Application evaluation of a rapid fluorescence quantitative PCR method for the detection of SARS-CoV-2 / 中华微生物学和免疫学杂志

Objective:To establish and evaluate a rapid nucleic acid detection method for SARS-CoV-2 based on COYOTE ? Flash20 real-time fluorescent quantitative PCR instrument. Methods:A rapid reaction system was constructed by using specific primer and probe sets targeting ORF1ab and N gene of SARS-CoV-2, and the sensitivity and specificity of the system were verified. At the same time, 108 clinical samples of COVID-19 were used to evaluate the application of this method.Results:The detection method did not require nucleic acid extraction, and the manual operation time was only one minute. After the sample was sent to the system, the test could be completed in 30 minutes. The detection limit of this method was 4×10 2 copies/ml. It had no cross-reactivity with other human coronaviruses (including HCoV-229E, HCoV-NL63, HCoV-OC43, HCoV-HKU1, SARS-CoV and MERS-CoV) and other respiratory viruses. The evaluation of clinical sample application showed that the total coincidence rate with the conventional RT-qPCR which required nucleic acid extraction was 98.15%. Conclusions:Through the application evaluation of the rapid fluorescent quantitative PCR method of SARS-CoV-2, it was found that the method was simple, fast, specific and sensitive, and it was suitable for real-time and rapid detection needs in varieties of situations.

Study of Aetiological and Endoscopic Profile of Patients with Upper Gastrointestinal Bleed in North East India - A Hospital Based Cross Sectional Study

Upper Gastro-Intestinal Bleeding (UGIB) is one of the common complaints with which patients present to casualty. It is associated with significant morbidity and mortality. The aetiological spectrum of UGIB is variable in different geographical regions. Our study aimed to analyse the aetiology, endoscopic profile, mortality, Rockall score and predictors of mortality in patients with UGIB, in North East India. METHODSThis cross-sectional study was conducted at Assam Medical College and Hospital in North East India. We enrolled patients with age 12 years and above, who were admitted between July 2019 and January 2020 with a history suggestive of UGIB. Demographic data of the patients was collected, after which they underwent clinical examination, and upper GI endoscopy. Mean ± standard deviation was used to express continuous variables. Frequency and percentage were used to express categorical variables. Test of significance for qualitative data was assessed by Chi-square test (for 2 x 2 tables). P value less than 0.05 was taken as statistically significant. RESULTSWe analysed 117 patients diagnosed with UGIB [80.34 % male, 19.60 % female], ratio of male to female of [4.08:1] was seen. The most common symptom was melena 87 patients (74.15 %), endoscopy finding showed that 48.71 % had oesophageal and / or gastric varices, 26.49 % had peptic ulcers, 17.94 % had gastric erosions / duodenal erosions / erosive gastritis, 1.7 % had Mallory-Weiss tear, 1.7 % had gastric malignancy, 1.7 % had GJ stoma bleed, 1.7 % had both oesophageal varices and peptic ulcer disease. Partial gastric outlet obstruction was observed in peptic ulcer disease in 2 patients (6.45 % of total peptic ulcer disease patients). 73.75 % patients had Rockall score < 5 and 26.49 % patients had Rockall score > 6. H. pylori infection (assessed by RUT) was an independent predictor of upper GI bleed in both variceal and non-variceal bleed [p < 0.001]. The mortality in our study was 7.69 %. Predictors of mortality in the study population were, patients with variceal bleed [p = < 0.001], Rockall score > 6 [p = 0.013], and chronic liver disease [p < 0.001]. The average duration of hospital admission of the study population is about 4.6 + / - 0.4 days. CONCLUSIONSThe study reported oesophageal varices was the most common cause of UGIB, followed by peptic ulcer in North East India. H. pylori was an independent predictor of both variceal and non-variceal bleed. Partial gastric outlet obstruction (GOO) was one of the common benign complication of peptic ulcer disease. Variceal bleed, Rockall score > 6, chronic liver disease were predictors of mortality.

