Following Trypanosoma cruzi RPA-DNA Interaction Using Fluorescent In Situ Hybridization Coupled with Immunofluorescence (FISH/IF).

Fluorescent in situ hybridization coupled with immunofluorescence (FISH/IF) is an assay that has been widely used to study DNA-protein interactions. The technique is based on the use of a fluorescent nucleic acid probe and fluorescent antibodies to reveal the localization of a DNA sequence and a specific protein in the cell. The interaction can be inferred by the quantification of the co-localization between the protein and the DNA. Here, we describe a detailed FISH/IF methodology that our group used to study RPA-telomere interaction in the pathogenic protozoa parasite Trypanosoma cruzi.

A high-density transcript linkage map with 1,845 expressed genes positioned by microarray-based Single Feature Polymorphisms (SFP) in Eucalyptus.

BACKGROUND: Technological advances are progressively increasing the application of genomics to a wider array of economically and ecologically important species. High-density maps enriched for transcribed genes facilitate the discovery of connections between genes and phenotypes. We report the construction of a high-density linkage map of expressed genes for the heterozygous genome of Eucalyptus using Single Feature Polymorphism (SFP) markers. RESULTS: SFP discovery and mapping was achieved using pseudo-testcross screening and selective mapping to simultaneously optimize linkage mapping and microarray costs. SFP genotyping was carried out by hybridizing complementary RNA prepared from 4.5 year-old trees xylem to an SFP array containing 103,000 25-mer oligonucleotide probes representing 20,726 unigenes derived from a modest size expressed sequence tags collection. An SFP-mapping microarray with 43,777 selected candidate SFP probes representing 15,698 genes was subsequently designed and used to genotype SFPs in a larger subset of the segregating population drawn by selective mapping. A total of 1,845 genes were mapped, with 884 of them ordered with high likelihood support on a framework map anchored to 180 microsatellites with average density of 1.2 cM. Using more probes per unigene increased by two-fold the likelihood of detecting segregating SFPs eventually resulting in more genes mapped. In silico validation showed that 87% of the SFPs map to the expected location on the 4.5X draft sequence of the Eucalyptus grandis genome. CONCLUSIONS: The Eucalyptus 1,845 gene map is the most highly enriched map for transcriptional information for any forest tree species to date. It represents a major improvement on the number of genes previously positioned on Eucalyptus maps and provides an initial glimpse at the gene space for this global tree genome. A general protocol is proposed to build high-density transcript linkage maps in less characterized plant species by SFP genotyping with a concurrent objective of reducing microarray costs. HIgh-density gene-rich maps represent a powerful resource to assist gene discovery endeavors when used in combination with QTL and association mapping and should be especially valuable to assist the assembly of reference genome sequences soon to come for several plant and animal species.

A comparative analysis of bleomycin-induced incomplete chromosome elements in two mammalian cell lines using a telomeric PNA probe.

Fluorescence in situ hybridization (FISH) with a telomeric peptide nucleic acid probe was employed to analyze the induction of incomplete chromosome elements (ICE; i.e., incomplete chromosomes and terminal fragments) by bleomycin (BLM) in two mammalian cell lines. Chinese hamster embryo cells (CHE cell line, average 2n = 23) and domestic rabbit cells (CPC cell line, average 2n = 44) were treated with 2.5 micro g/ml BLM; after 18 hr of incubation, first-division metaphases were stained with the telomeric probe, and ICE and other unstable chromosomal aberrations were scored. BLM induced ICE, dicentrics, and interstitial acentric fragments in CHE cells, but only ICE in CPC cells. About 50% of the metaphases in BLM-treated CHE cells contained one or more pairs of ICE, while only 20% of treated CPC cells contained ICE. Almost 100% of the BLM-induced ICE in both cell lines consisted of pairs formed by an incomplete chromosome and a terminal fragment. Our results confirm that ICE are the most frequent type of unstable chromosomal aberration induced by BLM in mammalian cells. Moreover, the present study shows that an increase in the chromosome number does not necessarily result in an increase in the frequency of BLM-induced ICE. The results also show that the difference in the chromosomal sensitivity to BLM in CHE and CPC cells is due to differences in the absolute frequency but not in the pattern (i.e., type and proportion) of ICE.

