Detection of Viable Streptococcus equi equi Using Propidium Monoazide Polymerase Chain Reaction.

Abstract

There is debate around the clinical significance of Streptococcus equi subsp. equi detection in low numbers using quantitative real-time PCR (qPCR). Propidium monoazide (PMA) qPCR has been used to differentiate DNA from viable and nonviable bacterial cells. The aim of this study was to evaluate the ability of PMA eqbE SEQ2190 triplex qPCR to differentiate DNA from viable and nonviable S. equi in positive and suspect positive clinical specimens. 57 stored (frozen and refrigerated) positive (36) or suspect positive (21) clinical specimens (determined via SeeI qPCR as the gold standard) were tested using eqbE SEQ2190 triplex qPCR with (+) and without (-) PMA pre-treatment. Cycle Thresholds (CT) were higher when using PMA indicating a mixture of heat killed (HK) and viable cells. Number of S. equi positive specimens were as follows: 6/57 eqbE +PMA, 13/57 eqbE -PMA (Chi- squared 3.1, p = 0.079); 10/57 SEQ2190 +PMA, 53/57 SEQ2190 -PMA (Chi- squared 65.6, p<0.0001). The mean CT were as follows: 23.88 eqbE -PMA, 29.89 eqbE + PMA (p=0.04); 24.9 SEQ2190 -PMA, 31.9 SEQ2190 +PMA (p<0.0001). PMA qPCR can be used to determine S. equi viability, but testing should be performed on fresh specimens.