Detection of single nucleotide polymorphisms within the Listeria genus using an 'asymmetric' fluorogenic probe set and fluorescence resonance energy transfer based-PCR

AIMS: We describe a novel and inexpensive fluorescence energy transfer (FRET)-based PCR protocol to distinguish single nucleotide polymorphisms (SNPs) within the genus Listeria. METHODS AND RESULTS: Sequence information for the 16S rRNA gene of representative Listeria species was used to design genus-specific primers and two species-specific probes that differed in sequence by one single nucleotide. The probes were 5' labelled with either fluorescein or Texas Red, quenched with a shorter yet complementary 3' dimethyl-amino-phenyloazo benzoic acid (DABCYL) labelled oligonucleotide, and then incorporated into a previously reported 'asymmetric' FRET-based PCR detection protocol. CONCLUSIONS: Listeria monocytogenes could be readily distinguished from other members of the Listeria genus after PCR amplification and measurement of endpoint fluorescence at two different wavelengths. SIGNIFICANCE AND IMPACT OF THE STUDY: The relatively low cost and high flexibility of this system will benefit laboratories in their efforts to develop rapid and specific methods to detect minor sequence differences between related microorganisms.     

Detection of DNA mutations by fluorescence resonance energy transfer-based preferential homoduplex formation assay

Molecularly targeted agents for cancer therapy are recognized as being effective and are gaining in popularity. However, the efficacy of the agents depends on the status of the targeted molecule such as the number of molecules expressed, activity, and mutation. Therefore, the use of companion diagnostics for investigating the status of the targeted molecule prior to therapy is highly important. We developed a simple and cost-effective somatic mutation detection method called the fluorescence resonance energy transfer-based preferential homoduplex formation assay (FRET–PHFA). By using double-stranded labeled DNA and fluorescence measurement with thermal control, this method provides higher reproducibility, easier handling, less risk for contamination, shorter assay time (only ∼15 min), and less cost compared with conventional PHFA. Here we report the evaluation of FRET–PHFA on the detection of multiallelic KRAS mutations in codons 12 and 13 compared with the TheraScreen clinical diagnostics kit. We found that FRET–PHFA detected KRAS mutations (1.25–50%) from all cell line DNA titration samples.     

微流控振荡流PCR快速检测单增李斯特氏菌

一种高通量振荡流PCR微流控技术已被成功开发,借此来快速检测食品致病菌-单增李斯特氏菌(L.monocytogenes).该PCR微流控装置主要由基于LabView的温度控制与采集系统、三个铜加热块以及聚四氟乙烯(PTFE)毛细管等构成.在该微流控装置上,三个铜块维持PCR反应所需要的三个温度,而包埋在铜块沟槽中的PT...     

Detection of asymmetric distribution of phospholipids by fluorescence resonance energy transfer

It is well established that the plasma membrane exhibits an asymmetric distribution of lipids between the inner and outer leaflets of the lipid bilayer. Recent studies suggest that the asymmetric distribution changes locally and temporarily, accompanied by cellular events. However, available methods to detect lipid asymmetry lack spatio-temporal resolution. As a technique of potential use for real-time imaging of lipid asymmetry, we a novel method that utilizes fluorescence resonance energy transfer (FRET) between NBD-labeled phospholipids (donor) and extracellular rhodamine (acceptor). When cell apoptosis was induced by staurosporine, the fluorescence intensity of NBD-labeled phosphatidylserine decreased owing to FRET from NBD to rhodamine. This method provides a simple way to detect lipid asymmetry and may be useful for observing dynamic changes in asymmetric distribution of lipids.     

Quantitative Detection of Listeria monocytogenes in Biofilms by Real-Time PCR

A quantitative method based on a real-time PCR assay to enumerate Listeria monocytogenes in biofilms was developed. The specificity for L. monocytogenes of primers targeting the listeriolysin gene was demonstrated using a SYBR Green I real-time PCR assay. The number of L. monocytogenes detected growing in biofilms was 6 × 10² CFU/cm².     

Establishment of a fluorescence resonance energy transfer-based bioassay for detecting dioxin-like compounds

Dioxins comprise a group of compounds which contain a double aromatic ring-like structure. They are among the most prevalent and toxic environmental pollutants. Accumulation of dioxins in human tissues poses a potential threat to human health. Currently, analytical chemical procedures dominate dioxin-detection protocols. In this study, we established a fluorescence resonance energy transfer (FRET)-based dioxin-detection bioassay. Aryl hydrocarbon receptor (AHR) and AHR nuclear translocator (ARNT) fused-cyan fluorescent protein (CFP) and -yellow fluorescent protein (YFP) constructed were transiently co-transfected into rat hepatoma cell line, H4IIEC3 cells. Our results showed that no FRET signals were detected in AHR-CFP- and ARNT-YFP-transfected H4IIEC3 cells. However, dioxin treatments upregulated FRET signals in these transfected cells in a dose-dependent manner. This work highlighted the potential of FRET technique in the detection of dioxin-like compounds.     

