Bacterial RNA Extraction and Purification from Whole Human Blood Using Isotachophoresis

ABSTRACT: We demonstrate a novel assay for physico-chemical extraction and isotachophoresis-based purification of 16S rRNA from whole human blood infected with Pseudomonas putida. This on-chip assay is unique in that the extraction can be automated using isotachophoresis in a simple device with no moving parts, it protects RNA from degradation when isolating from ribonuclease-rich matrices (such as blood), and produces a purified total nucleic acid sample that is compatible with enzymatic amplification assays. We show that the purified RNA is compatible with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and demonstrate a clinically relevant sensitivity of 0.03 bacteria per nanoliter using RT-qPCR. Nucleic acid amplification methods, including reversetranscription-quantitative polymerase chain reaction (RT-qPCR), targeting the 16S rRNA (16S rRNA) enable fast and specific detection of bacteria in complex samples such a