Modified pre-PCR protocol for detection of bacteria in blood samples of patients with sepsis

Abstract

Sepsis and septic shock are leading causes of deaths in hospitals. The most common cause of blood stream infection is bacteria. To reduce mortality, a rapid and accurate bacterial detection in sepsis patients is essential. It is necessary to identify pathogens based on their species for appropriate choice of antibiotics. In this study, we aimed to improve the pre-PCR procedure for detection of bacteria in blood samples and to compare the performance between real-time PCR, conventional PCR and blood culture. Blood samples from 16 sepsis patients were separated into whole blood, plasma, buffy coat and white blood cell. Bacterial nucleic acid was extracted and used to identify whether it is gram-positive or gram-negative bacteria using conventional PCR and real-time PCR with TaqMan probes. Two samples, whole blood and plasma components, were positive by real-time PCR using this protocol but negative by blood culture and conventional PCR. Results were in record with clinical data and therapeutic response. The limit of detection of real-time PCR with probe is 1,000 copies/µl of plasmid DNA. Therefore, blood separation is necessary to improve sensitivity of real-time PCR and TaqMan probe in identifying gram-positive and gram-negative bacteria this may be beneficial for early detection and proper antibiotic selection and may be useful to treat patients with sepsis.