RESUMEN
The combination of the molecular technique, the multivariate strategy and microchip capillary electrophoresis (MCE) was applied to rapid and sensitive analysis of genetically modified (GM) soybean in food samples. A multiplex-touchdown polymerase chain reaction (PCR) system was developed for simultaneously amplifying three target sequences in Roundup Ready soybean (RRS). Response surface methodology was introduced to determine the optimal separation condition in MCE with good resolution and short analytical time. The detection of the PCR products of RRS was completed within 4 min under the optimal conditions. The specificity of the method was evaluated by testing non-GM soybean materials and three GM maize varieties (MON810, Bt176 and Bt11). A sensitivity of 0.1% GM organisms content was obtained, which was remarkably lower than the labeling threshold for transgenic food defined as 0.9% in the European regulation. The relative standard deviation of migration time was in the range of 0.17-0.95%. The proposed method was rapid, sensitive and specific and can be used to identify and detect GM soybean in food samples.
Asunto(s)
ADN de Plantas/genética , Electroforesis Capilar/métodos , Glycine max/genética , Plantas Modificadas Genéticamente/genética , Reacción en Cadena de la Polimerasa/métodos , ADN de Plantas/análisis , Análisis de Regresión , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Cronobacter spp. (Enterobacter sakazakii) is an emerging opportunistic pathogen with a 40-80% mortality rate in infants and immunocompromised crowd resulting from the consumption of contaminated food. A novel method for detecting Cronobacter spp. in food samples by duplex polymerase chain reaction (PCR) in combination with capillary electrophoresis-laser induced fluorescence (CE-LIF) detector has been developed. The specific gene sequences of 16S-23S rDNA internal transcribed spacer (ITS) and the outer membrane protein A (OmpA) of Cronobacter spp. were amplified by duplex PCR. The PCR products were separated and determined sensitively by CE-LIF within 12min. The relative standard deviations of migration time for the detected DNA fragments were 2.01-2.91%. The detection limit was as low as 1.6×10(1)cfu/mL of Cronobacter spp. Besides, the specificity of the method was verified by 24 non-Cronobacter bacterial strains. A total of 120 commercial infant food formula were tested for the presence of Cronobacter spp. by using the proposed method. This current study demonstrates that the combination of CE-LIF method with duplex PCR is rapid, sensitive and environmental friendly, and has the potential to be adapted for the routine detection of Cronobacter spp. in food samples. To the best of our knowledge, this is the first use of CE-LIF for the detection of Cronobacter spp.
Asunto(s)
Cronobacter/aislamiento & purificación , Electroforesis Capilar/métodos , Microbiología de Alimentos , Reacción en Cadena de la Polimerasa/métodos , Cronobacter/genética , Humanos , Lactante , Alimentos Infantiles/microbiología , Fórmulas Infantiles , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: A pulsed amperometric detector-high performance anion chromatography method for the determination of L-carnitine in health food was established. METHODS: After samples were dissolved and dispersed with ether, L-carnitine in samples was extracted with 0. 1 mmol/L HC1. Using 200 mmol/L NaOH as the mobile phase, amino PAC PA10 Amino acid-separation column as separation column and pulse ampere detector with An as working electrode throughout the experiment. RESULTS: The linear was good in the range of 0.08 - 1.00 mg/ml. The intra-day RSDs of peak area and retention time for L-carnitine standard solution were 3.97% and 0.62% and the inter-day RSDs were 5.66% and 0.69%, respectively. The average recoveries were 95.6% - 113.5% and the RSD of sample analysis was 7.34%. The results of the proposed method for the analysis of L-carnitine in health foods were satisfactory. CONCLUSION: The proposed method is simple, accurate and reproducible. One analysis can be completed within 15 min. The method is adaptive for the analysis of L-carnitine in health food.
