Dot Blot Hybridization Assay Research Articles

Detection of Two Bipartite Geminiviruses Infecting Dicotyledonous Weeds in Trinidad.

Severe symptoms of suspected geminivirus etiology were manifested as intense yellow or golden mottling or mosaic of the lamina accompanied by mild leaf margin distortion on dicotyledonous weed species, Sida rhombifolia (L.) and Rhynchosia minima (L.), collected from 1999 to 2002 from the northeastern and central regions of Trinidad. S. rhombifolia is a common roadside weed while R. minima may have been introduced through restricted cultivation as a forage legume for livestock. Potato yellow mosaic virus-Trinidad isolate (PYMV-TT) has been implicated as the primary causal agent of begomoviral disease in large-scale tomato cultivation in Trinidad (2). It has been suggested that these weeds may be alternative hosts to PYMV-TT. However, all samples tested negative for PYMV-TT in dot blot hybridization assays using a PYMV-TT-specific DNA-A probe under high stringency. These results excluded the presence of PYMV-TT in these weeds. Polymerase chain reaction (PCR) amplification using clarified leaf extracts with degenerate primers for DNA-A (MP16 and MP82, PAL1v1978 and PAR1c715, and prV324 and prC889) and for DNA-B (PBL1v2040 and PCRc1) was performed on the weed samples (S. N. Rampersad and P. Umaharan, unpublished). Degenerate primers MP16 and MP82 target the 5' terminal region of the coat protein (cp) (2); PAL1v1978 and PAR1c715 direct amplification of the replication-associated protein gene (rep) and part of the cp gene (1); prV324 and prC889 amplify the core cp sequence (3). Primers PBL1v2040 and PCRc1 target the intergenic region and the 5' terminal of the BL1 ORF (1). PCR fragments obtained through amplification using this primer pair confirmed the presence of a DNA-B component for the unknown viruses. PCR fragments were sequenced and alignments were performed using DNASTAR (DNASTAR Inc., Madison, WI) and BLASTN ( www.ncbi.nlm.nih.gov/blast/ ) programs. None of the partial nucleotide sequences obtained for the viruses produced significant alignments with each other (5' terminal cp: 74% identity; core cp sequence: 78% identity), suggesting the detection of two distinct viruses. In addition, the partial sequences obtained were aligned to sequences of homologous regions of 11 New World begomoviruses (from the major representative clusters). The nearest match for R. minima, using alignments with 5' terminal cp (GenBank Accession No. AY221124), core cp (GenBank Accession No. AY217344), and 5' terminal BL1 region (GenBank Accession No. AY220490) was obtained for Rhynchosia golden mosaic virus (RhGMV, GenBank Accession Nos. AF408199 and AF442117) with 84 and 88% identity. There were no significant similarities found for sequence comparisons of the BL1 ORF. For S. rhombifolia, the highest homology using the 5' terminal cp (GenBank Accession No. AY220489), core cp (GenBank Accession No. AY217345), rep/cp region (GenBank Accession No. AY220488), and the 5' terminal BL1 region (GenBank Accession No. AY221125) was obtained for Sida golden mosaic virus (SiGMV, GenBank Accession Nos. AF049336, AF070923, and Y11100), with 82, 89, 84, and 87% identity. To our knowledge, this is the first report of geminivirus infection in these weed species in Trinidad. This may have substantial implications to future geminivirus disease outbreaks especially if there is expansion of the host range of these viruses to include economically important crops.

ITS probe development for specific detection of Rhizoctonia spp. and Suillus bovinus based on Southern blot and liquid hybridization-fragment length polymorphism

