scholarly journals Loop-Mediated Isothermal Amplification (LAMP): The Better Sibling of PCR?

Cells ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1931
Author(s):  
Marianna Soroka ◽  
Barbara Wasowicz ◽  
Anna Rymaszewska
Keyword(s):  
Dna Polymerase ◽  
Isothermal Amplification ◽  
Dna Denaturation ◽  
Japanese Company ◽  
Loop Mediated Isothermal Amplification ◽  
Naked Eye ◽  
Stable Temperature ◽  
Rna Targets ◽  
Eiken Chemical ◽  
Standard Techniques

In 1998, when the PCR technique was already popular, a Japanese company called Eiken Chemical Co., Ltd. designed a method known as the loop-mediated isothermal amplification of DNA (LAMP). The method can produce up to 109 copies of the amplified DNA within less than an hour. It is also highly specific due to the use of two to three pairs of primers (internal, external, and loop), which recognise up to eight specific locations on the DNA or RNA targets. Furthermore, the Bst DNA polymerase most used in LAMP shows a high strand displacement activity, which eliminates the DNA denaturation stage. One of the most significant advantages of LAMP is that it can be conducted at a stable temperature, for instance, in a dry block heater or an incubator. The products of LAMP can be detected much faster than in standard techniques, sometimes only requiring analysis with the naked eye. The following overview highlights the usefulness of LAMP and its effectiveness in various fields; it also considers the superiority of LAMP over PCR and presents RT-LAMP as a rapid diagnostic tool for SARS-CoV-2.

scholarly journals Room-temperature stable loop-mediated isothermal amplification (LAMP) reagents to detect leptospiral DNA

2021 ◽  
Vol 15 (4) ◽  
pp. 183-189
Author(s):  
Pui-Yuei Lee ◽  
Yien-Ping Wong ◽  
Shuhaidah Othman ◽  
Hui-Yee Chee
Keyword(s):  
Dna Polymerase ◽  
Cold Storage ◽  
Room Temperature ◽  
Rat Kidney ◽  
Isothermal Amplification ◽  
Storage Requirement ◽  
Loop Mediated Isothermal Amplification ◽  
Modified Method ◽  
Resource Limited ◽  
The Stability

Abstract Background Loop-mediated isothermal amplification (LAMP) is one of the most promising tools for rapidly detecting Leptospira spp. However, LAMP is hampered by cold storage to maintain the enzymatic activity of Bst DNA polymerase. Objective To overcome the drawback of cold storage requirement for LAMP reagents we modified the reagents by adding sucrose as stabilizer. We then sought to determine the stability at room temperature of the premixed LAMP reagents containing sucrose. Method Premixed LAMP reagents with sucrose and without sucrose were prepared. The prepared mixtures were stored at room temperature for up to 60 days, and were subjected to LAMP reactions at various intervals using rat kidney samples to detect leptospiral DNA. Results The premixed LAMP reagents with sucrose remained stable for 45 days while sucrose-free premixed LAMP reagents showed no amplification from day 1 of storage at room temperature up to day 14. Conclusion The LAMP reagent system can be refined by using sucrose as stabilizer, thus allowing their storage at room temperature without the need for cold storage. The modified method enables greater feasibility of LAMP for field surveillance and epidemiology in resource-limited settings.

scholarly journals Probing Loop-Mediated Isothermal Amplification (LAMP) targeting two gene-fragments of rose rosette virus

2021 ◽  
Author(s):  
Andrea Salazar ◽  
Francisco M. Ochoa-Corona ◽  
Jennifer D. Olson ◽  
Binoy Babu ◽  
Mathews Paret
Keyword(s):  
Dna Polymerase ◽  
Limit Of Detection ◽  
Rna Extraction ◽  
Cross Reactivity ◽  
Isothermal Amplification ◽  
Loop Mediated Isothermal Amplification ◽  
Capture Method ◽  
Visual Diagnosis ◽  
Rose Rosette ◽  
The Rose

