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Field surveys were carried out to assess the incidence of peach latent mosaic viroid in nine orchards of stone fruit trees in four growing regions of Poland. Forty of 119 peaches, six of 37 apricots, five of eight nectarines and one of four plums trees tested positive by RT-PCR for this viroid. Most of the infected stone fruit trees did not show any symptoms. Leaf yellow mosaic was only noticed on ‘Maycrest’ peach and ‘Saturn’ nectarine trees, and additional symptoms of cracked fruits with corky sutures and enlarged pits were observed on one ‘Maycrest’ tree. One or two isolates from each orchard and each species of infected tree were selected, and together, 11 isolates of peach latent mosaic viroid were used for genetic analyses. The nucleotide sequence similarity of selected isolates found in Poland was 93.6–100%. They shared 82.0–98.8% similarity with the viroid strains from different hosts and geographical regions. The phylogenetic analysis led to the clustering of all isolates detected during this study in the most numerous represented subgroup II-A, composed of peach latent mosaic viroid strains originating from different geographic regions and host plants. Although Polish isolates were clustered in the same subgroup, some differed in length and nucleotide sequences. It was shown that the genetic variability of isolates found in Poland was unrelated to the regions of their origin. A positive signal was obtained in dot-blot hybridization of all analyzed 11 isolates.

BackgroundInvasive 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.MethodsTo 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.ResultsOf 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.ConclusionsEffective 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.

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. [ABSTRACT FROM AUTHOR]

Objectives: Campylobacters are a major cause of gastroenteritis worldwide. These are fastidious in culture and false negative results are seen in many clinical laboratories. Among molecular methods, the dot-blot technique is widely used for a variety of purposes, especially diagnostics. So, the authors aimed to detect C. jejuni and C. coli simultaneously using a dot-blot assay., Methods: After evaluating the bioinformatics studies, a cadF -conserved fragment was selected for the design of primers and probe. DNAs from standard strains and a recombinant plasmid, prepared in this study, were used to assess the technique. The specificity of the method was also surveyed using DNAs from other enteric bacteria. The limit of detection was evaluated by recombinant plasmid and different concentrations of the designed probe., Results: A 95-bp fragment of cadF was selected, and in silico analysis studies showed that it is conserved between both species. Also, the non-specific annealing of the primers and probe with other bacteria was not seen theoretically. The technique with recombinant plasmid as well as DNAs of standard strains created black spots on the membrane, confirming that the probe was correctly synthesized. No non-specific reactions with other bacterial species were observed (specificity=100%). The limit of detection of the test was determined to be 50 µg/ml., Conclusions: This is the first study to simultaneously detect two important pathogens in the Campylobacter genus and was able to detect C. jejuni and C. coli with acceptable sensitivity and specificity., Competing Interests: The authors declare no conflicts of interest.Sponsorships or competing interests that may be relevant to content are disclosed at the end of this article., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Abstract 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.

Introgression of genetic material from wheat wild relatives into the common wheat genome remains important. This is a natural and inexhaustible source of enrichment of the wheat gene pool with genes that improve wheat's adaptive potential. Hexaploid lines F-F of wheat type were developed via hybridization of common wheat Aurora (AABBDD) and genome-substituted amphidiploid Aurotica (AABBTT). The hexaploid genome of the latter includes the diploid genome TT from wheat relative Aegilops mutica instead of subgenome DD of common wheat. F-F hybrids had limited self-fertility, which had substantially increased for some derivatives in F-F. For all generations, development of the lines was accompanied by cytogenetic control of the chromosome numbers. The chromosome numbers varied in general from 33 to 46 depending upon generation. In most descendants, that number was 42 chromosomes in F when plants with chromosome numbers 40-44 were selected in each generation. F lines originate from nine selffertile F plants, differ from Aurora according to some morphological characters, and have alien DNA in their genome as was demonstrated by DNA dot-blot hybridization with genomic DNA of Aegilops mutica as a probe. [ABSTRACT FROM AUTHOR]

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.

