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PubMed2001

Use of cloned DNA fragments as reference materials for event specific quantification of genetically modified organisms (GMOs).

For the quantification of genetically modified organisms (GMOs) in foods and feeds, real-time PCR is currently the most widely applied technique. To obtain a % of GMO, a GMO-specific target sequence is quantified relatively to a species-specific sequence. The correctness and reliability of the obtained quantitative results fully depend on the reference materials used as standards for setting up external calibration curves. We introduced a completely new type of standards for quantification of GMOs, based on cloned plasmid DNA solutions with well-known amounts of the sequences of interest, expressed as copy numbers. Moreover, the junction sequence between inserted DNA and plant DNA was used as 'unique identifier'. In this study, the model was applied for Roundup Ready soybean.

Calibration↗
PubMed2005

Progenitor cell trafficking in physiologic conditions and in myeloproliferative diseases: quantification of CD34+ cells by polymerase chain reaction.

BACKGROUND AND OBJECTIVES: Previous studies using flow cytometry have shown that CD34+ cell trafficking is increased in patients with chronic idiopathic myelofibrosis. Few data exist on physiologic CD34 + cell trafficking and the quantification of very low cell ranges requires reliable and sensitive measurement techniques. The aim of this study was to establish a quantitative polymerase chain reaction (PCR) technique for studying CD34+ cell trafficking in physiologic conditions, and in patients with myeloproliferative diseases. DESIGN AND METHODS: CD34+ cell trafficking was measured in 56 controls [(healthy controls (n=21), patients with ischemic cardiopathy (n=21), patients with secondary thrombocytosis or erythrocytosis (n=14)], and in 37 untreated patients with myeloproliferative diseases diagnosed according to the WHO-criteria [(essential thrombocythemia (n=10), polycythemia vera (n=14) and chronic idiopathic myelofibrosis (n=13)]. Quantitative PCR was used to determine CD34 mRNA expression in peripheral blood samples. RESULTS: Physiologic CD34 mRNA expression ranges were determined in the healthy control group. Mean CD34 mRNA expression was within the physiologic range in patients with ischemic cardiopathy, secondary thrombocytosis or erythrocytosis, essential thrombocythemia and polycythemia vera (p=0.146), but was significantly increased in patients with chronic idiopathic myelofibrosis (p<0.001). When analyzed individually, 12/13 patients with chronic idiopathic myelofibrosis and 3/14 patients with polycythemia vera showed CD34 mRNA expression above the physiologic range. INTERPRETATION AND CONCLUSIONS: This is a first report about CD34+ cell trafficking measured by quantitative PCR. Quantitative PCR is a reliable method suitable for the quantification of very low cell populations. Our study confirms the significant increase of CD34+ cell trafficking in patients with chronic idiopathic myelofibrosis, and in a subset of patients with polycythemia vera. Prospective studies are underway to characterize these circulating CD34+ cells and to investigate their role in the pathophysiology of myeloproliferative diseases.

Adult↗
PubMed1992

[Echocardiographic quantification of mitral regurgitation. Comparative study with transthoracic and transesophageal Doppler color echocardiography].

The quantification of mitral regurgitation by means of colour coded Doppler echocardiography still is a controversial issue. A prospective study on 86 consecutive patients with mitral regurgitation diagnosis was carried out by means of conventional transthoracic colour coded Doppler echocardiography in order to make a comparative study with results obtained by means of transoesophageal colour coded Doppler echocardiography. A systematic measurement of the following parameters was carried out in each patient: Colour coded area or mitral regurgitation jet, auricular depth of mitral regurgitation jet, severity degree of mitral regurgitation, left auricular area, mitral regurgitation ratio and transthoracic crossection plan of largest regurgitation area. Both echocardiographic techniques had the first three parameters in common. The mitral regurgitation area by transthoracic echocardiography was 370 +/- 41 mm2 and by transoesophagial technique 558 +/- 43 mm2 (p less than or equal to 0.001 - r = 0.71). The depth of mitral regurgitation by transthoracic path was 31 +/- 2.7 mm and by transoesophageal path 37 +/- 25 mm (p less than or equal to 0.01 - r = 0.74). The existing correlation between the mitral regurgitation area obtained by both techniques improved (r = 0.84) when a four camera apical transthoracic plan was used and worsened (r = 0.45) when the longitudinal parasternal path was used. We came to the conclusion that the valorization of mitral regurgitation made by both techniques is not accurately comparable, existing undervaluation in transthoracic echocardiography. The discrepancies between the two techniques are more patent and statistically more significant when longitudinal parasternal plan is used for transthoracic quantification of severity degree of mitral regurgitation.(ABSTRACT TRUNCATED AT 400 WORDS)