Analysis of streptonigrin-induced incomplete chromosome elements and interstitial fragments in Chinese hamster cells using a telomeric PNA probe.

We investigated the induction of incomplete chromosome elements (ICEs; i.e., elements with a telomeric signal at only one terminal end) and interstitial fragments induced by the antibiotic streptonigrin (SN) in a Chinese hamster embryo (CHE) cell line using FISH with a telomeric peptide nucleic acid probe. CHE cells were treated with 0-250 ng/ml SN and chromosomal aberrations were analyzed in the first mitosis after treatment using the telomeric probe. Exposure of CHE cells to SN resulted in a linear concentration-related increase in all of the aberration types analyzed (P < 0.05) except ring chromosomes. Depending on the SN concentration employed, 33-68% of the metaphases contained one or more pairs of ICEs (an incomplete chromosome accompanied by a terminal fragment or two incomplete chromosomes accompanied by a compound fragment). Pooled data from all SN concentrations revealed that 77.8% of the acentric fragments were terminal fragments, 18.8% interstitial fragments, and 3.4% compound fragments. Furthermore, it was estimated that about 80% of excess acentric fragments induced by SN originated from incomplete exchanges or terminal deletions and 20% from complete exchanges (interstitial deletions). These results show that incomplete chromosomes and terminal fragments are the most frequent asymmetrical chromosomal aberrations induced by SN and indicate that true incompleteness is a very common event following exposure to SN.

Clinical evaluation of the Gen-Probe Amplified Direct Test for detection of Mycobacterium tuberculosis complex organisms in cerebrospinal fluid.

Eighty-four cerebrospinal fluid (CSF) samples from different children who presented with signs and symptoms of meningitis were evaluated for the presence of Mycobacterium tuberculosis complex organisms by the Gen-Probe Amplified Mycobacterium tuberculosis Direct Test (MTD; Gen-Probe, San Diego, Calif.). All CSF samples had negative acid-fast smears by the Ziehl-Neelsen staining method. M. tuberculosis was recovered from five samples. M. tuberculosis did not grow from 19 additional samples, but the samples were from patients who fulfilled specific clinical and laboratory criteria for probable tuberculous meningitis (TBM). The remaining samples (n = 60) were from patients with other infections or noninfectious causes of meningitis. The results of the MTD were interpreted as positive or negative on the basis of recommended cutoff values for respiratory specimens. These results were interpreted as true or false positives or true or false negatives on the basis of the results of M. tuberculosis culture or whether the patient fulfilled criteria for probable TBM. The Gen-Probe MTD was 33% sensitive and 100% specific for detecting M. tuberculosis complex organisms in these 84 CSF samples. If the cutoff values for positive results were decreased for the MTD (> or = 11,000 versus > or = 30,000 relative light units), the sensitivity increased to 83% and the specificity remained 100%. These results for the MTD are encouraging considering that TBM is a highly fatal disease and difficult to diagnose by conventional laboratory techniques.

Malaria diagnosis: a review.

Malaria is the most prevalent endemic disease in large parts of the world and is subject to control by health authorities. Today, the goal of malaria control is to prevent mortality and reduce morbidity and socioeconomic losses through the progressive improvement and strengthening of local and national capabilities. The World Health Organization considers early diagnosis as the first basic element of the strategy to control the disease. Traditionally, laboratory diagnosis has been made using the thick blood film, which continues to be the gold standard test. However, this test has disadvantages such as the manner in which the film is prepared, the level of training of the observer, the adequacy of maintenance of materials and equipment and its only fair sensitivity. Thus, many research laboratories have concentrated their efforts on the development of alternative methods for malaria diagnosis. These include methods for the detection of Plasmodia within erythrocytes (fluorescent microscopy, Quantitative Buffy Coat (QBC), dark field microscopy, nucleic acid probes and immunofluorescence), methods for the detection of plasmodial antigens in body fluids (radioimmunoassay, enzyme immunoassay) and methods for the detection of anti-plasmodial antibodies in serum (indirect immunofluorescence, enzyme immunoassay, Western blotting). Here, we critically review the various methods for malaria diagnosis based on the world's literature and our experience with most of them, with emphasis on recent advances.

