Penggunaan Pelacak DNA untuk Deteksi Papaya ringspot virus dengan Metode Hibridisasi Asam Nukleat

Use of DNA Probe for Detection of Papaya ringspot virus Using Nucleic Acid Hybridization MethodPapaya ringspot caused by Papaya ringspot virus (PRSV) is one of the most destructive diseases of papaya. The disease had not been found in Indonesia, until disease outbreak in Nangroe Aceh Darussalam was reported in 2012. Since then, the disease spread rapidly in most papaya growing areas in Sumatera, Java and Bali. Papaya ringspot virus (PRSV) is generally detected using serological or polymerase chain reaction methods. Improvement in detection method is necessary to facilitate a more reliable tool for controlling the spread of PRSV. The aim of the research was to construct DNA probe for development of detection method based on nucleic acid hybridization. Molecular characterization based on HCPro gene sequence indicated high homology (97.88 to 99.05%) among PRSV isolates from Boyolali (Central Java), Medan (North Sumatera), Sleman (Yogyakarta) and Tabanan (Bali). Two DNA clones of HCPro gene were selected for probe construction and the probes were then labeled using PCR DIG-dioxigenin. Optimization of nucleic acid dot blot hybridization method to achieve strongest positive reaction was developed, i.e. using stringency washes at 1×SSC, 0.1% SDS, incubation at 60 oC for 15’. The DNA probe for PRSV has a high specificity and sensitifity; it could detect PRSV at the lowest concentration of nucleic acid (0.062 µg µL-1).

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Chemically synthesized non-radioactive biotinylated long-chain nucleic acid hybridization probes

Biochemical Journal ◽

10.1042/bj2510935 ◽

1988 ◽

Vol 251 (3) ◽

pp. 935-938 ◽

Cited By ~ 6

Author(s):

A H al-Hakim ◽

R Hull

Keyword(s):

Nucleic Acid ◽

Blot Hybridization ◽

Nucleic Acid Hybridization ◽

Cross Linking ◽

Dot Blot ◽

Hybridization Probes ◽

Target Dna ◽

Amino Derivatives ◽

Biotin Molecule ◽

Long Chain

A new method for the chemical labelling of nucleic acid with biotin to produce non-radioactive probes has been developed. NN'-Bis-(3-aminopropyl)butane-1,4-diamine (spermine) and long-chain diamino compounds (diaminohexane, diaminodecane and diaminododecane) were linked covalently to biotin and the resultant conjugates were attached to nucleic acid by using a cross-linking reagent (glutaraldehyde or diepoxyoctane). Iodoacetylation and biotinylation of the long-chain diamino compounds produced modified biotinylated conjugates that can be linked to DNA without the use of a cross-linking reagent. These types of probes attach one biotin molecule to each linker arm of spermine, diamino and iodoacetylated amino derivatives. Such probes have long linker arms separating the biotin moiety from the hybridization sites of the nucleic acid. These probes can detect 10 pg of target DNA by dot-blot hybridization.

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Spread and Increase of Ratoon Stunting Disease of Sugarcane and Comparison of Disease Detection Methods

Plant Disease ◽

10.1094/pdis.1999.83.12.1170 ◽

1999 ◽

Vol 83 (12) ◽

pp. 1170-1175 ◽

Cited By ~ 25

Author(s):

J. W. Hoy ◽

M. P. Grisham ◽

K. E. Damann

Keyword(s):

Yield Loss ◽

Detection Method ◽

Xylem Sap ◽

Disease Spread ◽

Detection Methods ◽

Disease Detection ◽

Correct Identification ◽

Dot Blot ◽

Ratoon Stunting Disease ◽

Enzyme Immunoassays

The spread and increase of ratoon stunting disease (RSD) resulting from two mechanical harvests were compared in eight sugarcane cultivars at two locations. RSD spread and increase were detected in the ratoon crops grown after each harvest and varied among cultivars and locations. Disease spread and increase were greater in plants grown from stalks collected at the first harvest than in the first ratoon growth from the harvested field. RSD infection was determined using five disease detection methods: alkaline-induced metaxylem autofluorescence; microscopic examination of xylem sap; and dot blot, evaporative-binding, and tissue blot enzyme immunoassays. The tissue blot enzyme immunoassay was the most accurate RSD detection method. The dot blot and evaporative-binding enzyme immunoassays were the least sensitive for detection of RSD-infected stalks, and alkaline-induced metaxylem autofluorescence was least accurate for correct identification of noninfected stalks. The results indicate that disease spread and increase are variable even among cultivars susceptible to yield loss due to RSD, and the greatest threat of disease spread and increase occurs at planting.

