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Abstract High sensitivity of qPCR assay can be compromised by the presence of PCR inhibitors in samples analyzed. The aim of this study was to analyze the RT-qPCR assay efficiency considering the RNA quality/quantity and the presence of PCR inhibitors in patients with chemotherapy and/or antibiotic therapy. We analyzed 60 samples using RT-qPCR from individuals suspected of leukemia and 44 samples were quantified by fluorimetry and spectrophotometry. The efficiency of the RT-qPCR assay was evaluated comparing the threshold cycle (Ct) from tested samples and the standard curve. The 260/280 and 260/230 ratios, the presence of PCR inhibitors and the amount of sample (ng) used in the RT-qPCR reaction can be associated with 56.8% (R²=0.56, p<0.05) in the Ct obtained. The decrease of the RT-qPCR efficiency can be explained in 42,8% due to the variation of the 260/280 ratio (R²=0.42,p<0.05). The presence of antibiotics in the blood sample can be associated in 11.3% with the variability of 260/280 ratio (R²=0.11,p<0.05). Presence of chemotherapeutic drugs in the blood sample was not correlated with Ct variation (p=0.17). The spectrophotometer determines a RNA quantification with 2.2 times higher than the fluorimeter (t=2.2, p=0,03) and this difference is correlated with the 260/280 ratio (R²=0.36, p<0.05). Samples with low purity had a reduction in the qPCR efficiency, although we did not observe false results.
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Humans , Polymerase Chain Reaction/methods , Nucleic Acid Synthesis Inhibitors , Molecular Diagnostic Techniques/methods , Spectrophotometry/instrumentation , Fluorometry/instrumentationABSTRACT
A Kunitz-type protease inhibitor (OPI, okra protease inhibitor) has been purified from okra (Abelmoschus esculentus) seedsby a combination of ammonium sulfate precipitation, anion-exchange chromatography and reverse-phase high-performanceliquid chromatography. The protein shows an apparent mass of 21 kDa on sodium dodecyl sulfate-polyacrylamide gelelectrophoresis under reducing condition. OPI exhibits inhibitory activity against trypsin. Analysis of the far-UV circulardichroism spectrum showed that the protein contains *39% b-sheets but only *5% a-helices. The protein is thermallyquite stable, and exhibits a cooperative thermal unfolding transition at *70C, as determined by circular dichroismspectroscopy and differential scanning fluorimetry. De novo sequencing of OPI by nanoESI-Q-ToF mass spectrometry (MS)allowed the assignment of about 83% of its primary structure, which indicated that the protein shares 43% sequence identitywith a putative 21 kDa trypsin inhibitor from Theobroma bicolor. An intramolecular disulfide linkage between Cys149 andCys156 was also detected. The protein showed *24 and *25% sequence identity with a-amylase/subtilisin inhibitor frombarley and soybean (Kunitz) trypsin inhibitor, respectively. Comparative structure modeling of OPI revealed a structuralfold similar to other Kunitz-type TIs. The presence of Cys149–Cys156 disulfide bond as detected by MS and a seconddisulfide bond connecting Cys44–Cys91, conserved in all Kunitz-type TIs, is also identified in the model.
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A novel fluorescence homogeneous biosensing strategy was developed for simple, rapid and sensitive detection of melamine in milk by using the polythymine oligonucleotide T24 and SYBR Green Ⅰ. In the absence of melamine, the fluorescence of SYBR Green Ⅰ was weak. The interactions between the single strand oligonucleotide T24 and SYBR Green Ⅰ were weak. In contrast, the presence of melamine drove the formation of T-melamine-T folded structure and enabled the SYBR Green Ⅰ to intercalate into double-strand DNA, resulting in the enhancement of fluorescence intensity. The results revealed that the method allowed a sensitive, simple, and rapid assay of melamine with a linear response range from 0. 1 μmol/L to 10 μmol/L and a detection limit of 35 nmol/L.
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Objective To explore the determination of nicotine in cigarettes by fluorescein and fluorimetry-basic possession of bonuses system.Methods Nicotine could interfere with fluorescence energy transfer between fluorimetry-basic possession of bonuses.A new way of energy transfer was established to determinate nicotine according to the fluorescence increment of fluorimetry.Results The linear range was 0.2-7.0 mg/L for the concentration of nicotine.The correlative coefficient was 0.9992.The detection limit was 0.18 mg/L.The recovery rates were 99.0 %-105 %,and RSDs were 1.1 %-4.8 % respectively.Conclusion This method is simple,fast,accurate and sensitive.It can be used to determinate nicotine in cigarettes.
