Sequence specific visual detection of LAMP reactions by addition of cationic polymers

BACKGROUND: Development of a practical gene point-of-care testing device (g-POCT device) requires innovative detection methods for demonstrating the results of the gene amplification reaction without the use of expensive equipment. We have studied a new method for the sequence-specific visual detection of minute amounts of nucleic acids using precipitation reaction by addition of cationic polymers to amplicons of Loop mediated isothermal Amplification (LAMP). RESULTS: Oligo DNA probes labeled with different fluorescent dyes were prepared for multiple nucleic acid templates, and the templates were amplified by the LAMP reactions under the existence of the probes. At completion of the LAMP reaction, an optimal amount of low molecular weight polyethylenimine (PEI) was added, resulting in the precipitation of the insoluble LAMP amplicon-PEI complex. The fluorescently labeled Oligo DNA probes hybridized to the LAMP product were incorporated into the precipitation, and the precipitate emitted fluorescence corresponding to the amplified nucleic acid templates. The color of emitted fluorescence can be detected easily by naked eye on a conventional UV illuminator. CONCLUSION: The presence or absence of minute amount of nucleic acid templates could be detected in a simple manner through visual assessment for the color of the LAMP amplicon-PEI complex precipitate. We conclude that this detection method may facilitate development of small and simple g-POCT device

Loop-mediated isothermal amplification (Lamp): A rapid, sensitive, specific, and cost-effective point-of-care test for coronaviruses in the context of covid-19 pandemic

The rampant spread of COVID-19 and the worldwide prevalence of infected cases demand a rapid, simple, and cost-effective Point of Care Test (PoCT) for the accurate diagnosis of this pandemic. The most common molecular tests approved by regulatory bodies across the world for COVID-19 diagnosis are based on Polymerase Chain Reaction (PCR). While PCR-based tests are highly sensitive, specific, and remarkably reliable, they have many limitations ranging from the requirement of sophisticated laboratories, need of skilled personnel, use of complex protocol, long wait times for results, and an overall high cost per test. These limitations have inspired researchers to search for alternative diagnostic methods that are fast, economical, and executable in low-resource laboratory settings. The discovery of Loop-mediated isothermal Amplification (LAMP) has provided a reliable substitute platform for the accurate detection of low copy number nucleic acids in the diagnosis of several viral diseases, including epidemics like Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS). At present, a cocktail of LAMP assay reagents along with reverse transcriptase enzyme (Reverse Transcription LAMP, RT-LAMP) can be a robust solution for the rapid and cost-effective diagnosis for COVID-19, particularly in developing, and low-income countries. In summary, the development of RT-LAMP based diagnostic tools in a paper/strip format or the integration of this method into a microfluidic platform such as a Lab-on-a-chip may revolutionize the concept of PoCT for COVID-19 diagnosis. This review discusses the principle, technology and past research underpinning the success for using this method for diagnosing MERS and SARS, in addition to ongoing research, and the prominent prospect of RT-LAMP in the context of COVID-19 diagnosis

Clinical Evaluation of Loop-Mediated Isothermal Amplification (LAMP) Assay for Rapid Detection of \u3cem\u3eNeisseria meningitidis\u3c/em\u3e in Cerebrospinal Fluid

Background Neisseria meningitidis (Nm) is a leading causative agent of bacterial meningitis in humans. Traditionally, meningococcal meningitis has been diagnosed by bacterial culture. However, isolation of bacteria from patients’ cerebrospinal fluid (CSF) is time consuming and sometimes yields negative results. Recently, polymerase chain reaction (PCR)-based diagnostic methods of detecting Nm have been considered the gold standard because of their superior sensitivity and specificity compared with culture. In this study, we developed a loop-mediated isothermal amplification (LAMP) method and evaluated its ability to detect Nm in cerebrospinal fluid (CSF). Methodology/Principal Findings We developed a meningococcal LAMP assay (Nm LAMP) that targets the ctrA gene. The primer specificity was validated using 16 strains of N. meningitidis (serogroup A, B, C, D, 29-E, W-135, X, Y, and Z) and 19 non-N. meningitidis species. Within 60 min, the Nm LAMP detected down to ten copies per reaction with sensitivity 1000-fold more than that of conventional PCR. The LAMP assays were evaluated using a set of 1574 randomly selected CSF specimens from children with suspected meningitis collected between 1998 and 2002 in Vietnam, China, and Korea. The LAMP method was shown to be more sensitive than PCR methods for CSF samples (31 CSF samples were positive by LAMP vs. 25 by PCR). The detection rate of the LAMP method was substantially higher than that of the PCR method. In a comparative analysis of the PCR and LAMP assays, the clinical sensitivity, specificity, positive predictive value, and negative predictive value of the LAMP assay were 100%, 99.6%, 80.6%, and 100%, respectively. Conclusions/Significance Compared to PCR, LAMP detected Nm with higher analytical and clinical sensitivity. This sensitive and specific LAMP method offers significant advantages for screening patients on a population basis and for diagnosis in clinical settings

Homogeneous Assays for Single-Nucleotide Polymorphism Genotyping : Exo-proofreading Assay based on Loop-mediated Isothermal Amplification

