Production and evaluation of egg derived hot start antibodies

BACKGROUND: Hot start can greatly improve specificity, sensitivity and yield of PCR. Non-specific amplification can occur in PCR when reaction mixture is prepared at room temperature, because Taq DNA polymerase is active and the primers can hybridize non-specifically. Hot start Taq DNA polymerases remain inactive at room temperature and are activated after heating at 95°C preventing non-specific amplification. Monoclonal antibodies against Taq DNA polymerase is the first line of reagents used for turn on regular Taq DNA polymerase into Hot start one. The goal of this research was to produce and evaluate Hot Start antibodies derived from chicken eggs. RESULTS: We performed affinity purification of yolk immunoglobulin (IgY) and obtained polyclonal Hot Start antibodies. The yield of specific antibodies was 0.36 mg per egg or 0.2% of total yolk antibodies. The protocol for real time measurement and Hot start IgY activity assessment was developed. We found that Hot start IgY can reversibly block Taq DNA polymerase activity at 50°C and have no negative impact neither on the Taq DNA polymerase activity after denaturation nor on the reverse transcriptase. We estimated that 1.0 µg of Hot start IgY effectively blocks 5 U activity of Taq DNA polymerase. CONCLUSIONS: Egg derived Hot Start polyclonal antibodies are the cheapest source of Hot start antibodies, from one immune egg we can isolate 0.36 mg IgY, this quantity is enough for producing 1800 U activity of Hot start Taq DNA Polymerase.

A simple and efficient method for extraction of Taq DNA polymerase

Background Thermostable DNA polymerase (Taq Pol ?) from Thermus aquaticus has been widely used in PCR, which was usually extracted with Pluthero's method. The method used ammonium sulfate to precipitate the enzyme, and it saved effort and money but not time. Moreover, we found that 30-40% activity of Taq Pol I was lost at the ammonium sulfate precipitation step, and the product contained a small amount of DNA. Results We provided a novel, simplified and low-cost method to purify the Taq Pol ? after overproduction of the enzyme in Escherichia coli, which used ethanol instead of ammonium sulfate to precipitate the enzyme. The precipitate can be directly dissolved in the storage buffer without dialysis. In addition, DNA and RNA contamination was removed with DNase I and RNase A before precipitation, and the extraction procedure was optimized. Our improvements increase recovery rate and specific activity of the enzyme, and save labor, time, and cost. Conclusions Our method uses ethanol, DNase I, and RNase A to purify the Taq Pol ?, and simplifies the operation, and increases the enzyme recovery rate and quality.

Association study between the Taq1A (rs1800497) polymorphism and schizophrenia in a Brazilian sample / Estudo de associação entre o polimorfismo genético Taq1A (rs1800497) e esquizofrenia em uma amostra brasileira

Schizophrenia is a severe psychotic disorder with recurrent relapse and functional impairment. It results from a poorly understood gene-environment interaction. The Taq1A polymorphism (located in the gene cluster NTAD) is a likely candidate for schizophrenia. Its rs1800497 polymorphism was shown to be associated with DRD2 gene expression. Therefore the present work aims to investigate a possible association between schizophrenia and such polymorphism. The compared distribution of the alleles and genotypes of the studied polymorphism was investigated in a Brazilian sample of 235 patients and 834 controls. Genotypic frequencies were in Hardy-Weinberg equilibrium. There was a trend of allelic association between the Taq1A polymorphism (rs1800497) with schizophrenia in the studied sample. However no statistically differences were found between cases and controls when analyzed by gender or schizophrenia subtypes.

A esquizofrenia é um grave transtorno psicótico que apresenta frequentes recaídas e incapacitação progressiva. Resulta de uma interação gene-ambiente ainda pouco compreendida. O polimorfismo Taq1A (localizado no grupamento genético NTAD) é considerado um possível candidato para esquizofrenia. O polimorfismo genético rs1800497 foi associado com alteração da expressão do gene do DRD2. Assim, o presente trabalho objetivou investigar a possível associação de tal polimorfismo com esquizofrenia. A distribuição de seus alelos e genótipos foi investigada em uma amostra brasileira composta de 235 pacientes e 834 controles. As frequências genotípicas estavam em equilíbrio de Hardy-Weinberg. Houve uma tendência de associação alélica entre o polimorfismo Taq1A (rs1800497) e esquizofrenia na amostra estudada. No entanto, não houve diferenças estatisticamente significantes entre os grupos de casos e controles, quando analisados por gênero e subtipos da esquizofrenia.

