Rapid indexing of the sunblotch disease of avocados using a complementary DNA probe to avocado sunblotch viroid

A routine procedure has been established for the sensitive and specific detection of avocado sunblotch viroid in partially purified nucleic acid extracts of avocado leaves by hybridisation analysis with ³²P-complementary DNA prepared against the purified viroid. Avocado sunblotch viroid was shown to be present in 12 avocado trees that had indexed positive in a biological test for sunblotch disease but was absent from 10 trees that indexed negative. The complete correlation between sunblotch disease and the presence of viroid indicates that the complementary DNA hybridisation assay procedure can be used for the indexing of sunblotch disease. The overall procedure of leaf extraction and hybridisation analysis can be completed in 5 days and is to be compared with up to 2 yr required for indexing by biological methods. The level of avocado sunblotch viroid in partially purified nucleic acid extracts of a number of different sources of sunblotch infected avocado leaves was found to vary 10 000-fold from 0.2% to 2 × 10^-5% by weight. The lower limit of detectability of the viroid by the hybridisation assay is considered to be about 10^-5% by weight; this is at least 10³ times more sensitive than the detection of the viroid by polyacrylamide gel electrophoresis of the leaf nucleic acid extracts followed by staining.     

In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus

Viroids, small single-stranded circular RNA molecules, are the smallest known infectious agents in Nature. The apparent inability of viroids to encode for proteins means that they must rely fully on host functions for their replication. The specific ultrastructural localization of viroids is fundamental to the determination of their replication strategies. In this paper the first in situ hybridization study to localize viroids within the cell at the electron microscope level is reported. Biotin-labelled RNA probes were used with subsequent detection by gold-labelled monoclonal anti-biotin antibodies to localize avocado sunblotch viroid and coconut cadang cadang viroid. Avocado sunblotch viroid was located in chloroplasts, mostly on the thylakoid membranes of cells from infected leaves of avocado (Persea americana). In contrast, coconut cadang cadang viroid was located in the nucleolus and nucleoplasm of cells of infected leaves of oil palm (Elaeis guineensis), with a higher concentration in the nucleolus. The results provide insight on the potential host RNA polymerases involved in the replication of these two viroids.     

Polyamines in plants infected by citrus exocortis viroid or treated with silver ions and ethephon

The levels of polyamines in leaves of Gynura aurantiaca DC and tomato, Lycopersicon esculentum Mill. cv Rutgers, infected with citrus exocortis viroid (CEVd) or treated with silver nitrate or ethephon (2-chloroethylphosphonic acid) were measured by HPLC in relation to development of symptoms. Previously it had been demonstrated that treatment of G. aurantiaca plants with silver nitrate or ethephon closely mimicked the effects of viroid infection in the plants. In the studies reported here, a marked decrease in putrescine level was observed in plants infected by CEVd or treated with silver ions or ethephon. There was no significant change in either spermidine or spermine levels. Treatment of G. aurantiaca plants with specific inhibitors of ethylene biosynthesis (aminoethoxyvinylglycine, Co²⁺) or ethylene action (norbornadiene) prevented the decrease of putrescine associated with silver nitrate treatment and had no effect on spermidine or spermine levels. The development of viroid-like symptoms, the production of associated pathogenesis-related proteins, and the rise in protease activity induced by silver nitrate, were all suppressed by exogenous application of putrescine. The decreased level of putrescine as an ethylene-mediated step in the transduction of the viroid and silver or ethephon signaling is discussed.     

Highly sensitive fluorescent-labeled probes and glass slide hybridization for the detection of plant RNA viruses and a viroid

In this study, a modified method of the conventional RNA dot-blot hybridization was established, by replacing ~(32)p labels with CY5 labels and replacing nylon membranes with positive-charged glass slides, for detecting plant RNA viruses and a viroid. The modified RNA dot-blot hybridization method was named glass slide hybridization. The optimum efficiency of RNA binding onto the surfaces of activated glass slide was achieved using aminosilane-coated glass slide as a solid matrix and 5×saline sodium citrate (SSC) as a spotting solution. Using a CY5-labeled DNA probe prepared through PCR amplification, the optimized glass slide hybridization could detect as little as 1.71 pg of tobacco mosaic virus (TMV) RNA. The sensitivity of the modified method was four times that of dot-blot hybridization on nylon membrane with a ~(32)P-labeled probe. The absence of false positive within the genus Potyvirus [potato virus A, potato virus Y (PVY) and zucchini yellow mosaic virus] showed that this method was highly specific. Furthermore, potato spindle tuber viroid (PSTVd) was also detected specifically. A test of 40 field potato samples showed that this method was equivalent to the conventional dot-blot hybridization for detecting PVY and PSTVd. To our knowledge, this is the first report of using dot-blot hybridization on glass slides with fluorescent-labeled probes for detecting plant RNA viruses and a viroid.     

