Beating the reaction limits of biosensor sensitivity with dynamic tracking of single binding events

The clinical need for ultrasensitive molecular analysis has motivated the development of several endpoint-assay technologies capable of single-molecule readout. These endpoint assays are now primarily limited by the affinity and specificity of the molecular-recognition agents for the analyte of interest. In contrast, a kinetic assay with single-molecule readout could distinguish between low-abundance, high-affinity (specific analyte) and high-abundance, low-affinity (nonspecific background) binding by measuring the duration of individual binding events at equilibrium. Here, we describe such a kinetic assay, in which individual binding events are detected and monitored during sample incubation. This method uses plasmonic gold nanorods and interferometric reflectance imaging to detect thousands of individual binding events across a multiplex solid-phase sensor with a large area approaching that of leading bead-based endpoint-assay technologies. A dynamic tracking procedure is used to measure the duration of each event. From this, the total rates of binding and debinding as well as the distribution of binding-event durations are determined. We observe a limit of detection of 19 fM for a proof-of-concept synthetic DNA analyte in a 12-plex assay format.First author draf

Steps and Tools for PCR-Based Technique Design

The identity and clonal differences within bacterial populations have been broadly explored through PCR-based techniques. Thus, bacterial identification and elucidation of DNA fingerprinting have provided insights regarding their phenotypic and genotypic variations. Indeed, some diversity of rates may reflect changes among subpopulations that have their own ecological dynamic and individual traits on coexisting genotypes. Therefore, identification of polymorphic regions from nucleic acid sequences is based on the identification of both conserved and variable regions. Advantages of PCR-based methods are high sensitivity, specificity, speed, cost-effectiveness, and the opportunity for simultaneous detection of many microbial agents or variants. Fingerprint information might allow the tracking of certain outbreaks globally in several reference databases containing valuable genotyping information. In this chapter, we will review applications from Web resources and computational tools online for the designing of PCR-based methods to identify bacterial species. We will also focus on lab applications and key conditions for technique standardization

Development of a multiplex real-time PCR assay using two thermocycling platforms for detection of major bacterial pathogens associated with bovine respiratory disease complex from clinical samples

Bovine respiratory disease complex (BRDC) is one of the most significant diseases of cattle. Bacterial pathogens involved in BRDC include Mannheimia haemolytica, Mycoplasma bovis, Histophilus somni, and Pasteurella multocida. We developed and evaluated a multiplexed real-time hydrolysis probe (rtPCR) assay using block-based Peltier and rotary-based thermocycling on lung tissue, nasal swabs, and deep nasopharyngeal swabs. The rtPCR results were compared to culture or a gel-based M. bovis PCR using statistical analysis to determine optimum quantification cycle (Cq) cutoffs to maximize agreement. The limits of detection were 1.2–12 CFU/reaction for each pathogen. M. haemolytica was the most prevalent organism detected by rtPCR, and was most frequently found with P. multocida. The rtPCR assay enabled enhanced levels of detection over culture for all pathogens on both thermocycling platforms. The rotary-based thermocycler had significantly lower Cq cutoffs (35.2 vs. 39.7), which maximized agreement with gold standard culture or gel-based PCR results following receiver operating characteristic analysis to maximize sensitivity (Se) and specificity (Sp). However, overall assay Se and Sp were similar on both platforms (80.5% Se, 88.8% Sp vs. 80.1% Se, 88.3% Sp). Implementation of these tests could enhance the detection of these pathogens, and with high-throughput workflows could reduce assay time and provide more rapid results. The assays may be especially valuable in identifying coinfections, given that many more antemortem samples tested in our study were positive for 2 or more pathogens by rtPCR (n = 125) than were detected using culture alone (n = 25)

