Open Access Highly Accessed Methodology article

A novel, nested, multiplex, real-time PCR for detection of bacteria and fungi in blood

Tomasz Gosiewski1*, Danuta Jurkiewicz-Badacz3, Agnieszka Sroka1, Monika Brzychczy-Włoch1 and Małgorzata Bulanda2

Author Affiliations

1 Department of Bacteriology, Microbial Ecology and Parasitology, Chair of Microbiology Jagiellonian University Medical College, Czysta 18 Str, 31-121 Kraków, Poland

2 Department Epidemiology of Infection, Chair of Microbiology Jagiellonian University Medical College, Czysta 18 Str, 31-121 Kraków, Poland

3 The John Paul II Hospital in Krakow, Pradnicka 80 Str, 31-202 Kraków, Poland

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BMC Microbiology 2014, 14:144  doi:10.1186/1471-2180-14-144

Published: 4 June 2014

Abstract

Background

The study describes the application of the PCR method for the simultaneous detection of DNA of Gram-negative bacteria, Gram-positive bacteria, yeast fungi and filamentous fungi in blood and, thus, a whole range of microbial etiological agents that may cause sepsis. Material for the study was sterile blood inoculated with four species of microorganisms (Escherichia coli, Staphylococcus aureus, Candida albicans and Aspergillus fumigatus) and blood collected from patients with clinical symptoms of sepsis. The developed method is based on nested-multiplex real-time PCR .

Results

Analysis of the obtained data shows that sensitivity of nested-multiplex real-time PCR remained at the level of 101 CFU/ml for each of the four studied species of microorganisms and the percentage of positive results of the examined blood samples from the patients was 70% and 19% for the microbiological culture method. The designed primers correctly typed the studied species as belonging to the groups of Gram-positive bacteria, Gram-negative bacteria, yeast fungi, or filamentous fungi.

Conclusions

Results obtained by us indicated that the designed PCR methods: (1) allow to detect bacteria in whole blood samples, (2) are much more sensitive than culture method, (3) allow differentiation of the main groups of microorganisms within a few hours.

Keywords:
Nested PCR; Multiplex PCR; Sepsis; Bacteria; Fungi; Gram differentiation