RESUMEN
OBJECTIVES: The usefulness of cases diagnosed in administrative registers for research purposes is dependent on diagnostic validity. This study aimed to investigate the validity and inter-rater reliability of recorded diagnoses of tic disorders and obsessive-compulsive disorder (OCD) in the Swedish National Patient Register (NPR). DESIGN: Chart review of randomly selected register cases and controls. METHOD: 100 tic disorder cases and 100 OCD cases were randomly selected from the NPR based on codes from the International Classification of Diseases (ICD) 8th, 9th and 10th editions, together with 50 epilepsy and 50 depression control cases. The obtained psychiatric records were blindly assessed by 2 senior psychiatrists according to the criteria of the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision (DSM-IV-TR) and ICD-10. PRIMARY OUTCOME MEASUREMENT: Positive predictive value (PPV; cases diagnosed correctly divided by the sum of true positives and false positives). RESULTS: Between 1969 and 2009, the NPR included 7286 tic disorder and 24,757 OCD cases. The vast majority (91.3% of tic cases and 80.1% of OCD cases) are coded with the most recent ICD version (ICD-10). For tic disorders, the PPV was high across all ICD versions (PPV=89% in ICD-8, 86% in ICD-9 and 97% in ICD-10). For OCD, only ICD-10 codes had high validity (PPV=91-96%). None of the epilepsy or depression control cases were wrongly diagnosed as having tic disorders or OCD, respectively. Inter-rater reliability was outstanding for both tic disorders (κ=1) and OCD (κ=0.98). CONCLUSIONS: The validity and reliability of ICD codes for tic disorders and OCD in the Swedish NPR is generally high. We propose simple algorithms to further increase the confidence in the validity of these codes for epidemiological research.
Asunto(s)
Trastorno Obsesivo Compulsivo/diagnóstico , Trastornos de Tic/diagnóstico , Adolescente , Adulto , Estudios de Casos y Controles , Manual Diagnóstico y Estadístico de los Trastornos Mentales , Femenino , Humanos , Incidencia , Clasificación Internacional de Enfermedades , Masculino , Variaciones Dependientes del Observador , Trastorno Obsesivo Compulsivo/epidemiología , Sistema de Registros , Reproducibilidad de los Resultados , Suecia/epidemiología , Trastornos de Tic/epidemiologíaRESUMEN
Direct detection of PCR product via hybridisation assay, would facilitate the development of rapid tools for genetic analysis. Here, a PCR primer designed to generate a PCR amplicon tagged with single stranded DNA tails at each end of the duplex, which can be used for direct hybridisation with a surface immobilised probe and an enzyme labelled reporter probe is presented. Four modified sequence specific primers (SSP) pairs were designed for the selective amplification of coeliac disease associated alleles (DQA1*05, DQB1*02, DQB1*03:02 alleles), and human growth hormone (positive control). Multiplex PCR products were electrochemically detected in less than 5 min at 37 °C via direct hybridisation to short probes immobilised on individual electrodes of a genosensor array, and subsequent hybridisation to an enzyme labelled reporter probe. The developed electrochemical genosensor array exploiting the modified primers for the direct detection of PCR products was applied to the genotyping of real patient samples.
Asunto(s)
Enfermedad Celíaca/genética , Enfermedad Celíaca/inmunología , Técnicas Electroquímicas/métodos , Antígenos HLA/genética , Alelos , Secuencia de Bases , Técnicas Biosensibles , Cartilla de ADN/genética , Técnicas de Genotipaje , Humanos , Dispositivos Laboratorio en un Chip , Reacción en Cadena de la Polimerasa MultiplexRESUMEN
Celiac disease is an auto-immune disorder induced by ingestion of gluten in genetically predisposed individuals. Its diagnostics is more accurate using a combination of immunologic and genetic tests to detect of high levels of certain auto-antibodies and the presence human leukocyte antigen HLA-DQ2 or HLA-DQ8 genetic markers. In this work, we report the design and testing of automated microsystems combining sample treatment, storage, fluidic transport, and detection in a single platform able to carry out genetic or serologic analysis for detection of celiac disease markers. These microsystems share a common footprint and many fluidic features and are thus able to perform a complete assay. The microsystem for the genetic assay extracts and amplifies the DNA prior to detection, while the serology microsystem contains a filter and chamber for the generation and subsequent dilution of plasma. The performance of both platforms is demonstrated and compared with reference methods with an excellent correlation, which makes the developed platform amenable for clinical studies.
