RESUMO
In Taiwan, the number of applications for inspecting imported food has grown annually and noncompliant products must be accurately detected in these border sampling inspections. Previously, border management has used an automated border inspection system (import food inspection (IFI) system) to select batches via a random sampling method to manage the risk levels of various food products complying with regulatory inspection procedures. Several countries have implemented artificial intelligence (AI) technology to improve domestic governmental processes, social service, and public feedback. AI technologies are applied in border inspection by the Taiwan Food and Drug Administration (TFDA). Risk management of border inspections is conducted using the Border Prediction Intelligent (BPI) system. The risk levels are analyzed on based on the noncompliance records of imported food, the country of origin, and international food safety alerts. The subjects of this study were frozen fish products, which have been under surveillance by the BPI system. The purpose of this study was to investigate the relevance between the noncompliant trend of frozen fish products using the adoption of the BPI system and the results of postmarket sampling inspections. The border inspection and postmarket sampling data were divided into two groups: IFI and BPI groups (corresponding to before and after the adoption of the BPI system, respectively). The Chi-square test was employed to analyze the noncompliant differences in products between before and after the BPI system adoption. Despite the number of noncompliance batches being statistically insignificant after the adoption of the BPI system, the noncompliance rate of frozen fish products at the border increased from 3.0% to 4.7%. Meanwhile, the noncompliance rate in the postmarket decreased from 2.1% to 1.9%. The results indicate that the BPI system improves the effectiveness of interception of noncompliant products at the border, thereby preventing the entrance of noncompliant products to the postmarket. The variables were further classified and organized according to the scope of this study and product characteristics. Furthermore, ordinal logistic regression (OLR) was employed to determine the correlations among border, postmarket, and major influencing factors. Based on the analysis of major influencing factors, small fish and fish internal organ products exhibited significantly high risk for fish body type and product type, respectively. The BPI system effectively utilizes the large amount of data accumulated from border inspections over the years. Additionally, real-time information on bilateral data obtained from the border and postmarket should be bidirectionally shared for effectively intercepting noncompliance products and used for improving the border management efficiency.
Assuntos
Inteligência Artificial , Produtos Pesqueiros , Estados Unidos , Animais , Humanos , Taiwan , Peixes , Inocuidade dos AlimentosRESUMO
Due to rapid development of biotechnology in recent years, the field of regenerative medicine has attracted considerable attention. Regenerative medicine-related regulations have been established in several countries to ensure the quality, safety, and efficacy of innovative treatments. Considering the diversity of regenerative medicine, the regulatory framework in Taiwan has been adjusted in response to global trend and local demand. Before 2010, cell and gene therapies were regarded as "new medical practice" under the "Medical Care Act." Along with the establishment of Taiwan Food and Drug Administration (TFDA) in 2010, regenerative medicine was regulated as "medicinal products" under the "Pharmaceutical Affairs Act." Then, the Ministry of Health and Welfare (MOHW) established a new dual-track regulatory pathway for regenerative medicine in 2016. The dual-track pathway divided regenerative medicine into medical practices and medicinal products, aiming to improve the accessibility of new treatments to patients and maintain the flexibility for clinical operations. In order to refine the regulation, the MOHW proposed two draft Acts for regenerative medicine in 2022. The two draft Acts are currently under legislative process. It is expected that the research and development of regenerative medicine can be further accelerated, thus providing early access to innovative therapies for patients in the future.