Development Of A Sensor By Electro-Polymerization Of Erichrome Black-T On Glassy Carbon Electrode And Determination Of An Anti-Inflammatory Drug Diclofenac

Objective: The aim of this study was to develop a simple, reliable voltammetric method and its validation for determination of nonsteroidal anti-inflammatory drug diclofenac (DFC). Methods: The proposed method was based on electro-oxidation of DFC at poly (erichrome black T) modified glassy carbon electrode (PEBT/GCE) in 0.2 M phosphate buffer solution of pH 7.0. Cyclic voltammetry and differential pulse voltammetric techniques were employed to study electro-oxidation behavior. Under the optimal conditions, variations of EBT concentration, effect of pH, scan rate on the oxidation of DFC was studied. Results: A well-defined oxidation peak at about +0.59 V vs. standard calomel electrode was observed for voltammetric detection of DFC. pH effect shows the participation of an equal number of protons and electrons in the mechanism. The relation between a logarithm of peak current with the logarithm of scan rate indicated adsorption controlled behavior of electrode process. Concentration variations show a good linear response in the range 0.05 µM to 40 µM with the detection limit of 5.25 × 10-8 M. Conclusion: The prepared sensor exhibited good selectivity, sensitivity, and stability for the detection of DFC in the pharmaceutical dosage form and real samples. The developed method could possibly be adopted for pharmacokinetic studies and also in clinical and quality control laboratories where time and economy were important.

Effect of different types of microplate and loading volumes on the detection results of multi-function microplate reader / 中国职业医学

OBJECTIVE: To evaluate the effect of different types of microplate and loading volumes on the detection results of multi-function microplate reader, and to optimize the analysis method. METHODS: A multi-function microplate reader was used to perform spectrum scanning on each of 5 detection holes of common and ultraviolet(UV) microplates, and the applicable detection wavelength range was those with light transmittance greater than 80.00%. The optical density measurement was carried out on each 12 detection holes of common and UV microplates at different wavelengths, then the matching of the detection holes was compared. Potassium permanganate was quantitatively analyzed by common microplate and UV microplate, while acetone was analyzed by UV microplate, and then detection limit, lower limit of quantitation(LLQ), accuracy and precision at different loading volumes and concentrations were calculated and compared. RESULTS: The shortest applicable analyzing wavelengths for common and UV microplates were(362±2) and(230±3) nm respectively, while the longest applicable analyzing wavelengths were both 1 000 nm. The light transmittance of UV microplate was higher than that of common microplate when the analyzing wavelengths were lower than 400 nm(P<0.01). The deviation and range of light transmittance of detection holes analyzed by UV microplates were smaller than that of common microplates when the analyzing wavelengths were 350-1 000 nm(P<0.05). The detection limit and LLQ of potassium permanganate by multi-function microplate reader was not associated with the types of microplate. The adding standard recoveries of potassium permanganate by UV microplate was higher than that by common microplate(P<0.05). The adding standard recoveries of potassium permanganate by loading volumes of 200 and 250 μL was lower than that by loading volumes of 150 μL(P<0.01), while adding standard recovery of acetone by loading volumes of 200 μL was lower than that by loading volumes of 150 μL(P<0.05). CONCLUSION: When using a multi-function microplate reader to detect chemicals, it is recommended to use UV microplate with wavelengths at the range of 230-1 000 nm, and loading volumes of 200-250 μL.

Discussion on calculation method of detection limit and quantitative limit of occupational health biological monitoring method / 中华劳动卫生职业病杂志

Objective@#To discuss calculation method of detection limit and quantitative limit of occupational health biological monitoring.@*Methods@#The detection limit and the quantitative limit of phenyl glyoxylic acid and Mandelic acid were calculated by using three different methods of IUPAC, NIOSH and OSHA respectively.@*Results@#The IUPAC, NIOSH and OSHA methods were used to calculate the detection limit and the quantitative limit of the phenyl glyoxylic acid and Mandelic acid, and the results are different.@*Conclusion@#To calculate the detection limit and quantitative limit of occupational health biological monitoring methods, the standard curve method is adopted to ensure that the rate of detection in the vicinity of detection limit and more than 75% of the quantitative limits are used.

