Multiplex primer prediction software for divergent targets

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

We demonstrated the application of the algorithm experimentally using purified vaccinia DNA, amplifying the expected band with a Poxviridae short primer multiplex PCR, and confirmed the band's expected sequence. We provided an example of the effect of background nucleic acids by spiking human DNA into the PCR reaction over a series of mass ratios. While a band for vaccinia was visualized by slab gel electrophoresis for all mass ratios, background DNA smears were also clear at the higher ratios. At a mass ratio of 1 : 1 vaccinia:human DNA, there are 2.7 pg or 0.82 copies of the human genome present in the reaction. To place this into context, there are $6.58 pg or two copies of genomic DNA in one human cell. Thus, the results from the 1 : 1 mass ratio reaction represents the impact that $41% of the total DNA content from one human cell could have on the results, when analyzed by slab gel electrophoresis. In a clinical sample, there could be a much greater number of human cells and DNA. Amplification with these family level primer sets will depend on viral titers in the actual sample, and these experiments show that there are cases where amplification will either not be possible or require additional sample purification steps. While short primer PCR multiplexes may enable amplification of diverse target sets, they will not match the specificity of longer primers. Combination of these short primer multiplexes with digitized, microfluidic-based picoliter reactions (40) and/or next-generation microfluidic-based sample purification technologies that have the ability to isolate target from contaminating nucleic acids may help to overcome this limitation. However, even in the absence of such technologies, a highly conserved short primer multiplex could enrich amplification products for members of a particular viral family compared to randomly amplified or unamplified sample. Then it would need to be followed by product sequencing or array hybridization, such as TaqMan Õ or Luminex bead-based suspension arrays (http://www.luminexcorp.com/), to provide more specific information about the organism(s) present, since electrophoretic banding patterns may contain unexpected bands, particularly if the sample contains a significant amount of nonviral nucleic acids.