Real-time Fluorescence Resonance Energy Transfer Research Articles

Chirality-Dependent Dynamic Evolution of the Protein Corona on the Surface of Quantum Dots.

Elucidating the biological behavior of engineered nanoparticles, for example, the protein corona, is important for the development of safe and efficient nanomedicine, but our current understanding is still limited due to its highly dynamic nature and lack of adequate analytical tools. In the present work, we demonstrate the establishment of a fluorescence resonance energy transfer (FRET)-based platform for monitoring the dynamic evolution behavior of the protein corona in complex biological media. With human serum albumin and lysozyme as the model serum proteins, the protein exchange process of the preformed corona on the surface of chiral quantum dots (QDs) upon feeding either individual protein or human serum was monitored in situ by FRET. Important parameters characterizing the evolution process of protein corona could be obtained upon quantitative analysis of FRET data. Further combining real-time FRET monitoring with gel electrophoresis experiments revealed that the nature of the protein initially adsorbed on the surface of QDs significantly affects the subsequent dynamic exchange behavior of the protein corona. Furthermore, our results also revealed that only a limited proportion of proteins are involved in the protein exchange, and the exchange process exhibits a significant dependence on the surface chirality of QDs. This work demonstrates the feasibility of FRET as a powerful tool to exploit the dynamic evolution process of the protein corona, which can provide theoretical guidance for further design of advanced nanomaterials for biomedical applications.

Simultaneous Real-Time Three-Dimensional Localization and FRET Measurement of Two Distinct Particles.

Many biological processes employ mechanisms involving the locations and interactions of multiple components. Given that most biological processes occur in three dimensions, the simultaneous measurement of three-dimensional locations and interactions is necessary. However, the simultaneous three-dimensional precise localization and measurement of interactions in real time remains challenging. Here, we report a new microscopy technique to localize two spectrally distinct particles in three dimensions with an accuracy (2.35σ) of tens of nanometers with an exposure time of 100 ms and to measure their real-time interactions using fluorescence resonance energy transfer (FRET) simultaneously. Using this microscope, we tracked two distinct vesicles containing t-SNAREs or v-SNARE in three dimensions and observed FRET simultaneously during single-vesicle fusion in real time, revealing the nanoscale motion and interactions of single vesicles in vesicle fusion. Thus, this study demonstrates that our microscope can provide detailed information about real-time three-dimensional nanoscale locations, motion, and interactions in biological processes.

Real-Time Fluorescence-Based Approaches to Disentangle Mechanisms of a Protein's RNA Chaperone Activity.

RNA-binding proteins with an RNA chaperone activity exert either one or both of the following catalytic activities: (1) RNA annealing, i.e., the protein supports intra- as well as intermolecular RNA-RNA interactions and (2) strand displacement, i.e., the protein mediates the exchange of individual strands of a preexisting RNA duplex. To discriminate and further characterize these activities, it requires defined assay systems. These are based on entirely or partially complementary RNA oligonucleotides that are labeled with fluorescent and/or quencher dyes. The non-catalyzed and the protein-supported associations of the RNA molecules are followed by a real-time fluorescence resonance energy transfer (FRET) system. By site-specific modification of the RNAs or the protein, the substrate- and protein-specific parameters of the RNA chaperone activity can be explored and identified.In this chapter, we present strategies on the design of labeled RNA molecules to be used to characterize the activities of an RNA-binding protein and explain how to monitor progress curves of RNA annealing and strand displacement reactions in single cuvette or well-plate scales. We provide sets of equations and models to determine and analyze different types of reactions, e.g., by calculation of first- and second-order rate constants. Likewise, we demonstrate how to exploit these simple experimental setups to elucidate elementary principles of the reaction mechanisms performed by the protein of interest by applying basic kinetic applications, such as ARRHENIUS and linear free energy relationship analyses. These approaches will be explained by providing example plots and graphs from experiments investigating the RNA chaperone activities of the RNA-binding proteins NF90-NF45 and AUF1 p45.

Molecular detection of mutations involved in Helicobacter pylori antibiotic resistance in Algeria.