Verification of the linear range and the minimum detection limit in real-time fluorescence quantitative PCR for HBV DNA / 国际检验医学杂志

Objective Use real-time fluorescence quantitative polymerase chain reaction(PCR)to determine HBV DNA,then calculate the linear range and the minimum detection limit,which are the main performance indicators in the laboratory verification. Methods According to the related documents,by serial dilution of high concentration samples,samples of serial concentrations were obtained which were out of the linear range mentioned in the instructions,then verifid the new linear range.By serial dilution of low concentration,samples were obtained,the concentrations of which were lower than the minimum detection limit of provide by the manufacturer,then the new minimum detection limit was verified.Results The linear range of HBV DNA detection was 8.58× 102 -8.41×107 IU/mL,and the minimum detection limit of HBV DNA detection was 4.07 ×102 IU/mL.Conclusion The linear range and the minimum detection limit of Real-time fluorescence quantitative PCR assessed reaches the expected requirement,and the method and validation scheme are simple and feasible.

Determinação do limite mínimo de detecção da técnica de Nested-PCR para os poliomavírus JC e BK / Assessment of minimum detection limit of Nested-PCR for JC and BK polyomaviruses

Introduction: Polyomaviruses (BKV and JCV) cause infection mainly in immunocompromised adults. A sensitive and specific diagnosis tool is fundamental to demonstrate the BKV and JCV infections. Nowadays many laboratories are using a PCR technique for detecting polyomaviruses genome in clinical samples. In this context, the purpose of this study is to determine the threshold of detection of the nested-PCR for polyomaviruses JC and BK. Methods: Serial dilutions of the samples of BKV and JCV of known concentration (100 copies/mL, 50 copies/mL, 25 copies/mL, 10 copies/mL, 5 copies/mL, and 1 copy/ml) were subjected to the technique of nested-PCR. All dilutions were tested 11 times to determine the minimum detection limit. Results: The minimum detection limit of the nested-PCR for JC and BK viruses was 25 copies/mL. This dilution (25 copies/mL) showed 100% PCR positive reaction. Furthermore, we found that weak positive results were obtained at dilutions of 1,5 and 10 copies/mL in some repetitions. Dilutions of 25, 50, and 100 copies/mL always had very positive results. Conclusions: These values are similar to those reported in other studies, contributing to the indication of this PCR for potential diagnostic purposes.

Introdução: Os poliomavírus (JCV e BKV) causam infecções principalmente em adultos imunocomprometidos. Um diagnóstico sensível e específico é de fundamental importância para os pacientes portadores de JCV e BKV. Atualmente alguns laboratórios têm utilizado a técnica de PCR para a detecção do material genético destes vírus em amostras clínicas. Assim, o objetivo deste estudo é determinar o limite mínimo de detecção da técnica de nested-PCR para os poliomavírus JC e BK. Métodos: Diluições seriadas (100 cópias/mL; 50 cópias/mL; 25 cópias/mL; 10 cópias/mL; 5 cópias/mL e 1 cópia/mL) de controles positivos comerciais de JCV e BKV com concentrações conhecidas foram submetidas à técnica de nested-PCR semi-duplex. Todas as diluições foram testadas 11 vezes para determinação do limite mínimo de detecção. Resultados: O limite mínimo de detecção da reação de nested-PCR para os vírus JC e BK foi de 25 cópias/mL para ambos, com 100% de positividade das diluições testadas na reação de PCR. Ainda, pudemos observar que resultados positivos fracos foram obtidos nas diluições de 1, 5 e 10 cópias/mL em algumas das repetições realizadas. As diluições de 25, 50 e 100 cópias/mL sempre obtiveram resultado rancamente positivo. Conclusões: Estes valores são semelhantes aos relatados em outros estudos, contribuindo para a indicação desta reação de PCR para potenciais fins diagnósticos.

Optimization of Near Infrared Variable Selection Method Based on Multivariate Detection Limit / 世界科学技术-中医药现代化