Background: Northern Thailand is one of the highest a-thalassemia incidence areas where 30-40% of inhabitants have been reported to carry aberrant α-globin genes. However, all previous α-thalassemia prevalence surveys in northern Thailand have been undertaken without consideration of ethnicity. Here we report the prevalence of a-thalassemia genes in 4 Tai (Yong, Yuan, Khuen, Lue) and 4 Mon-Khmer speaking populations (Blang, Mon, Paluang, Lawa). Methods: DNA extracted from 141 individuals was genotyped for 4 α-thalassemia deletional types (--SEA, --THAI, -α3.7, -α4.2) using MultiplexGap-PCR analysis and 2 non-deletional types (Hb CS, Hb Pakse) using dot-blot hybridization technique. Results and discussion: A total of 33 α-thalassemia carrying individuals (23.4%) were detected of which 32 were heterozygotes and one was a homozygote. The most common α-thalassemia detected were -α3.7 (17.7%) and --SEA (3.5%), while Hb CS was detected in 2.1% of cases. No occurrence of --THAI, -α4.2 and Hb Pakse was observed. The prevalence of α-thalassemia carriers varied between the different ethnic groups, with the Yuan having the highest prevalence of α-thalassemia carriers (50%) while the Lawa had the lowest prevalence (0%). The Paluang had a high prevalence (42%) of a single deletion type (-α3.7) possibly related to the endogamous marriage traditions of this ethnic group. Conclusion: The extreme variation of a-thalassemia prevalence among the different ethnic groups highlights the significantly different genetic backgrounds found in these peoples, as consequences of dissimilar cultures. Our study suggests that ethnicity must be considered in any of the disease-causing allele prevalence surveys in this region. [ABSTRACT FROM AUTHOR]

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.

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.

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 qRTPCR 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. [ABSTRACT FROM AUTHOR]

Nucleocapsid (N) protein of infectious bronchitis virus (IBV), one of the viral structural proteins, induces strong antibody response in natural infection. In this study, a simple, recombinant N protein-based dot-blot test was developed to serologically examine chicken serum samples for the presence of IBV antibody. Initially, 72 serum samples were tested for the presence of IBV antibody using a commercial enzyme linked immunosorbent assay (ELISA) kit. Forty six IBV positive serum samples (group A) produced strong signals in the dot-blot assay. Seven negative serum samples (group B) produced weak but specific signals using the dot-blot assay in conjunction with Western blot analysis. The remaining 19 samples (group C) from IBV negative specific pathogen free (SPF) chickens did not produce visible signals using the dot-blot assay. In conclusion, the above results suggest that the dot-blot assay is a reliable, sensitive, and specific test for serological detection of IBV positive chickens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Field surveys were carried out in the main peach-growing areas of China to assess the virus or viroid disease status of peach trees. A total of 505 leaf samples were tested by ELISA or RT-PCR for the presence of nine major peach viruses. The viruses most frequently detected were Apple chlorotic leaf spot virus (ACLSV) (71.8%), followed by Prunus necrotic ringspot virus (PNRSV) (20.2%), Cherry green ring mottle virus (CGRMV) (8.1%), and Apricot pseudo-chlorotic leaf spot virus (APCLSV) (1.6%). The overall average of virus infection level in peach trees was 24.6%. Apple mosaic virus (ApMV), Plum pox virus (PPV), Prune dwarf virus (PDV), Cherry virus A (CVA) and Cherry leaf roll virus (CLRV) were not detected. Of another 583 leaf samples tested for the presence of the known major peach viroids, by dot-blot hybridization, 444 (76.2%) were infected. Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) were detected in 443 and 18 samples, respectively. This is the first large-scale study on major viruses and viroids that infect peach trees in China. [ABSTRACT FROM AUTHOR]

Ear rot, caused by Fusarium verticillioides (FV), is a destructive disease of maize as it reduces grain yield and increases risks of mycotoxin production, thus endangering livestock. To identify genes differentially expressed during FV infection, four cDNA libraries were constructed for suppression subtractive hybridization using RNA isolated from bracts of an FV-resistant inbred maize line, Bt-1, as well as an FV-susceptible maize inbred line, Ye478. A total of 145 clones were obtained following reverse dot-blot hybridization, and these were sequenced from these libraries. Similarity analysis revealed that of these genes, 93 were unique, including 68 of known function, 24 of unknown function, and a single novel gene. Most genes of known function were predominantly involved in plant defense such as cell defense, transcription regulation, signal transduction, and metabolism. Expression profiles of eight representative genes, using semiquantitative reverse transcription-polymerase chain reaction, confirmed that differential gene regulation during FV infection was involved. These findings suggested that these differentially expressed genes might be involved in FV defense responses in maize. [ABSTRACT FROM AUTHOR]

