Identification of sepsis-causing bacteria from whole blood without culture using primers with no cross-reactivity to human DNA

Abstract

Sepsis is a major health concern globally, and identification of the causative organism usually takes several days. Furthermore, molecular amplification using whole blood from patients with sepsis remains challenging because of primer cross-reactivity with human DNA, which can delay appropriate clinical intervention. To address these concerns, we designed primers that could reduce cross-reactivity. By evaluating these primers against human DNA, we confirmed that the cross-reactivity observed with conventional primers was notably absent. In silico PCR further demonstrated the specificity and efficiency of the designed primers across 23 bacterial species that are often associated with sepsis. When tested using blood samples from sepsis patients, the designed primers showed moderate sensitivity and high specificity. Surprisingly, our method identified bacteria even in samples that were detected at other sites but tested negative using conventional blood culture methods. Although we identified some challenges, such as contamination with Acetobacter aceti due to the saponin pretreatment of samples, the developed method demonstrates remarkable potential for rapid identification of the causative organisms of sepsis and provides a new avenue for diagnosis in clinical practice.