Malaria diagnosis: a review

Malaria is the most prevalent endemic disease in large parts of the world and is subject to control by health authorities. Today, the goal of malaria control is to prevent mortality and reduce morbidity and socioeconomic losses through the progressive improvement and strengthening of local and national capabilities. The World Health Organization considers early diagnosis as the first basic element of the strategy to control the disease. Traditionally, laboratory diagnosis has been made using the thick blood film, which continues to be the gold standard test. However, this test has disadvantages such as the manner in which the film is prepared, the level of training of the observer, the adequacy of maintenance of materials and equipment and its only fair sensitivity. Thus, many research laboratories have concentrated their efforts on the development of alternative methods for malaria diagnosis. These include methods for the detection of Plasmodia within erythrocytes (fluorescent microscopy, Quantitative Buffy Coat (QBC(), dark field microscopy, nucleic acid probes and immunofluorescence), methods for the detection of plasmodial antigens in body fluids (radioimmunoassay, enzyme immunoassay) and methods for the detection of anti-plasmodial antibodies in serum (indirect immunofluorescence, enzyme immunoassay, Western blotting). Here, we critically review the various methods for malaria diagnosis based on the world's literature and our experience with most of them, with emphasis on recent advances.

The use of molecular techniques in the epidemiology and control of infectious diseases.

The ability to identify specific strains within a given species of pathogen is an important aid in the rational development of effective measures to prevent and control nosocomial infections. The efforts of both microbiologist and hospital epidemiologic are facilitated greatly by the availability of the newer molecular epidemiologic typing techniques. Although these methods clearly have limitations, they generally are a significant improvement over the more conventional typing methods, many of which are too cumbersome, insensitive, and time-consuming to be of practical value for epidemiologic evaluations. Based on current experience, the molecular typing methods that appear to be the most practical and useful for both large and small scale epidemiologic studies are the DNA-based methods of REAP and PFGE. Many questions remain concerning the appropriate role of molecular typing methods in the clinical microbiology laboratory. Often, molecular typing may be performed more efficiently in a reference laboratory. In contrast, selected methods such as plasmid analysis are well within the scope of clinical microbiology laboratories and may be an important adjunct to the hospital infection control effort. Given the limitations of the available methods and the complex nature of patients at risk for nosocomial infections, it is imperative that these methods be employed with clear epidemiologic objectives in mind. Typing should always include unrelated as well as epidemiologically related isolates and, whenever possible, all organisms should be typed under identical conditions, preferably within the same test run. In addition, results are most effectively used to supplement rather than to replace hypotheses and questions thoughtfully developed by the clinician or epidemiologist. Ideally, typing is performed independently by the laboratory to avoid bias, but the results are applied collaboratively to ensure that both the potential insights and the unavoidable ambiguities presented by the results are clearly appreciated. Additional studies based upon sound epidemiologic principles will help clarify the role of the various molecular typing methods as epidemiologic markers and advance our understanding of the epidemiology of nosocomial infections.

In-situ hybridization for the diagnosis and typing of human papillomavirus.

The human papillomavirus (HPV) has been associated with the production of many skin and mucosal lesions, the development of squamous cell carcinoma of the genital areas, skin and aerodigestive tracts, and possibly adenocarcinoma of the uterine cervix. There are more than 60 known genotypes of HPV. Some genotypes have been associated with lesions that have none or minimal chances of malignant transformation; while other genotypes (especially types 16, 18, 31, 35, and 51) have been found in mild, moderate, and severe dysplasia, carcinoma in-situ or frank invasive carcinoma. HPV cannot be propagated in tissue cultures. The presence of the virus can be demonstrated by immunologic techniques, which are not sensitive enough with the present methodology, or by searching for the presence of the viral DNA by DNA or RNA hybridization techniques. Determining the viral genotype in the tissue involved will permit the separation of those lesions supposedly to be low risk from those associated with the high risk types. This knowledge may be helpful in the future to determine the appropriate management of patients infected with HPV.

DNA hybridization with oligodeoxyribonucleotide probes for identifying enterotoxin-producing Escherichia coli.

The DNA hybridization method using oligodeoxyribonucleotide probes was compared with the GM, ELISA and the infant mouse assay for determining production of LT and ST, respectively, by 2000 strains of Escherichia coli. Sensitivity and specificity by DNA hybridization for LT were 98.7 and 99.8%, and for ST were 78.8 and 99.6%.