Development of a Multiplexed Microsphere PCR for Culture-Free Detection and Gram-Typing of Bacteria in Human Blood Samples

Abstract

Bloodstream infection is a significant clinical problem, particularly in vulnerable patient groups such as those undergoing chemotherapy and bone marrow transplantation. Clinical diagnostics for suspected bloodstream infection remain centered around blood culture (highly variable timing, in the order of hours to days to become positive), and empiric use of broad-spectrum antibiotics is therefore employed for patients presenting with febrile neutropenia. Gram-typing provides the first opportunity to target therapy (e.g., combinations containing vancomycin or teicoplanin for Gram-positives; piperacillin-tazobactam or a carbapenem for Gram-negatives); however, current approaches require blood culture. In this study, we describe a multiplexed microsphere-PCR assay with flow cytometry readout, which can distinguish Gram-positive from Gram-negative bacterial DNA in a 3.5 h time period. The combination of a simple assay design (amplicon-dependent release of Gram-type specific Cy3-labeled oligonucleotides) and the Luminex-based readout (for quantifying each specific Cy3-labeled sequence) opens opportunities for further multiplexing. We demonstrate the feasibility of detecting common Gram-positive and Gram-negative organisms after spiking whole bacteria into healthy human blood prior to DNA extraction. Further development of DNA extraction methods is required to reach detection limits comparable to blood culture.

An Analytically and Diagnostically Sensitive RNA Extraction and RT-qPCR Protocol for Peripheral Blood Mononuclear Cells

“…The ssoAdvanced TM master-mix, identified herein as optimal of those tested, appears to be one such master-mix facilitating PCR efficiency in the presence of co-extracted inhibitors. Optimization of mastermix reagents will likely continue to be important in improving blood-based PCR analysis and diagnostics (59,60); especially for accurate amplification of relatively low abundant targets, comparisons between populations with high variability, or amplification from inhibitor-enriched mediums (i.e., whole blood extractions) (10,59). We tested three RNA extraction kits by evaluating extraction quality and efficiency: RNeasy R Mini Kit, RNeasy R Micro Kit, and MagMAX TM mirVana TM Kit; in combination with the RNA purification and concentration kit: RNeasy R MiniElute Cleanup Kit.…”

“…Additionally, when compared to silica-column extractions, we found MagMAX was more cost and time efficient when running larger number of samples (e.g., 96 samples in ∼2 h). We therefore expect magnetic bead-based extractions will become increasingly common within bloodbased nucleic acid isolations (59,61,62). In addition to extraction techniques (e.g., silica column, phase separation), other factors that could impact RNA quality, yield and concentration include sample collection, storage, and transportation.…”

Developing Recombinant Antibodies by Phage Display Against Infectious Diseases and Toxins for Diagnostics and Therapy

“…The many drawbacks of these methods are the low throughput and high costs caused by their inherently long experimental duration, the higher risk of exposure to pathogenic organisms and the need of highly trained personnel. Real-time PCR and other molecular diagnostics approaches have been introduced to reduce the time needed for the identification of many bacteria ( Liang et al., 2018 ), while offering high detection sensitivity and specificity. Nevertheless, such methods most often depend on the availability of laboratory infrastructure and, if portable, are less amenable to high throughput parallelized measurements ( Xu et al., 2020 ).…”

Dark-Field Microscopic Detection of Bacteria using Bacteriophage-Immobilized SiO2@AuNP Core–Shell Nanoparticles

“…In recent years, numerous culture-independent techniques have been developed for the rapid and sensitive detection of pathogenic bacteria. Among them, molecular biological (DNA microarray, polymerase chain reaction (PCR) detection, and derivatives − ) and immunological methods (enzyme-linked immunosorbent assays (ELISA), − lateral flow immunoassays, − and immunomagnetic separation − ) are the most commonly employed. However, these methods require expensive equipment and highly trained laboratory analysts .…”