New real-time quantitative PCR procedure for quantification of bifidobacteria in human fecal samples

The application of a real-time quantitative PCR method (5' nuclease assay), based on the use of a probe labeled at its 5' end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 x 10(4) cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces.     

New Real-Time Quantitative PCR Procedure for Quantification of Bifidobacteria in Human Fecal Samples

The application of a real-time quantitative PCR method (5′ nuclease assay), based on the use of a probe labeled at its 5′ end with a stable, fluorescent lanthanide chelate, for the quantification of human fecal bifidobacteria was evaluated. The specificities of the primers and the primer-probe combination were evaluated by conventional PCR and real-time PCR, respectively. The results obtained by real-time PCR were compared with those obtained by fluorescent in situ hybridization, the current gold standard for intestinal microbiota quantification. In general, a good correlation between the two methods was observed. In order to determine the detection limit and the accuracy of the real-time PCR procedure, germfree rat feces were spiked with known amounts of bifidobacteria and analyzed by both methods. The detection limit of the method used in this study was found to be about 5 × 10⁴ cells per g of feces. Both methods, real-time PCR and fluorescent in situ hybridization, led to an accurate quantification of the spiked samples with high levels of bifidobacteria, but real-time PCR was more accurate for samples with low levels. We conclude that the real-time PCR procedure described here is a specific, accurate, rapid, and easy method for the quantification of bifidobacteria in feces.     

Microchamber array based DNA quantification and specific sequence detection from a single copy via PCR in nanoliter volumes

A novel method for DNA quantification and specific sequence detection in a highly integrated silicon microchamber array is described. Polymerase chain reaction (PCR) mixture of only 40 nL volume could be introduced precisely into each chamber of the mineral oil layer coated microarray by using a nanoliter dispensing system. The elimination of carry-over and cross-contamination between microchambers, and multiple DNA amplification and detection by TaqMan chemistry were demonstrated, for the first time, by using our system. Five different gene targets, related to Escherichia coli were amplified and detected simultaneously on the same chip by using DNA from three different serotypes as the templates. The conventional method of DNA quantification, which depends on the real-time monitoring of variations in fluorescence intensity, was not applied to our system, instead a simple method was established. Counting the number of the microchambers with a high fluorescence signal as a consequence of TaqMan PCR provided the precise quantification of trace amounts of DNA. The initial DNA concentration for Rhesus D (RhD) gene in each microchamber was ranged from 0.4 to 12 copies, and quantification was achieved by observing the changes in the released fluorescence signals of the microchambers on the chip. DNA target could be detected as small as 0.4 copies. The amplified DNA was detected with a CCD camera built-in to a fluorescence microscope, and also evaluated by a DNA microarray scanner with associated software. This simple method of counting the high fluorescence signal released in microchambers as a consequence of TaqMan PCR was further integrated with a portable miniaturized thermal cycler unit. Such a small device is surely a strong candidate for low-cost DNA amplification, and detected as little as 0.4 copies of target DNA.     

Development of a lab-on-a-chip device for diagnosis of plant pathogens

A lab-on-a-chip system for rapid nucleic acid-based analysis was developed that can be applied for diagnosis of selected Phytophthora species as a first example for use in plant pathology. All necessary polymerase chain reaction process (PCR) and hybridization steps can be performed consecutively within a single chip consisting of two components, an inflexible and a flexible one, with integrated microchannels and microchambers. Data from the microarray is collected from a simple electrical measurement that is based on elementary silver deposition by enzymatical catalyzation. Temperatures in the PCR and in the hybridization zone are managed by two independent Peltier elements. The chip will be integrated in a compact portable system with a pump and power supply for use on site. The specificity of the lab-on-a-chip system could be demonstrated for the tested five Phytophthora species. The two Pythium species gave signals below the threshold. The results of the electrical detection of the microarray correspond to the values obtained with the control method (optical grey scale analysis).     

A real-time PCR-based method for determining the surface coverage of thiol-capped oligonucleotides bound onto gold nanoparticles

Here we report a real-time PCR-based method for determining the surface coverage of dithiol-capped oligonucleotides bound onto gold nanoparticles alone and in tandem with antibody. The detection of gold nanoparticle-bound DNA is accomplished by targeting the oligonucleotide with primer and probe binding sites, amplification of the oligonucleotide by PCR, and real-time measurement of the fluorescence emitted during the reaction. This method offers a wide dynamic range and is not dependant on the dissociation of the oligonucleotide strands from the gold nanoparticle surface; the fluorophore is not highly quenched by the gold nanoparticles in solution during fluorescence measurements. We show that this method and a fluorescence-based method give equivalent results for determining the surface coverage of oligonucleotides bound onto 13 or 30 nm gold nanoparticles alone and in tandem with antibody. Quantifying the surface coverage of immobilized oligonucleotides on metallic nanoparticle surfaces is important for optimizing the sensitivity of gold nanoparticle-based detection methods and for better understanding the interactions between thiol-functionalized oligonucleotides and gold nanoparticles.     