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Characterization of a fusion cDNA (RARA/myl) transcribed from the t(15;17) translocation breakpoint in acute promyelocytic leukemia

Molecular and Cellular Biology ◽

10.1128/mcb.12.2.800-810.1992 ◽

1992 ◽

Vol 12 (2) ◽

pp. 800-810

Author(s):

K S Chang ◽

S A Stass ◽

D T Chu ◽

L L Deaven ◽

J M Trujillo ◽

...

Keyword(s):

Cdna Library ◽

Acute Promyelocytic Leukemia ◽

Blot Analysis ◽

Blot Hybridization ◽

Promyelocytic Leukemia ◽

Dna Probe ◽

Translocation Breakpoint ◽

Chromosome 15 ◽

Dot Blot ◽

Mrna Species

A nonrandom chromosomal translocation breakpoint, t(15;17)(q22;q21), is found in almost all patients with acute promyelocytic leukemia (APL). Most of these breakpoints occur within the second intron of the retinoic acid receptor-alpha (RARA) gene. We screened a cDNA library of APL and have identified and sequenced a cDNA transcribed from the t(15;17) translocation breakpoint. The 5' end of cDNA p1715 consists of 503 bp of the RARA exon II sequence. A 1.76-kb cDNA without homology to any known gene available in GenBank was found truncated downstream. This cDNA sequence was assigned to chromosome 15 by dot blot hybridization of the flow cytometry-sorted chromosomes. We designate this fusion cDNA RARA/myl, which is different from myl/RARA reported by de The et al. (H. de The, C. Chomienne, M. Lanotte, L. Degos, and A. Dejean, Nature (London) 347:558-561, 1990). This result demonstrates that the two different types of hybrid mRNA can be transcribed from this breakpoint. We screened a non-APL cDNA library and identified a 2.8-kb myl cDNA. This cDNA is able to encode a polypeptide with a molecular weight of 78,450. Alternative splicing of the myl gene which resulted in myl proteins with different C terminals was found. Southern blot analysis of the genomic DNA isolated from 17 APL patients by using the myl DNA probe demonstrated that the myl gene in 12 samples was rearranged. Northern (RNA) blot analysis of RARA gene expression in two APL RNA samples showed abnormal mRNA species of 4.2 and 3.2 kb in one patient and of 4.8 and 3.8 kb in another patient; these were in addition to the normal mRNA species of 3.7 and 2.7-kb. The myl DNA probe detected a 2.6-kb abnormal mRNA in addition to the normal mRNA species of 3.2, 4.2, and 5.5 kb. Using the polymerase chain reaction, we demonstrated that both RARA/myl and myl/RARA were coexpressed in samples from three different APL patients. From this study, we conclude that the t(15;17) translocation breakpoint results in the transcription of two different fusion transcripts which are expected to be translated into fusion proteins.

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Evidence for Association of a Viroid with Tapping Panel Dryness Syndrome of Rubber (Hevea brasiliensis)

Plant Disease ◽

10.1094/pdis.2000.84.10.1155c ◽

2000 ◽

Vol 84 (10) ◽

pp. 1155-1155 ◽

Cited By ~ 10

Author(s):

P. Ramachandran ◽

S. Mathur ◽

L. Francis ◽

A. Varma ◽

J. Mathew ◽

...

Keyword(s):

