Ribosomal RNA Gene Probe Research Articles

Carbon quantum dots functionalized tapered optical fiber for highly sensitive and specific detection of Leptospira DNA

• An infectious disease called leptospirosis has caused human mortality, alarming health professionals. • This biosensor acts as an alternate leptospirosis diagnostic tool by using tapered optical fiber as a sensing transducer. • Leptospira DNA detection was enhanced by functionalizing tapered optical fibre with carbon quantum dots (CQDs). • This article suggests the development of a more sensitive, specific, and reliable leptospirosis diagnostic tool. In this paper, we report on the development of DNA biosensor using carbon quantum dots (CQDs) to enhance Leptospira DNA detection. Leptospira is a bacterium that invades human body and cause leptospirosis disease. Leptospirosis has attracted attention mainly due to several cases of human mortality which have distressed health professionals. This biosensor system employs tapered optical fiber as a sensing transducer. The CQDs were functionalized at the tapered region via in situ deposition (drop-casted) before incubation using commercialized chemicals to link the 16 s ribosomal RNA gene (rrs) probe DNA. Detection was achieved with the occurrence of hybridization between the probe DNA and different concentrations of complementary DNA ( Leptospira DNA). This biosensor with limit of detection (LOD) of 1.0 fM demonstrated better sensitivity at 1.8295 nm/nM and higher affinity as its dissociation constant (K d ) of 0.552 × 10 −09 M is lower compared to previous reported biosensors without CQDs. High specificity biosensor is validated when minimal shift was observed for non-complementary DNA. This novel biosensor was also able to distinguish between genomic DNA of Leptospira serovars and Clostridium difficile as control sample. The proposed biosensor has successfully exhibited the prominent role of nanomaterial for sensor enhancement. Consequently, the biosensor approach in this study has emphasized the potential for the development of more sensitive and reliable leptospirosis diagnostic testing in the future.

Karyological, Molecular and Cytogenetical Studies on Tree Plants in V.N. Sukachev Institute of Forest

Aim. The aim of the article is to find out the karyological features and conduct molecular and cytogenetic analysis on tree plants for biological diversity studies, solve problems of taxonomics, evolutionary and population genetics. Methods. Classic methods with acetohematoxylin staining of slides and new method of fluorescent in situ hybridization (FISH) had been used. Results . More than 250 populations and provenances of representatives of different genera of gymnosperms from the families Pinaceae, Cupressaceae, Ephedraceae and tree angiosperms from the families Rosaceae, Aceraceae, Polygonaceae, Nitrariaceae, Rhamnaceae, Fabaceae, Ericaceae were studied. Investigations had been conducted in natural populations and during the introduction, in optimal and extreme conditions, in disturbed ecosystems, botanical gardens and parks; in addition, various intraspecific forms and unique trees had been studied. The variability of chromosome numbers and a wide range of chromosomal mutations had been revealed. Use of fluorescence in situ hybridization (FISH) with the 45S and 5S ribosomal RNA gene probes and DAPI staining allowed to identify of homologous chromosome pairs in the karyotypes of conifers and to facilitate the comparative karyotype analysis of those species. Analysis of the chromosome numbers of woody angiosperms showed a significant role of polyploidy in their evolution. Conclusions. The studies on karyotypes in tree species showed their diversity, variability of chromosome numbers, higher level of chromosomal anomalies and their wide spectrum in extreme conditions, under introduction and also in cultivars and abnormal form of trees. The use of molecular cytogenetic markers made it possible to obtain new information on the structure of conifer chromosomes. The role of polyploidy in the evolution of woody angiosperms was shown.