Integration of amplified differential gene expression (ADGE) and DNA microarray

Amplified Differential Gene Expression (ADGE) provides a new concept that the ratios of differentially expressed genes are magnified before detection in order to improve both sensitivity and accuracy. This technology is now implemented with integration of DNA reassociation and PCR. The ADGE technique can be used either as a stand-alone method or in series with DNA microarray. ADGE is used in sample preprocessing and DNA microarray is used as a displaying system in the series combination. These two techniques are mutually synergistic: the quadratic magnification of ratios of differential gene expression achieved by ADGE improves the detection sensitivity and accuracy; the PCR amplification of templates enhances the signal intensity and reduces the requirement for large amounts of starting material; the high throughput for DNA microarray is maintained.     

Label-free electrical detection of DNA hybridization for the example of influenza virus gene sequences

Microarrays based on DNA-DNA hybridization are potentially useful for detecting and subtyping viruses but require fluorescence labeling and imaging equipment. We investigated a label-free electrical detection system using electrochemical impedance spectroscopy that is able to detect hybridization of DNA target sequences derived from avian H5N1 influenza virus to gold surface-attached single-stranded DNA oligonucleotide probes. A 23-nt probe is able to detect a 120-nt base fragment of the influenza A hemagglutinin gene sequence. We describe a novel method of data analysis that is compatible with automatic measurement without operator input, contrary to curve fitting used in conventional electrochemical impedance spectroscopy (EIS) data analysis. A systematic investigation of the detection signal for various spacer molecules between the oligonucleotide probe and the gold surface revealed that the signal/background ratio improves as the length of the spacer increases, with a 12- to 18-atom spacer element being optimal. The optimal spacer molecule allows a detection limit between 30 and 100 fmol DNA with a macroscopic gold disc electrode of 1 mm radius. The dependence of the detection signal on the concentration of a 23-nt target follows a binding curve with an approximate 1:1 stoichiometry and a dissociation constant of KD=13+/-4 nM at 295 K.     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

Detection of Mycobacterium avium subspecies paratuberculosis in Swiss dairy cattle by real-time PCR and culture: a comparison of the two assays

AIMS: To compare the two different diagnostic assays for the detection of Mycobacterium avium ssp. paratuberculosis, the aetiological agent of paratuberculosis. METHODS AND RESULTS: Faecal samples were derived from 310 cows, representing 13 commercial dairy herds in various locations in Switzerland with expected increased risk because of a past history of disease. Detection assays for M. avium ssp. paratuberculosis were culture (gold standard) and a newly designed real-time PCR. Real-time PCR identified 31 of 310 animals as positive within this risk population whereas culture identified 20 positive animals. The specificity of real-time PCR was confirmed by DNA sequencing of the PCR product. Depending on the test used, the paratuberculosis prevalence in our tested risk population ranged from 6.5 to 10%. CONCLUSIONS: Real-time PCR and culture data were in good agreement, and real-time PCR generates data in a short time in contrast to culture. SIGNIFICANCE AND IMPACT OF THE STUDY: We consider real-time PCR as a suitable alternative method to culture for the detection of M. avium ssp. paratuberculosis in a national surveillance programme.     

DNA supercoiling suppresses real-time PCR: a new approach to the quantification of mitochondrial DNA damage and repair

As a gold standard for quantification of starting amounts of nucleic acids, real-time PCR is increasingly used in quantitative analysis of mtDNA copy number in medical research. Using supercoiled plasmid DNA and mtDNA modified both in vitro and in cancer cells, we demonstrated that conformational changes in supercoiled DNA have profound influence on real-time PCR quantification. We showed that real-time PCR signal is a positive function of the relaxed forms (open circular and/or linear) rather than the supercoiled form of DNA, and that the conformation transitions mediated by DNA strand breaks are the main basis for sensitive detection of the relaxed DNA. This new finding was then used for sensitive detection of structure-mediated mtDNA damage and repair in stressed cancer cells, and for accurate quantification of total mtDNA copy number when all supercoiled DNA is converted into the relaxed forms using a prior heat-denaturation step. The new approach revealed a dynamic mtDNA response to oxidative stress in prostate cancer cells, which involves not only early structural damage and repair but also sustained copy number reduction induced by hydrogen peroxide. Finally, the supercoiling effect should raise caution in any DNA quantification using real-time PCR.     

