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PubMed2002

Mapping of an origin of DNA replication near the transcriptional promoter of the human HPRT gene.

A quantitative PCR method was used to map a functional origin of DNA replication in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene in normal human fibroblasts. This PCR method measures the abundance of specific sequences in short fragments of newly replicated DNA from logarithmically growing cells. Quantitative measurements rely on synthetic molecules (competitors) that amplify with the same primer sets as the target molecules, but generate products of different sizes. This method was first utilized to determine the position of the replication origin near the lamin B2 gene (Giacca et al. [1994] Proc. Natl. Acad. Sci. U S A. 91:7119-7123). In the present study, primer sets were tested along a 16-kb region near exon 1 of the HPRT gene. The most abundant fragment was found to be located in the first intron of HPRT, just downstream of the promoter and exon 1 of the gene, and approximately 3.5 kb upstream of a previously reported autonomously replicating sequence (Sykes et al. [1988] Mol. Gen. Genet. 212:301-309).

Chromosome Mapping↗

PubMed2002

Prognostic factors influencing feasibility of cytogenetic and molecular analysis in leukapheresis products in chronic myelogenous leukemia patients.

Conventional cytogenetic (CC) study and molecular analysis were performed in 150 leukapheresis products from 36 patients diagnosed with chronic myelogenous leukemia who were included in an autologous stem cell transplantation program. The aims of the study were to evaluate the effectiveness of these two methods for the detection of residual disease in the harvest and to identify the factors influencing the number of cycling cells present in the apheresis products. Progenitor cell mobilization procedures performed late after diagnosis (>12 months), a short interval between interferon-alpha discontinuation and mobilization (<3.5 months), and an intensive mobilization regimen (idarubicin, cytarabine, and etoposide, ICE protocol) were associated with a low probability of obtaining 25 metaphases, which was achieved in only 41 instances (25% of the samples). In 38 samples, less than ten metaphases were obtained; a peripheral blood leukocyte count <1.0x10(9)/l at mobilization and mononuclear cell counts in the bag <0.5x10(8)/kg significantly increased the probability to obtain less than ten metaphases for CC analysis. Previous interferon-alpha treatment during > or =12 months and low mononuclear cell counts in the bag (<0.5x10(8)/kg) increased the probability of not obtaining mitosis for cytogenetic analysis. Molecular analysis by the polymerase chain reaction (PCR) technique did not give discriminate information in the samples not evaluable by cytogenetics due to the high frequency of PCR-positive results. We conclude that new techniques such as hypermetaphase fluorescence in situ hybridization (FISH), interphase FISH, or quantitative PCR need to be routinely employed in the study of leukapheresis samples of chronic myelogenous leukemia patients for a better assessment of the neoplastic contamination of the infused products.

Adult↗

PubMed2002

Quantitative real-time polymerase chain reaction for detection of circulating cells with t(14;18) in volunteer blood donors and patients with follicular lymphoma.

The chromosomal rearrangement t(14;18)(q32;21) involves the major (MBR) or minor (mcr) breakpoint cluster regions and the immunoglobulin heavy chain joining regions (JH) in most follicular lymphomas. As a first step towards determining the clinical significance of circulating cells with t(14;18), we detected and quantitated circulating cells in samples obtained from volunteer blood donors and follicular lymphoma patients. The t(14;18) was co-amplified with beta-actin with real-time quantitative PCR (QRT-PCR) in reactions containing 1 microg of DNA from peripheral blood or bone marrow aspirates. The cell number was quantitated using linear regression and an external standard of serially diluted DNA from cell lines with MBR/JH or mcr/JH rearrangements. At dilutions of 10(5) and 106, sensitivity was 100 and 55% for MBR/JH, and 100 and 10% for mcr/JH rearrangements. Among 102 volunteer blood donors MBR/JH vs. mcr/JH amplicons were detected in 22 vs. 4% with duplicate 1 microg DNA reactions, and in 41 vs. 6% with a total 10 microg DNA analyzed in multiple reactions. Among volunteer blood donors the mean number of circulating cells with MBR/JH vs. mcr/JH rearrangements were 0.8 vs. 0.1/microg DNA, and exceeded the upper normal limit (defined as the mean of all volunteer samples plus two standard deviations) in 3% vs. 2%, respectively. Analysis for MBR/JH rearrangements revealed that follicular lymphoma patients vs. volunteer blood donors were positive in 76% vs. 22% (p = 0.008 by Fisher's exact test); that the mean number of MBR/JH cells per microg of DNA was 91 vs. 0.8 (p = 0.0002 by Mann-Whitney test); and the number of the MBR/JH cells exceeded the upper normal limit in 32% vs. 3% of subjects (p = 0.0001 by Fisher's exact test). Circulating cells with mcr/JH were not detected among any of these 25 lymphoma patients. We conclude that patients with follicular lymphoma are more frequently positive, have higher numbers of circulating cells with t(14;18), which exceed upper normal limit more frequently than in volunteer blood donors.

