Melt curve analysis of the HIV-1 and β-actin biplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay.

<p>Melt analysis showed products specific to HIV-1 at 78°C and β-actin at 89°C as seen in the singleplex assays. Specificity was confirmed in the presence of normal human plasma (NHP) without HIV-1 or β-actin (negative control) and showed no amplification (n = 3).</p

PATH NINA heater.

<p>(Right) Cross-section showing the internal components of the heater. Approximate dimensions of the heater are 80 mm diameter by 120 mm height.</p

Average temperature profiles of 74 runs with nine prototype non-instrumented nucleic acid amplification (NINA) heaters.

<p>Error bars show one standard deviation. Four devices were removed from testing when a failure mode rendered them no longer able to maintain temperature within specification. Final run data are not included in the graph. Mean time between failures (MTBF) for the failed devices is 14 runs.</p

A comparison of the effects on the limit of detection (LOD) of assays heated by the non-instrumented nucleic acid amplification (NINA) heater.

<p>Combination of assays run with and without warm-up ramp and with or without oil are evaluated. Table entries show the number of replicates that returned a positive result as the numerator of a fraction showing the total number of replicates run in the denominator.</p><p>A comparison of the effects on the limit of detection (LOD) of assays heated by the non-instrumented nucleic acid amplification (NINA) heater.</p

Three non-instrumented nucleic acid amplification (NINA) heaters were conditioned in an environmental chamber, and the performance of each heater was determined at multiple temperatures (12°C, 14°C, 16°C, 30°C, 31°C, and 32°C).

<p>Averages are shown. The green lines show averages that were within the specification of 61.5°C +/−1.5°C. The red lines show runs that over-heated, and the blue lines show runs that fell below the test specifications.</p

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) HIV-1 assay specifications.

<p>Reverse transcription loop-mediated isothermal amplification (RT-LAMP) HIV-1 assay specifications.</p

HIV LAMP amplicon detection via Milenia test strips.

<p>(A) Milenia strips used for the detection of fluorescein isothiocyanate (FITC)/biotin-labeled amplicons. Image to the right shows a positive result while image to the left is a negative result. (B) Best cassettes for the dual detection of FITC/biotin (HIV assay) and digoxigenin (DIG)/biotin (β-actin assay)–labeled amplicons. Image shows a positive result for both FITC/biotin amplicons and DIG/biotin amplicons.</p

An evaluation of the non-instrumented nucleic acid amplification (NINA) heaters as compared to a real-time PCR thermocycler by measuring the performance of the HIV LAMP assay with a range of HIV RNA target concentrations.

<p>All results were confirmed by NALF, agarose gel electrophoresis, and melt curve analysis. Table entries show the number of replicates that returned a positive result as the numerator of a fraction showing the total number of replicates run in the denominator.</p><p>An evaluation of the non-instrumented nucleic acid amplification (NINA) heaters as compared to a real-time PCR thermocycler by measuring the performance of the HIV LAMP assay with a range of HIV RNA target concentrations.</p

Details of the primer sets used for reverse transcription loop-mediated isothermal amplification (RT-LAMP) including the primers tagged for nucleic acid lateral flow (NALF) detection.

<p>The HIV and β-actin primer sets are modified for lateral flow detection by the addition of biotin, fluorescein isothiocyanate (FITC), and digoxigenin (DIG).</p><p>Details of the primer sets used for reverse transcription loop-mediated isothermal amplification (RT-LAMP) including the primers tagged for nucleic acid lateral flow (NALF) detection.</p

Results for normal human plasma (NHP) containing HIV-1 RNA diluted to 580, 290, 145, 73, and 0 (negative control) total copies in a 25 µl reaction.

<p>Extracted genomic nucleic acid at 2.2 ng/µL was in all samples except the negative control and HIV-positive control. In this comparison, samples and controls assayed by the non-instrumented nucleic acid amplification (NINA)/nucleic acid lateral flow (NALF) system and the Stratagene system were aliquoted from the same sample extraction and handled identically up to amplification. Since the NINA heater does not have top-heating, oil was added to these samples for amplification to control for any condensation artifacts. The samples in the top-heated Stratagene were amplified as per the manufacturer’s instructions (no mineral oil) to reflect a standardized reference method.</p><p>Results for normal human plasma (NHP) containing HIV-1 RNA diluted to 580, 290, 145, 73, and 0 (negative control) total copies in a 25 µl reaction.</p