Corrigendum: Electrical Detection of Dengue Biomarker Using Egg Yolk Immunoglobulin as the Biological Recognition Element.

This corrects the article DOI: 10.1038/srep07865.

Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes.

In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.

Evaluation of recombinant Cryptosporidium hominis GP60 protein and anti-GP60 chicken polyclonal IgY for research and diagnostic purposes / Avaliação da proteína recombinante GP60 de Cryptosporidium hominis e da IgY policlonal anti-GP60 recombinante para uso em pesquisa e diagnóstico

Abstract In this study, a method for expressing Cryptosporidium hominis GP60 glycoprotein in Escherichia coli for production of polyclonal anti-GP60 IgY in chickens was developed aiming future studies concerning the diagnosis, prevention and treatment of cryptosporidiosis. The full-length nucleotide sequence of the C. hominis gp60 gene was codon-optimized for expression in E. coli and was synthesized in pET28-a vector. Subcloning was performed on several different strains of BL21 E. coli. Temperature, time and inducer IPTG concentration assays were also performed and analyzed using SDS-PAGE. The optimal conditions were observed at a temperature of 37 °C, with overnight incubation and 1 mM of IPTG. Purification was performed by means of affinity chromatography using the AKTA Pure chromatography system and the Hi-Trap™ HP column (GE Healthcare). The recombinant protein GP60 (rGP60) thus generated was used to immunize laying hens owing the production of polyclonal IgY. Western blot and indirect immunofluorescence showed that the polyclonal antibody was capable of binding to rGP60 and to Cryptosporidium parvum sporozoites, respectively. The rGP60 and the IgY anti-rGP60 generated in this study may be used as templates for research and for the development of diagnostic methods for cryptosporidiosis.

Resumo Neste trabalho, foi desenvolvido um método de expressão da glicoproteína GP60 de Cryptosporidium hominis em Escherichia coli visando produzir anticorpos IgY anti-GP60 em galinhas para utilização em estudos futuros com os objetivos de diagnóstico, prevenção e tratamento da criptosporidiose. A sequência completa de nucleotídeos do gene gp60 de C. hominis foi códon-otimizada para expressão em E. coli e sintetizada no vetor pET28-a. A subclonagem foi realizada em várias estirpes diferentes de E. coli BL21. Os ensaios de concentração do indutor IPTG, temperatura e tempo foram realizados e analisados por SDS-PAGE. As condições ótimas de expressão foram observadas em temperatura de 37 °C, incubação durante a noite e 1 mM de IPTG. A purificação da proteína foi realizada por cromatografia de afinidade utilizando o sistema de cromatografia AKTA Pure e a coluna Hi-Trap™ HP (GE Healthcare). A proteína recombinante GP60 (rGP60) foi utilizada para imunizar galinhas poedeiras para produzir IgY policlonal anti-rGP60. Verificou-se por Western blot e por imunofluorescência indireta que o anticorpo policlonal apresentou reatividade com a rGP60 e com esporozoítos de Cryptosporidium parvum, respectivamente. A rGP60 e a IgY anti-rGP60 geradas neste estudo podem ser utilizadas como modelos para o desenvolvimento de ensaios para pesquisa e diagnóstico da criptosporidiose.

Electrical detection of dengue biomarker using egg yolk immunoglobulin as the biological recognition element.

Nonstructural protein 1 (NS1) is secreted by dengue virus in the first days of infection and acts as an excellent dengue biomarker. Here, the direct electrical detection of NS1 from dengue type 2 virus has been achieved by the measurement of variations in open circuit potential (OCP) between a reference electrode and a disposable Au electrode containing immobilized anti-NS1 antibodies acting as immunosensor. Egg yolk immunoglobulin (IgY) was utilized for the first time as the biological recognition element alternatively to conventional mammalian antibodies in the detection of dengue virus NS1 protein. NS1 protein was detected in standard samples in a 0.1 to 10 µg.mL(-1) concentration range with (3.2 ± 0.3) mV/µg.mL(-1) of sensitivity and 0.09 µg.mL(-1) of detection limit. Therefore, the proposed system can be extended to detect NS1 in real samples and provide an early diagnosis of dengue.