Blood Cells↗

PubMed2004

Development of an exonuclease protection mediated PCR bioassay for sensitive detection of Ah receptor agonists.

The aromatic hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates many of the biological and toxicological effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) and related chemicals. Here we developed a novel method to detect the presence of AhR ligands using Exonuclease Protection Mediated PCR bioassay (EPM-PCR). This assay measures the ability of a chemical to activate AhR DNA binding in vitro. In the presence of AhR ligand, an expected length PCR product was observed on electrophoresis, but no signal was detected in the absence of ligand. Real-time quantitative PCR was performed to quantify DNA bound to ligand:AhR complex. We obtained a standard curve with TCDD concentration to bound DNA copies in the range of 0.01 pM-10 nM of TCDD. Minimal detection limit of the assay was below 0.01 pM TCDD, and the whole detection time was less than 5 h. In comparison to the chemical-activated luciferase gene expression (CALUX) bioassay, EPM-PCR bioassay is more sensitive and easier to perform. These results suggest that this assay is useful for detection and quantification of TCDD and related AhR ligands in a cell-free system without the use of radioactivity.

Animals↗

PubMed1999

Saccharomyces cerevisiae RNA polymerase I terminates transcription at the Reb1 terminator in vivo.

We have mapped transcription termination sites for RNA polymerase I in the yeast Saccharomyces cerevisiae. S1 nuclease mapping shows that the primary terminator is the Reb1p terminator located at +93 downstream of the 3' end of 25S rRNA. Reverse transcription coupled with quantitative PCR shows that approximately 90% of all transcripts terminate at this site. Transcripts which read through the +93 site quantitatively terminate at a fail-safe terminator located further downstream at +250. Inactivation of Rnt1p (an RNase III involved in processing the 3' end of 25S rRNA) greatly stabilizes transcripts extending to both sites and increases readthrough at the +93 site. In vivo assay of mutants of the Reb1p terminator shows that this site operates in vivo by the same mechanism as has previously been delineated through in vitro studies.

Base Sequence↗

PubMed2006

AIB1 gene amplification and the instability of polyQ encoding sequence in breast cancer cell lines.