Use of a fluorogenic probe in a PCR-based assay for the detection of Listeria monocytogenes.

A PCR-based assay for Listeria monocytogenes that uses the hydrolysis of an internal fluorogenic probe to monitor the amplification of the target has been formatted. The fluorogenic 5' nuclease PCR assay takes advantage of the endogenous 5' --> 3' nuclease activity of Taq DNA polymerase to digest a probe which is labelled with two fluorescent dyes and hybridizes to the amplicon during PCR. When the probe is intact, the two fluorophores interact such that the emission of the reporter dye is quenched. During amplification, the probe is hydrolyzed, relieving the quenching of the reporter and resulting in an increase in its fluorescence intensity. This change in reporter dye fluorescence is quantitative for the amount of PCR product and, under appropriate conditions, for the amount of template. We have applied the fluorogenic 5' nuclease PCR assay to detect L. monocytogenes, using an 858-bp amplicon of hemolysin (hlyA) as the target. Maximum sensitivity was achieved by evaluating various fluorogenic probes and then optimizing the assay components and cycling parameters. With crude cell lysates, the total assay could be completed in 3 h with a detection limit of approximately 50 CFU. Quantification was linear over a range of 5 x 10(1) to 5 x 10(5) CFU.     

Quantitative monitoring of 2-oxoglutarate in Escherichia coli cells by a fluorescence resonance energy transfer-based biosensor

2-Oxoglutarate (2OG) is a metabolite from the highly conserved Krebs cycle and not only plays a critical role in metabolism but also acts as a signaling molecule in a variety of organisms. Environmental inorganic nitrogen is reduced to ammonium by microorganisms, whose metabolic pathways involve the conversion of 2OG to glutamate and glutamine. Tracking of 2OG in real time would be useful for studies on cell metabolism and signal transduction. Here, we developed a genetically encoded 2OG biosensor based on fluorescent resonance energy transfer by inserting the functional 2OG-binding domain GAF of the NifA protein between the fluorescence resonance energy transfer (FRET) pair YFP/CFP. The dynamic range of the sensors is 100 μM to 10 mM, which appeared identical to the physiological range observed in E. coli. We optimized the peptide lengths of the binding domain to obtain a sensor with a maximal ratio change of 0.95 upon 2OG binding and demonstrated the feasibility of this sensor for the visualization of metabolites both in vitro and in vivo.     

Detection of epitope-tagged proteins in flow cytometry: fluorescence resonance energy transfer-based assays on beads with femtomole resolution.

Epitope tagging of expressed proteins is a versatile tool for the detection and purification of the proteins. This approach has been used in protein-protein interaction studies, protein localization, and immunoprecipitation. Among the most popular tag systems is the FLAG epitope tag, which is recognized by three monoclonal antibodies M1, M2, and M5. We describe novel approaches to the detection of epitope-tagged proteins via fluorescence resonance energy transfer on beads. We have synthesized and characterized biotinylated and fluorescein-labeled FLAG peptides and examined the binding of FLAG peptides to commercial streptavidin beads using flow cytometric analysis. A requirement of assay development is the elucidation of parameters that characterize the binding interactions between component systems. We have thus compiled a set of Kd values determined from a series of equilibrium binding experiments with beads, peptides, and antibodies. We have defined conditions for binding biotinylated and fluoresceinated FLAG peptides to beads. Site occupancies of the peptides were determined to be on the order of several million sites per bead and Kd values in the 0.3-2.0 nM range. The affinity for antibody attachment to peptides was determined to be in the low nanomolar range (less than 10 nM) for measurements on beads and solution. We demonstrate the applicability of this methodology to assay development, by detecting femtomole amounts of N-terminal FLAG-bacteria alkaline phosphatase fusion protein. These characterizations form the basis of generalizable and high throughput assays for proteins with known epitopes, for research, proteomic, or clinical applications.     

Rapid Quantitative Detection of Listeria monocytogenes in Meat Products by Real-Time PCR

We describe a quick and simple method for the quantitative detection of Listeria monocytogenes in meat products. This method is based on filtration, Chelex-100-based DNA purification, and real-time PCR. It can detect as few as 100 CFU/g and quantify as few as 1,000 CFU/g, with excellent accuracy compared to that of the plate count method. Therefore, it is a promising alternative for the detection of L. monocytogenes in meat products.     

