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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.

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.

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.

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.

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.

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.

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.

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.