This study was aimed to optimize the near infrared (NIR) variable selection method based on multivariate detection limit (MDL). Using Qing-Kai-Ling (QKL) injection as object, three variable selection methods (interval par-tial least-squares, iPLS; backward interval partial least squares, BiPLS; moving window interval partial least squares, mwPLS) were used to establish the PLS models of baicalin in QKL injection, respectively. The prediction ability of different variable selection method was compared. MDL of all models were calculated in contrast to the MDL value of full spectra PLS model, to select optimal variable selection method. The results showed that different variable selec-tion methods had different prediction ability. Among them, iPLS had the best performance which determination coef-ficient of prediction (Rpre2) and the root mean square errors of prediction (SEP) were 0.996 5 and 602.3 μg·mL-1, re-spectively. All MDLs of different variable selection methods were reduced compared with the full spectra PLS model. The value of iPLS was the lowest comes to be 1.19 μg·mL-1. The results above indicated that the best variable se-lection method for baicalin in QKL injection was iPLS. MDL theory took the error of calibration and validation set and the leverage of external sample into account, which can comprehensively evaluate model detection performance compared to the classic chemical indicator parameters. This method was particularly suitable for the variable selec-tion method optimization of NIR quantitative model of low concentration sample such as Chinese herbal medicine.

Loop-mediated isothermal amplification assay for the detection of Salmonella spp. in pig feces / 대한수의학회지

Salmonella are causative agents of gastroenteritis and systemic disease in animals. The invA gene was selected as a target sequence of loop-mediated isothermal amplification (LAMP) assay for diagnosis of Salmonella infection. The detection limits for broth dilution, spiked feces and enrichment were 10(4), 10(5) and 10(2) CFUs/mL, respectively. The LAMP assay developed in the present study may be a reliable method for detection of Salmonella spp. in pig feces.

Validación e implementación de una metodología para el análisis microbiológico de un producto líquido preservado, elaborado en una industria farmacéutica / Validation and implementation of a methodology for the microbiological analysis of a preserved liquid product produced in a pharmaceutical industry

Objetivo: validar e implementar una metodología para el análisis microbiológico de un producto líquido preservado con parabenos, elaborado en una industria farmacéutica, según las técnicas de análisis propuestas por la United States Pharmacopeia (USP), versión XXXIV, 2011. Métodos: para los ensayos cuantitativos se trabajó con Staphylococcus aureus y Candida albicans, y para los ensayos cualitativos con Escherichia coli, Staphylococcus aureus y Pseudomonas aeruginosa. Resultados: se obtuvieron resultados acordes con lo establecido por la USP. La metodología descrita se consideró reproducible y robusta al tener la capacidad de no ser afectada por variaciones al desarrollar la técnica, lo cual genera resultados confiables y precisos. Conclusiones: todos los parámetros de validación cumplen la especificación según la USP, lo que muestra conformidad en la totalidad de los parámetros evaluados.

Objective: to validate and to implement a methodology for microbiological analysis of a liquid product preserved with parabens produced by a pharmaceutical company, based on the United States Pharmacopeia analytical methods, XXXIV version, 2011. Methods: the quantitative tests were carrying out for Staphylococcus aureus and Candida albicans, and qualitative tests for Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa. Results: the results were consistent with those established by the USP. The described methodology was considered reproducible and robust because of its capacity of being unaffected by variations when implementing the technique, thus generating reliable and accurate results. Conclusions: all validation parameters met the USP specification, showing compliance with all the evaluated parameters.

Verification of an improved method for determination of urinary iodine by As3+-Ce4+ catalytic spectrophotometry / 中国地方病学杂志

Objective To verify the feasibility and application value of an improved method for determination of urinary iodine by As3+-Ce4+ catalytic spectrophotometry.Methods Adults urine samples were collected,iodine calibration curves of 0-300 μg/L and 300-1200 μg/L were prepared,and urinary iodine was determined by the improved As3+-Ce4+ catalytic spectrophotometric method.Lyophilized human urinary iodine ingredient standards were used to validate linearity and range,limit of detection,precision and accuracy of this improved detection method.Results The linear range of the calibration curve was 0-300 μg/L,the detection limit was 1.8 μg/L,and the range of correlation coefficient was-0.9995--0.9997.When measuring urinary iodine at 40-80,100-149,200-280 μg/L,the relative standard deviations were 1.5％,0.8％ and 0.5％.When measuring urinary iodine at 40-80,100-149,150-180 μg/L,the average recoveries were 97.8％,99.8％ and 96.6％.Two given values of urinary iodine of national standard samples were (73.0± 9.0) and (206.0± 10.0)μg/L,and the results determined by this method were (75.5 + 0.9) and (207.5 ± 1.9)μg/L,and the relative deviation was 3.4％ and 0.7％,respectively,the results determined were all within the given value range.The linear range of the calibration curve was 300-1200 μg/L,the detection limit was 305.2 μg/L,the range of the correlation coefficient was-0.9996--0.9999.When measuring urinary iodine at 300-400,500-600 and 1000-1200μg/L,the relative standard deviations were 0.6％,1.0％ and 0.7％.When measuring urinary iodine at 300-499,500-599 and 600-700 μg/L,the average recoveries were 99.7％,99.2％ and 100.4％.Two given values of urinary iodine of national standard samples were (558.3 ± 3.5) and (884.8 ± 4.7)μg/L,the results determined by this method were (556.0 + 17.0) and (883.0 ± 28.0)μg/L,and the relative deviation was 0.4％ and 0.2％,respectively,the results were all within the given value range.Conclusions This method extends the detection range of iodine concentration,and improves precision and accuracy.This method greatly reduces the amount of arsenic used therefore reduces environmental pollution,which is suitable for promotion.

