Table 1.
Sequences of the BmHha I, OvGST1a and WbLDR LAMP primers.
Fig 1.
Temperature profile of the NINA heater.
The temperature of 25 μl (LAMP reaction volume) of water in PCR tubes containing a T type thermocouple was monitored in four separate heaters using NI Signal Express software. The target temperature of 63°C is denoted by a black line.
Fig 2.
Color change of neutral red and phenol red indicator dyes in LAMP reactions.
The WbLDR primer set was used to amplify genomic W. bancrofti (Wb) DNA using the colorimetric master mix containing either neutral red or phenol red dye and Bst 2.0 WS. Before amplification (T0), reactions containing neutral red are colorless. Samples turn pink if positive or a brownish yellow if negative as shown after a sixty minute (T60) amplification. Reactions containing phenol red are pink at T0 and remain pink if negative but turn yellow if positive as shown here after a sixty minute (T60) amplification.
Fig 3.
Sensitivity and specificity of the BmHha I colorimetric NINA-LAMP assay with neutral red dye.
Comparison of sensitivity using Bst DNA polymerase, Large Fragment (wt Bst LF), Bst 2.0 DNA polymerase (Bst 2.0) or Bst 2.0 WarmStart DNA polymerase (Bst 2.0 WS) on a 10X serial dilution of genomic B. malayi DNA (A). Species-specificity using 100 pg of various genomic DNAs (B). Detection of B. malayi DNA in mosquitoes (C). Species names are abbreviated as follows: B. malayi (Bm), W. bancrofti (Wb), O. volvulus (Ov), Homo sapiens (Hs), Simulium vittatum (Sv) and Aedes aegypti (Aa). The non-template controls (NTCs) contain water.
Fig 4.
Sensitivity and specificity of the OvGST1a colorimetric NINA-LAMP assay using Bst 2.0 WS.
Sensitivity comparison of neutral red and phenol red dyes on a 10X dilution series of genomic O. volvulus DNA (A). Species-specificity using 1 ng of various genomic DNAs (B). Detection of O. volvulus in black flies (C). Species names are abbreviated as follows: O. volvulus (Ov); W. bancrofti (Wb); Loa loa (Ll); B. malayi (Bm); Aedes aegypti (Aa); Simulium vittatum (Sv); Simulium squamosum (Sa) and Homo sapiens (Hs). NTC = Non-template control.
Fig 5.
Sensitivity and specificity of the WbLDR colorimetric NINA-LAMP assay using Bst 2.0 WS.
(A) LAMP reactions incubated in the NINA heater exhibit the same sensitivity as those incubated in a qPCR machine with DNA amplification monitored in real time using a dsDNA binding dye. Genomic W. bancrofti DNA dilutions equivalent to 1/5 mf (1), 1/50 mf (2), 1/500 mf (3), 1/5000 mf (4), 1/50,000 mf (5), 1/500,000 mf (6), 1/5,000,000 mf (7) and H2O (8) were used as template. The end point color change of the LAMP reactions incubated in the NINA heater are shown in the upper panel. Real time amplification curves and end point color change of LAMP reactions are shown in the lower panel. (B) Species-specificity using 100 pg of various genomic DNAs as template. (C) Detection of W. bancrofti DNA in blood samples from 8 different LF positive patients (S1-S8). Species names are abbreviated as follows: W. bancrofti (Wb), B. malayi (Bm), O. volvulus (Ov), Aedes aegypti (Aa) and Homo sapiens (Hs). All experiments were performed in triplicate. One representative of each is shown.