Asunto(s)
Carnitina/análisis , Cromatografía por Intercambio Iónico/métodos , Suplementos Dietéticos/análisis , Alimentos Orgánicos/análisis , Cromatografía Líquida de Alta Presión/métodos , Técnicas Electroquímicas/métodosRESUMEN
It is very rare that a foramen magnum arachnoid cyst induces compression of the spinal cord and syringomyelia, and currently there are few treatment experiences available. Here we reported the case of a 43-year-old male patient who admitted to the hospital due to weakness and numbness of all 4 limbs, with difficulty in urination and bowel movement. MRI revealed a foramen magnum arachnoid cyst with associated syringomyelia. Posterior fossa decompression and arachnoid cyst excision were performed. Decompression was fully undertaken during surgery; however, only the posterior wall of the arachnoid cyst was excised, because it was almost impossible to remove the whole arachnoid cyst due to toughness of the cyst and tight adhesion to the spinal cord. Three months after the surgery, MRI showed a reduction in the size of the arachnoid cyst but syrinx still remained. Despite this, the symptoms of the patient were obviously improved compared to before surgery. Thus, for the treatment of foramen magnum arachnoid cyst with compression of the spinal cord and syringomyelia, if the arachnoid cyst could not be completely excised, excision should be performed as much as possible with complete decompression of the posterior fossa, which could result in a satisfying outcome.
Asunto(s)
Quistes Aracnoideos/complicaciones , Foramen Magno/patología , Compresión de la Médula Espinal/etiología , Siringomielia/etiología , Adulto , Quistes Aracnoideos/patología , Quistes Aracnoideos/cirugía , Descompresión Quirúrgica/métodos , Humanos , Imagen por Resonancia Magnética , Masculino , Médula Espinal/patología , Compresión de la Médula Espinal/complicaciones , Compresión de la Médula Espinal/patología , Compresión de la Médula Espinal/cirugía , Siringomielia/complicaciones , Siringomielia/patología , Resultado del TratamientoRESUMEN
OBJECTIVE: To determine residues of multiple biphenyl ether herbicides simultaneously in water using high performance liquid chromatography (HPLC) with cloud-point extraction. METHODS: The residues of eight biphenyl ether herbicides (including bentazone, fomesafen, acifluorfen, aclonifen, bifenox, fluoroglycofenethy, nitrofen, oxyfluorfen) in water samples were extracted with cloud-point extraction of Triton X-114. The analytes were separated and determined using reverse phase HPLC with ultraviolet detector at 300 nm. Optimized conditions for the pretreatment of water samples and the parameters of chromatographic separation applied. RESULTS: There was a good linear correlation between the concentration and the peak area of the analytes in the range of 0.05-2.00 mg/L (r = 0.9991-0.9998). Except bentazone, the spiked recoveries of the biphenyl ether herbicides in the water samples ranged from 80.1% to 100.9%, with relative standard deviations ranging from 2.70% to 6.40%. The detection limit of the method ranged from 0.10 microg/L to 0.50 microg/L. CONCLUSION: The proposed method is simple, rapid and sensitive, and can meet the requirements of determination of multiple biphenyl ether herbicides simultaneously in natural waters.
Asunto(s)
Compuestos de Bifenilo/análisis , Cromatografía Líquida de Alta Presión/métodos , Herbicidas/análisis , Residuos de Plaguicidas/análisis , Contaminantes Químicos del Agua/análisis , Benzamidas/análisis , Benzotiadiazinas/análisis , Nitrobenzoatos/análisis , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To develop an gas chromatography-electrophoretic capture detection (GC-ECD)method for determining dimethyl sulphate (DMS) in environmental samples. METHODS: The environmental samples were dissolved with acetone and the DMS was reacted with KI. The reaction product of methyl iodide was extracted with Hexane and determined by GC-ECD, with HP-5 (30.0 m x 0.32 mm x 0.25 microm) as column, high purity nitrogen as carries, 60 degrees C for column temperature and 240 degrees C for ECD and injector temperature, and 5:1 for split ratio. RESULTS: The linear range appeared from 0.05 microg/mL to 2.0 microg/mL. The detection limit was 0.0011 microg/mL. The precision [measured by relative standard deviation (RSD)] of peak area and reserved time for the standard solution was 4.95% and 0.15% respectively for intra-day detections, and 5.99% and 0.83% respectively for inter-day detections. Satisfactory results were obtained for the analysis of environmental samples. The RSD for the water and soil samples were 9.6% and 7.5% respectively and the recoveries were 76.0%-85.0% and 82.0%-110.0% respectively. CONCLUSION: The method was simple, accurate, sensitive and applicable for DMS analysis in environmental samples.
