Universal Primers for Detection of Novel Plant Capsid-Less Viruses: Papaya Umbra-like Viruses as Example

For diagnosis of positive-sense single-stranded RNA viruses, primers are usually raised against the sequence encoding capsid proteins, since structural proteins are more conserved. This chapter focuses on the design of primers for a group of novel viruses lacking a capsid, known as papaya Umbra-like viruses (unassigned genus) associated with Papaya Sticky Disease, which represent a threat to papaya production. Based on sequence alignments of a region encoding the RNA-dependent RNA Polymerase, universal primers to detect all the known viruses from four countries are proposed. The Forward universal primer can be used in combination with clade- and subclade-specific primers for rapid virus identification. We walk the reader through downloading sequences from nucleotide databases, doing sequence alignments and phylogenetic tree construction to identify conserved and variable regions as valid primer targets; we also show how to design and analyze the primers.