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Parietaria mottle virus (PMoV) is considered an emerging virus in many countries of the Mediterranean basin. Outbreaks of tomato necrotic disease caused by the tomato strain PMoV-T have been recorded in Italy, France, Spain, and Greece. No resistant or tolerant tomato cultivars are available and no cases of resistance have been reported in the section Lycopersicon of the Solanum genus so far. Therefore, wild relatives of tomato were evaluated to identify potential sources of resistance to PMoV infection. In all, 16 germplasm accessions from six wild Solanum spp. of the section Lycopersicon, along with susceptible near-isogenic tomato lines (Monalbo, Momor, and Mospomorist), were evaluated for resistance against two different PMoV-T strains, TI1 (from Italy) and FR1 (from France), under greenhouse conditions using mechanical inoculations. Local and systemic virus infections were determined by enzyme-linked immunosorbent assay (ELISA) 15 and 30 days after mechanical inoculation and confirmed by dot-blot hybridization of viral RNA with a PMoV-specific digoxigenin-labelled riboprobe. No local or systemic symptoms were observed in three accessions of S. peruvianum and one accession of S. corneliomuelleri. Moreover, the virus was never detected in the inoculated and upper noninoculated leaves of these accessions, either with ELISA or the specific riboprobe. The accession of S. lycopersicum var. cerasiformae was resistant only to the TI1 strain. This is the first description of sources of extreme resistance to PMoV in the section Lycopersicon of the Solanum genus.

BACKGROUND Invasive cervical carcinoma (ICC) accounts for 23% of all cancer-related deaths in Zimbabwean women. Trials for a national program of genotype-specific human papillomavirus (HPV) vaccines are underway to prevent cervical carcinoma, but the distribution of HPV types among women with ICC according to HIV status is unknown. METHODS To determine prevalence and distribution of high-risk HPV genotypes by HIV status in women with ICC, we performed a cross-sectional study on women referred for ICC testing at 4 urban referral hospitals in Zimbabwe from June 2014 to December 2015. Cervical biopsies were obtained for histology and HPV genotyping. HIV serology testing was performed. HPV testing was performed using MY09/MY11 polymerase chain reaction followed by typing using dot-blot hybridization. RESULTS Of 107 participants with histologically proven ICC, HIV prevalence was 49.5% (53/107). HIV-positive women tended to be younger (median age 44 years) than HIV-negative women (median age 59 years). HPV prevalence was 94% (101/107), ranging from 1 to 5 genotypes per participant. HPV 16 (81.5%), 18 (24%), 33 (13%), 35 (11%), 56 (9%), and 45 (7.4%) were the most prevalent genotypes among HIV-negative participants; HPV 16 (67.9%), 18 (43.4%), 56 (18.9%), 45 (15.1%), 33 (11.3%), and 58 (9.4%) were the most prevalent among HIV-positive participants. Eighty-three percent of women were infected with either HPV-16 or HPV-18. CONCLUSIONS Effective vaccination programs against HPV 16 and HPV 18 could prevent up to 83% of cases of cervical cancer in Zimbabwe. HIV may influence distribution of some HPV genotypes given the significant increase in prevalence of HPV 18 among HIV-positive participants.

With the help of the Dot-blot hybridization the difference in steps of homology between mRNA of control plants and small regulatory si/mi RNA isolated from second-generation plantlets of wheat, corn, soybeans, sugar beets, chickpea, etc. cultivated from the seeds of plants infected and processed by new polycomponent plant growth regulators Regoplant® and Stimpo® in the first generation was found. It is proved that this difference is related to a partial reprogramming of the cell genome under the influence of biostimulators on growing plants with infected backgrounds that turns out in induction of low-molecular si/miRNA with antipathogenic and antiparasitic properties, which are the components of the immune system of a living organism.

Cultivars of sugarcane (Saccharum) are hybrids between species S. officinarum (x = 10, 2n = 8x = 80) and S. spontaneum (x = 8, 2n = 5 - 16x = 40 - 128). These accessions have 100 to 130 chromosomes, 80-85% of which are derived from S. officinarum, 10-15% from S. spontaneum, and 5-10% are possible recombinants between the two genomes. The aim of this study was to analyze the repetition of DNA sequences in S. officinarum and S. spontaneum. For this purpose, genomic DNA from S. officinarum was digested with restriction enzymes and the fragments cloned. Sixty-eight fragments, approximately 500 bp, were cloned, sequenced and had their identity analyzed in NCBI, and in the rice, maize, and sorghum genome databases using BLAST. Twelve clones containing partial transposable elements, one single-copy control, one DNA repetitive clone control and two genome controls were analyzed by DNA hybridization on membrane, using genomic probes from S. officinarum and S. spontaneum. The hybridization experiment revealed that six TEs had a similar repetitive DNA pattern in the genomes of S. officinarum and S. spontaneum, while six TEs were more abundant in the genome of S. officinarum. We concluded that the species S. officinarum and S. spontaneum have differential accumulation LTR retrotransposon families, suggesting distinct insertion or modification patterns.