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Biomarcadores/sangre , Enfermedad Celíaca/sangre , Técnicas Electroquímicas/instrumentación , Técnicas Genéticas/instrumentación , Inmunoensayo/instrumentación , Dispositivos Laboratorio en un Chip , Autoanticuerpos/sangre , Enfermedad Celíaca/genética , Técnicas Electroquímicas/métodos , Diseño de Equipo , HumanosRESUMEN
Coeliac disease is a small intestinal disorder, induced by ingestion of gluten in genetically predisposed individuals. Coeliac disease has been strongly linked to human leukocyte antigens (HLA) located on chromosome 6, with almost 100 % of coeliac disease sufferers carrying either a HLA-DQ2 or HLA-DQ8 heterodimer, with the majority carrying HLA-DQ2 encoded by the DQA1*05:01/05:05, DQB1*02:01/02:02 alleles, whereas the remaining carry the HLA-DQ8 encoded by the DQA1*03:01, DQB1*03:02 alleles. In this work, we present the development of a multiplex electrochemical genosensor array of 36 electrodes, housed within a dedicated microfluidic platform and using a total of 10 sequence-specific probes for rapid medium-high resolution HLA-DQ2/DQ8 genotyping. An evaluation of the selectivity of the designed probes was carried out with the target sequences and 44 potentially interfering alleles, including single base mismatch differentiations; good selectivity was demonstrated. The performance of the electrochemical genosensor array was validated, analyzing real human samples for the presence of HLA-DQ2/DQ8 alleles, and compared with those obtained using laboratory-based HLA typing, and an excellent correlation was obtained.
Asunto(s)
Enfermedad Celíaca/diagnóstico , Enfermedad Celíaca/genética , Técnicas Electroquímicas/métodos , Antígenos HLA-DQ/genética , Análisis por Micromatrices/métodos , Alelos , Susceptibilidad a Enfermedades , Genotipo , Antígenos HLA-DQ/análisis , HumanosRESUMEN
Coeliac disease is an inflammation of the small intestine, occurring in genetically susceptible individuals triggered by the ingestion of gluten. Human Leukocyte Antigens (HLA) DQ2 and DQ8 gene have been identified as key genetic factors in coeliac disease as they are presented in almost 100 % of the patients. These genes are encoded by the combination of certain alleles in the DQA and DQB region of chromosome 6. Specifically, DQA1*05:01 and DQB1*02:01 alleles for serologically defined leukocyte antigen DQ2 cis, DQA1*05:05 and DQB1*02:02 for DQ2 trans and DQA1*03:01 and DQB1*03:02 alleles for the DQ8. Specific identification of these alleles is a challenge due to the high number of alleles that have been identified so far: 46 in the DQA region and 160 in the DQB region (as of IMGT/HLA Database 10/2011 release). In the reported work, the development of a multiplex colorimetric assay for the low to medium HLA typing of the DQ2 and DQ8 genes is presented. The optimisation of probe design and assay conditions, performed by both surface plasmon resonance and enzyme-linked oligonucleotide assay, are reported. Finally, the performances of the developed typing platform were validated by the analysis of real patient samples and HLA typing, compared with those obtained using hospital based typing technology and an excellent correlation obtained.
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Enfermedad Celíaca/genética , Técnicas de Genotipaje/métodos , Antígenos HLA-DQ/genética , Alelos , Secuencia de Bases , Colorimetría/métodos , Sondas de ADN/genética , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Hibridación de Ácido Nucleico/métodos , Sensibilidad y Especificidad , Resonancia por Plasmón de SuperficieRESUMEN
Post-transcriptional regulation via the 3' untranslated region (3' UTR) of mRNA is an important factor in governing eukaryotic gene expression. Achieving detailed understanding of these processes requires highly quantitative systems in which comparative studies can be performed. To this end, we have developed a plasmid reporter system for Saccharomyces cerevisiae, in which the 3' UTR can be easily replaced and modified. Accurate quantification of the tandem affinity purification tag (TAP)-reporter protein and of TAP-mRNA is achieved by immuno-QPCR and by RT-QPCR, respectively. We have used our reporter system to evaluate the consequences on gene expression from varying the 3' UTR, a problem often encountered during C-terminal tagging of proteins. It was clear that the choice of 3' UTR was a strong determinant of the reporter expression, in a manner dependent on the growth conditions used. Mutations affecting either decapping (lsm1Delta) or deadenylation (pop2Delta) were also found to affect reporter gene expression in a highly 3' UTR-dependent manner. Our results using this set-up clearly indicate that the common strategy used for C-terminal tagging, with concomitant replacement of the native 3' UTR, will very likely provide incorrect conclusions on gene expression.
