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PubMed1996

Improved rapid detection of the PML/RARalpha fusion gene in acute promyelocytic leukemia.

Acute promyelocytic leukemia (APL) is a medical emergency which requires rapid diagnosis and tailored treatment. Detection of the PML/RARalpha fusion gene in APL blasts is critical to start promptly the specific therapy with all-trans retinoic acid (ATRA). APL lacking this genetic lesion have been reported as being ATRA resistant. Reverse-transcription polymerase chain reaction (RT-PCR) has been extensively used to detect the PML/RARalpha cDNA. The reported PML/RARalpha amplification techniques are laborious and time consuming, and include conventional RNA extraction, cDNA synthesis and a two-round (nested) PCR. We hereby describe a few variations of the commonly adopted RNA extraction and PML/RARalpha RT-PCR protocols which allow a molecular diagnosis of APL to be carried out in less than 5 h. Processing of small volumes of leukemic cell lysate (0.5 ml) in a microfuge allows extraction of good quality RNA in 1 h. After reverse transcription to obtain cDNA, a 'hot start' PCR procedure was adopted which enabled us to amplify clearly visible and specific products after a single (not nested) amplification round. The PML/RARalpha fusion gene was detected in the blasts of six consecutive APL at diagnosis, and an APL-tailored protocol including ATRA was started in each case within 6 h of admission. On repeated experiments, the assay proved highly specific and sensitive for the rapid detection of all PML/RARalpha transcript types. Our data should encourage the use of this rapid procedure for the diagnosis of both typical APL and, particularly, less typical cases awaiting urgent therapeutic intervention.

Base Sequence↗

PubMed2002

A multiplex allele specific polymerase chain reaction (MAS-PCR) on the dihydrofolate reductase gene for the detection of Cryptosporidium parvum genotypes 1 and 2.

A multiplex allele specific polymerase chain reaction (MAS-PCR) based on the Cryptosporidium parvum dihydrofolate reductase (dhfr) gene sequence differentiates genotype 1 ('Human') from 2 ('Cattle') in a 1-step reaction. The MAS-PCR was validated on a panel of 34 microscopically positive C. parvum faecal samples of human and animal origin in comparison with 2 published PCR-restriction fragment length polymorphism (RFLP) methods targeting dhfr and the oocyst wall protein (cowp) genes. A validation panel of 37 negative faecal samples of human and animal origin was also tested in comparison with the cowp PCR-RFLP. MAS-PCR was found to be as sensitive for species detection as the most sensitive of the other tests, and detected more mixed genotype infections than the two other tests combined. In addition the MAS-PCR showed equivalent detection sensitivity in comparison with a published nested RFLP targeting the SSU rRNA gene, on a panel of prepared mixed genotype samples. The 1-step reaction is simpler and less expensive to perform than the RFLP methods, while the C. parvum specific amplicons and those for genotypes 1 and 2 (575, 357 and 190 bp respectively) can be easily distinguished on agarose gel.

Alleles↗

PubMed1992

Targeted misexpression of Hox-4.6 in the avian limb bud causes apparent homeotic transformations.

In the limb bud the 5' members of the Hox-4 gene cluster are expressed in a nested set of overlapping domains which are progressively restricted in the posterior and distal directions. These domains arise early in limb bud development and come to approximate the primordia of the major structural elements of the limb along the anterior/posterior axis (Fig. 1). This pattern, and the fact that surgical manipulations which lead to mirror image duplications along the anterior/posterior axis give rise to mirror image duplications of the domains of expression of these genes, have led to the proposal that these transcription factors specify positional identity along the anterior/posterior axis. Here we test this hypothesis directly using replication-competent retroviral vectors to expand the domain of expression of the Hox-4.6 gene anteriorly during limb development in vivo. We report that alteration of the domain of expression of the Hox-4.6 gene in the developing limb leads to reproducible pattern alterations consistent with a posterior homeotic transformation.