Adult↗
PubMed2005

Quantification of 123I-PE2I binding to dopamine transporter with SPECT after bolus and bolus/infusion.

UNLABELLED: The aim of the present study was to describe a method combining easy implementation in a clinical setting with accuracy and precision in quantification of 123I-labeled N-(3-iodoprop-(2E)-enyl)-2beta-carboxymethoxy-3beta-(4'-methylphenyl)nortropane (PE2I) binding to brain dopamine transporter. METHODS: Five healthy subjects (mean age, 50 y; range, 40-68 y) were studied twice. In the first experiment, dynamic SPECT data and arterial plasma input curves obtained after 123I-PE2I bolus injection were assessed using Logan, kinetic, transient equilibrium, and peak equilibrium analyses. Accurate and precise determination of BP1 (binding potential times the free fraction in the metabolite-corrected plasma compartment) and BP2 (binding potential times the free fraction in the intracerebral nonspecifically bound compartment) was achieved using Logan analysis and kinetic analysis, with a total study time of 90 min. In the second experiment, (123)I-PE2I was administrated as a combined bolus and constant infusion. The bolus was equivalent to 2.7 h of constant infusion. RESULTS: The bolus-to-infusion ratio of 2.7 h was based on the average terminal clearance rate from plasma in the bolus experiments. Steady state was attained in brain and plasma within 2 h, and time-activity curves remained constant for another 2 h. Even when an average bolus-to-infusion ratio was used, the striatal BP1 and BP2 values calculated with kinetic analysis (BP1 = 21.1 +/- 1.1; BP2 = 4.1 +/- 0.4) did not significantly differ from those calculated with bolus/infusion analysis (BP1 = 21.0 +/- 1.2; BP2 = 4.3 +/- 0.3). Computer simulations confirmed that a 2-fold difference in terminal clearance rate from plasma translates into only a 10% difference in BP1 and BP2 calculated from 120 to 180 min after tracer administration. CONCLUSION: The bolus/infusion approach allows accurate and precise quantification of 123I-PE2I binding to dopamine transporter and is easily implemented in a clinical setting.

Adult↗
PubMed2005

A duplex real-time qPCR assay for the quantification of human nuclear and mitochondrial DNA in forensic samples: implications for quantifying DNA in degraded samples.

A duplex real-time qPCR assay was developed for quantifying human nuclear and mitochondrial DNA in forensic samples. The nuclear portion of the assay utilized amplification of a approximately 170-190 bp target sequence that spans the repeat region of the TH01 STR locus, and the mitochondrial portion of the assay utilized amplification of a 69 bp target sequence in the ND1 region. Validation studies, performed on an ABI 7000 SDS instrument using TaqMan detection, demonstrated that both portions of the duplex assay provide suitable quantification sensitivity and precision down to 10-15 copies of each genome of interest and that neither portion shows cross-reactivity to commonly encountered non-human genomes. As part of the validation studies, a series of DNase-degraded samples were quantified using three different methods: the duplex nuclear-mitochondrial qPCR assay, the ABI Quantifiler Human DNA Quantification Kit qPCR assay, which amplifies and detects a 62 bp nuclear target sequence, and slot blot hybridization. For non-degraded and moderately degraded samples in the series, all three methods were suitably accurate for quantifying nuclear DNA to achieve successful STR amplifications to yield complete profiles using the ABI AmpFlSTR Identifiler kit. However, for highly degraded samples, the duplex qPCR assay provided better estimates of nuclear template for STR amplification than did either the commercial qPCR assay, which overestimated the quantity of STR-sized DNA fragments, leading to an increased proportion of undetected alleles at the larger STR loci, or slot blot hybridization, which underestimated the quantity of nuclear DNA, leading to an increased proportion of STR amplification artifacts due to amplification of excess template.

