Specific discrimination of three pathogenic salmonella enterica subsp enterica serotypes using CarB-based oligonuceotide microarray

It is important to rapidly and selectively detect and analyze pathogenic Salmonella enterica subsp. enterica in contaminated food to reduce the morbidity and mortality of Salmonella infection and to guarantee food safety. In the present work, we developed an oligonucleotide microarray containing duplicate specific capture probes based on the carB gene, which encodes the carbamoyl phosphate synthetase large subunit, as a competent biomarker evaluated by genetic analysis to selectively and efficiently detect and discriminate three S. enterica subsp. enterica serotypes: Choleraesuis, Enteritidis, and Typhimurium. Using the developed microarray system, three serotype targets were successfully analyzed in a range as low as 1.6 to 3.1 nM and were specifically discriminated from each other without nonspecific signals. In addition, the constructed microarray did not have cross-reactivity with other common pathogenic bacteria and even enabled the clear discrimination of the target Salmonella serotype from a bacterial mixture. Therefore, these results demonstrated that our novel carB-based oligonucleotide microarray can be used as an effective and specific detection system for S. enterica subsp. enterica serotypes.open117Nsciescopu

Identification and Characterization of Phytoplankton in Barnegat Bay, New Jersey

Barnegat Bay is a marine estuary that encompasses 670-square miles of the eastern coast of New Jersey. The bay serves as a home to thousands of species of plants, animals, and microorganisms. During the summer months, the population of the bay increases from 576,000 to over 1.5 million residents. In 2011, the Barnegat Bay Partnership reported that environmental conditions of the bay are declining due to increased pollutants from nutrient fertilizers and runoff. Increased nitrogen levels have led to eutrophication and proliferation of phytoplankton algal blooms. Harmful algal blooms (HABs) cause a decrease in dissolved oxygen levels in the water as well as the release of harmful toxins that affect organisms inhabiting the bay, including humans. To research and develop methods to prevent HABs from occurring, 16 Barnegat Bay sites ranging from the northern to southern regions of the bay were studied in order identify species of cyanobacteria, such as Synechococcus and Prochlorococcus, and other phytoplankton such as diatoms and dinoflagellates. Polymerase chain reaction (PCR), microscopy, and flow cytometry were utilized to generate profiles of each site. Collectively, the utilization of the three methods detected phytoplankton species such as Synechococcus, Prochlorococcus, and Cylindrotheca fusiformis. Viral plaque assays using viral lysate from each site detected the presence of cyanophage as a natural control for phytoplankton populations

A rapid staining-assisted wood sampling method for PCR-based detection of pine wood nematode Bursaphelenchus xylophilus in Pinus massoniana wood tissue

For reasons of unequal distribution of more than one nematode species in wood, and limited availability of wood samples required for the PCR-based method for detecting pinewood nematodes in wood tissue of Pinus massoniana, a rapid staining-assisted wood sampling method aiding PCR-based detection of the pine wood nematode Bursaphelenchus xylophilus (Bx) in small wood samples of P. massoniana was developed in this study. This comprised a series of new techniques: sampling, mass estimations of nematodes using staining techniques, and lowest limit Bx nematode mass determination for PCR detection. The procedure was undertaken on three adjoining 5-mg wood cross-sections, of 0.5 · 0.5 · 0.015 cm dimension, that were cut from a wood sample of 0.5 · 0.5 · 0.5 cm initially, then the larger wood sample was stained by acid fuchsin, from which two 5-mg wood cross-sections (that adjoined the three 5-mg wood cross-sections, mentioned above) were cut. Nematode-staining-spots (NSSs) in each of the two stained sections were counted under a microscope at 100· magnification. If there were eight or more NSSs present, the adjoining three sections were used for PCR assays. The B. xylophilus – specific amplicon of 403 bp (DQ855275) was generated by PCR assay from 100.00% of 5-mg wood cross-sections that contained more than eight Bx NSSs by the PCR assay. The entire sampling procedure took only 10 min indicating that it is suitable for the fast estimation of nematode numbers in the wood of P. massonina as the prelimary sample selections for other more expensive Bx-detection methods such as PCR assay

Labeling of Unique Sequences in Double-Stranded DNA at Sites of Vicinal Nicks Generated by Nicking Endonucleases

We describe a new approach for labeling of unique sequences within dsDNA under nondenaturing conditions. The method is based on the site-specific formation of vicinal nicks, which are created by nicking endonucleases (NEases) at specified DNA sites on the same strand within dsDNA. The oligomeric segment flanked by both nicks is then substituted, in a strand displacement reaction, by an oligonucleotide probe that becomes covalently attached to the target site upon subsequent ligation. Monitoring probe hybridization and ligation reactions by electrophoretic mobility retardation assay, we show that selected target sites can be quantitatively labeled with excellent sequence specificity. In these experiments, predominantly probes carrying a target-independent 3′ terminal sequence were employed. At target labeling, thus a branched DNA structure known as 3′-flap DNA is obtained. The single-stranded terminus in 3′-flap DNA is then utilized to prime the replication of an externally supplied ssDNA circle in a rolling circle amplification (RCA) reaction. In model experiments with samples comprised of genomic λ-DNA and human herpes virus 6 type B (HHV-6B) DNA, we have used our labeling method in combination with surface RCA as reporter system to achieve both high sequence specificity of dsDNA targeting and high sensitivity of detection. The method can find applications in sensitive and specific detection of viral duplex DNA.Wallace A. Coulter Foundatio