Animals↗

PubMed2006

Nested polymerase chain reaction for detection of pathogenic leptospires.

Leptospirosis is a widespread zoonosis caused by pathogenic members of the genus Leptospira that has a great impact on human and veterinary public health. Early diagnosis of leptospirosis is important because severe lepto spiral infection can have a fulminant course. The available serological techniques for the diagnosis of leptospirosis have low sensitivity during the early stage of the disease. Efforts are being made to develop simpler, effective, efficient, and inexpensive diagnostic methods. In this work, we first evaluate a polymerase chain reaction (PCR) based method for diagnosis of leptospirosis. Primers were designed to amplify a 264 bp region within the lipL32 gene that is conserved among pathogenic Leptospira and absent in nonpathogenic species. The sensitivity and specificity of the assay were evaluated using 7 saprophytic serovars, 37 pathogenic serovars, and 15 other microorganisms. The method was very specific for pathogenic serovars, however, it lacked sensitivity. To enhance the sensitivity, another primer pair was designed to amplify a 183 bp region within the 264 bp region of the lipL32 gene and was used in a nested PCR assay. This approach was much more sensitive than conventional PCR.

Animals↗

PubMed2001

Glucose catabolism in cancer cells: identification and characterization of a marked activation response of the type II hexokinase gene to hypoxic conditions.

One of the most common signatures of highly malignant tumors is their capacity to metabolize more glucose to lactic acid than their tissues of origin. Hepatomas exhibiting this phenotype are dependent on the high expression of type II hexokinase, which supplies such tumors with abundant amounts of glucose 6-phosphate, a significant carbon and energy source especially under hypoxic conditions. Here we report that the distal region of the hepatoma type II hexokinase promoter displays consensus motifs for hypoxia-inducible factor (HIF-1) that overlap E-box sequences known to be related in other gene promoters to glucose response. Moreover, we show that subjecting transfected hepatoma cells to hypoxic conditions activates the type II hexokinase promoter almost 3-fold, a value that approaches 7-fold in the presence of glucose. Consistent with these findings is the induction under hypoxic conditions of the HIF-1 protein. Reporter gene analyses with a series of nested deletion mutants of the hepatoma type II hexokinase promoter show that a significant fraction of the total activation observed under hypoxic conditions localizes to the distal region where the overlapping HIF-1/E-box sequences are located. Finally, DNase I footprint analysis with a segment of the promoter containing these elements reveals the binding of several nuclear proteins. In summary, these novel studies identify and characterize a marked glucose-modulated activation response of the type II hexokinase gene to hypoxic conditions within highly glycolytic hepatoma cells, a property that may help assure that such cells exhibit a growth and survival advantage over their parental cells of origin.

Amino Acid Motifs↗

PubMed1994

Recombinant limbs as a model to study homeobox gene regulation during limb development.

Recombinant limb buds constructed of an ectodermal jacket containing dissociated anterior wing bud mesoderm cells form limb-like structures lacking anteroposterior polarity. Polarity can be restored by implanting a small piece of polarizing region into the recombinant limb. We used recombinant limbs to study expression of Msx1 and Msx2 and 5' Hoxd genes in limb development. Detectable mesodermal expression of Msx1 and Msx2 was lost during preparation of the recombinants. When expression was restored both genes were expressed in a symmetrical fashion in the apical mesoderm whether or not the recombinant was polarized. The normal anterior pattern of Msx1 and Msx2 was never reestablished. The roles Msx1 and Msx2 play in limb patterning are at present not clear. In nonpolarized recombinant limbs the 5' Hoxd genes were sequentially and uniformly expressed in the subridge mesoderm across the anteroposterior axis. They exhibited nested domains of expression showing exclusively distal restriction. In polarized recombinants the 5' Hoxd genes were expressed progressively in an asymmetric, nested fashion adjacent to the implanted polarizing region; there was also a distal mesodermal region further from the implant that did not express the most 5' Hoxd genes. Both of these observations are similar to the normal limb. Our results demonstrate that the polarizing region is not necessary for the activation or maintenance of 5' Hoxd gene expression but that its presence is required in order for the normal pattern to be established. We propose that the expression of the Hoxd genes is an intrinsic property of rapidly growing limb cells and that their expression is related to the limb bud proximodistal outgrowth. Our data also support the influence of the polarizing region upon Hoxd gene expression in two ways. First there is a short-range action responsible for the normal asymmetric expression and second a long-range effect suppressing the expression of 5'-Hoxd genes by the anterior cells.

