Development of a quantitative SYBR Green real-time PCR and microscopic slide culture for in vitro enumeration of Mycoplasma hyopneumoniae

Abstract

Mycoplasma hyopneumoniae's slow growth makes enumeration challenging using the traditional colony-forming unit (CFU) method. We introduced an innovative CFU-slide culture (CFU-SC) approach to address this issue. CFU-SC allows accurate counting of M. hyopneumoniae microcolonies, ranging from 7 to 50 µm, using high magnification. While comparing the accuracy of enumeration among CFU, CFU-SC, quantitative polymerase chain reaction (qPCR), and colour change unit (CCU), we found a robust positive correlation between qPCR and CFU-SC with colourCCU method for quantifying M. hyopneumoniae. Newly isolated strains exhibited the lowest Pearson correlation coefficient (r = 0.52) when comparing CFU and real-time PCR, while the highest coefficient (r = 0.86) was observed for the type strain J and new isolates using real-time PCR and CFU-SC. A consensus growth pattern was observed among all quantification methods, except CFU, between newly isolated strains and the type strain J. Notably, new isolates of M. hyopneumoniae showed no senescence phase after 228 h of cultivation, differing from the type strain J. The disparity in growth rate and pattern between new isolates and the type strain J is evident in the smaller agar microcolonies (7-10 µm) of the isolates, contrasting with the larger colonies (100-200 µm) of type strain J.

Keywords: Mycoplasma hyopneumoniae; CFU-slide culture; colony-forming unit (CFU); colour change unit (CCU); enumeration; quantitative polymerase chain reaction (qPCR).