Analysis of viable Enterococcus faecalis using Propidium Monoazide and real-time PCR

• Grant number: 13/24063-1
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Effective date (Start): March 01, 2014
• Effective date (End): December 31, 2014
• Field of knowledge: Health Sciences - Dentistry - Endodontics
• Principal Investigator: Ericka Tavares Pinheiro
• Grantee: Viviane Danelon Neves
• Host Institution: Faculdade de Odontologia (FO). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

DNA-based molecular methods for detection of microbial species offer advantages over culturing methods. However, one of their disadvantages is the inability to differentiate DNA between viable and dead cells. To overcome this problem, an assay was developed to discriminate between viable or dead bacteria using DNA intercalating substance, propidium monoazide (PMA) dye, in association with quantitative PCR (qPCR). The aim of this investigation is to test in vitro the use of PMA in combination with qPCR for detection and quantification of Enterococcus faecalis. Three different concentrations of PMA (50, 100 e 240 µM) were added to suspensions of 106 CFU/mL of viable/dead E. faecalis. After DNA isolation, qPCR was carried out using specific primers for E. faecalis. PMA was further tested with different mixtures containing varying ratios of viable and dead cells. The efficacy of PMA to detect viable/dead cells was tested by analysis of variance.

Scientific publications

(References retrieved automatically from Web of Science and SciELO through information on FAPESP grants and their corresponding numbers as mentioned in the publications by the authors)

PINHEIRO, ERICKA T.; NEVES, VIVIANE D.; REIS, CAROLINE C.; LONGO, PRISCILA L.; MAYER, MARCIA P. A.. Evaluation of the Propidium Monoazide-quantitative Polymerase Chain Reaction Method for the Detection of Viable Enterococcus faecalis. JOURNAL OF ENDODONTICS, v. 42, n. 7, p. 1089-1092, JUL 2016. (13/24063-1)