Karyological and cytogenetic research of the conifers of boreal zone by classic and new methods

Aim. Establishing of karyological features and conducting of cytogenetic analysis on conifer plants for biological diversity studies, solving of problems of taxonomics, evolutionary and population genetics. Methods. Classic methods with acetohematoxylin staining of slides and fluorescent in situ hybridization (FISH). Results. More than 150 populations and provenances of representatives of different conifer genera from the Pinaceae and Cupressaceae families were studied. The studies were carried out in natural populations and during the introduction, in optimal and extreme conditions, in disturbed ecosystems, botanical gardens and parks; in addition, various intraspecific forms have been studied. The variability of chromosome numbers and a wide range of chromosomal mutations have been revealed. Fluorescence in situ hybridization (FISH) with the 45S and 5S ribosomal RNA gene probes and DAPI staining allows to identify of homologous chromosome pairs in the karyotypes of conifers and to facilitate the comparative karyotype analysis of these species. Conclusions. The studies of chromosomes in species of the Pinaceae and Cupressaceae families showed a karyotypic diversity and chromosomal anomalies in extreme conditions and under introduction. The use of molecular cytogenetic markers made it possible to obtain new information on the structure of conifer chromosomes.
 Keywords: chromosomes, nucleolar loci, chromosome mutations, Pinaceae, Cupressaceae.

Karyotype and B chromosome variation in Lilium amabile Palibin.

Lilium amabile Palibin (2n = 2x = 24) is an endemic lily species in Korea. B chromosomes are supernumerary chromosomes and the presence of B chromosome in L. amabile was known by previous researches. The current research was conducted to characterize the genetical and cytological features of the B chromosome plants in L. amabile. Karyotype and B chromosome cytotype analyses were carried out among 135L. amabile accessions that were collected from six geographical locations in Korea using conventional aceto-carmine staining as well as FISH technique with ribosomal RNA gene probes. The karyotype of L. amabile genome consisted of two large metacentric, four intermediate subtelocentric, and six intermediate to small acrocentric chromosomes in which chromosomes 1, 6 and 7 carried the 45S rRNA gene loci and chromosome 3 carried the 5S rRNA gene. There were 4 types of B chromosomes, two large B chromosomes and two small B chromosomes. The ribosomal RNA gene loci were not present in the B chromosomes. The 135 accessions were classified into 13 cytotypes including diploids and different B chromosome aneuploids. Among the aneuploids, the most frequent cytotype was 24 + 1B, which was followed by 24 + 2B, 24 + 1b, 24 + 1B + 2b, 24 + 1B + 4b, and 24 + 2B + 4b. The karyotype of L. amabile was consistent with other species in the genus Lilium without polyploids. The B chromosome cytotypes were highly variable and the occurrences of different cytotypes were random among the six populations, implying that the B and b chromosome occurrence was random in each population.

Resolving the Taxonomic Status of Chamelea gallina and C. striatula (Veneridae, Bivalvia): A Combined Molecular Cytogenetic and Phylogenetic Approach.

The striped venus clams Chamelea gallina and C. striatula are commercially important bivalves inhabiting European and North African coastal waters. The taxonomic status of these taxa has been the subject of debate for decades. In order to elucidate this issue, we generated 5S and 28S ribosomal RNA and H3 histone gene probes and mapped them by fluorescent in situ hybridization to the chromosomes of morphologically identified striped venus clams, collected from four geographically distant Atlantic and Mediterranean populations. The nucleotide variation at the three DNA markers, that is, the nuclear internal transcribed spacer 2 (ITS2), the mitochondrial cytochrome c oxidase subunit I (COI), and the large ribosomal subunit rRNA (16S) fragments, was also studied and the resultant phylogenetic trees were evaluated. Striking differences in both the chromosome distribution of these genes and the clustering of the samples on the phylogenetic trees observed provide clear evidence that C. gallina and C. striatula are separated species.

