| Grant number: | 13/24063-1 |
| Support Opportunities: | Scholarships in Brazil - Scientific Initiation |
| Start date: | March 01, 2014 |
| End date: | 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. | |
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