A Microfluidic Approach to Selection and Enrichment of Aptamers for Biomolecules and Cells

This thesis presents microfluidic devices for selection and amplification of nucleic acids (aptamers) that bind to specific targets. Aptamers are very attractive molecules in many biological applications due to their interesting properties including high target binding affinities and stability. Using conventional platforms for aptamer generation (SELEX, systematic evolution of ligands by exponential enrichment) is labor-intensive and time consuming. Microfluidic devices have been developed to improve the aptamer enrichment efficiency. However, aptamer generation using these devices is still inefficient because they require complicated flow control components for sample and reagent handling and additional off-chip processes. We developed microfluidic SELEX platforms for rapid isolation of aptamers that possess greatly simplified designs which enable easy chip fabrication and operation. The simplicity of the devices is achieved by utilizing a combination of bead-based selection and amplification of target binding nucleic acids, and gel-based electrokinetic transfer of nucleic acids. In the devices, nucleic acids that bind to targets are isolated on target-functionalized microbeads or target cells in a microchamber and electrokinetically transported to another chamber through a gel-filled microchannel by an electric field. The strands are then hybridized onto reverse primers immobilized on microbeads and amplified via polymerase chain reaction (PCR) using on-chip temperature control. The amplified strands are separated from the beads and electrophoretically transferred back into the selection chamber for subsequent SELEX rounds. Using the devices, we demonstrated enrichment of target-binding nucleic acids against human immunoglobulin E (IgE), the glucose-boronic acid complex, and MCF-7 cancer cells. With the physical and functional integration allowed by the monolithic design realized in our devices, the total process time for selection of aptamers was drastically reduced compared with that required by conventional aptamer selection platforms. Moreover, the binding affinities of the selected strands using our devices are comparable to those of aptamers obtained using the conventional platforms

A new electrophoresis technique to separate microsatellite alleles*

Analysis of large numbers of SSR (simple sequence repeats: microsatellites) reactions can be tedious, time-consuming and expensive. The objective of this study was to report a new electrophoresis method to analyze and visualize SSR data quickly and accurately and compare it to the ability of four other electrophoresis methods. Individual PCR reactions consisting of DNA from several Cornus florida L. (flowering dogwood) cultivars and two SSR primer pairs were assembled for analysis using the following three methods: agarose gel, polyacrylamide gel and QIAxcel System. Two separate PCRreactions consisting of the same components plus a fluorescent-labeled primer were set up for analyses using the CEQTM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer. These fiveelectrophoretic methods were assessed for advantages and disadvantages. Polyacrylamide gels had highest resolution of alleles, whereas agarose gels had the lowest. However, with both separationmedia, it was difficult to score the size of alleles. Capillary electrophoresis with the CEQTM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer easily separated products and determined allelic size, but was more expensive than electrophoresis using either agarose or polyacrlamide gels. The QIAxcel System had lower  esolution than CEQTM 8000 Genetic Analysis System and ABI 3130xl DNA Sequencer. However, QIAxcel System was rapid and cost effective compared to the two widely used capillary sequencers, and also provided a computer generated gel image. For researchers in small to intermediate-sized laboratories, the QIAxcel System using a twelve channel, sieving-gel cartridge is an affordable device for SSR assays used for mapping and population diversity analysis

Targeted disruption of py235ebp-1: Invasion of erythrocytes by Plasmodium yoelii using an alternative Py235 erythrocyte binding protein

Plasmodium yoelii YM asexual blood stage parasites express multiple members of the py235 gene family, part of the super-family of genes including those coding for Plasmodium vivax reticulocyte binding proteins and Plasmodium falciparum RH proteins. We previously identified a Py235 erythrocyte binding protein (Py235EBP-1, encoded by the PY01365 gene) that is recognized by protective mAb 25.77. Proteins recognized by a second protective mAb 25.37 have been identified by mass spectrometry and are encoded by two genes, PY01185 and PY05995/PY03534. We deleted the PY01365 gene and examined the phenotype. The expression of the members of the py235 family in both the WT and gene deletion parasites was measured by quantitative RT-PCR and RNA-Seq. py235ebp-1 expression was undetectable in the knockout parasite, but transcription of other members of the family was essentially unaffected. The knockout parasites continued to react with mAb 25.77; and the 25.77-binding proteins in these parasites were the PY01185 and PY05995/PY03534 products. The PY01185 product was also identified as erythrocyte binding. There was no clear change in erythrocyte invasion profile suggesting that the PY01185 gene product (designated PY235EBP-2) is able to fulfill the role of EBP-1 by serving as an invasion ligand although the molecular details of its interaction with erythrocytes have not been examined. The PY01365, PY01185, and PY05995/PY03534 genes are part of a distinct subset of the py235 family. In P. falciparum, the RH protein genes are under epigenetic control and expression correlates with binding to distinct erythrocyte receptors and specific invasion pathways, whereas in P. yoelii YM all the genes are expressed and deletion of one does not result in upregulation of another. We propose that simultaneous expression of multiple Py235 ligands enables invasion of a wide range of host erythrocytes even in the presence of antibodies to one or more of the proteins and that this functional redundancy at the protein level gives the parasite phenotypic plasticity in the absence of differences in gene expression