Development of specific DNA probes targeting rDNA internal transcribed spacer (ITS-1 or -2) sequences is described for the detection of strains representing uninucleate and binucleate Rhizoctonia spp. and Suillus bovinus. Discriminatory taxon/species-specific target sequences were identified following full length ITS sequence alignment of the test fungal sequences and those of other root associated pathogenic or mycorrhizal fungi. Both long (124-151 bp) and shorter (20-25 bp) probes were generated for assessment in Southern dot blot and liquid hybridization ITS capture-fragment length polymorphism assays. Fungal genomic DNA was presented as the target in dot blot protocols using the longer DIG (digoxigenin) labelled probes whilst the shorter 3' biotin-labelled oligonucleotide probes were hybridized to PCR amplified full length ITS (ITS1-5.8S-ITS2) in both dot blot and liquid hybridization assays. The optimal hybridization temperatures for dot blot detection also produced maximal target specific signals in the liquid hybridization protocol. The latter protocol was found to be more discriminatory as target fungi were detected on the basis of combined probe hybridization-ITS capture and 5' Cy-5 labelled ITS length polymorphism analysis (+/- 5 bp) following denaturing sequencing gel electrophoresis in a ALFexpress DNA sequencer.

Human parvovirus Bb19 detection in asymptomatic blood donors: association with increased neopterin concentrations.

Serum neopterin concentrations were determined in 20,000 blood donations. For the 400 donations with neopterin concentrations above the 98 th percentile and another 1200 donations with neopterin concentrations in the lower range, results of human parvovirus (HPV) B19 tests were compared. Infectious specimens were identified by dot blot hybridization assay and polymerase chain reaction (PCR) that used the outer primers and detected 1 pg of HPV B19 DNA, corresponding to approximately 10(5) copies of the genome, in the specimens and by a nested PCR that detected 1-10 fg of DNA, corresponding to 10(2)-10(3) copies of the genome. Of 400 specimens with neopterin concentrations > or =12 nmol/L (98th percentile, current cutoff), 10 tested positive by dot blot hybridization assay (9 of these were confirmed by nested PCR). Among 1200 specimens with low neopterin concentrations (<12 nmol/L), no specimen containing HPV B19 DNA was detected. These findings suggest an association between elevated neopterin concentrations and HPV B19 infectivity.

Analysis of atrazine-degrading microbial communities in soils using most-probable-number enumeration, DNA hybridization, and inhibitors

The purpose of this study was to determine whether there was an association between kinetics of atrazine mineralization, the number and type of atrazine-degrading microorganisms, and the presence of three genes representing different steps in the degradative pathway of atrazine in three soils with different histories of atrazine application. Composite soil samples were collected from two agricultural fields and one riparian zone soil. The samples were amended with atrazine, and mineralization was measured in biometers as 14CO 2 evolution in samples spiked with [ring-U- 14C]-labeled atrazine. Atrazine-degrading microorganisms were enumerated by a most-probable-number (MPN) method, which was based on loss of atrazine (HPLC assay) in acetate media. Dot-blot hybridization assays were performed on total DNA extracted from the MPN samples. The DNA probes used in dot-blot assays were the genes atzA (atrazine chlorohydrolase from Pseudomonas ADP), atrA (cytochrome P-450 from Rhodococcus TE1), and trzD (cyanuric acid amidohydrolase from Pseudomonas NRRLB-12228). The herbicide amendment enhanced the subsequent rate of mineralization of atrazine in all three soil samples. The MPN numbers were in the range of 10 0–10 2 cells g −1 dry wt. soil, indicating that atrazine-degrading microorganisms could not be quantitatively enumerated by this technique. Positive hybridization signals of DNA extracted from MPN samples were frequent with the atzA probe; the atrA dot blots had fewer positive signals. The trzD signals were negligible or undetectable, although the parallel mineralization studies showed fast and extensive 14CO 2 evolution from [ring-U- 14C]-atrazine. The results suggested that trzD, the only gene known to encode s-triazine ring-cleavage, is not dominant among the atrazine-degrading populations of these soils. Streptomycin and cycloheximide were added to soil samples in biometers to determine the relative contributions of bacteria and fungi in the soils to the mineralization of atrazine. The relative suppression of mineralization in the presence of the bacterial or fungal specific inhibitor was approximately the same, indicating that both groups contributed to the mineralization of atrazine.

