Polymerase chain reaction as a sensitive and rapid method for specific detection of Mycoplasma pneumoniae in clinical samples

The fast diagnosis of Mycoplasma primary atypical pneumonia is impaired by the lack of routinely available culture methods for isolation of Mycoplasma pneumoniae from clinical specimens. Likewise, serological methods commonly used for diagnosis are insensitive and non-specific. In this study, we have established and applied the polymerase chain reaction (PCR) technique to detect M. pneumoniae DNA in clinical samples originating from the respiratory tract. The PCR results were compared with those from culture and serology tests. To standardize the detection of M. pneumoniae by PCR, we first used DNA from culture grown organisms and clinical samples seeded with M. pneumoniae. PCR amplification was performed with M. pneumoniae-specific primers to amplify 144, 153 and 631 bp DNA fragments by using primer pairs MP5-1/MP5-2, P1-178/P1-331 and P1-178/P1-809, respectively. The amplification of the 631 bp DNA fragment was found to be most sensitive for the detection of M. pneumoniae. Using the most sensitive PCR, a total of 47 respiratory specimens from patients suspected of community acquired pneumonia were tested. While none of the specimens were positive for M. pneumoniae in culture, 6 specimens gave positive results by PCR. In 4 out of the 5 PCR positive samples tested serologically, the results were supported by elevated levels of anti-mycoplasma IgG/IgM/IgA. Thus, these results suggest that PCR is the most sensitive method to detect M. pneumoniae in clinical specimens.     

Improved detection limit for a direct determination of 8-hydroxy-2'-deoxyguanosine in untreated urine samples by capillary electrophoresis with optical detection

Method for a direct determination of 8-hydroxy-2'-deoxyguanosine (8OHdG) in untreated urine samples by capillary electrophoresis with optical detection was developed. Optimisation of conditions resulted in a significant lowering of the limit of detection (LOD) by a factor of 400 as compared to our previous study. Optimum separation of 8OHdG from other urine components was achieved using the separation electrolyte containing 80 mM 2-(cyclohexylamino)ethanesulfonic acid, 9 mM LiOH (pH 8.6), and 0.1 mM cetyltrimethylammonium bromide ensuring the electro-osmotic flow inversion. In the model aqueous samples, these conditions allow separating 8OHdG and 2'-deoxyguanosine (dG) from other nucleosides/nucleotides including 2'-deoxycitidine 5'-monophosphate (dCMP), thymidine 5'-monophosphate (TMP), adenosine (A), and thymidine (T). On the other hand, 2'-deoxyadenosine 5'-monophosphate (dAMP) and 2'-deoxyguanosine 5'-monophosphate (dGMP) migrate together, and guanosine (G), 2'-deoxyadenosine (dA), 2'-deoxycytidine (dC) are transported as neutral species with the electro-osmotic flow. In the spiked urine samples, 8OHdG and dG are well separated from each other and from other urine components and exhibit a linear calibration over the concentration range of 0.1-2.0 microM for 8OHdG (LOD = 42 nM) and 0.2-5.0 microM for dG (LOD = 86 nM), but urine metabolites interfere with the determination of dCMP, TMP, A and T. Method is applicable to untreated urine samples with slightly enhanced levels of 8OHdG compared to that found in healthy individuals.     

A rapid and sensitive method for the detection of Yersinia enterocolitica strains from clinical samples

AIMS: To compare three culture methods to detect Yersinia enterocolitica from oral or rectal swabs from experimentally infected pigs. METHODS AND RESULTS: The three methods used were: direct plating on Cefsulodin-Irgasan-Novobiocin (CIN) agar, cold enrichment in phosphate buffered saline (PBS) followed by plating on CIN agar and selective enrichment with Luria-Bertani-Bile Salts Irgasan (LB-BSI) followed by plating on CIN agar. Selective enrichment with LB-BSI produced the highest recovery rate (63%), when compared with cold enrichment (52%) and plating on CIN agar alone (43%). Selective enrichment with LB-BSI was significantly (P < 0.02) more sensitive than direct plating on CIN agar and more sensitive than cold enrichment (P < 0.1). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Selective enrichment with LB-BSI was more sensitive than the widely accepted method of cold enrichment and it reduced the time required for detection of Y. enterocolitica by three weeks. Selective enrichment with LB-BSI was also compatible with a multiplex PCR technique.     