Gene mutations and polymorphisms of TP53 and FHIT in chronic esophagitis and esophageal carcinoma.

AIM: The aim of this study was to investigate genetic changes of the TP53 (tumor protein p53) and FHIT (fragile histidine triad) genes in precursor lesion such as chronic esophagitis (CE) and in esophageal squamous cell carcinoma (ESCC). MATERIALS AND METHODS: PCR-Single Strand Conformation Polymorphism (SSCP) analysis and DNA sequencing in 30 CE and 10 ESCC specimens were performed. RESULTS: DNA sequencing indicated two novel mutations in the TP53 exons 5 (codon 147) and 6 (codon 197) in 2/9 SSCP positive cases of ESCC, but no mutation was found in the CE. The SIFT (Sorting Intolerant From Tolerant) web-based program showed that missense variant at codon 197 of TP53 could affect the protein function. Additionally, polymorphisms of the TP53 exon 4 (codon 36 and 72) and of the FHIT exon 7 (codon 88) were observed in 4/11 (36%) cases of CE and 6/9 (67%) SSCP positive cases of ESCC after DNA sequencing. CONCLUSION: TP53 gene mutations are not common events in CE, but are frequent in ESCC, and as polymorphisms of TP53 and FHIT may confer a greater risk for the development of esophageal carcinoma, further studies are required.

Taenia solium and Taenia saginata: identification of sequence characterized amplified region (SCAR) markers.

Cysticercosis is one of the most important zoonosis, not only because of the effects on animal health and its economic consequences, but also due to the serious danger it poses to humans. The two main parasites involved in the taeniasis-cysticercosis complex in Brazil are Taenia saginata and Taenia solium. Differentiating between these two parasites is important both for disease control and for epidemiological studies. The purpose of this work was to identify genetic markers that could be used to differentiate these parasites. Out of 120 oligonucleotide decamers tested in random amplified polymorphic DNA (RAPD) assays, 107 were shown to discriminate between the two species of Taenia. Twenty-one DNA fragments that were specific for each species of Taenia were chosen for DNA cloning and sequencing. Seven RAPD markers were converted into sequence characterized amplified region (SCAR) markers with two specific for T. saginata and five specific for T. solium as shown by agarose gel electrophoresis. These markers were developed as potential tools to differentiate T. solium from T. saginata in epidemiological studies.

Detecção de Leptospira pomona em sêmen bovino por eletroforese capilar fluorescente / Detection of Leptospira pomona in bovine semen by fluorescent capillary eletrophoresis

Este estudo pretendeu avaliar o limiar de detecção da técnica de PCR aliada à eletroforese capilar para diagnóstico da Leptospira pomona em sêmen bovino. Doses inseminantes livres de patógenos foram contaminadas experimentalmente com Leptospira pomona em escalas que variavam de 10(elevado a 0) a 10(elevado a 7) bactérias/ml e submetidas à extração de DNA pelo método de fenol/clorofórmio. Após a reação de PCR, a visualização dos fragmentos foi realizada em três tipos de eletroforese: agarose 2% sob luz UV, acrilamida 8% corado com prata e eletroforese capilar fluorescente. A detecção de DNA de Leptospira pomona em sêmen bovino através de eletroforese capilar fluorescente foi possível a partir de concentração de 10(elevado a 2 )bactérias/ml. Nos métodos de eletroforese em agarose 2%, observou-se limite de detecção de 10(elevado a 4 )bactérias/ml e em gel de poliacrilamida 8% o limite de detecção foi de 10(elevado a 2 )bactérias/ml. A eletroforese capilar demonstrou ser uma alternativa eficaz e rápida na detecção de DNA de Leptospira em sêmen bovino podendo ser uma valiosa ferramenta para controle de qualidade do sêmen produzido em centrais de inseminação artificial dada a facilidade de automação desse processo.

