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@#In this paper, the uncertainties of correction factors of fluconazole impurities determined by HPLC standard curve method were evaluated, and the main common factors affecting the accuracy of standard curve method were found, so as to improve the accuracy of the method.In this study, the corresponding fitting lines of fluconazole and its impurities A, B, C, D, F and I were established respectively, and the ratio of the slope of fitting lines of each impurity and its corresponding principal component was calculated as the correction factor of the impurity.Then on the basis of GUM method, the uncertainty of each impurity correction factor determined by standard curve method was evaluated according to the established uncertainty evaluation scheme of correction factor determination process.The correction factor and uncertainty of fluconazole impurities A, B, C, D, F and I were 1.068 ± 0.046, 0.102 ± 0.005, 0.0582 ± 0.0031, 1.382 ± 0.121, 0.802 ± 0.067 and 1.383 ± 0.119, respectively, and the coverage factor k was 2.Finally, the contribution rate of each uncertainty component was calculated.In the relative combined standard uncertainties urel(f) of fluconazole impurities A, B, C, D, F and I correction factors, the sum of contribution rate of slope uncertainty urel(K) of the linear equation of principal component and its impurity is more than 85%; in the slope uncertainties urel(K) of linear equation, the contribution rates of uncertainties of solution concentration in 8 of 12 data groups are more than 80%, and the contribution rates of uncertainties introduced by reference substance content in solution concentration are about 80%.It can be seen that the preparation of linear solution concentration is the most influential factor in the determination of impurity correction factor by standard curve method, followed by the linear fitting process.In the preparation process of linear solution concentration, the purity of reference substance is the most influential factor, followed by weighing and pipetting times.The conclusion can help the experimenters to better formulate experimental plans and ensure the accuracy of the results when doing similar work.
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Objective To investigate the linear relationship and standard curve equation between acidic concentrated solution added KCl and the changes of K+ concentration in dialysate,and to apply it in personalized dialysis.Methods The speed of concentrated liquid pump of Fresenius 4008S hemodialysis machine was calibrated,the ratio of the concentration solution to the reverse osmosis water was determined,KCl was added to the concentrated A solution by an equal increment method to detect K+ concentration in the corresponding dialysate,and the K+ concentration standard curve of dialysate was mapped.This study is based on blood K+ concentration of adams-stokes syndrome patients before dialysis,referring to the standard curve,the most suitable dialysate K+ concentration was selected to personalized dialysis,the blood K+ concentration of the patients was measured after dialysis,and ECG monitoring and clinical symptoms observation were carried out.Results There was a linear relationship between acidic concentrated solution added KCl and the changes of K+ concentration in dialysate,the curve equation was y =0.384 lx + 0.002 3,R2 =0.999 4.There was no obvious change in the concentration of other electrolyte ions in the dialysate.Referring to the standard curve,the concentration of dialysate K+ could be adjusted accurately.The blood K+ concentration of adams-stokes syndrome patients could be corrected in time after several times of K+ concentration of personalized dialysis,and ECG recovered eventually,and arrhythmia,syncope,chest tightness and other symptoms disappeared.Conclusion There is a linear relationship between the concentration of dialysate K + and the concentration of KCl added in acidic concentrated solution in the Fresenius 4008S hemodialysis machine.Personalized dialysis is performed by the standard curve with obvious clinical application value,and references are provided for precise regulation of dialysate ion concentration.
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Objective To investigate the linear relationship and standard curve equation between acidic concentrated solution added KCl and the changes of K+ concentration in dialysate,and to apply it in personalized dialysis.Methods The speed of concentrated liquid pump of Fresenius 4008S hemodialysis machine was calibrated,the ratio of the concentration solution to the reverse osmosis water was determined,KCl was added to the concentrated A solution by an equal increment method to detect K+ concentration in the corresponding dialysate,and the K+ concentration standard curve of dialysate was mapped.This study is based on blood K+ concentration of adams-stokes syndrome patients before dialysis,referring to the standard curve,the most suitable dialysate K+ concentration was selected to personalized dialysis,the blood K+ concentration of the patients was measured after dialysis,and ECG monitoring and clinical symptoms observation were carried out.Results There was a linear relationship between acidic concentrated solution added KCl and the changes of K+ concentration in dialysate,the curve equation was y =0.384 lx + 0.002 3,R2 =0.999 4.There was no obvious change in the concentration of other electrolyte ions in the dialysate.Referring to the standard curve,the concentration of dialysate K+ could be adjusted accurately.The blood K+ concentration of adams-stokes syndrome patients could be corrected in time after several times of K+ concentration of personalized dialysis,and ECG recovered eventually,and arrhythmia,syncope,chest tightness and other symptoms disappeared.Conclusion There is a linear relationship between the concentration of dialysate K + and the concentration of KCl added in acidic concentrated solution in the Fresenius 4008S hemodialysis machine.Personalized dialysis is performed by the standard curve with obvious clinical application value,and references are provided for precise regulation of dialysate ion concentration.