A new electrochemical biosensor for DNA detection based on molecular recognition and lead sulfide nanoparticles

In this paper, we constructed a new electrochemical biosensor for DNA detection based on a molecule recognition technique. In this sensing protocol, a novel dual-labeled DNA probe (DLP) in a stem–loop structure was employed, which was designed with dabcyl labeled at the 3′ end as a guest molecule, and with a Pb nanoparticle labeled at the 5′ end as electrochemical tag to indicate hybridization. One α-cyclodextrin-modified electrode (α-CD/MCNT/GCE) was used for capturing the DNA hybridization. Initially, the DLP was in the “closed” state in the absence of the target, which shielded dabcyl from the bulky α-CD/MCNT/GCE conjugate due to a steric effect. After hybridization, the loop sequence (16 bases) formed a rigid duplex with the target, breaking the relatively shorter stem duplex (6 bases). Consequently, dabcyl was forced away from the Pb nanoparticle and became accessible by the electrode. Therefore, the target hybridization event can be sensitively transduced via detecting the electrochemical reduction current signal of Pb. Using this method, as low as 7.1 × 10⁻¹⁰ M DNA target had been detected with excellent differentiation ability for even a single mismatch.     

Esterase 2-oligodeoxynucleotide conjugates as sensitive reporter for electrochemical detection of nucleic acid hybridization

A thermostable, single polypeptide chain enzyme, esterase 2 from Alicyclobacillus acidocaldarius, was covalently conjugated in a site specific manner with an oligodeoxynucleotide. The conjugate served as a reporter enzyme for electrochemical detection of DNA hybridization. Capture oligodeoxynucleotides were assembled on gold electrode via thiol-gold interaction. The esterase 2-oligodeoxynucleotide conjugates were brought to electrode surface by DNA hybridization. The p-aminophenol formed by esterase 2 catalyzed hydrolysis of p-aminophenylbutyrate was amperometrically determined. Esterase 2 reporters allows to detect approximately 1.5 x 10(-18)mol oligodeoxynucleotides/0.6 mm2 electrode, or 3 pM oligodeoxynucleotide in a volume of 0.5 microL. Chemically targeted, single site covalent attachment of esterase 2 to an oligodeoxynucleotide significantly increases the selectivity of the mismatch detection as compared to widely used, rather unspecific, streptavidin/biotin conjugated proteins. Artificial single nucleotide mismatches in a 510-nucleotide ssDNA could be reliably determined using esterase 2-oligodeoxynucleotide conjugates as a reporter.     

A simple and direct electrochemical detection of interferon-gamma using its RNA and DNA aptamers

Tuberculosis is the most frequent cause of infection-related death worldwide. We constructed a simple and direct electrochemical sensor to detect interferon (IFN)-gamma, a selective marker for tuberculosis pleurisy, using its RNA and DNA aptamers. IFN-gamma was detected by its 5'-thiol-modified aptamer probe immobilized on the gold electrode. Interaction between IFN-gamma and the aptamer was recorded using electrochemical impedance spectroscopy and quartz crystal microbalance (QCM) with high sensitivity. The RNA-aptamer-based sensor showed a low detection limit of 100 fM, and the DNA-aptamer-based sensor detected IFN-gamma to 1 pM in sodium phosphate buffer. With QCM analysis, the aptamer immobilized on the electrode and IFN-gamma bound to the aptamer probe was quantified. This QCM result shows that IFN-gamma exists in multimeric forms to interact with the aptamers, and the RNA aptamer prefers the high multimeric state of IFN-gamma. Such a preference may describe the low detection limit of the RNA aptamer shown by impedance analysis. In addition, IFN-gamma was detected to 10 pM by the DNA aptamer in fetal bovine serum, a mimicked biological system, which has similar components to pleural fluid.     