ROLE OF EPSTEIN-BARR VIRUS IN THE PATHOGENESIS OF MULTIPLE SCLEROSIS

Multiple sclerosis (MS) is a chronic pathology of the central nervous system, characterized by inflammation, demyelination, and neurodegeneration. The prevalence and incidence rates of MS are on the rise worldwide. What causes MS is unknown. However, it is widely accepted that MS occurs in genetically susceptible individuals exposed to certain environmental factors. Sero-epidemiological data suggest a strong association between Epstein-Barr virus (EBV) and MS. EBV is one of the commonest viruses in the human population with ~90% global seropositivity. EBV infects naïve B cells and immortalizes them. While some postmortem studies have shown EBV in MS lesions, others have failed to reach similar observation. Variation in technical approaches and sample size could be the reason for reported inconsistencies. Thus, we have assessed the hypothesis that EBV may enter the MS brain and contribute to disease pathogenesis. Four objectives were highlighted in this thesis: to investigate EBV presence in MS brain using a large sample size, quantify EBV viral load in brain tissues, determine the possible route of virus entry to the brain, and determine viral gene expression in MS brain. In a case-control design, 122 MS and non-MS cases were studied and compared. Formalin stored brain coronal slices were received from Rocky Mountain MS Centre, US. DNA extraction was optimized and PCR amplification of EBV BamHI was performed to determine EBV presence/ absence in the brain. EBV viral load was quantified in infected cases using qPCR. Localization of infected cells was achieved using EBER in situ hybridization, and viral gene expression was determined using immunohistochemistry. The phenotype of EBV infected cells was characterized using immunohistochemistry for cellular markers. EBV DNA has been detected in 90% MS and 24% non-MS cases. EBV infected cells were more prevalent in brain parenchyma than in meninges, expressing latent EBNA1 and lytic BZLF1. EBV-infected cells were likely to have a B lymphocyte phenotype. We concluded that EBV differential presence in the MS brain reflected a pathogenic contribution to MS. Further studies are warranted to determine the mechanism of EBV involvement in M

Rapid screening of Salmonella enterica serovars Enteritidis, Hadar, Heidelberg and Typhimurium using a serologically-correlative allelotyping PCR targeting the O and H antigen alleles

<p>Abstract</p> <p>Background</p> <p>Classical <it>Salmonella </it>serotyping is an expensive and time consuming process that requires implementing a battery of O and H antisera to detect 2,541 different <it>Salmonella enterica </it>serovars. For these reasons, we developed a rapid multiplex polymerase chain reaction (PCR)-based typing scheme to screen for the prevalent <it>S. enterica </it>serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.</p> <p>Results</p> <p>By analyzing the nucleotide sequences of the genes for O-antigen biosynthesis including <it>wb</it>a operon and the central variable regions of the H1 and H2 flagellin genes in <it>Salmonella</it>, designated PCR primers for four multiplex PCR reactions were used to detect and differentiate <it>Salmonella </it>serogroups A/D1, B, C1, C2, or E1; H1 antigen types i, g, m, r or z₁₀; and H2 antigen complexes, I: 1,2; 1,5; 1,6; 1,7 or II: e,n,x; e,n,z₁₅. Through the detection of these antigen gene allele combinations, we were able to distinguish among <it>S. enterica </it>serovars Enteritidis, Hadar, Heidelberg, and Typhimurium. The assays were useful in identifying <it>Salmonella </it>with O and H antigen gene alleles representing 43 distinct serovars. While the H2 multiplex could discriminate between unrelated H2 antigens, the PCR could not discern differences within the antigen complexes, 1,2; 1,5; 1,6; 1,7 or e,n,x; e,n,z₁₅, requiring a final confirmatory PCR test in the final serovar reporting of <it>S. enterica</it>.</p> <p>Conclusion</p> <p>Multiplex PCR assays for detecting specific O and H antigen gene alleles can be a rapid and cost-effective alternative approach to classical serotyping for presumptive identification of <it>S. enterica </it>serovars Enteritidis, Hadar, Heidelberg, and Typhimurium.</p

Microfluidic PCR with plasmonic imaging for rapid multiplexed characterization of DNA from microbial pathogens

Bloodstream infections (BSIs) are a critical concern in modern medicine due to their continued prevalence in modern hospitals, along with high costs and attributable mortality, particularly among those who are immunocompromised. The current gold standard for detection and characterization of causative pathogens involves cell culture, which can take 24-48 hours to complete, increasing time to adequate treatment and thus mortality. The rise of antimicrobial resistance in hospital acquired infections has reduced the effectiveness of broad spectrum antimicrobial treatments, resulting in a clear need for a rapid, sensitive technique for characterization of resistance markers in microbial pathogens without cell culture. Here we present the development of a microfluidic platform for polymerase chain reaction (PCR) mediated amplification of microbial gene targets in a continuous flow system for potential coupling with sample preparation systems to reduce time to diagnosis from days to within two hours. This culminated in a thermoelectric cooler mediated fluidic thermocycler with a recirculating assay region for real-time hybridization measurements to minimize assay time. We subsequently demonstrated development of a low-cost optical system for localized surface plasmon resonance imaging using a digital micromirror device and tuned nanoprism monolayers for DNA hybridization with a spectral resolution of 2nm. This LSPR imaging system was integrated in-flow into the microfluidic thermocycler, enabling detection of input E. coli DNA samples at a minimum concentration of 5fg/ [microliter]. We further demonstrated multiplex detection of target markers, indicating potential for assaying target panels for characterization of pathogens. Overall, the studies in this dissertation demonstrate a microfluidic PCR system with built-in sensitive LSPR detection of DNA hybridization. It should serve as a starting point for exploration of and expansion with fluidic sample preparation with a focus on rapid characterization of pathogens.Biomedical Engineerin