Dataset Information

Early detection of Trichinella spiralis in muscle of infected mice by real-time fluorescence resonance energy transfer PCR.

ABSTRACT: 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.

SUBMITTER: Tantrawatpan C

PROVIDER: S-EPMC3777295 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Early detection of Trichinella spiralis in muscle of infected mice by real-time fluorescence resonance energy transfer PCR.

Tantrawatpan Chairat C Intapan Pewpan M PM Thanchomnang Tongjit T Sanpool Oranuch O Janwan Penchom P Boonmars Thidarut T Morakote Nimit N Maleewong Wanchai W

Vector borne and zoonotic diseases (Larchmont, N.Y.) 20130629 9

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 ...[more]

PMID: 23808975

Similar Datasets

Project description:The present study compares the immunogenic patterns of muscle larvae excretory-secretory proteins (ML E-S) from T. spiralis and T. britovi recognized by Trichinella-infected human sera. Samples were analyzed using two-dimensional electrophoresis (2-DE) coupled with 2D-immunoblot and liquid chromatography-tandem mass spectrometry LC-MS/MS analysis, two ELISA procedures and a confirmatory 1D-immunoblot test. Sera were obtained from nine patients with a history of ingestion of raw or undercooked meat who presented typical clinical manifestations of trichinellosis and from eleven healthy people. Specific anti-Trichinella IgG antibodies were detected in all samples tested with the Home-ELISA kits, but in only four samples for the commercially-available kit. The 1D-immunoblot results indicated that all nine serum samples were positive for T. spiralis ML E-S antigens, expressed as the presence of specific bands. In contrast, eight of the serum samples with T. britovi E-S ML antigens were positive, with one serum sample taken from a patient at 33dpi (days post infection) being negative. To identify immunoreactive proteins that are specifically recognized by host antibodies, both species of ML E-S proteins were subjected to 2D-immunoblotting with human serum taken at 49 dpi. The sera recognized 22 protein spots for T. spiralis and 18 for T. britovi in 2D-immunoblot analysis. Their molecular weights (MW) ranged from 50 to 60 kDa. LC-MS/MS analysis identified both common and specifically-recognized immunoreactive proteins: transmembrane serine protease 9, serine protease, antigen targeted by protective antibodies and Actin-1 partial were shared for both Trichinella species; hypothetical protein T01_7775 and P49 antigen, partial those specific to T. spiralis; deoxyribonuclease-2-alpha and hypothetical protein T03_17187/T12_7360 were specific to T. britovi. Our results demonstrate the value of 2-DE and 2D-immunblot as versatile tools for pinpointing factors contributing to the parasite-host relationship by comparing the secretomes of different Trichinella species.

Project description:BackgroundToxoplasmosis is an infectious disease caused by the parasitic protozoan Toxoplasma gondii. It is endemic worldwide and, depending on the geographic location, 15 to 85% of the human population are asymptomatically infected. Routine diagnosis is based on serology. The parasite has emerged as a major opportunistic pathogen for immunocompromised patients, in whom it can cause life-threatening disease. Moreover, when a pregnant woman develops a primary Toxoplasma gondii infection, the parasite may be transmitted to the fetus and cause serious damage. For these two subpopulations, a rapid and accurate diagnosis is required to initiate treatment. Serological diagnosis of active infection is unreliable because reactivation is not always accompanied by changes in antibody levels, and the presence of IgM does not necessarily indicate recent infection. Application of quantitative PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii DNA in amniotic fluid, blood, tissue samples, and cerebrospinal fluid.MethodsTwo separate, real-time fluorescence PCR assays were designed and evaluated with clinical samples. The first, targeting the 35-fold repeated B1 gene, and a second, targeting a newly described multicopy genomic fragment of Toxoplasma gondii. Amplicons of different intragenic copies were analyzed for sequence heterogeneity.ResultsComparative LightCycler experiments were conducted with a dilution series of Toxoplasma gondii genomic DNA, 5 reference strains, and 51 Toxoplasma gondii-positive amniotic fluid samples revealing a 10 to 100-fold higher sensitivity for the PCR assay targeting the newly described 529-bp repeat element of Toxoplasma gondii.ConclusionWe have developed a quantitative LightCycler PCR protocol which offer rapid cycling with real-time, sequence-specific detection of amplicons. Results of quantitative PCR demonstrate that the 529-bp repeat element is repeated more than 300-fold in the genome of Toxoplasma gondii. Since individual intragenic copies of the target are conserved on sequence level, the high copy number leads to an ultimate level of analytical sensitivity in routine practice. This newly described 529-bp repeat element should be preferred to less repeated or more divergent target sequences in order to improve the sensitivity of PCR tests for the diagnosis of toxoplasmosis.

Project description:Conjugated polydiacetylene (PDA) possessing stimuli-responsive properties has been intensively investigated for developing efficient sensors. We report here fluorescence resonance energy transfer (FRET) in liposomes synthesized using different molar ratios of dansyl-tagged diacetylene and diacetylene-carboxylic acid monomers. Photopolymerization of diacetylene resulted in cross-linked PDA liposomes. We used steady-state electronic absorption, emission, and fluorescence anisotropy (FA) analysis to characterize the thermal-induced FRET between dansyl fluorophores (donor) and PDA (acceptor). We found that the monomer ratio of acceptor to donor ( R ad) and length of linkers (functional part that connects dansyl fluorophores to the diacetylene group in the monomer) strongly affected FRET. For R ad = 10 000, the acceptor emission intensity was amplified by more than 18 times when the liposome solution was heated from 298 to 338 K. A decrease in R ad resulted in diminished acceptor emission amplification. This was primarily attributed to lower FRET efficiency between donors and acceptors and a higher background signal. We also found that the FRET amplification of PDA emissions after heating the solution was much higher when dansyl was linked to diacetylene through longer and flexible linkers than through shorter linkers. We attributed this to insertion of dansyl in the bilayer of the liposomes, which led to an increased dansyl quantum yield and a higher interaction of multiple acceptors with limited available donors. This was not the case for shorter and more rigid linkers where PDA amplification was much smaller. The present studies aim at enhancing our understanding of FRET between fluorophores and PDA-based conjugated liposomes. Furthermore, receptor tagged onto PDA liposomes can interact with ligands present on proteins, enzymes, and cells, which will produce emission sensing signal. Therefore, using the present approach, there exist opportunities for designing FRET-based highly sensitive and selective chemical and biochemical sensors.

Dataset Information

Early detection of Trichinella spiralis in muscle of infected mice by real-time fluorescence resonance energy transfer PCR.

Publications

Early detection of Trichinella spiralis in muscle of infected mice by real-time fluorescence resonance energy transfer PCR.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets