from the conferences organized by TANGER Ltd. provisional website
Viral-like particles that express Ebola glycoprotein on surface are very sophisticated and risky techniques for developing diagnostics and study of the virus biology. On the other hand, liposome nanoparticles are easy to prepare and have customized lipid content to encapsulate preferred cargo inside while chemically modified to carry other molecules on their surface. The aim of this study was to prepare Ebola liposome viral-like nanoparticles encapsulated with nucleic acid and to develop a polymerase chain reaction (PCR) method to quantitate liposomes without extraction. Liposomes containing equal ratios of three lipids (cholesterol, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) and phosphatidylcholine) and carrying two fragments of Ebola partial and whole GP gene (700 bp and 2031 bp, respectively) were synthesized. Liposome size was estimated in the range of 80-100 nm. Zeta potential analysis showed a negative charge of liposome particles in the ranges of -30 and -70 mV. Direct PCR method was developed to avoid fragment loss during extraction. Several additives were tested to improve PCR detection of liposomes including DMSO, glycerol, triton X-100, tween 20 and tween 80. The addition of triton X-100 (as low as 0.5% per reaction) has showed significant improvement in amplification. The addition of MgCl2 (>50 mM per reaction) in presence of triton X-100 also improved amplification. Although a complete specific product was not obtained, the amplification was semi-quantitative at 5 orders of serial dilution. By employing PCR with shorter product sizes, we believe it is possible to develop more accurate method in the future.Keywords: Liposome, Ebola, Viral-Like Particles, PCR, PCR additives
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