Cuantificación de glibenclamida en muestras de limpieza de equipos farmacéuticos mediante cromatografía líquida de alta resolución / Quantification of glibenclamide in cleaning samples of pharmaceutical equipment through high performance liquid chromatography

Objetivo: proponer un procedimiento analítico selectivo para la cuantificación de glibenclamida en muestras de limpieza de equipos farmacéuticos mediante cromatografía líquida de alta resolución. Métodos: la fase móvil consistió en una mezcla equivalente de volúmenes de acetonitrilo y solución amortiguadora KH2PO4 de concentración 0,037 mol/L a pH 5,25 y flujo 1,5 mL/min, en una columna Nucleosil 100 C8. La glibenclamida se inyectó con progesterona como estándar interno y empleando detector UV a una l= 230 nm. Resultados: el método resultó lineal en el intervalo de concentraciones de 0,4-150 mg/mL, teniendo como límites de detección y cuantificación 10 y 40 ng/mL respectivamente y siendo específico al analito en presencia del placebo, sus productos de degradación y a otros ingredientes farmacéuticamente activos. Se consideraron potenciales de interferencias para el método propuesto: captopril, clortalidona, dexametasona, digoxina, 8-cloroteofilina, difenhidramina HCl, fenobarbital, haloperidol, hidroclorotiazida, ácido fumárico, ketotifeno, metoclopramida HCl, piridoxina HCl, piroxicam, prednisona y nifedipino. Se identificaron: ibuprofeno, indometacina, trifluoperazina HCl, tioridazina HCl e imipramina HCl, como interferentes del procedimiento en concentraciones cercanas a 10 mg/mL. Conclusiones: el método desarrollado es sensible, rápido y especialmente selectivo para la evaluación de residuales del principio activo glibenclamida en equipos de producción de tabletas, empleando un muestreo por hisopado, y pudiera utilizarse potencialmente cuando exista sospecha de contaminación cruzada de glibenclamida con otros fármacos de los aquí descritos.

Objective: to submit a selective analytical method for quantization of glibenclamide in cleaning samples of pharmaceutical equipment using high performance liquid chromatography. Methods: the mobile phase consisted of an equal mixing of acetonitrile/phosphate buffer KH2PO4; with 0.037 mol/L concentration pH 5.25 and flow of 1.5 mL/min, in a Nucleosil 100 C8 column. Glibenclamide was injected with progesterone as internal standard and using an UV detector= 230 nm Results: the method was linear in the 0.4-150 mg/mL concentration interval having a detection and quantization limits of 10 and 40 ng/mL respectively. It was specific to analyte when placebo is present, to degradation products and to other active ingredients. Possible interferences with the proposed method was considered for captopril, chlortalidone, dexametasone, diphenhydramin HCl, digoxine, 8-chlortheophylline, diphenhydramina HCl, phenobarbital, haloperidol, hydrochlorothiazide, fumaric acid, ketotifen, metoclopramide HCl, piridoxine HCl, piroxicam, prednisone and nifedipine, On the other hand, ibuprofen, indometacin, trifluoperazine HCl, thioridazine HCl and imipramine were identified as interferences in the procedure at concentration figures close to 10 mg/mL. Conclusions: the present method is sensitive, quick and selective for the evaluation of residues of active pharmaceutical principle glibenclamide in tablet production equipment after a swap sampling and it could be potentially used in case of cross-contamination of glibenclamide and other drugs already described.