Asunto(s)
Cromatografía de Gases/métodos , Electroforesis/métodos , Contaminantes Ambientales/análisis , Ésteres del Ácido Sulfúrico/análisis , Mutágenos/análisisRESUMEN
OBJECTIVE: To establish a high performance anion chromatography method for the determination of adenosine in food. METHODS: Adenosine in the food sample was extracted with Acetonitrile-water, and separated with Amino PAC PA10 Amino acid-separation column, with 0.25 mol/L NaOH as the mobile phase. A pulse ampere detector with Au as working electrode was used throughout the experiment. RESULTS: A good linear result was produced for adenosine in the range of 1.00-20.00 microg/mL. The inner-day RSD of peak area and retention time for the standard adenosine solution were 4.97% and 0.47%, respectively. The intra-day RSD of peak area and retention time for the standard adenosine solution were 6.08% and 0.52%, respectively. The proposed method was applied to food and satisfactory results were obtained, with and average recoveries of 93.3%-106.7% and 8.86% of RSD. CONCLUSION: The high performance anion exchange chromatography method is a simple, accurate, and reliable method for measuring adenosine in food, which can be completed within 10 minutes.
Asunto(s)
Adenosina/análisis , Cromatografía Líquida de Alta Presión/métodos , Aditivos Alimentarios/análisisRESUMEN
A rapid and sensitive method based on microchip capillary electrophoresis with condition optimization of genetic algorithm-support vector regression (GA-SVR) was developed and applied to simultaneous analysis of multiplex PCR products of four foodborne pathogenic bacteria. Four pairs of oligonucleotide primers were designed to exclusively amplify the targeted gene of Vibrio parahemolyticus, Salmonella, Escherichia coli (E. coli) O157:H7, Shigella and the quadruplex PCR parameters were optimized. At the same time, GA-SVR was employed to optimize the separation conditions of DNA fragments in microchip capillary electrophoresis. The proposed method was applied to simultaneously detect the multiplex PCR products of four foodborne pathogenic bacteria under the optimal conditions within 8 min. The levels of detection were as low as 1.2 x 10(2) CFU mL(-1) of Vibrio parahemolyticus, 2.9 x 10(2) CFU mL(-1) of Salmonella, 8.7 x 10(1) CFU mL(-1) of E. coli O157:H7 and 5.2 x 10(1) CFU mL(-1) of Shigella, respectively. The relative standard deviation of migration time was in the range of 0.74-2.09%. The results demonstrated that the good resolution and less analytical time were achieved due to the application of the multivariate strategy. This study offers an efficient alternative to routine foodborne pathogenic bacteria detection in a fast, reliable, and sensitive way.
Asunto(s)
Algoritmos , Bacterias/aislamiento & purificación , Electroforesis por Microchip/métodos , Microbiología de Alimentos , Reacción en Cadena de la Polimerasa/métodos , Bacterias/genética , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Reproducibilidad de los Resultados , Salmonella/genética , Salmonella/aislamiento & purificación , Sensibilidad y Especificidad , Shigella/genética , Shigella/aislamiento & purificación , Factores de Tiempo , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/aislamiento & purificaciónRESUMEN
OBJECTIVE: To develop a rapid method for determination of nicotine and its metabolite cotinine in human hair with capillary gas chromatography with flame ionization detector. METHODS: The hair sample was digested by 1.5 mol/L sodium hydroxide solution. The nicotine and cotinine in the hair sample were extracted with a mixed solvent of dichloromethane-methanol (3:1). Aliquot of the extraction solution was vaporized with nitrogen flow and then methanol was added to dissolve the analysts. The analysts were tested with capillary gas chromatography. RESULTS: The detection limits (signal to noise ratio of 3:1) were 4.3 ng/mL for nicotine and 10 ng/mL for cotinine, respectively. The spiked recoveries were 90.33%-113.1% for nicotine with relative standard deviation (RSD) of 2.1%-7.0% and 92.92%-117.4% for cotinine with relative standard deviation (RSD) of 4.4%-8.9%, respectively. CONCLUSION: The proposed method is sensitive, simple, easy and rapid. It can be easily extended to large scale applications in tobacco epidemiology studies.
