UNASSIGNED: The well-known @entity1 pathogenic bacterium, @entity1838 , can enter a physiologically viable but non-culturable (VBNC) state under stress conditions. The differentiation of VBNC cells and nonviable cells is essential for both disease prevention and basic research. Among all the methods for detecting viability, @entity15777 ( @entity4515 ) combined with real-time PCR is popular because of its specificity, sensitivity, and speed. However, the effect of @entity4515 treatment is not consistent and varies among different species and conditions. In this study, with an initial cell concentration of 1*10(8)CFU/ml, time and dose-effect relationships of different @entity4515 treatments were evaluated via quantitative real-time PCR using live cell suspensions, dead cell suspensions and VBNC cell suspensions of @entity1838 O1 El Tor strain C6706. The results suggested that a @entity4515 treatment of 20 M @entity4515 for 20min was optimal under our conditions. This treatment maximized the suppression of the PCR signal from membrane-compromised dead cells but had little effect on the signal from membrane-intact live cells. In addition to the characteristics of @entity4515 treatment itself, the initial concentration of the targeted bacteria showed a significant negative influence on the stability of @entity4515 -PCR assay in this study. We developed a strategy that mimicked a 1*10(8)CFU/ml cell concentration with dead bacteria of a different bacterial species, the DNA of which cannot be amplified using the real time PCR primers. With this strategy, our optimal approach successfully overcame the impact of low cell density and generated stable and reliable results for counting viable cells of @entity1838 in the VBNC state.

Enumeration of viable non-culturable @entity1838 using XXXX combined with quantitative PCR.

@entity1 :: ('9606', 'Species') :: ['human'] @entity15777 :: ('MESH:C533957', 'Chemical') :: ['propidium monoazide'] @entity4515 :: ('CHEBI:60755', 'Chemical') :: ['PMA'] @entity1838 :: ('666', 'Species') :: ['V. cholerae', 'Vibrio cholerae']
@entity15777 :: (MESH:C533957,Chemical) :: ['propidium monoazide']
