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Dot-blot Hybridization (dot-blot + hybridization)
Selected AbstractsSensitive and Specific Digoxigenin-labelled RNA Probes for Routine Detection of Citrus tristeza virus by Dot-blot HybridizationJOURNAL OF PHYTOPATHOLOGY, Issue 6 2006L. Barbarossa Abstract 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. [source] A comparison of molecular methods for the routine detection of viroids,EPPO BULLETIN, Issue 3-4 2000R. A. Mumford Viroids, such as Chrysanthemum stunt viroid (CSVd) and Potato spindle tuber viroid (PSTVd), are important plant pathogens. However, because of their unique biological properties, viroids have proved, in the past, difficult to diagnose. The use of molecular methods has now changed this and this paper reports the comparison of three such methods (dot-blot hybridization using DIG-labelled cRNA probes, reverse transcription-polymerase chain reaction (RT-PCR) and TaqMan), which have been developed for routine detection of CSVd. Sensitivity comparisons show that the TaqMan assay is more sensitive than either RT-PCR (100 times) and hybridization (1000 times). RT-PCR and TaqMan assays have also been developed to detect PSTVd. In addition to the development of sensitive detection methods, considerable emphasis has been placed on making these assays amenable to mass-scale detection through the use of internal controls and the development of a rapid, reliable probe capture extraction system. [source] Distribution and diversity of type III secretion system-like genes in saprophytic and phytopathogenic fluorescent pseudomonadsFEMS MICROBIOLOGY ECOLOGY, Issue 3 2004Sylvie Mazurier Abstract Type three secretion systems (TTSSs) are protein translocation mechanisms associated with bacterial pathogenicity in host plants, and hypersensitive reactions in non-host plants. Distribution and diversity of TTSS-like genes within a collection of saprophytic and phytopathogenic fluorescent pseudomonads were characterized. This collection included 16 strains belonging to 13 pathogenic species, and 87 strains belonging to five saprophytic species isolated from plant rhizosphere and soil. Presence of conserved hypersensitive reaction/pathogenicity (hrp) genes (hrc RST) was assessed both by PCR using primers designed to amplify the corresponding sequence and by dot-blot hybridization using a PCR-amplified hrc RST fragment as a probe. PCR allowed the detection of TTSS-like genes in 75% and 32% of the phytopathogenic and saprophytic strains, respectively, and dot-blot hybridization in 100% and 49% of the phytopathogenic and saprophytic strains, respectively. The restriction fragment length polymorphism (RFLP) of 26 amplified hrc RST fragments revealed a considerable diversity. Twenty-one distinct RFLP types were identified and one hrc RST fragment was sequenced per RFLP type. The obtained hrc RST sequences clustered into three groups. Two of these groups included both phytopathogenic and saprophytic strains. The diversity of 16S rRNA genes, commonly used as an evolution marker, was characterized using PCR-RFLP. Polymorphism of the 16S rRNA genes corresponded to that of hrc RST genes, suggesting that these genes have followed a similar evolution. However, the occurrence of few mismatches suggests that sometimes TTSS-like genes might have undergone horizontal genetic transfer. [source] Study of Short- and Long-Term Storage of Teeth and Its Influence on DNAJOURNAL OF FORENSIC SCIENCES, Issue 6 2009Leticia Rubio M.D. Abstract:, DNA degradation can interfere with the resolution of forensic cases. Allelic dropout often reduces the opportunity for adequate comparisons between degraded and reference samples. This study analyzed DNA degradation in 24 extracted teeth after storage at room temperature for 0, 2, 5, and 10 years. DNA concentration, quantified by dot-blot hybridization, declined significantly for the first 2 years, but there was no significant further degradation from the second to the tenth year of storage. COfilerÔ analysis was used and the allelic dropout ratio for the amelogenin locus relative to CSF1PO locus was also estimated. Statistically significant differences were found between fresh teeth and teeth from the 2- and 5-year groups but not from the 10-year group. Under our storage conditions most of the DNA degradation occurred during the first 2 years. Further research is needed to control for individual and external factors that could affect DNA. [source] Population-based type-specific prevalence of high-risk human papillomavirus infection in middle-aged Swedish WomenJOURNAL OF MEDICAL VIROLOGY, Issue 4 2002Ola Forslund Abstract Human papillomavirus (HPV) DNA testing can be used to identify women at risk of the development of cervical cancer. The cost-effectiveness of HPV screening is dependent on the type-specific HPV prevalence in the general population. The present study describes the prevalence and spectrum of high-risk HPV types found in a large real-life population-based HPV screening trial undertaken entirely within the cervical screening program offered to middle-aged Swedish women. Cervical brush samples from 6,123 women aged 32,38 years were analyzed using a general HPV primer (GP5+/6+) polymerase chain reaction-enzyme immunoassay (PCR-EIA) combined with reverse dot-blot hybridization for confirmation and HPV typing by a single assay. In this study, 6.8% (95% CI 6.2,7.5) (417/6,123) were confirmed as high-risk HPV positive. Infections with 13 different high-risk HPV types were detected, of which HPV 16 was the most prevalent type (2.1%; 128/6,123), followed by HPV 31 (1.1%; 67/6,123). Any one of the HPV types 18, 33, 35, 39, 45, 51, 52, 56, 58, 59, or 66 was detected in 3.6% (223/6,123) of the women. Infection with two, three, and five types simultaneously was identified in 32, 5, and 1 women, respectively. The combination of PCR-EIA as a screening test and reverse dot-blot hybridization as a confirmatory test, was found to be readily applicable to a real-life population-based cervical screening. The type-specific HPV prevalence found support in previous modeling studies suggesting that HPV screening may be a favorable cervical screening strategy. J. Med. Virol. 66:535,541, 2002. © 2002 Wiley-Liss, Inc. [source] Sensitive and Specific Digoxigenin-labelled RNA Probes for Routine Detection of Citrus tristeza virus by Dot-blot HybridizationJOURNAL OF PHYTOPATHOLOGY, Issue 6 2006L. Barbarossa Abstract 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. [source] Limited movement of Cucumber mosaic virus (CMV) in yellow passion flower in BrazilPLANT PATHOLOGY, Issue 2 2002R. Gioria 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. [source] A sensitive method for detecting bamboo mosaic virus (BaMV) and establishment of BaMV-free meristem-tip culturesPLANT PATHOLOGY, Issue 1 2000A sensitive method was used to detect bamboo mosaic virus (BaMV) and its associated satellite RNA (satBaMV) by 32P- and digoxigenin (Dig)-labelled probes synthesized from cDNA clones of BaMV genomic (L probe) and satBaMV (S probe) RNA. Both the 32P- and Dig-labelled L and S probes could detect as little as 490 pg of BaMV viral RNA by slot- and dot-blot hybridization. In infected leaf extracts, 32P-labelled L and S probes detected virus at 25-fold higher dilutions than Dig-labelled probes, which were also successfully used to detect BaMV infection in plants derived from meristem-tip culture. However, immunoassays failed to detect BaMV in meristem culture. By dot-blot hybridization assays, 25% of the seedlings were shown to be virus-free. These results suggest that a highly sensitive method for the detection of BaMV infection is required for the establishment of BaMV-free cultures. Meristem-tip culture also provides an efficient method for obtaining virus-free bamboo plants. [source] | ||||||||||