电化学方法在李斯特氏菌毒力基因检测中的应用

单核李斯特氏菌(Listeria monocytogenes, LM)是食源性李斯特氏病的病源菌, 该病可引起败血病、脑膜炎、流产等。李斯特氏菌的毒力因子listeriolysin O (LLO)是引发李斯特氏病的主要原因。文章使用一种特殊的电化学方法从样品中检测编码LLO的hlyA基因。该方法以化合物Nhydroxysulfosuccinimide (NHS) 和 N-(3-dimethylamion) propyl- N'-ethyl carbodiimidehydrochloride (EDC) 作为激活剂, 使单链DNA探针结合到金电极表面组成工作电极, 以[Co(phen)₃](ClO₄)₃ 作为指示剂来检测循环伏安曲线(Cyclic voltammetry , CV), 通过CV峰值的变化来估算hlyA基因的含量, 从而确定LM的污染情况。这种新颖的电化学方法用于免标记的目标DNA的杂交检测, 具有快速和方便的特点。     

Detection of bacterial pathogens in municipal wastewater using an oligonucleotide microarray and real-time quantitative PCR

As a first step toward building a comprehensive microarray, two low density DNA microarrays were constructed and evaluated for the accurate detection of wastewater pathogens. The first one involved the direct hybridization of wastewater microbial genomic DNA to the functional gene probes while the second involved PCR amplification of 23S ribosomal DNA. The genomic DNA microarray employed 10 functional genes as detection targets. Sensitivity of the microarray was determined to be approximately 1.0 microg of Esherichia coli genomic DNA, or 2 x 10(8) copies of the target gene, and only E. coli DNA was detected with the microarray assay using municipal raw sewage. Sensitivity of the microarray was enhanced approximately by 6 orders of magnitude when the target 23S rRNA gene sequences were PCR amplified with a novel universal primer set and allowed hybridization to 24 species-specific oligonucleotide probes. The minimum detection limit was estimated to be about 100 fg of E. coli genomic DNA or 1.4 x 10(2) copies of the 23S rRNA gene. The PCR amplified DNA microarray successfully detected multiple bacterial pathogens in wastewater. As a parallel study to verify efficiency of the DNA microarray, a real-time quantitative PCR assay was also developed based on the fluorescent TaqMan probes (Applied Biosystems).     

Development and evaluation of a new detection tool—visual DNA microarray for simultaneous and specific detection of human immunodeficiency virus type-1 and hepatitis C virus

Human immunodeficiency virus type-1 (HIV-1) and hepatitis C virus (HCV) are transfusion-transmitted human pathogens that have a major impact on blood safety and public health. Based on multiplex asymmetrical PCR and coupled with gold labelled silver stain (GLSS), we developed the visual DNA microarray for sensitive and specific detection of these two viruses. Capturing probes of 5′-end-amino-modified oligonucleotides were immobilized on glass surface to bind the complement biotinylated target DNA. The Au–streptavidin probe was introduced to the microarray for specific binding to biotin. Black images of microarray spots which result from the precipitation of silver onto Au–streptavidin probes, were visualized by naked eyes. In order to improve the efficiency of microarray hybridization, triplex asymmetrical PCR of HIV-1, HCV and Human enterovirus 71 (EV-71, used as positive control) were performed to prepare abundant biotinylated single-stranded target DNA. The sensitivity of visual DNA microarray (10³ copies/ml) was higher than conventional PCR (10⁴ copies/ml) and was identical to FQ-PCR (10³ copies/ml). Total 152 blood samples containing the two viruses were tested using the DNA microarray and fluorescence quantitative real-time PCR (FQ-PCR). The results were identical (P > 0.05). So this system has high sensitivity and may have potential in clinical applications.     

Electrochemical immunoanalysis of cardiac myoglobin

Methods of myoglobin determination based on electrochemical analysis by means of analysis of electrochemical parameters of modified electrodes have been proposed. The method of direct detection is based on interaction of myoglobin with anti-myoglobin with subsequent electrochemical registration of this hemoprotein. The electrode surface was modified by a membrane-like synthetic didodecyldimethylammonium bromide (DDAB), gold nanoparticles and antibodies to human cardiac myoglobin the electrochemical reduction of myoglobin heme was registered provided that the antigen (myoglobin) was present in the samples. The reaction of myoglobin binding to antibodies immobilized on the electrode surface was also registered using electrochemical impedance spectroscopy. The study of electro analytical characteristics revealed high specificity and sensitivity of the developed method. The biosensor was characterized by low detection limit and a high working range of the detected concentrations from 17.8 to 1780 ng/ml (from 1 to 100 nM). The method of myoglobin determination based on a signal of gold nanoparticles has also been proposed. The signal was detected with stripping voltammetry. There was a change in the cathodic peak area and the peak height of gold oxide reduction for the electrodes with antibodies and the electrodes with the antibody-myoglobin complex.     

A novel electrochemical detection method for aptamer biosensors

A beacon aptamer-based biosensor for the detection of thrombin was developed using electrochemical transduction method. Gold surface was modified with a beacon aptamer covalently linked at 5'-terminus with a linker containing a primary aliphatic amine. Methylene blue (MB) was intercalated into the beacon sequence, and used as an electrochemical marker. When the beacon aptamer immobilized on gold surface encounters thrombin, the hairpin forming beacon aptamer is conformationally changed to release the intercalated MB, resulting a decrease in electrical current intensity in voltamogram. The peak signal of the MB is clearly decreased by the binding of thrombin onto the beacon aptamer. The linear range of the signal was observed between 0 and 50.8 nM of thrombin with 0.999 correlation factor. This method was able to linearly and selectively detect thrombin with a detection limit of 11 nM.     

Real-time PCR in virology

The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5′ endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.