Molecular cytogenetic analysis of Siberian Larix species by fluorescence in situ hybridization

Karyotypes of three Larix species (L. sibirica, L. gmelinii, and L. cajanderi) were analyzed using fluorescence in situ hybridization (FISH) with 45S and 5S ribosomal RNA gene probes and 4′,6-diamidino-2-phenylindole (DAPI) staining. Two major 45S ribosomal DNA (rDNA) loci (per haploid genome) have been observed in the intercalary regions of two metacentric chromosomes, III and IV, of L. sibirica; in addition to them, minor nucleolus organizing regions (NORs) were mapped in pericentromeric regions of chromosomes I, II, VI, and XII. Two closely related species, L. gmelinii and L. cajanderi, showed similar hybridization patterns; both species possessed an additional major locus of 45S rDNA in the distal region of the long arm of submetacentric chromosome VII that is absent in L. sibirica. Only one locus of the 5S rDNA was found in all larch species we studied; it was located in the distal region of the chromosome III short arm, which also carried the major NOR in the opposite arm. This chromosome containing major loci of the two ribosomal RNA gene families can serve as a marker of the genus Larix. The intra- and interspecific karyotype diversity in the genus Larix is discussed.

Microorganisms in Root Canal–treated Teeth from a German Population

Posttreatment apical periodontitis is usually associated with persistent or secondary intraradicular infection. This study evaluated the presence and relative levels of 28 bacterial taxa in treated root canals of teeth with posttreatment apical periodontitis from German patients using 16S ribosomal RNA (rRNA) gene probes in a reverse-capture checkerboard hybridization assay. Species-specific polymerase chain reaction (PCR) was also performed to detect Enterococcus faecalis and Candida albicans. Bacterial DNA was detected in all samples. Twenty of the 28 taxon-specific probes tested were reactive with at least one sample. Taxa detected more frequently included Streptococcus species (47%), Lactobacillus species (35%), Dialister invisus (29%), Eubacterium infirmum (29%), Prevotella intermedia (29%), Selenomonas sputigena (29%), Synergistes oral clone BA121 (29%), and Treponema denticola (29%). Only eight taxa were present at levels >10(5). Of these, streptococci and T. denticola were the most prevalent. Species-specific PCR detected E. faecalis in 47% of the cases and C. albicans in 6%. Findings of this study confirm the strong association between persistent/secondary intraradicular infection and posttreatment apical periodontitis. Most cases harbored a mixed infection, and E. faecalis, if present, was never the most dominant species in the consortium. Several other bacterial taxa were detected, and an involvement with the etiology of posttreatment apical periodontitis is suspected.

Centromeric localization of an S-RNase gene in Petunia hybrida Vilm.

S-RNase has been identified to be an S-allele-specific stylar determinant contributing to the self-incompatibility response in Solanaceae. In order to examine the physical location of the S-RNase gene, multi-color fluorescence in situ hybridization (FISH) using the S (B1) -RNase cDNA probe and ribosomal RNA gene (rDNA) probe was performed on an S (B1) S (B2 )heterozygote of Petunia hybrida. The S (B1) -RNase gene was detected as a doublet signal close to the centromere of chromosome III. Next, we performed FISH using a large genome probe prepared from a λSB1-311 clone (20 kb) which contains the S (B1) -RNase gene and its 3´ flanking region. This probe hybridized to the centromeric regions of all P. hybrida chromosomes. Sequence analysis of the λSB1-311 clone revealed the presence of a repetitive sequence consisting of a novel 666 bp unit sequence. A subclone (pBS-SB1B5) containing this unit sequence also hybridized to all of the centromeric regions, confirming that this unit is the centromeric specific repetitive sequence. These data suggested that the S ( B1 ) -RNase gene is located very close to (within a distance of 12 kb from) the centromeric-specific repetitive sequence. Likewise, the pBS-SB1B5 probe hybridized to the centromeric regions of all chromosomes in P. littoralis, another Petunia species. However, the probe did not hybridize to the centromere of the chromosomes from other species in Solanaceae. These results suggested that this centromeric repetitive sequence might be a genus-specific one.

Evaluation of 16S, map1 and pCS20 probes for detection of Cowdria and Ehrlichia species.

