A rapid and simple quantitative method for specific detection of smaller coterminal RNA by PCR (DeSCo-PCR): application to the detection of viral subgenomic RNAs

Abstract

ABSTRACT: RNAs that are 5'-truncated versions of a longer RNA but share the same 3' terminus can be generated by alternative promoters in transcription of cellular mRNAs or by replicating RNA viruses. These truncated RNAs cannot be distinguished from the longer RNA by a simple two-primer RT-PCR because primers that anneal to the cDNA from the smaller RNA also anneal to-and amplify-the longer RNA-derived cDNA. Thus, laborious methods, such as northern blot hybridization, are used to distinguish shorter from longer RNAs. For rapid, low-cost, and specific detection of these truncated RNAs, we report detection of smaller coterminal RNA by PCR (DeSCo-PCR). DeSCo-PCR uses a nonextendable blocking primer (BP), which outcompetes a forward primer (FP) for annealing to longer RNA-derived cDNA, while FP outcompetes BP for annealing to shorter RNA-derived cDNA. In the presence of BP, FP, and the reverse primer, only cDNA from the shorter RNA is amplified in a single-tube reaction containing both RNAs. Many positive strand RNA viruses generate 5'-truncated forms of the genomic RNA (gRNA) called subgenomic RNAs (sgRNA), which play key roles in viral gene expression and pathogenicity. We demonstrate that DeSCo-PCR is easily optimized to selectively detect relative quantities of sgRNAs of red clover necrotic mosaic virus from plants and Zika virus from human cells, each infected with viral strains that generate different amounts of sgRNA. This technique should be readily adaptable to other sgRNA-producing viruses, and for quantitative detection of any truncated or alternatively spliced RNA