Assuntos
Terapia Baseada em Transplante de Células e Tecidos , Medicina Regenerativa , Humanos , Taiwan , Terapia Genética , BiotecnologiaRESUMO
Tobacco-specific nitrosamines (TSNAs) as carcinogens endanger our health and life from cigarette products. However, the safe range of TSNAs levels in commercial cigarette products has not yet been established. For the purpose of safety and supervision, a three-step stacking approach including field amplified sample injection (FASI), sweeping, and analyte focusing by micelle collapse (AFMC), was developed for the simultaneous determination of five TSNAs levels in cigarette products. This approach also involved aspects of chemometric experimental design, including fractional factorial design and central composite design. After the multilevel optimization of the experimental design, the five TSNAs were well separated. The LOD (S/N = 3) values of the N´-nitrosonornicotine (NNN), N´-nitrosoanatabine (NAT), N´-nitrosoanabasine (NAB), 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the FASI-sweeping-AFMC CE approach were 1.000 ng/mL, 0.500 ng/mL, 0.125 ng/mL, 1.000 ng/mL, and 0.500 ng/mL respectively. The results of relative standard deviation (RSD) and relative error (RE) were all less than 3.35%, demonstrating good precision and accuracy. Finally, this novel approach was further applied to monitor three commercial cigarette products, and a range of 250.1-336.6 ng/g for NNN, 481.6-526.7 ng/g for NAT, 82.2-247.6 ng/g for NAB, 167.7-473.7 ng/g for NNAL, and 39.4-246.7 ng/g for NNK could be observed among these. Based on these results, the novel CE stacking strategy was successfully applied for the analysis of five TSNAs levels in cigarette products and could serve as a tool for assays of quality control of nitrosamines.
Assuntos
Nitrosaminas , Produtos do Tabaco , Carcinógenos/análise , Quimiometria , Eletroforese Capilar , Nitrosaminas/análise , Projetos de Pesquisa , Nicotiana , Produtos do Tabaco/análiseRESUMO
A three-step stacking capillary electrophoresis (CE) composed of field-amplified sample injection, sweeping, and analyte focusing by micellar collapse (FASI-sweeping-AFMC) was developed to determine dabigatran (D) and its major active metabolite, dabigatran acyl-beta-d-glucuronide (DAG), in human plasma. After optimization and validation, this novel approach was further applied to monitor 5 real samples, and the 25.2-186.8 ng mL-1 D could be observed among those. Based on these results, the novel CE stacking strategy was successfully applied for the analysis of D and DAG in human plasma and could be served as a tool for clinical assays.
Assuntos
Dabigatrana , Micelas , Eletroforese Capilar/métodos , HumanosRESUMO
Congenital red and green color blindness is the most X-linked recessive disorder in humans caused by deletions or gross structural rearrangements of the visual pigment gene array that lead to altered the functions of visual pigments in their retina differ from normal. The incidence is about 7-10% in male and close association of X-linked recessive disorders (such as: hemophilia A, hemophilia B, duchenne muscular dystrophy). However, the traditional genetic analysis methods are time-consuming and low-efficiencies. Therefore, the purpose of the study is to develop a rapid method for genotyping of red and green pigment genes. We describe herein the first method for simultaneous evaluation of ten exons in the red and green pigment genes for genetic analysis. A forward specific primers with identifiable universal fluorescent multiplex PCR (FSIUFM-PCR) method utilized one universal primer (containing two universal non-human sequences) and forward specific primers in the multiplex PCR reaction system for simultaneously fluorescent labeling of eleven gene fragments (ten exons in red and green pigment genes and one internal standard). All the PCR products were analyzed on capillary electrophoresis with short-end injection, which had the advantage of high resolution and rapid separation. Of all 80 detected individuals, 7 subjects with color vision deficiencies (including 3 subjects only had red exons 1-5, 4 subjects had a specific red-green or green-red hybrid gene and 73 subjects with normal color vision). All genotyping results showed good agreement with DNA sequencing data. This method provided a better potential technique for genotyping and identifying of red and green pigment genes. In addition, FSIUFM-PCR method will be useful in many fields, such as diagnosis of diseases, analysis of polymorphisms and quantitative assay.