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Regiones no Traducidas 3' , Regulación de la Expresión Génica , Biología Molecular/métodos , Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero , Saccharomyces cerevisiae/fisiología , Vectores Genéticos , PlásmidosRESUMEN
Laser-assisted microdissection has mainly been used in cancer studies to excise pure cell populations from heterogeneous tissues. Cancer and normal cells selected by laser-assisted microdissection have frequently been used for mRNA expression studies usually by reverse transcriptase-quantitative polymerase chain reaction (qPCR). Recently, real time immuno-qPCR was developed as a new tool for highly sensitive measurements of proteins. Using reverse transcriptase-qPCR and immuno-qPCR, we measured the amounts of prostate-specific antigen mRNA and its corresponding protein in homogeneous and comparable cell populations, collected from normal and cancer prostates by laser-assisted microdissection. With these techniques, prostate-specific antigen mRNA and protein were quantified over a wide range of concentrations with a sensitivity sufficient to analyze single prostate cells (LNCaP). We did not find significant differences in prostate-specific antigen protein and mRNA between normal and cancer cells. The expression of prostate-specific antigen protein and mRNA was highly correlated in both normal and pathological cells. In microdissected peritubular stromal areas of prostate cancers, the concentration of prostate-specific antigen protein was about 100 times higher than in normal prostate, indicating an increased transit of secreted prostate-specific antigen. In the same samples, prostate-specific antigen mRNA was not detectable. Our data demonstrate, for the first time, the feasibility of simultaneous application of reverse transcriptase-qPCR and immuno-qPCR in studies of homogeneous cell populations, collected by laser-assisted microdissection. The approach is expected to become a very powerful tool for expression studies in human cancers at both mRNA and protein levels.
Asunto(s)
Adenocarcinoma/genética , Regulación Neoplásica de la Expresión Génica , Antígeno Prostático Específico/genética , Antígeno Prostático Específico/metabolismo , Neoplasias de la Próstata/genética , ARN Mensajero/metabolismo , Adenocarcinoma/metabolismo , Adenocarcinoma/patología , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Humanos , Rayos Láser , Masculino , Microdisección , Reacción en Cadena de la Polimerasa/métodos , Próstata/metabolismo , Próstata/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , ARN Neoplásico/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The tandem affinity purification (TAP)-tag has rapidly gained a wide popularity, mostly in studies on protein interactions, but lately also in large-scale protein quantification studies. We have developed an immuno-quantitative real-time PCR (qPCR) method to achieve rapid, sensitive and accurate quantification of TAP-tagged (and protein A-tagged) proteins in yeast with a detection range between 10(7) and 10(10) molecules. The immuno-qPCR protein quantification showed an excellent correlation to the published in vivo fluorescent protein (GFP)-based large-scale protein quantifications, but allowed for a much higher sensitivity. The correlation with published data from the large-scale Western blotting-based quantification of the TAP-tag was lower, but the sensitivity of detection was on roughly the same level. The practical use of the immuno-qPCR approach was demonstrated by analysis of osmo-regulated proteins, where the 2000-fold increase in expression of Catalase (Ctt1p), from an extremely low basal expression, could be accurately quantified. All steps of the method, from cell growth, to protein extraction and determination and the immuno-qPCR reaction itself are potentially amenable to automatization. Therefore, since the TAP-tag and protein A are useful in most model organisms, the immuno-qPCR method is both generic and suitable for large-scale studies.
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Cromatografía de Afinidad/métodos , Reacción en Cadena de la Polimerasa/métodos , Proteínas de Saccharomyces cerevisiae/análisis , Saccharomyces cerevisiae/química , Western Blotting , Medios de Cultivo , Genes Fúngicos , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Estándares de Referencia , Saccharomyces cerevisiae/genéticaRESUMEN
Currently, in real-time PCR, one often has to choose between using a sequence-specific probe and a nonspecific double-stranded DNA (dsDNA) binding dye for the detection of amplified DNA products. The sequence-specific probe has the advantage that it only detects the targeted product, while the nonspecific dye has the advantage that melting curve analysis can be performed after completed amplification, which reveals what kind of products have been formed. Here we present a new strategy based on combining a sequence-specific probe and a nonspecific dye, BOXTO, in the same reaction, to take the advantage of both chemistries. We show that BOXTO can be used together with both TaqMan probes and locked nucleic acid (LNA) probes without interfering with the PCR. The probe signal reflect formation of target product, while melting curve analysis of the BOXTO signal reveals primer-dimer formation and the presence of any other anomalous products.
