Construção de um controle interno derivado de bacteriófago para utilização em um teste de detecção do vírus da Hepatite C baseado em PCR em tempo real

Orientador : Prof. Dr. Marco Aurélio KriegerCo-orientadora : Profa. Dra. Daniela Parada PavoniDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduaçao em Processos Biotecnológicos. Defesa: Curitiba,13/04/2010Bibliografia: fls. 178-185Resumo: A hepatite C hoje representa um sério problema de saúde pública no Brasil e no mundo. Pelo menos 3% da população mundial está infectada. Dos casos existentes, 15% são curados e 85% tornam-se crônicos. 20% dos crônicos evoluem para cirrose hepatica e 4% para formas mais graves de doenças do fígado. Não há vacinas disponíveis e os tratamentos usados são de eficácia limitada. Desta forma um diagnóstico correto é essencial e somente a utilização de técnicas bem padronizadas e monitoradas com controles internos podem trazer esta garantia e assegurar a interpretação conclusiva dos achados obtidos evitando a liberação de resultados falso-positivos ou falso-negativos que podem comprometer um diagnóstico final. Baseando-se nestas informações foi feita a proposta deste trabalho cujo objetivo principal foi o desenvolvimento de um controle interno derivado de um bacteriófago estável e não infeccioso para um teste de detecção do vírus da hepatite C baseado em PCR em tempo real. Este controle por ser um vírus RNA como o HCV, pode controlar a metodologia desde a extração até a amplificação. Na construção desse controle foi utilizado o RNA comercial do bacteriófago MS2. O genoma inteiro do fago amplificado por PCR foi inserido no plasmídeo pGEM®-T Easy originando o pGEM®-T Easy - MS2, cuja clonagem produziu quantidade suficiente do genoma para a contrução do pET-47b(+) - MS2. Neste plasmídeo foi inserido, no gene da replicase do MS2, uma sequência sintética com 144 bases derivada da região 5' UTR do HCV originando o pET-47b(+) - MS2 - SS. Nesta sequência algumas bases foram alteradas permitindo o desenho de uma sonda para distinguir na PCR em tempo real o controle interno do HCV. Este plasmídeo foi transformado na bactéria BL21(DE3)pLyS e foi usado para expressar o bacteriófago MS2 recombinante que é o controle interno competitivo (CIC). Simultaneamente, expressou-se o MS2 a partir do vetor pET-47b(+) - MS2 que serviu como padrão na quantificação do M 2 recombinante e que também foi testado como um controle nterno não competitivo na PCR em Tempo Real. Padronizou-se reações multiplex "two step" e "one step" para as combinações CIC/HCV usando os mesmos "primers" para amplificar o HCV e o CIC e sondas para distinguir os dois alvos. Para as combinações MS2/HCV utilizou-se "primers" e sondas específicas para cada um dos alvos. Foram estudadas diversas concentrações destes controles e definiu-se que 0,025 PFU eq. do CIC e 0,1 PFU do MS2 são as quantidades ideais para serem adicionadas à amostra clínica antes de extração do RNA, porque não interferem na detecção de amostras com cargas virais altas e baixas. Foram conduzidos também estudos para avaliar-se a estabilidade e verificou-se que a estocagem a 4ºC tanto do CIC como do MS2 garante a manutenção da carga viral ao longo do tempo analisado. Portanto o presente trabalho mostrou a viabilidade técnica do desenvolvimento e da utilização de dois controles internos em uma metodologia diagnóstica e abriu perspectivas para a criação de novos controles, uma vez que os plasmídeos construídos podem servir como base para o desenvolvimento de controles específicos para metodologias diagnósticas de outras doenças causadas por vírus RNA.Abstract: Hepatitis C is a serious public health problem in Brazil and in the world. At least 3% of world population is infected, there is no vaccine available and the treatment used is of limited effectiveness. Around 85% of patients evolve to chronic phase, 20% of them develop liver cirrhosis and 4% other serious hepatic diseases. In this way a diagnostic tool with high sensitivity and specificity and quality control measures are instruments to ensure the correct interpretation of the diagnosis and prevention of false positives and false negatives results. Therefore the main objective of this work was to develop a stable internal control derived from non-infectious bacteriophage for hepatitis C virus detection test based on real-time PCR. This control is a RNA virus that can control the methodology from extraction to amplification. In the construction of this control it was used a commercial bacteriophage MS2 RNA. The entire genome of the phage amplified by PCR was inserted into plasmid pGEM®-T Easy resulting in the pGEM®-T Easy - MS2, which produced enough genome for the construction of pET-47b(+) - MS2. It was inserted into the MS2 replicase gene a synthetic sequence of 144 bases derived from 5' UTR region of HCV resulting in the pET-47b(+) - MS2 - SS. In this sequence a few bases were exchanged allowing the design of a probe to distinguish in the real-time PCR the internal control from HCV. This plasmid was transformed in E. coli BL21(DE3)pLyS that expressed the recombinant bacteriophage MS2, the competitive internal control (CIC). At the same time the wild type MS2 was expressed from the pET-47b(+) - MS2 and used as the standard quantification of recombinant MS2 (CIC). Wild type MS2 was also tested as a non-competitive internal co trol in real-time PCR. Multiplex reactions "two step" and "one step" were standardized for combinations of CIC/HCV using the same primers to amplify the HCV and CIC and probes to distinguish the two targets. For the combination MS2/HCV we used primers and probes specific for each target. We studied different concentrations of these controls and set 0.025 PFU eq. of CIC and 0.1 PFU of MS2 as the ideal amount for be added to the clinical sample prior to extraction because they do not interfere with the detection of high and low viral loads samples. We conducted studies to evaluate the stability of phage solution and found that the storage at 4°C of both CIC and the MS2 ensures the viral load maintence over the assessed time. Therefore this study has demonstrated the technical feasibility of the development and use of two internal controls in a diagnostic method and allows the creation of other controls since the plasmids may serve as a basis for developing controls for specific diagnostic methods for other RNA viral diseases