BACKGROUND: The poly Q polymorphism in AIB1 (amplified in breast cancer) gene is usually assessed by fragment length analysis which does not reveal the actual sequence variation. The purpose of this study is to investigate the sequence variation of poly Q encoding region in breast cancer cell lines at single molecule level, and to determine if the sequence variation is related to AIB1 gene amplification. METHODS: The polymorphic poly Q encoding region of AIB1 gene was investigated at the single molecule level by PCR cloning/sequencing. The amplification of AIB1 gene in various breast cancer cell lines were studied by real-time quantitative PCR. RESULTS: Significant amplifications (5-23 folds) of AIB1 gene were found in 2 out of 9 (22%) ER positive cell lines (in BT-474 and MCF-7 but not in BT-20, ZR-75-1, T47D, BT483, MDA-MB-361, MDA-MB-468 and MDA-MB-330). The AIB1 gene was not amplified in any of the ER negative cell lines. Different passages of MCF-7 cell lines and their derivatives maintained the feature of AIB1 amplification. When the cells were selected for hormone independence (LCC1) and resistance to 4-hydroxy tamoxifen (4-OH TAM) (LCC2 and R27), ICI 182,780 (LCC9) or 4-OH TAM, KEO and LY 117018 (LY-2), AIB1 copy number decreased but still remained highly amplified. Sequencing analysis of poly Q encoding region of AIB1 gene did not reveal specific patterns that could be correlated with AIB1 gene amplification. However, about 72% of the breast cancer cell lines had at least one under represented (<20%) extra poly Q encoding sequence patterns that were derived from the original allele, presumably due to somatic instability. Although all MCF-7 cells and their variants had the same predominant poly Q encoding sequence pattern of (CAG)3CAA(CAG)9(CAACAG)3(CAACAGCAG)2CAA of the original cell line, a number of altered poly Q encoding sequences were found in the derivatives of MCF-7 cell lines. CONCLUSION: These data suggest that poly Q encoding region of AIB1 gene is somatic unstable in breast cancer cell lines. The instability and the sequence characteristics, however, do not appear to be associated with the level of the gene amplification.

Acetyltransferases↗

PubMed1996

Comparison of polymerase chain reaction and pp65 antigen test for early detection of human cytomegalovirus in blood leukocytes of cardiac transplant recipients.

OBJECTIVE: To establish whether polymerase chain reaction (PCR) for cytomegalovirus deoxyribonucleic acid (DNA) can provide clinical information for the management of the infection. METHODS: Leukocytes in 30 heart transplant recipients were monitored by pp65 antigen testing and PCR for 82 to 365 days after transplantation. RESULTS: Of the 30 patients, 26 developed cytomegalovirus infection, nine of whom were symptomatic. Altogether, 300 leukocyte samples were examined. The concordance between PCR and pp65 antigen test was 82.6%. In symptomatic patients after surgery, PCR detected cytomegalovirus infection after 38 plus minus 16 days and the pp65 antigen test, after 48 plus minus 15 days. Symptomatic infection correlated with a higher number of pp65-positive leukocytes than did asymptomatic infection: 310 plus minus 356 vs 24 plus minus 35 (p < 0.005)/200,000 examined, respectively. Clearance of virus was observed by PCR after 125 plus minus 73 days (range 29 to 225) in symptomatic, and after 82 plus minus 70 days (range 16 to 301) in asymptomatic, cases of infection. CONCLUSIONS: The positive predictive value of PCR for symptomatic infection was 34.6%. Our findings correlate with previous reports and show that the qualitative detection of cytomegalovirus DNA is not associated with overt disease whereas quantitation of pp65-positive leukocytes closely correlate with symptom onset. Insofar as the results are not quantitative, PCR is not a marker of clinically apparent infection. Careful monitoring of cytomegalovirus infection based on quantitative pp65 antigen assay can fulfill all clinical needs for early diagnosis and proper management of the infection

Journal Article↗

PubMed1994

Preferential usage of T-cell receptor alpha beta variable regions among tumor-infiltrating lymphocytes in primary human malignant melanomas.

The usage of T-cell-receptor (TCR) alpha beta variable (V) regions among tumor infiltrating lymphocytes (TILs) in primary human malignant melanomas was characterized using a method based on the polymerase chain reaction (PCR). A panel of 57 different variable-region primers specific for the TCR V alpha I-29 and V beta I-28 was designed, and a semi-quantitative PCR method applicable to formalin-fixed, paraffin-embedded tissues was developed. This semi-quantitative method was demonstrated to be reproducible and to be useful for the assessment of V alpha- and V beta-gene-family usage in formalin-fixed, paraffin-embedded tissue samples. A total of 9 different histopathologically characterized primary tumors were analyzed in this study. The TILs in these tumors were found to preferentially express certain TCR V alpha and V beta regions. The differential usage of certain V alpha regions was very pronounced as illustrated by V alpha 4, which was highly expressed in 3/8 tumors, and V alpha 22, which was highly expressed in 4/8 tumors. For comparison, specific highly expressed V alpha regions in control samples of peripheral-blood lymphocytes rarely exceeded 10%. The most highly expressed V beta region was V beta 8, which was highly expressed in 2/8 tumors. For the highly expressed V alpha 4 and V alpha 22 and V beta 8 regions, the high levels may be explained by the in situ clonal or oligoclonal expansion of TIL. In one specific case, the high expression of V beta 8 was due to expansion of a single clone of TILs, as evidenced by a fully readable sequence of the CDR3 (V-D-J) region, determined by direct sequencing of the PCR product corresponding to V beta 8. In contrast, sequence analysis of V alpha 22, which was expressed in the same tumor sample at similar levels, demonstrated the simultaneous presence of 3 different CDR3 (V-J) sequences derived from V alpha 22 transcripts of exactly the same length. The observed preferential use of TCR V alpha and V beta regions suggests the in situ clonal expansion of specific populations of T-cells, possibly reactive with melanoma-associated peptides presented by HLA molecules. The preferential use of TCR V alpha and V beta regions may imply the involvement of a limited number of shared melanoma-associated peptides.