Assuntos
Defeitos da Visão Cromática , Reação em Cadeia da Polimerase Multiplex , Defeitos da Visão Cromática/diagnóstico , Defeitos da Visão Cromática/genética , Eletroforese Capilar/métodos , Éxons/genética , Genótipo , Humanos , MasculinoRESUMO
Highly stable and facile one-pot copper nanoclusters (Cu NCs) coated with poly(allylamine hydrochloride) (PAH) have been synthesized for selectively sensing deferasirox (DFX) in ß-thalassemia plasma. DFX is an important drug used for treating iron overloading in ß-thalassemia, but needs to be monitored due to certain toxicity. In this study, the PAH-Cu NCs showed highly stable fluorescence with emission wavelengths at 450 nm. The DFX specifically interacted with the copper nanocluster to turn off the fluorescence of the PAH-Cu NCs, and could be selectively quantified through the fluorescence quenching effect. The linear range of DFX in plasma analyzed by PAH-Cu NCs was 1.0-100.0 µg/mL (r = 0.985). The relative standard deviation (RSD) and relative error (RE) were lower than 6.51% and 7.57%, respectively, showing excellent reproducibility of PAH-Cu NCs for sensing DFX in plasma. This method was also successfully applied for an analysis of three clinical plasma samples from ß-thalassemia patients taking DFX. The data presented high similarity with that obtained through a capillary electrophoresis method. According to the results, the PAH-Cu NCs could be used as a tool for clinically sensing DFX in human plasma for clinical surveys.
RESUMO
In this study, a simple fluorescent detection of survival motor neuron gene (SMN) in diagnosis of spinal muscular atrophy (SMA) based on nucleic acid amplification test and the poly-T luminescent copper nanoclusters (CuNCs) was established. SMA is a severely genetic diseases to cause infant death in clinical, and detection of SMN gene is a powerful tool for pre- and postnatal diagnosis of this disease. This study utilized the molecular inversion probe for recognition of nucleotide variant between SMN1 (c.840 C) and SMN2 (c.840 C > T) genes, and rolling circle amplification with a universal primer for production of poly-T single-strand DNA. Finally, the fluorescent CuNCs were formed on the poly-T single-strand DNA template with addition of CuSO4 and sodium ascorbate. The fluorescence of CuNCs was only detected in the samples with the presence of SMN1 gene controlling the disease of SMA. After optimization of experimental conditions, this highly efficient method was performed under 50 °C for DNA ligation temperature by using 2U Ampligase, 3 h for rolling circle amplification, and the formation of the CuNCs by mixing 500 µM Cu2+ and 4 mM sodium ascorbate. Additionally, this highly efficient method was successfully applied for 65 clinical DNA samples, including 4 SMA patients, 4 carriers and 57 wild individuals. This label-free detection strategy has the own potential to not only be a general method for detection of SMN1 gene in diagnosis of SMA disease, but also served as a tool for detection of other single nucleotide polymorphisms or nucleotide variants in genetic analysis through designing the different sensing probes.
Assuntos
Corantes Fluorescentes/química , Atrofia Muscular Espinal/diagnóstico por imagem , Técnicas de Amplificação de Ácido Nucleico , Compostos Organometálicos/química , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Cobre/química , Fluorescência , Corantes Fluorescentes/síntese química , Humanos , Atrofia Muscular Espinal/genética , Nanoestruturas/química , Compostos Organometálicos/síntese química , Tamanho da Partícula , Poli T/química , Propriedades de Superfície , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
The beta-adrenergic agonists (ß-agonists) working as repartitioning agents that make the carcass leaner and enhance the feeding efficiency in animals have been banned in the European Union, China and Taiwan. Here, traditional anionic surfactants, such as sodium dodecyl sulfate (SDS) were replaced with sodium di-(2-ethylhexyl)-sulfosuccinate (AOT) in field-amplified sample injection and sweeping-micellar electrokinetic chromatography (FASI-sweeping MEKC) for simultaneous analysis of eight ß-agonists in animal feeds. The AOT vesicles provided a better resolution of ß-agonists than micelles of SDS. The detection limits of the eight ß-agonists were above 5ng/mL by using this stacking capillary electrophoresis (CE) method. In comparison of traditional MEKC method (sample injection, 1psi for 5s), the stacking strategy provided 400-2000 fold sensitivity enhancement. After method validation, this method was successfully applied for analyzing four animal feeds, and none ß-agonist was detected. This strategy possessing good resolution of eight ß-agonists was suitable for serving as a tool for routine analysis of animal feeds.
