Short communication

SYBR Green I DNA staining increases the detection sensitivity of viruses by polymerase chain reaction

Microbes are ubiquitously present in nature and their presence in environment or food materials is natural and unavoidable. Presence of microbes in food products may be beneficial or may cause serious threat to health upon consumption. With reference to European Commission (EC), Food and Drug Administration (FDA) and Hazard Analysis Critical Control Point (HACCP) guidelines, detection of illness causing foodborne microbes/pathogens and/or presence of GMOs has become a burning issue that needs to be attended on priority basis. This chapter is an attempt to describe various molecular techniques for detection of foodborne pathogens and GMOs. Emphasis has been made to elaborate basic to next-generation techniques developed related to PCR, microarrays, and DNA sequencing that have achieved unprecedented level of performance in terms of sensitivity, speed, precision, and accuracy.

A series of nanogel particles with the growing cross-linking degree was synthesized in reverse micelles by copolymerization of a cationic monomer N,N-dimethylaminoethylmethacrylate with increasing amounts of N,N′-methylenebisacrylamide (MBA). The growth of MBA content in the studied range of 0.2–15% (mol) resulted in a regular decrease of the particles’ size with a narrowing in their size distribution which occurred at the first of the cross-linker addition. In the second region, where the MBA content was more than 5% (mol), the size distribution was very narrow. The cross-linked polymers prepared at 2–5% of the cross-linker amount demonstrated a minimum size of the polyplexes and enhancement in the transfection with plasmid DNA and small interfering RNA targeted to the luciferase gene. The revealed changes in the properties of nanogels occurring upon the changes in the cross-linking degree appear to be a platform for a controlled manufacturing of efficient cross-linked cationic vectors.

Bovine herpes virus-1 (BoHV-1) is a serious viral pathogen of domestic and wild cattle. Herein, we report development of a new molecular diagnostic assay for rapid and sensitive detection of BoHV-1 utilizing the loop-mediated isothermal amplification (LAMP) technique. BoHV-1-LAMP assay was optimized to amplify the target DNA by incubation the Bst-DNA polymerase enzyme with a set of specially constructed six primers, based on the gE-gene of BoHV-1 virus, at 65 °C for 60 min. BoHV-1-LAMP products were detected by visual inspection using SYBR Green-I stain and had a ladder-like appearance by gel electrophoresis analysis. Negative results obtained with DNA from other tested fish viruses confirmed the specificity of the assay. The analytical sensitivity of the BoHV-1-LAMP assay was 1 fg of BoHV-1 DNA (dilution of 10⁶). The developed assay could successfully detect BoVH-1 DNA from clinical samples. Results of this study indicate that the developed BoHV-1-LAMP is rapid and highly sensitive assay not only for detection of BoHV-1 in clinical samples, but also for differentiation between wild-type (gE-positive) and gE-negative BoHV-1 viruses, which will improve the control programs of BoHV-1 in Egypt.

A novel two-step, SYBR Green I based real-time RT-PCR assay was developed for detection and quantification of BCoV using ABI PRISM 7500 sequence detection system. The assay was carried out using two sets of primers designed to amplify highly conserved sequences of the nucleocapsid gene of BCoV and the internal control, bovine glyceraldehyde-3-phosphate dehydrogenase, RNA. Specific identification of both targets was elucidated by melt curve analysis, in which the BCoV amplified product generated a melt peak at 78.35 ± 0.26 °C and the internal control RNA at 82.54 ± 0.32 °C. The assay was highly specific since all negative controls and other viruses of clinical and structural relevance failed to develop any positive results. The detection limit of the reaction was 10³ plasmid copies and 1.17 × 10⁻³ TCID₅₀ of the tissue culture propagated virus. Standard deviation and coefficient of variation was low for both intra-assay and inter-assay variability. The assay performance on field samples was evaluated on 103 (68 fecal and 35 nasal) swab specimens and compared with the conventional RT-PCR assay. The results of both assays matched for the diagnosis of 65 fecal and 33 nasal samples. However, three fecal and two nasal samples tested negative in gel-based assay were positive for the real-time RT-PCR. The robustness and a high-throughput performance of the developed assay make it a powerful tool in diagnostic applications and in BCoV research.

Carbohydrate structures attached to glycoproteins, glycolipids, and proteoglycans play key roles in a variety of biological recognition events. This chapter discusses the range of proteins and activities that are relevant to the broad-based profiling of glycan-related gene expression. The development of tools to determine the presence and abundance of transcripts encoding the full spectrum of glycan-related proteins in cells and tissues is the first step in the broader goal of determining the global regulation of glycan structures in animal systems. The techniques for detection of transcript abundance in biological samples can be categorized as hybridization, sequence, or amplification-based methods. An overview of each method along with their benefits and drawbacks is presented with examples of information gained by the application of these techniques within the field of glycobiology. Broad-based transcriptional profiling of glycan-related genes provides insights into the global regulation of glycan synthesis and degradation in animal systems. The relevant pathways provide a frame of reference for the scope of target genes that need to be profiled in order to fully understand the global regulation of glycan structures in animal systems. These pathways include enzyme systems required for activated sugar precursor biosynthesis and transport, glycan biosynthesis, modification, and catabolism, and glycan recognition by lectins. Cloning strategies are employed to identify the respective glycan-related genes. The use of biochemical pathway information as a bridge between the two disparate data sets is discussed.