DIG-labeled DNA Probes Research Articles

A novel species of RNA virus associated with root lesion nematode Pratylenchus penetrans.

The root lesion nematode Pratylenchus penetrans is a migratory species that attacks a broad range of plants. While analysing transcriptomic datasets of P. penetrans, we have identified a full-length genome of an unknown positive-sense single-stranded RNA virus, provisionally named root lesion nematode virus 1 (RLNV1). The 8614-nucleotide genome sequence encodes a single large polyprotein with conserved domains characteristic for the families Picornaviridae, Iflaviridae and Secoviridae of the order Picornavirales. Phylogenetic, BLAST and domain search analyses showed that RLNV1 is a novel species, most closely related to the recently identified sugar beet cyst nematode virus 1 and potato cyst nematode picorna-like virus. In situ hybridization with a DIG-labelled DNA probe confirmed the presence of the virus within the nematodes. A negative-strand-specific RT-PCR assay detected RLNV1 RNA in nematode total RNA samples, thus indicating that viral replication occurs in P. penetrans. To the best of our knowledge, RLNV1 is the first virus identified in Pratylenchus spp.

FIRST REPORT OF TOMATO INFECTIOUS CHLOROSIS VIRUS IN GLOBE ARTICHOKE ECOTYPES IN APULIA, SOUTHERN ITALY

Apulia (southern Italy) hosts a rich variety of globe artichoke ecotypes of high interest for breeding programs and industrial production. Most of the ecotypes are at risk of genetic erosion because of the high incidence of plant pathogens, which include infections from taxonomically different viruses. In the early autumn of 2014 and 2016 five samples were collected from each of the early-flowering ecotypes, Brindisino, Carciofo di Lucera, Francesina di Brindisi, Francesina di Trinitapoli, Locale di Mola, Nero di Ostuni, Tricasino, Troianella, Verde di Putignano, Violetto di Putignano and the late-flowering ecotypes Bianco di Taranto and Locale di Mola tardivo, and tested for viral infections by dot blot hybridization with Dig-labeled DNA probes and RT-PCR (Minutillo et al., 2012). In the five samples of the ecotypes Verde di Putignano, Violetto di Putignano, Nero di Ostuni and Troianella the hybridization signal revealed the presence of tomato infectious chlorosis virus (TICV) that in the ecotype Troianella was in mixed infection with artichoke Italian latent virus and turnip mosaic virus. TICV infection was confirmed by RT-PCR with TICV-specific primer pair for 5'- TCAGTGCGTACGTTAATGGG-3' and rev 5'-CACAGTATACAGCAGCGGCAG-3' (Minutillo et al. 2012). Amplicons from the ecotypes Verde di Putignano and Troianella were sequenced. Nucleotide sequence analyses revealed 99% identity with a TICV isolate from USA (accession No. FJ542306). Previous surveys detected TICV in globe artichoke in Liguria, Sardinia, Latium, Campania and Sicily but this is the first report of TICV in artichoke in Apulia and in the ecotypes Verde di Putignano, Violetto di Putignano, Nero di Ostuni and Troianella. Artichoke plants infected by TICV did not show symptoms and thus may act as perennial TICV reservoirs.

<em>In Situ</em> Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

In Situ Detection of Bacteria within Paraffin-embedded Tissues Using a Digoxin-labeled DNA Probe Targeting 16S rRNA.

The presence of bacteria within the pocket epithelium and underlying connective tissue in gingival biopsies from patients with periodontitis has been reported using various methods, including electron microscopy, immunohistochemistry or immunofluorescence using bacteria-specific antibodies, and fluorescent in situ hybridization (FISH) using a fluorescence-labeled oligonucleotide probe. Nevertheless, these methods are not widely used due to technical limitation or difficulties. Here a method to localize bacteria within paraffin-embedded tissues using DIG-labeled DNA probes has been introduced. The paraffin-embedded tissues are the most common form of biopsy tissues available from pathology banks. Bacteria can be detected either in a species-specific or universal manner. Bacterial signals are detected as either discrete forms (coccus, rod, fusiform, and hairy form) of bacteria or dispersed forms. The technique allows other histological information to be obtained: the epithelia, connective tissue, inflammatory infiltrates, and blood vessels are well distinguished. This method can be used to study the role of bacteria in various diseases, such as periodontitis, cancers, and inflammatory immune diseases.

First Report of Okra enation leaf curl virus and Associated Cotton leaf curl Multan betasatellite and Cotton leaf curl Multan alphasatellite Infecting Cotton in Pakistan: A New Member of the Cotton Leaf Curl Disease Complex.