Specific detection of Stenotrophomonas maltophilia strains in albacore tuna ( Thunnus alalunga) by reverse dot-blot hybridization

A reverse dot-blot DNA/DNA hybridization method coupled with a non-radioactive nucleic acid detection system was evaluated for the direct detection of the emerging pathogen Stenotrophomonas maltophilia in albacore tuna, a fish species of high commercial value in Europe and the US. Probes consisting of total genomic DNA of S. maltophilia, when used in dot-blot hybridization assays, differed in a sufficient way with respect to Morganella morganii, Enterobacter aerogenes Enterobacter agglomerans, Klebsiella planticola, Acinetobacter baumani and other bacteria frequently isolated from spoiled tuna fish species, as to allow its specific detection in extracts of albacore tuna. The introduction of an enrichment step prior to DNA isolation and labelling allowed the successful detection of 10 2 viable cells of S. maltophilia in 1 ml of artificially-contaminated albacore muscle extracts with no cross-hybridization with other Gram-negative competing microflora being observed. The detection strategy described in this work may be useful for the detection and control of S. maltophilia in tuna fish species and seafood products.

Production of canthaxanthin by Haloferax alexandrinus under non-aseptic conditions and a simple, rapid method for its extraction.

The production of carotenoids from Haloferax alexandrinus strain TM(T) was investigated at various concentrations of NaCl (10-25%) in culture media under non-aseptic conditions. PCR and dot blot hybridization assays were employed to monitor the growth of Hfx. alexandrinus in the culture under aseptic and non-aseptic conditions. The amplified PCR products of 16S rDNA from Hfx. alexandrinus grown under aseptic conditions were used as specific probes, which bound with amplified PCR products of 16S rDNA dots from both aseptic and non-aseptic conditions (20-25% NaCl). The results indicated that contamination of the culture was precluded at high NaCl concentrations (20-25%). Therefore, it is not necessary to perform asepsis during the biotechnological processes of carotenoid production by Hfx. alexandrinus. A 1-l-scale cultivation of the cells in flask cultures under non-aseptic conditions produced 3.12+/-0.5 g dry weight, 6.34+/-2.5 mg total carotenoids and 2,156.67+/-0.1 microg canthaxanthin. Further experiments in a batch fermenter, under non-aseptic conditions, also demonstrated increases in the biomass concentration and carotenoid production. When grown in a standard growth medium at 25% NaCl, the cells of Hfx. alexandrinus lysed spontaneously in fresh water and hence carotenoids could be extracted directly from the cells without any mechanical disintegration. These results demonstrate the feasibility and simplicity of commercial production of carotenoids using Hfx. alexandrinus.

Prevalence of Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV) and White Spot Syndrome Virus (WSSV) in Litopenaeus vannamei in the Pacific Ocean off the Coast of Panama

Abstract.— In March 2000, 104 wild caught Litopenaeus wannamei broodstock, captured off the Pacific coast of Panama, were screened for the following penaeid viruses: infectious hypodermal and hematopoietic necrosis virus (IHHNV) and white spot syndrome virus (WSSV). The purpose of this study was to determine the prevalence of IHHNV and WSSV in wild shrimp in this area of the Western Hemisphere and to acquire specific pathogen free (SPF) L. vannamei for inclusion into the Oceanic Institute's genetic breeding program. The prevalence of the viruses was determined using the dot blot hybridization format, which is a commercially available molecular method for detecting these viruses. Dot blot hybridization assays can be used as an initial screening method to detect moderately to highly infected shrimp. The results from the dot blot assays indicated the prevalence of IHHNV in 28% and WSSV in 2% of the 104 hemolyrnph samples tested. Results from this study were used to establish the initial candidate SPF status of the animals that were assessed and to determine the prevalence of two serious pathogens of penaeid shrimp captured from the wild of the Pacific Ocean in the Central American region off the coast of Panama.