Identification and detection of Cordyceps sinensis with LAMP / 中草药

Objective: The method of loop-mediated isothermal amplification (LAMP) was employed to detect and identify Cordyceps sinensis rapidly. Methods: Specific LAMP primers were designed according to the CS2 serine protease (csp2) gene of Cordyceps sinensis. C. sinensis DNA was extracted using CTAB method. The reaction conditions of LAMP were optimized. Specificity of LAMP reaction was validated by six different strains and using restriction enzyme Taq1 digested the LAMP products. Sensitivity of LAMP was tested with diluted C. sinensis solution with 10-fold gradient. LAMP products were shown by gel electrophoresis or adding SYBR Green I. Results: The method of LAMP for detecting C. sinensis was effective and specific. The detection limit of LAMP assay was up to 6 pg/mL. Conclusion: LAMP protocol is a promising method for the identification and detection of C. sinensis and Chinese materia medica as well.

Montaje y validación del método de análisis poscombustión y detección por infrarrojo no dispersivo para determinación de carbono orgánico total (cot) en agua / Assembly and validation by the method of combustion analysis and ondispersive innfrared detection for determining total organic carbon (toc) in water

La técnica de análisis por combustión y detección por infrarrojo no dispersivo (IRND) fue validada para la determinación de materia orgánica en agua, cuantificada como carbono orgánico total (COT). Previamente se optimizó la eliminación del carbono inorgánico (CI) de la muestra con un tiempo óptimo de 1,5 min y una relación ácido de 5%. Se estableció un rango dinámico lineal (RDL) entre 3 y 20 mg/L de COT, en el cual la recta de regresión cumplió con los parámetros que acreditaron su linealidad según el análisis por el método de los mínimos cuadrados, mostrando un coeficiente de correlación de 0,9994. La sensibilidad expresada por la pendiente de la recta de regresión indicó una variación de aproximadamente 5 unidades en la respuesta del detector por cada mg/L de COT. Los límites de detección y cuantificación obtenidos a partir de la recta de regresión fueron 0,517 y 1,722 mg/L de COT, respectivamente. La precisión de la técnica, teniendo como meta un 5% en coeficiente de variación (CV), fue mejor en el agua potable y en concentraciones cercanas a ésta (aprox. 7 mg/L de COT), mientras que las mayores desviaciones se presentaron en concentraciones cercanas a los límites inferior y superior del RDL. Las recuperaciones de concentraciones conocidas sobre muestras reales adicionadas fueron del 85% con baja adición y del 83% con adición alta, valores que sugieren una reevaluación del desempeño de la técnica con respecto a su exactitud, no obstante se alcanzó la meta de recuperación comprendida entre el 70 y el 130%. La técnica así establecida es por tanto apta para el análisis de aguas crudas, potables y residuales y es viable su utilización en el seguimiento a procesos de oxidación avanzada aplicados en el tratamiento de agua para consumo humano.

The technique of combustion analysis and detection by non-dispersive infrared (NDIR) was validated for the determination of organic matter in water, quantified as total organic carbon (TOC). Previously optimized the elimination of inorganic carbon (IC) of the sample with an optimum time sparge of 1.5 min and an acid ratio of 5%. It established a linear dynamic range (LDR) between 3 and 20 mg/L of TOC, in which the regression line that met the parameters credited its linearity as analyzed by the method of least squares, showing a correlation coefficient of 0,9994. The sensitivity expressed by the slope of the regression line indicated a variation of about 5 units in the detector response for each mg/L of TOC. The detection and quantification limits obtained from the regression line were 0.517 and 1.722 mg/L of TOC, respectively. The precision of the technique, aiming at 5% coefficient of variation (CV) was better in the drinking water at concentrations close to it (about 7 mg/L of TOC), whereas larger deviations were presented at concentrations near the lower and upper limits of LDR. The recoveries of known concentrations of actual samples of 85% additions were low addition and 83% with high added values that suggest a reevaluation of the performance of the technique with respect to its accuracy, however the goal was reached between recovery 70 to 130%. The technique is well established thereby capable of analyzing raw water, drinking and waste and feasible to use in monitoring advanced oxidation processes applied in the treatment of drinking water.