Base Sequence↗

PubMed1997

Diagnostic tests for hepatitis C.

Diagnostic tests for hepatitis C can be divided into the following two general categories: 1) serological assays that detect antibody to hepatitis C virus (anti-HCV); and 2) molecular assays that detect, quantify, and/or characterize HCV RNA genomes within an infected patient. Serological assays have been subdivided into screening tests for anti-HCV, such as the enzyme immunoassay (EIA), and supplemental tests such as the recombinant immunoblot assay (RIBA). Three generations of anti-HCV tests have been developed, and each generation has resulted in an improvement in the sensitivity of detecting anti-HCV. Supplemental anti-HCV tests are designed to resolve false-positive testing by EIA, and are appropriately used in low-prevalence settings in which false-positive anti-HCV tests remain a problem. Third-generation anti-HCV tests (EIA-3 and RIBA-3, respectively) contain antigens from the HCV core, nonstructural 3, nonstructural 4, and nonstructural 5 genes. Detection of HCV RNA in patient specimens by polymerase chain reaction (PCR) provides evidence of active HCV infection and is potentially useful for confirming the diagnosis and monitoring the antiviral response to therapy. Optimal HCV PCR assays at present have a sensitivity of less than 100 copies of HCV RNA per milliliter of plasma or serum. Standardization and proficiency testing of diagnostic laboratories performing HCV PCR remains an important problem for future study. Two main technologies exist for assessing HCV RNA levels or viral load. Quantitative PCR is the most sensitive test for determining hepatitis C viral load, whereas the branched-chain DNA test appears to be the most precise method. Major limitations of the current tests are inadequate dynamic range and high variability of PCR-based assays, and poor sensitivity of the branched-chain DNA test. Molecular tests have also been developed to classify HCV into distinct genotypes; the clinical importance of HCV genotype determination remains a subject for future investigation.

Genotype↗

PubMed2005

Investigation of human brain tumors for the presence of polyomavirus genome sequences by two independent laboratories.

JC virus (JCV), BK virus (BKV) and simian virus 40 (SV40) may be associated with human brain tumors. These polyomaviruses have been shown to induce brain tumors in experimentally infected animals. Several studies have found polyomavirus genomic sequences in human brain tumor tissues by using polymerase chain reaction (PCR), while others have not. Inconsistencies in previous findings may be due in part to small sample sizes and differences in underlying patient populations, laboratory techniques and quality control measures. To assess the role of polyomaviruses in human brain tumors and address inconsistencies of previous reports, we investigated the prevalence of viral sequences in a series of 225 brain tumor tissue specimens in 2 independent laboratories. PCR followed by Southern hybridization was performed at the National Institute of Neurological Disorders and Stroke (NINDS). Real-time quantitative PCR was performed on the same tissues at Johns Hopkins University (JHU). Only those tumors with amplifiable DNA were tested further for polyomavirus sequences. Positive and negative control tissues were included, and all specimens were masked. Amplifiable DNA was detected in 225/225 (100%) tumors at NINDS, 9 (4%) of which contained polyomavirus sequences (3 JCV-positive, 3 BKV-positive and 3 SV40-positive). The JHU laboratory amplified DNA from 165/225 (73%) tumors, of which 1 tumor tested positive (for SV40). No tumors tested positive in both laboratories. Results for masked quality control tissues were concordant between laboratories. Nucleotide sequences for JCV, BKV and SV40 are rarely present in a large series of adult and pediatric brain tumors.