A panel of 16S ribosomal RNA gene probes has been developed for the study of the epidemiology of heartwater; five of these detect different cowdria genotypes, one detects five distinct genotypes; one detects any Group III Ehrlichia species other than Cowdria and one detects any Group II Ehrlichia species. These probes have been used on PCR-amplified rickettsial 16S rRNA genes from over 200 Amblyomma hebraeum ticks. Control ticks were laboratory-reared and either uninfected or fed on sheep experimentally infected with different cowdria isolates, field ticks were collected from animals in heartwater-endemic areas. All tick-derived DNA samples were also examined by PCR amplification and probing for two other cowdria genes (map1 and pCS20) which have previously been used for heartwater epidemiology. This paper describes the first direct comparison of all currently available DNA probes for heartwater-associated organisms.

Comparative evaluation of 16S, map1 and pCS20 probes for the detection of Cowdria and Ehrlichia species in ticks.

We have developed a panel of 16S ribosomal RNA gene probes for heartwater epidemiology; five of these detect different Cowdria genotypes (Ball3, Senegal, Omatjenne, Crystal Springs, and Mara 87/7); one detects all five of these genotypes; one detects any Group III Ehrlichia species other than Cowdria; one detects any Group II Ehrlichia species. We have used these probes on PCR-amplified rickettsial 16S rRNA genes from over 200 Amblyomma ticks. Control ticks were laboratory-reared and either uninfected or fed on sheep experimentally infected with different Cowdria isolates, field ticks were harvested from animals in heartwater-endemic and heartwater-free areas. All the samples were also examined by PCR amplification and probing for two other Cowdria genes (map1 and pCS20) which have been used for heartwater epidemiology. This paper describes the first direct comparison of all the currently available DNA probes for heartwater-associated organisms.

Babesia bovis: Genome size, number of chromosomes and telomeric probe hybridisation

Pulsed field gel electrophoresis of intact chromosomes of Babesia bovis revealed four chromosomes in the haploid genome. A telomere probe, derived from Plasmodium berghei, hybridised to eight SfiI restriction fragments of genomic B. bovis DNA digests indicating the presence of four chromosomes. A small subunit (18S) ribosomal RNA gene probe hybridised to the third chromosome only. The genome size of B. bovis is estimated to be 9.4 million base pairs. The sizes of chromosomes 1, 2, 3 and 4 are estimated to be 1.4, 2.0, 2.8 and 3.2 million base pairs, respectively.

Molecular analysis of enrichment cultures of marine methane oxidising bacteria

Abstract Methane oxidising bacteria (methanotrophs) are an important group of bacteria in the global cycling of methane and may act as a sink for methane in freshwater, soil and marine environments. Their ecology is fairly well documented in terrestrial and freshwater environments but marine methanotrophs have been less-well characterised, partly due to the difficulties in the isolation, cultivation and identification of these organisms. Molecular ecological techniques are being developed to aid the characterisation and enumeration of marine methane oxidising bacteria and to assess their role in the turn-over of methane in the marine environment. 16S ribosomal RNA gene probes have been developed to detect and identify marine methanotrophs. The use of these probes, coupled with fluorescence microscopy techniques, to aid enrichment, cultivation and subsequent characterisation of marine methanotrophs is described. The use of the polymerase chain reaction to amplify methane monooxygenase genes from enrichments of marine methanotrophs is also described.

Ribotyping ofMycoplasma gallisepticumstrains with a 16S ribosomal RNA gene probe

Ribotyping with a 16S ribosomal RNA gene (rDNA) probe was applied to 25 Mycoplasma gallisepticum strains composed of nine originally isolated in the US and UK, and 16 field isolates from Japan. Four distinct ribotypes were identified among the M. gallisepticum strains on the basis of sizes of the bands produced by digesting genomic DNA with restriction enzymes HindIII, EcoRI, BglII and EcoRV. Three ribotypes were recognized among the 16 Japanese isolates. The original and vaccine F strains were classified as the same ribotype, although they produced different banding patterns with BglII, suggesting that the vaccine strain may be a mutant of the original. Other avian mycoplasma species produced banding patterns different from each other and from M. gallisepticum strains. Characterization of M. gallisepticum strains by ribotyping with a 16S rDNA probe may benefit surveillance and epidemiological investigations on M. gallisepticum infection.