Nucleic Acid ◽

Polyacrylamide Gel Electrophoresis ◽

Blot Hybridization ◽

Potato Spindle Tuber Viroid ◽

Low Molecular Weight ◽

Total Nucleic Acid ◽

Dot Blot ◽

Dot Blot Hybridization ◽

Low Salt ◽

Tapping Panel Dryness

Tapping panel dryness (TPD) is one of the most destructive maladies affecting rubber plantations and is becoming a matter of serious concern. Reduced latex yield leading to total drying of the tapping panel is the obvious symptom. The cause of TPD syndrome is unknown but has been mostly attributed to abiotic causes. In India, the high yielding commercial clone RRII 105 is affected by TPD, leading to enormous losses. We have observed that TPD-affected trees show symptoms of bark scaling, cracking, drying, necrotic streaking, and browning of internal bark leading to the decay of internal tissues. Often prominent abnormal bulges on the lower part of tree trunks occur where the first panel begins to dry. Investigations on TPD-affected rubber samples did not reveal the association of fungus, bacterium, virus, or a protozoan. Total nucleic acid extracts purified from leaf and bark tissues of affected samples and analyzed by polyacrylamide gel electrophoresis under denaturing conditions of low salt and high temperature showed the presence of nucleic acids similar in electrophoretic mobility to low molecular weight (LMW) RNA, of ~359 nucleotides such as potato spindle tuber viroid (PSTVd). The LMW nucleic acid detected from TPD-affected samples was found to be RNA based on its sensitivity to RNase and insensitivity to DNase, phenol, and heat treatments. The LMW RNA was purified and cloned in a pUC 19-derived vector by using primers specific to PSTVd (1). The cloned DNA, when random labeled and used as probe reacted specifically to nucleic acid extracts from TPD-affected rubber trees but not from healthy tissue in dot-blot hybridization assays. Based on the above findings, a viroid etiology for TPD syndrome is proposed. Reference: (1) R. A. Owens, A. T. Candresse, and T. O. Diener. Virology 175:238, 1990.

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Detection and identification of poliovirus in environmental samples using nucleic acid hybridization

Canadian Journal of Microbiology ◽

10.1139/m90-113 ◽

1990 ◽

Vol 36 (9) ◽

pp. 664-669 ◽

Cited By ~ 2

Author(s):

David R. Preston ◽

G. Rasul Chaudhry ◽

Samuel R. Farrah

Keyword(s):

Tissue Culture ◽

Nucleic Acid ◽

Environmental Samples ◽

Nucleic Acid Hybridization ◽

Dot Blot ◽

Polio Virus ◽

Detection And Identification ◽

Specificity And Sensitivity ◽

Hybridization Procedure ◽

Great Specificity

A procedure was developed to effectively extract viral RNA from poliovirus tissue-culture lysates while eliminating the hybridization background associated with tissue cultures uninfected with poliovirus. Poliovirus cDNA cloned into a pUC vector was used as probe. Both the recombinant plasmids and the cDNA showed great specificity towards poliovirus. However, both probes hybridized with the single-stranded DNA coliphage [Formula: see text]. Tissue culture was found to be an effective method to increase the number of viruses found in environmental samples to a level detectable by hybridization procedures, whereas direct hybridization of RNA from unamplified and highly concentrated raw wastewater showed poor hybridization signals. The specificity and sensitivity of the hybridization procedure developed during these studies indicate that this method may be best suited for the identification rather than the detection of viruses isolated from environmental samples. Key words: nucleic acid hybridization, polio virus, water, dot blot.

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A Rapid Protocol for Evaluating Prunus Germplasm for Tomato Ringspot Virus Resistance

HortScience ◽

10.21273/hortsci.29.9.1068 ◽

1994 ◽

Vol 29 (9) ◽

pp. 1068-1070

Author(s):

J.M. Halbrendt ◽

E.V. Podleckis ◽

A. Hadidi ◽

R. Scorza ◽

R. Welliver

Keyword(s):

Prunus Persica ◽

Blot Hybridization ◽

Control Plant ◽

Chenopodium Quinoa ◽

Ringspot Virus ◽

Prunus Domestica ◽

Dot Blot ◽

Tomato Ringspot Virus ◽

Leaf Tissues ◽

And Control

Rooted cuttings of `Halford' and `Redhaven' peaches [Prunus persica (L.) Batsch] and `Stanley' (Prunus domestica L.) and `Marianna 2624' (P. cerasifera × P. munsoniana) plums were planted in soil containing ≈38 tomato ringspot virus-(TomRSV) infested nematodes (Xiphinema americanum sensu lato Cobb) per 100 cc. Test- and control-plant sap extracts were made from root and leaf tissues after 10, 22, and 34 weeks. Aliquots of these samples were assayed by mechanical inoculation to Chenopodium quinoa Willd. Total nucleic-acid extracts prepared from the remainder of each sample were analyzed by dot blot hybridization using a cRNA probe for TomRSV. The bioassay identified one `Stanley' and two `Redhaven' infected plants. Hybridization results indicated that two of two `Stanley', three of three `Halford', five of five `Redhaven', and zero of six `Marianna 2624' were infected. Our results demonstrate the sensitivity of molecular hybridization for TomRSV detection in Prunus and substantiate the TomRSV resistance of `Marianna 2624'.

