Feasibility of probiotic lactobacillus and yeast as oral vaccine carrier against coronaviruses

Ph.DDOCTOR OF PHILOSOPH

Establishment of one-step SYBR green-based real time-PCR assay for rapid detection and quantification of chikungunya virus infection

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus and one of the prevalent re-emerging arbovirus in tropical and subtropical regions of Asia, Africa, and Central and South America. It produces a spectrum of illness ranging from inapparent infection to moderate febrile illness as well as severe arthralgia or arthritis affecting multiple joints. In this study, a quantitative, one-step real-time SYBR Green-based RT-PCR system for the non-structural protein 2 (nsP2) of CHIKV that can quantify a wide range of viral RNA concentrations was developed. Comparisons between the conventional semi-quantitative RT-PCR assay, immunofluorescence detection method and the one-step SYBR Green-based RT-PCR assay in the detection of CHIKV infection revealed much rapid and increase sensitivity of the latter method. Furthermore, this newly developed assay was validated by in vitro experiments in which ribavirin, a well-known RNA virus inhibitor, showed a dose-dependent inhibition of virus replication on cells that was assessed by viral infectivity and viral RNA production. Our results demonstrate the potential of this newly developed one-step SYBR Green I-based RT-PCR assay may be a useful tool in rapid detection of CHIKV and monitoring the extent of viral replication possibly in patients' samples

Development of 2, 7-Diamino-1, 8-Naphthyridine (DANP) Anchored Hairpin Primers for RT-PCR Detection of Chikungunya Virus Infection - Fig 1

<p>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.</p

Optimization of number of PCR cycles.

<p>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.</p

DANP-anchored hairpin primer sequences used for detection of CHIKV.

<p>DANP-anchored hairpin primer sequences used for detection of CHIKV.</p

The detection limit of a DANP-anchored RT-PCR assay was determined using 10-fold serial diluted CHIKV genomic RNA.

<p>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.</p

DANP-anchored RT-PCR primer was evaluate.

<p>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.</p