Epidemiological profile of Zika, Dengue and Chikungunya virus infections identified by medical and molecular evaluations in Rondonia, Brazil

Several arboviruses have emerged and/or re-emerged in North, Central and SouthAmerican countries. Viruses from some regions of Africa and Asia, such as the Zika and Chikungunya virus have been introduced in new continents causing major public health problems. The aim of this study was to investigate the presence of RNA from Zika, Dengue and Chikungunya viruses in symptomatic patients from Rondonia, where the epidemiological profile is still little known, by one-step real-time RT-PCR. The main clinical signs and symtoms were fever (51.2%), headache (78%), chills (6.1%), pruritus (12.2%), exanthema (20.1%), arthralgia (35.3%), myalgia (26.8%) and retro-orbital pain (19.5%). Serum from 164 symptomatic patients were collected and tested for RNA of Zika, Dengue types 1 to 4 and Chikungunya viruses, in addition to antibodies against Dengue NS1 antigen. Direct microscopy for Malaria was also performed. Only ZIKV RNA was detected in 4.3% of the patients, and in the remaining 95.7% of the patients RNA for Zika, Dengue and Chikungunya viruses were not detected. This finding is intriguing as the region has been endemic for Dengue for a long time and more recently for Chikungunya virus as well. The results indicated that medical and molecular parameters obtained were suitable to describe the first report of symptomatic Zika infections in this region. Furthermore, the low rate of detection, compared to clinical signs and symptoms as the solely diagnosis criteria, suggests that molecular assays for detection of viruses or other pathogens that cause similar symptoms should be used and the corresponding diseases could be included in the compulsory notification list

Construção de um controle interno derivado de bacteriófago para utilização em um teste de detecção do vírus da Hepatite C baseado em PCR em tempo real