This study was performed in order to evaluate the detection limit of PCR with fluorescent capillary electrophoresis for Leptospira pomonadiagnosis in bovine semen. Negative bovine semen samples were artificially contaminated with Leptospira pomona 10(involution 0) to 10(involution 7 ) bacteria/ml) and DNA was extracted by phenol/chloroform protocol. DNA fragments visualization was done by three electrophoresis methods: under UV light in 2 % agarose gel, silver staining 8% polyacrylamide gel and fluorescent capillary electrophoresis. The detection limit of capillary electrophoresis for Leptospira pomona was 10(involution 2) bacteria/ml. Under UV light, in 2 % agarose gel, the detection limit was of 10(involution 4) bacteria/ml while for silver stained 8 % polyacrylamide gel it was 10(involution 2) bacteria/ml. PCR with fluorescent capillary electrophoresis is an efficient andrapid diagnostic test for DNA detection of Leptospira in bovine semen and this can be an important tool for herd and semen sanitary controlin artificial insemination centers.

Desenvolvimento de novas metodologias para a identificação e caracterização genética de Herpesvírus Bovino tipo 1 (BHV-1) / Indentification and genetic characterization of bovine herpesvirus type 1 (BHV-1)

O Herpesvírus bovino tipo 1 (BHV-1), membro da subfamília Alphaherpesvirinae, gênero Varicellovirus, está disseminado mundialmente e tem sido motivo de grande preocupação para criadores e autoridades sanitárias de vários países. Este vírus pode causar rinotraqueíte infecciosa bovina (IBR), vulvovaginite pustular infecciosa (IPV), balanopostite pustular infecciosa (IPB), conjuntivite e infecções sistêmicas, sendo o aborto uma das consequências mais frequentes. O impacto econômico desta enfermidade é observado pelo retardo do crescimento de animais jovens, menor produção leiteira, morte embrionária e fetal, abortamento, com maior frequência no segundo e terceiro trimestres de gestação, eficiência reprodutiva de matrizes e touros reduzidas, além das restrições ao comércio internacional de animais vivos e seus produtos tais como sêmen e embriões, previstas no Código Internacional de Saúde Animal. Planos de controle e erradicação do vírus têm sido aplicados em alguns países da União Européia através da utilização de vacinas com genes deletados, que permitem a diferenciação entre animais naturalmente infectados e vacinados. O presente estudo teve como objetivo desenvolver ferramentas de diagnóstico e caracterização do BHV-1 baseadas na análise do DNA, que auxiliassem no aprimoramento dos programas de controle e erradicação desse vírus nos rebanhos bovinos. Para isso, foi desenvolvido um teste de nested PCR fluorescente para um fragmento do gene timidina quinase (TK) que permite a detecção e discriminação entre BHV-1 e BHV-5. Foram colhidas 100 amostras de gânglios trigemelares e leucócitos de fêmeas bovinas abatidas em frigorífico, sem histórico de vacinação prévia para BHV-1 e que não apresentavam sinais clínicos da doença. Foi possível diagnosticar por nested PCR fluorescente em 32 amostras de leucócitos e BHV-1 e/ou BHV-5 em 56 gânglios trigemelares, posteriormente confirmados por sequenciamento direto...

Taenia saginata: differential diagnosis of human taeniasis by polymerase chain reaction-restriction fragment length polymorphism assay.

Speciation of Taenia in human stool is important because of their different clinical and epidemiological features. DNA analysis has recently become possible which overcomes the problems of differentiating human taeniid cestodes morphologically. In the present study, we evaluated PCR coupled to restriction fragment length polymorphism to differentiate Taenia solium from Taenia saginata eggs present in fecal samples from naturally infected patients. A different DraI-RFLP pattern: a two-band pattern (421 and 100 bp) for T. saginata and a three-band pattern (234, 188, and 99 bp) for T. solium was observed allowing the two species to be separated. The lower detection limit of the PCR-RFLP using a non-infected fecal sample prepared with a given number of T. saginata eggs was 34 eggs in 2 g stool sediment. The 521 bp mtDNA fragment was detected in 8 out of 12 Taenia sp. carriers (66.6%). Of these, three showed a T. solium pattern and five a T. saginata pattern.