Effect of cyanuric acid amendment on atrazine mineralization in surface soils and detection of the s-triazine ring-cleavage gene trzD

Cyanuric acid amendment was tested for potential enhancement of atrazine mineralization activity because this compound is the metabolite in the degradation pathway of s-triazines prior to ring-cleavage. The effect of cyanuric acid on: (i) subsequent atrazine mineralization kinetics and (ii) the detection of a hybridization signal to the s-triazine ring-cleavage gene trzD in surface soils with different histories of atrazine application were examined in this study. Composite soil samples from two agricultural soils and one riparian zone soil were amended with cyanuric acid. Mineralization was measured as 14CO 2 evolution in samples spiked with [ 14C-U-ring]-labeled atrazine. Dot-blot hybridization assays were performed on total genomic DNA extracted from the soils using the gene trzD (cyanuric acid amidohydrolase) as a probe. The source of this gene is Pseudomonas NRRLB-12228, which can cleave the s-triazine ring, but cannot grow with atrazine. The cyanuric acid amendment had a variable effect on the subsequent mineralization of atrazine in all three soils. The trzD hybridization signals in all three soils were negligible or undetectable, although parallel mineralization studies of one of the agricultural soils showed fast and extensive 14CO 2 evolution from [ring-U- 14C]-atrazine. The results suggest that the trzD gene, which is currently the only known s-triazine ring-cleavage gene, is not dominant among the atrazine-degrading populations of these soils.

Garlic viral complex: identification of Potyviruses and Carlavirus in Central Brazil

Garlic viruses often occur in complex infections in nature. In this study, a garlic virus complex, collected in fields in Brazil, was purified. RT-PCR was performed using specific primers designed from the consensus regions of the coat protein genes of Onion yellow dwarf virus, a garlic strain (OYDV-G) and Leek yellow stripe virus (LYSV). cDNA of Garlic common latent virus (GCLV) was synthesized using oligo-dT and random primers. By these procedures individual garlic virus genomes were isolated and sequenced. The nucleotide sequence analysis associated with serological data reveals the presence of two Potyvirus OYDV-G and LYSV, and GCLV, a Carlavirus, simultaneously infecting garlic plants. Deduced amino acid sequences of the Brazilian isolates were compared with related viruses reported in different geographical regions of the world. The analysis showed closed relations considering the Brazilian isolates of OYDV-G and GCLV, and large divergence considering LYSV isolate. The detection of these virus species was confirmed by specific reactions observed when coat protein genes of the Brazilian isolates were used as probes in dot-blot and Southern blot hybridization assays. In field natural viral re-infection of virus-free garlic was evaluated.

A system to enhance the sensitivity of digoxigenin-labelled probe: detection of B19 DNA in serum samples

A highly sensitive dot-blot hybridisation assay for the routine screening of numerous samples is described, using parvovirus B19 as a model. Digoxigenin-labelled B19 DNA probe was constructed by PCR, hybrids were detected by an anti-digoxigenin monoclonal antibody followed by a second step, using anti-mouse antibodies conjugated to an alkaline phosphatase-dextran complex (EnVision™, Dako) was carried out. The sensitivity of the assay was evaluated using both colourimetric and chemiluminescent substrates for the alkaline phosphatase and was compared with a dot-blot hybridisation assay using the digoxigenin-labelled probe and a standard detection system. With the colourimetric substrate, the EnVision™ system was able to detect 10 fg of B19 DNA, while with the chemiluminescent substrate the sensitivity increased by up to 2 fg (6×10 2 genome copies). This detection system was shown to increase the sensitivity of the assay compared to the standard colourimetric visualisation for the digoxigenin-labelled probe, which could detect 0.1 pg. On account of its sensitivity and specificity the dot-blot hybridisation assay together with the chemiluminescent substrate for the EnVision™ detection system was used to analyse 760 serum samples; the same sera were tested for B19 DNA with the standard colourimetric visualisation for the digoxigenin-labelled probe used routinely in the diagnostic laboratory.

CHEMILUMINESCENCE ASSAY FOR BACTERIUM HELICOBACTER PYLORI DNA

A chemiluminescence dot blot hybridization assay for Helicobacter pylori (H. pylori) DNA was established. A 20-mer oligo-nucleotide probe labeled directly alkaline phosphatase (AP) and highly sensitive chemiluminescence substrate for AP were used to detect H. pylori DNA. Preliminary H. pylori DNA testing was carried out on gastric biopsy specimens of 24 cases from patients. The results of detection were compared with that of histological examination and rapid urease test, showing the chemiluminescence dot blot hybridization assay is specific and more sensitive in eliminating false-negative. This method has much shorter detection period and simpler detection protocol than reported probe hybridization assays. It may be used in the H. pylori infection diagnosis as an alternative method for contrast.