Assuntos
Cromatografia Capilar Eletrocinética Micelar , Ração Animal , Animais , Micelas , Tensoativos , MagrezaRESUMO
X-inactive-specific transcript (XIST), a long non-coding RNA, is essential for the initiation of X-chromosome inactivation. However, little is known about other roles of XIST in the physiological process in eukaryotic cells. In this study, the bioinformatics approaches revealed XIST could be processed into a small non-coding RNA XPi2. The XPi2 RNA was confirmed by a northern blot assay; its expression was gender-independent, suggesting the role of XPi2 was beyond X-chromosome inactivation. The pull-down assay combined with LC-MS-MS identified two XPi2-associated proteins, nucleolin and hnRNP A1, connected to the formation of G-quadruplex. Moreover, the microarray data showed the knockdown of XPi2 down-regulated the KRAS pathway. Consistently, we tested the expression of ten genes, including KRAS, which was correlated with a G-quadruplex formation and found the knockdown of XPi2 caused a dramatic decrease in the transcription level of KRAS among the ten genes. The results of CD/NMR assay also supported the interaction of XPi2 and the polypurine-polypyrimidine element of KRAS. Accordingly, XPi2 may stimulate the KRAS expression by attenuating G-quadruplex formation. Our present work sheds light on the novel role of small RNA XPi2 in modulating the G-quadruplex formation which may play some essential roles in the KRAS- associated carcinogenesis.
Assuntos
Quadruplex G , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas p21(ras)/genética , RNA Longo não Codificante/genética , Pequeno RNA não Traduzido/genética , Inativação do Cromossomo X/genética , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica/métodos , Ribonucleoproteína Nuclear Heterogênea A1/genética , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Humanos , Células MCF-7 , Masculino , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Ligação Proteica , Interferência de RNA , RNA Longo não Codificante/metabolismo , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/genética , NucleolinaRESUMO
A field-amplified sample stacking-sweeping micellar electrokinetic chromatography with short-end injection was established for determination of deferasirox (DFX) in plasma. DFX was extracted from plasma and reconstituted with deionized water (lower conductivity solution). Capillary (effective length, 10cm) was filled with background electrolyte (40mM phosphate buffer, pH 4.5, containing 20% methanol). After sample loading from outlet end at 5psi for 15s, separation was carried out by applying high voltage at 15kV for 10min. Sodium dodecyl sulfate (SDS) was used to sweep DFX for enhancing sensitivity. The optimal CE separation conditions were 40mM phosphate buffer at pH 4.5 containing 100mM SDS and 20% methanol. The analysis time was about 3.5min for DFX. The calibration curve of DFX was ranged from 1 to 20µg/ml. The linearity (r) was more than 0.998. RSD and RE in intra- and inter-day assays were all below 12.14%. The limit of detection (LOD, S/N=3) for DFX was 0.3µg/ml. The sensitivity enhancement factor between sweeping-FASS MEKC and capillary zone electrophoresis is 3.3. Finally, the method was applied for determination of DFX in ß-thalassemia patients.
Assuntos
Benzoatos/sangue , Cromatografia Capilar Eletrocinética Micelar/métodos , Quelantes de Ferro/metabolismo , Triazóis/sangue , Adulto , Benzoatos/análise , Cromatografia Capilar Eletrocinética Micelar/normas , Deferasirox , Feminino , Humanos , Quelantes de Ferro/análise , Masculino , Triazóis/análise , Adulto JovemRESUMO
One CE method was established for detecting deferoxamine (DFO) and deferiprone (DFR) in plasma. For ß-thalassemia patients, DFO and DFR are major medicines to treat the iron overload caused by blood transfusion. Field-amplified sample injection combined with sweeping was used for sensitivity enhancement in CE. This method was performed on an uncoated fused-silica capillary. After liquid-liquid extraction, the plasma samples were electrokinetically injected into capillary at +10 kV for 180 s. The phosphate buffer (100 mM) containing 50 mM triethanolamine was used as the BGE (pH 6.6). Separation buffer was phosphate buffer (100 mM, pH 3.0) containing 150 mM SDS. This method showed good linearity (r ≥ 0.9960). Precision and accuracy were evaluated by the results of RSD and relative error of intrabatch and interbatch analyses, and all of the absolute values were less than 6.12%. The LODs (S/N = 3) were 200 ng/mL for DFO, and 25 ng/mL for DFR. The LOQ (S/N = 10) of DFO and DFR were 600 and 75 ng/mL, respectively. This method was applied for clinical applications of five ß-thalassemia patients.