Serological methods cannot detect viroids because they do not code for any proteins. It is not surprising, therefore, that viroids were the first obligate plant pathogen for which molecular hybridization was used as a detection tool (Owens and Diener, 1981). Molecular hybridization has also facilitated considerable progress in identifying viroid replication intermediates, ribozyme activities, and RNA structure. Many laboratories have replaced molecular hybridization as a routine diagnostic tool with reverse transcription polymerase chain reaction (RT-PCR) (see Hadidi et al., 2011; James et al., 2006 for review). However, hybridization remains a useful technique because it balances sensitivity with ease of use, time, and cost. The basic principles of nucleic acid hybridization on solid supports are described in previous reviews (Hull, 1993; Mu¨ hlbach et al., 2003; Palla´s et al., 1998, 2011) and will not be repeated here. In this chapter we update the last two decades of progress using molecular hybridization methodologies (dot-blot, gel-blot, multiplex, tissue-printing, and in situ hybridization) to detect and study viroids.

Background and Objective: High-level gentamicin resistance (HLGR: MIC ≥ 500 µg/ml) in Enterococci is mediated by aminoglycoside modifying enzymes which is mainly encoded by aac(6′)-Ie-aph(2′′)-Ia gene. The aim of this study was to evaluate the frequency of aac(6′)-Ie-aph(2′′)-Ia gene in clinical isolates of Enterococcus facium and Enterococcus faecalis collected from hospitals in northwest of Iran. Materials and methods: In the present study a total of 111 enterococcus isolates were collected from 4 hospitals during a two year period (July 2009-August 2011). Bacterial identification and species determination were carried out by standard biochemical tests. Antimicrobial susceptibility was evaluated by Kirby Bauer disc diffusion method. MICs were determined by agar dilution method. The frequency of aac(6′)Ie-aph(2″)Ia gene in the isolates was determined by PCR. The carriage of resistance gene on Tn5281 transposon was identified by long PCR and dot-blot hybridization methods. Results: Antibiotic susceptibility tests revealed that the highest resistance was against streptomycin (74.77%) and erythromycin (67.58%) whereas the highest susceptibility was observed to vancomycin (81.1%). 36 isolates (32.43%) were identified as HLGR, 34(94.44%) of them had resistant gene in their genome. Long PCR studies revealed that 88 % of HLGR clinical isolates harboured Tn5281. The aac(6′)Ie-aph(2″)Ia resistance gene was present on Tn5281 transposon in all 32 isolates according to dot blot hybridization test. Conclusion: The results of this study indicated that aac(6′)Ie-aph(2″)Ia resistance gene is highly prevalent in gentamicin resistant isolates. Carriage of aac(6′)Ie-aph(2″)Ia resistance gene on Tn5281 transposable element suggests possible contribution of this transposone on dissemination of resistance gene among enterococcus isolates.

Recent years have seen an explosion in the development and application of molecular tools for identifying microbes and analyzing their activity. These tools are increasingly applied to strains of lactic acid bacteria (LAB), including those used in fermentation and as well as those marketed as probiotics, for identification and analysis of their activity. Many of these tools are based on 16S ribosomal DNA sequences and exploit either hybridization or PCR techniques. Furthermore, complete or partial genomes of various LAB and bifidobacteria have been determined and offer omics-based approaches to analyze the activity of the bacteria provided that the mechanisms of their action are known. Finally, fluorescent probes coupled to flow cytometry are used to monitor the physiological capacity of bacterial cells in situ. All these approaches can be used for the screening and selection of LAB, assessing their role in fermentation and flavor development in fermented products. Additional aspects of probiotic LAB include their viability and vitality during processing and analysis of their presence, persistence, and performance in the gastrointestinal tract. An overview of these approaches is provided, and specific examples of their application to lactic cultures are presented. Because of their abundant use in tracing and tracking of LAB, a complete listing of 16S ribosomal RNA probes for lactobacilli and bifidobacteria is provided.

A non-radioactive dot-blot hybridization assay for the successful detection of Citrus tristeza virus (CTV) RNA in total nucleic acid extracts of infected citrus was developed. Two digoxigenin (DIG)-labelled minus-sense riboprobes, complementary to the coat protein gene sequence of a Chinese and an Apulian CTV isolate were synthesized. Several citrus tissues were evaluated as optimal virus source and leaf petioles were found appropriate material for reliable detection. The hybridization assay showed a detection limit corresponding to 0.2 mg of fresh infected tissue. The riboprobes allowed CTV detection in isolates from different geographical areas, grown in the screenhouse or in the field, resulting in similar hybridization patterns. The infected trees were tested during different seasons with positive results, although from July to August most of the samples gave a weaker hybridization signal, compared to other seasons. The high sensitivity and reliability of the molecular hybridization assay described make it a good alternative to serological methods for CTV detection.