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ObjectiveTo evaluate the uncertainty for the determination of ethanol in human blood by auto-headspace gas chromatography (HS-GC) with internal standard curve method.Methods Each source of uncertainty, arising from the procedure of testing, was analyzed and conifrmed according to the guidelines of the uncertainty in measurement . After each uncertainty component was quantized, the combined standard uncertainty and the expanded uncertainty of the result were calculated.Results The relative uncertainty brought from the measurement repeatability, standard solution of the ethanol, the sample of blood, internal standard solution of the tert-butyl alcohol, the calibration cure, gas chromatography were 3.4%,0.71%,0.61%,0.41%,1.1% and 1.3% respectively; the relative expanded uncertainty of ethanol in blood was 3.9%.Conclusion The measurement uncertainty of the concentration of ethanol was came primarily from the measurement repeatability of sample, HS-GC and standard curve of ethanol.
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Objective To establish the real-time fluorescence quantitative PCR method for the detection of bifidobacteria in human fecal samples, and to provide an effective means for measuring intestinal bacteria. Methods Total DNA of bacteria was extracted from 60 cases of children's fecal samples. Three primers of bifidobacteria based on the 16S ribosomal RNA (16SrRNA)which possessed specialities of bacteria as amplified region were designed.The part of amplified 16SrRNA gene sequences was used as standard production.The serial dilution of standard was analyzed to build an absolute quantitative standard curve with SYBR GreenⅠ dye method, and the bifidobacterium contents in sixty human fecal samples were calculated. The sensitivity of the reaction was calculated by detecting the lowest detectable standard which determined the sensitivity of the reaction. The PCR products’melting curve was used to evaluate the specificity.The coefficient of variation (CV)of different batches of standard with the same concentration was used to evaluate the stability of reaction.Results The length of PCR product fragment which was used to build the standard curve was about 6 1 3 bp, the sequencing result was consist with the goals, and the standard sample of bifidobacteria was successfully established in real-time fluorescence quantitative PCR.The standard curve showed a good linear relationship with R2=0.999.The minimum detection value was 1.48×102 copies per reaction.The melting curve of real-time fluorescence quantitative PCR was a single peak.The test samples were batched and then examined by fluorescence quantitative PCR.The CV of standards’ Ct values which calculated from the standard (1.48 × 103 -1.48 × 107 copies · μL-1 )were 2.94%, 3.39%, 3.54%,3.08%,and 3.34%,respectively.The contents of bifidobacteria in fecal from 60 children was 7.77± 0.86(copies · g-1 wet fecal)transformed by logarithmic.Conclusion The established real-time fluorescence quantitative PCR method has high sensitivity, strong specificity and good repeatability, which is suitable for detection of human fecal bifidobacteria content.
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Foi avaliada a eficiência de uma fitase (FT) bacteriana na liberação de fósforo fítico utilizando-se curvas de calibração para características ósseas e de desempenho em frangos de corte. O delineamento experimental foi inteiramente ao acaso, com seis tratamentos e seis repetições até 28 dias de idade. O tratamento-controle foi uma dieta à base de milho e farelo de soja deficiente em fósforo (P). Dois tratamentos corresponderam às dietas basais acrescidas de P suplementar, 0,05 por cento e 0,10 por cento, e os outros à dieta basal com 66, 99 e 131 FTU/kg de ração. A curva padrão é definida pelo efeito da adição de P suplementar consumido sobre características ósseas e de desempenho, e os resultados dos tratamentos com fitase são confrontados com a curva para cálculo de P liberado. A adição de P suplementar influenciou o ganho de peso, o peso vivo e o consumo de ração de forma quadrática, bem como miligramas de cinzas ósseas de forma linear. A curva padrão adotada foi da variável miligramas de cinzas ósseas, pois a resposta linear melhor descreve a curva. As inclusões de 66, 99 e 131 FTU/kg liberaram, respectivamente, 0,048 por cento, 0,049 por cento e 0,062 por cento de P. A fitase bacteriana é eficiente na liberação de fósforo fítico e possui viabilidade econômica.