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FUNDAMENTOS: O organismo humano possui eficientes mecanismos de defesa contra os radicais livres. A relação causal observada entre estresse oxidativo e diversos processos degenerativos despertou o interesse para a exploração de diversos antioxidantes. OBJETIVOS: Este trabalho propõe um método in vivo para comprovação da eficácia de um novo complexo de alta atividade anti-radicalar (acetato de tocoferila, licopeno e mistura de ácidos clorogênicos rica em ácido caféico). MÉTODOS: Neste ensaio, não invasivo e placebo controlado, a medida da taxa de peróxido cutâneo realizou-se em diferentes áreas - três após a incidência da radiação UV, duas tratadas, uma não tratada, e uma não tratada e não irradiada. A presença do peróxido foi detectada pela aplicação de sonda fluorescente em adesivo específico, que retirou uma amostra do estrato córneo dos sítios supracitados. O cálculo da proteção anti-radicalar dá-se em função das unidades fluorimétricas obtidas. RESULTADOS: Tomando-se como base áreas controles não irradiadas, as áreas irradiadas e tratadas com o complexo estudado apresentaram concentrações 116 por cento menores (p=0,02 por cento) de peróxidos cutâneos, com significância estatística em relação às áreas apenas irradiadas. Já as áreas irradiadas e tratadas com o placebo apresentaram concentrações apenas 49 por cento menores (p=0,501), o que não é estatisticamente significativo em comparação às áreas irradiadas. CONCLUSÃO: Os resultados obtidos indicam que o complexo estudado possui significativa capacidade protetora da pele contra a ação de radicais livres formados a partir da exposição solar.
BACKGROUND: Our organism has important defense mechanisms against free radicals. The causal relationship observed among the oxidative stress and degenerative problems in humans, are getting attention for the exploration of antioxidant agents. OBJECTIVES: This study proposes an in vivo methodology for proving the efficacy of a new high-activity antioxidant complex (tocopheryl acetate, licopene and a pool of chlorogenic acids rich in cafeic acid). METHODS: In this non-invasive and placebo controlled assay, the measure of the peroxide rates was performed in different areas, three after UV radiation, two treated, one non-treated, and an other non-treated and non-irradiated. The peroxide presence was detected through a fluorescent probe on samples stripped form the sites above mentioned. The free-radical scavenger activity is calculated through the fluorimetry results. RESULTS: Based on non-irradiated areas dataÆs, the irradiated and complex-treated areas presented skin-peroxide concentration 116 percent lower, statistically significant (p=0,02 percent) compared to the non-treated irradiated areas. Although, placebo-treated irradiated areas presented skin-peroxide concentration 49 percent lower, a non-statistically significant rate (p=0,501 percent) compared to the irradiated areas. CONCLUSION: The results indicate that studied-complex, have significant skin-protective action against the free-radicals, usually formed after sun exposure.
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Objective To establish a new kinetic fluorimetry method for the determination of trace nickel in human hair samples. Methods The method was based on the catalysis of nickel on the oxidation of fluorescein(CF) by hydrogen peroxide in an almost neutral medium. As nickel is present, the fluorescence intensity of the solution decreases. Results The wavelength of excitation was 492 nm and the wavelength of emission was 516 nm. The reaction was optimized at 100 ℃ for 10 min. The linear range of determination was 4.0-80.0 ?g/L and the detection limit was 2.2 ?g/L . The RSDs and recovery rates of the method were 2.6%-3.2%, and 96.7%-102.8% respectively. Conclusion This method is simple, rapid and sensitive for the determination of trace nickel in human hair samples.
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The authors used high sensitive micellar enhanced fluorimetric method to monitor amiodarone blood-medical concentration. SLS severed as micellar reagent. Under experimental condition,we used 930 fluorophotommeter which made in China as the main analytic instrument,and obtained a series pharmacokinetic parameter ,eg. t_(1/2) (?) was 1.18 h, t_(1/2)(?)was 40.75 h, K_(21) was 0.278 l/h,K_(10) was 0.036 l/h,K_(12) was 0.291 l/h. The linear range of this method was 2 ? 10_(-9)～8 ? 10_(-6) mol/L,determined limit was 1.3 ? 10~(-9)mol/L, average recovery was 99.93%.
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Three methods C2, 3-diaminonaphathalene molecular fluorimetry, hydride generation atomic fluorescence spectrometry (HG-AFS) and hydride generation atomic absorption spectrometry (HG-AAS)] for the determination of selenium in food samples were compared with one another. The absolute detection limits of HG-AFS and HG-AAS are 1.2 and 0.9ng respectively, lower than that of molecular fluorimetry(3.9ng). The precisions and accuracies of HG-AFS and HG-AAS are similar to molecular fluorimetry. When these methods were used to determine selenium in 6 standard reference materials, there was no significant difference among the mean values from each of the methods.
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A rapid method for directly determining tryptophan contents in foods and feedstuffs was described. l-5mg dry sample could be completely hydro-lyzed in a teflon tube containing 5N NaOH with 0.5% soluble starch under 145℃ for 4 hours in vacuum (760mmHg). The hydrolysate was then adjusted to neutral with 6N HCL in icebath and diluted by five times with 4M urea solution (pH = 11), and was measured at excitation wavelength 280nm and fluorescence wavelength 368nm. The sensitivity of the method was 5ng /10ml, and the recovery rate of 97-107% were obtained in the range of 1-5 ?g/10ml standard tryptophan. The value of a number of foods and feed-stuffs obtained were in agreement with the literatature values.