Fig 1.
A. Excitation-emission spectrum of DANP-DNA complexes. 365 nm UV-light is selected for excitation because only basal level of absorbance by DANP-C-bulge complex can be seen while the DANP-dsDNA complex absorbed substantially at this wavelength. Similarly, emission light at 430 nm is measured mainly because it generates the most significant difference when DANP-dsDNA and DANP-C-bulge complexes. B. Illustration of the chemical binding change happens to DANP molecule during PCR procedure.
Table 1.
DANP-anchored hairpin primer sequences used for detection of CHIKV.
Fig 2.
Optimization of number of PCR cycles.
DANP-anchored RT-PCR is carried out with and without the presence of CHIKV RNA template and the fluorescence intensity is measured after every 5 PCR cycles from both before and after PCR reactions. The fluorescence intensity starts to increase significantly after 20 cycles when CHIKV RNA is present and reaches saturation after 30 cycles, while that of NTC also starts to increase slowly from 25 to 30 cycles and become more obvious afterwards. As a result, the maximum difference in fluorescence intensity can be achieved after 30 cycles of PCR reaction. Data are shown as means SEM of five experiments. ***P < 0.001, **P < 0.01 by multiple t-test.
Fig 3.
DANP-anchored RT-PCR primer was evaluate.
A: Specific PCR product with expected size (296 bp) is observed only in reaction with CHIKV RNA template after 30 PCR cycles. Lane 1, DNA ladder (GeneRuler Ultra Low Range, Thermal Fisher Scientific, Waltham, Massachusetts, USA); lane 2, CHIKV RNA+ after 30 PCR cycles; lane 3, CHIKV RNA+ before PCR; lane 4, NTC after 30 PCR cycles; and lane 5, NTC before PCR. B: The fluorescence intensity from the PCR reactions with and without CHIKV RNA before and after 30 PCR cycles. Only the reaction with CHIKV RNA shows significant increment in fluorescence intensity (approximately 2000 AU) after 30 cycles of PCR reaction. Data are shown as means SEM of five experiments. ***P < 0.001 by Student’s t-test.
Fig 4.
The detection limit of a DANP-anchored RT-PCR assay was determined using 10-fold serial diluted CHIKV genomic RNA.
The detection limit for the assay was equivalent to 0.001 PFU per reaction of CHIKV. Data are shown as means SEM of three experiments. ***P < 0.001, **P < 0.01 by Student’s t-test.
Fig 5.
Cross-reactivity of DANP-anchored RT-PCR was evaluated with a panel of positive-sense RNA viruses.
There is no significant increase in fluorescence intensity found in any one of the viruses tested showing no cross-reactivity of the assay to these viruses. Data are shown as means SEM of three experiments. ***P < 0.001 by Student’s t-test.
Table 2.
Performance of DANP-ANCHORED on Serum samples.