In Algeria, there are limited data regarding the pattern of Helicobacter pylori primary antibiotic resistance. The aim of this study was to evaluate the primary resistance of H. pylori to clarithromycin, ciprofloxacin, tetracycline and rifampicin and to determine the molecular mechanisms involved in the resistance. Two hundred and seventy Algerian adults who had never received H. pylori treatment were enrolled in this study. Human biopsies were obtained for culture and antimicrobial susceptibility testing was performed by Etest for clarithromycin, ciprofloxacin, tetracycline and rifampicin. Real-time fluorescence resonance energy transfer (FRET)-PCR was also performed in all cases to assess primary clarithromycin resistance and point mutations involved, real-time PCR was used to detect mutations involved in tetracycline primary resistance and sequencing of the QRDR of gyrA was performed to detect mutations involved in quinolone resistance. No resistance to rifampicin was detected. Resistance to clarithromycin and ciprofloxacin was found in 29.7% and 17.9%, respectively. Results of real-time FRET-PCR showed that A2143G was the most frequent point mutation, A2142C was not found and 42 patients (15.5%) were infected by both resistant and susceptible genotypes. Only two isolates were resistant to tetracycline and exhibited an A926G mutation. Four mutations were found to be responsible for resistance to ciprofloxacin [N87K (44.73%), D91N (23.68%), N87I (18.42%) and D91G (7.89%)]. Local data regarding the primary resistance of H. pylori to clarithromycin, ciprofloxacin, tetracycline and rifampicin and the main genetic mutations involved in the resistance are necessary for a periodic evaluation of antibiotic consumption and new therapeutic strategies in Algeria.

Characterization of MazF-Mediated Sequence-Specific RNA Cleavage in Pseudomonas putida Using Massive Parallel Sequencing.

Under environmental stress, microbes are known to alter their translation patterns using sequence-specific endoribonucleases that we call RNA interferases. However, there has been limited insight regarding which RNAs are specifically cleaved by these RNA interferases, hence their physiological functions remain unknown. In the current study, we developed a novel method to effectively identify cleavage specificities with massive parallel sequencing. This approach uses artificially designed RNAs composed of diverse sequences, which do not form extensive secondary structures, and it correctly identified the cleavage sequence of a well-characterized Escherichia coli RNA interferase, MazF, as ACA. In addition, we also determined that an uncharacterized MazF homologue isolated from Pseudomonas putida specifically recognizes the unique triplet, UAC. Using a real-time fluorescence resonance energy transfer assay, the UAC triplet was further proved to be essential for cleavage in P. putida MazF. These results highlight an effective method to determine cleavage specificity of RNA interferases.

A singleplex real-time fluorescence resonance energy transfer PCR with melting curve analysis for the differential detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs in faeces.

Because the eggs of Paragonimus, Echinostoma and Fasciola are very similar in size and shape, it is difficult to distinguish and accurately identify species by the morphology of their eggs, which is a standard diagnostic method. In this study, a novel assay combining a real-time fluorescence resonance energy transfer PCR and melting curve analysis using one set of primers and fluorophore-labelled hybridization probes specific for the 28S rDNA region was developed for the molecular detection of Paragonimus heterotremus, Echinostoma malayanum and Fasciola gigantica eggs. This assay could detect and distinguish P. heterotremus, E. malayanum and F. gigantica DNA with the distinct melting temperature (Tm) values of 57.99±0.08, 62.12±0.15 and 74.10±0.18, respectively. The assay can also be used to detect and distinguish DNA from P. bangkokensis, P. harinasutai, P. machorchis, E. revolutum, Hypodereum conoideum and F. hepatica, which have different Tm values. The sensitivity of this assay enabled the detection of one egg of P. heterotremus, E. malayanum or F. gigantica per 100 mg of faeces. In addition, the specificity testing showed no fluorescence signal for other parasites. Due to the sensitivity and specificity of our assay in detecting P. heterotremus, E. malayanum and F. gigantica, our method could be used to accurately diagnose these three medically important parasitic groups and has potential implications for molecular epidemiological investigations of human and/or animal infections.

New real-time-PCR method to identify single point mutations in hepatitis C virus.