Screening wild plants of Capsicum annuum for resistance to pepper huasteco virus (PHV): Presence of viral DNA and differentiation among populations

Plants collected in thirteen wild populations of Capsicum annuum from Northwest Mexico were tested for resistance to the pepper huasteco begomovirus (formerly subgroup III) (PHV) that is transmitted by the white fly Bemisia tabaci Genadius. Plants were inoculated using both grafting and biolistic methods. Presence of viral DNA was detected by dot blot hybridization and densitometry. Populations varied in their resistance to PHV. Plants of only two of the populations either did not develop disease symptoms or showed very light symptoms after inoculation. In some cases, symptoms appeared several days after inoculation. In plants of these populations viral DNA was detected by dot-blot hybridization assays but they appear to be a good source of resistance (symptomless) for use in breeding programmes.

Competition between Wild-Type and Recombinant Nucleopolyhedroviruses in a Greenhouse Microcosm

Abstract Wild-type Autographa californica nucleopolyhedrovirus (AcNPV or AcNPV.WT), AcNPV expressing a scorpion toxin (AcNPV.AaIT), and AcNPV expressing a mutated juvenile hormone esterase (AcJHE.SG) were compared in their capability to produce epizootics in larvae of Trichoplusia ni infesting collards in a greenhouse microcosm. Larvae treated in four different ways were released into 1.8-m 2 microplots in week 1. The four treatments included (1) uninfected larvae (control), (2) 100% AcNPV.WT-infected larvae (WT), (3) 100% AcNPV.AaIT-infected larvae (AaIT), and (4) 1:1 ratio of AcNPV.WT-infected and AcNPV.AaIT-infected larvae (WT+AaIT). On a weekly basis, larvae were sampled and new, uninfected larvae were added to all plots. Sampled larvae were reared until death and then subjected individually to DNA–DNA dot-blot hybridization assay to determine the proportion of insects infected with each virus in each plot. The entire experiment was repeated with AcJHE.SG in the place of AcNPV.AaIT. Epizootics of AcNPV.WT lasted 8 weeks after a single viral release in the replicated greenhouse microplots. AcJHE.SG epizootics also lasted 8 weeks after viral release, but this virus and AcNPV.AaIT were both out-competed by AcNPV.WT. AcNPV.AaIT was no longer detected in the T. ni population by the fourth week after release. AcNPV.WT also increased to greater numbers in soil than AcNPV.AaIT or AcJHE.SG after 8 weeks. Thus, it was possible to induce 8-week epizootics of AcNPV.WT in replicated microplots under artificial greenhouse conditions, and the wild-type virus out-competed the recombinant virus for a niche in this greenhouse microcosm, which reduces the probability that the recombinant virus will persist in an agroecosystem.

DNA hybridization assay for detection of nucleopolyhedrovirus in whitemarked tussock moth (Orgyia leucostigma) larvae

DNA dot-blot hybridization assays utilizing a horseradish peroxidase-labelled whole genomic DNA probe and enhanced chemiluminescence were conducted to quantify detection thresholds of nucleopolyhedrovirus (NPV) in whitemarked tussock moth (Orgyia leucostigma) larvae. The minimum detection thresholds for an aqueous suspension of occlusion bodies (OBs), OBs added to macerates of non-infected larvae and OBs in macerates of diseased larvae were 7.8 × 103, 7.8 × 103, and 1.5 × 103 OBs, respectively. Purified viral DNA was detected at a concentration of 1.6 × 10−1 ng in a 20 µl volume. The presence of pre-occluded viral nucleocapsids and DNA, inherent to infected larvae, improved the detection threshold five-fold compared with OBs alone. Larval tissues did not block the detection system utilized, nor did they bind non-specifically to the probe. Detection thresholds, upon sequential hybridization of the same membrane, on average deteriorated two-fold between the first and second hybridization and an additional six-fold between the second and third hybridization. NPV infection was detected two days post-inoculation (pi) in about one-third of the larvae examined and in almost all larvae three days pi. Microscopic analysis of stained larval smears missed NPV infection in almost all larvae two days pi and about two-thirds of the larvae three days pi. Results from the two methods of analysis were not comparable until four days pi. The detection system utilized is a reliable, efficient and simple method for the early detection of NPV infection in large numbers of larvae and may be used for further studies quantifying the role of this baculovirus in the ecology of whitemarked tussock moth populations. © 2001 Society of Chemical Industry

