시각화시켰으며, 기존의 PCR과 비교 분석함으로써, S. parauberis에 대한 신속하고 정확한 진단법을 확립하였다. Loop-mediated isothermal amplification (LAMP) technique relies on autocycling strand displacement DNA sysnthesis without template denaturation steps under isothermal conditions. LAMP has more advantages than modern PCR, as it requires only basic laboratory equipment like an isothermal water bath, oven, and heating cabinet. Hence, in this study, five random primers were designed with Streptococcus parauberis, shikimate kinase Arok gene sequences (Genbank accession number: CP0024711). Two primers were selected based on the ladder pattern. Other optimum reaction conditions like temperature, time, and sensitivity were established and confirmed with conventional SYBR-green PCR. Results confirmed that the designed random primers were species specific, without any non-target DNA amplification under isothermal conditions. Hence, this study suggests that LAMP techniques could be used in the diagnosis of fish pathogen, specifically S. parauberis.

Development of a high-throughput centrifugal loop-mediated isothermal amplification microdevice for multiplex foodborne pathogenic bacteria detection

Seo, Ji Hyun,Park, Byung Hyun,Oh, Seung Jun,Choi, Goro,Kim, Do Hyun,Lee, Eun Yeol,Seo, Tae Seok Elsevier 2017 Sensors and actuators. B, Chemical Vol.246 No.-

<P><B>Abstract</B></P> <P>Since the foodborne pathogenic bacteria cause serious diseases and socio-economic losses throughout the world, the molecular diagnosis of foodborne poisoning pathogen in the early stage is essential for preventing excessive damages. In this study, a colorimetric based foodborne pathogen detection on the centrifugal microdevice was demonstrated in a high throughput manner. The developed centrifugal microdevice consists of a sample reservoir, a spiral shaped sample injection microchannel, 24 aliqouting chambers (2.5μL each), cross capillary valves, and 24 reaction chambers on a single device. Once the sample was loaded, the rotation speed at 850rpm allowed the sample solution to be divided into 24 aliqouting chambers. Then, the samples loaded in the aliqouting chambers could be injected into the reaction chambers at 5000rpm evenly. The centrifugal device was placed in an oven at 66°C for target gene amplification. As a gene amplification method, loop mediated isothermal amplification (LAMP) was used in which specific primer sets targeting three kinds of foodborne pathogens (<I>Escherichia coli</I> O157:H7, <I>Salmonella typhimurium</I>, and <I>Vibrio parahaemolyticus</I>) were included. The presence of each pathogen was identified by Eriochrome Black T (EBT)-mediated colorimetric change from purple to sky blue with the naked eye, and the color of each reaction chamber was analyzed by a ratiometric image processing method. Green/Red (G/R) and Blue/Red (B/R) ratios were set as the criteria to differentiate negative results from positive ones. The threshold values for G/R (0.915<G/R<1.098) and B/R (B/R=1.2723) ratios were determined from 216 experimental data (84 negative and 132 positive) using the RGB-based image processing method. The entire genetic analytical process was completed on the high-throughput centrifugal microdevice within 1h. The extracted DNA of each pathogen was successfully amplified and detected at a 500 copy level. Furthermore, the bacterial cell could be directly used as a sample and analyzed at a 100 cell level, thereby demonstrating the great potential for the sample-to-answer generic analysis of pathogens on a chip even in resource limited environments.</P> <P><B>Highlights</B></P> <P> <UL> <LI> A centrifugal LAMP microdevice was developed for the multiplex pathogen detection. </LI> <LI> The cultured bacterial cells were directly used. </LI> <LI> The entire genetic analysis was completed on the microdevice within 1h. </LI> <LI> The gene amplification was verified by the EBT-mediated colorimetric change. </LI> <LI> A RGB-based image processing was conducted to improve the colorimetric assay. </LI> </UL> </P>

Loop-mediated isothermal amplification assay for detection of four immunosuppressive viruses in chicken

Song, HyeSoon,Bae, YouChan,Park, SeokChan,Kwon, HyukMan,Lee, HeeSoo,Joh, SeongJoon Elsevier 2018 JOURNAL OF VIROLOGICAL METHODS Vol.256 No.-

<P><B>Abstract</B></P> <P>Loop-mediated isothermal amplification (LAMP) methods to detect chicken infectious anemia virus (CIAV), reticuloendotheliosis virus (REV), and Marek’s disease virus (MDV), and a reverse transcription (RT)-LAMP assay to detect infectious bursal disease virus (IBDV), were developed. The CIAV-LAMP, REV-LAMP, MDV-LAMP, and IBDV-RT-LAMP methods were performed using four sets of six primers targeting the VP1 gene of CIAV, the gp90 gene of REV, the Meq gene of MDV, and the VP2 gene of IBDV. The results (a change in color) were observed visually. The methods showed high specificity and sensitivity. The detection limits were 50 genomic copies of CIAV, 16 genomic copies of REV, 20 genomic copies of MDV, and 250 genomic copies of IBDV. When used to test clinical samples, the results of the LAMP assays were in 100% agreement with a previously described PCR. Therefore, the LAMP assays are simple, rapid, highly sensitive, and specific methods for detecting four immune-suppressive viruses.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Four immunosuppressive viruses in chicken can be detected with LAMP. </LI> <LI> The results of detection can be observed visually by a change in color. </LI> <LI> The detection methods show high specificity and sensitivity. </LI> </UL> </P>