AIMTo develop a fast, low-cost diagnostic strategy to identify single point mutations in highly variable genomes such as hepatitis C virus (HCV).METHODSIn patients with HCV infection, resistance-associated amino acid substitutions within the viral quasispecies prior to therapy can confer decreased susceptibility to direct-acting antiviral agents and lead to treatment failure and virological relapse. One such naturally occurring mutation is the Q80K substitution in the HCV-NS3 protease gene, which confers resistance to PI inhibitors, particularly simeprevir. Low-cost, highly sensitive techniques enabling routine detection of these single point mutations would be useful to identify patients at a risk of treatment failure. LightCycler methods, based on real-time PCR with sequence-specific probe hybridization, have been implemented in most diagnostic laboratories. However, this technique cannot identify single point mutations in highly variable genetic environments, such as the HCV genome. To circumvent this problem, we developed a new method to homogenize all nucleotides present in a region except the point mutation of interest.RESULTSUsing nucleotide-specific probes Q, K, and R substitutions at position 80 were clearly identified at a sensitivity of 10% (mutations present at a frequency of at least 10% were detected). The technique was successfully applied to identify the Q80K substitution in 240 HCV G1 serum samples, with performance comparable to that of direct Sanger sequencing, the current standard procedure for this purpose. The new method was then validated in a Catalonian population of 202 HCV G1-infected individuals. Q80K was detected in 14.6% of G1a patients and 0% of G1b in our setting.CONCLUSIONA fast, low-cost diagnostic strategy based on real-time PCR and fluorescence resonance energy transfer probe melting curve analysis has been successfully developed to identify single point mutations in highly variable genomes such as hepatitis C virus. This technique can be adapted to detect any single point mutation in highly variable genomes.

Abstract 12646: Oxidation-Dependent Phosphomimetic Effect of a Human Heart Failure Mutation of Phospholamban

Rationale: A naturally-occurring, missense Arg9-to-Cys (R9C) mutation of phospholamban (PLB) triggers cardiomyopathy and premature death in humans. However, the fundamental molecular mechanism underlying the cardiotoxic role of R9C-PLB in sarco/endoplasmic reticulum Ca 2+ -ATPase (SERCA) regulation and cardiomyocyte Ca 2+ handling is not clear. Objective: The goal of this study was to investigate the acute physiological consequences of R9C-PLB mutation on cardiomyocyte Ca 2+ kinetics and contractility and identify the molecular mechanism underlying R9C pathology. Methods and Results: We measured the physiological consequences of R9C-PLB mutation on Ca 2+ transients and sarcomere shortening in adult cardiomyocytes at increasing pacing frequencies. In contrast to studies of chronic R9C-PLB expression in transgenic mice, we found that acute expression of R9C-PLB exerts a positively inotropic and lusitropic effect in cardiomyocytes. Importantly, R9C-PLB exhibited blunted sensitivity to frequency potentiation and β-adrenergic stimulation, two major physiological mechanisms for the regulation of cardiac performance. To identify the molecular mechanism of R9C pathology, we fused fluorescent protein tags to PLB and SERCA, and compared the effect of R9C and pentamer-destabilizing mutation (SSS) on PLB oligomerization and PLB-SERCA interaction. Fluorescence resonance energy transfer (FRET) measurements in live cells revealed that R9C exhibited an increased affinity of PLB oligomerization, and a decreased binding affinity to SERCA due to an oxidative modification which mimics phosphorylation. Real-time FRET analysis in cardiomyocytes revealed that R9C-PLB exhibits enhanced sensitivity to oxidative stress, which is a prevailing condition in heart failure. Conclusions: We conclude that the primary mechanism of R9C pathology is a phosphomimetic effect of PLB cysteine oxidation, manifested as increased oligomerization and a change in the structure of the PLB-SERCA regulatory complex.

Adenosine A2A Receptor Ligand Binding Experiments by Using Real-time Single-cell FRET

We designed a fluorescence resonance energy transfer (FRET)-based approach to study the ligand binding constants of the adenosine A2A receptor (A2AR). Our assay is based in the interaction of a fluorescent A2AR agonist ligand (MRS5424) with an A2AR tagged with the cyan fluorescent protein (CFP) at the N-terminus (i.e. A2ARCFP) and expressed in living cells. Thus, upon fast superfusion of the A2ARCFP expressing cells with MRS5424, the ligand-receptor interaction is determined by single-cell FRET in a real-time mode. Accordingly, our approach allowed immediate ‘real-time’ readout of the ligand-receptor interaction, thus allowing kinetic binding experiments, a feature impossible to achieve using conventional radioisotope-labelled ligands. In addition, since our assay permitted the visual confirmation of receptor localization it also allowed localized saturation binding experiments.