AbstractThis study explores the development of Loop-mediated isothermal amplification of DNA (LAMP) for detection of rose rosette emaravirus (RRV), a technique with the potential to be translated to rose nurseries. RRV is a negative-sense single-stranded RNA Emaravirus and causal agent of the rose rosette disease (RRD). Transmission of RRV is by Phyllocoptes fructiphilus, an eriophyid mite. Although RRV symptoms are characteristics, early visual diagnosis of RRD can be misleading and confusing since it may appear similar to herbicide damage. Two sets of RRV gene sequences composed of twenty-two accessions of RRV-P3 (RNA 3) and another twenty-four from RRV-P4 (RNA 4) were analyzed and two sets of four LAMP primers were designed for broad-range detection of RRV isolates. The direct antigen-capture method for direct trapping of RRV in plastic was used for RNA extraction followed by cDNA synthesis. LAMP reactions were optimized for Bst 2.0 DNA polymerase using the outer RRV-F3/RRV-B3 primers, and internal RRV-FIP/RRV-BIP primers. LAMP reactions were for 1 hour at 64°C (RRV-P3) and 66.5°C (RRV-P4) using either a thermocycler or a portable dry bath. LAMP was also optimized using DNA polymerase GspSSD LD using the same RRV sets of primers. RRV was detected in symptomatic and non-symptomatic RRD tissue from Oklahoma. The limit of detection (LoD) using Bst 2.0 LAMP was 1pg/μL and 1 fg/μL with GspSSD LD quantitative LAMP. The LoD of pre-reaction hydroxy naphthol blue (HNB, 120 μM) for colorimetric (visual) reactions was 10 pg/μL and 0.1 pg/μL using SYBR green I (1:10 dilution) in colorimetric post-reactions. No cross-reactivity was detected in LAMP reaction testing cDNAs of eight commonly co-infecting rose viruses (INSV, ArMV, MSpV, TSWV, ApMV, PNRSV, ToRSV, and TMV), and one virus taxonomically related to RRV (HPWMoV). RNA from healthy rose tissues and non-template controls (water) were included in all LAMP assays.

scholarly journals Loop-Mediated Isothermal Amplification Allows Rapid, Simple and Accurate Molecular Diagnosis of Human Cutaneous and Visceral Leishmaniasis Caused by Leishmania infantum When Compared to PCR

2021 ◽  
Vol 9 (3) ◽  
pp. 610
Author(s):  
Ana Victoria Ibarra-Meneses ◽  
Carmen Chicharro ◽  
Carmen Sánchez ◽  
Emilia García ◽  
Sheila Ortega ◽  
...  
Keyword(s):  
Visceral Leishmaniasis ◽  
Real Time ◽  
Parasite Load ◽  
Isothermal Amplification ◽  
Excellent Correlation ◽  
Loop Mediated Isothermal Amplification ◽  
Real Time Quantitative Pcr ◽  
Specific Amplification ◽  
Polymerase Chain ◽  
Eiken Chemical

Loop-mediated isothermal amplification allows the rapid, sensitive and specific amplification of DNA without complex and expensive equipment. We compared the diagnostic performance of Loopamp™ Leishmania Detection Kit (Eiken Chemical Co., Ltd., Tokyo, Japan) with conventional and real-time polymerase chain reaction (PCR) for human cutaneous and visceral leishmaniasis caused by L. infantum. A total of 230 DNA samples from cutaneous (CL) and visceral (VL) leishmaniasis cases and controls from Spain, characterized by Leishmania nested PCR (LnPCR) were tested by: (i) the Loopamp™ Leishmania Detection Kit (Loopamp), run on Genie III real-time fluorimeter (OptiGene, UK); and (ii) real-time quantitative PCR (qPCR). The Loopamp test returned 98.8% (95% confidence interval—CI: 96.0–100.00) sensitivity and specificity of 97.7% (95% CI: 92.2–100) on VL samples, and 100% (95% CI: 99.1–100) sensitivity and 100.0% (95% CI: 98.8–100.0) specificity on CL samples. The Loopamp time-to-positivity (Tp) obtained by real-time fluorimetry showed excellent concordance (C = 97.91%) and strong correlation (r = 0.799) with qPCR’s cycle threshold (Ct). The performance of Loopamp is comparable to that of LnPCR and qPCR in the diagnosis of cutaneous and visceral leishmaniasis due to L. infantum. The excellent correlation between the Tp and Ct should be further investigated to determine the accuracy of Loopamp to quantify parasite load in tissues.

scholarly journals Feasibility and Operational Performance of Tuberculosis Detection by Loop-Mediated Isothermal Amplification Platform in Decentralized Settings: Results from a Multicenter Study

2016 ◽  
Vol 54 (8) ◽  
pp. 1984-1991 ◽  
Author(s):  
Christen M. Gray ◽  
Achilles Katamba ◽  
Pratibha Narang ◽  
Jorge Giraldo ◽  
Carlos Zamudio ◽  
...  
Keyword(s):  
Isothermal Amplification ◽  
Nucleic Acid Amplification ◽  
Smear Microscopy ◽  
Eligibility Criteria ◽  
Loop Mediated Isothermal Amplification ◽  
Root Cause ◽  
Operational Characteristics ◽  
Reaction Volumes ◽  
Nonspecific Amplification ◽  
Eiken Chemical