Adolescent↗

PubMed2003

Ultrastructural localization of vimentin immunoreactivity and gene expression in tanycytes and their alterations in hamsters kept under different photoperiods.

Tanycytes are located in the basolateral walls of the third ventricle. By light- and electron-microscopic immunohistochemistry, we demonstrated that the tanycytes of Djungarian hamsters were intensely immunostained with the vimentin monoclonal antibody V9. The cells always extended long radial processes in which aggregations of vimentin-immunoreactive intermediate filaments were prominent. The tanycytes showed photoperiod-dependent changes. The population of vimentin-immunoreactive tanycytes was increased in hamsters exposed to continuous lighting for 1 month or to a long photoperiod (16 h light:8 h dark) for 2 months. On the other hand, the immunoreactive cells were significantly decreased in hamsters exposed to complete darkness for 1 month or to a short photoperiod (8 h light:16 h dark) for 2 months. The pericapillary spaces of the primary plexus of the portal circulation system were lined by the end-feet of tanycytic processes. Electron microscopy confirmed that the tanycytic processes were markedly decreased in number and size after exposure to complete darkness. Expression of vimentin mRNA in the hamster mediobasal hypothalamus was detected by the reverse transcription-polymerase chain reaction (RT-PCR). The alterations of vimentin mRNA expression under different photoperiods were analyzed using laser capture microdissection and real-time quantitative PCR. The level of vimentin mRNA in the mediobasal hypothalamus was enhanced after exposure to continuous lighting for 1 month or to a long photoperiod for 2 months, whereas it was significantly diminished after exposure to constant darkness or short photoperiod. These changes in the tanycytes under different photoperiods may influence the portal circulation system and also the cell activity of the pars tuberalis, and may thus participate in photoperiodic regulation of the endocrine system.

Animals↗

PubMed2025

Effect of transgene on salt tolerance of tobacco.

To explore the effects of salt-tolerance gene accumulation on salt tolerance in transgenic plant, we used four types of plant expression vector (N27, N28, N29, and N30) carrying mtlD, mtlD + gutD, mtlD + gutD + BADH, mtlD + gutD + BADH + sacB genes respectively, to transform tobacco through Agrobacterium-mediated method. Transgenic lines were identified through polymerase chain reaction (PCR) detection. Transgenic lines and non-transgenic plant (CK) were subjected to 6‰ sodium chloride solution stress; then, fluorescence quantitative PCR (FQ-PCR) and salt tolerance indexes were used to assess characteristics. PCR showed the exogenous genes had been integrated into the tobacco genome. FQ-PCR showed under clean water treatment the target genes were expressed in all transgenic plants at the transcriptional level. The transcript abundances of target genes changed with the number of genes increased, and improved following salt stress. Comparative analyses of salt tolerance indexes showed height growth, biomass (except for N29), chlorophyll content, net photosynthetic rate, Fv/Fm, and PI of all transgenic plants and CK were lower under salt stress than under clean water treatment, to varying degrees. However, the descent ratio was smaller in transgenic plants. A comprehensive evaluation of multiple salt-tolerance indicators performed using the membership function method showed the average salt tolerance of each vector transgenic line was higher than that of CK, and salt tolerance was greater in transgenic polyvalent gene lines than in transgenic monovalent gene lines. The average salt tolerance was N29 > N28 > N30 > N27 > CK. This study provides a theoretical and practical reference for salt tolerance breeding in other plants.

Plants, Genetically Modified↗

PubMed2006

Detection of Escherichia coli O157 using equal-length double-stranded fluorescence probe in a real-time polymerase chain reaction assay.

