ABSTRACT
Rapid detection and characterization of pathogens in patients with bloodstream infections (BSIs) is a persistent problem for modern medicine, as current techniques are slow or provide incomplete diagnostic information. Real-time polymerase chain reaction (qPCR) allows specific detection of a wide range of targets and quantification of pathogenic burdens to aid in treatment planning. However, new technological advances are required for a rapid and multiplex implementation of qPCR in clinical applications. In this paper, the feasibility of a novel microfluidic platform for qPCR is presented, integrating highly sensitive, label-free localized surface plasmon resonance (LSPR) imaging of DNA hybridization into a recirculating chip design for real-time analysis. Single target and multiplex detection of DNA target amplification are demonstrated, with a limit of detection of 5 fg µL-1 of E. coli DNA for single target PCR, correlating with approximately 300 bacteria per mL. The results of this study demonstrate the potential of this platform for simultaneous real-time detection of multiple target genes within 15 minutes that could provide live saving benefits in patients with BSIs.
Subject(s)
Microfluidic Analytical Techniques/instrumentation , Real-Time Polymerase Chain Reaction/instrumentation , Surface Plasmon Resonance/instrumentation , DNA, Bacterial/analysis , DNA, Bacterial/genetics , Equipment Design , Escherichia coli/genetics , Limit of Detection , Microfluidic Analytical Techniques/methods , Real-Time Polymerase Chain Reaction/methods , Surface Plasmon Resonance/methodsABSTRACT
Two examples of a "new" IgM saline-agglutinating auto-antibody are described. The antibodies bind complement, have the ability to cause in vivo hemolysis, and are most active at room temperature at a pH of about 6.5. Despite tests on more than 5,000 people, no nonreactive cell sample has been found. The reactive antigen is not denatured by neuraminidase, papain, or ficin, and is present on i adult red blood cells. The antibodies appear to be slightly inhibited by human saliva and milk, and more convincingly inhibited by urine from Sd(a+) persons. They are not inhibited by urine from Sd(a-) persons, but are strongly inhibited by guinea pig urine. The serologic characteristics indicate a relationship to the Sda blood group and the auto-antibody has been named antiSdx. Sdx antigen is present on red blood cells from some higher primates and is absent from rabbit, rhesus monkey, dog and sheep cells.