Animals↗

PubMed2006

Molecular diagnostics in sepsis: from bedside to bench.

BACKGROUND: Based on recent in vitro data, we tested the hypothesis that microarray expression profiles can be used to diagnose sepsis, distinguishing in vivo between sterile and infectious causes of systemic inflammation. STUDY DESIGN: Exploratory studies were conducted using spleens from septic patients and from mice with abdominal sepsis. Seven patients with sepsis after injury were identified retrospectively and compared with six injured patients. C57BL/6 male mice were subjected to cecal ligation and puncture, or to IP lipopolysaccharide. Control mice had sham laparotomy or injection of IP saline, respectively. A sepsis classification model was created and tested on blood samples from septic mice. RESULTS: Accuracy of sepsis prediction was obtained using cross-validation of gene expression data from 12 human spleen samples and from 16 mouse spleen samples. For blood studies, classifiers were constructed using data from a training data set of 26 microarrays. The error rate of the classifiers was estimated on seven de-identified microarrays, and then on a subsequent cross-validation for all 33 blood microarrays. Estimates of classification accuracy of sepsis in human spleen were 67.1%; in mouse spleen, 96%; and in mouse blood, 94.4% (all estimates were based on nested cross-validation). Lists of genes with substantial changes in expression between study and control groups were used to identify nine mouse common inflammatory response genes, six of which were mapped into a single pathway using contemporary pathway analysis tools. CONCLUSIONS: Sepsis induces changes in mouse leukocyte gene expression that can be used to diagnose sepsis apart from systemic inflammation.

Adolescent↗

PubMed1996

Detection of t(2;5)(p23;q35) translocation by reverse transcriptase polymerase chain reaction and in situ hybridization in CD30-positive primary cutaneous lymphoma and lymphomatoid papulosis.

The t(2;5) generates a chimeric NPM-ALK transcript encoded by the nucleophosmin NPM gene fused to the anaplastic lymphoma kinase gene ALK. Using a reverse transcriptase nested polymerase chain reaction assay we have detected NPM-ALK transcripts within CD30+ primary cutaneous lymphoma and lymphomatoid papulosis (LP). The t(2;5) was identified in 4 out of 9 CD30+ anaplastic lymphomas and in 1 out of 4 CD30+ pleomorphic lymphomas. Moreover, the t(2;5) was detected in 3 out of 10 LPs. All NPM-ALK-positive lymphomas and 1 NPM-ALK-positive LP exhibited a clonal rearrangement of the T cell receptor gamma-chain gene. The t(2;5) was detected in 2 cases of LP without other evidence for a clonal lymphoid population. To identify cells carrying the t(2;5) translocation, we used immunohistochemistry to detect the ALK-encoded p80 protein and in situ hybridization for the specific detection of NPM-ALK transcripts. Both p80 protein and NPM-ALK transcripts were expressed by anaplastic or large CD30+ lymphoma cells with positive NPM-ALK amplification. The presence of t(2;5) in a subset of CD30+ cutaneous lymphoma and LP may indicate a common pathogenesis with a subset of anaplastic nodal lymphoma.

Adult↗

PubMed1997

Detection and identification of ruminant and porcine pestiviruses by nested amplification of 5' untranslated cDNA regions.