Analysis of real time PCR amplification efficiencies from three genomic region of dengue virus / Análisis de las eficiencias de amplificación por PCR en tiempo real de tres regiones genómicas del virus dengue

Early diagnosis of dengue virus (DENV) infection represents a key factor in preventing clinical complications attributed to the disease. The aim of this study was to evaluate the amplification efficiencies of an in-house quantitative real time-PCR (qPCR) assay of DENV, using the non-structural conserved genomic region protein-5 (NS5) versus two genomic regions usually employed for virus detection, the capsid/pre-membrane region (C-prM) and the 3’-noncoding region (3’NC). One-hundred sixty seven acute phase serum samples from febrile patients were used for validation purposes. Results showed that the three genomic regions had similar amplification profiles and correlation coefficients (0.987-0.999). When isolated viruses were used, the NS5 region had the highest qPCR efficiencies for the four serotypes (98-100%). Amplification from acute serum samples showed that 41.1% (67/167) were positive for the universal assay by at least two of the selected genomic regions. The agreement rates between NS5/C-prM and NS5/3’NC regions were 56.7% and 97%, respectively. Amplification concordance values between C-prM/NS5 and NS5/3’NC regions showed a weak (k= 0.109; CI 95%) and a moderate (k= 0.489; CI 95%) efficiencies in amplification, respectively. Serotyping assay using a singleplex NS5-TaqMan® format was much more sensitive than the C-prM/SYBR Green® I protocol (76%). External evaluation showed a high sensitivity (100%), specificity (78%) and high agreement between the assays. According to the results, the NS5 genomic region provides the best genomic region for optimal detection and typification of DENV in clinical samples.

El diagnóstico precoz de la infección por el virus dengue (DENV) constituye un elemento clave para la prevención de las complicaciones clínicas propias de la enfermedad. El objetivo del estudio fue evaluar la detección de DENV mediante un ensayo cuantitativo de PCR-tiempo real (qPCR), desarrollado localmente, utilizando la región no-estructural-5 (NS5), versus dos regiones tradicionalmente empleadas para la detección del virus, la región cápside/pre-membrana (C-prM), y la región noncodificante-3’ (3’NC). Se recolectaron 167 muestras de suero de pacientes en fase aguda de la enfermedad. Las tres regiones génicas tuvieron perfiles de amplificación/coeficientes de correlación similares (0,987-0,999). Sin embargo, la región NS5 tuvo la eficiencia de amplificación más elevada para los cuatro serotipos (98-100%). Durante el proceso de validación, 41,1% (67/167) de las muestras de suero resultaron positivas para DENV al menos por dos de las regiones genómicas empleadas. Los valores de concordancia entre las regiones NS5/C-prM y NS5/3’NC fueron de 56,7% y 97%, respectivamente. La concordancia fue débil entre las regiones NS5/C-prM (k= 0,109; CI 95%), sin embargo, fue moderada entre las regiones NS5/3’NC (k= 0,489; CI 95%). El ensayo de tipificación uniplex en formato NS5/TaqMan® mostró alta sensibilidad (100%) que el protocolo C-prM/SYBRGreen®-I (76%). La validación externa del ensayo mostró una alta sensibilidad (100%), especificidad (78%) y acuerdo alto entre los ensayos utilizados. De acuerdo a los resultados obtenidos, la región NS5 ofrece la mayor opción para la detección y serotipificación del DENV en muestras clínicas.