The lack of a rapid and reliable means for routine pathogen identification has been one of the main limitations in plant disease management, and has pushed the development of culture-independent, molecular approaches. Currently, DNA array technology is the most suitable technique for high-throughput detection and identification, as well as quantification, of multiple pathogens in a single assay. Closely related pathogens that may have completely different host ranges or pathogenicity often differ in only a single to a few base pairs in genes that may be targeted for identification. Therefore, the ability to discriminate single nucleotide polymorphisms (SNPs) should be pursued in any diagnostic assay. In this paper, we demonstrate the utility of DNA array technology to detect SNPs while accounting for specific criteria such as the position of the mismatch, the sequence of the oligonucleotide, and the length and amount of labeled amplicons that are hybridized. When disregarding mismatches at the extreme ends of the oligonucleotides, cross hybridization to single mismatch oligonucleotides is rare when processing environmental samples that contain genetic material from unknown sources. In addition to plant pathology, this study is relevant for any field of research where DNA arrays are used to detect mutations or polymorphisms, ranging from human diagnostics to environmental microbiology and microbial ecology.

The failure to adequately identify plant pathogens from culture-based morphological techniques has led to the development of culture-independent molecular approaches. Increasingly, diagnostic laboratories are pursuing fast routine methods that provide reliable identification, sensitive detection, and accurate quantification of plant pathogens. In addition, since plants or parts thereof can be infected by multiple pathogens, multiplex assays that can detect and quantify different pathogens simultaneously are highly desirable. Technologies that can meet these requirements, especially those involving polymerase chain reaction, are being developed and implemented in horticultural and agricultural practice. Currently, DNA array technology is the most suitable technique for multiplex detection of plant pathogens. Recently, a quantitative aspect was added to this technology, making DNA arrays highly attractive for various research and practical applications. Here, we review the most important recent advances in molecular plant pathogen diagnostics, with special attention to fungal molecular diagnostics. In addition to their applicability in practice, the different criteria that have to be fulfilled for developing robust detection procedures that can routinely be used by diagnostic laboratories are discussed.

An assay for the detection of Strawberry vein banding virus (SVBV) in Fragaria spp. based on nucleic acid sequence based amplification (NASBA) and real-time detection using molecular beacons (real-time NASBA) is described. This assay was compared both with biological indexing, the current method for certification of SVBV, and a newly optimised PCR-based detection method. Performance of the assay was tested on three SVBV isolates in Fragaria indicator plants and 317 field strawberries from five European countries. The assay was shown to be SVBV-specific, testing negative for other common aphid-borne strawberry viruses. The virus was detected in purified total RNA preparations diluted a millionfold, which is an amount equivalent to 1ng of fresh material. The real-time NASBA method developed here offers the potential of a fast, sensitive and reliable approach for the routine diagnosis of strawberry stock material

Fusarium wilt, caused by Fusarium oxysporum f. sp. lycopersici, and Verticillium wilt, caused by either Verticillium albo-atrum or Verticillium dahliae, are devastating diseases of tomato (Lycopersicon esculentum) found worldwide. Monitoring is the cornerstone of integrated pest management of any disease. The lack of rapid, accurate, and reliable means by which plant pathogens can be detected and identified is one of the main limitations in integrated disease management. In this paper, we describe the development of a molecular detection system, based on DNA array technology, for rapid and efficient detection of these vascular wilt pathogens. We show the utility of this array for the sensitive detection of these pathogens from complex substrates like soil, plant tissues and irrigation water, and samples that are collected by tomato growers in their greenhouses.

Acute and latent infections with the Brazilian LA031 strain of Aujeszky’s disease virus (ADV) were established in mice. Ultraviolet irradiated ADV administered subcutaneously was a successful way to establish latent infection. The presence of ADV was detected by PCR. Two sets of 22-mer primers were synthesized and used to amplify gG glycoprotein gene sequences in acute and latent infected trigeminal nerve ganglia. The specificity of the amplification was verified by dot-blot hybridization.

