Molecular beacon

Nanoparticles (NPs) are playing a progressively more significant role in multimodal and multifunctional molecular imaging. The agents like Superparamagnetic iron oxide (SPIO), manganese oxide (MnO), gold NPs/nanorods and quantum dots (QDs) possess specific properties like paramagnetism, superparamag-netism, surface plasmon resonance (SPR) and photoluminescence respectively. These specific properties make them able for single/multi-modal and single/multi-functional molecular imaging. NPs generally have nanomolar or micromolar sensitivity range and can be detected via imaging instrumentation. The distinctive characteristics of these NPs make them suitable for imaging, therapy and delivery of drugs. Multifunctional nanoparticles (MNPs) can be produced through either modification of shell or surface or by attaching an affinity ligand to the nanoparticles. They are utilized for targeted imaging by magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), positron emission tomography (PET), computed tomography (CT), photo acoustic imaging (PAI), two photon or fluorescent imaging and ultra sound etc. Toxicity factor of NPs is also a very important concern and toxic effect should be eliminated. First generation NPs have been designed, developed and tested in living subjects and few of them are already in clinical use. In near future, molecular imaging will get advanced with mul-timodality and multifunctionality to detect diseases like cancer, neurodegenerative diseases, cardiac diseases , inflammation, stroke, atherosclerosis and many others in their early stages. In the current review, we discussed single/multifunctional nanoparticles along with molecular imaging modalities. Statement of Significance The present article intends to reveal recent avenues for nanomaterials in multimodal and multifunctional molecular imaging through a review of pertinent literatures. The topic emphasises on the distinctive characteristics of nanomaterial which makes them, suitable for biomedical imaging, therapy and delivery of drugs. This review is more informative of indicative technologies which will be helpful in a way to plan, understand and lead the nanotechnology related work.

Pork identification in raw meat using real-time polymerase chain reaction (PCR) was developed. Total DNA from meat samples were successfully extracted and found to be of high quality and produced clear PCR products. Porcine-specific molecular beacon probe and primers that amplifies 119 bp of the cytochrome b gene fragment of swine (Sus scrofa domestica) was used. Analysis of data showed that the C q (quantification cycle) from 10 ng/μl porcine DNA is (18.70 ± 0.12 to 19.08 ± 0.06). Meanwhile, the other samples exhibited negative result, which confirmed the specificity of the primers. The method also showed that the limit of detection of pork was 0.0001 ng. Based on the regression analysis of the standard curve, the 96% efficiency of real-time PCR was achieved with high correlation coefficient (r 2 = 0.9989). Sensitivity of the assay in discriminating pork as low as 0.1% (w/w) pork in pork–beef mixtures was also obtained. Reproducibility of the assay was successfully validated by applying sample and experimental replicates in every assay being conducted. Thus, this methodology could serve as a fast and sensitive method for detection of pork for meat species verification.

Aptamers are artificial nucleic acid ligands, specifically generated against certain targets, such as amino acids, drugs, proteins or other molecules. In nature they exist as a nucleic acid based genetic regulatory element called a riboswitch. For generation of artificial ligands, they are isolated from combinatorial libraries of synthetic nucleic acid by exponential enrichment, via an in vitro iterative process of adsorption, recovery and reamplification known as systematic evolution of ligands by exponential enrichment (SELEX). Thanks to their unique characteristics and chemical structure, aptamers offer themselves as ideal candidates for use in analytical devices and techniques. Recent progress in the aptamer selection and incorporation of aptamers into molecular beacon structures will ensure the application of aptamers for functional and quantitative proteomics and high-throughput screening for drug discovery, as well as in various analytical applications. The properties of aptamers as well as recent developments in improved, time-efficient methods for their selection and stabilization are outlined. The use of these powerful molecular tools for analysis and the advantages they offer over existing affinity biocomponents are discussed. Finally the evolving use of aptamers in specific analytical applications such as chromatography, ELISA-type assays, biosensors and affinity PCR as well as current avenues of research and future perspectives conclude this review.