Assuntos
Desferroxamina/sangue , Eletroforese Capilar/métodos , Piridonas/sangue , Talassemia beta/sangue , Deferiprona , Humanos , Limite de Detecção , Reprodutibilidade dos TestesRESUMO
Gold nanoparticles (AuNPs) are useful for diagnostic and biomedical applications, mainly because of their ease in preparation and conjugation, biocompatibility, and size-dependent optical properties. However, bare AuNPs do not possess specificity for targets. AuNPs conjugated with DNA aptamers offer specificity for various analytes, such as proteins and small molecules/ions. Although DNA aptamers themselves have therapeutic and target-recognizing properties, they are susceptible to degradation in vivo. When DNA aptamers are conjugated to AuNPs, their stability and cell uptake efficiency both increase, making aptamer-AuNPs suitable for biomedical applications. Additionally, drugs can be efficiently conjugated with DNA aptamer-AuNPs to further enhance their therapeutic efficiency. This review focuses on the applications of DNA aptamer-based AuNPs in several biomedical areas, including anticoagulation, anticancer, antibacterial, and antiviral applications.
Assuntos
DNA/química , Ouro/química , Nanopartículas Metálicas/química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Anticoagulantes/química , Anticoagulantes/farmacologia , Antineoplásicos/química , Antineoplásicos/toxicidade , Aptâmeros de Nucleotídeos/química , Transporte Biológico , Coagulação Sanguínea/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Portadores de Fármacos/química , HumanosRESUMO
Real applications in clinical diagnosis of label-free fluorescent copper nanoclusters (CuNCs) are demonstrated. Double-strand DNA (dsDNA) can act as an effective template for the formation of CuNCs, which can be used to distinguish the deletion or duplication genotypes of Duchenne muscular dystrophy (DMD) due to different fluorescent intensities. After PCR, the DMD amplicons reacted with copper ion by reduction of ascorbic acid and generated fluorescence. The exons of the DMD gene were taken as the model analytes for genetic diagnosis. In this sensing system, the deletion type does not show fluorescence; on the other hand, the duplication type emits higher fluorescence than normal type. Parameters of this sensing system were optimized, including PCR conditions, levels of copper ion and ascorbate, and reaction time. The DMD-dominated exons 45, 46, and 47 were detected, and the method was applied to six samples of DMD patients. The results were consistent with those of the multiplex ligation-dependent probe amplification method. This strategy was feasible to detect all exons of this disease.
Assuntos
Cobre/química , Corantes Fluorescentes/química , Deleção de Genes , Duplicação Gênica , Genótipo , Nanopartículas Metálicas/química , Distrofia Muscular de Duchenne/genética , Técnicas Biossensoriais , DNA/genética , Éxons , Humanos , Distrofia Muscular de Duchenne/diagnóstico , Tamanho da Partícula , Espectrometria de FluorescênciaRESUMO
For the first time, structural information regarding the role of simvastatin in bone anabolism is described, and a bone-specific statin is introduced. Polyaspartate-conjugated simvastatin was synthesized by solid-phase synthesis with the assistance of microwave irradiation. It displays significant bone targeting and bone formation with less toxicity than simvastatin.
Assuntos
Acil Coenzima A/metabolismo , Anabolizantes , Inibidores de Hidroximetilglutaril-CoA Redutases , Sinvastatina , Anabolizantes/síntese química , Anabolizantes/química , Anabolizantes/farmacologia , Animais , Modelos Animais de Doenças , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/síntese química , Inibidores de Hidroximetilglutaril-CoA Redutases/química , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Fígado/metabolismo , Estrutura Molecular , Osteogênese/efeitos dos fármacos , Sinvastatina/análogos & derivados , Sinvastatina/síntese química , Sinvastatina/química , Sinvastatina/farmacologia , Técnicas de Síntese em Fase Sólida , Peixe-ZebraRESUMO
One rapid CE method was established to diagnose Duchenne muscular dystrophy (DMD). DMD is a severe recessive inherited disorder frequently caused by gene deletions. Among them, exons 1-20 account for nearly 30% of occurrences. In this study, the universal multiplex PCR was used to enhance the fluorescently labeling efficiency, which was performed only by one universal fluorescent primer. After PCR, a short-end injection CE (short-end CE) speeded up the genotyping of the DMD gene. This method involved no extra purification, and was completed within 9 min. The CE conditions contained a polymer solution of 1.5% hydroxylethylcellulose in 1× TBE buffer at 6 kV for separation. This method was applied to test six DMD patients and one healthy male person. The results showed good agreement with those of multiplex ligation-dependent probe amplification. This method can be applied for clinical diagnosis of DMD disease. Accurate diagnosis of the DMD gene is the best way to prevent the disease.