Cotton (Gossypium hirsutum L.) is an important and widely cultivated crop in Pakistan, upon which many rely for economic security. Cotton leaf curl disease (CLCuD) is caused by a complex comprising of more than eight species in the genus Begomovirus (family Geminiviridae) with associated betasatellite and alphasatellites. During 2011, characteristic symptoms of leaf curl disease were widespread (>40%), and the whitefly Bemisia tabaci (Genn.) vector of the leaf curl complex was abundant in commercial cotton fields in Burewala, Pakistan. Symptoms included vein thickening, upward or downward leaf curling, and foliar enations. To test for the presence of a begomovirus(es), total DNA was extracted from 100 mg of symptomatic leaf tissues from five different plants (isolates CLCuDBur1 to 5) using the CTAB method (1). Total DNA extracts were used for rolling circle amplification (RCA) using TempliPhi DNA Amplification Kit (GE Healthcare). Of the five field isolates, the RCA product for only one, CLCuDBur3, digested with HindIII, produced an apparently full-length ~2.7 kb fragment, suggesting that CLCuD-Bur3 represented a distinct isolate. The 2.7-kb fragment was cloned into the plasmid vector pGEM-3Zf+ (Promega, Madison, WI). To test for the presence of associated alphasatellites and betasatellites, the PCR primers, AlphaF/R and BetaF/R (2), were used to amplify the putative 1.4-kbp molecules. The resultant 1.4-kb PCR products were ligated into the pGEMT-Easy vector and cloned. Cloned inserts for each were subjected to DNA sequencing, bidirectionally. The cloned monopartite, helper begomovirus genome (HF567945), one betasatellite (HF567946), and one alphasatellite (HF567947) sequences were determined and found to be 2,742, 1,358, and 1,376 bases long, respectively. Pairwise sequence comparisons were carried out for each using the 10 most closely related species or strains (identified in GenBank using BLASTn) using MEGA5 software. The CLCuDBur3 genome sequence shared its highest identity (99.6%) with Okra enation leaf curl virus (OELCuV) (KC019308), so CLCuDBur3 is a variant of OELCuV, a begomovirus reported previously from Abelmoschus esculentus (L.) (okra) plants in India. The betasatellite and alphasatellite shared their highest nt identity at 96 and 98.7% with Cotton leaf curl Multan betasatellite (CLCuMB) (AM774311) and Cotton leaf curl Multan alphasatellite (CLCuMA), respectively (misnamed as CLCuBuA in GenBank) (FN658728). Additionally, the HindIII-digested RCA products were analyzed by Southern blot hybridization using a DIG-labeled DNA probe specific for the intergenic region of either Cotton leaf curl Burewala virus (CLCuBuV) or OELCuV. The OELCuV, but not the CLCuBuV, probe hybridized with HindIII digested RCA products (CLCuDBur3 genome), confirming the presence of OELCuV and the absence of CLCuBuV, the latter being the most prevalent begomovirus species infecting cotton in Pakistan. This is the first report of OELCuV infecting cotton plants in Pakistan, underscoring the discovery of yet another begomovirus member of the CLCuD complex. Further, the possible co-infection of cotton by OELCuV and other recognized species of the CLCuD complex could facilitate further diversification (potentially, through recombination) and lead to the emergence of new variants with the potential to cause damage to the cotton crop in Pakistan.

Simple and nonradioactive detection of microRNAs using digoxigenin (DIG)-labeled probes with high sensitivity

The discovery of microRNAs (miRNAs), which are ∼21-23 nucleotides that can regulate targeted mRNA by transcript cleavage or protein translation suppression, has changed the landscape of biomedical field greatly. At present, Northern blot analysis based on radioisotopes is still the most popular method on the detection of miRNAs for its high sensitivity. However, radioisotopes have been known for certain disadvantages, such as instability, expense, and safety; thus, developing a nonradioactive and highly sensitive method is needed. Here, we report a simple, nonradioactive, and sensitive method for miRNAs detection based on 5'-phos-3'-DIG-labeled probes prepared through splinted ligation and EDC cross-linking (DSLE). The method was more sensitive than traditional Northern blots with a DIG-labeled DNA probe and can detect as low as 2 fmol of miRNAs. The whole procedure can be completed within 6-8 h. DSLE method is very convenient, cost-effective, time-saving, and highly sensitive.

DNA Fragmentation Analysis

DNA fragmentation with length corresponding to multiple integer of approximately 180 base pairs is a distinct feature of apoptosis in animals and programmed cell death in plants. This feature can simply be detected by DNA gel electrophoresis followed by ethidium bromide staining, although in some cases it is difficult to distinguish the DNA laddering. We herein describe a protocol to detect a programmed cell death-associated DNA laddering of plant tissues. After agarose-gel electrophoresis of genomic DNA, Southern hybridization using DIG-labeled genomic DNA probe is performed, that improves detection of DNA laddering.

