A Lateral Flow Strip Based on a Truncated Aptamer-Complementary Strand for Detection of Type-B Aflatoxins in Nuts and Dried Figs

Type-B aflatoxins (AFB1 and AFB2) frequently contaminate food, especially nuts and fried figs, and seriously threaten human health; hence, it is necessary for the newly rapid and sensitive detection methods to prevent the consumption of potentially contaminated food. Here, a lateral flow aptasensor for the detection of type-B aflatoxins was developed. It is based on the use of fluorescent dye Cy5 as a label for the aptamer, and on the competition between type-B aflatoxins and the complementary DNA of the aptamer. This is the first time that the complementary strand of the aptamer has been used as the test line (T-line) to detect type-B aflatoxins. In addition, the truncated aptamer was used to improve the affinity with type-B aflatoxins in our study. Therefore, the lengths of aptamer and cDNA probe were optimized as key parameters for higher sensitivity. In addition, binding buffer and organic solvent were investigated. The results showed that the best pair for achieving improved sensitivity and accuracy in detecting AFB1 was formed by a shorter aptamer (32 bases) coupled with the probe complementary to the AFB1 binding region of the aptamer. Under the optimal experimental conditions, the test strip showed an excellent linear relationship in the range from 0.2 to 20 ng/mL with a limit of detection of 0.16 ng/mL. This aptamer-based strip was successfully applied to the determination of type-B aflatoxins in spiked and commercial peanuts, almonds, and dried figs, and the recoveries of the spiked samples were from 93.3%−112.0%. The aptamer-complementary strand-based lateral flow test strip is a potential alternative tool for the rapid and sensitive detection of type-B aflatoxins in nuts and dried figs. It is of help for monitoring aflatoxins to avoid the consumption of unsafe food.

Femtomolar and selective dopamine detection by a gold nanoparticle enhanced surface plasmon resonance aptasensor

ABSTRACTUltrasensitive and selective detection and quantification of dopamine (DA) plays a key role in monitoring neurodegenerative diseases. However, the detection limit reported for DA detection is typically in the lower nM range. Pushing the detection limit to pM or lower for this particular target to cover the physiological levels (< 130 pM) is significant. Herein, DA DNA aptamer (DAAPT) gold nanoparticle (AuNP) conjugate is utilized to enhance the surface plasmon resonance (SPR) signal, which enables to detect and quantify DA in the femtomolar (200 fM) to picomolar range. To the best of our knowledge, this is the lowest detection limit achieved for SPR sensing of dopamine. The as-prepared 10 nm DAAPT-AuNP conjugate demonstrates strong binding affinity (Kd = 3.1 ± 1.4 nM) to the complementary DNA (cDNA) probe on gold chip. The cDNA probe is immobilized to the chip via polydopamine surface chemistry, which allows the Michael addition of any primary amine-terminated biomolecules. By adjusting the concentration of the DAAPT-AuNP conjugate, two calibration curves are generated with dynamic ranges from 100 µM to 2 mM, and from 200 fM to 20 nM, respectively. Both calibration curves have negative slopes, showing good agreement to a dose-response curve in an enzyme inhibition assay. In addition, the sensing strategy is evaluated to be specific for DA detection using a series of DA analogs and other metabolites as potential interferences.

Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus)

We conducted a large-scale gene expression screen using the 3,200 cDNA probe microarray developed specifically for Ursus americanus to detect expression differences in liver and skeletal muscle that occur during winter hibernation compared with animals sampled during summer. The expression of 12 genes, including RNA binding protein motif 3 ( Rbm3), that are mostly involved in protein biosynthesis, was induced during hibernation in both liver and muscle. The Gene Ontology and Gene Set Enrichment analysis consistently showed a highly significant enrichment of the protein biosynthesis category by overexpressed genes in both liver and skeletal muscle during hibernation. Coordinated induction in transcriptional level of genes involved in protein biosynthesis is a distinctive feature of the transcriptome in hibernating black bears. This finding implies induction of translation and suggests an adaptive mechanism that contributes to a unique ability to reduce muscle atrophy over prolonged periods of immobility during hibernation. Comparing expression profiles in bears to small mammalian hibernators shows a general trend during hibernation of transcriptional changes that include induction of genes involved in lipid metabolism and carbohydrate synthesis as well as depression of genes involved in the urea cycle and detoxification function in liver.

