Application of Synthetic Standard Curves for Absolute Quantification of Hepatitis A and E by Real-Time PCR

Abstract

observe that, despite displaying reliable results, using plasmid DNA as template is more expensive and takes longer to synthetize than DNA oligonocleotides. The advantage of using DNA oligonucleotides is that it can be custom synthesized, and only requires the nucleotide sequence information. Over the past decade, synthetic biology has taken advantage of a comprehensive inventory of biomolecular parts [7] and it is providing alternative diagnostic tools for viral detection and monitoring. This report highlights the application of synthetics standard curves for quantification of hepatitis A and E genomes by real-time PCR. The aim of this study was to examine the potential of two DNA oligos for hepatitis A (HAV) and hepatitis E virus (HEV) genomes quantification that could be adopted readily into established testing protocols. The HPLC-pure oligonucleotides specifying the 89bp HAV (5' non-coding region) and 74bp HEV (ORF3 region) amplicons were custom synthesized by IDT® (CoralVille, USA) (Table 1), corresponding to the same genome fragments used for plasmid DNA standard curve construction. Following manufacturer's specifications, the DNA oligo was dissolved in DNase/ RNase-free distilled water to 100pmol/uL which is equivalent, approximately, to 10 13 DNA molecules/uL according to the Avogadro number calculation-([DNAg/uL]/ Plasmid size (bp) x 660) x (6,022x10 23). The standard curves were generated by performing a TaqMan real time PCR, previously described by De Paula et al. and by Jothikumar et al. for HEV [8,9]. In this study, the plasmid standard curve was replaced to ultramer template tenfold dilution series (10-1-10-20). The specific primers and probes were used according to the literature above. After determining the dilution range for each synthetic curve, they were