Based on published gene sequences of bovine viral diarrhoea virus (BVDV) type I and classical swine fever virus (CSFV), genus- and species-specific primers were designed to detect and identify pestivirus cDNA sequences in a nested polymerase chain reaction (PCR). The PCR primers were validated using cDNA synthesized from 146 pestivirus isolates, comprising representatives of all four so far described genotypes (BVDV type I, BVDV type II, CSFV and border disease virus), as well as others of uncertain classification. PCR products of the predicted size were amplified from all viruses with the genus-specific primers. All 53 cattle isolates, including 5 typed antigenically as BVDV type II were amplified by the internal BVDV-specific primers, but not the CSFV-specific primers. The same result was found for other BVDV type I and II viruses isolated from sheep and pigs. Seventy-seven CSF viruses were amplified by their respective internal primers. Available information strongly indicate that 4 CSF viruses also amplified by the BVDV-specific primers had been contaminated with BVDV in cell cultures. Border disease viruses were mostly not detected by the BVDV-specific primers, but were detected weakly by the CSFV-specific primer pair. Using carrier RNA for extraction of viral RNA, the sensitivity of detection of the single and nested PCR was, respectively, 5 and 50 times higher than obtained with a cell culture assay. The RT-PCR also detected BVDV in all of 15 commercial batches of fetal calf serum examined, and verified three earlier diagnoses of CSFV by detecting specific gene sequences in 30 year old frozen archival organ samples.

Animals↗

PubMed2006

Phylogeography of postglacial range expansion in Nigronia serricornis Say (Megaloptera: Corydalidae).

We have examined the effects of post-Wisconsinan glacial range expansion on the phylogeography of the saw-combed fishfly, Nigronia serricornis Say (Megaloptera: Corydalidae) because aquatic insects are under-represented in postglacial studies (and in phylogeography in general), and because the effects of ecological degradation on the population genetics of environmental indicator species like N. serricornis cannot be measured unless the underlying phylogeography is understood. Sequence data from a 630-base fragment of the mitochondrial cytochrome oxidase I (COI) gene were subjected to amova and nested clade analysis for 30 populations (n = 344) of N. serricornis. Both the amova and nested clade analysis revealed substantial population structure; 44.4% of the variance occured among populations. Three northward migrations are apparent: one from Tennessee into Illinois, Indiana, Wisconsin, Michigan and Ontario, a second that radiated eastward from Pennsylvania, and a third that moved along the coast from North Carolina into Connecticut, Massachusetts, Maine and then into New York. The latter two of these migrations were the result of contiguous range expansions, while the former expansion, out of Tennessee, appears to have been rapid with little gene flow from the source population. Additional clades included a group of haplotypes in central Kentucky that appear to have expanded along preglacial drainages, and clades in North Carolina and Georgia that have remained centrally located. Haplotype diversity decreased from south to north, a pattern that has been widely reported for animal and plant populations that expanded with the retreat of the Wisconsinan glaciation.

Animals↗

PubMed1999

Polymerase chain reaction amplification of bacterial 16s rRNA genes in prostate biopsies from men without chronic prostatitis.

OBJECTIVES: A previously reported study using nested polymerase chain reaction (PCR) analysis indicated the presence of DNA from a variety of prokaryotic microorganisms in 77% of transperineal prostate biopsies from patients with chronic nonbacterial prostatitis. Because that study did not include a control group, we investigated whether microbial DNA could also be found in transperineal prostate biopsies obtained from men who did not have a history of prostatitis. METHODS: Transperineal biopsies of both lobes of the prostate were obtained under ultrasound guidance from 9 patients with localized adenocarcinoma of the prostate. DNA was extracted from the prostatic tissue and two-round amplification performed using nested primers from a highly conserved region of the bacterial 16s rRNA gene. Amplified DNA was purified and sequenced, and sequences obtained were compared to bacterial rRNA genes recorded in GenBank. Results. Eleven of 18 biopsy specimens from 8 of 9 patients were positive for bacterial DNA by PCR. Sequence data indicated a predominant organism in 8 of 11 specimens, with greater than 95% homology to DNA from several different genera of bacteria, including Escherichia and Bacteroides. All 9 control samples from the instruments before biopsy were negative. CONCLUSIONS: The presence of bacterial 16s rRNA genes in prostatic tissue is not specific for chronic prostatitis and occurred in most of our patients with localized prostate cancer. Whether the presence of such bacteria is related to the development of prostatic diseases such as prostatitis or prostatic cancer will require carefully controlled trials, including appropriate control groups examined identically.