Human Parvovirus B19, Cytomegalovirus Infections and ThyroidAutoimmunity in Women with Recurrent First Trimester Abortion

OBJECTIVE: The goal of this study was to modify and optimize the screening and diagnostic procedures of unexplained recurrent abortion. Antithyroid antibodies, parvovirus B19 (B19V) and cytomegalovirus (CMV) infections, which were usually overlooked in clinical practice of handling cases of recurrent abortion, were studied to evaluate their role in the pathogenesis of recurrent first trimester abortion. Also , we tried to elucidate the importance of detection of these agents and whether it should be routine prenatal investigations of women with unexplained recurrent abortion. STUDY DESIGN: sixty women with unexplained recurrent first trimester abortion were the study group and thirty term healthy pregnant women, were selected as a control. Placental and fetal tissues were analyzed for B19V and CMV deoxyribonucleic acids using nested polymerase chain reaction (PCR) assay. Also sera were analyzed for T3, T4 and TSH levels, the presence of antithyroid antibodies (thyroglobulin and thyroid peroxidase), CMV specific IgM antibodies and parvovirus B19-DNA by dot blot hybridization assay. RESULTS: Parvovirus B19-DNA was demonstrated in nearly one third of the aborted products (19/60 - 31.67 %) and 6.67% of the placentas of the control group, that represent highly significant difference (P 0.05). In contrast, only 5 aborting women (8.33 %) and a woman of the control group (3.33%) were CMV-IgM seropositive, that also represent non significant changes (P > 0.05). Antithyroglobulin antibody levels were significantly higher in aborting women (7/60) than control (P 0.05). We did not find any significant relation between thyroid autoimmunity and viral infections (B19V & CMV) in women with unexplained recurrent first trimester miscarriage. CONCLUSION: Parvovirus B19 infection was found to be an important pathogenic agent in recurrent first trimester abortion. It is essential to investigate for B19Vinfection among pregnant women with recurrent abortion. This significant finding demonstrates the usefulness of a sensitive molecular tool, PCR assay of viral DNA to aid in the diagnosis. However, testing for cytomegalovirus infection and antithyroid antibodies are not essential as routine prenatal investigations and should be restricted to certain patients with unexplained recurrent pregnancy loss.

Human Parvovirus B19, Cytomegalovirus Infections and ThyroidAutoimmunity in Women with Recurrent First Trimester Abortion