RFLP analysis of Mycobacterium malmoense strains using ribosomal RNA gene probes: an addition tool to examine intraspecies variation

Abstract To examine the intraspecies variation of Mycobacterium malmoense, 29 clinical isolates, chemotyped by thin-layer chromatography of their surface glycolipids into five subgroups, were typed using RFLP analysis. Two 32P-labelled ribosomal RNA (rRNA) gene fragments were used as probes for chromosomal DNA digested with BanI. By comparison of the fingerprint patterns, the strains could be divided into five ribotypes, two major and three minor ones. The chemotypes were not connected to any of the ribotypes. Combination of the results of the two techniques produced a considerable resolution. This method is a useful additional tool when studying the epidemiological behaviour of M. malmoense.

Differential screening of mitochondrial cDNA libraries from male-fertile and cytoplasmic male-sterile sugar-beet reveals genome rearrangements at atp6 and atpA loci

As part of a strategy to define differences in genome organization and expression between cytoplasmic male-sterile (CMS) and male-fertile (MF) sugar-beet mitochondria, cDNA libraries from both mitochondrial genotypes were constructed. Preliminary screening with ribosomal RNA gene probes identified candidate cDNA clones corresponding to structural genes. In addition, reciprocal hybridization experiments were performed using labelled first-strand cDNA to identify uniquely transcribed sequences. One cDNA clone (pYC700) is unique to CMS mitochondria and is located upstream of the F0F1-ATPase subunit 6 gene (atp6). Another cDNA clone (pYC130), when used as a probe in northern hybridization analysis, revealed novel transcript profiles in CMS sugar-beet mitochondria. Sequence analysis of this cDNA showed strong homology with the F0F1-ATPase subunit alpha (atpA) coding sequences from several higher plants. The atp6 and atpA loci from each genotype were cloned and the genomic organization, DNA sequence and transcription of each locus was studied. Differences in the transcript profiles of each gene are a consequence of genomic rearrangements 5' to the coding sequence.

Analysis of Sitka spruce–interior spruce introgression in British Columbia using cytoplasmic and nuclear DNA probes

Restriction fragment length polymorphisms of the nuclear ribosomal RNA genes were used to define species-specific patterns for interior spruce (a complex of white spruce, Piceaglauca (Moench) Voss, and Engelmann spruce, Piceaengelmannii Parry) and Sitka spruce (Piceasitchensis (Bong.) Carr.) populations in British Columbia. These patterns are highly conserved among seed lots and individual trees of each species from diverse geographic origins. Both the ribosomal RNA gene probe and species-specific probes for the chloroplast and mitochondrion, described previously, were used to analyze 164 dominant trees from 19 locations in a zone of presumed Sitka–interior spruce introgression in northwestern British Columbia. Hybrid fraction was estimated from an index based on the relative abundance of polymorphic ribosomal RNA hybridizing bands for each tree and was in good agreement with the proportion of trees exhibiting either interior or Sitka spruce specific chloroplast bands. The results demonstrate that complex hybrids occur frequently in this zone, indicative of true introgression. Furthermore, the trend of variation in species mix is consistent with the topographical trends. It is anticipated that the results of this survey will be useful in defining seed transfer guidelines for reforestation of this region.