The objective was to determine the efficiency of a bacterial phytase to release phytate phosphorus using calibration curves for performance and bone characteristics in broiler chickens. A completely randomized design with 6 treatments and 6 replicates was used in an experiment with chickens from 1 to 28 days of age. The control treatment was a diet based on corn and soybean meal deficient in phosphorus. Two treatments consisted of the basal diet supplemented with additional phosphorus (0.05 percent and 0.10 percent), and the other treatments received 66, 99 and 131 FTU/kg of feed. The standard curves represented the effect of the levels of additional P intake on performance and bone variables. Then, the responses of the phytase treatments were compared to the standard curves to calculate the P released. The increasing levels of supplemental P had a quadratic effect on weight gain, live weight and feed intake, and linear effect on mg of bone ash. The standard curve elected was mg of bone ash because linear response better represents the curve. Inclusion of 66, 99 and 131 FTU/kg released 0.048 percent, 0.049 percent and 0.062 percent. The bacterial phytase is efficient in releasing phytate, which may be of economical significance.
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PURPOSE: Radioimmunoassay (RIA) was usually performed by the batch assay. To improve the efficiency of RIA without increase of the cost and time, random assay could be a choice. We investigated the possibility of the random assay using automatic dispenser by assessing the agreement between batch assay and random assay. MATERIALS AND METHODS: The experiments were performed with four items; Triiodothyronine (T3), free thyroxine (fT4), Prostate specific antigen (PSA), Carcinoembryonic antigen (CEA). In each item, the sera of twenty patients, the standard, and the control samples were used. The measurements were done 4 times with 3 hour time intervals by random assay and batch assay. The coefficient of variation (CV) of the standard samples and patients' data in T3, fT4, PSA, and CEA were assessed. ICC (Intraclass correlation coefficient) and coefficient of correlation were measured to assessing the agreement between two methods. RESULTS: The CVs (%) of T3, fT4, PSA, and CEA measured by batch assay were 3.2+/-1.7%, 3.9+/-2.1%, 7.1+/-6.2%, 11.2+/-7.2%. The CVs by random assay were 2.1+/-1.7%, 4.8+/-3.1%, 3.6+/-4.8%, and 7.4+/-6.2%. The ICC between the batch assay and random assay were 0.9968 (T3), 0.9973 (fT4), 0.9996 (PSA), and 0.9901 (CEA). The coefficient of correlation between the batch assay and random assay were 0.9924(T3), 0.9974 (fT4), 0.9994 (PSA), and 0.9989 (CEA) (p<0.05). CONCLUSION: The results of random assay showed strong agreement with the batch assay in a day. These results suggest that random assay using automatic dispenser could be used in radioimmunoassay.
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Humans , Carcinoembryonic Antigen , Prostate-Specific Antigen , Radioimmunoassay , Thyroxine , TriiodothyronineABSTRACT
Objective To establish a fluorescent quantitative polymerase chain reaction method for quantifying the ICP4 gene expression of herpes simplex virus type 2(HSV2).Methods According to the HSV2 ICP4 gene sequence,we designed and synthesized PCR primer.The purified PCR product was sequenced after connecting with pMD-18 T plasmid.According to the sequence assay results,the primer and probe of fluorescent quantitative PCR was designed and synthesized.Standard recombinant plasmid extracted from the positive bacteriumclone was used as standard substance.The plasmid as standard substance was diluted for 10 times,then PCR reaction proceeded.The sensitivity and dependability of the real-time fluorescent quantitative PCR were analyzed.Results The sequence result indicated that there was non-sense mutation of A-G and T-C.And the detection sensitivity was 101 copy.The Ct value were 14.275土0.137,17.988?0.162,22.081土0.259,25.957土0.345,29.565?0.203,33.269土0.287,37.737?0.698,respectively with 107~ 101 copies/?l.The coefficient of variability were 0.965%,0.902%,1.174%,1.329%,0.686%,0.862% and1.851%,respectively.There was a good linear function in statistics between the Ct value and the concentration gradient of standard plasmid DNA specimen.The coefficient of regression was 0.998.Conclusion The method of quantification of ICP4 gene of HSV2 with real-time fluorescent quantitative PCR is successfully established,and the method has good sensitivity and dependability,which can be used to quantitative detecting HSV2 ICP4.