The molecular characterization of an Eimeria and Cryptosporidium detected in Asian seabass (Lates calcarifer) cultured in Vietnam

An intestinal Eimeria was previously reported as a significant pathogen of Asian seabass (Lates calcarifer) in nurseries in Vietnam. In the present study, both Eimeria and Cryptosporidium were detected by sequence analyses of fragments of the 18S rRNA gene amplified from these Vietnamese L. calcarifer tissues. Based on these analyses, the Eimeria from the Vietnamese L. calcarifer formed clades with the Eimeria detected in L. calcarifer tissues from Australia, but clustered separately from other known Eimeria and Goussia species. The Cryptosporidium detected in L. calcarifer from Vietnam clustered closest with C. parvum and C. hominis. In situ hybridization using DIG-labeled DNA probes generated from 18S PCR products on the Vietnamese L. calcarifer wax block tissues showed that this method could not be used to distinguish between Eimeria and Cryptosporidium, due to the conserved nature of the 18S locus. A previously published study on the morphology of parasite developmental stages and oocysts in the Vietnamese L. calcarifer tissues showed only an intestinal Eimeria infection. The Cryptosporidium could be present at very low levels undetectable by microscopy in intestines, or being ubiquitous, was a possible contaminant from feed or water. While molecular analysis is a very useful tool in the study of disease and identification of aetiological agents, this study reiterates the importance of demonstrating organisms in situ in tissues.

Development of a probe-based blotting technique for the detection of Tobacco streak virus

Digoxigenin (DIG)-labeled DNA probe was developed for a sensitive and rapid detection of the tobacco streak virus (TSV) isolates in India by dot-blot and tissue print hybridization techniques. DIG-labeled DNA probe complementary to the coat protein (CP) region of TSV sunflower isolate was designed and used to detect the TSV presence at field levels. Dot-blot hybridization was used to check a large number of TSV isolates with a single probe. In addition, a sensitivity of the technique was examined with the different sample extraction methods. Another technique, the tissue blot hybridization offered a simple, reliable procedure and did not require a sample processing. Thus, both non-radioactively labeled probe techniques could facilitate the sample screening during TSV outbreaks and offer an advantage in quarantine services.

Response of Various Tomato Genotypes to Begomovirus Infection and Its Improved Diagnostic

Begomovirus infection was identified from tomato growing areas in West Java (Bogor), Central Java (Boyolali), and D.I. Yogyakarta (Kaliurang). Efforts to reduce the infection among others are planting resistance varieties. This research was undertaken to evaluate 14 tomato genotypes for their response to the infection. Dot blot hybridization using nonradioactive (digoxigenin) DNA probe was employed to determine the presence of begomovirus in inoculated plants. Polymerase chain reaction-amplified product of DNA clone of tobacco leaf curl virus A¢â‚¬â€œIndonesia was used as a source of DNA probe. All of tomato genotypes evaluated in this study was infected separately by three strain of begomovirus (GVPSlm, GVABy, GVCBgr). Tomato genotypes Bonanza, Jelita, Safira, Permata, Presto , PSPT 8, PSPT 5B, Apel-Belgia, Karibia, Mitra , PSPT 9, Marta , and PSPT 2, showed susceptible or highly susceptible response to the three strains of begomovirus. Exception to those was shown by cv. Intan which resulted in moderate resistance when inoculated with GVCBgr although it resulted susceptible response with the other two strains. Dot-blot hybridization technique was proved to be a powerful tool to detect begomovirus infection in plants showing symptom as well as symptom-less plants. Accumulation of the virus in those plants was relatively high, except in cv. Bonanza and Apel-Belgia . Dot-blot hybridization technique using DIG-labeled DNA probe was able to detect begomovirus DNA in infected tissue up to 10-2 dilution factor. Key words: Geminivirus, hybridization, resistance, tomato

Development of a Babesia bigemina Specific Probe from Monomorphic Fragment of RAPD-PCR

Ravindran, R., Mishra, A.K. and Rao, J.R. 2007. Development of a Babesia bigemina specific probe from monomorphic fragment of RAPD-PCR. J. Appl. Anim. Res., 31: 173–176. A highly species specific Dig labeled DNA probe has been developed for detection of Babesia bigemina with an analytical sensitivity of 2 μg B. bigemina DNA from a monomorphic fragment of RAPD-PCR.