A DNA hybridization dot-blot assay using a radioactive and a non-radioactive probe has been developed for the detection of Aujeszky's disease virus (ADV). The Bam H I 7 fragment of ADV genomic DNA was labeled by nick translation using 32P-dCTP and the 196bp polymerase chain reaction (PCR) gG glycoprotein gene amplified fragment was also labeled by nick translation but using biotin d-7-dATP. This technique provides a fast and effective means of detecting acute cases of ADV infection but it was unable to detect ADV nucleic acid sequences in trigeminal nerve ganglia of latent infected pigs and mice.Uma sonda radioativa e uma outra biotinilada foram produzidas para detecção do vírus da doença de Aujeszky (VDA). O fragmento de Bam H I 7 do DNA genômico do VDA foi marcado por "nick translation" empregando P32dCTP e um fragmento de 196pb, resultante de uma amplificação pela reação em cadeia pela polimerase marcado com biotina 7-dATP. Essas sondas, de uma maneira rápida e específica, prestaram-se para a detecção da infecção aguda pelo VDA. Entretanto, não se mostraram sensíveis o suficiente para detectar seqüências genômicas de VDA no gânglio trigêmio de suínos e camundongos com infecção latente.

Мета роботи – підсилити стійкість рослин огірків і картоплі до нематод за допомогою нових ефективних мікробних препаратів на основі авермектинів та визначити молекулярно-генетичні механізми їх захисної дії. Застосовані методи польових і молекулярно-генетичних досліджень: виділення si/miРНК з клітин рослин, Дотблот гібридизація si/miРНК з популяціями цитоплазматичних мРНК, дослідження функціональної активності si/miРНК у безклітинній системі білкового синтезу. В польових та тепличних дослідах у оброблених новими мікробними препаратами Аверком (продуцент штам Streptomyces avermitilis УКМ Ас-2179) та його модифікаціями рослин огірків і картоплі встановлені зменшення ураження нематодами і збільшення врожайності культур. Методом Дот-блотингу встановлено різницю (6–23%) у ступеню гомології si/miРНК та мРНК між дослідними та контрольними рослинами. У безклітинній системі білкового синтезу з проростків пшениці підтверджено високий рівень (38–65%) антинематодної сайленсингової активності малих регуляторних si/miРНК із клітин дослідних рослин. Підвищення стійкості до паразитичних нематод інфікованих рослин огірків і картоплі за обробки препаратами на основі Аверкому та встановлені різниці у відсотку гомології між популяціями малих регуляторних si/ miРНК, виділеними з цих рослин, свідчать, що імуномодулювальна дія препаратів виявляється в напрямку індукції в клітинах рослин синтезу антинематодних si/miРНК, унаслідок цього знижується їх ураження паразитичними нематодами та підвищується урожайність., Цель работы – усилить устойчивость растений огурцов и картофеля к нематодам с помощью новых эффективных микробных препаратов на основе авермектинов и определить молекулярно-генетические механизмы их защитного действия. Использованы методы полевых и молекулярно-генетических исследований: выделение si/miРНК из клеток растений, Дот-блот гибридизация si/miРНК с популяциями цитоплазматических мР НК, исследование функциональной активности si/miРНК в бесклеточной системе белкового синтеза. В полевых и тепличных опытах у обработанных новыми микробными препаратами Аверком (продуцент штамм Streptomyces avermitilis УКМ Ас-2179) и его модификациями растений огурцов и картофеля установлены уменьшение поражения нематодами и увеличение урожайности культур. Методом Дот-блотинга установлена разница (6–23%) в степени гомологии si/miРНК и мР НК между опытными и контрольными растениями. В бесклеточной системе белкового синтеза из проростков пшеницы подтвержден высокий уровень (38–65%) антинематодной сайленсинговой активности малых регуляторных si/miРНК из клеток опытных растений. Повышение устойчивости к паразитическим нематодам инфицированных растений огурцов и картофеля при обработке препаратами на основе Аверкома и установленные разницы в проценте гомологии между популяциями малых регуляторных si/miРНК, выделенных из этих растений, свидетельствуют, что имм уномодулирующее действие этих препаратов проявляется в направлении индукции в клетках растений синтеза антинематодных si/miРНК, вследствие чего снижается их поражение паразитическими нематодами и повышается урожайность., Aim. To strengthen the resistance of cucumbers and a potato plants to nematodes by means of new effective microbic preparations on the basis of avermectines and to determine the molecular-genetic mechanisms of their protective action. Methods. Methods of field and molecular-genetic experiments, such as: isolation of si/miRNA from plant cells, the Dotblot hybridization si/miRNA with populations of cytoplasmic mRNA, investigation of functional activity si/miRNA in cell free system of protein synthesis. Results. In the field and greenhouse experiments at cucumbers and potato plants treated by new microbic preparations Averkom (producer strain Streptomyces avermitilis UKM Ас-2179) and its modifications the reduction of affection by parasitic nematodes and increase in productivity of cultures are determined. By Dot-blot method the difference (6–23%) is found in the degree of homology si/miRNA and mRNA between the experimental and control plants. In the cell free systems of protein synthesis from wheat sprouts the high level (38–65%) antinematode silencing activity of small regulatory si/miRNA from cells of experimental plants is confirmed. Conclusions. Increase of resistance to parasitic nematodes of the infected cucumbers and potato plants at the treatment by preparations on the basis of Averkom and the established differences in homology percent betweenpopulations of small regulatory si/miRNA, isolated from these plants, evidence that immunomodulating action of these preparations is occurred in the direction of induction in plant cells of antinematode si/miRNA synthesis, as a result their affection by parasitic nematodes decreases and productivity raises.

