The Potential of TaqMan Array Cards for Detection of Multiple Biological Agents by Real-Time PCR

The TaqMan Array Card architecture, normally used for gene expression studies, was evaluated for its potential to detect multiple bacterial agents by real-time PCR. Ten PCR assays targeting five biological agents (Bacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis, and Yersinia pestis) were incorporated onto Array Cards. A comparison of PCR performance of each PCR in Array Card and singleplex format was conducted using DNA extracted from pure bacterial cultures. When 100 fg of agent DNA was added to Array Card channels the following levels of agent detection (where at least one agent PCR replicate returned a positive result) were observed: Y. pestis 100%, B. mallei & F. tularensis 93%; B. anthracis 71%; B. pseudomallei 43%. For B. mallei & pseudomallei detection the BPM2 PCR, which detects both species, outperformed PCR assays specific to each organism indicating identification of the respective species would not be reproducible at the 100 fg level. Near 100% levels of detection were observed when 100 fg of DNA was added to each PCR in singleplex format with singleplex PCRs also returning sporadic positives at the 10 fg per PCR level. Before evaluating the use of Array Cards for the testing of environmental and clinical sample types, with potential levels of background DNA and PCR inhibitors, users would therefore have to accept a 10-fold reduction in sensitivity of PCR assays on the Array Card format, in order to benefit for the capacity to test multiple samples for multiple agents. A two PCR per agent strategy would allow the testing of 7 samples for the presence of 11 biological agents or 3 samples for 23 biological agents per card (with negative control channels)

Host immunity in the protective response to vaccination with heat-killed Burkholderia mallei

<p>Abstract</p> <p>Background</p> <p>We performed initial cell, cytokine and complement depletion studies to investigate the possible role of these effectors in response to vaccination with heat-killed <it>Burkholderia mallei </it>in a susceptible BALB/c mouse model of infection.</p> <p>Results</p> <p>While protection with heat-killed bacilli did not result in sterilizing immunity, limited protection was afforded against an otherwise lethal infection and provided insight into potential host protective mechanisms. Our results demonstrated that mice depleted of either B cells, TNF-α or IFN-γ exhibited decreased survival rates, indicating a role for these effectors in obtaining partial protection from a lethal challenge by the intraperitoneal route. Additionally, complement depletion had no effect on immunoglobulin production when compared to non-complement depleted controls infected intranasally.</p> <p>Conclusion</p> <p>The data provide a basis for future studies of protection via vaccination using either subunit or whole-organism vaccine preparations from lethal infection in the experimental BALB/c mouse model. The results of this study demonstrate participation of B220⁺cells and pro-inflammatory cytokines IFN-γ and TNF-α in protection following HK vaccination.</p

Developing and understanding biofluid vibrational spectroscopy : a critical review

Vibrational spectroscopy can provide rapid, label-free, and objective analysis for the clinical domain. Spectroscopic analysis of biofluids such as blood components (e.g. serum and plasma) and others in the proximity of the diseased tissue or cell (e.g. bile, urine, and sputum) offers non-invasive diagnostic/monitoring possibilities for future healthcare that are capable of rapid diagnosis of diseases via specific spectral markers or signatures. Biofluids offer an ideal diagnostic medium due to their ease and low cost of collection and daily use in clinical biology. Due to the low risk and in vasiveness of their collection they are widely welcomed by patients as a diagnostic medium. This review under scores recent research within the field of biofluid spectroscopy and its use in myriad pat hologies such as cancer and infectious diseases. It highlights current progresses, advents, and pitfalls within the field and discusses future spectroscopic clinical potentials for diagnostics. The requirements and issues surrounding clinical translation are also considered

Polymerase chain reaction on a viral nanoparticle

The field of synthetic biology includes studies that aim to develop new materials and devices from biomolecules. In recent years much work has been carried out using a range of biomolecular chassis including α-helical coiled coils, α-sheet amyloids and even viral particles. In this work we show how hybrid bionanoparticles can be produced from a viral M13 bacteriophage scaffold through conjugation to DNA primers that can template a polymerase chain reaction (PCR). This unprecedented example of a PCR on a virus particle has been studied by flow aligned linear dichroism spectroscopy, which gives information on the structure of the product as well as a new protototype methodology for DNA detection. We propose that this demonstration of PCR on the surface of a bionanoparticle is a useful addition to ways in which hybrid assemblies may be constructed using synthetic biology

A high-sensitivity electrochemiluminescence-based ELISA for the measurement of the oxidative stress biomarker, 3-nitrotyrosine, in human blood serum and cells