We have developed a new method Proofreading-LAMP (PR-LAMP) for genotyping single-nucleotide polymorphisms (SNPs) in combination with the use of nucleic acid amplification by loop-mediated isothermal amplification (LAMP) and the 3\u27-5\u27 exonuclease proofreading (exo-proofreading) activity of DNA polymerase. Using as a model a detection system for the SNP (G1951A) found in the human aldehyde dehydrogenase 2 (ALDH2) gene, typing primers with fluorescent-labeled 3\u27 ends, and cloned DNA as a template, consisting of ALDH2^ * 1, ALDH2 ^* 2, or a mixture of ALDH2^ * 1 and ALDH2^ * 2, and 3\u27-5\u27 exo+ Pwo DNA polymerase were added to a LAMP reaction system, and amplification and the exo-proofreading reaction proceeded at 60℃ for 30 minutes, at the same time as which SNPs were detected by fluorescence polarization (FP). This procedure permitted evaluation by means of a single-step reaction at 60℃ for 30 minutes for ALDH2^ * 1, ALDH2 ^* 1/ * 2, and ALDH2 ^* 2, and allowed a single nucleotide change to be distinguished with excellent reproducibility. Furthermore, the typing of human genomic samples, similar to the procedure for cloned DNA, yielded results that were in complete agreement with the results of PCR-RFLP. PR-LAMP is accurate, simple, rapid, and robust, and is a flexible method that may be used in applications ranging from point-of-care testing (POCT) to high-throughput screening (HTS)

Sequence specific visual detection of LAMP reactions by addition of cationic polymers-1

<p><b>Copyright information:</b></p><p>Taken from "Sequence specific visual detection of LAMP reactions by addition of cationic polymers"</p><p>BMC Biotechnology 2006;6():3-3.</p><p>Published online 10 Jan 2006</p><p>PMCID:PMC1373654.</p><p>Copyright © 2006 Mori et al; licensee BioMed Central Ltd.</p>lution. After LAMP reaction in the presence of both FITC-labeled HBV probes and ROX-labeled HCV probes followed by addition of the prescribed amount (0.2 μmol as monomer) of PEI (Mw = 600), it was centrifuged for several seconds using a desk-top, low-speed centrifuge. The tube was then visually observed as is on a UV illuminator (365 nm). It was possible to differentiate the LAMP reaction by visualizing the presence of precipitate fluorescence and the color of the fluorescence. 1, LAMP reaction negative. 2, When LAMP reaction with PSA amplification (unrelated LAMP reaction) occurred. 3, When it contained HBV template nucleic acid. 4, When it contained HCV template nucleic acid. 5, When it contained both HBV and HCV template nucleic acids. (B) Diagram of principle of sequence-specific visual detection method that utilizes precipitation titration of LAMP products by adding PEI. First, a LAMP reaction is carried out using a LAMP primer set for two types of template nucleic acid and fluorescently labeled probes, which can hybridize to loop segments of each LAMP products. When a LAMP reaction corresponding to a certain fluorescently labeled probe progresses, the probe will sequentially hybridize to the loop segment generated during the reaction. On the other hand, an unrelated probe remains free in the solution. When an optimized amount of PEI is added after reaction for a set length of time, the positive charge of PEI neutralizes the negative charge of the DNA to form an insoluble LAMP product-PEI complex. At this stage, fluorescently labeled probes hybridized to LAMP products are taken up by the LAMP product-PEI complex together with the LAMP products. Since most of the PEI added is used for formation of the LAMP product-PEI complex, free oligo DNA probes cannot form a complex with PEI. When the generated insoluble complex is pelletized by centrifugation and the pellet is irradiated with excitation light, the labeled fluorescent dye hybridized to LAMP products produces fluorescence

Sequence specific visual detection of LAMP reactions by addition of cationic polymers-2

<p><b>Copyright information:</b></p><p>Taken from "Sequence specific visual detection of LAMP reactions by addition of cationic polymers"</p><p>BMC Biotechnology 2006;6():3-3.</p><p>Published online 10 Jan 2006</p><p>PMCID:PMC1373654.</p><p>Copyright © 2006 Mori et al; licensee BioMed Central Ltd.</p>NA recognition probes by DNA-PEI complex (Mw of PEI is 600). When 0.2 μmol to 1.0 μmol of PEI was added as a monomer, almost 100% of labeled probes hybridized to the LAMP products for lambda DNA was taken up by the DNA-PEI complex. On the other hand, when 0.4 μmol to 0.8 μmol of PEI was added as a monomer to a reaction solution in which an amplification reaction did not take place, a small amount

Sequence specific visual detection of LAMP reactions by addition of cationic polymers-0

<p><b>Copyright information:</b></p><p>Taken from "Sequence specific visual detection of LAMP reactions by addition of cationic polymers"</p><p>BMC Biotechnology 2006;6():3-3.</p><p>Published online 10 Jan 2006</p><p>PMCID:PMC1373654.</p><p>Copyright © 2006 Mori et al; licensee BioMed Central Ltd.</p>ng amplification step, and elongation and recycling step) by the primers depicted in the enclosure. In the starting material production step, the starting material (6) is generated by primers (forward inner primer (FIP) and backward inner primer (BIP)). A complementary strand (11) of the starting material (6) is synthesized from the starting material (6) by a reaction that uses itself as a template and by a reaction from an FIP annealed to the loop segment, thus making up the cycle amplification step. During this step, probes (probe F and probe B, respectively) designed for the region between the F1 and F2 region or the B1 and B2 region can hybridize to the loop segment. As the cycle reaction progresses, an elongation and recycling step takes place, during which elongated products (8, 13, etc.) with an inverted repeat structure are generated. Numbers 14 and 15, which have a cauliflower structure, are also generated. They have many loop structures to which probes can hybridize