Over a period of two years, more than 5,000 citrus trees were tested for the presence of Citrus tristeza virus (CTV) on the island of Crete, resulting in thirty eight positives. Comparisons of the relative transcript levels of CTV p23, coat protein (CP), polymerase (POL) and an intergenic (POL/p33) region using quantitative RT-PCR, revealed consistent differences in abundance for each of these RNAs among flowers, stems, young fruits and leaves of infected orange trees. CTV p23 RNAs accumulated at highest levels, reaching a maximum in the flowers and the lowest levels in the leaves, while POL RNAs consistently accumulated at low levels in all tissues tested. A PCR-amplified dig-labelled CTV p23 DNA probe was applied to stem and leaf prints, and to crude and total RNA leaf extracts, using non isotopic hybridization. This technique, when applied to stem or leaf prints, and particularly to total RNA, unequivocally provided strong signals with minimal backgrounds. Moreover, an antiserum with high sensitivity and specificity of CTV detection as shown by DAS and immuno-print ELISA was produced against bacterially-expressed CTV CP. In the former method, stems and flowers contained higher levels of CTV CP when compared to leaf extracts. Taking into account Cretan geography and the importance of citrus to the island, systematic surveys for CTV eradication, sustainable control measurements and epidemiological studies need to be undertaken. The observations, materials and methods presented here may assist all three tasks at local and national level.

Telomeres, which form the protective ends of eukaryotic chromosomes, are a ubiquitous and conserved structure of eukaryotic genomes but the basic structural unit of most telomeres, a repeated minisatellite motif with the general consensus sequence T(n)A(m)G(o), may vary between eukaryotic groups. Previous studies on several species of green algae revealed that this group exhibits at least two types of telomeric sequences, a presumably ancestral type shared with land plants (Arabidopsis type, TTTAGGG) and conserved in, for example, Ostreococcus and Chlorella species, and a novel type (Chlamydomonas type, TTTTAGGG) identified in Chlamydomonas reinhardtii. We have employed several methodical approaches to survey the diversity of telomeric sequences in a phylogenetically wide array of green algal species, focusing on the order Chlamydomonadales. Our results support the view that the Arabidopsis-type telomeric sequence is ancestral for green algae and has been conserved in most lineages, including Mamiellophyceae, Chlorodendrophyceae, Trebouxiophyceae, Sphaeropleales, and most Chlamydomonadales. However, within the Chlamydomonadales, at least two independent evolutionary changes to the Chlamydomonas type occurred, specifically in a subgroup of the Reinhardtinia clade (including C. reinhardtii and Volvox carteri) and in the Chloromonadinia clade. Furthermore, a complex structure of telomeric repeats, including a mix of the ancestral Arabidopsis-type motifs and derived motifs identical to the human-type telomeric repeats (TTAGGG), was found in the chlamydomonadalean clades Dunaliellinia and Stephanosphaeria. Our results indicate that telomere evolution in green algae, particularly in the order Chlamydomonadales, is far more dynamic and complex than thought before. General implications of our findings for the mode of telomere evolution are discussed.

A one-step reverse transcription-polymerase chain reaction (RT-PCR) followed by a restriction analysis has been developed for the simultaneous detection and identification of five different strains of Pepino mosaic virus (PepMV).

Aims: To detect the possible coexistence of striped jack nervous necrosis virus (SJNNV) and red-spotted grouper nervous necrosis virus (RGNNV) genotypes in a single fish, a methodology based on the combination of PCR amplification and blot hybridization has been developed and applied in this study. Methods and Results: The degenerate primers designed for the PCR procedure target the T4 region within the capsid gene, resulting in the amplification of both genotypes. The subsequent hybridization of these amplification products with two different specific digoxigenin-labelled probes resulted in the identification of both genotypes separately. The application of the RT-PCR protocol to analyse blood samples from asymptomatic wild meagre (Argyrosomus regius) specimens has shown a 46·87% of viral nervous necrosis virus carriers. The combination of RT-PCR and blot hybridization increases the detection rate up to 90·62%, and, in addition, it has shown the coexistence of both genotypes in 18 out of the 32 specimens analysed (56·25%). Conclusions: This study reports the coexistence of betanodaviruses belonging to two different genotypes (SJNNV and RGNNV) in wild fish specimens. Significance and Impact of the Study: This is the first report demonstrating the presence of SJNNV and RGNNV genotypes in the same specimen. This study also demonstrates a carrier state in this fish species for the first time.