Cucurbit leaf crumple virus Identified in Common Bean in Florida

Virus-like symptoms of leaf deformation and rugosity, especially of younger leaves, and a mild mosaic were observed on fresh market common (green) bean (Phaseolus vulgaris L.) plants in Hendry County in southwest Florida in December of 2007 and again in February of 2008. All bean fields were adjacent to watermelon fields in which Cucurbit leaf crumple virus (CuLCrV), Squash vein yellowing virus (SqVYV), and Papaya ringspot virus type W (PRSV-W) infections had previously been confirmed (fall of 2007) by PCR, reverse transcription (RT)-PCR, and/or ELISA. Whiteflies, Bemisia tabaci, were observed on both bean and watermelon plants in December and February. Fifteen samples (eleven with symptoms) were collected in December and two (both with symptoms) in February. Initial ELISA assays using commercially available antisera for potyviruses or Cucumber mosaic virus (Agdia, Elkhart, IN) were negative. Total nucleic acids were extracted and used for PCR testing. All samples tested negative by RT-PCR using specific primers for SqVYV, PRSV-W, and Cucurbit yellow stunting disorder virus, and degenerate primers for potyviruses. Ten of fifteen December samples (ten of eleven symptomatic samples) and both February samples yielded PCR products of the expected size with the degenerate begomovirus primers, PAR1c496/PAL1v1978, which amplify a portion of the begomovirus A component (3). PCR products from three December and both February samples were cloned and sequenced. The 1,159-nt PCR products shared 99% identity with each other and 96% identity with the corresponding region of A component sequences of Arizona and California CuLCrV isolates (GenBank Accession Nos. AF256200 and AF224760, respectively). Additional degenerate begomovirus primers PBL1v2040/PCRc154, which amplify a 381-nt portion of the hypervariable region of the begomovirus B component (3), and AC1048/AV494, which amplify a 533-nt portion of a conserved region of the coat protein gene (4), were used to confirm the identity of CuLCrV in the three December samples. The PBL1v2040/PCRc154 PCR products shared 98 to 99% identity with each other and 94 to 95% identity with the corresponding region of B component sequences of Arizona and California CuLCrV isolates (GenBank Accession Nos. AF327559 and AF224761, respectively), whereas the AC1048/AV494 PCR products shared 99% identity with each other and 97% identity with the corresponding region of A component sequences of Arizona and California CuLCrV isolates. Nucleic acid dot-blot hybridization assays of sap from homogenized leaves of the three December samples (from which the PCR product clones were obtained) with a digoxigenin-labeled CuLCrV cDNA probe also confirmed the presence of CuLCrV. Although CuLCrV has been reported to experimentally infect common bean and tobacco (2), to our knowledge, this is the first report of CuLCrV infecting any noncucurbit host in Florida. This finding suggests that CuLCrV may be more widely distributed than previously known in Florida (1) and that common bean (and potentially other legumes) are potential reservoirs for CuLCrV. References: (1) F. Akad et al. Plant Dis. 92:648, 2008. (2) J. K. Brown et al. Phytopathology 92:734, 2002. (3) M. R. Rojas et al. Plant Dis. 77:340, 1993. (4) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.

Identification of a Naegleria fowleri Membrane Protein Reactive with Anti-Human CD59 Antibody

ABSTRACT Naegleria fowleri, the causative agent of primary amebic meningoencephalitis, is resistant to complement lysis. The presence of a complement regulatory protein on the surface of N. fowleri was investigated. Southern blot and Northern blot analyses demonstrated hybridization of a radiolabeled cDNA probe for CD59 to genomic DNA and RNA, respectively, from pathogenic N. fowleri. An 18-kDa immunoreactive protein was detected on the membrane of N. fowleri by Western immunoblot and immunofluorescence analyses with monoclonal antibodies for human CD59. Complement component C9 immunoprecipitated with the N. fowleri “CD59-like” protein from amebae incubated with normal human serum. In contrast, a gene or protein similar to CD59 was not detected in nonpathogenic, complement-sensitive N. gruberi amebae. Collectively, our studies suggest that a protein reactive with antibodies to human CD59 is present on the surface of N. fowleri amebae and may play a role in resistance to lysis by cytolytic proteins.