A multiplex real-time PCR method using hybridization probes for the detection and the quantification of Fusarium proliferatum, F. subglutinans, F. temperatum, and F. verticillioides

Maize contamination with Fusarium species is one of the major sources of mycotoxins in food and feed derivates. In the present study, a LightCycler^® real-time PCR method using hybridization probes was developed for the specific identification, detection, and quantification of Fusarium proliferatum, Fusarium subglutinans, Fusarium temperatum, and Fusarium verticillioides, four mycotoxin-producing pathogens of maize. Primers and hybridization probes were designed to target the translation elongation factor 1α (EF-1α) gene of F. subglutinans and F. temperatum or the calmodulin (Cal) gene of F. proliferatum and F. verticillioides. The specificity of the real-time PCR assays was confirmed for the four Fusarium species, giving no amplification with DNA from other fungal species commonly recovered from maize. The assays were found to be sensitive, detecting down to 5 pg and 50 pg of Fusarium DNA in simplex and multiplex conditions respectively, and were able to quantify pg-amounts of Fusarium DNA in artificially Fusarium-contaminated maize samples. The real-time PCR method developed provides a useful tool for routine identification, detection, and quantification of toxigenic Fusarium species in maize.     

Development and Testing of a DNA Macroarray To Assess Nitrogenase (nifH) Gene Diversity

A DNA macroarray was developed and evaluated for its potential to distinguish variants of the dinitrogenase reductase (nifH) gene. Diverse nifH gene fragments amplified from a clone library were spotted onto nylon membranes. Amplified, biotinylated nifH fragments from individual clones or a natural picoplankton community were hybridized to the array and detected by chemiluminescence. A hybridization test with six individual targets mixed in equal proportions resulted in comparable relative signal intensities for the corresponding probes (standard deviation, 14%). When the targets were mixed in unequal concentrations, there was a predictable, but nonlinear, relationship between target concentration and relative signal intensity. Results implied a detection limit of roughly 13 pg of target ml⁻¹, a half-saturation of signal at 0.26 ng ml⁻¹, and a dynamic range of about 2 orders of magnitude. The threshold for cross-hybridization varied between 78 and 88% sequence identity. Hybridization patterns were reproducible with significant correlations between signal intensities of duplicate probes (r = 0.98, P < 0.0001, n = 88). A mixed nifH target amplified from a natural Chesapeake Bay water sample hybridized strongly to 6 of 88 total probes and weakly to 17 additional probes. The natural community results were well simulated (r = 0.941, P < 0.0001, n = 88) by hybridizing a defined mixture of six individual targets corresponding to the strongly hybridizing probes. Our results indicate that macroarray hybridization can be a highly reproducible, semiquantitative method for assessing the diversity of functional genes represented in mixed pools of PCR products amplified from the environment.     

Multiplex Fluorogenic Real-Time PCR for Detection and Quantification of Escherichia coli O157:H7 in Dairy Wastewater Wetlands

Surface water and groundwater are continuously used as sources of drinking water in many metropolitan areas of the United States. The quality of water from these sources may be reduced due to increases in contaminants such as Escherichia coli from urban and agricultural runoffs. In this study, a multiplex fluorogenic PCR assay was used to quantify E. coli O157:H7 in soil, manure, cow and calf feces, and dairy wastewater in an artificial wetland. Primers and probes were designed to amplify and quantify the Shiga-like toxin 1 (stx1) and 2 (stx2) genes and the intimin (eae) gene of E. coli O157:H7 in a single reaction. Primer specificity was confirmed with DNA from 33 E. coli O157:H7 and related strains with and without the three genes. A direct correlation was determined between the fluorescence threshold cycle (C_T) and the starting quantity of E. coli O157:H7 DNA. A similar correlation was observed between the C_T and number of CFU per milliliter used in the PCR assay. A detection limit of 7.9 × 10⁻⁵ pg of E. coli O157:H7 DNA ml⁻¹ equivalent to approximately 6.4 × 10³ CFU of E. coli O157:H7 ml⁻¹ based on plate counts was determined. Quantification of E. coli O157:H7 in soil, manure, feces, and wastewater was possible when cell numbers were ≥3.5 × 10⁴ CFU g⁻¹. E. coli O157:H7 levels detected in wetland samples decreased by about 2 logs between wetland influents and effluents. The detection limit of the assay in soil was improved to less than 10 CFU g⁻¹ with a 16-h enrichment. These results indicate that the developed PCR assay is suitable for quantitative determination of E. coli O157:H7 in environmental samples and represents a considerable advancement in pathogen quantification in different ecosystems.     