Currently available nucleic acid amplification platforms for tuberculosis (TB) detection are not designed to be simple or inexpensive enough to implement in decentralized settings in countries with a high burden of disease. The loop-mediated isothermal amplification platform (LAMP) may change this. We conducted a study in adults with symptoms suggestive of TB in India, Uganda, and Peru to establish the feasibility of using TB-LAMP (Eiken Chemical Co.) in microscopy laboratories compared with using smear microscopy against a reference standard of solid and liquid cultures. Operational characteristics were evaluated as well. A total of 1,777 participants met the eligibility criteria and were included for analysis. Overall, TB-LAMP sensitivities among culture-positive samples were 97.2% (243/250; 95% confidence interval [CI], 94.3% to 98.2%) and 62.0% (88/142; 95% CI, 53.5% to 70.0%) for smear-positive and smear-negative TB, respectively, but varied widely by country and operator. Specificities ranged from 94.5% (446/472; 95% CI, 92.0% to 96.4%) to 98.0% (350/357; 95% CI, 96.0% to 99.2%) by country. A root cause analysis identified high temperatures, high humidity, and/or low reaction volumes as possible causes for false-positive results, as they may result in nonspecific amplification. The study was repeated in India with training focused on vulnerable steps and an updated protocol; 580 participants were included for analysis. Specificity in the repeat trial was 96.6% (515/533; 95% CI, 94.7% to 97.9%). To achieve acceptable performance of LAMP at the microscopy center level, significant training and infrastructure requirements are necessary.

Rapid detection of orf virus by loop-mediated isothermal amplification based on the DNA polymerase gene

2012 ◽  
Vol 158 (4) ◽  
pp. 793-798 ◽  
Author(s):  
Jida Li ◽  
Deguang Song ◽  
Wenqi He ◽  
Yingfu Bao ◽  
Rongguang Lu ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Rapid Detection ◽  
Isothermal Amplification ◽  
Orf Virus ◽  
Polymerase Gene ◽  
Loop Mediated Isothermal Amplification ◽  
Dna Polymerase Gene

scholarly journals Loop-mediated isothermal amplification–fluorescent loop primer assay for the genotyping of a single nucleotide polymorphism at position 2254 in the viral DNA polymerase gene of equid alphaherpesvirus 1

2019 ◽  
Vol 31 (4) ◽  
pp. 640-644 ◽  
Author(s):  
Koji Tsujimura ◽  
Hiroshi Bannai ◽  
Manabu Nemoto ◽  
Hiroshi Kokado
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Dna Polymerase ◽  
Isothermal Amplification ◽  
Polymerase Gene ◽  
Nucleotide Polymorphism ◽  
Viral Dna ◽  
Single Nucleotide ◽  
Loop Primer ◽  
Loop Mediated Isothermal Amplification ◽  
Annealing Curve

We developed a loop-mediated isothermal amplification (LAMP)–fluorescent loop primer (FLP) assay for genotyping the A/G2254 single nucleotide polymorphism (SNP) in the viral DNA polymerase gene of species Equid alphaherpesvirus 1 (EHV-1), which is associated with the neuropathogenic potential of this virus. In addition to the use of regular LAMP primers to amplify the target region, a 5’-FAM–labeled backward loop primer (FLB) and 3’-dabcyl–labeled quencher probe (QP) were designed for annealing curve analysis of the amplification product. The QP, which contacts the FLB, is located at the SNP site and has the A2254 allele. LAMP reactions were performed at 63°C for 40 min, and the subsequent annealing curve analyses were accomplished within 20 min. The LAMP-FLP assay could clearly differentiate A2254 and G2254 genotypes according to the difference in the annealing temperature of the QP between the 2 genotypes. Good agreement between the LAMP-FLP and the real-time PCR for genotyping of this SNP was observed in the detection of EHV-1 in equine clinical samples. The newly developed assay is a simple and rapid method for detecting and differentiating EHV-1 strains with A2254 and G2254 polymorphisms and would be suitable for clinical use.

scholarly journals Specific and rapid identification of the Pheretima aspergillum by loop-mediated isothermal amplification

2019 ◽  
Vol 39 (2) ◽  
Author(s):  
Qing Huang ◽  
Zhiwu Li ◽  
Zhiguo Ma ◽  
He Li ◽  
Runqian Mao
Keyword(s):  
Dna Polymerase ◽  
Rapid Identification ◽  
Isothermal Amplification ◽  
Coi Gene ◽  
Conventional Pcr ◽  
Lamp Product ◽  
Loop Mediated Isothermal Amplification ◽  
Animal Medicine ◽  
Site Identification ◽  
Dna Template

Abstract Guang-dilong (Pheretima aspergillum) is a traditional Chinese animal medicine that has been used for thousands of years in China. In the present study, we purposed to establish a new rapid identification method for Guang-dilong. We provided a useful technique, loop-mediated isothermal amplification (LAMP), to differentiate Guang-dilong from other species. Four specific LAMP primers were designed based on mitochondrial cytochrome c oxidase I (COI) gene sequences of Guang-dilong. LAMP reaction, containing DNA template, four primers, 10× Bst DNA polymerase reaction buffer, dNTPs, MgSO4, and Bst DNA polymerase, was completed within 60 min at 63°C. The LAMP product can be visualized by adding SYBR Green I or detected by 2% gel electrophoresis. LAMP technology was successfully established for rapid identification of Guang-dilong. In addition, DNA template concentration of 675 fg/μl was the detection limit of LAMP in Guang-dilong, which was 1000-times higher than conventional PCR. The simple, sensitive, and convenient LAMP technique is really suited for on-site identification of Guang-dilong in herbal markets.

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