Dot-blot hybridization has been successfully used for the construction of single nucleotide polymorphism (SNP)-based linkage maps, quantitative trait locus analysis, marker-assisted selection, and the identification of species and cultivars. This method is, however, time-consuming, even for a small number of plant samples. We propose a method in which streptavidin-coated magnetic beads replace the nylon membrane for immobilization of the PCR products and are hybridized with allele-specific oligonucleotide probes and a digoxigenin-labeled oligonucleotide hybridized with the allele-specific oligonucleotide probe. After amplification of plant DNA by PCR with the biotinylated primers, those oligonucleotide probes having species-specific or allele-specific sequences were mixed together with the digoxigenin-labeled oligonucleotide and the streptavidin-coated magnetic beads at a temperature suitable for each probe. Species-specific internal transcribed spacer 1 (ITS1) sequences and allele-specific sequences of the hypervariable region I of S-locus receptor kinase ( SRK) specifically detected ITS1 sequences and SRK alleles in Brassica species, respectively. SNPs were also successfully analyzed by using allele-specific oligonucleotide probes and competitive oligonucleotides. In the SNP analysis, PCR products were indirectly captured by magnetic beads. SNP alleles of eight cultivars each of Brassica rapa and Raphanus sativus were analyzed using streptavidin-coated magnetic beads. The genotyping results corresponded well with those of dot-blot-SNP analysis. Although allele-specific hybridization using streptavidin-coated magnetic beads is somewhat costly, it is easier and more rapid than dot-blot hybridization. This method is suitable for the analysis of a small number of plant samples with a large number of DNA markers. [ABSTRACT FROM AUTHOR]

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 TnAmGo, 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. [ABSTRACT FROM AUTHOR]

Tomato Yellow Leaf Curl virus (TYLCV-Eg) was isolated from whiteflies-infected tomato (Lycopersicon esculentum cv.Castle rock) plants growing in Nubaria and El-Behera Governorate. The infected plants exhibited systemic viral symptoms in the form of sever leaf curling, leaf crinkle with marginal yellowing, stem upright, twisted and stunted. TYLCV-Eg reacted positively with polyclonal antibodies specific to TYLCV using DAS-ELISA. It was transmitted by both syringe injection and whiteflies with transmission efficiency of about 80% and 100%, respectively. TYLCV-Eg isolate was transmitted to different species belonging to families Cucurbitaceae, Fabaceae, Solanaceae and Chenopodiaceae. TYLCV had TIP of 70ºC, DEP of 10-7 and LIV of about 6 days. Electron micrograph of the partially purified TYLCV revealed the presence of monomer and dimmer gemini particles with dimensions of 22 nm and 20 × 30 nm to 24 × 30 nm, respectively when negatively stained with uranyl acetate. Using degenerate oligonucleotide primers, the viral coat protein gene was amplified successfully by PCR, producing ∼ 500 bp fragment from tomato infected plants. The viral genome was detected by specific DNA probe using dot blot hybridization technique. Comparative nucleotide sequence analysis showed a similarity of 98% between TYLCV-Eg and other isolates. [ABSTRACT FROM AUTHOR]

Abstract: A simple and sensitive dot blot hybridization assay using a digoxigenin-labeled cRNA polyprobe was developed for the simultaneous detection of six viroids that infect pome and stone fruit trees. The polyprobe was constructed by cloning sequentially partial sequences of each viroid into a single vector, with run-off transcription driven by the T7 promoter. All six viroids were detectable within a dilution range of 5−3 to 5−4 in total nucleic acid extracts from infected trees. Individual trees were co-inoculated to create mixed infections and all four pome fruit viroids and both stone fruit viroids could be detected in pear and peach trees, respectively, using the polyprobe. The results of the assays using the polyprobe were comparable to those using single probes. The methods were validated by testing geographically diverse isolates of viroids, as well as field samples from several collections in the US. The assay offers a rapid, reliable and cost-effective approach to the simultaneous detection of six fruit trees viroids and has the potential for routine use in quarantine, certification, and plant genebank programs where many samples are tested and distributed worldwide. [ABSTRACT FROM AUTHOR]