DNA↗
PubMed2003

Interference of contaminating DNA in the quantification of a toluene-induced tod gene in Pseudomonas putida.

To determine the extent of interference of co-extracted DNA contamination in the quantification of the tod gene transcript, two different concentrations of RNA (high, 500 ng/microl; low, 250 ng/microl) from a toluene-induced culture of Pseudomonas putida were treated with different amounts of DNase (2, 4, 6 and 8 U) and incubated for 30 and 60 min. The highly sensitive and reproducible TaqMan system was used to quantify the transcript of the tod gene, the tod gene in contaminating DNA and the 16S rRNA gene in DNase-treated RNA samples. For the high RNA concentration, the shorter incubation time (30 min) lowered the level of contaminating DNA as evidenced by the presence of 2.5 x 10(6) copies of the tod gene before treatment to 1.4 x 10(5) copies/microl (8 U), whereas, irrespective of the DNase units used, the longer incubation time (60 min) considerably lowered the level of DNA contamination (2.5 x 10(6) to 6.5 x 10(2) copies of the tod gene/microl). However, for the low RNA concentration, DNase treatment was found to be equally effective in lowering the level of contaminating DNA (10(6) to 10(2) copies of the tod gene/mu), irrespective of the incubation time and the amount of DNase used. Although the results of gel electrophoresis of conventional PCR amplification of the low RNA concentration revealed the absence of the target gene in contaminating DNA, the results of the TaqMan PCR indicated that a very low amount of contaminating DNA (less than 10(3) copies of the tod gene/mul) was still present in RNA samples, even after the DNase treatment. The number of copies of the tod gene transcript in RNA samples did not show any marked variation because of the DNase treatment. However, the proportion of contaminating DNA in RNA samples considerably decreased due to the treatment (0.01 to 0.000001). Furthermore, these results suggested that the extent of the removal of contaminating DNA from RNA samples depends on the concentration of RNA, the amount of DNase used and the incubation time. It is also suggested that the copies of the catabolic genes in contaminating DNA have to be quantified along with the target genes in RNA samples to have a more accurate quantification of the target genes for better understanding of their roles in many microbial processes.

Journal Article↗
PubMed2005

[Qualification and quantification of 10 sulfonamides in animal feedstuff by high performance liquid chromatography-electrospray tandem mass spectrometry].

The presence of sulfonamide (SA) residues in foods is largely due to the raising of animals with sulfonamide antibiotics added or polluted feedstuff. Because of interference from the matrices, the commonly used immunoassay or chromatographic method is not suitable for the analysis of multi-SAs in feedstuff. A high performance liquid chromatographic-electrospray tandem mass spectrometric (HPLC/ESI-MS-MS) method has been established for the simultaneous determination of multi-SAs including sulfadiazine (SD), sulfapyridine (SPD), sulfamerazine (SM1), sulfameter (SM), sulfamethazine (SM2), sulfamethoxypyridazine (SMP), sulfamethoxazole (SMZ), sulfamonomethoxine (SMM), sulfadimethoxine (SDM) and sulfaquinoxaline (SQX). After solvent extraction, solid phase extraction, dilution and reversed-phase HPLC separation, SAs were detected by ESI-MS-MS under multi-reaction monitoring mode. The qualification analysis was done by using retention time and distribution of diagnostic ion pairs, and the quantification was based on the peak intensity of common fragment ion m/z 156. The limits of quantification for 10 SAs were 0.5 - 2.0 microg/kg (S/N = 10). The correlation coefficient of linear calibration curve was over 0.9995 within the SAs concentration range 2.0 - 200 microg/L except for SDM and SQX. At the spiked level of 1.0 mg/kg, the average recoveries for the 10 SAs were between 70% and 92%, the relative standard deviations were under 10% for intra-day and under 15% for inter-day. Routine tests showed the method was fast, sensitive, specific, and practical for the SAs determination in feedstuff.