BACKGROUND: Enterohemorrhagic Escherichia coli (E. coli) O157 is a dangerous pathogen, which causes bloody diarrhea and severe hemolytic uremic syndrome (HUS). Although several assay systems based on real-time polymerase chain reaction (PCR) have been integrated to detect this pathogen, most of them are not specific. We report a real-time quantitative PCR method targeting rfbE, a gene specifically expressed in E. coli O157. This method can therefore be used to diagnose enterohemorrhagic Escherichia coli (E. coli) O157. METHODS: A nucleic acid based diagnostic assay system, combining equal-length double-stranded fluorescence probe technique and real-time PCR, was developed to detect E. coli O157. This assay system take advantage of the highly conserved rfbE O-antigen synthesis gene, and a pair of fluorescence-quenching probes complementary to rfbE gene were used in a real-time PCR to quantify the presence of the pathogen. RESULTS: The specificity of the diagnostic method was assessed by comparing test results on 14 different related pathogens including common E. coli, enteroinvasive Escherichia coli (EIEC), Salmonella, Shigella and E. coli O157. The detection limit of the method was determined using 10-fold serial dilutions of an E. coli O157 standard sample, and as few as 1.49 x 10(3) CFU/ml could be detected. All E. coli with serotype O157, which expresses rfbE gene, were positive in this assay, while all other species without rfbE gene expression were negative. CONCLUSIONS: By combining equal-length double-stranded fluorescence probe technique and real-time PCR, we have developed a simple, rapid, specific and sensitive method to detect E. coli O157.

DNA↗

PubMedDate not supplied

Quantification of a low cellular immune response to aid in identification of pediatric liver transplant recipients at high-risk for EBV infection.

OBJECTIVE: Uncontrolled EBV infection leading to lymphoproliferative disease is a significant cause of morbidity in pediatric orthotopic liver transplant (OLT) recipients. Herein, we describe the use of a novel immune assay, which quantifies the lymphocyte immune response and correlates the value to risk for EBV infection. METHODS: All patient data were prospectively collected between 2003 and 2005. The study included 18 pediatric liver transplant recipients, seven males and 11 females with a mean follow-up of 47 months post-OLT. Patient EBV load was monitored using real-time quantitative PCR (qPCR). The ATP release (ng/mL) of CD3+ lymphocytes after mitogenic stimulation with phytohemagluttinin (PHA; Cylex Corporation) was used to quantitate patient immune response. Patients were stratified by EBV load: low (<1000 copies/microg DNA), medium (1000-4000 copies/microg DNA), and high (>4000 copies/microg DNA). RESULTS: Patients with low EBV loads had a significantly (p < 0.04) stronger immune response to PHA than patients with EBV load >1000 copies/microg DNA. Further analysis demonstrated that patients with ATP level <125 ng/mL had 100% probability of an EBV titer >4000 copies/microg DNA, when compared with 22% if the ATP level was between 125 and 400 ng/mL or only 15% if >400 ng/mL (p < 0.05). When immunosuppression was reduced, we observed an increase of the ATP release that correlated with a decrease of the EBV viral load. CONCLUSION: In conclusion, this study investigates the use of a lymphocyte activation assay to closely measure the immunosuppression status of pediatric liver transplant recipients. Because measurement of EBV DNA load as a single parameter has a poor positive predictive value for development of PTLD, the association of these assays may be of help in the identification of patients at risk for PTLD.

Adenosine Triphosphate↗

PubMed1997

Paraffin-embedded tissue as a source of RNA for gene expression analysis in oral malignancy.