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Sondas de ADN/genética , ADN/análisis , ADN/genética , Colorantes Fluorescentes , Hibridación Fluorescente in Situ/métodos , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodos , Microscopía Fluorescente/métodos , Desnaturalización de Ácido Nucleico , Temperatura de TransiciónRESUMEN
The scientific, medical, and diagnostic communities have been presented the most powerful tool for quantitative nucleic acids analysis: real-time PCR [Bustin, S.A., 2004. A-Z of Quantitative PCR. IUL Press, San Diego, CA]. This new technique is a refinement of the original Polymerase Chain Reaction (PCR) developed by Kary Mullis and coworkers in the mid 80:ies [Saiki, R.K., et al., 1985. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350], for which Kary Mullis was awarded the 1993 year's Nobel prize in Chemistry. By PCR essentially any nucleic acid sequence present in a complex sample can be amplified in a cyclic process to generate a large number of identical copies that can readily be analyzed. This made it possible, for example, to manipulate DNA for cloning purposes, genetic engineering, and sequencing. But as an analytical technique the original PCR method had some serious limitations. By first amplifying the DNA sequence and then analyzing the product, quantification was exceedingly difficult since the PCR gave rise to essentially the same amount of product independently of the initial amount of DNA template molecules that were present. This limitation was resolved in 1992 by the development of real-time PCR by Higuchi et al. [Higuchi, R., Dollinger, G., Walsh, P.S., Griffith, R., 1992. Simultaneous amplification and detection of specific DNA-sequences. Bio-Technology 10(4), 413-417]. In real-time PCR the amount of product formed is monitored during the course of the reaction by monitoring the fluorescence of dyes or probes introduced into the reaction that is proportional to the amount of product formed, and the number of amplification cycles required to obtain a particular amount of DNA molecules is registered. Assuming a certain amplification efficiency, which typically is close to a doubling of the number of molecules per amplification cycle, it is possible to calculate the number of DNA molecules of the amplified sequence that were initially present in the sample. With the highly efficient detection chemistries, sensitive instrumentation, and optimized assays that are available today the number of DNA molecules of a particular sequence in a complex sample can be determined with unprecedented accuracy and sensitivity sufficient to detect a single molecule. Typical uses of real-time PCR include pathogen detection, gene expression analysis, single nucleotide polymorphism (SNP) analysis, analysis of chromosome aberrations, and most recently also protein detection by real-time immuno PCR.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Reacción en Cadena de la Polimerasa/métodos , Programas Informáticos , Animales , Sistemas de Computación , Femenino , Colorantes Fluorescentes/química , Amplificación de Genes , Expresión Génica , Reacción en Cadena de la Polimerasa/instrumentación , Sensibilidad y Especificidad , Xenopus laevisRESUMEN
Real-time PCR is a very sensitive technique to measure DNA concentrations. In real-time immuno-PCR, it is used as the detection system for quantification of proteins. Many ways to perform and assemble real-time immuno-PCR are possible. We have tested three different approaches for the detection of prostate specific antigen (PSA) and compared them with each other and with ELISA. We also demonstrate the applicability of real-time immuno-PCR to classify clinical PSA samples. Assemblage I is performed stepwise attaching the capture antibody to the vessel surface by adsorption and having the DNA-label linked to the detection antibody through biotin and streptavidin. In assemblage II, capture antibody is also adsorbed to the vessel surface but the detection antibody is pre-conjugated to the DNA-label. Assemblage III uses the pre-conjugated detection antibody/DNA-label but binds the capture antibody through biotin to surface immobilized streptavidin. We found assemblage II to be the most sensitive, with a detection limit of 4.8 x 10(5) PSA molecules. This can be compared to the detection limit of the ELISA, which is 5.7 x 10(7) molecules. Assemblage III was the most reproducible with a standard deviation (SD) of 0.21 cycles, while assemblage I was the least reproducible (SD=0.45 cycles). The SD of assemblage II was 0.25 cycles. We conclude that using pre-conjugated detection antibody/DNA-label enhances both the sensitivity and the reproducibility of real-time immuno-PCR. Measurements of PSA in serum samples using real-time immuno-PCR correlated well with measurements performed with ELISA. The real-time immuno-PCR measurements were more sensitive and the dynamic range was larger than with the ELISA.