Asunto(s)
Cotinina/análisis , Ionización de Llama/métodos , Cabello/química , Nicotina/análisis , Humanos , Límite de Detección , Sensibilidad y EspecificidadRESUMEN
OBJECTIVE: To develop a rapid approach of refractive index detection with high performance liquid chromatography (HPLC) for the determination of rhamnose, fructose, glucose, surose and maltose in Lycium barbarum L. METHODS: The sample was extracted with water and the analyte was separated by Zorbax Carbohydrate column with acetonitrile-water as mobile phase. RESULTS: Good linear correlations between the concentrations and the peak areas of the analyte were found, with the correlation coefficients ranging from 0.9976 to 0.9998. The spiked recovery rates ranged from 92.27% to 101.93%, with 1.85%-3.27% of relative standard deviations (n=5). The limits of detection (S/N =3) were 4-6 mg/kg. CONCLUSION: The proposed method is suitable for the determination of monosaccharide and oligosaccharide in Lycium barbarum L.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Lycium/química , Monosacáridos/análisis , Oligosacáridos/análisis , Límite de DetecciónRESUMEN
A method for monitoring foodborne pathogenic bacteria by multiplex polymerase chain reaction (PCR)--capillary electrophoresis (CE) with a laser induced fluorescence detector was developed. Three sets of primers were designed to amplify the gene segments of uidA gene in E. coli. O157:H7, invA gene in salmonella and ipaH gene in Shigella, individually. The multiple PCR system and the separation conditions of CE were optimized. Using a capillary coated with linear polyacrylamide and sieving buffer of 7.0 g/L methyl cellulose (MC) under 8.3 kV of electric voltage, the proposed method was able to simultaneously detect the PCR products of specific genes existing in the three kinds of pathogenic bacteria within 22 min. The relative standard deviations of migration time for the detected DNA fragments were ranging from 1.47% to 2.07%. In comparison with agarose gel electrophoresis, the proposed method is rapid, sensitive and accurate.
Asunto(s)
Electroforesis Capilar/métodos , Microbiología de Alimentos , Reacción en Cadena de la Polimerasa/métodos , Espectrometría de Fluorescencia/métodos , Antígenos Bacterianos/genética , Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , ADN Bacteriano/genética , Electroforesis en Gel de Agar , Reproducibilidad de los ResultadosRESUMEN
A high performance liquid chromatographic (HPLC) method for the simultaneous determination of sulfonylurea herbicide and diphenylether herbicide residues in soybean and rice samples was developed and evaluated. The analytes in soybean and rice samples were extracted with acetonitrile. The extract was cleaned up using an SPE-C18 cartridge, separated and quantitatively determined by reversed-phase HPLC with an ultraviolet detector. The conditions of the pretreatment for soybean and rice samples as well as the parameters of chromatographic separation were optimized. The method had a good linearity in the range of 0.05-2.0 mg/L. The quantitative limits (LOQ) of the 8 herbicide residues were 0.01-0.02 mg/kg. The spiked recoveries were in the range of 91.6%-116.1% for soybean samples and 76.6%-110.8% for rice samples with the relative standard deviations of 1.0%-12.2%. It is concluded that the proposed method is rapid, accurate and sensitive and can be used for the determination of the eight herbicide residues in soybean and rice.