Hb Constant Spring [Hb CS, α142(H19)Term] and Hb Paksé [α142(H19)Term] occur from the mutation in the termination codon of the α2-globin gene, TAA>CAA (→Gln) and TAA>TAT (→Tyr), respectively. They are the most common nondeletional α-thalassemia (α-thal) variants causing Hb H disease in Southeast Asia. In this study, 587 cord blood samples were screened for the Hb CS and Hb Paksé mutations by a dot-blot hybridization technique using oligonucleotide probes specific for each mutation. The results showed that the prevalence of Hb CS and Hb Paksé in Central Thailand are 5.80 and 0.51%, respectively, which is in concordance with the results from previous studies. [ABSTRACT FROM AUTHOR]

Simple, reliable methods for identification of species are required for management of many species and lines in a plant gene bank. Species-specific probes were designed from published sequences of the ITS1 region in rDNA of 16 species in Brassica and its related genera, and used as probes for dot-blot hybridization with plant genomic DNA. All the probes detected species-specific signals at dot-blots of genomic DNAs of the 16 species in Brassica, Diplotaxis, Eruca, and Raphanus. Signals of the Brassica digenomic species in the U’s triangle, i.e., B. napus, B. juncea, and B. carinata, were detected by the probes of their parental monogenomic species, i.e., B. rapa, B. nigra, and B. oleracea. The probe for B. oleracea showed signals of B. balearica, B. cretica, B. incana, B. insularis, and B. macrocarpa, which have the C genome as B. oleracea. Eruca vesicaria DNA was detected by the probe for E. sativa, which has been classified as a subspecies of E. vescaria. DNA of leaf tissue extracted by an alkaline solution and seed DNA prepared by the NaI method can be used directly for dot-blotting. Misidentification of species was revealed in 20 accessions in the Tohoku University Brassica Seed Bank. These results indicate dot-blot hybridization to be a simple and efficient technique for identification of plant species in a gene bank. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A rapid and efficient protocol for the extraction of genomic DNA from plant pathogenic fungi was developed. Key features of the protocol include the SDS-assisted lysis of fungal mycelium with inclusion of a glass bead to help break hyphal walls, followed by isopropanol precipitation of the DNA. The protocol was used to extract genomic DNA from a collection of 26 fungal species, representing many important plant pathogens. Yield of DNA ranged from 2.1-4.9 μg per 20 mg of mycelium or 0.4-0.6 μg per 20 mg of spores. The DNA was of sufficient purity to be digested by restriction enzymes, to serve as a template in the PCR-amplification of genomic fragments as large as 4.9 kb, and to be used in dot-blot hybridization for the detection of multiple- and single-copy genes. [ABSTRACT FROM AUTHOR]

Abstract: The goal of this study was to develop a field diagnosis system based on isothermal reverse transcription-loop-mediated amplification (RT-LAMP) for shrimp Taura syndrome virus (TSV), placing emphasis on specific and simple detection of the LAMP amplicons. After a single-tube RT-LAMP reaction for TSV was established, colorimetric dot–blot hybridization (DBH) was adopted to detect signals only from the target-derived amplicons. The results showed that the modified DBH offered unambiguous and sensitive detection of the TSV RT-LAMP amplicons without the UV cross-linking and denaturation steps. Together, TSV RT-LAMP-DBH assay reached the same dilution point as reverse transcription-nested polymerase chain reaction-agarose gel electrophoresis (RT-nPCR-AGE) for TSV detection. Specificity of the assay was demonstrated by the absence of DBH signal from yeast tRNA and various shrimp viruses. TSV RT-LAMP-DBH was applied to 125 Penaeus vannamei and demonstrated a very good concordance (kappa value, 0.823) with RT-nPCR-AGE assay in detection efficiency. Furthermore, a one-step guanidinium thiocyanate (GuSCN) homogenization method was established to provide RNA extraction efficiency comparable to that of the TRIzol Reagent for RT-LAMP. Requiring simply a heating apparatus, the GuSCN RNA extraction-isothermal RT-LAMP-DBH protocol has the potential for further development for diagnosis of diseases in the field. [Copyright &y& Elsevier]