Recent years have seen an explosion in the development and application of molecular tools for identifying microbes and analyzing their activity. These tools are increasingly applied to strains of lactic acid bacteria (LAB), including those used in fermentation and as well as those marketed as probiotics, for identification and analysis of their activity. Many of these tools are based on 16S ribosomal DNA sequences and exploit either hybridization or PCR techniques. Furthermore, complete or partial genomes of various LAB and bifidobacteria have been determined and offer omics-based approaches to analyze the activity of the bacteria provided that the mechanisms of their action are known. Finally, fluorescent probes coupled to flow cytometry are used to monitor the physiological capacity of bacterial cells in situ. All these approaches can be used for the screening and selection of LAB, assessing their role in fermentation and flavor development in fermented products. Additional aspects of probiotic LAB include their viability and vitality during processing and analysis of their presence, persistence, and performance in the gastrointestinal tract. An overview of these approaches is provided, and specific examples of their application to lactic cultures are presented. Because of their abundant use in tracing and tracking of LAB, a complete listing of 16S ribosomal RNA probes for lactobacilli and bifidobacteria is provided.

Marginale Parodontitis, die multikausale Erkrankung des Parodonts ist eine Infektionskrankheit, modifiziert durch Wirtsfaktoren und äußere Einflüße. Die als pathogene Mischflora bezeichnete Kombination kommensaler Mikroorganismen spielt die primäre Rolle in der Ätiopathogenese der Parodontitis. In der Aufstellung des Studienziels wurden einzelne Bakterienarten (T. forsythensis, P. gingivalis, A. actinomycetemcomitans, C. rectus, F. nucleatum, Fusobacterium spp., P. intermedia, E. corrodens, V. parvula und C. ochracea) ausgewählt, die eventuell als "Markerkeime" in der aggressiven Form der Parodontitis betrachtet werden können. Dazu wurde eine Kontrollgruppe untersucht, die eine gesunde parodontale Flora besitzt. Die angewandte Nachweismethode basiert auf der PCR-Amplifikation von 16S rDNA und darauffolgender dot-blot Hybridisierung mit Oligonukleotidsonden. Die entsprechenden Sonden wurden hergestellt, optimiert und evaluiert. Für die epidemiologische Untersuchung wurde subgingivale Plaque von vier Parodontaltaschen und einer Kontrollstelle von 45 Patienten mit aggressiver Parodontitis, sowie an fünf Stellen von 21 Senioren entnommen. Die Prävalenz der einzelnen Bakterienarten wurde mit Hilfe des Chi-Quadrat Test verglichen. Obgleich eine hohe interindividuelle Variabilität der Kolonisationsmuster zu beobachten war, konnten T. forsythensis, P. gingivalis, C. rectus und F. nucleatum signifikant häufiger in den Parodontaltaschen als an den gesunden Stellen nachgewiesen werden und können deswegen als "Leitkeime" der aggressiven Parodontitis angesehen werden. A. actinomycetemcomitans konnte nur bei einzelnen Patienten mit aggressiver Parodontitis festgestellt werden. Die Ergebnisse für P. intermedia und E. corrodens ließen keine eindeutige Assoziation sowohl mit der aggressiven Parodontitis als auch mit dem gesunden Parodontalzustand zu. Bei Senioren wurde C. ochracea besonders häufig nachgewiesen. Die Ergebnisse dieser Studie bewiesen die erfolgreiche Einsetzbarkeit der hergestellten Oligonukleotidsonden. A multifactorial risk pattern of periodontitis has been recognized, where in addition to host and environmental factors, a pathogenic microbiota plays a primary role. The purpose of the current research was to analyze the prevalence of periodontitis-associated microorganisms in patients with aggressive periodontitis and periodontally healthy elders by using molecular-biologic detection methods like eubacterial PCR-amplification of 16S rDNA in combination with dot-blot hybridization. The oligonucleotide probes for the detection of T. forsythensis, P. gingivalis, A. actinomycetemcomitans, C. rectus, F. nucleatum, Fusobacterium spp., P. intermedia, E. corrodens, V. parvula and C. ochracea were designed and evaluated. The PCR products of 42 cultivated target and closely related bacteria were used for the optimization of hybridization conditions. For the epidemiological study, subgingival plaque was sampled from four pockets and one healthy site of 45 aggressive periodontitis patients as well as from five sites of 21 elderly. The differences in the prevalence of bacterial species were analyzed by the chi-square test. The data revealed frequent colonization by T. forsythensis, P. gingivalis, F. nucleatum and C. rectus in patients with aggressive periodontitis, however individual variations were obvious. These species could be predominantly identified in periodontal pockets, but were significantly less common in the healthy sites of the periodontitis patients and in the elderly. These putative pathogens can be conclusively determined as the key-bacteria in patients with aggressive periodontitis. No direct association for P. intermedia and E. corrodens with aggressive periodontitis or periodontal health could be seen. A. actinomycetemcomitans could be detected in only a few patients, reducing its suspected importance in the etiology of aggressive periodontitis. C. ochracea was highly prevalent in the well-maintained elderly, suggesting its association with healthy flora. The results of the study confirmed the reliability of the oligonucleotide probes in a specific and sensitive detection of the respective oral species.

