Multiplex primer prediction software for divergent targets

Gardner, Shea N.; Hiddessen, Amy L.; Williams, Peter L.; Hara, Christine; Wagner, Mark C.; Colston, Bill W.

Previous studies have shown the utility of short primer singleplex PCR using 9-mers or 10-mers followed by gel electrophoresis for genetic fingerprinting of eukaryotes and bacteria (41, 42) . For viruses, with much smaller and more diverse genomes, the large numbers of 9-mer or 10-mer primers required to generate at least one band from every virus as predicted by our analyses implies that primer size would need to be as short as 5-mers to rely on a gel banding pattern using only one priming sequence for fingerprinting viruses (unpublished data). However, the analyses here predict that imperfect sample purification to eliminate eukaryotic nucleic acids could be problematic for universal viral priming using primers shorter than 15 bases, particularly for multiplexes of 10 or more primers. Nanda et al. (43, 44) were able to achieve sufficient viral isolation from cell culture samples to allow viral identification using viral PCR with priming sequences as short as pentamers, so the problem of contaminating host nucleic acids for specific, short primer PCR of viruses is not insurmountable. They found specific pentamer PCR to be several logs more sensitive than nonspecific amplification, provided that they purified encapsidated viral nucleic acids prior to PCR. Another method that has been used for virus discovery is VIDISCA (Virus discovery cDNA-AFLP) using restriction enzyme digestion, adaptor ligation, and PCR by priming with the adaptor sequence (45, 46) . This method, like the pentamer priming used by (43, 44) , requires prior separation of encapsidated viral nucleic acids, as it generates fragments from any DNA present, viral, host or otherwise. Multiplex PCR with primers 10-15 nt in length may be yet another alternative strategy lying between these nonspecific methods and PCR with standard primers of at least 18 nt, as we have shown that it can add some measure of specificity for a viral family.