Animal Feed↗
PubMed1992

[Quantification of the levels of p24 antigen and antibodies in human immunodeficiency virus infection].

BACKGROUND: Quantification and clinical evaluation of p24 antigen and anti-HIV antibody levels. METHODS: Follow up of 13 HIV infected patients (53 sera) by determination of p24 antigen, total anti-HIV antibodies, anti-p24 and anti-env antibodies by ELISA and their semiquantitation. IgG and IgM class antibody determination by immunoblot techniques. RESULTS: The highest levels of p24 have been found in WR 1 and WR 6 stages. Commonly accepted serologic pattern has not been found in 100% of our cases. Western-blot is more sensitive for the detection of anti-p24 antibodies. CONCLUSIONS: p24 antigen appearance and a decrease in anti-p24 antibody level is related to a worse clinical prognosis. p24 antigen quantification is a usefull test for monitoring AZT therapy in patients suffering from AIDS.

Acquired Immunodeficiency Syndrome↗
PubMed2005

Development of a competitor DNA template of the 38 kDa gene for molecular quantification of M. tuberculosis.

The molecular quantification of Mycobacterium tuberculosis (TB) from clinical samples can improve the management of TB. Competitive polymerase chain reaction (C-PCR) is an accepted technique often used for this purpose, and IS6110 is the most popular target in such studies. As the number of these elements varies from 0 to 16 in clinical isolates, it is prone to give inconsistent results. A simple PCR-based approach is described in this study to generate a novel competitor for a single copy 38 kDa gene for the development of C-PCR for the quantification of the M. tuberculosis genome.

Adolescent↗
PubMed2006

Automated quantification of myocardial ischemia and wall motion defects by use of cardiac SPECT polar mapping and 4-dimensional surface rendering.

SPECT of cardiac perfusion and blood pools provides ungated 3-dimensional and gated 4-dimensional (4D) datasets of the ventricular myocardium. Modern reconstruction and review software is used to reorient the transverse thoracic slices into cardiac short-axis slices. Several validated algorithms are used to analyze these data. These programs segment out the left ventricle, determine the apical and basal limits, and then contour the endo- and epicardial surfaces. From these, 4D images of cardiac function that enable a dynamic review of wall motion are obtained. Global function in gated studies can be quantified by automatic computation of stroke volume and ejection fraction. Remapping of myocardial perfusion and wall motion into polar maps enables standardized quantification of the extent and severity of heart disease after comparison with databases of healthy hearts (normal databases). Several validated software packages make processing of these SPECT datasets comparatively easy and operator independent. The objectives of this review article are to describe the steps in the processing of a cardiac SPECT dataset for viewing and quantification, to explain the underlying algorithms used for automated processing, to compare the features of various software packages, to demonstrate how to read polar maps, and to identify and correct artifacts resulting from errors in automated processing.

Gated Blood-Pool Imaging↗
PubMedDate not supplied

Simultaneous quantification of terpenelactones and flavonol aglycones in hydrolyzed ginkgo biloba extract by liquid chromatography with inline ultraviolet and evaporative light scattering detection.

We report here a liquid chromatography (LC) method with inline ultraviolet/evaporative light scattering (UV/ELS) detection for the simultaneous quantification of the terpenelactones and flavonol aglycones in a single sample of hydrolyzed Ginkgo biloba extract (GBE). The sample is hydrolyzed by a rapid and convenient oven heating method for 1 h at 90 degrees C with 10% hydrochloric acid. The 1 h hydrolysis was found to be equivalent to the 2.25 h reflux treatment for dry powder extract, where total flavonol glycosides were 28.4 and 28.1%, respectively. Acceptable precision was achieved for total terpenelactones [relative standard deviation (RSD) = 4.8%] by ELS detection, and total flavonol aglycones (RSD = 2.3%) by UV detection. The analytical range was 1.5 to 7.3% (w/w) for the individual terpenelactones (ELS) and 2.5 to 15.0% (w/w) for the individual glycosides (UV) calculated from the aglycones quercetin, kaempferol, and isorhamnetin. This improved method allows for the first time high throughput sample preparation coupled with the quantification of the predominant compounds generally used for quality control of GBE in a single assay.