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Primers and a Specific DNA Probe for Detecting Lactic Acid Bacteria Producing 3-Hydroxypropionaldehyde from Glycerol in Spoiled Ciders

Journal of Food Protection ◽

10.4315/0362-028x-64.6.833 ◽

2001 ◽

Vol 64 (6) ◽

pp. 833-837 ◽

Cited By ~ 22

Author(s):

OLIVIER CLAISSE ◽

ALINE LONVAUD-FUNEL

Keyword(s):

Lactic Acid ◽

Lactic Acid Bacteria ◽

Polymerase Chain Reaction Amplification ◽

Klebsiella Oxytoca ◽

Blot Hybridization ◽

Dna Probe ◽

Dot Blot ◽

Glycerol Dehydratase ◽

Polymerase Chain ◽

Dna Primers

Of the 40 strains isolated from several spoiled ciders where glycerol was degraded, 36 were identified as Lactobacillus collinoides, three were Lactobacillus hilgardii, and one was Lactobacillus mali. However, only 30 L. collinoides and two L. hilgardii could degrade glycerol. The glycerol dehydratase activity was shown. The main product of the transformation was 1,3 propanediol. Two DNA primers GD1 and GD2 were chosen in the region encoding one of the subunits of glycerol dehydratase of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella Typhimurium, and Clostridium pasteurianum. A 279-bp amplicon in polymerase chain reaction amplification was obtained with the genomic L. collinoides IOEB 9527 DNA as template. The amino acid sequence deduced from the amplicon DNA sequence showed a very high similarity and identity with the gene of gram-negative and C. pasteurianum species. After labeling, the amplicon was used as DNA probe in dot-blot hybridization with the genomic DNA of all the tested strains. Only strains that could degrade glycerol hybridized. Moreover, polymerase chain reactions using GD1 and GD2 revealed only glycerol dehydratase genes of positive L. collinoides and L. hilgardii strains. The primers and the amplicon proved to be suitable and reliable tools to detect the lactic acid bacteria involved in the deterioration of cider.

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Time-resolved fluorescence detection of enzyme-amplified lanthanide luminescence for nucleic acid hybridization assays

Clinical Chemistry ◽

10.1093/clinchem/37.9.1506 ◽

1991 ◽

Vol 37 (9) ◽

pp. 1506-1512 ◽

Cited By ~ 23

Author(s):

E F Templeton ◽

H E Wong ◽

R A Evangelista ◽

T Granger ◽

A Pollak

Keyword(s):

Alkaline Phosphatase ◽

Nucleic Acid ◽

Fluorescence Detection ◽

Dna Hybridization ◽

Nucleic Acid Hybridization ◽

Time Resolved Fluorescence ◽

Dot Blot ◽

Time Resolved ◽

Lanthanide Luminescence ◽

Hybridization Assays

Abstract A new nonisotopic detection method based on time-resolved fluorescence for nucleic acid hybridization assays with alkaline phosphatase labels has been developed: enzyme-amplified lanthanide luminescence (EALL). EALL combines the amplification of an enzyme label with the sensitivity and background elimination of time-resolved fluorescence detection of lanthanide ion luminescence. The detection system for alkaline phosphatase makes use of a phosphorylated salicylic acid derivative that, upon dephosphorylation, gives a product capable of forming a luminescent terbium chelate. We demonstrate DNA hybridization assays by using two substrates, one for membrane and one for solution-based formats. Using the substrate that produces a more adhesive product allows performance of dot-blot and Southern blot assays on nylon membranes; results can be recorded with a time-resolved photographic camera system, or with an ultraviolet transilluminator-based system. Less than 4 pg of target sequence can be detected in a dot-blot assay after incubation with substrate for 2-4 h. DNA microwell-plate hybridization assays with the more soluble substrate/product pair can be quantified with time-resolved fluorescence plate readers, giving a similar detection sensitivity. EALL is thus a practical time-resolved fluorescence-based alternative to other detection systems for DNA hybridization assays.

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