Detection of polymorphisms among Theileria parva stocks using repetitive, telomeric and ribosomal DNA probes and anti-schizont monoclonal antibodies

A total of 21 Theileria parva stocks from 6 countries were characterized using T. parva repetitive and ribosomal DNA probes, a Plasmodium berghei telomeric oligonucleotide and a panel of anti-schizont monoclonal antibodies (MAbs). Hybridization of the repetitive DNA probe to Southern blots of EcoRI-digested T. parva DNA revealed 20 different restriction fragment patterns among DNA samples isolated from infections initiated using 16 parasite stocks. The panel of anti-schizont MAbs defined 8 different profiles among schizont-infected lymphoblastoid cell-cultures infected with the same 16 T. parva stocks. Many stocks, which were differentiated by the repetitive DNA probe, could not be distinguished using the anti-schizont MAbs. A cloned T. parva small subunit ribosomal RNA (SSUrRNA) gene probe separated 17 T. parva stocks into 2 groups, exhibiting either 1 or 2 restriction fragments, when hybridized to EcoRI-digested T. parva DNA. When hybridized to PvuII-digested DNA from 8 T. parva stocks, the ribosomal probe identified 4 groups with similar restriction fragment patterns. A synthetic oligonucleotide derived from a P. berghei telomeric sequence hybridized to 7 or 8 size-polymorphic restriction fragments in the EcoRI-digested DNA of most T. parva stocks. The telomeric and ribosomal probes defined the same 4 groups among 8 T. parva stocks as assessed by similarities in restriction fragment patterns. Based on the comparison of repetitive DNA sequences from the T. parva Uganda and Muguga stocks, a synthetic oligonucleotide was developed which distinguished the DNA of the T. parva Uganda stock from that of 4 other T. parva stocks on a positive/negative basis.

The use of a chemiluminescence-linked universal bacterial ribosomal RNA gene probe and blood gas analysis for the rapid detection of bacterial contamination in white cell-reduced and nonreduced platelets.

Because of the rising incidence of bacterial growth and septic platelet transfusions in aging units, platelet storage is currently limited in the United States to 5 days. This approved shelf life of platelets might be altered if methods were devised to rapidly detect infected units and/or to decrease the incidence of bacterially contaminated platelets. An investigation was conducted on the effect of a prototype blood collection system with an in-line filter for the production of white cell-reduced platelet-rich plasma on the growth of bacteria in platelets prepared from whole blood that had been inoculated with Staphylococcus epidermidis. Additional studies were conducted with a chemiluminescence-linked ribosomal RNA (rRNA) gene probe and with blood gas analysis to identify possible methods for the rapid detection of bacterial contamination. All units were followed for 9 days of storage. The filtration of the platelet-rich plasma resulted in an approximate 2 log10 reduction in white cells with an average loss of 6.7 percent of platelets. Filtration did not appear to alter bacterial growth. In all platelet units that supported growth, pO2 dropped to negligible values and pCO2 rose relative to culture-negative units. The changes were most sensitive and specific beyond 5 days of storage. The universal bacterial rRNA probe assay was able to detect S. epidermidis in concentrations as low as 1 x 10(3) colony-forming units per mL in some cases and reliably detected all units contaminated at a concentration of 1 x 10(4) colony-forming units per mL.(ABSTRACT TRUNCATED AT 250 WORDS)

Restriction fragment length polymorphism analysis of Fusobacterium necrophorum using a novel repeat DNA sequence and a 16S rRNA gene probe.

A repeated DNA sequence was isolated from Fusobacterium necrophorum biotype AB, strain FnS1. The repeated sequence shared considerable homology with the transposase gene from the Pseudomonas syringiae insertion sequence IS801. The repeat sequence was used together with a 16S ribosomal RNA gene probe to type F. necrophorum isolates using restriction fragment length polymorphisms. The probes revealed differences between several clinical isolates and will be useful tools to study the epidemiology of ovine foot abscess and other diseases caused by F. necrophorum.

Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genus Verticillium

Restriction fragment length polymorphisms in mitochondrial DNA and ribosomal RNA gene complexes as an aid to the characterization of species and sub-species populations in the genus Verticillium