Laboratory diagnosis of viral respiratory infections is generally performed by virus isolation in cell culture and immunofluorescent assays. Reverse transcriptase PCR is now recognized as a sensitive and specific alternative for detection of respiratory RNA viruses. A rapid real-time multiplex PCR assay was developed for the detection of influenza A and influenza B viruses, human respiratory syncytial virus (RSV), parainfluenza

In this study, a molecular beacon-based duplex real-time PCR assay was developed for the simultaneous detection of Listeria monocytogenes and Salmonella spp. by targeting their virulence genes hly and invA, respectively. The detection sensitivity of the assay in reconstituted non-fat dried milk (11%) was 3 and 4 log cfu mL−1 for L. monocytogenes and Salmonella spp., respectively, without any pre-enrichment. However, after pre-enrichment of the samples in brain heart infusion broth for 6 h, the assay could detect as low as 1 log cfu of both the pathogens. The assay was quantifiable for the respective pathogens over 5 and 4 log cfu mL−1 with regression coefficient of 0.9956 and 0.9905. On application of the developed assay on 60 dairy products, one sample of raw milk was positive for L. monocytogenes, while Salmonella spp. was detected in one sample of ice cream. The performance of the duplex assay was validated using microbiological methods and commercial individual pathogen detection kits, which further affirmed the performance of the assay by positively detecting the same samples. The developed assay can be used to monitor the quality of dairy products in context of L. monocytogenes and Salmonella spp. 本研究以李斯特单胞菌毒力基因 hly 和沙门氏菌的毒力基因 invA 为靶点, 建立了同时检测上述两种病原菌的分子信标-双重实时PCR (qPCR) 的分析方法。该方法对未经富集的还原脱脂乳 (11%) 中李斯特单胞菌和沙门氏菌的检测灵敏度分别是1×103和1×104cfu mL-1。当样品在脑心浸液肉汤 (BHI) 培养基中富集培养6小时候, 该方法对两种病原菌的最低检测线可达1×101cfu mL-1。该方法对李斯特单胞菌和沙门氏菌两种致病菌的定量范围分别为1×105和1×104, 线性回归系数分别为0.9956和0.9905。采用该方法检对60个乳制品样品进行了检测, 其中一份鲜奶样品中检测出李斯特单胞菌, 而在一份冰激凌样品中检测出沙门氏菌。根据微生物学方法和商业生产的试剂盒法的验证结果, 证明本研究建立的双重实时 PCR 方法是有效的, 可以通过检测李斯特单胞菌和沙门氏菌来监控乳制品的质量。

Multiphoton tomography for in vivo high-resolution multidimensional imaging has been used in clinical investigations and small animal studies. The novel femtosecond laser tomographs have been employed to detect cosmetics and pharmaceutical components in situ as well as to study the interaction of drugs with intratissue cells and the extracellular matrix under physiological conditions. Applications include the intra-tissue accumulation of sunscreen

Molecular beacons (MBs) are specifically designed DNA hairpin structures that are widely used as fluorescent probes. Applications of MBs range from genetic screening, biosensor development, biochip construction, and the detection of single-nucleotide polymorphisms to mRNA monitoring in living cells. The inherent signal-transduction mechanism of MBs enables the analysis of target oligonucleotides without the separation of unbound probes. The MB stem–loop structure holds the fluorescence-donor and fluorescence-acceptor moieties in close proximity to one another, which results in resonant energy transfer. A spontaneous conformation change occurs upon hybridization to separate the two moieties and restore the fluorescence of the donor. Recent research has focused on the improvement of probe composition, intracellular gene quantitation, protein–DNA interaction studies, and protein recognition.

Several techniques have been developed for detection and quantification of genetically modified organisms, but quantitative real-time PCR is by far the most popular approach. Among the most commonly used real-time PCR chemistries are TaqMan probes and SYBR green, but many other detection chemistries have also been developed. Because their performance has never been compared systematically, here we present an extensive evaluation of some promising chemistries: sequence-unspecific DNA labeling dyes (SYBR green), primer-based technologies (AmpliFluor, Plexor, Lux primers), and techniques involving double-labeled probes, comprising hybridization (molecular beacon) and hydrolysis (TaqMan, CPT, LNA, and MGB) probes, based on recently published experimental data. For each of the detection chemistries assays were included targeting selected loci. Real-time PCR chemistries were subsequently compared for their efficiency in PCR amplification and limits of detection and quantification. The overall applicability of the chemistries was evaluated, adding practicability and cost issues to the performance characteristics. None of the chemistries seemed to be significantly better than any other, but certain features favor LNA and MGB technology as good alternatives to TaqMan in quantification assays. SYBR green and molecular beacon assays can perform equally well but may need more optimization prior to use.