Detection limits of LAMP assays.

<p>(A-C) <i>TP</i> LAMP assay involving (A) <i>TPA</i> strain Nichols with copy numbers 10E+5 to 10E+0, (B) <i>TPE</i> strain Gauthier tested in a range of 10E+6 to 10E+0 copies, and (C) <i>TEN</i> strain Bosnia A in 10E+6 to 10E+0 copy numbers. (D-E) <i>TPA</i> LAMP assay run with <i>TPA</i> strain Nichols from 10E+5 to 10E+0 copy numbers, and the same LAMP assay run with (E) <i>TEN</i> strain Bosnia A in copy numbers ranging from 10E+6 to 10E+0. (F-G) <i>TPE</i>/<i>TEN</i> LAMP assay involving (E) <i>TPE</i> strain Gauthier tested in 10E+6 to 10E+0 copy numbers, and (F) <i>TEN</i> strain Bosnia A tested in 10E+6 to 10E+0 copies. Red cross = Negative control, <i>TPA</i> strain Nichols (blue), <i>TPE</i> strain Gauthier (green), <i>TEN</i> strain Bosnia A (black); symbols represent copy numbers: circle with cross = 10E+6, hexagon = 10E+5, square turned = 10E+4, square = 10E+3, circle = 10E+2, down-pointing triangle = 10E+1, up-pointing triangle = 10E+0.</p

Gene target selection for loop-mediated isothermal amplification for rapid discrimination of <i>Treponema pallidum</i> subspecies

<div><p>We show proof of concept for gene targets (<i>polA</i>, <i>tprL</i>, and TP_0619) that can be used in loop-mediated isothermal amplification (LAMP) assays to rapidly differentiate infection with any of the three <i>Treponema pallidum</i> subspecies (<i>pallidum</i> (<i>TPA</i>), <i>pertenue</i> (<i>TPE</i>), and <i>endemicum</i> (<i>TEN</i>)) and which are known to infect humans and nonhuman primates (NHPs). Four <i>TPA</i>, six human, and two NHP <i>TPE</i> strains, as well as two human <i>TEN</i> strains were used to establish and validate the LAMP assays. All three LAMP assays were highly specific for the target DNA. Amplification was rapid (5–15 min) and within a range of 10E+6 to 10E+2 of target DNA molecules. Performance in NHP clinical samples was similar to the one seen in human <i>TPE</i> strains. The newly designed LAMP assays provide proof of concept for a diagnostic tool that enhances yaws clinical diagnosis. It is highly specific for the target DNA and does not require expensive laboratory equipment. Test results can potentially be interpreted with the naked eye, which makes it suitable for the use in remote clinical settings.</p></div

Simplified Real-Time Multiplex Detection of Loop-Mediated Isothermal Amplification Using Novel Mediator Displacement Probes with Universal Reporters

A variety of real-time detection techniques for loop-mediated isothermal amplification (LAMP) based on the change in fluorescence intensity during DNA amplification enable simultaneous detection of multiple targets. However, these techniques depend on fluorogenic probes containing target-specific sequences. That complicates the adaption to different targets leading to time-consuming assay optimization. Here, we present the first universal real-time detection technique for multiplex LAMP. The novel approach allows simple assay design and is easy to implement for various targets. The innovation features a mediator displacement probe and a universal reporter. During amplification of target DNA the mediator is displaced from the mediator displacement probe. Then it hybridizes to the reporter generating a fluorescence signal. The novel mediator displacement (MD) detection was validated against state-of-the-art molecular beacon (MB) detection by means of a HIV-1 RT-LAMP: MD surpassed MB detection by accelerated probe design (MD: 10 min, MB: 3–4 h), shorter times to positive (MD 4.1 ± 0.1 min shorter than MB, <i>n</i> = 36), improved signal-to-noise fluorescence ratio (MD: 5.9 ± 0.4, MB: 2.7 ± 0.4; <i>n</i> = 15), and showed equally good or better analytical performance parameters. The usability of one universal mediator-reporter set in different multiplex assays was successfully demonstrated for a biplex RT-LAMP of HIV-1 and HTLV-1 and a biplex LAMP of Haemophilus ducreyi and Treponema pallidum, both showing good correlation between target concentration and time to positive. Due to its simple implementation it is suggested to extend the use of the universal mediator-reporter sets to the detection of various other diagnostic panels

Detection limits for the three LAMP assays.

<p>Corresponding graphs can be found in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006396#pntd.0006396.g002" target="_blank">Fig 2</a>. + = exponential amplification, (+) = no exponential amplification,— = no amplification.</p

LAMP assays will simplify the existing test algorithm for human treponematoses.

<p>The test algorithm proposed by Ratnam 2005 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006396#pntd.0006396.ref042" target="_blank">42</a>] is shown in thin lines. The new LAMP assays for diagnosis of yaws is shown in bold lines and will simplify and accelerate yaws diagnosis. Furthermore, it enables the clinician to rapidly discriminate <i>TPE</i> infection from infection with other <i>TP</i> subspecies or pathogens.</p

Performance characteristics of the three LAMP assays.

<p>(A) LAMP targeting the <i>polA</i> gene. All <i>TP</i> strains become positive. (B) LAMP assay targeting the <i>tprL</i> locus, which results in amplification of <i>TPA</i> and <i>TEN</i> strains. (C) Only <i>TPE</i> and <i>TEN</i> strains generate positive results in the LAMP assay targeting the TP_0619 locus. Note that the strain material in the three assays had different <i>TP</i> copy numbers as indicated in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0006396#pntd.0006396.s004" target="_blank">S1 Table</a>. Data points are presented as mean±SEM values. Red cross = Negative control; <i>TPA</i> strains (blue) up-pointing triangle = Mexico A, down-pointing triangle = SS14, circle = Nichols, square = Seattle 81–4; <i>TPE</i> strains (green) square = Gauthier, square turned = Sei Geringing K403, hexagon = Kampung Dalan K363, circle = Samoa D, circle with cross = CDC-1, square with cross CDC-2; <i>TPE</i> simian strains (purple) cross = Fribourg-Blanc, star = RNP; <i>TEN</i> strains (black) square = Bosnia A, circle with cross = Iraq B.</p

Oligonucleotide primers used in this study.

<p>Oligonucleotide primers used in this study.</p

Primers designed for the RT-LAMP.

<p>Primers designed for the RT-LAMP.</p

Specificity of the RT-LAMP assay to detect CHIKV RNA (black line).

<p>There was no amplification of the negative control (continuous grey line) and RNA of other viruses assayed (discontinuous grey lines). (A) shows the amplification profile for the RT-LAMP, and (B) represents the annealing curve for specificity.</p

CHIKV RNA standard curve quantified by absolute one-step qRT-PCR.

<p>CHIKV RNA standard curve quantified by absolute one-step qRT-PCR.</p