Branched DNA amplification multimers for the sensitive, direct detection of human hepatitis viruses.

Branched oligonucleotides (bDNA) have been synthesized containing a unique primary segment and a set of identical secondary fragments covalently attached to the primary sequence through branch points. The primary sequence is designed to hybridize (directly or indirectly) to a target nucleic acid, such as hepatitis B virus (HBV) or hepatitis C virus (HCV) genomic DNA or RNA, respectively. The secondary fragments are used to direct the binding of multiple copies of a small oligonucleotide labelled with alkaline phosphatase. Assays for the presence of HBV and HCV based on the application of these branched amplification multimers have been devised. It is possible to detect as few as 1,000 hepatitis viral genomes directly.     

Disposable DNA electrochemical sensor for hybridization detection

A disposable electrochemical sensor for the detection of short DNA sequences is described. Synthetic single-stranded oligonucleotides have been immobilized onto graphite screen printed electrodes with two procedures, the first involving the binding of avidinbiotinylated oligonucleotide and the second adsorption at a controlled potential. The probes were hybridized with different concentrations of complementary sequences. The formed hybrids on the electrode surface were evaluated by differential pulse voltammetry and chronopotentiometric stripping analysis using daunomycin hydrochloride as indicator of hybridization reaction. The probe immobilization step, the hybridization event and the indicator detection, have been optimized. The DNA sensor obtained by adsorption at a controlled potential was able to detect 1 microgram/ml of target sequence in the buffer solution using chronopotentiometric stripping analysis.     

Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen

In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP–Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP–Au NRs nanocomposites and the labeling of secondary antibody (Ab₂) were performed by one-pot assembly of HRP and Ab₂ on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab₁) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen–antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H₂O₂) and 3,3′,5,5′-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1–100 ng ml⁻¹, and the limit of detection was 30 pg ml⁻¹ (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications.     

Highly sensitive direct detection and quantification of Burkholderia pseudomallei bacteria in environmental soil samples by using real-time PCR

The soil bacterium and potential biothreat agent Burkholderia pseudomallei causes the infectious disease melioidosis, which is naturally acquired through environmental contact with the bacterium. Environmental detection of B. pseudomallei represents the basis for the development of a geographical risk map for humans and livestock. The aim of the present study was to develop a highly sensitive, culture-independent, DNA-based method that allows direct quantification of B. pseudomallei from soil. We established a protocol for B. pseudomallei soil DNA isolation, purification, and quantification by quantitative PCR (qPCR) targeting a type three secretion system 1 single-copy gene. This assay was validated using 40 soil samples from Northeast Thailand that underwent parallel bacteriological culture. All 26 samples that were B. pseudomallei positive by direct culture were B. pseudomallei qPCR positive, with a median of 1.84 × 10(4) genome equivalents (range, 3.65 × 10(2) to 7.85 × 10(5)) per gram of soil, assuming complete recovery of DNA. This was 10.6-fold (geometric mean; range, 1.1- to 151.3-fold) higher than the bacterial count defined by direct culture. Moreover, the qPCR detected B. pseudomallei in seven samples (median, 36.9 genome equivalents per g of soil; range, 9.4 to 47.3) which were negative by direct culture. These seven positive results were reproduced using a nested PCR targeting a second, independent B. pseudomallei-specific sequence. Two samples were direct culture and qPCR negative but nested PCR positive. Five samples were negative by both PCR methods and culture. In conclusion, our PCR-based system provides a highly specific and sensitive tool for the quantitative environmental surveillance of B. pseudomallei.     