Infectious hypodermal and haematopoietic necrosis virus (IHHNV) infections in giant freshwater prawn, Macrobrachium rosenbergii

Between July 2004 and January 2005, high mortalities (up to 80–100%) were frequently encountered in postlarvae of Macrobrachium rosenbergii in southern Taiwan. Pathologically, eosinophilic intranuclear inclusion bodies (INIs) were found only in the hepatopancreatic tubular epithelial cells of the infected postlarvae from hatchery farms. No lesions could be detected in tissue of ectodermal or mesodermal origin. Interestingly, different lesions were found in sub-adults collected from a grow-out farm. Atrophic changes in abdominal muscles from the fourth to sixth segment and tail fan, associated with a reddish discoloration, were prominent features in these shrimp, but there was no unusual mortality or INI formation. In PCR assays for the detection of infectious hypodermal and haematopoietic necrosis virus (IHHNV), hepatopancreatic parvovirus (HPV), white spot syndrome virus (WSSV), Taura syndrome virus (TSV), yellowhead virus (YHV), M. rosenbergii nodavirus (MrNV) and extra small virus (XSV), only an expected 389-bp product, specific for the IHHNV nonstructural protein gene, was obtained from all postlarvae and sub-adults examined. Positive reactions to in situ hybridization, using a DIG-labelled DNA probe, further confirmed IHHNV as the causative agent. In a comparison of our strains with Taiwanese (GenBank accession no. AY 355306 and AY 355308) and American strains (GenBank accession no. AF218266 and AF273215), nucleotide sequence identities were up to 99.7%. This is the first report concerning natural infection of IHHNV in postlarvae and sub-adults of M. rosenbergii.

Purification and molecular cloning of a major allergen from Anisakis simplex.

A heat-stable allergen with a molecular weight of 21 k was purified from larvae of the nematode Anisakis simplex by gel filtration, anion-exchange FPLC and reverse-phase HPLC. When analyzed by immunoblotting and ELISA, seven of eight patient sera reacted to the 21 k allergen, demonstrating that this protein is a major allergen of A. simplex. A full-length cDNA encoding the 21 k allergen was cloned by a combination of 3'RACE and screening of an expression library with DIG-labeled DNA probes. The precursor of the 21 k allergen was judged to be composed of a signal peptide (23 residues) and a mature protein (171 residues). As compared to the N-terminal amino acid sequence (up to the 17th residue) of Ani s 1 previously identified as the major allergen, the 21 k allergen has only one replacement, suggesting that the 21 k allergen belongs to the same protein family of Ani s 1. Although the 21 k allergen was found to have 30-40% sequence identity with Kunitz-type trypsin inhibitor domain containing hypothetical proteins of Caenorhabditis elegans, it lacked inhibitory activity against trypsin. The 21 k allergen was successfully expressed in Escherichia coli as a GST-fusion protein showing reactivity with IgE in patient sera.

PCR detection ofclavibacter michiganensis subsp.sepedonicus- infected tuber samples in a plate capture assay

The speed and sensitivity of PCR-based assays allow shorter turnaround times for the detection of pathogens for which culture and serological methods are difficult or unavailable. PCR was performed with primer sets Cms50 and Cms72, designed previously by Millset al. (1997) through subtractive hybridization to detectClavibacter michiganensis subspeciessepedonicus (Cms). In bacterial suspensions, fewer than three cells/10 ul reaction were detected after PCR amplicons were hybridized with specific DIG-labeled DNA probes in an enzyme-linked oligonucleosorbent assay (ELOSA). In naturally infected tuber samples representing three cultivars of potato, the diagnostic sensitivity of PCR/ ELOSA was 96%, while the specificity exceeded 99%. PCR/ELOSA detectedCms in infected tuber samples with equal sensitivity regardless of colony morphology, potato cultivar, or primer sets.

Isolation and characterization of a purC(orf)QLF operon from Lactococcus [correction of Lactobacillus] lactis MG1614.

We have isolated genes encoding enzymes of the de novo purine nucleotide biosynthesis pathway from Lactococcus lactis MG1614 by colony hybridization using DIG-labeled DNA probes. The organization of the genes needed for the de novo biosynthesis of purine nucleotides in L. lactis differs from that found in other organisms. In L. lactis there is a gene cluster, which contains five out of the 11 genes needed for the de novo biosynthesis of IMP, namely purC, orf, purQ, purL and purF. These genes were shown to be transcribed as a single transcription unit by Northern hybridization analysis. The 5' end of the transcript of the purC(orf)QLF operon was determined by primer extension analysis using fluorescently end-labeled probes. The purC(orf)QLF operon of L. lactis is transcribed in Escherichia coli, and the gene product of the purF gene, glutamine phosphoribosylpyrophosphate amidotransferase (glutamine PRPP ATase, EC 2.4.2.14), can functionally complement the E. coli purF mutant strain TX158. We also show that the promoter of the purC(orf)QLF operon is regulated in response to exogenously added purines.

