Table 1.
Reverse transcription loop-mediated isothermal amplification (RT-LAMP) HIV-1 assay specifications.
Figure 1.
(Right) Cross-section showing the internal components of the heater. Approximate dimensions of the heater are 80 mm diameter by 120 mm height.
Table 2.
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.
Figure 2.
HIV LAMP amplicon detection via Milenia test strips.
(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.
Figure 3.
Average temperature profiles of 74 runs with nine prototype non-instrumented nucleic acid amplification (NINA) heaters.
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.
Figure 4.
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).
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.
Table 3.
A comparison of the effects on the limit of detection (LOD) of assays heated by the non-instrumented nucleic acid amplification (NINA) heater.
Table 4.
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.
Figure 5.
Melt curve analysis of the HIV-1 and β-actin biplex reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay.
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).
Table 5.
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.