Aged↗

PubMed1992

Molecular analysis of pseudorabies viral vaccines and their rapid differentiation from wild-type isolates using DNA-amplified glycoprotein I and thymidine kinase gene segment polymorphisms.

Several conventional and genetically recombinant modified-live viral (MLV) vaccines are used to control pseudorabies virus infections (Aujeszky's disease, PRV) in swine. Differentiating vaccinal PRV (V-PRV) from wild PRV (WT-PRV) is important for herd health, regulatory and forensic purposes, and for studies of PRV latency and epidemiology. All PRV vaccines used currently contain glycoprotein I (gI) and/or thymidine kinase (TK) gene deletions, whereas WT-PRV typically contain intact gI and TK genes. Utilizing these differences we developed an effective but simple differential polymerase chain reaction (PCR) approach based upon the amplification of gI and TK gene polymorphisms. The primary immunoreactive epitope-encoding region of the gI gene and nearly the entire TK gene were amplified and analyzed using nested PCR procedures. TK and gI PCR products were cleaved with Sal I and Sac I, and Nco I restriction enzymes respectively. PCR product and restriction fragment length polymorphisms enabled most V-PRV to be clearly distinguished from each other, and all of them, as a group, clearly differentiated from typical WT-PRV. Mixtures of V-PRV and WT-PRV could be identified as such. The uncommon but occasional occurrence of atypical WT-PRV containing altered gI and/or TK genes indicates the need for interpretive caution, particularly if aberrant gene segment polymorphisms are observed. This rapid and precise molecular approach will facilitate regulatory monitoring, epidemiological investigations, diagnostic differentiation, purity testing and latency/recrudescence studies with the class of biologicals and offers a model for similar analyses of other MLV biologicals as well.

Animals↗

PubMed1997

Comparison of nested PCR with immunofluorescent-antibody assay for detection of Ehrlichia canis infection in dogs treated with doxycycline.

A partial 16S rRNA gene was amplified in Ehrlichia canis-infected cells by nested PCR. The assay was specific and did not amplify the closely related Ehrlichia chaffeensis, Ehrlichia muris, Neorickettsia helminthoeca, and SF agent 16S rRNA genes. The assay was as sensitive as Southern hybridization, detecting as little as 0.2 pg of E. canis DNA. By this method, all blood samples from four dogs experimentally infected with E. canis were positive as early as day 4 postinoculation, which was before or at the time of seroconversion. One hundred five blood samples from dogs from Arizona and Texas (areas of E. canis endemicity) and 30 blood samples from dogs from Ohio (area of E. canis nonendemicity) were examined by nested PCR and immunofluorescent-antibody (IFA) test. Approximately 84% of dogs from Arizona and Texas had been treated with doxycycline before submission of blood specimens. Among Arizona and Texas specimens, 46 samples were PCR positive (44%) and 80 were IFA positive (76%). Forty-three of 80 IFA-positive samples (54%) were PCR positive, and 22 of 25 IFA-negative samples (88%) were negative in the nested PCR. None of the Ohio specimens were IFA positive, but 5 specimens were PCR positive (17%). Our results indicate that the nested PCR is highly sensitive and specific for detection of E. canis and may be more useful in assessing the clearance of the organisms after antibiotic therapy than IFA, especially in areas in which E. canis is endemic.

Animals↗

PubMed2005

Occult hepatitis B in renal transplant patients.