The generation of 3-nitrotyrosine, within proteins, is a post-translational modification resulting from oxidative or nitrative stress. It has been suggested that this modification could be used as a biomarker for inflammatory diseases. Despite the superiority of mass spectrometry-based determinations of nitrotyrosine, in a high-throughput clinical setting the measurement of nitrotyrosine by an enzyme-linked immunosorbent assay (ELISA) is likely to be more cost-effective. ELISAs offer an alternative means to detect nitrotyrosine, but many commercially available ELISAs are insufficiently sensitive to detect nitrotyrosine in healthy human serum. Here, we report the development, validation and clinical application of a novel electrochemiluminescence-based ELISA for nitrotyrosine which provides superior sensitivity (e.g. a 50-fold increase in sensitivity compared with one of the tested commercial colorimetric ELISAs). This nitrotyrosine ELISA has the following characteristics: a lower limit of quantitation of 0.04 nM nitrated albumin equivalents; intra- and inter-assay coefficients of variation of 6.5% and 11.3%, respectively; a mean recovery of 106 ± 3% and a mean linearity of 0.998 ± 0.001. Far higher nitration levels were measured in normal human blood cell populations when compared to plasma. Mass spectrometry was used to validate the new ELISA method. The analysis of the same set of chemically modified albumin samples using the ELISA method and mass spectrometry showed good agreement for the relative levels of nitration present in each sample. The assay was applied to serum samples from patients undergoing elective surgery which induces the human inflammatory response. Matched samples were collected before and one day after surgery. An increase in nitration was detected following surgery (median (IQR): 0.59 (0.00–1.34) and 0.97 (0.00–1.70) nitrotyrosine (fmol of nitrated albumin equivalents/mg protein) for pre- and post-surgery respectively. The reported assay is suitable for nitrotyrosine determination in patient serum samples, and may also be applicable as a means to determine oxidative stress in primary and cultured cell populations

Combining vaccination and postexposure CpG therapy provides optimal protection against lethal sepsis in a biodefense model of human melioidosis.

The Gram-negative bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a major cause of lethal sepsis and morbidity in endemic areas of Southeast Asia and a potential bioterrorism threat. We have used susceptible BALB/c mice to evaluate the potential of targeting vaccination and generic immunotherapy to the lung for optimal protection against respiratory challenge. Intranasal vaccination with live attenuated B. pseudomallei increased survival and induced interferon-γ-secreting T cells in the lung. Intranasal delivery of CpG oligodeoxynucleotides also provided significant protection; however, combining preexposure vaccination with CpG treatment at the time of infection or up to 18 hours after infection, provided significantly greater protection than either treatment alone. This combination prolonged survival, decreased bacterial loads by >1000-fold, and delayed the onset of sepsis. This novel approach may be applicable to other potential biodefense agents for which existing countermeasures are not fully effective

Observations on the Inactivation Efficacy of a MALDI-TOF MS Chemical Extraction Method on Bacillus anthracis Vegetative Cells and Spores.

A chemical (ethanol; formic acid; acetonitrile) protein extraction method for the preparation of bacterial samples for matrix assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF MS) identification was evaluated for its ability to inactivate bacterial species. Initial viability tests (with and without double filtration of the extract through 0.2 μM filters), indicated that the method could inactivate Escherichia coli MRE 162 and Klebsiella pneumoniae ATCC 35657, with or without filtration, but that filtration was required to exclude viable, avirulent, Bacillus anthracis UM23CL2 from extracts. Multiple, high stringency, viability experiments were then carried out on entire filtered extracts prepared from virulent B. anthracis Vollum vegetative cells and spores ranging in concentration from 10(6)-10(8) cfu per extract. B. anthracis was recovered in 3/18 vegetative cell extracts and 10/18 spore extracts. From vegetative cell extracts B. anthracis was only recovered from extracts that had undergone prolonged Luria (L)-broth (7 day) and L-agar plate (a further 7 days) incubations. We hypothesise that the recovery of B. anthracis in vegetative cell extracts is due to the escape of individual sub-lethally injured cells. We discuss our results in view of working practises in clinical laboratories and in the context of recent inadvertent releases of viable B. anthracis

CpG-DNA protects against a lethal orthopoxvirus infection in a murine model.

CpG-DNA has been described as a potent activator of the innate immune system, with potential to protect against infection caused by a range of pathogens in a non-specific manner. Here two classes of CpG-DNA (CpG-A and CpG-B) have been investigated for their abilities to protect mice from infection with an orthopoxvirus (vaccinia virus). Dosing with either CpG-A or B by the intraperitonal or intranasal route protected mice against a subsequent intranasal challenge with vaccinia virus. To our knowledge, this is the first time CpG-mediated protection has been demonstrated at the lung surface. The level of protection was greater when CpG-DNA was administered intranasally demonstrating a clear relationship between the route of CpG dosing and infection route. Treatment with CpG-B reduced viral titer in the lung by 10,000-fold at day 3 post-infection. The CC chemokines RANTES and MIP-1beta were elevated in the broncho-alveolar lavage from animals treated intranasally with CpG-B compared to untreated and intraperitoneally dosed controls, and it is possible that these chemokines play a role in the clearance of intranasally delivered vaccinia virus

Summary of <i>B</i>. <i>anthracis</i> recovery, from dried MALDI protein extracts derived from vegetative cells, in QUAL and QUANT experiments.

<p>Summary of <i>B</i>. <i>anthracis</i> recovery, from dried MALDI protein extracts derived from vegetative cells, in QUAL and QUANT experiments.</p