Detection, identification and quantification of plant pathogens are the cornerstones of preventive plant disease management. To detect multiple pathogens in a single assay, DNA array technology currently is the most suitable technique. However, for sensitive detection, polymerase chain reaction (PCR) amplification before array hybridization is required. To evaluate whether DNA array technology can be used to simultaneously detect and quantify multiple pathogens, a DNA macroarray was designed and optimized for accurate quantification over at least three orders of magnitude of the economically important vascular wilt pathogens Verticillium albo-atrum and Verticillium dahliae. A strong correlation was observed between hybridization signals and pathogen concentrations for standard DNA added to DNA from different origins and for infested samples. While accounting for specific criteria like amount of immobilized detector oligonucleotide and controls for PCR kinetics, accurate quantification of pathogens was achieved in concentration ranges typically encountered in horticultural practice. Subsequently, quantitative assessment of other tomato pathogens (Fusarium oxysporum, Fusarium solani, Pythium ultimum and Rhizoctonia solani) in environmental samples was performed using DNA array technology and correlated to measurements obtained using real-time PCR. As both methods of quantification showed a very high degree of correlation, the reliability and robustness of the DNA array technology is shown.

An assay for the detection of Strawberry vein banding virus (SVBV) in Fragaria spp. based on nucleic acid sequence based amplification (NASBA) and real-time detection using molecular beacons (real-time NASBA) is described. This assay was compared both with biological indexing, the current method for certification of SVBV, and a newly optimised PCR-based detection method. Performance of the assay was tested on three SVBV isolates in Fragaria indicator plants and 317 field strawberries from five European countries. The assay was shown to be SVBV-specific, testing negative for other common aphid-borne strawberry viruses. The virus was detected in purified total RNA preparations diluted a millionfold, which is an amount equivalent to 1ng of fresh material. The real-time NASBA method developed here offers the potential of a fast, sensitive and reliable approach for the routine diagnosis of strawberry stock material

Peach latent mosaic viroid (PLMVd) was found in naturally infected sweet cherry trees grown in commercial orchards in southern Italy. The viroid was detected in nucleic acid extracts of symptomless leaves by molecular hybridization with a PLMVd cRNA probe. The viroid was transmitted by grafting from sweet cherry to peach seedlings and identified in peach by molecular hybridization.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2014-02-26T17:13:17Z No. of bitstreams: 1 WOS000175209200002.pdf: 505294 bytes, checksum: 5d5013a6fe49d8e6329ea86822fd5e6d (MD5) Made available in DSpace on 2014-02-26T17:13:17Z (GMT). No. of bitstreams: 1 WOS000175209200002.pdf: 505294 bytes, checksum: 5d5013a6fe49d8e6329ea86822fd5e6d (MD5) Previous issue date: 2002-04-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T14:03:19Z No. of bitstreams: 1 WOS000175209200002.pdf: 505294 bytes, checksum: 5d5013a6fe49d8e6329ea86822fd5e6d (MD5) Made available in DSpace on 2014-05-20T14:03:19Z (GMT). No. of bitstreams: 1 WOS000175209200002.pdf: 505294 bytes, checksum: 5d5013a6fe49d8e6329ea86822fd5e6d (MD5) Previous issue date: 2002-04-01 Symptoms of Cucumber mosaic virus (CMV) on yellow passion flower (Passiflora edulis f. flavicarpa) are characterized by bright yellow mottling on leaves, starting at random points on the vine and diminishing in intensity towards the tip, which becomes symptomless as it grows. To determine whether symptomless portions of vines are CMV-free or represent latent infection, leaves with and without symptoms were collected from infected vines in the field. Biological, serological (plate-trapped antigen enzyme-linked immunosorbent assay, PTA-ELISA), Western blot and dot-blot hybridization assays showed that portions of the vines without symptoms were CMV-free. Vegetatively propagated vines with symptoms showed remission of symptoms on newly developed leaves. One year later, no CMV was detected in the upper leaves of these plants. Mechanically inoculated passion flower seedlings behaved similarly; symptoms were shown by few leaves after inoculation. Afterwards, plants became symptomless and CMV was not detected in the upper leaves or root system, 40 or 85 days after inoculation. The mechanism responsible for remission of symptoms accompanied by CMV disappearance is not known. USP, ESALQ, Dept Entomol Fitopatol & Zool Agr, BR-13418900 Piracicaba, SP, Brazil UNESP, IBILCE, Dept Bot, BR-15054000 Sao Jose do Rio Preto, SP, Brazil UNESP, IBILCE, Dept Bot, BR-15054000 Sao Jose do Rio Preto, SP, Brazil