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Glycine max/química , Herbicidas/análisis , Herbicidas/química , Oryza/química , Compuestos de Sulfonilurea/análisis , Compuestos de Sulfonilurea/química , Estructura Molecular , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVE: To develop a method of detecting retinol (vitamin A), vitamin D3, alpha-tocopherol (vitamin E) and beta-carotene in human serums with HPLC. METHODS: Proteins were precipitated with anhydrous ethanol. Fat-solutable vitamins in human serums were extracted with aether/petroleum ether mixture and determined with HPLC. RESULTS: The linear ranges for the retinol, VD3, alpha-tocopherol and beta-carotene were 0.012 microg/mL-500 microg/mL, 0.030 microg/mL-500 microg/mL, 0.12 microg/mL-500 microg/mL, and 0.015 microg/mL-500 microg/mL, respectively. The detection limits for the retinol, VD3, alpha-tocopherol and beta-carotene were 0.012 microg/mL, 0.030 microg/mL, 0.12 microg/mL and 0.015 microg/mL, respectively. The relative standard derivations (RSD) for the retinol, VD3, alpha-tocopherol and beta-carotene were 0.75%, 0.54%, 2.06% and 2.74%, respectively. The proposed method recovered 92%-116%, 98%-112%, 84.8%-106%, and 90%-105% retinol, VD3, alpha-tocopherol and beta-carotene in human serums respectively. CONCLUSION: The method is simple, quick and applicable to all of the four fat-solutable vitamins.
Asunto(s)
Colecalciferol/análisis , Cromatografía Líquida de Alta Presión/métodos , Vitamina A/análisis , alfa-Tocoferol/análisis , beta Caroteno/análisis , Humanos , Límite de DetecciónRESUMEN
A flow injection-CCD-diode array detection spectrophotometric method using partial least squares (PLS) algorithm for the simultaneous determination of iron, copper and cobalt in food samples has been established. The method was based on the chromogenic reaction between metallic ions and 5-Br-PADAP in the presence of acetic acid-acetic sodium buffer solution (pH 5) containing 30 g x L(-1) ascorbic acid and 2% (phi) Triton X-100. The overlapped spectra of these complexes were collected by CCD diode array detector and the multi-wavelength absorbance data were processed using partial least squares algorithm. The reaction conditions and analytical parameters of FIA were investigated. The food samples can be analyzed without any separation after digestion, and the sampling rate was 45 x h(-1). The linear ranges of Fe2+, Cu+ and Co2+ were 0.2-10.0 microg x mL(-1), 0.1-5.0 microg x mL(-1), and 0.01-1.0 microg x mL(-1) and the detection limits were 0.2, 0.1 and 0.01 microg x mL(-1), respectively. The average recoveries of spiked samples were 89.4%-110.8% for the three elements. The relative standard deviation (RSD) of samples was in the range of 1.1%-12.1%. Comparing the proposed method with ICP-AES, the relative error was below 12.1%. Above all, this method is simple, quick, sensitive, selective, and easy to be apply and generalize.