Molecular diagnosis of Arcobacter and Campylobacter in diarrhoeal samples among Portuguese patients

The present study was conducted to investigate the prevalence and diversity of Arcobacter and Campylobacter spp. in 298 stool samples of patients with diarrhoea, collected from 22 Portuguese hospitals, between September and November 2012. Detection of Arcobacter and Campylobacter spp. was performed using molecular-based detection techniques, such as real-time fluorescence resonance energy transfer PCR, species-specific PCR, and sequencing of amplified PCR products. Overall, 1.3% of the samples were positive for Arcobacter butzleri and 0.3% for Arcobacter cryaerophilus. Campylobacter spp. were found in 31.9% of diarrhoeic faeces. Campylobacter jejuni and Campylobacter concisus were the most prevalent species (13.7% and 8.0%, respectively). The prevalence of Arcobacter and Campylobacter spp. was significantly different between children and adults (39.7% versus 22.8%, P = 0.003). We underline the high prevalence of these pathogens in diarrhoeal samples among Portuguese patients, with particular relevance in the paediatric age group.

Application of a real-time fluorescence resonance energy transfer polymerase chain reaction assay with melting curve analysis for the detection of Paragonimus heterotremus eggs in the feces of experimentally infected cats

Paragonimus heterotremus is a medically important lung fluke that causes human and animal paragonimiasis in Southeast Asia, including Thailand. In the current study, a real-time fluorescence resonance energy transfer polymerase chain reaction (real-time FRET PCR) with melting curve analysis was developed and evaluated to detect P. heterotremus eggs in the feces of experimentally infected cats. The detection limit of this method for the P. heterotremus DNA sequence was 3 × 10(2) copies of the positive control plasmid and 10(-3) ng of P. heterotremus genomic DNA. The assay system could detect 10 eggs of P. heterotremus per gram of cat feces. No fluorescence signal was observed when DNA purified from 16 other organisms or genomic DNA from cats and human beings were tested. Real-time FRET PCR yielded positive results for all fecal samples from 17 P. heterotremus-infected cats and showed a negative relationship (r = -0.852, P < 0.001) between the number of parasite eggs in feces and the number of PCR cycles. The assay could detect genomic DNA from P. heterotremus, P. westermani, P. macrorchis, P. siamensis, P. harinasutai, and P. bangkokensis and can differentiate P. heterotremus from the other 5 species. The 6 Paragonimus species examined were divided into 4 groups by melting peak analysis. This assay can be useful for the detection of, and epidemiological studies on, P. heterotremus infection in endemic areas.

Early Detection ofTrichinella spiralisin Muscle of Infected Mice by Real-Time Fluorescence Resonance Energy Transfer PCR

Real-time fluorescence resonance energy transfer (FRET) PCR and melting curve analysis using newly developed fluorophore-labeled hybridization probes were applied for the detection of Trichinella spiralis DNA in muscle of mice following oral inoculation with 300 T. spiralis larvae. The developed assay could detect and differentiate T. spiralis, Trichinella papuae, and Trichinella pseudospiralis DNAs by the different melting temperatures (Tm). The assay had a detection limit of 5 × 10(2) positive control plasmid copies, which was equivalent to 1 ng of T. spiralis DNA spiked into 250 mg of muscle sample. No fluorescence signal was detected when the technique was applied to the DNA of 27 parasites other than Trichinella spp. The assay could detect T. spiralis DNA in muscle at 7, 14, and 21 days postinoculation. The range, mean ± standard deviation, and median of the Tm values of all positive muscle tissue samples were 60.4-60.8, 60.6 ± 0.2, and 60.5, respectively. This assay provides an effective tool for the specific, sensitive, and high-throughput detection of T. spiralis DNA in muscle during the early stage of infection. In addition, the technique can be useful for epidemiologic surveillance in naturally infected wildlife.