Chromatography↗
PubMed2005

[Significance of quantification of WT1 mRNA for monitoring minimal residual disease in acute myeloid leukemia patients].

OBJECTIVE: To evaluate the significance of quantification of WT1 mRNA for monitoring minimal residual disease (MRD) in patients with acute myeloid leukemia (AML). METHODS: WT1 mRNA level was detected with real-time quantitative RT-PCR (RQ-PCR) technique in bone marrow samples from 15 normal subjects (NBM) and 123 AML patients. Sixty-two AML samples were also detected AML1-ETO mRNA expression by RQ-PCR. Simultaneously follow-up of WT1 and AML1-ETO levels were carried out in 50 samples from 8 AML patients. WT1 and AML1-ETO levels were normalized by internal control ABL gene. RESULTS: All correlation co-efficiencies were over 0.99 for WT1, AML1-ETO and ABL standard curves. Co-efficiencies of both interassay and intraassay variation were below 4%. The WT1 expression levels in NBM were 0.001 to 0.019 with a median level of 0.008. Higher levels of WT1 expression were found in 61 of 67 (91%) newly diagnosed AML patients compared with NBMs and 37 of the 67 (55.2%) showed 100-fold higher WT1 levels than that in NBMs. WT1 mRNA levels were highest in M(4EO) and M(3) and lowest in M(1) and M(5) patients. There was an excellent correlation between WT1 and AML1-ETO gene expression levels (r = 0.88, P < 0.001). WT1 expression levels in three patients who were in continuous complete hematological remission (CHR) were within normal range. In three of four relapsing patients, WT1 expression levels increased 31.4, 11.4 and 4.0 fold respectively one month before hematological relapse. CONCLUSIONS: Quantification of WT1 expression level by RQ-PCR may be used to monitor MRD for most AML patients, but it is less sensitive than fusion gene. Continuous or significant increase of WT1 expression in CHR patients predicts an impending relapse.

Adolescent↗
PubMedDate not supplied

Quantification of proteins in dairy products using an optical biosensor.

Two recent techniques using optical immunosensor technology were developed for the quantification of milk proteins in dairy products. The first application is the simultaneous quantification of the 3 major caseins (alpha(s1), beta, and kappa). This assay consists of a 2-step sandwich strategy, with 2 monoclonal antibodies directed against the N- and C-terminal extremities of each of the caseins, respectively. This strategy permits only intact caseins to be quantified, and not their degradation products. The technique is fast (10 min), sensitive (detection limit about 0.87 microg/mL), and has been applied successfully to raw and drinking milks. In the second application, the severity of the heat treatment sustained by a milk of unknown origin is determined by quantifying separately the native and heat-denatured forms of alpha-lactalbumin with specific monoclonal antibodies. The technique allows discrimination of the different heat treatments studied (pasteurization, direct and indirect ultra-high temperature, sterilization), is fast (4 min), repeatable, fully automated, and requires no pretreatment of the milk sample.

Animals↗
PubMed2006

Liquid chromatographic-electrospray tandem mass spectrometric method for the quantification of nimodipine in human plasma.

A simple, sensitive and rapid liquid chromatography/electrospray ionization tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of nimodipine, a calcium channel blocker, in human plasma. Following liquid-liquid extraction, the analytes were separated using an isocratic mobile phase on a reverse phase C18 column and analyzed by MS in the multiple reaction monitoring mode using the respective [M + H]+ ions, m/z 419/343 for nimodipine and m/z 409/228 for the IS. The assay exhibited a linear dynamic range of 0.2-50 ng/mL for nimodipine in human plasma. The lower limit of quantification was 200 pg/mL with a relative standard deviation of less than 8%. Acceptable precision and accuracy were obtained for concentrations over the standard curve range. A run time of 3 min for each sample made it possible to analyze more than 250 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.

Biological Availability↗
PubMed1991

[Quantification of mitral valve diseases by Doppler].