OBJECTIVE: The goal of this study was to modify and optimize the screening and diagnostic procedures of unexplained recurrent abortion. Antithyroid antibodies, parvovirus B19 (B19V) and cytomegalovirus (CMV) infections, which were usually overlooked in clinical practice of handling cases of recurrent abortion, were studied to evaluate their role in the pathogenesis of recurrent first trimester abortion. Also , we tried to elucidate the importance of detection of these agents and whether it should be routine prenatal investigations of women with unexplained recurrent abortion. STUDY DESIGN: sixty women with unexplained recurrent first trimester abortion were the study group and thirty term healthy pregnant women, were selected as a control. Placental and fetal tissues were analyzed for B19V and CMV deoxyribonucleic acids using nested polymerase chain reaction (PCR) assay. Also sera were analyzed for T3, T4 and TSH levels, the presence of antithyroid antibodies (thyroglobulin and thyroid peroxidase), CMV specific IgM antibodies and parvovirus B19-DNA by dot blot hybridization assay. RESULTS: Parvovirus B19-DNA was demonstrated in nearly one third of the aborted products (19/60 - 31.67 %) and 6.67% of the placentas of the control group, that represent highly significant difference (P 0.05). In contrast, only 5 aborting women (8.33 %) and a woman of the control group (3.33%) were CMV-IgM seropositive, that also represent non significant changes (P > 0.05). Antithyroglobulin antibody levels were significantly higher in aborting women (7/60) than control (P 0.05). We did not find any significant relation between thyroid autoimmunity and viral infections (B19V & CMV) in women with unexplained recurrent first trimester miscarriage. CONCLUSION: Parvovirus B19 infection was found to be an important pathogenic agent in recurrent first trimester abortion. It is essential to investigate for B19Vinfection among pregnant women with recurrent abortion. This significant finding demonstrates the usefulness of a sensitive molecular tool, PCR assay of viral DNA to aid in the diagnosis. However, testing for cytomegalovirus infection and antithyroid antibodies are not essential as routine prenatal investigations and should be restricted to certain patients with unexplained recurrent pregnancy loss.

A New Tomato-Infecting Begomovirus in Barbados.

In September 1998, tomato plants in Barbados exhibited symptoms of severe leaf curling without marginal chlorosis. These symptoms were often associated with an increase in whitefly (Bemisia tabaci) populations. DNA was extracted from leaf tissue from symptomatic tomato plants. Polymerase chain reaction (PCR) was performed with DNA-A degenerate primer pair PAC1v1978/PAV1c715, which amplifies part of the rep gene, the cp gene, and the common region (CR), and with DNA-B primer pair PBC1v2039/PBV1c800, which amplifies part of the bc1 and bv1 genes and the CR (2). The amplified PCR fragments of DNA-A and DNA-B were 1.3 and 1.4 kb, respectively, which are the expected sizes from bipartite, whitefly-transmitted geminiviruses of the Western Hemisphere (2). DNA sequence of the cloned fragments of DNA-A and DNA-B are available as GenBank No. AF213013 and AF213014, respectively. The 181 nucleotides of the CR of DNA-A had a nucleotide identity of 96% with the CR of DNA-B, which indicates that this is a bipartite begomovirus. Pairwise comparisons using DNASTAR (DNASTAR, Madison, WI) of the sequenced part of DNA-A was most similar to Cabbage leaf curl virus (CaLCuV, 69%, U65529) and Squash leaf curl virus extended host range isolate (SqLCV-E, 64%, M38183), and <59% to 13 other bipartite Western Hemisphere geminiviruses and Tomato yellow leaf curl virus from Israel (X15656). Pairwise comparisons of the DNA-B fragment sequence was 59 and 55% similar to CaLCuV (U65530) and SqLCV-E (M38182), respectively. Phylogenetic analysis of DNA-A of the major groups of Western Hemisphere begomoviruses placed the Barbados tomato-infecting geminivirus in the cluster with CaLCuV and SqLCV-E (1), while DNA-B analysis placed it with CaLCuV. The DNA-A amplified fragment was used as a probe at high stringency with the dot blot hybridization assay using the Genius II labeling and detection kit (Boeringer Mannheim) to detect this geminivirus in tomato and several other plant species, which had typical geminiviral symptoms. Strong hybridization signals were obtained for all 23 tomato plants with symptoms, weak signals were observed for two of three muskmelon and two of seven watermelon plants, all with leaf curling symptoms. No hybridization signals were observed for peppers with leaf curling symptoms and two weed species, Macroptilium lathyroides and Rhynchosia minima, with golden mosaic symptoms or with the symptomless plant species used as negative controls. The weak signals observed from watermelon and muskmelon samples indicated the presence of low virus titer or geminiviruses distinct from this tomato virus. The presence of viral DNA in these two plant species was confirmed by PCR with degenerate primers described above. Resulting database searches of sequences in the GenBank revealed that the Barbados tomato virus appears to be a previously unreported virus. This new virus is given the provisional name Tomato leaf curl Barbados virus (ToLCBBV).