Assuntos
Eletroforese Capilar/métodos , Técnicas de Genotipagem/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Distrofia Muscular de Duchenne/genética , Éxons , Humanos , MasculinoRESUMO
An inverse-shifting polymerase chain reaction (IS-PCR) combined with short-end capillary gel electrophoresis (CGE) was developed for genotyping of intron 22 inversion Type 1 (Inv22-1) and Type 2 (Inv22-2) of hemophilia A (HA). Severe HA cases are affected by intron 22 inversion around 45-50%. Inv22-1 has higher frequency than Inv22-2. The aim of this study is to distinguish them by genotyping. In order to improve Inv22 genotyping efficiency, five primers were designed and applied to differentiate the wild type, Inv22-1, Inv22-2 and carrier. Three amplicons of 405, 457 and 512 bp were recognized for wild type; 333, 457 and 584 bp for Inv22-1; 385, 405 and 584 bp for Inv22-2. The Inv22-1 carrier has 5 amplicons including 333, 405, 457, 512, 584 bp and Inv22-2 carrier is differentiated by 385, 405, 457, 512 and 584 bp. The amplicons between Inv22-1 and Inv22-2 carriers are only different in 333 bp for Inv22-1 carrier and 385 bp for Inv22-2 carrier. Capillary gel electrophoresis (CGE) was used for separation within 5 min. The separation voltage was set at 8 kV (cathode at detector), and the temperature was kept at 25°C. The sieving matrix was 89 mM Tris, 89 mM boric acid, 2mM EDTA containing 0.4% (w/v) HPMC and 1 µM of YO-PRO(®)-1 Iodide. Total of 50 HA patients (including 35 non-Inv22, 14 Inv22-1, and one Inv22-2 patients) and 7 HA carriers were diagnosed in the application. Seven random samples (5 patients and 2 carriers) were subjected to comparison and gave identical results of DNA sequencing and this modified IS-PCR.
Assuntos
Inversão Cromossômica/classificação , Inversão Cromossômica/genética , Eletroforese Capilar/métodos , Hemofilia A/genética , Íntrons/genética , Reação em Cadeia da Polimerase/métodos , Estudos de Casos e Controles , Rearranjo Gênico , Genótipo , HumanosRESUMO
This is the first capillary electrophoresis (CE) analysis for diagnosis of hemophilia A (HA). The intron 22 inversion of factor VIII gene (F8) causes 40-50 % of severe bleeding disorder of HA in all human populations. Consequently, identification of the disease-causing mutations is becoming increasingly important for accurate genetic counseling and prenatal diagnosis. In this study, the key steps of inverse-shifting polymerase chain reaction (IS-PCR) and of short-end injection capillary electrophoresis were used for more specific and rapid genotyping of intron 22 inversion of F8. In IS-PCR, three specific primers were used to amplify 512-bp amplicon for wild type and 584-bp amplicon for patients with intron 22 inversion. The capillary gel electrophoresis (CGE) system was performed using 1× Tris-borate-EDTA (TBE) buffer containing 0.3 % (w/v) polyethylene oxide (PEO). The PCR amplicons were electrokinetically injected at 10 kV for 10 s at a temperature of 25 °C. The optimal short-end injection CGE was applied to detect the F8 gene of HA patients and carriers within 5 min. Intron 22 inversion was indeed found on some HA patients (13/35, 37.1 %). All genotyping results showed good agreement with DNA sequencing method and long-distance polymerase chain reaction (LD-PCR). The IS-PCR combined with short-end injection CGE method was feasible and efficient for intron 22 inversion screening of F8 in the HA populations.