Estabilidad por cromatografía en capa delgada de mezclas de tinturas de Quassia amara y Maytenus ilicifolia / Stability by thin-layer chromatography from Quassia amara and Maytenus ilicifolia tinctures

Una línea de investigación que ha tomado auge en los últimos tiempos, es aquella que se refiere a los productos fitoterapéuticos, por lo que se hizo necesaria una evaluación integral para su posterior utilización. Este trabajo describe un método por cromatografía en capa delgada, para evaluar la estabilidad de tinturas obtenidas a partir de un proceso de percolación de 2 plantas de origen brasileño: Quassia amara y Maytenus ilicifolia. El estudio se realizó teniendo en cuenta 3 temperaturas de almacenamiento: refrigeración, ambiente y 40 ºC, durante un tiempo de 90 días. Se seleccionaron las condiciones cromatográficas, se determinó el límite de detección y se demostró la selectividad del método, para lo cual se sometió el extracto a 4 condiciones de estrés. Se usó como fase móvil n-butanol-acido acético-agua (4:1:5) y como revelador la luz ultravioleta a una l de 366 nm. Los mejores resultados se observan cuando el extracto analizado se conserva en refrigeración durante el tiempo de estudio. Bajo condiciones de estrés, solo existen resultados favorables cuando es expuesto a la luz ultravioleta. Se establece como límite de detección el correspondiente a 7 µL.

In past years, a research field with boom is that related to phytotherapeutical products, being necessary an integral research for its latter utilization. Present paper describes a thin layer chromatography (TLC) method to assess the stability of tinctures obtained from a percolation process of two Brazilian plants: Quassia amara and Maytenus ilicifolia. Study took into account three storage temperatures: refrigeration, room-temperature and at 40 ºC during 90 days. Chomatographic conditions were selected, detection limit was determined, and method selectivity was demonstrated where the extract undergoes four stress-conditions. As a mobile phase we used n-butanol-acetic acid-water (4:1:5), and as UV at a l of 366 nm. The better results are obtained when analyzed extract is stored in refrigeration during study-time. Under stress-conditions only it is possible to obtain favorable results under UV. Detection limit is that corresponding to 7 µL.

Trends in detection methods of ricin / 军事医学科学院院刊

Ricin is a plant-derived ribosome-inhibitor which can be easily purified in large quantities from castor beans. It is a potent irreversible inhibitor of protein synthesis. The mode of intoxication could be inhalation, ingestion, intravenous injection.Ricin has been classified as a schedule 1 threat agent by the Chemical Weapons Convention.A fast and sensitive method for the detection of this threat agent is an important tool for preventing or dealing with the consequences of intoxication. An ideal method should be highly sensitve, highly selective, and well capable of identifying ricin in a short assay time. Several methods have been established for ricin detection. This review summaries the development of detection methods for ricin in recent years.

Determinação do limite mínimo de detecção da técnica de PCR “nested” para o vírus da hepatite B (HBV) / Evaluation of minimum detection limit to hepatitis B virus (HBV) PCR “nested”

Mundialmente, a hepatite pelo vírus B (HBV) é considerada um dos maiores problemas de saúde pública, apesar da vacinação. A Organização Mundial da Saúde (OMS) estima que mais de 2 bilhões de pessoas estejam infectadas pelo HBV. O Brasil é classificado como área de incidência intermediária pela OMS. No entanto, estudos de prevalência detectaram diferenças de índices de infecção nas regiões geográficas: 8% na região Amazônica, 2,5% nas regiões Centro-Oeste e Nordeste, 2% na Sudeste e 1% na região Sul. Um diagnóstico sensível e específico é de fundamental importância para os pacientes portadores do HBV. O objetivo deste estudo foi determinar o limite mínimo de detecção da técnica de PCR “nested” “in house” para o HBV. Diluições seriadas de uma amostra quantificada de HBV (1000 cópias/mL; 750 cópias/mL; 500 cópias/mL; 250 cópias/mL) foram submetidas à técnica de PCR “nested”. O alvo da amplificação por PCR foi a região do core e pré-core do vírus. Para extração dos ácidos nucléicos da amostra foi empregado o kit comercial QIAmp. O limite mínimo de detecção encontrado foi de 500 cópias/mL ou 10 cópias por reação de PCR.