Echo Doppler makes a fundamental contribution to the non-invasive quantification of mitral valve disease. It enables calculation of gradients and of orifice surface area in mitral stenosis. The quantification of mitral insufficiency is also possible though more difficult: it is based upon the combination of semi-quantitative and quantitative methods, in particular the calculation of regurgitation fraction obtained by comparison of flow rates at different orifices in cases of pure regurgitation. Finally, the calculation of pulmonary artery pressures from a Doppler record in tricuspid and pulmonary insufficiency can be used to assess the effects of mitral valve disease on the pulmonary circulation. Echo Doppler thus provides a reliable non-invasive hemodynamic assessment of mitral valve disease and should lead to a reduction in the number of invasive investigations in this type of valve disease.

Blood Pressure↗
PubMedDate not supplied

Detection and quantification of Pneumocystis carinii using a sandwich ELISA.

Antigenic sites on Pneumocystis carinii, the basis for organism enumeration by an enzyme-linked immunosorbent assay (ELISA) were adversely affected by incubation in detergents. However, stronger detergent concentrations were needed to eliminate high levels of non-specific background. Good P. carinii quantification was obtained with low non-specific background when the detergent was used only in the washing steps and not in cell suspension solutions. Formalin fixation of the cells resulted in good ELISA quantification of organism numbers with low non-specific background. No adverse effects were observed using a detergent on fixed cells. Although the system's range of accuracy needs to be expanded, a reduction in the number of organisms in response to the effects of pentamidine in vitro could be demonstrated by ELISA.

Animals↗
PubMed1991

Predictive, error-compensating kinetic method for enzymatic quantification of creatinine in serum.

Here we describe an error-compensating kinetic-based method for the enzymatic quantification of creatinine in serum. The method, which has a large linear range and very low dependency on experimental variables that influence enzyme activity, is based on the use of creatinine amidohydrolase in a four-step coupled reaction sequence to generate a product that is monitored photometrically. We collected data for absorbance vs time during two to four half-lives of each reaction and fit a first-order model to the data to compute the total absorbance change that would be measured if the reaction were monitored to completion. Computed values of absorbance change agreed well with measured values and varied linearly with creatinine concentration in the sample throughout the range examined: 44 to 1326 mumol/L. A twofold change in enzyme activity in the final reaction mixture (3.3 to 6.6 kU/L) produced changes of only 12% and 4% for creatinine concentrations of 44 and 353 mumol/L, respectively. Results (y) for 39 serum samples that contained creatinine concentrations between 20 and 1800 mumol/L agreed well with liquid-chromatographic results (x), yielding linear least-squares statistics of y = (1.03 +/- 0.01) x + (5 +/- 5) mumol/L (r = 0.995, Sy.x = 37 mumol/L). We conclude that the predictive kinetic approach is a robust method for the quantification of creatinine in serum.

Chromatography, High Pressure Liquid↗
PubMed1990

[Quantification and clinical significance of plasma apolipoproteins].

The apolipoproteins are important determinants of the structure and metabolism of plasma lipoproteins. This paper reviews analytical methods and the clinical significance of plasma apolipoproteins. Our data on apo VLDL and apo HDL analysis using fast protein liquid chromatography (FPLC), monoclonal antibody against apo VLDL, especially apo C-I, apo B isoproteins (apo B-100 and apo B-48) and plasma apolipoprotein concentrations in the patients with diabetes mellitus and coronary heart disease, were described. Among the methods of apolipoprotein quantification, single radial immunodiffusion (SRID) is widely used in Japan and plasma concentrations of apo A-I, A-II, B, C-II, C-III and E in healthy adults were reported. We showed the usefulness of FPLC for fractionation of human apo VLDL and apo HDL. We prepared several monoclonal antibodies against human apo VLDL, especially apo C-I, which were used for quantification and structural analysis of plasma apo C-I. Apo B-48 was found to be a good metabolic marker of exogenous lipoproteins (chylomicron and chylomicron remnant) and apo B-48 containing lipoproteins were increased in the poorly controlled diabetic patients.

Antibodies, Monoclonal↗