Assuntos
Eletroforese Capilar , Fator VIII/genética , Genótipo , Hemofilia A/genética , Íntrons , Reação em Cadeia da Polimerase , Soluções Tampão , DNA/química , Análise Mutacional de DNA , Ácido Edético/química , Feminino , Hemofilia A/diagnóstico , Heterozigoto , Humanos , Masculino , Mutação , Polietilenoglicóis/química , Polímeros/química , Manejo de EspécimesRESUMO
This is the first ligase chain reaction used for diagnosis of spinal muscular atrophy (SMA). Universal fluorescent tri-probe ligation (UFTPL), a novel strategy used for distinguishing the multi-nucleotide alternations at single base, is developed to quantitatively analyze the SMN1/SMN2 genes in diagnosis of SMA. Ligase chain reaction was performed by adding three probes including universal fluorescent probe, connecting probe and recognizing probe to differentiate single nucleotide polymorphisms in UFTPL. Our approach was based on the two UFTPL products of survival motor neuron 1 (SMN1) and SMN2 genes (the difference of 9 mer) and analyzed by capillary electrophoresis (CE). We successfully determined various gene dosages of SMN1 and SMN2 genes in homologous or heterologous subjects. By using the UFTPL-CE method, the SMN1 and SMN2 genes were fully resolved with the resolution of 2.16±0.37 (n=3). The r values of SMN1 and SMN2 regression curves over a range of 1-4 copies were above 0.9944. Of the 48 DNA samples, the data of gene dosages were corresponding to that analyzed by conformation sensitive CE and denatured high-performance liquid chromatography (DHPLC). This technique was found to be a good methodology for quantification or determination of the relative genes having multi-nucleotide variants at single base.
Assuntos
Eletroforese Capilar/métodos , Corantes Fluorescentes/química , Atrofia Muscular Espinal/diagnóstico , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Sequência de Bases , Primers do DNA , Humanos , Atrofia Muscular Espinal/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genéticaRESUMO
A "turn on/off" biosensor for diagnosis of exon 7 of the SMN1 gene was developed by employing a "scorpion primer". This scorpion primer was based on the principle of fluorescence resonance energy transfer using a fluorophore, a blocker and a quencher. It was successfully applied to detect 10 volunteer samples, and not only to in vitro testing.
Assuntos
Técnicas Biossensoriais , Atrofia Muscular Espinal/diagnóstico , Atrofia Muscular Espinal/genética , Mutação Puntual , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Éxons/genética , Transferência Ressonante de Energia de Fluorescência , Humanos , Reação em Cadeia da Polimerase , Sensibilidade e EspecificidadeRESUMO
Microemulsion electrokinetic chromatography (MEEKC) is proposed for analysis of di-n-butyl phthalate (DBP) and di-(2-ethylhexyl) phthalate (DEHP) in soft drinks. However, the instability of microemulsion is a critical issue. In this research, a novel material, Pluronic® F-127, which has the properties of polymer and surfactant, was added for stabilizing the microemulsion in the MEEKC system. Our data demonstrate that the presence of Pluronic® F-127 (0.05-0.30%) also helps enhance resolution of highly hydrophobic compounds, DBP and DEHP. The electrokinetic injection of sodium dodecyl sulphate (SDS) including sample (-10 kV, 20 s) was introduced in this MEEKC system and this yielded about 25-fold sensitivity enhancement compared with hydrodynamic injection (1 psi, 10 s). During method validation, calibration curves were linear (r≥0.99), within a range of 75-500 ng/mL for DBP and 150-1000 ng/mL for DEHP. As the precision and accuracy assays, absolute values of relative standard deviation (RSD) and relative error (RE) in intraday (n=3) and interday (n=5) observations were less than 4.93%. This method was further applied for analyzing six commercial soft drinks and one was found containing 453.67 ng/mL of DEHP. This method is considered feasible for serving as a tool for analysis of highly hydrophobic molecules.