Preliminary studies on the use of the Cascade Rolling Circle Amplification technique for Plum pox virus detection

Isothermal techniques for the amplification of nucleic acids have emerged in the last years. In contrast to the Polymerase chain reaction (PCR), the most prevalent method to amplify DNA in vitro, the reactions can be run at constant temperatures. Specificity and sensitivity are at least as high as that obtained by using PCR and the methods are less time consuming. Therefore, the isothermal amplification of nucleic acids provides a powerful tool for the detection of Plum pox virus (PPV), the causal agent of the Sharka disease. The cascade rolling circle amplification (CRCA), first described by Thomas et al. (1999), is based on the rolling circle mechanism that many viruses use to replicate their genome multiplicatively. Circular Probes, also called Padlock probes (PLP), which arise from the ligation of the terminal region of DNA probes upon side by side hybridization to the target serve as template (Nilsson et al. 1994).For detecting PPV by CRCA, RNA was extracted and reverse transcribed to cDNA using a PPV specific primer. Several PLPs with varying lengths and sequences complementary region to the cDNA were designed and tested. Furthermore, different pairs of primers for the subsequent amplification were developed. For specific ligation Ampligase and T4 DNA Ligase were tested. In CRCA, two polymerases with strong strand displacement activity were compared: Phi29 DNA Polymerase and Bst DNA Polymerase. These enzymes differ in their optimal reaction temperature.Ligation as well as amplification do occur, but there is high background amplification also in negative and no template controls. Discrimination was possible after a restriction digestion is carried out. As proven by sequencing of reaction products non-specific signals were a result of primer polymerization. Current work focuses on the reduction of the background amplification and improvement of the sensitivity. Keywords: Cascade Rolling Circle Amplification, CRCA, isothermal amplification of DNA, Plum pox virus, PPV