Dopamine D2 receptor-mediated modulation of adenosine A2A receptor agonist binding within the A2AR/D2R oligomer framework

The molecular interaction between adenosine A2A and dopamine D2 receptors (A2ARs and D2Rs, respectively) within an oligomeric complex has been postulated to play a pivotal role in the adenosine–dopamine interplay in the central nervous system, in both normal and pathological conditions (e.g. Parkinson’s disease). While the effects of A2AR challenge on D2R functioning have been largely studied, the reverse condition is still unexplored, a fact that might have impact in therapeutics. Here, we aimed to examine in a real-time mode the D2R-mediated allosteric modulation of A2AR binding when an A2AR/D2R oligomer is established. Thus, we synthesized fluorescent A2AR agonists and evaluated, by means of a flow cytometry homogeneous no-wash assay and a real-time fluorescence resonance energy transfer (FRET)-based approach, the effects on A2AR binding of distinct antiparkinsonian drugs in current clinical use (i.e. pramipexole, rotigotine and apomorphine). Our results provided evidence for the existence of a differential D2R-mediated negative allosteric modulation on A2AR agonist binding that was oligomer-formation dependent, and with apomorphine being the best antiparkinsonian drug attenuating A2AR agonist binding. Overall, the here-developed methods were found valid to explore the ability of drugs acting on D2Rs to modulate A2AR binding, thus serving to facilitate the preliminary selection of D2R-like candidate drugs in the management of Parkinson’s disease.

Real-time detection of cellular death receptor-4 activation by fluorescence resonance energy transfer

Targeted therapy involving the activation of death receptors DR4 and/or DR5 by its ligand, TRAIL, can selectively induce apoptosis in certain tumor cells. In order to profile the dynamic activation or trimerization of TRAIL-DR4 in live cells in real-time, the development of an apoptosis reporter cell line is essential. Fluorescence resonance energy transfer (FRET) technology via a FRET pair, cyan fluorescence protein (CFP) and yellow fluorescence protein (YFP), was used in this study. DR4-CFP and DR4-YFP were stably expressed in human lung cancer PC9 cells. Flow cytometer sorting and limited dilution coupled with fluorescence microscopy were used to select a monoclonal reporter cell line with high and compatible expression levels of DR4-CFP and DR4-YFP. FRET experiments were conducted and FRET efficiencies were monitored according to the Siegel's YFP photobleaching FRET protocol. Upon TRAIL induction a significant increase in FRET efficiencies from 5% to 9% demonstrated the ability of the DR4-CFP/YFP reporter cell line in monitoring the dynamic activation of TRAIL pathways. 3D reconstructed confocal images of DR4-CFP/YFP reporter cells exhibited a colocalized expression of DR4-CFP and DR4-YFP mainly on cell membranes. FRET results obtained during this study complements the use of epi-fluorescence microscopy for FRET analysis. The real-time FRET analysis allows the dynamic profiling of the activation of TRAIL pathways by using the time-lapse fluorescence microscopy. Therefore, DR4-CFP/YFP PC9 reporter cells along with FRET technology can be used as a tool for anti-cancer drug screening to identify compounds that are capable of activating TRAIL pathways.

Abstract 18309: Familial Atrial Fibrillation - Common Atrial Fibrillation Susceptibility Alleles and Response to Catheter Ablation

Background: Familial atrial fibrillation (AF) is common but AF-causing rare genetic variants are infrequently found supporting the multigenetic nature of this arrhythmia. On the other hand, genome-wide association studies have identified common AF susceptibility alleles, but their possible relation with familial AF is unknown. The aim of this study was to compare familial and sporadic AF with respect to common alleles on chromosomes 1q21 (rs13376333), 4q25 (rs10033464 and rs2200733) and 16q22 (rs7193343) and rhythm outcome after radiofrequency catheter ablation (CA). Methods: A total of 393 patients (mean age 59 +/- 10 years, 68 % males, 38 % persistent AF), who underwent CA were studied. The family history of AF was considered positive if at least one of the first-degree family members had AF based on patient's interviews, chart and ECG reviews. Genotypes were determined using real-time polymerase chain reaction and fluorescence resonance energy transfer. AF recurrence &gt; 30 sec was detected using serial 7-day Holter ECG recordings after a blanking period between 3 and 12 months after radiofrequency CA. Results: Familial AF was found in 14.2 % and was associated with the absence of structural heart disease (OR 1.888, 95 % CI 1.017 - 3.505, p=0.042) and age (OR 0.964, 95 % CI 0.938 - 0.991, p=0.009). In familial AF, minor allele frequency of AF risk alleles on chromosomes 1q21 (40.0 % vs. 32.8 %) and 4q25 (rs10033464: 26.4 % vs. 11.0 % and rs2200733: 39.4 % vs. 27.2 %) tended to be higher compared with non-familial AF. Using a dominant genetic model, rs2200733 on chromosome 4q25 showed the strongest association with familial AF (OR 2.222, 95 % CI 1.015 - 4.865, p=0.042). AF recurrence was observed in 27.6 % of non-familial AF and in 30.9 % of familial AF patients (p=0.621). Conclusion: This study, for the first time, shows a dominant modulating function of a common AF susceptibility allele on chromosome 4q25 also in familial AF. Ablation outcome is comparable in familial and non-familial AF highlighting the role of the pulmonary veins in AF development.