Profile-based direct kernels for remote homology detection and fold recognition

MOTIVATION: Protein remote homology detection is a central problem in computational biology. Supervised learning algorithms based on support vector machines are currently one of the most effective methods for remote homology detection. The performance of these methods depends on how the protein sequences are modeled and on the method used to compute the kernel function between them. RESULTS: We introduce two classes of kernel functions that are constructed by combining sequence profiles with new and existing approaches for determining the similarity between pairs of protein sequences. These kernels are constructed directly from these explicit protein similarity measures and employ effective profile-to-profile scoring schemes for measuring the similarity between pairs of proteins. Experiments with remote homology detection and fold recognition problems show that these kernels are capable of producing results that are substantially better than those produced by all of the existing state-of-the-art SVM-based methods. In addition, the experiments show that these kernels, even when used in the absence of profiles, produce results that are better than those produced by existing non-profile-based schemes. AVAILABILITY: The programs for computing the various kernel functions are available on request from the authors.     

Nanoelectrode ensembles as recognition platform for electrochemical immunosensors

In this study we demonstrate the possibility to prepare highly sensitive nanostructured electrochemical immunosensors by immobilizing biorecognition elements on nanoelectrode ensembles (NEEs) prepared in track-etch polycarbonate membranes. The gold nanodisk electrodes act as electrochemical transducers while the surrounding polycarbonate binds the antibody-based biorecognition layer. The interaction between target protein and antibody is detected by suitable secondary antibodies labelled with a redox enzyme. A redox mediator, added to the sample solution, shuttles electrons from the nanoelectrodes to the biorecognition layer, so generating an electrocatalytic signal. This allows one to fully exploit the highly improved signal-to-background current ratio, typical of NEEs. In particular, the receptor protein HER2 was studied as the target analyte. HER2 detection allows the identification of breast cancer that can be treated with the monoclonal antibody trastuzumab. NEEs were functionalized with trastuzumab which interacts specifically with HER2. The biorecognition process was completed by adding a primary antibody and a secondary antibody labelled with horseradish peroxidase. Hydrogen peroxide was added to modulate the label electroactivity; methylene blue was the redox mediator generating voltammetric signals. NEEs functionalized with trastuzumab were tested to detect small amounts of HER2 in diluted cell lysates and tumour lysates.     

Cu@Au alloy nanoparticle as oligonucleotides labels for electrochemical stripping detection of DNA hybridization

Synthesis of the novel Cu@Au alloy nanoparticle and its application in an electrochemical DNA hybridization detection assay is described in this article. We report a low-temperature method for generating core-shell particles consisting of a core of Cu and a thin layer of Au shell that can be readily functionalized with oligonucleotides. Core-shell Cu@Au particles were successfully labeled to a 5'-alkanethiol capped oligonucleotides probe that is related to the colitoxin gene. The DNA genetic sensing assay relies on the electrostatic adsorption of target oligonucleotides onto conducting polypyrrole (PPy) surface at the glassy carbon electrode (GCE), and its hybridization to the alloy particle-oligonucleotides DNA probe. Hybridization events between probe and target were monitored by the release of the copper metal atoms anchored on the hybrids by oxidative metal dissolution and the indirectly determination of the solubilized Cu2+ ions by sensitive anodic stripping voltammetry (ASV). The detection limit is 5.0 pmol l(-1) of target oligonucleotides. The Cu@Au core-shell nanoparticles combining the surface modification properties of Au with the good electrochemical activity of Cu core shows their perspective application in the electrochemical DNA hybridization analysis assay.     

Nanoparticle assembly for sensitive DNA detection using SERRS

SERRS (surface-enhanced resonance Raman scattering) is a vibrational technique, whereby a relatively weak Raman scattering effect is enhanced through the use of a visible chromophore and a roughened metal surface. The direct analysis of DNA by SERRS requires the modification of a nucleic acid sequence to incorporate a chromophore, and adsorption of the modified sequence on to a roughened metal surface. Aggregated metallic nanoparticles are commonly used in the analysis of dye-labelled DNA by SERRS, allowing for detection levels that rival those gained from standard fluorescence-based techniques. In the present paper, we report on how SERRS can be exploited for the analysis of clinically relevant DNA samples. We also report on the ability of nanoparticles to aggregate as the result of a biologically significant event, as opposed to the use of an external charge-modifying agent. The self-assembly of metallic nanoparticles is shown to be a promising new technique in the move towards extremely sensitive methods of DNA analysis by SERRS.     