Acute and latent infections with the Brazilian LA031 strain of Aujeszky’s disease virus (ADV) were established in mice. Ultraviolet irradiated ADV administered subcutaneously was a successful way to establish latent infection. The presence of ADV was detected by PCR. Two sets of 22-mer primers were synthesized and used to amplify gG glycoprotein gene sequences in acute and latent infected trigeminal nerve ganglia. The specificity of the amplification was verified by dot-blot hybridization. Infecções dos tipos aguda e latente foram obtidas em camundongos infectados com a amostra brasileira LA031 do vírus da doença de Aujeszky (ADV). A administração subcutânea da amostra de ADV irradiada com luz ultravioleta foi indispensável para se estabelecer a infecção latente. A presença do ADV em animais foi detectada por PCR. Oligonucleotídeos de 22 mer foram sintetizados e usados para amplificar sequências do gene da glicoproteína gG no gânglio do nervo trigêmio em camundongos com infecção aguda e latente. A especificidade da amplificação foi verificada por hibridização do tipo dot-blot.

A DNA hybridization dot-blot assay using a radioactive and a non-radioactive probe has been developed for the detection of Aujeszky's disease virus (ADV). The Bam H I 7 fragment of ADV genomic DNA was labeled by nick translation using 32P-dCTP and the 196bp polymerase chain reaction (PCR) gG glycoprotein gene amplified fragment was also labeled by nick translation but using biotin d-7-dATP. This technique provides a fast and effective means of detecting acute cases of ADV infection but it was unable to detect ADV nucleic acid sequences in trigeminal nerve ganglia of latent infected pigs and mice. Uma sonda radioativa e uma outra biotinilada foram produzidas para detecção do vírus da doença de Aujeszky (VDA). O fragmento de Bam H I 7 do DNA genômico do VDA foi marcado por "nick translation" empregando P32dCTP e um fragmento de 196pb, resultante de uma amplificação pela reação em cadeia pela polimerase marcado com biotina 7-dATP. Essas sondas, de uma maneira rápida e específica, prestaram-se para a detecção da infecção aguda pelo VDA. Entretanto, não se mostraram sensíveis o suficiente para detectar seqüências genômicas de VDA no gânglio trigêmio de suínos e camundongos com infecção latente.

Since the nucleotide sequence of cholecystokinin (CCK) cDNA was found in the rat gene, we applied cDNA to quantitate the CCK mRNA. The size of the mRNA for a CCK precursor was 850 nucleotides in length using brain cytoplasmic RNA. There were no bands except CCK mRNA by Northern blot analysis. We also examined the developmental changes and regional distribution of CCK mRNA in rat brains by dot-blot and gel-blot hybridization using CCK cDNA as a probe. CCK mRNA was barely detectable in the fetal brain, but started to increase postnatally and attained the plateau level after 20–30 days. Further, the level of CCK mRNA was highest in the frontal cortex, followed by those of the hippocampus and striatum. The cerebellum contained only negligible CCK mRNA. These results are in agreement with those of CCK concentration in the corresponding brain areas and suggest a transcriptional control of CCK concentration.

Background It is well known that different Eimeria maxima strains exhibit significant antigenic variation. However, the genetic basis of these phenotypes remains unclear. Methods Total RNA and mRNA were isolated from unsporulated oocysts of E. maxima strains SH and NT, which were found to have significant differences in immunogenicity in our previous research. Two subtractive cDNA libraries were constructed using suppression subtractive hybridization (SSH) and specific genes were further analyzed by dot-blot hybridization and qRT-PCR analysis. Results A total of 561 clones were selected from both cDNA libraries and the length of the inserted fragments was 0.25–1.0 kb. Dot-blot hybridization revealed a total of 86 differentially expressed clones (63 from strain SH and 23 from strain NT). Nucleotide sequencing analysis of these clones revealed ten specific contigs (six from strain SH and four from strain NT). Further analysis found that six contigs from strain SH and three from strain NT shared significant identities with previously reported proteins, and one contig was presumed to be novel. The specific differentially expressed genes were finally verified by RT-PCR and qRT-PCR analyses. Conclusions The data presented here suggest that specific genes identified between the two strains may be important molecules in the immunogenicity of E. maxima that may present potential new drug targets or vaccine candidates for coccidiosis.