Virp1 is a host protein with a major role in Potato spindle tuber viroid infection in Nicotiana plants

Viroids are small, circular, single-stranded RNA molecules that, while not coding for any protein, cause several plant diseases. Viroids rely for their infectious cycle on host proteins, most of which are likely to be involved in endogenous RNA-mediated phenomena. Therefore, characterization of host factors interacting with the viroid may contribute to the elucidation of RNA-related pathways of the hosts. Potato spindle tuber viroid (PSTVd) infects several members of the Solanaceae family. In an RNA ligand screening we have previously isolated the tomato protein Virp1 by its ability to specifically interact with PSTVd positive-strand RNA. Virp1 is a bromodomain-containing protein with an atypical RNA binding domain and a nuclear localization signal. Here we investigate the role of Virp1 in the viroid infection cycle by the use of transgenic lines of Nicotiana tabacum and Nicotiana benthamiana that either overexpress the tomato Virp1 RNA or suppress the orthologous Nicotiana genes through RNA silencing. Plants of the Virp1-suppressed lines were not infected by PSTVd or Citrus exocortis viroid through mechanical inoculation, indicating a major role of Virp1 in viroid infection. On the other hand, overexpression of tomato Virp1 in N. tabacum and N. benthamiana plants did not affect PSTVd KF 440-2 infectivity or symptomatology in these species. Transfection experiments with isolated protoplasts revealed that Virp1-suppressed cells were unable to sustain viroid replication, suggesting that resistance to viroid infection in Virp1-suppressed plants is likely the result of cell-autonomous events.     

Ultrasensitive staining of nucleic acids with silver

A method for ultrasensitive detection of proteins on polyacrylamide gels by staining with silver, recently described by C. R. Merril, D. Goldman, S. A. Sedman, and M. H. Ebert (Science211, 1437–1438 (1981)), was applied with slight modifications to staining nucleic acids. Silver staining of double-stranded DNA was at least 100 times as sensitive as fluorescence staining with ethidium bromide, and at least 20 times as sensitive as staining with ammoniacal silver. The limit of detection of double-stranded DNA was approximately 25–50 pg/band with a cross-sectional area of 5 mm². The intensities of silver staining of double-stranded fragments 271 bp or longer from HaeIII endonuclease digests of φX174 RF DNA were linear over a concentration range of 0.25 to 4 ng DNA/band. RNA and single-stranded DNA species as short as 10 to 20 nucleotides were detected with high sensitivity after electrophoresis on denaturing gels containing urea, suggesting that silver staining may be applicable to the sequencing of a few micrograms of unlabeled DNA. Methods for staining DNA using ammoniacal silver were relatively insensitive for small DNA fragments.     

Replication of in vitro constructed viroid mutants: location of the pathogenicity-modulating domain of citrus exocortis viroid

Sequence variants from field isolates of citrus exocortis viroid (CEV) that cause either mild or severe symptoms on tomato plants have previously been classified into two groups, A and B. These groups differ primarily in two domains, P_L and P_R, of the proposed native structure. Infectivity studies with full-length cDNA clones of variants from each class have now directly confirmed the original correlation between Class A sequences and the severe phenotype and between Class B sequences and the mild phenotype. Direct evidence for this correlation could only be obtained by using individual sequence variants since field isolates of CEV have been shown to contain a mixture of RNA species. The construction and infectivity of chimaeric cDNA clones derived from mild and severe sequence variants of CEV has demonstrated that novel, infectious viroid molecules can be generated in vitro, and that P_L is the pathogenicity-modulating domain. The role of the P_R domain is not known but infectivity experiments with one chimaeric cDNA clone suggest that it may influence the efficiency of the infection or replication process of the viroid in the plant.     