Random amplified polymorphic DNA for the specific detection of bubaline Echinococcus granulosus by hybridization assay

The polymerase chain reaction (PCR) method to randomly amplify polymorphic DNA (RAPD) was used to differentiate the bubaline and bovine strains of Echinococcus granulosus and buffalo host DNA. Four random oligonucleotide primers of 10–11 mer were analyzed for their ability to direct the amplification of polymorphic DNA fragments from parasites and bovine DNA. Significant DNA polymorphism was observed between the E. granulosus isolates. A selectively amplified DNA fragment 0.9 kilobase (kb) from E. granulosus buffalo isolate by primer AP2 (5′-TGCCGAGCTG-3′) was reamplified and used as a DIG-labelled DNA probe. Dot-blot hybridization of total genomic DNA differentiated buffalo E. granulosus isolate from bovine isolate and bubaline host DNA with no detectable cross-hybridization signal.

Detection of Y-bearing porcine spermatozoa by in situ hybridization using digoxigenin-labeled, porcine male-specific DNA probe produced by polymerase chain reaction.

This study was carried out to determine whether Y-bearing porcine spermatozoa could be detected by in situ hybridization using a digoxigenin (Dig)-labelled DNA probe specific to the Y chromosome produced by polymerase chain reaction (PCR). A conventional PCR (with Dig-dUTP) was performed using a set of oligonucleotide primers (5'-AAGTGGTCAGCGTGTCCATA-3' and 5'-TTTCTCCTGTATCCTCCTGC-3') for 236 bp fragment of porcine male-specific DNA sequence and 1.25 x 10(4) template white blood cells obtained from a boar. When fluorescence in situ hybridization with the Dig-labelled DNA probe was applied to the metaphase chromosome spreads prepared from both boar and gilts, the fluorescein signal was only detected on the long arm of the Y chromosome. In addition, immunocytochemical detection with the Dig-labelled DNA probe and alkaline phosphatase-labeled anti-Dig was applied to both sperm nuclei pretreated with dithiothreitol and white blood cells; 51% of sperm nuclei and 96% of white blood cells obtained from boar were labelled, whereas none of white blood cells obtained from gilts were labelled with the Dig-labelled DNA probe. The results indicated that in situ hybridization with porcine male-specific DNA probe produced by PCR made possible the direct visualization of Y-bearing porcine spermatozoa by in situ hybridization.

Gene Diagnosis of Viroids: Comparisons of Return-PAGE and Hybridization Using DIG-labeled DNA and RNA Probes for Practical Diagnosis of Hop Stunt, Citrus Exocortis and Apple Scar Skin Viroids in Their Natural Host Plants.

Return-PAGE was reliable for diagnosis of HSVd in hop, CEVd in citron and ASSVd in apple, but was not reliable for HSVd in grapevine and citron, and ASSVd in pear, because of their low concentration. A disadvantage of return-PAGE was possible appearance of double or triple bands in case of mix-infecting samples such as hop or citron. Although DIG-labeled DNA probe was 2.5 to 25 times more sensitive than return-PAGE, the practical reliability of this method was not so superior to return-PAGE. DIG-labeled RNA probe was the most sensitive of the three methods examined in this experiment, which was 25-125 times and 100-625 times more sensitive than DIG-DNA probe and return-PAGE, respectively. Furthermore, DIG-RNA probe was sensitive enough to detect HSVd in grapevine, a symptomless host of the pathogen, although negative sample was emphasized to be re-examined by the other sensitive method such as cucumber bioassay or RT-PCR. DIG-RNA probe, however, is not sensitive enough to detect HSVd in citrus and ASSVd in pear. Based on the results obtained in this experiment, we proposed appropriate amounts of plant tissues for the successful diagnosis of the three viroids.

Detection of parvovirus B19 in a skin biopsy of a patient with erythema infectiosum

We report the findings on a skin biopsy taken from a child acutely infected with parvovirus B19 showing the typical exanthematous rash. By indirect immunofluorescence with a monoclonal antibody to B19, viral capsid proteins were detected in epidermal cells localized mainly in the stratum basale. Additionally, B19 DNA was detected in epidermal cells of the stratum basale by in situ hybridization using a Dig-labelled B19 DNA probe. The detection of viral capsid proteins and viral DNA suggests the presence of complete viral particles. It is therefore concluded that B19 plays a direct role in the formation of the exanthematous rash in erythema infection.