BACKGROUND: Occult hepatitis B (HB) is characterized by the presence of HBV-DNA in patients who do not have HB surface antigen (HBsAg) detectable in sera, and is frequently described in patients with hepatitis C virus (HCV) infection. These viral liver diseases are common and may have a negative impact on the survival of renal transplant patients, especially if they are both present. In this study we aimed to evaluate the prevalence of occult HB in renal transplant patients either with or without HCV infection. PATIENTS AND METHODS: In a cross-sectional survey 101 HbsAg-negative renal transplant patients were evaluated; 51 were anti-HCV positive. Sera were analyzed for the presence of the S and core genes of the HBV-DNA by a nested polymerase chain reaction technique. Markers of HBV infection and liver function tests were also analyzed. RESULTS: The core gene was identified in 1 HCV-infected patient and 1 anti-HCV-negative patient who also presented the S gene (prevalence: 2% and 1% for each gene, respectively). HCV-infected patients had longer pre-transplant dialysis time (50.8 +/- 34.6 vs. 32.0 +/- 20.9; P < 0.001). Liver function tests were also increased in the HCV-infected group: alanine aminotransferase (P < 0.001), aspartate aminotransferase (P < 0.05), gamma-glutamyl transpeptidase (P<0.02), and alkaline phosphatase (P < 0.04). Multivariate analysis revealed that HCV infection was the only determinant of the altered results of the liver function tests. CONCLUSION: We found that occult HB is a condition present in our population of renal transplant patients and that HCV infection does not seem to be associated with occult HB infection in this setting.

Adult↗

PubMed2007

A polymorphism in the 3' untranslated region of the gene encoding prostaglandin endoperoxide synthase 2 is not associated with an increase in breast cancer risk: a nested case-control study.

INTRODUCTION: Prostaglandins are integral components in the cellular response to inflammation, promoting cellular proliferation and angiogenesis. The enzyme responsible for the conversion of arachidonic acid to prostaglandins in response to inflammation is prostaglandin endoperoxide synthase 2/cyclo-oxygenase 2 (PTGS2/COX2). Polymorphisms in the PTGS2 gene have been associated with various diseases, including inflammatory bowel disease and cancer of the lung, colorectum, and breast. METHODS: We genotyped the five most common polymorphisms (rs20417, rs5277, rs20432, rs5275, and rs4648298) in the Nurses' Health Study (1,270 cases, 1,762 controls) to test the hypothesis that polymorphisms in PTGS2 are associated with breast cancer risk, using logistic regression analyses. The Nurses' Health Study 2 (317 cases, 634 controls) and Harvard Women's Health Study (702 cases, 703 controls) were used to further examine putative associations. RESULTS: The rs5275 polymorphism in the 3' untranslated region of the PTGS2 gene was associated with a decrease in breast cancer risk. We therefore genotyped this single-nucleotide polymorphism in the Nurses' Health Study 2 and Harvard Women's Health Study. Similar results were observed in these subsequent analyses, with no statistically significant heterogeneity in risk estimates between studies. In pooled analyses, women homozygous for the T allele at rs5275 had a 20% lower risk of breast cancer than those homozygous for the C allele (odds ratio 0.80, 95% confidence interval 0.66 to 0.97). CONCLUSION: Although this polymorphism may be associated with a decrease in breast cancer risk among Caucasian women, we provide strong evidence that it is not associated with an increased risk of breast cancer.

3' Untranslated Regions↗

PubMed1997

In-cell assembly of scFv from human thyroid-infiltrating B cells.

The construction of a large library of single-chain Fv (scFv) antibody fragments involves a random assortment of heavy and light chains. Although useful for the production of recombinant antibodies, this method is not adapted to the study of the autoantibody repertoire formed in vivo during autoimmune diseases. To attain this objective, we describe the use of the in-cell PCR together with Cre-recombination applied, to our knowledge, for the first time to human B cells to obtain in situ pairing of the variable (V) region genes of the immunoglobulin heavy (H) and light (L) chains. Our method is based on amplification and recombination of the VH and VL genes within CD19+ B cells isolated from human thyroid tissue. Nested primers were designed to amplify the known major human VH and VL gene families. After reverse transcription PCR and three rounds of PCR including recombination between VH and VL using the Cre-loxP system, we obtained a unique 800-bp band corresponding in size to scFv fragments. We provide evidence that recombination between VH and VL genes occurred inside the same cell. This in-cell amplification and association procedure is a potentially useful tool for the study of autoantibody gene families and the VH/VL pairing that occurs during the autoimmune process.