In order to assess the presence of yellow head virus (YHV) in shrimp farms along the Pacific coast of Mexico, 39 samples from 26 randomly chosen farms were analysed by means of reverse transcriptase-polymerase chain reaction (RT-PCR) and dot-blot hybridization. Eleven samples were positive for YHV. The disease was reproduced by means of an infectivity bioassay performed with an extract of pleopods from the positive samples. Cumulative mortality reached 50% in 14 days. Four pairs of primers which amplified several YHV genome regions were designed and used to test dead and surviving shrimp from the bioassay by RT-PCR, resulting in positive results for every expected amplicon. The results of this study provide presumptive evidence of the presence of YHV in Mexican shrimp farms at least during 1999–2000. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Apple stem grooving virus (ASGV) and apple chlorotic leaf spot virus (ACLSV) are two major viruses of pear. In this study, in vitro thermotherapy was carried out at 37°C for 25, 30 and 35 days followed by subculturing of meristem tips of different sizes to eliminate ASGV and ACLSV from pear plants. Virus titers in heat-treated shoot tips were evaluated by ELISA testing of regenerated plants. Results showed that thermotherapy for 35 days significantly decreased the titer of ASGV and ACLSV in cultures regenerated from tips of main and axillary shoots, especially in those from explants 1 mm in length from the lip of meristems. Dot-blot hybridization of biotinylated cDNA probes derived from ACLSV and ASGV was used to detect these viruses in crude tissue extracts of in vitro-grown pear plants. Intense signals were consistently detected in untreated plant samples equivalent to less than 0.5 mg tissue. Comparison of signals from dot-blot hybridization and ELISA absorbance values (A405) confirmed that dot-blot hybridization had a higher sensitivity than PAS-ELISA. Dot-blot hybridization could detect viruses with a titer below the threshold level of ELISA. These results indicate that dot-blot hybridization is a useful tool for large-scale surveys of viruses, which facilitates the production of virus-free propagation materials in certification and sanitation programs. Results of PAS-ELISA and dot-blot hybridization showed that high virus elimination efficiency was achieved by a combination of thermotherapy for 35 days and in vitro culture of 1 mm meristem tips. [ABSTRACT FROM AUTHOR]

Abstract: Soybean dwarf virus (SbDV) is divided into four strains (YS, YP, DS, and DP) on the basis of host symptoms in infected soybean plants and on aphid vector specificity. To detect and discriminate each strain of SbDV by dot-blot hybridization, probes Y, D, S, and P were prepared. Probes Y and D, covering most of the 3′-noncoding region of the viral genome containing the sequence of small subgenomic RNA, could discriminate strains in accord with the host symptoms. Probes S and P were derived from the 5′-half of open reading frame 5 encoding the N-terminal half of the readthrough domain which is closely related to the aphid vector specificity of each strain. Thus, the four SbDV strains could be discriminated by the combination of these probes. This method, based on a procedure specific to the SbDV sequence, is a good alternative for routine examination of infected plants in soybean breeding programs for evaluation of resistance to SbDV and for assessment of the distribution of each strain in epidemiological studies. [Copyright &y& Elsevier]

Examines the internal fruit rot caused by Fusarium subglutinans in sweet peppers. Development of a dot-blot hybridization technique for rapid and accurate detection and diagnosis of the plant disease; Differentiation of the disease from fruit rots of peppers caused by other pathogens; Capability of the dot-blot assay to detect F. subglutinans infection in the frozen and fresh host tissues.

An experiment was carried out with plants grown in containment in order to determine potential effects on metabolically active microbial populations as well as on soil enzyme activities in the rhizosphere of genetically modified Basta-tolerant oilseed rape. Transgenic as well as isogenic wild-type plants were grown in combination with the associated herbicide Basta (active ingredient glufosinate) or the herbicide Butisan S (active ingredient metazachlor), respectively. In control treatments, weeds were mechanically removed. Rhizosphere soil was sampled at the early and late flowering stage as well as at senescence. RNA was isolated and 16S rRNA was amplified by reverse transcription and PCR. Amplicons were subjected to denaturating gradient gel electrophoresis in order to generate community fingerprints of metabolically highly active bacteria. Additionally, RNA was hybridized with group-specific probes. Furthermore, bacterial biomass and activities of invertase, alkaline phosphatase, urease and arylsulfatase were determined. Results showed that oilseed rape rhizosphere bacteria were affected by the genetic modification as well as by the herbicide application, however, these effects were minor compared to the influence of the plant growth stage. At senescence, invertase, urease and alkaline phosphatase activities were significantly enhanced in the rhizospheres of transgenic plants as compared to wild-type plants propably due to an altered root exudation in comparison to the wildtype plant. Dot blot hybridizations indicated altered activities and/or abundances of various phylogenetic groups at all sampling times. The transformation process may have unintentionally altered the physiology of plants (e.g. root exudation) leading to changes in rhizosphere community structures and bacterial activities. [ABSTRACT FROM AUTHOR]