Direct electrochemical sensor for fast reagent-free DNA detection

A novel reagentless direct electrochemical DNA sensor has been developed using ultrathin films of the conducting polymer polypyrrole doped with an oligonucleotide probe. Our goal was to develop a prototype electrochemical DNA sensor for detection of a biowarfare pathogen, variola major virus. The sensor has been optimized for higher specificity and sensitivity. It was possible to detect 1.6 fmol of complementary oligonucleotide target in 0.1 ml in seconds by using chronoamperometry. The sensitivity of the developed sensor is comparable to indirect electrochemical DNA sensors, which use electrochemical labels and reagent-intensive amplification. The developed sensing electrode is reusable, highly stable and suitable for storage in solution or in dry state.     

Sequence-specific electrochemical detection of nucleic acids in real samples

This paper reviews past and current developments in the field of electrochemical biosensors with a focus on the sequence-specific detection of nucleic acids in real samples. After electrochemical hybridization sensors had been first described in 1993, it took nearly a decade until some of the many proposed protocols were indeed applied to real samples like blood or tissue. Electrochemical transduction schemes used either rely on electroactive moieties such as intercalators, groove binders, covalently attached labels, and products of enzyme markers or they are completely indicator free like impedance-based detection principles. Most detection schemes require a polymerase chain reaction amplification step to allow for sufficient selectivity and sensitivity. Today, several companies develop electrochemical microarrays able to detect dozens to many thousands of sequences in a single experiment.     

A new gel tube method for the direct detection, identification and susceptibility testing of bacteria in clinical samples

We recently developed a simple new method which is designed to separate and concentrate bacteria from a sample by centrifugation in a gel system. Bacterial enzyme activity is then detected inside the gel without further manipulation using a colorimetric or fluorogenic substrate. The method provides a rapid, direct means of detecting bacteria in clinical samples, dispensing with the 24-h period normally required to isolate colonies on agar. Various applications of the method are described below, e.g. screening of negative urine samples, identification of Escherichia coli in urine samples, identification of Staphylococcus aureus in blood culture broths and detection of oxacillin-resistant S. aureus in blood culture broths. The advantages of the gel system and other applications are discussed.     

A sensitive analytical method for the detection and quantitation of adenosine in biological samples

A new method for the measurement of adenosine in biological materials has been developed. The method is based on the combined principles of isotope dilution and enzymatic catalysis using a highly specific adenosine kinase isolated from rat heart. By differential centrifugation and gel filtration, this adenosine kinase was obtained free of adenosine deaminase and other enzymes which would have been a source of error in the use of this enzyme in the adenosine assay. The cardiac adenosine kinase was shown to be highly specific and to exhibit an apparent K_m for adenosine of 0.35 μM. Using this enzyme, unknown quantities of adenosine could be detected by measuring the effect of their addition on the conversion of radioisotopic adenosine to 5′-AMP in the kinase reaction. In this procedure, as little as 20 pmoles of adenosine could be detected. To test the applicability of the assay, measurements of the tissue content of this nucleoside were made in samples of dog and rat hearts frozen in situ under control, hypoxic, or ischemic conditions. The assay has several advantageous features when compared to other existing methods used to measure adenosine: a minimum of sample preparation is required before the actual assay procedure; many samples can be processed per day by a single operator; single determinations can be done on as little as 5 μl of sample, and the specificity of the assay can be readily checked by treatment of samples with adenosine deaminase.     

A direct solubilization procedure for the determination of DNA and protein in cultured fibroblast monolayers

A procedure for the direct solubilization of fibroblast monolayers allowing precise determination of DNA and protein content has been developed. This simple procedure is applicable to the analysis of these macromolecules in fibroblast cultures containing small numbers of cells (<1 × 10⁵).