B-Lymphocytes↗

PubMed2004

Coexistence of tetrasomy 8 and trisomy 8 in acute promyelocytic leukemia (AML-M3) with t(15;17)(q22;q12).

This study was purposed to characterize the first case of acute promyelocitic leukemia (AML-M(3a)) with t(15;17), trisomy 8 and tetrasomy 8, and explore its characteristics of morphology, cytogenetics, molecular biology, immunology and clinical features. Morphological changes of peripheral blood and bone marrow smears were observed under microscope. Chromosome specimen was prepared by 24 h short-term culture of bone marrow cell, RHG-banding technique was used for karyotypic analysis. PML-RARa fusion gene transcript was detected by nested-reverse transcription-polymerase chain reaction (nested RT-PCR). Interphase fluorescence in situ hybridization (FISH) using chromosome 8 centromere specific probe were carried out to detect abnormal numbers of chromosome 8. Immunophenotypic analysis was performed by flow cytometry. The results showed that peripheral blood smear revealed 65% promyelocyte, and bone marrow aspirate was hypercellular with 72.4% promyelocyte, moderately basophilic cytoplasm with numerous azurophilic granules. Karyotype analysis demonstrated 48, XY, +8, +8, t(15;17)(q22;q12) [16]/47, XY, +8, t(15;17)(q22;q12) [3]/46, XY, t(15;17)(q22;q12) [1]. RT-PCR assay revealed PML-RARa fusion gene transcript (+). FISH showed that the percentages of cells exhibiting 1, 2, 3, 4, 5, 6 green fluorescence signals were 0.5, 7, 19, 55, 18 and 0.5, respectively. This confirmed the presence of tetrasomy 8 and trisomy 8 and also revealed a low percentage of a pentasomy 8 clone. Immunophenotypes of the blasts displayed that CD13 (96.2%), CD33 (55.9%), CYMPO (93.5%) were positive. All the lymphoid markers tested were negative. The patient survival time was just 10 days. It is concluded that tetrasomy 8 is secondary cytogenetic event after t(15;17) in this case. It may be a consequence of clonal evolution of trisomy 8. t(15;17) AML-M(3) with tetrasomy 8 heralds a poor prognosis.

Chromosomes, Human, Pair 15↗

PubMed2006

Genotyping of single Cryptosporidium oocysts in sewage by semi-nested PCR and direct sequencing.

This study describes an approach for genotyping individual Cryptosporidium oocysts obtained from sewage. We isolated single immunofluorescent assay (IFA)-stained Cryptosporidium oocysts from sewage concentrate using glass capillary pipettes and inverted epifluorescence microscopy. Each isolated Cryptosporidium oocyst was analyzed by semi-nested PCR for the 18S rRNA gene and direct sequencing of the PCR products. A total of 74 of 107 oocysts isolated from sewage were genotyped successfully. Of the 74 genotyped isolates, 51% (38 oocysts) were identified as C. parvum genotype 1, 4% (3 oocysts) of C. parvum VF383 human isolates, 20% (15 oocysts) of C. parvum genotype 2, 14% (10 oocysts) of C. meleagridis, 7% (5 oocysts) of C. sp. Pig 1, 3% (2 oocysts) of C. sp PG1-26 pig isolates and 1% (1 oocyst) of C. parvum CPM1 isolated from mouse. The results of this study demonstrate that 18S rRNA-based semi-nested PCR and direct sequencing can be used to characterize individual Cryptosporidium oocysts and also to reveal the distribution of Cryptosporidium genotypes in environmental waters.

Animals↗