A nested-polymerase chain reaction (PCR) has been set up to be performed in a single closed tube for the detection of Pseudomonas savastanoi pv. savastanoi. Nested-PCR coupled with dot-blot hybridization was able to detect up to one cell of the target per ml of olive extract, showing the greatest sensitivity compared with all previously reported detection assays. Validation of the developed procedure for diagnosis and epidemiological purposes was achieved by testing ca. 240 asymptomatic plant samples from olive trees. When performing the other previously reported techniques (bacterial isolation and single PCR), P. savastanoi was detected in 50 of the analyzed samples, while with the new developed nested-PCR assay, the bacterium was detected in 82 samples. [Copyright &y& Elsevier]

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 102 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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.

Exfoliated cervical cells from 321 Japanese women were examined for human papillomavirus (HPV) DNA types 6, 11, 16, 18, 31, 33 and 35 using polymerase chain reaction (PCR) and dot-blot hybridization methods. HPV DNA was present in 9.3% of patients with normal cervixes, 72.7% of patients with cervical intraepithelial neoplasia (CIN) and 77.8% of patients with invasive carcinoma. Younger patients (29 years) with normal cervixes had a 18.5% incidence of HPV DNA, whilst similar older patients (50 years) had a 1.9% incidence, a significant difference (χ2= 6.478, P < 0.01). In the CIN I and II groups, an incidence of 11.1% of types 16 and 18 was found, while in the CIN III or invasive carcinoma group the incidence was 58.1%, again a significant difference (χ2 = 12.075, P < 0.01). Furthermore, persistence or progression of CIN showed a significant correlation with infections by types 16 and 33 (χ2= 4.904, P < 0.01). However, no significantly different incidence of HPV infection was found between the CIN and the invasive carcinoma groups. It is suggested that (a) younger patients with normal cervixes have a higher incidence of HPV infection than do older patients; (b) HPV types 16, 18 and 33 are important etiologic agents of CIN III and invasive carcinoma, as well as in the persistence and progression of CIN; (c) progression of CIN to invasive carcinoma may depend on factors other than HPV infection in the cervix. [ABSTRACT FROM AUTHOR]

Morphology, the extent of elimination of donor chromosomes and the organelle composition of highly asymmetric somatic hybrid plants between a interspecific tomato hybrid Lycopersicon esculentum x L. pennellii (EP) as donor and a Solarium melongena, eggplant (E), recipient, were studied. Morphologically, the somatic hybrids most resemble eggplant but, due to polyploidy, growth is slower relative to both fusion parents. The somatic hybrids produce flowers that are characterized by abnormal styles, stigmas and by anthers which do not produce pollen. Limited amounts of donor EP genomic DNA were found in the three somatic hybrid plants (H18-1, H18-2 and H18-3), by dot-blot hybridization with probe pTHG2, equivalent to 6.23,5.41, and 5.95% EP, respectively. These percentages translated to the presence of 3.59, 2.90 and 3.19 average-size EP chromosomes in plants H1 8-1,-2 and-3, respectively. RFLP determination of L. esculentum- and L. pennellii-specific chromosomes revealed that only fragments of eight to ten out of the 24 EP chromosomes (EP has 12 L. esculentum and 12 L. pennellii chromosomes) are present in the asymmetric somatic hybrid plants. Loci of L. esculentum and L. pennellii were evenly represented in plants H18-1, -2, and -3: four to five from L. esculentum and four to five from L. pennellii. All somatic hybrid plants retained locus TG22, chromosome 4, from both EP species. Although the regeneration of plants, H18-1, -2 and-3 was from one callus, loci TG31 and TG79 of L. esculentum chromosome 2 and L. pennellii chromosome 9, respectively, were missing in hybrid plant H18-1. The three somatic hybrid plants all had chloroplast DNA fragments specific for S. melongena. The mitochondrial genome (mtDNA) in the asymmetric somatic hybrids showed predominantly the pattern of eggplant; however, some eggplant-specific polymorphic bands were not present in the three plants. [ABSTRACT FROM AUTHOR]

Discusses the use of DNA hybridization assays in the detection of piscirickettsia salmonis in salmonid fish. Difference between the dot-blot hybridization and in situ hybridization approaches; Optimum dilution of the anti-digoxigenin alkaline phosphatase conjugated antibody; Efficacy of the DNA hybridization approaches in detecting piscirikettsia salmonis.