Kinetics of Precursor Interactions with the Bacterial Tat Translocase Detected by Real-time FRET

The Escherichia coli twin-arginine translocation (Tat) system transports fully folded and assembled proteins across the inner membrane into the periplasmic space. Traditionally, in vitro protein translocation studies have been performed using gel-based transport assays. This technique suffers from low time resolution, and often, an inability to distinguish between different steps in a continuously occurring translocation process. To address these limitations, we have developed an in vitro FRET-based assay that reports on an early step in the Tat translocation process in real-time. The natural Tat substrate pre-SufI was labeled with Alexa532 (donor), and the fluorescent protein mCherry (acceptor) was fused to the C terminus of TatB or TatC. The colored Tat proteins were easily visible during purification, enabling identification of a highly active inverted membrane vesicle (IMV) fraction yielding transport rates with NADH almost an order of magnitude faster than previously reported. When pre-SufI was bound to the translocon, FRET was observed for both Tat proteins. FRET was diminished upon addition of nonfluorescent pre-SufI, indicating that the initial binding step is reversible. When the membranes were energized with NADH, the FRET signal was lost after a short delay. These data suggest a model in which a Tat cargo initially associates with the TatBC complex, and an electric field gradient is required for the cargo to proceed to the next stage of transport. This cargo migration away from the TatBC complex requires a significant fraction of the total transport time.

Constitutive K-RasG12D Activation of ERK2 Specifically Regulates 3D Invasion of Human Pancreatic Cancer Cells via MMP-1

Pancreatic ductal adenocarcinomas (PDAC) are highly invasive and metastatic neoplasms commonly unresponsive to current drug therapy. Overwhelmingly, PDAC harbors early constitutive, oncogenic mutations in K-Ras(G12D) that exist prior to invasion. Histologic and genetic analyses of human PDAC biopsies also exhibit increased expression of extracellular signal-regulated kinase (ERK) 1/2 and proinvasive matrix metalloproteinases (MMP), indicators of poor prognosis. However, the distinct molecular mechanisms necessary for K-Ras/ERK1/2 signaling and its influence on MMP-directed stromal invasion in primary human pancreatic ductal epithelial cells (PDEC) have yet to be elucidated in three-dimensions. Expression of oncogenic K-Ras(G12D) alone in genetically defined PDECs reveals increased invadopodia and epithelial-to-mesenchymal transition markers, but only when cultured in a three-dimensional model incorporating a basement membrane analog. Activation of ERK2, but not ERK1, also occurs only in K-Ras(G12D)-mutated PDECs cultured in three-dimensions and is a necessary intracellular signaling event for invasion based upon pharmacologic and short hairpin RNA (shRNA) inhibition. Increased active invasion of K-Ras(G12D) PDECs through the basement membrane model is associated with a specific microarray gene expression signature and induction of MMP endopeptidases. Specifically, MMP-1 RNA, its secreted protein, and its proteolytic cleavage activity are amplified in K-Ras(G12D) PDECs when assayed by real-time quantitative PCR, ELISA, and fluorescence resonance energy transfer (FRET). Importantly, shRNA silencing of MMP-1 mimics ERK2 inhibition and disrupts active, vertical PDEC invasion. ERK2 isoform and MMP-1 targeting are shown to be viable strategies to attenuate invasion of K-Ras(G12D)-mutated human pancreatic cancer cells in a three-dimensional tumor microenvironment.