Asunto(s)
Análisis de los Alimentos/métodos , Análisis de los Mínimos Cuadrados , Metales Pesados/análisis , Espectrofotometría/métodos , Calibración , Cobalto/análisis , Cobre/análisis , Fabaceae/química , Análisis de Inyección de Flujo , Análisis de los Alimentos/instrumentación , Concentración de Iones de Hidrógeno , Hierro/análisis , Reproducibilidad de los Resultados , Espectrofotometría/instrumentación , Espectrofotometría Atómica/métodos , Té/químicaRESUMEN
A method for determining collagen in tendon by reversed-phase high performance liquid chromatography (RP-HPLC) was developed. After hydrolysis with hydrochloric acid, the collagen in samples was decomposed into hydroxyproline which hydroxyproline can be derivatized with 2, 4-dinitrochlorobenzene for the determination by HPLC (reversed-phase C18 column, 0.01 mol/L NaAc-HAc (pH 6.0)-CH3CN (80: 20, v/v) as mobile phase, detection at 360 nm). The factors influencing hydrolysis, derivatization and HPLC analysis were studied and optimized. Sixty samples were analyzed with the proposed method. The linear range was from 3 microg/L to 100 mg/L and the detection limit was 3 microg/L. The relative standard deviation (RSD) of determination was 1.95%. The recoveries of spiked samples were 98.4%-110.8%. The results show that the method is sensitive, accurate and suitable for tendon determination.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Cromatografía de Fase Inversa/métodos , Colágeno/análisis , Tendones/química , Estabilidad de Medicamentos , Matriz Extracelular/metabolismo , Análisis de Inyección de Flujo , Humanos , Hidroxiprolina/análisis , Límite de Detección , Propionatos/análisis , EstereoisomerismoRESUMEN
OBJECTIVE: To develop a capillary gas chromatographic method for the determination of mannitol in health food. METHODS: Mannitol was extracted from the sample with water by ultrasonic wave. The aliquot of sample solution was acetylated to form mannitol hexacetate with lower boiling point. Then the derivative was separated with the use of SPB-608 capillary chromatographic column and determined by flame ionization detector (FID) at the column temperature of 180 degrees C, the detector temperature of 210 degrees C and the nitrogen flow rate of 30 mL/min. RESULTS: The linear range of this method was 0.05-10.00 mg/mL and the limit of detection of was 0.01 mg/mL. The within-day and between-days precisions were 4.4% and 7.3%-7.6%, respectively. The average recoveries were 84.6%-107.6%. Mannitol could be separated from glucose, inositol and sorbitol under the optimum chromatographic condition. CONCLUSION: The results demonstrate that the method has the advantages of simplicity, accuracy and sensitivity. It is suitable for the determination of mannitol in health food.
Asunto(s)
Alimentos Orgánicos/análisis , Manitol/análisis , Cromatografía de Gases/métodos , HumanosRESUMEN
A sensitive and simple method based on solid-phase extraction (SPE) and HPLC with fluorescence detection for the determination of bisphenol A (BPA) and 4-nonylphenol (4-NP) in rat serum, liver and testis tissues has been developed. The chromatographic conditions consisted of a C18 column and mobile phase composition of acetonitrile and water with flow rate of 1.0 ml/min. The fluorescence detection was performed at excitation and emission wavelengths of 227 nm and 313 nm, respectively. Under these conditions, BPA and 4-NP were well separated and showed good linearities in the ranges of 0.01-50.0 microg/ml for BPA and 0.15-150.0 microg/ml for 4-NP with correlation coefficients greater than 0.999. The detection limits of serum and tissue samples were 2.8 ng/ml and 1.4 ng/g for BPA and 5.6 ng/ml and 2.8 ng/g for 4-NP at a signal-to-noise ratio (S/N) of 3. The intra-assay and the inter-assay precisions were better than 11.4%. Recoveries of BPA and 4-NP were 78.6-95.0% and 80.2-93.4%, respectively. The proposed method was applied to a toxicokinetic study of BPA and 4-NP including individual and combined oral administration to rats. The results showed that 4-NP remarkably altered the toxicokinetic parameters of BPA in testis, while parameters of BPA were not obviously altered in serum and liver under the experimental conditions investigated. On the other hand, there was no significant difference in the toxicokinetics of 4-NP when administered with BPA.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Hígado/química , Fenoles/análisis , Testículo/química , Administración Oral , Animales , Área Bajo la Curva , Compuestos de Bencidrilo , Interacciones Farmacológicas , Hígado/metabolismo , Masculino , Tasa de Depuración Metabólica , Fenoles/sangre , Fenoles/farmacocinética , Ratas , Ratas Wistar , Reproducibilidad de los Resultados , Testículo/metabolismo , Distribución TisularRESUMEN
A method for the determination of chlorhexidine acetate in disinfectors using capillary electrophoresis (CE) was developed using the following conditions: detection wavelength, 254 nm; applied voltage, 15 kV; uncoated fused silica capillary column, 50 cm x 75 microm i.d.; buffer, 15 mmol/L phosphate and acetonitrile (60:40, v/v). Under the optimum conditions, chlorhexidine acetate in disinfectors was determinated in 4 min. The effects of different parameters, such as buffer component, buffer concentration, buffer pH value and electrophoretic voltage, on the CE analysis of chlorhexidine acetate were studied in detail. The linear range of the proposed method was 0.01-0.10 g/L. The detection limit was 0.004 mg/L. The relative standard deviation (RSD) of absorbance was 3.97% and the RSD of migration time was 2.99%. The spiked recoveries of samples were 91.4%-116.6%. The method was compared with the high performance liquid chromatographic method and the relative error was less than 4.0%. The proposed method was simple, quick and suitable for disinfector analysis.