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Objective To discuss a convenient and pragmatic method of fitting and optimizing standard curve for determining concentration of serum hepatitis B virus large surface protein(HBV-LP).MethodsEnzyme-linked immunosorbent assay(ELISA)was used to measure the absorbance of standard preparation of HBV-LP.Concentration and absorbance of standard preparation of HBV-LP was carried out curve fitting with 4-parameter formula model and linear model and log-linear model and quadratic polynomial model and cubic polynomial model and S model by program solution of Excel,respectively.The most standard curve for determining concentration of serum HBV-LP was determined with coefficient of determination of regression model.ResultsThe scatterplot of standard preparation of HBV-LP submited nonlinear tendency.There were all significance to regression equation of 4-parameter formula model and linear model and log-linear model and quadratic polynomial model and cubic polynomial model and S model(P
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BACKGROUND: Although relative quantification by real-time PCR may be easier to perform than absolute quantification, there is a risk of errors associated with standard curve construction. The aim of this study was to evaluate the effects of standard curves on relative quantification using real-time PCR in Candida albicans. METHODS: The reproducibility of real-time PCR-based standard curves for target genes and a reference gene generated from PCR amplicons (10-fold serial dilution, 10(-4) to 10(-9)) was evaluated. In addition, the effects on standard curves were evaluated by running the same cDNA samples. RESULTS: The within-run variation (CV) by crossing point (Cp) was 0.12-1.05% for ERG11 and ACT1, whereas the between-run CV was 2.07-6.84% for ERG11, CDR1, MDR1 (target gene) and ACT1 (ref-erencegene). The differences in PCR efficiency between targets and reference may be attributable to variations in relative quantification. CONCLUSIONS: To achieve reliable relative quantification of mRNA in real-time PCR, a feasible guide-line and standardization are of major importance.
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Candida albicans , DNA, Complementary , Polymerase Chain Reaction , Real-Time Polymerase Chain Reaction , RNA, Messenger , RunningABSTRACT
Objective:To construct the standard curve for real-time PCR detecting the tissue expression level of Angptl3 in SD rat.Methods:The total RNA of the SD rat's liver was the template.We got lots of the Angptl3 partial DNA sequence by RT-PCR.The products were cloned into the pUCm-T vector,Then we diluted the solution of plasmid and used real-time PCR(SYBR GreenⅠ)to construct the standard curve.Results:The correlation coefficient of the standard curve was 0.997 6.The reaction efficiency was 0.862 3.The correlation and the amplify efficiency were good.Conclusion:It is successful to construct the standard curve for real-time PCR detecting the tissue expression level of Angptl3 in SD rat.
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Objective To observe the distribution of dopaminergic neurons in SNpc and to establish standard curve in normal mice so as to measure the changes of dopaminergic neurons in number in SNpc of the mice toxicated with MPTP. Methods Ten male C57BL6 mice aged 8-12 weeks, weight 20-22 g, were randomized to receive 20 mg/kg MPTP or physical saline every 3 h for 4 times, then killed 7 d later. The mouse mesocerebrum was taken out and fixed, frozen, sectioned. All sections containing SNpc were observed under the guide of mouse brain atlas. Every other sections were chosen to stain tyrosine hydroxylase (TH) to show dopaminergic neurons immunohistochemically. The TH positive cells in SNpc were counted in each section and the standard curve was established. Results The standard curve of SNpc compact part position and TH positive cells was established. By comparing the standard curves for the MPTP intoxicated mice and the saline mice, TH positive cells in SNpc from MPTP toxicated mice decreased significantly, which confirmed the validity and feasibility of the standard curve. Conclusion The establishment of standard curve greatly facilitates the comparison of specimen from different groups and makes the assessment of dopaminergic neuron loss more accurate and efficient. The standard curve can serve as an excellent reference curve for the assessment of dopaminergic neurons in SNpc in normal C57BL6 mice.