TGFA/Taq I polymorphism and environmental factors in non-syndromic oral clefts in Southern Brazil

We report a study of TGFA/ Taq I polymorphisms and environmental factors in non-syndromic oral cleft in Southern Brazil. Nonsyndromic cleft case-parent triads were recruited to participate. Clinical data was collected with an emphasis on tobacco and alcohol use during pregnancy. DNA was extracted from peripheral blood and TGFA/ Taq I polymorphisms were analyzed by PCR/RFLP with Taq I restriction enzyme. Association of clefts and TGFA/ Taq I polymorphisms was determined using a transmission disequilibrium test (TDT). Association of environmental factors, clefts, and genotypes was evaluated with Fisher's exact test. The minor allele frequency was 0.064. We found no evidence of association between TGFA/ Taq I polymorphisms and clefting (TDT p = 0.335). We also found no association between TGFA/ TaqI polymorphisms and environmental factors (alcohol and/or tobacco). Therefore, no evidence was found that TGFA/ Taq I polymorphisms play a role in clefting in this population. No evidence was found that tobacco or alcohol exposure during pregnancy was related to clefting, however a larger sample size is needed to confirm these results.

Relación del polimorfismo TaqI del gen del receptor de la vitamina D con la lepra lepromatosa en población mexicana / Association between the TaqI polymorphism of Vitamin D Receptor gene and lepromatous leprosy in a Mexican population sample

OBJETIVO: Determinar la relación del polimorfismo TaqI del gen del receptor de la vitamina D (RVD) con la lepra lepromatosa (LL) en individuos originarios de Sinaloa, México. MATERIAL Y MÉTODOS: Se amplificó un fragmento de 740 pb del gen RVD en muestras de ADN de 71 pacientes con LL y 144 controles en el Hospital General de Culiacán durante el periodo 2004-2007. El polimorfismo se identificó mediante la endonucleasa TaqI. RESULTADOS: Se observó un aumento de relevancia estadística del genotipo TT en pacientes con LL en comparación con los controles (p= 0.040; RM= 1.82). CONCLUSIÓN: Se demuestra un nexo entre el genotipo TT y la susceptibilidad a la LL.

OBJETIVE: To establish the association of the vitamin D receptor gene TaqI polymorphism with lepromatous leprosy (LL) in individuals from Sinaloa, Mexico. MATERIAL AND METHODS: A 740 bp fragment was amplified from the VDR gene in DNA samples of 71 patients with LL and 144 controls in the Hospital General de Culiacán during 2004-2007. Polymorphism was identified through TaqI endonuclease. RESULTS: A significant increase in the genotype TT of the VDR gene was observed in patients when compared to controls (p = 0.040; OR = 1.82). CONCLUSIONS: Our data support the association between the TT genotype and susceptibility to LL in this Mexican population.

Monitoramento de doença residual mínima em leucemia mielóide crônica por PCR em tempo real: [revisão] / Monitoring minimal residual disease in chronic myeloid leukemia by real time PCR: [review]

O monitoramento de doença residual mínima (DRM) em leucemia mielóide crônica é extremamente importante, pois possibilita o diagnóstico precoce de eventuais recidivas da doença. Este estudo visa apresentar uma revisão bibliográfica sobre a aplicação e a avaliação da eficácia do método de PCR em tempo real no monitoramento da doença residual mínima em pacientes com leucemia mielóide crônica. O método fornece informações a respeito do número e cinética das células tumorais residuais, sendo atualmente o padrão ouro para seu monitoramento. O uso de transcrição reversa associada à PCR em tempo real tornou a quantificação de mRNA mais simples e precisa. A metodologia mais utilizada é a TaqMan, que emprega a atividade exonucleásica 5'-3' da Taq DNA Polimerase. O monitoramento da DRM é feito após transplante de medula óssea ou após terapias baseadas em drogas, como o interferon-a e o mesilato de imatinibe. Entretanto, os estudos sobre o assunto apresentam os dados de maneiras conflitantes, dificultando a interpretação e comparação dos resultados. A determinação e uniformização dos cut-offs em diversas condições se fazem necessárias a fim de que a metodologia de PCR em tempo real seja aplicada na clínica.