Asunto(s)
Clorhexidina/análisis , Desinfectantes/análisis , Electroforesis CapilarRESUMEN
A new method for rapidly detecting restriction enzyme pattern of mycobacterium deoxyribonucleic acid (DNA) by capillary electrophoresis with laser induced fluorescence detection (CE-LIFD) was developed. Polymerase chain reaction was used to amplify a 439 bp fragment of 65,000 (Mr) heat shock protein gene (hsp65) of mycobacterium. After digesting the amplification products by BstE II and Hae III respectively, the patterns of enzyme cleavaged products were detected by both CE-LIFD and agarose gel electrophoresis (AGE). The experimental parameters of CE were optimized. The restriction enzyme patterns of mycobacterium DNA can be detected under the optimum electrophoresis conditions: a coated capillary column with the length of 50 cm and 100 microm i. d., electrophoresis buffer of 45 mmol/L TBE (trihydroxymethyl aminomethane (Tris)-boric acid-ethylenediaminetetraacetic acid (EDTA)) and 11 kV running voltage. The restriction enzyme patterns for eight species of mycobacteria were studied. Relative standard deviations of the relative migration times of the DNA segments were less than 3.6%. Compared with AGE, CE is more outstanding in resolution and detection time, and it can be applied as a more effective means for DNA restriction enzyme pattern analysis.
Asunto(s)
Enzimas de Restricción del ADN/análisis , Electroforesis Capilar/métodos , Mycobacterium/genética , Mapeo Restrictivo/métodos , Espectrometría de Fluorescencia , Animales , Proteínas Bacterianas/análisis , Chaperonina 60/análisis , Enzimas de Restricción del ADN/genética , ADN Bacteriano/genética , Desoxirribonucleasas de Localización Especificada Tipo II/análisis , Electroforesis en Gel de Agar , Rayos Láser , Reacción en Cadena de la PolimerasaRESUMEN
OBJECTIVE: To develop a rapid detection method for genetically modified soybean resistant to glyphosate. METHODS: A duplex PCR was performed with primers designed in this study to simultaneously amplify heterogenous genes in transgenic soybean: CaMV-35S promoter, NOS terminator and CP4-EPSPS gene. And a simple capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was developed and applied to the rapid analysis of the above PCR products by using a 50 cm length x 100 microm i.d. capillary coated with linear polyacrylamide and an 8 g/L HPMC-4000 sieving buffer under 200 V/cm electric field strength. RESULTS: The proposed method was able to simultaneously detect the three heterogenous genes existing in genetically modified soybean under the optimization conditions of PCR and capillary electrophoresis. The measured sequences of the duplex PCR products were identical with the original genes' sequences. Moreover, the sample volumes required were not more than 5 nl and the detection could be completed in less than 24 min. The relative standard deviations (R. S. D.) of the migration times for the PCR products were < or = 3.2%. CONCLUSION: In comparison with agarose gels electrophoresis, the duplex PCR-based capillary electrophoretic method with laser-induced fluorescence detection is rapid, sensitive and accurate, and it is suitable for detection of genetically modified soybean.