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-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-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

Sensitive and less invasive confirmatory diagnosis of visceral leishmaniasis in Sudan using loop-mediated isothermal amplification (LAMP)

<div><p>Background</p><p>Confirmatory diagnosis of visceral leishmaniasis (VL), as well as diagnosis of relapses and test of cure, usually requires examination by microscopy of samples collected by invasive means, such as splenic, bone marrow or lymph node aspirates. This causes discomfort to patients, with risks of bleeding and iatrogenic infections, and requires technical expertise. Molecular tests have great potential for diagnosis of VL using peripheral blood, but require well-equipped facilities and trained personnel. More user-friendly, and field-amenable options are therefore needed. One method that could meet these requirements is loop-mediated isothermal amplification (LAMP) using the Loopamp <i>Leishmania</i> Detection Kit, which comes as dried down reagents that can be stored at room temperature, and allows simple visualization of results.</p><p>Methodology/Principal findings</p><p>The Loopamp <i>Leishmania</i> Detection Kit (Eiken Chemical Co., Japan), was evaluated in the diagnosis of VL in Sudan. A total of 198 VL suspects were tested by microscopy of lymph node aspirates (the reference test), direct agglutination test-DAT (in house production) and rK28 antigen-based rapid diagnostic test (OnSite <i>Leishmania</i> rK39-Plus, CTK Biotech, USA). LAMP was performed on peripheral blood (whole blood and buffy coat) previously processed by: i) a direct boil and spin method, and ii) the QIAamp DNA Mini Kit (QIAgen). Ninety seven of the VL suspects were confirmed as cases by microscopy of lymph node aspirates. The sensitivity and specificity for each of the tests were: rK28 RDT 98.81% and 100%; DAT 88.10% and 78.22%; LAMP-boil and spin 97.65% and 99.01%; LAMP-QIAgen 100% and 99.01%.</p><p>Conclusions/Significance</p><p>Due to its simplicity and high sensitivity, rK28 RDT can be used first in the diagnostic algorithm for primary VL diagnosis, the excellent performance of LAMP using peripheral blood indicates that it can be also included in the algorithm for diagnosis of VL as a simple test when parasitological confirmatory diagnosis is required in settings that are lower than the reference laboratory, avoiding the need for invasive lymph node aspiration.</p></div

Role of Loopamp <i>Leishmania</i> Detection Kit in the VL diagnostic algorithm.

<p>Role of Loopamp <i>Leishmania</i> Detection Kit in the VL diagnostic algorithm.</p

Identification of positive and negative LAMP results using blue LED light illumination with a visualization unit that is integral to the Loopamp LF-160 incubator.

<p>Identification of positive and negative LAMP results using blue LED light illumination with a visualization unit that is integral to the Loopamp LF-160 incubator.</p

Details and summary of the diagnostic performance of different studies using LAMP for VL diagnosis compared to our study.

<p>Details and summary of the diagnostic performance of different studies using LAMP for VL diagnosis compared to our study.</p

Evaluation of clinical specimens using LAMP assay.

<p>A. Visualization by Naked Eye: 1. positive control; 2. CCD6 Chronic Chagas Disease (case 6); 3. NI8: non-infected patient, (case 8); 4. AI-TxRID 2: acute infection of transplanted recipient from infected donor (case 2); 5. RCD 1: reactivated Chagas disease (case 1); 6. CCD1: chronic Chagas disease 1 (case 1); 7. CI 4: congenital Chagas disease (case 4); 8. negative control. B. Detection of LAMP reaction using Genie III Fluorimeter. 1: positive control; 2 to 7: clinical specimens indicated in A; 7: Negative control. The Y Axis denotes Fluorescence and X axis denotes Tt (time when fluorescence passes the threshold).</p

Inclusivity of LAMP assay tested in purified DNA samples from <i>T</i>. <i>cruzi</i> strains representative of the different discrete type units.

<p>Left panels: Visualization by the naked eye of <i>T</i>. <i>cruzi</i> DNA stocks representative of different DTUs. Tc I (A: 1.0 x10^-2 fg/test; B: 1.0 x10^-3 fg/test); Tc II (C: 2.5 fg/test; D: 2.5x10^-1 fg/test); Tc III (E: 7.5 x 10⁻² fg/test; F: 7.5 x 10⁻³ fg/test); Tc IV (G: 5.0 x 10⁻¹ fg/test, H: 5.0 x 10⁻² fg/test); Tc V (I: 1.5 x 10⁻¹ fg/test; J: 1.5 x 10⁻² fg/test); Tc VI (K: 1.0 x 10⁻¹ fg/test; L. 1.0 x 10⁻² fg/test) Right panels: Amplification plots obtained in the LAMP reaction after analyzing the samples in a Rotor Gene 3000 thermocycler. Y axis represents fluorescence and x axis represents Cts (Threshold cycles). Only the highest dilution giving amplification and the next dilution giving non detectable results are shown.</p

Clinical specimens results from LAMP test and qPCR as a comparator.

<p>Clinical specimens results from LAMP test and qPCR as a comparator.</p