Quantification and optimization of Candida albicans DNA in blood samples using Real- Time PCR

Background:

Candida albicans (C. albicans) is a major cause of candidaemia in people with impaired immunity. Blood culture is a “gold standard” for candidaemia detection but is time-consuming and relatively insensitive. We established a real-time PCR assay for C. albicans detection in blood by LightCycler PCR and melting curve analysis.

Methods:

Five milliliter blood samples from healthy volunteers were spiked with 10⁰-10⁶ C. albicans cells to determine the detection limit of our method. DNA was extracted from whole blood using glass beads and the QIAamp DNA Blood Mini Kit (Qiagen, Hilden Germany). DNA from C. albicans isolates were amplified with primers and inserted into Escherichia coli (E. coli) DH5α.1 cells with the TA cloning vector (Invitrogen). The plasmid was used for standardization and optimization. A quantitative PCR assay with the LightCycler amplification and detection system based on fluorescence resonance energy transfer (FRET) with two different specific probes was established. To assess the precision and reproducibility of real-time PCR the intra-assay precision was determined in six consecutive assays.

Results:

No cross-reactivity of the hybridization probes with the DNA of non-C. albicans species or human genomic DNA was observed, which confirmed its 100% specificity. The minimum limit detected was one C. albicans cell or 10⁰ CFU/ml (10 fg) per PCR reaction. The real-time PCR efficiency rate for Candida was high (E = 1.95). Melting curve analysis of C. albicans showed a specific melting peak temperature of 65.76 °C.

Conclusion:

The real-time PCR assay we developed is highly specific and sufficiently sensitive to detect the fungal load for early diagnosis of invasive candidiasis. Key Words: Invasive candidiasis, Real-time PCR, Candida albicans     

Polymerase Chain Reaction Assay for Rapid, Sensitive Detection, and Identification of Colletotrichum gloeosporioides causing Greater Yam Anthracnose

Anthracnose caused by Colletotrichum gloeosporioides is an economically important disease which affects greater yam (Dioscorea alata L.) worldwide. Apart from airborne conidia, the pathogen propagules surviving in soil and planting material are the major sources of inoculum. A nested PCR assay has been developed for specific detection of C. gloeosporioides in soil and planting material. In conventional (single-round) PCR, the limit of detection was 20?pg, whereas in nested PCR the detection limit increased to 0.2?pg of DNA. The primers designed were found to be highly specific and could be used for accurate identification of the pathogen up to species level. The protocol was standardized for detection of the pathogen in artificially and naturally infected field samples.     

Molecular hybridization between rat liver deoxyribonucleic acid and complementary ribonucleic acid

RNA (cRNA) was synthesized in vitro on a template of rat liver DNA and its hybridization with rat liver DNA was studied by using the nitrocellulose-filter method. Sonication of the DNA diminished its apparent capacity to hybridize with RNA by about 50%. This is not due to cross-linkage of DNA molecules, because it could be shown that less than 2% of the sonicated DNA was cross-linked. The effect is due instead to the small size of the sonicated DNA molecules. Below a single-stranded molecular weight of 5×10⁵ the DNA showed a progressive loss of capacity to hybridize with decrease in molecular weight. Evidence is presented suggesting that the apparently diminished capacity of the DNA to hybridize is due to loss of hybridized DNA from the membrane filters. When cRNA at concentrations of up to 25μg/ml is annealed with sonicated total DNA, an apparent hybridization saturation value is found at which about 2.5% of the DNA is hybridized with RNA. Increasing the cRNA concentration tenfold brought about the hybridization of a second component of the DNA approximately equal in amount to the first. The renaturation of rat liver DNA was studied by measuring the fall in the extinction at 260nm and two different components of renaturation were observed within the reiterated fraction of DNA. By hybridizing cRNA with different fractions of rat DNA the two components of the hybridization curve are shown to correspond to the two components of the renaturation curve. The conclusion is drawn that at a cRNA concentration of 250μg/ml most of the reiterated fraction of rat liver DNA is hybridized after annealing for 16h under standard conditions (0.30m-sodium chloride–30mm-sodium citrate at 65°C). Even with such a high cRNA concentration little or no hybridization of the slowly renaturing DNA fraction occurs. It is suggested that the most highly reiterated DNA component is poorly transcribed in vitro.     