All over the world, the hepatitis B virus (HBV) is considered one of the major problems of public health, despite vaccination. World Health Organization (WHO) estimates that more than 2 billions of persons are infected by HBV. Brazil is classified as an area of intermediary incidence by WHO. However, prevalence studies have detected differences of infection indexes in geographic regions: 8% in the Amazonian region, 2,5% in middle-west and Northeast, 2% in Southeast and 1% in South. A sensitive and specific diagnosis is very important to the HBV carrier patients. The aim of this study was to determine the minimum limit of detection of the nested PCR in house technique for HBV. Serial dilutions of one quantified sample of HBV (1000 copies/mL; 750 copies/mL; 500 copies/mL; 250 copies/mL) were submitted to a nested PCR. The target of PCR was viral core and pre-core region. Commercial kit, QiAmp, was employed to purify nucleic acids from the sample. The minimum detection limit found was 500 copies/mL or 10 copies per PCR reaction.

Oligonucleotide Hybridization Detection Based on Surface Plasmon Resonance Technology / 航天医学与医学工程

Objective To establish a kind of detection technique of nucleic acid based on surface plasmon resonance(SPR) and to set up the foundation of real-time, online space microbial detection. Methods A portable online bio-molecules analyzer based on SPR biosensor was applied. The probe was mercapto-modified at the 5’ end and immobilized on the sensor surface. Then the target sequences in the solution were monitored and sensitivity, specificity and reproducibility of the method were investigated. Results The results showed that detection method with good specificity and sensitivity could realize online detection of target sequence. The system could detect 2.3 nmol/L target sequence, CV value of nine detections was 3.5% and that of thirty detections was 14.7%. Conclusion The established nucleic acid detection method has the advantage of high sensitivity, good specificity and reproducibility, which can be applied in the field of nucleic acid detection.

Evaluation of a Bacillus stearothermophilus tube test as a screening tool for anticoccidial residues in poultry

A Bacillus stearothermophilus var. calidolactis C953 tube test was evaluated for its ability in detecting the residue of selected anticoccidial drugs in poultry, specically sulfamethazine, furazolidone, and amprolium. Various concentrations of each drug were injected into chicken liver and kidney tissues and these tissues were tested to determine the drug detection limits for each drug. The detection limit was defined as the drug concentration at which 95% of the test results were interpreted as positive. The limits of detection in liver tissue were 0.35 microgram/ml for furazolidone, 0.70 microgram/ml for sulfamethazine and 7.80 microgram/ ml for amprolium. In kidney tissues, they were 0.30 microgram/ml for furazolidone, 0.54 microgram/ml for sulfamethazine, and 7.6 microgram/ml for amprolium. It was concluded that this tube test could be used to screen for the residue of these three drugs in poultry.

Development of an Immuno-PCR Protocol for Detection of a Small Amount of Antigen / 대한진단검사의학회지

BACKGROUND: Immuno-PCR has been known as a highly sensitive and specific method, yet no standardized protocol is available. We analyzed each step of immuno-PCR to develop a reliable standardized method. METHODS: We made a protocol modified from several methods reported previously, and performed immuno-PCR, but false positive reactions were noted. To reduce the false positivity, we investigated the buffer reagents and biotin-labelled oligo-nucleotide probe. Using a finally determined protocol, we compared the detection-limits of the immuno-PCR and ELISA methods. RESULTS: Streptavidin was identified as a main reagent causing a non-specific binding, thus it was replaced by neutravidin. The employment of CAS block as a dilution buffer for the biotin-labelled oligo-nucleotide probe and Casein block as a buffer for the detection antibodies resulted in a dramatic reduction in the false positive reactions. The standardized immuno-PCR detected angiogenin antigen at a concentration as low as 5 fg/mL, while an ELISA method detected 5 pg/mL. CONCLUSIONS: The immuno-PCR procedure newly described in this study was ultra-sensitive with no false positivity. This method can be utilized as an epochal tool for detection of a small amount of antigen which would not be discovered by ELISA method.