Role of chemokines, neuronal projections, and the blood-brain barrier in the enhancement of cerebral EAE following focal brain damage.

The role of focal brain damage as a trigger for autoimmune inflammation in multiple sclerosis (MS) is unclear. In this study we examine mechanisms by which experimental autoimmune encephalomyelitis (EAE) is enhanced by focal brain damage. EAE was produced in Lewis rats by footpad inoculation; focal brain damage, in the form of a cortical cryolesion (cryolesion-EAE), was induced 8 days post-inoculation (d.p.i.). The distribution of inflammation and chemokine production in cryolesion-EAE and EAE-only were compared. Inflammation in the brain, measured by immunocytochemistry for T lymphocytes (W3/13) and microglial activation (MHC class II -OX6), was significantly enhanced in cryolesion-EAE 11-15 d.p.i. (p < 0.01-0.05) but by 20-40 d.p.i., equated with EAE-only. Inflammation in cryolesion-EAE related to breakdown of the blood-brain barrier (BBB) at the site of the cryolesion and also to the corticospinal tracts and thalamus, reflecting the afferent and efferent neuronal connections with the cryolesioned cortex. Semiquantitative RT/PCR dot-blot hybridization assay showed a 6-fold increase in mRNA for specific chemokines in the brain in cryolesion-EAE at 9 d.p.i. (MCP-1) and 11 d.p.i. (MCP-1 and MCP-5) with no significant increase in RANTES, GRO-alpha, or MIP-1alpha. By 14 d.p.i., the levels of MCP-1 and MCP-5 mRNA equated with EAE-only animals. These results suggest that enhancement and location of autoimmune inflammation in the brain following focal cortical injury initially involve chemokines such as the macrophage chemoattractants MCP-1 and MCP-5, and the activities of afferent and efferent neuronal connections with the site of damage. By analogy, similar factors may modulate or reactivate autoimmune inflammation in MS.

Tyramide signal amplification of biotinylated probe in dot-blot hybridization assay for the detection of parvovirus B19 DNA in serum samples

Highly sensitive assay systems are necessary for large-scale virological screenings. We evaluated the use of tyramide signal amplification (TSA) for biotinylated probe in dot-blot hybridization assay to detect B19 DNA in serum samples. The probe was constructed by PCR and directly labeled with biotin during amplification reaction. The sensitivity of the dot-blot hybridization assay with TSA detection method was evaluated in comparison with a hybridization assay using the direct detection of biotinylated probe by streptavidin–biotin–alkaline phosphatase substrate. The TSA detection was able to detect 1 pg of B19 DNA and proved to be 10–50 times more sensitive than the hybridization assay with the direct detection of biotinylated probe. The analysis of 720 serum samples by TSA detection of biotinylated probe showed that the assay may be a valid diagnostic tool in routine testing of B19 DNA in serum samples.

Use of high specific activity StarFire oligonucleotide probes to visualize low-abundance pre-mRNA splicing intermediates in S. pombe.

An oligonucleotide labeling system was developed that can produce radiolabeled hybridization probes with tenfold or more higher specific activity than is obtained by traditional 5'-end-labeling with polynucleotide kinase. Yet the system is as rapid and simple as kinase labeling. The reaction uses the Klenow fragment of E. coli DNA polymerase to add alpha-32P-dA residues to the 3'-end of an oligonucleotide in a primer-extension reaction. Unlike other methods of radioactive tailing (e.g., terminal transferase), a single species is produced of both known length and known specific activity. The reaction is efficient, and over 90% of probe molecules are routinely labeled. Using this method of labeling, an oligonucleotide was shown to be tenfold more sensitive in detecting target DNA sequences in a dot blot hybridization assay, compared to the same oligonucleotide labeled using polynucleotide kinase. Northern blots of Schizosaccharomyces pombe RNA were probed with an oligonucleotide specific for intron 1 of the tf2d gene, a TATA-box binding transcription factor. Kinase-labeled tf2d probe detected only unspliced RNA, while the same oligonucleotide labeled using the new method detected both unspliced tf2d RNA and rare pre-mRNA splicing intermediates.