Detection of viable and dead Listeria monocytogenes on gouda-like cheeses by real-time PCR

AIMS: Surface contamination by Listeria monocytogenes of gouda-like cheeses during processing represents a potential public health problem. The aim of this work was to develop novel real-time PCR diagnostics to detect the presence of viable, dead or viable but not culturable (VBNC) cells on gouda-like cheeses. METHODS AND RESULTS: We used ethidium monoazide bromide (EMA)-PCR for direct quantification of viable and dead cells, while semiquantitative detection of culturable cells below the PCR detection limit (c. 100 CFU g(-1)) was obtained by combining growth and real-time PCR. We were able to quantify the fraction of >0.5% viable cells in a background of dead cells by EMA-PCR, given that the viable cell concentration was above the PCR detection limit. The combined growth and real-time PCR complemented the EMA-PCR, and enabled semiquantitative detection of low levels of culturable cells (10 and 100 CFU g(-1)). SIGNIFICANCE AND IMPACT OF THE STUDY: The significance of this work is that we have developed a novel concept for detection of viable and potentially infectious L. monocytogenes.     

Acid-tolerant Listeria monocytogenes persist in a model food system fermented with nisin-producing bacteria

AIMS: To investigate the induction of the acid tolerance response (ATR) in Listeria monocytogenes and to assess the persistence of the pathogen in broth fermented using a nisin-producing starter culture. METHODS AND RESULTS: Lactic, acetic and hydrochloric acids were used to induce the ATR in L. monocytogenes growing at early exponential phase. Cells were then challenged in medium acidified to pH 3.5 with the same acid. Only lactic acid induced a detectable ATR. ATR+ cells maintained their initial numbers after 1 h exposure while ATR- were reduced by c. 4 log10 CFU. ATR+ or ATR- cells were also inoculated in M17G broth fermented with nisin-producing (nis+) or control (nis-) Lactococcus lactis. When exposed to nisin, the numbers of ATR+ cells were c. 2 log10 CFU higher than non detectable ATR- cells at day 3. In the absence of nisin (nis- culture), L. monocytogenes was recovered from all ATR+ and ATR- samples after 30 days. In contrast, no L. monocytogenes were recovered from any nis+ATR- samples but four of five nis+ATR+ samples were positive for L. monocytogenes after 30 days. CONCLUSIONS: The ATR confers cross-resistance to nisin for at least 30 days in a system fermented by nisin-producing bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: The cross-resistance induced by the ATR should be considered for the safety of foods fermented with bacteriocin-producing cultures.     

Mutational and inhibitive analysis of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer-based assays

The 3C-like protease (3CL(pro)) of severe acute respiratory syndrome coronavirus (SARS-CoV) plays key roles in viral replication and is an attractive target for anti-SARS drug discovery. In this report, a fluorescence resonance energy transfer (FRET)-based method was developed to assess the proteolytic activity of SARS-CoV 3CL(pro). Two internally quenched fluorogenic peptides, 1NC and 2NC, corresponding to the N-terminal and the C-terminal autocleavage sites of SARS-CoV 3CL(pro), respectively, were used as substrates. SARS-CoV 3CL(pro) seemed to work more efficiently on 1NC than on 2NC in trans-cleavage assay. Mutational analysis demonstrated that the His41 residue, the N-terminal 7 amino acids, and the domain III of SARS-CoV 3CL(pro) were important for the enzymatic activity. Antibodies recognizing domain III could significantly inhibit the enzymatic activity of SARS-CoV 3CL(pro). The effects of class-specific protease inhibitors on the trans-cleavage activity revealed that this enzyme worked more like a serine protease rather than the papain protease.     

Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe

A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.     

Detection of Listeria monocytogenes by direct colony hybridization on hydrophobic grid-membrane filters by using a chromogen-labeled DNA probe.

A DNA probe specific for Listeria monocytogenes was isolated from a beta-hemolytic recombinant clone of an L. monocytogenes gene bank. It was labeled with horseradish peroxidase and used in a direct colony hybridization method on hydrophobic grid-membrane filters for the detection of the organism. Following color development of the chromogen, a commercial counter (HGMF Interpreter) was able to detect and count the organisms electronically. The method gave a positive reaction with 70 L. monocytogenes strains, while showing a negative reaction with 10 strains of other Listeria spp. and with 20 organisms of other genera.