Rapid detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples using a duplex real-time fluorescence resonance energy transfer PCR and melting curve analysis

We developed a single step duplex real-time fluorescence resonance energy transfer (FRET) PCR merged with melting curve analysis for the fast detection and differentiation of Clonorchis sinensis and Opisthorchis viverrini eggs in human fecal samples. Two species of mitochondrial NADH dehydrogenase subunit 2 (nad2) DNA elements, the 165-bp nad2 product of C. sinensis and the 209-bp nad2 product of O. viverrini, were amplified by species-specific primers, and the fluorescence melting curve analyses were generated from hybrid of amplicons and two pairs of species-specific fluorophore-labeled probes. By their different fluorescence channels and melting temperatures, both C. sinensis and O. viverrini eggs in infected human fecal samples were detected and differentiated with high (100%) sensitivity and specificity. Detection limit was as little as a single C. sinensis egg and two O. viverrini eggs in 100 mg of fecal sample. The assay could distinguish the DNA of both parasites from the DNA of negative fecal samples and fecal samples with other parasitosis, as well as from the well-defined genomic DNA of human leukocytes and other parasites. It can reduce labor time of microscopic examination and is not prone to carry over contamination of agarose electrophoresis. Our duplex real-time FRET PCR method would be useful to determine the accurate range of endemic areas and/or to discover the co-endemic areas of two liver flukes, C. sinensis and O. viverrini, in Asia. This method also would be helpful for the differential diagnosis of the suspected cases of liver fluke infections among travelers who had visited the endemic countries of those parasites.

Real-time PCR improve detection of Trichomonas vaginalis compared to conventional techniques

Trichomonas vaginalis is the cause of one of the most common types of vaginitis, trichomoniasis. Clinical manifestations of symptomatic women are generally nonspecific, but include vaginal discharge, vaginitis and irritation. T. vaginalis infection has also been linked to the increased risk of human immunodeficiency virus transmission. The aim of the study was to compare a real-time polymerase chain reaction (PCR) assay with culture and wet-mount examination for the detection of T. vaginalis. This is a descriptive analytical study. Vaginal swabs from 504 female patients with suspected vaginitis were included in the study. They were subjected to culture, wet-mount microscopic examination (WM), and real-time PCR. Real-time fluorescence resonance energy transfer hybridization probe PCR assays were used in this study to test for T. vaginalis DNA, targeting the b-tubulin genes. The result of the PCR was compared with culture and wet-mount microscopy. WM were positive for T. vaginalis in 60/504 cases (11.9%) and cultures were positive for T. vaginalis in 116/504 cases (23%). Real-time PCR was done on 50/504 specimens which was randomly chosen, 33/50 (66%) cases were positive for T. vaginalis (78% sensitivity), 25/50 (50%) of them were culture and wet-mount examination positive. Of them, 1/33 (3%) were culture positive and wet examination negative, 7/33 (21%) real-time positive were negative by culture while 17/50 (34%) real-time PCR negative cases were also negative by both techniques. The time taken for PCR assay was 4 to 8 h whereas positive protozoal cultures took up to 4 days. The Real-time PCR done in this study, provide a rapid, sensitive and specific diagnosis of T. vaginalis infection.

Molecular detection of Schistosoma japonicum in infected snails and mouse faeces using a real-time PCR assay with FRET hybridisation probes

A real-time polymerase chain reaction (PCR) assay with fluorescence resonance energy transfer (FRET) hybridisation probes combined with melting curve analysis was developed to detect Schistosoma japonicum in experimentally infected snails and in faecal samples of infected mice. This procedure is based on melting curve analysis of a hybrid between an amplicon from the S. japonicum internal transcribed spacer region 2 sequence, which is a 192-bp S. japonicum-specific sequence, and fluorophore-labelled specific probes. Real-time FRET PCR could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails and a single egg inoculated in 100 mg of non-infected mouse faeces. All S. japonicum-infected snails and all faecal samples from infected mice were positive. Non-infected snails, non-infected mouse faeces and genomic DNA from other parasites were negative. This assay is rapid and has potential for epidemiological S. japonicum surveys in snails, intermediate hosts and faecal samples of final hosts.