Monitoring minimal residual disease (MRD) in chronic myeloid leukemia is a very important issue, because it makes early diagnosis of relapse of the disease possible. The objective of this study is to present a review on the use and evaluation of real time PCR in monitoring minimal residual disease in patients with chronic myeloid leukemia. The method provides information on the number and kinetics of tumour residual cells which currently it is the gold standard for monitoring MRD. The use of reverse transcription associated to real time PCR has made the quantification of mRNA easier and more accurate. TaqMan methodology, that exploits the 5'-3' exonuclesase activity of Taq DNA Polymerase, is the most common method used for this purpose. Monitoring MRD is required after stem cell transplantation and after drug-based therapies including Interferon-a and Imatinib Mesylate. So far, the studies published on this issue present differing and conflicting data, making the interpretation and comparison of the results very difficult. In order to use real time PCR to monitor MRD in chronic myeloid leukemia patients it is necessary to determine and to standardize cut-off points for results obtained under different conditions. This procedure will certainly be helpful for better clinical decision making.

Obtención de ADN polimerasa de Thermus aquaticus en un laboratorio de mediana complejidad / Thermus aquaticus polymerase DNA's production in a medium complexity laboratory

La PCR (reaccion en cadena de la polimerasa) es una técnica de diagnóstico molecular muy sensible, capaz de revelar tan sólo una copia de ADN en una mexcla compleja del mismo. El punto central para la difusión de esta metodología fue el descubrimiento de una ADN polimerasa estable al calor aislada de Thermus aquaticus (taq ADN pol), lo cual permitió automatizar el proceso de PCR usando un ciclador térmico, sin la necesidad de una adición continua de ADN polimerasa fresca. El objetivo del trabajo fue adaptar un método para la obtención de taq ADN pol en un laboratorio hospitalario de mediana complejidad usando el equipamiento y los medios de cultivo disponibles. Con este mètodo se consiguió producir un extracto crudo de 10.000 unidades de enzima, de fácil purificación, que mostró tener muy buena actividad enzimática por lo cual es posible utilizarla en una amplia variedad de aplicaciones (PCR clásica, PCR anidada, PCR transcriptasa reversa). La actividad enzimática para estos casos fue comparable con la de la taq polimerasa que se adquiere en el mercado.

Evaluation of a QIAamp DNA stool purification kit for Shiga-toxigenic Escherichia coli detection in bovine fecal swabs by PCR / Evaluación del kit QIAamp DNA stool purification para la detección de Escherichia coli productor de toxina Shiga en hisopados de materia fecal bovina por PCR[

A commercial kit intended for Taq polymerase inhibitor removal was tested to detect Shiga-toxigenic Escherichia coli (STEC) by polymersase chain reaction (PCR) directly from cattle fecal samples. Forty-five samples were analysed for the presence of stx genes. Results were compared to those obtained by two other methods: amplification of DNA purified by a non-commercial procedure (heat lysis protocol), and amplification of DNA from samples cultured in solid media, commonly used in our lab. Identical numbers of positive samples (33/45, 73 %) were obtained with the QIAamp DNA stool purification kit and the culturing procedure, suggesting an adequate removal of inhibitors that interfere in PCR amplification from the feces. Besides, the number of positive samples detected using DNA purified by the non-commercial protocol was lower, 25/39 (64%) than that achieved by using the kit. In conclusion, the use of the QIAamp DNA stool purification kit provided a rapid stx gene detection by PCR in bovine fecal samples.

Un kit comercial diseñado para la eliminación de inhibidores de la polimerasa Taq fue ensayado para la detección de STEC por PCR en muestras fecales de bovinos. Cuarenta y cinco muestras fueron evaluadas por la presencia de genes stx. Los resultados fueron comparados con aquéllos obtenidos por otros dos métodos: amplificación de ADN purificado por un procedimiento no comercial (protocolo de lisis por calor), y amplificación de ADN de muestras cultivadas en medio sólido, comúnmente usado en nuestro laboratorio. El mismo número de muestras positivas (33/45, 73 %), fueron obtenidas con el QIAamp DNA stool purification kit y el procedimiento de cultivo, sugiriendo una eliminación adecuada de inhibidores que interfieren con la amplificación en materia fecal. Por otro lado, el número de muestras positivas detectadas usando ADN purificado por el protocolo no comercial fue menor, 25/39 (64%). En conclusión, el uso del kit QIAamp DNA stool purification permitió una detección rápida de genes stx por PCR en muestras fecales bovinas.