Pyrosequencing with di-base addition for the identification of pathogenic bacteria

Abstract

We proposed a novel method for the identification of pathogenic bacteria, in which a partial amplicon of the molecular markers was targeted by using pyrosequencing with di-base addition (PDBA). PDBA was conducted by synchronously adding di-base instead of one base into a reaction, and a set of highly sequence-specific encodings containing the type and the number of incorporated nucleotide(s) (peak height) were obtained. By comparing the encoding sizes of each isolate and the number of incorporated nucleotide(s) in each cycle, moving from first to last, various kinds of bacteria could be unambiguously identified. To verify its feasibility, we simulated PDBA results from thirteen isolates of Mycobacterium species and compared their encoding sizes and the number of incorporated nucleotide(s) in each cycle with those predicted by a homemade software. The thirteen isolates were successfully differentiated. We also targeted partial RNase P RNA gene (rnpB) of cultured M. paratubercuosis and M. celatum to differentiate the two isolates. By comparing the encoding size of each isolate and the number of incorporated nucleotide(s) in each cycle, the two Mycobacterium isolates were successfully differentiated. In conclusion, PDBA enabled to reliably identify pathogenic bacteria.