Determination of potato spindle tumor viroid and chrysanthenum stund viroid using biotinylated olideoxyribonucleotides]

A 26 base long oligodeoxyribonucleotide complementary to a common RNA sequence of potato spindle tuber viroid (PSTV) and chrysantemum stunt viroid (CSV) was synthesized. The 3'-end biotinylated one was used for the detection of PSTV and CSV RNA immobilized on nitrocellulose filters by nucleic acid hybridization. Visualization of hybridization results was performed by two ways, either by streptavidin-alkaline phosphatase conjugate or streptavidine and biotinylated alkaline phosphatase. It was possible to detect 0.65 ng of purified CSV and PSTV RNA. The suggested system of viroid diseases detection can be used by agricultural and horticultural enterprises.     

Linear oligomeric potato spindle tuber viroid (PSTV) RNAs are accurately processed in vitro to the monomeric circular viroid proper when incubated with a nuclear extract from healthy potato cells

A nuclear extract for the processing of oligomeric viroid RNA in vitro has been prepared from nuclei isolated from healthy potato cells grown in suspension culture. Linear RNA molecules containing concatameric units of (+) or (−) strands, respectively, of the potato spindle tuber viroid (PSTV) were synthesized in vitro with the aid of the SP6 RNA polymerase and used as substrates for processing. When oligomeric linear PSTV (+)RNAs are incubated with the nuclear extract, monomeric linear molecules are accurately excised from them, and ligated to monomeric PSTV (+)RNA circles representing the viroid proper. Oligomeric PSTV (−)RNAs are likewise processed but with a much lower efficiency. Viroid-processing operates although other nucleolytic activities are still present in the extract. These results substantiate our previous finding that oligomeric PSTV does not process autocatalytically under in vitro conditions where certain introns and other RNAs do. This is the first report of an in vitro RNA processing system derived from higher plants.     

Electrochemical detection of Pseudomonas aeruginosa 16S rRNA using a biosensor based on immobilized stem-loop structured probe

We have designed an electrochemical DNA biosensor based on stem-loop structured probes for enzymatic detection of Pseudomonas aeruginosa 16S ribosomal RNA (rRNA) in composting degradation. The probe modified with a thiol at its 5′ end and a biotin at its 3′ end was immobilized on a gold electrode through self-assembly. The stem-loop structured probes were “closed” when target was absent, then the hybridization of the target induced the conformational changes to “open”, along with the biotin at its 3′ end binding with streptavidin-horseradish peroxidase (HRP), and subsequent quanti?cation of the target was detected via electrochemical detecting the enzymatic product in the presence of substrate. Under the optimum experiment conditions, the amperometric current response to HRP-catalyzed reaction was linearly related to the logarithm of the target nucleic acid concentration, ranging from 0.3 and 600 pg/μL, with the detection limit of 0.012 pg/μL. A correlation coefficient of 0.9960 was identified. The 16S rRNA extracted from P. aeruginosa was analyzed by this proposed sensor. The results were in agreement with the reference values deduced from UV spectrometric data. The biosensor was indicative of good precision, stability, sensitivity, and selectivity.     

Large-scale purification of viroid RNA using Cs2SO4 gradient centrifugation and high-performance liquid chromatography

A procedure for the purification of viroid RNA from tomato plants is described which yields up to a milligram of viroid RNA of gel electrophoretic homogeneity within 2 days. This technique is at least three times as fast as previous methods and is generally applicable to other RNA species. Plant material was homogenized and phenol extracted. In a Cs₂SO₄ density gradient, viroid RNA together with low-molecular-weight RNA, was separated from large single-stranded RNA, DNA, polysaccharides, polyphenols, and other compounds. The separation is based on the differences in the buoyant density and on the selective precipitation of large single-stranded RNA in Cs₂SO₄. Further purification of viroid RNA was achieved by HPLC over a weak anion exchanger linked to silica gel of optimized pore size. The elution was carried out by a salt gradient with complete exclusion of divalent metal ions. The procedures were applied to whole plants, leaves, stems, roots, cells, and protoplasts. The yields of nucleic acids at the different steps of purification are given for leaves, stems, and roots.