Table 1.
Primer and probe sequences.
Table 2.
Dissection of W. bancrofti infected Cx. pipiens.
Table 3.
W. bancrofti Cuticle Genes Evaluated for Expression Onset.
Figure 1.
Primer and probe alignment with cut-1.2 sequences of W. bancrofti and B. malayi.
A portion of the cut-1.2 sequences from B. malayi and W. bancrofti have been aligned with the primers and probes designed for the qRT-PCR cut-1.2 detection assay. The nucleotides in red (red arrows) represent single nucleotide polymorphisms between the Brugia and Wuchereria transcripts that provide the specificity of the target. The exons are differentiated in the W. bancrofti sequence by lower and upper case letters (vertical red bar). The probe spans the exon-exon boundary to prevent detection of any contaminating genomic DNA.
Table 4.
W. bancrofti cut-1.2 Expression Timeline.
Table 5.
Sensitivity of W. bancrofti cut-1.2 L3-Detection Assay.
Figure 2.
Conventional RT-PCR detection of W. bancrofti tph-1 and cut-1.2 in a mosquito time-course.
Time-course set C illustrates no amplification of the L3-activated cut-1.2 transcript (123 bp) in time-points prior to L3 development, while the control tph-1 transcript (153 bp) is detected in all time-points indicating that parasite RNA was present. Panel A shows mosquitoes collected from 0–8 days dPBM and panel B shows mosquitoes collected 9–13, and 16 dPBM. dPBM = the number of days post blood meal, Un Cxp = Unfed Cx. pipiens mosquitoes, Wb gDNA = W. bancrofti genomic DNA, NTC = No template control.
Figure 3.
Sensitivity testing of the W. bancrofti multiplex L3-detection assay by conventional RT-PCR.
The tph-1 transcript (‘any-stage’ detection) is detected in all samples, while the cut-1.2 transcript (L3-detection) is only detected in samples of pool size up to 20 mosquitoes. The L3-detection sensitivity limit by conventional RT-PCR is one infective mosquito in a pool of 20 mosquitoes. 1∶10 = one bloodfed mosquito (day 16 post blood meal) in a pool of 10 mosquitoes, 1∶15 = one bloodfed mosquito in a pool of 15 mosquitoes, etc., 5∶10 = 5 bloodfed mosquitoes in a pool of 10 mosquitoes. Un Cxp = Unfed Cx. pipiens, NTC = no template control.