OBJECTIVE: To assess the feasibility of using archival oral mucosal tissue to examine gene expression at the ribonucleic acid (RNA) level. MATERIALS AND METHODS: We describe the isolation of RNA from 8 microns sections of formalin-fixed paraffin-embedded oral mucosal tissue. RNA was reverse transcribed and three candidate genes amplified by polymerase chain reaction (PCR). The ribosomal protein S14 gene is a housekeeping gene which has been used as an internal standard in several quantitative PCR protocols. The thymidine kinase (TK) gene is expressed at low levels in most tissues and, with a well-documented genomic organisation, is a useful tool for discrimination between genomic DNA and cDNA. The RI alpha gene is reported to be overexpressed in many cancer cell lines, in malignant tissue and in vitro transformed cells. RESULTS: The S14 gene, the TK gene and the RI alpha gene of the cAMP-dependent protein kinase (PKA) were amplified successfully from formalin-fixed paraffin-embedded tissue sections. The TK primer pair is a useful additional tool in the unambiguous identification of RNA-derived species. CONCLUSION: RNA suitable for reverse transcribed (RT)-PCR was extracted from archival oral mucosal tissue. This should permit rapid sequence analysis of transcribed tumor suppressor genes and oncogenes in this material. Furthermore, the RT-PCR approach described may allow quantification of gene expression in oral mucosal archival material processed in a standard fashion.

Carcinoma, Squamous Cell↗

PubMed2004

Detection of four Plasmodium species in blood from humans by 18S rRNA gene subunit-based and species-specific real-time PCR assays.

There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the "gold standard" in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four species-specific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.

Animals↗

PubMed2004

Rapid quantification of Yersinia enterocolitica in pork samples by a novel sample preparation method, flotation, prior to real-time PCR.

The development of real-time PCR thermal cycles in the late 1990s has opened up the possibility of accurate quantification of microorganisms in clinical, environmental, and food samples. However, a lack of suitable sample preparation methods that allow rapid quantification of the nucleic acids, remove PCR inhibitors, and prevent false-positive results due to DNA originating from dead cells has limited the use of quantitative PCR. We have used for the first time a new variant of density gradient centrifugation, called flotation, as a user-friendly sample preparation method prior to PCR. This paper describes the use of this sample preparation method, without DNA purification, for direct detection and quantification of Yersinia enterocolitica in PCR-inhibitory meat juice from pork. Flotation combined with qPCR could overcome PCR interference in juice from pork, as was shown by amplification efficiencies of 1.006 +/- 0.021 and 1.007 +/- 0.025, which are comparable to the amplification efficiency obtained for purified DNA samples (1.005 +/- 0.059). Applying flotation to meat juice samples containing natural background flora and spiked with different levels of Y. enterocolitica showed that direct quantification of Y. enterocolitica was possible down to a level of at least 4.2 x 10(3) CFU per ml of meat juice, even in the presence of 10(6) CFU of background flora per ml. Finally, the results showed that samples containing large amounts of Y. enterocolitica DNA did not result in a positive PCR signal. This indicates that the risk of false-positive results due to detection of DNA originating from dead cells can be greatly reduced by using flotation prior to PCR.

Animals↗

PubMed2004

Altered gene expression and increased bursting activity of colonic smooth muscle ATP-sensitive K+ channels in experimental colitis.

The ATP-sensitive K(+) channel (K(ATP)) is a complex composed of an inwardly rectifying, pore-forming subunit (Kir 6.1 and Kir 6.2) and the sulfonylurea receptor (SUR1 and SUR2). In gastrointestinal smooth muscle, these channels are important in regulating cell excitability. We examined the molecular composition of the K(ATP) channel in mouse colonic smooth muscle and determined its activity in the pathophysiological setting of experimental colitis. Following 7 days of dextran sulfate sodium (DSS) treatment in drinking water, colonic inflammation was scored by histology and physical signs. In whole cell recordings, levcromakalim-induced currents were significantly larger in inflamed cells. In cell-attached patch recordings of single-channel events, levcromakalim enhanced the bursting duration in inflamed cells. The single-channel conductance of approximately 42 pS was not altered with inflammation. mRNA for both Kir 6.1 and 6.2 were detected by RT-PCR. Kir 6.1 was localized to the plasma membrane, whereas Kir 6.2 was mainly detected in the cytosol by immunohistochemistry. Quantitative PCR showed that Kir 6.1 gene expression was upregulated by almost 22-fold, whereas SUR2B was downregulated by threefold after inflammation. Thus decreased motility of the colon during inflammation may be associated with changes in the transcriptional regulation of Kir 6.1 and SUR2B gene expression.

ATP-Binding Cassette Transporters↗