Orientador : Prof. Dr. Marco Aurélio KriegerCo-orientadora : Profa. Dra. Daniela Parada PavoniDissertação (mestrado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduaçao em Processos Biotecnológicos. Defesa: Curitiba,13/04/2010Bibliografia: fls. 178-185Resumo: A hepatite C hoje representa um sério problema de saúde pública no Brasil e no mundo. Pelo menos 3% da população mundial está infectada. Dos casos existentes, 15% são curados e 85% tornam-se crônicos. 20% dos crônicos evoluem para cirrose hepatica e 4% para formas mais graves de doenças do fígado. Não há vacinas disponíveis e os tratamentos usados são de eficácia limitada. Desta forma um diagnóstico correto é essencial e somente a utilização de técnicas bem padronizadas e monitoradas com controles internos podem trazer esta garantia e assegurar a interpretação conclusiva dos achados obtidos evitando a liberação de resultados falso-positivos ou falso-negativos que podem comprometer um diagnóstico final. Baseando-se nestas informações foi feita a proposta deste trabalho cujo objetivo principal foi o desenvolvimento de um controle interno derivado de um bacteriófago estável e não infeccioso para um teste de detecção do vírus da hepatite C baseado em PCR em tempo real. Este controle por ser um vírus RNA como o HCV, pode controlar a metodologia desde a extração até a amplificação. Na construção desse controle foi utilizado o RNA comercial do bacteriófago MS2. O genoma inteiro do fago amplificado por PCR foi inserido no plasmídeo pGEM®-T Easy originando o pGEM®-T Easy - MS2, cuja clonagem produziu quantidade suficiente do genoma para a contrução do pET-47b(+) - MS2. Neste plasmídeo foi inserido, no gene da replicase do MS2, uma sequência sintética com 144 bases derivada da região 5' UTR do HCV originando o pET-47b(+) - MS2 - SS. Nesta sequência algumas bases foram alteradas permitindo o desenho de uma sonda para distinguir na PCR em tempo real o controle interno do HCV. Este plasmídeo foi transformado na bactéria BL21(DE3)pLyS e foi usado para expressar o bacteriófago MS2 recombinante que é o controle interno competitivo (CIC). Simultaneamente, expressou-se o MS2 a partir do vetor pET-47b(+) - MS2 que serviu como padrão na quantificação do M 2 recombinante e que também foi testado como um controle nterno não competitivo na PCR em Tempo Real. Padronizou-se reações multiplex "two step" e "one step" para as combinações CIC/HCV usando os mesmos "primers" para amplificar o HCV e o CIC e sondas para distinguir os dois alvos. Para as combinações MS2/HCV utilizou-se "primers" e sondas específicas para cada um dos alvos. Foram estudadas diversas concentrações destes controles e definiu-se que 0,025 PFU eq. do CIC e 0,1 PFU do MS2 são as quantidades ideais para serem adicionadas à amostra clínica antes de extração do RNA, porque não interferem na detecção de amostras com cargas virais altas e baixas. Foram conduzidos também estudos para avaliar-se a estabilidade e verificou-se que a estocagem a 4ºC tanto do CIC como do MS2 garante a manutenção da carga viral ao longo do tempo analisado. Portanto o presente trabalho mostrou a viabilidade técnica do desenvolvimento e da utilização de dois controles internos em uma metodologia diagnóstica e abriu perspectivas para a criação de novos controles, uma vez que os plasmídeos construídos podem servir como base para o desenvolvimento de controles específicos para metodologias diagnósticas de outras doenças causadas por vírus RNA.Abstract: Hepatitis C is a serious public health problem in Brazil and in the world. At least 3% of world population is infected, there is no vaccine available and the treatment used is of limited effectiveness. Around 85% of patients evolve to chronic phase, 20% of them develop liver cirrhosis and 4% other serious hepatic diseases. In this way a diagnostic tool with high sensitivity and specificity and quality control measures are instruments to ensure the correct interpretation of the diagnosis and prevention of false positives and false negatives results. Therefore the main objective of this work was to develop a stable internal control derived from non-infectious bacteriophage for hepatitis C virus detection test based on real-time PCR. This control is a RNA virus that can control the methodology from extraction to amplification. In the construction of this control it was used a commercial bacteriophage MS2 RNA. The entire genome of the phage amplified by PCR was inserted into plasmid pGEM®-T Easy resulting in the pGEM®-T Easy - MS2, which produced enough genome for the construction of pET-47b(+) - MS2. It was inserted into the MS2 replicase gene a synthetic sequence of 144 bases derived from 5' UTR region of HCV resulting in the pET-47b(+) - MS2 - SS. In this sequence a few bases were exchanged allowing the design of a probe to distinguish in the real-time PCR the internal control from HCV. This plasmid was transformed in E. coli BL21(DE3)pLyS that expressed the recombinant bacteriophage MS2, the competitive internal control (CIC). At the same time the wild type MS2 was expressed from the pET-47b(+) - MS2 and used as the standard quantification of recombinant MS2 (CIC). Wild type MS2 was also tested as a non-competitive internal co trol in real-time PCR. Multiplex reactions "two step" and "one step" were standardized for combinations of CIC/HCV using the same primers to amplify the HCV and CIC and probes to distinguish the two targets. For the combination MS2/HCV we used primers and probes specific for each target. We studied different concentrations of these controls and set 0.025 PFU eq. of CIC and 0.1 PFU of MS2 as the ideal amount for be added to the clinical sample prior to extraction because they do not interfere with the detection of high and low viral loads samples. We conducted studies to evaluate the stability of phage solution and found that the storage at 4°C of both CIC and the MS2 ensures the viral load maintence over the assessed time. Therefore this study has demonstrated the technical feasibility of the development and use of two internal controls in a diagnostic method and allows the creation of other controls since the plasmids may serve as a basis for developing controls for specific diagnostic methods for other RNA viral diseases

External control viral-like particle construction for detection of emergent Arboviruses by real-time reverse-transcription PCR

Submitted by Manoel Barata ([email protected]) on 2019-12-04T17:17:15Z No. of bitstreams: 1 BMRI2019-25604ok.pdf: 1048630 bytes, checksum: a8f1a3ae8fad241e2707cfd63c41f3c7 (MD5)Approved for entry into archive by Manoel Barata ([email protected]) on 2020-02-12T14:23:15Z (GMT) No. of bitstreams: 1 BMRI2019-25604ok.pdf: 1048630 bytes, checksum: a8f1a3ae8fad241e2707cfd63c41f3c7 (MD5)Made available in DSpace on 2020-02-12T14:23:15Z (GMT). No. of bitstreams: 1 BMRI2019-25604ok.pdf: 1048630 bytes, checksum: a8f1a3ae8fad241e2707cfd63c41f3c7 (MD5) Previous issue date: 2019Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil / Universidade Federal do Paraná. Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Fundação Oswaldo Cruz. Escritório Rondônia. Porto Velho, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil / Universidade Federal do Paraná. Programa de Pós-Graduação em Engenharia de Bioprocessos e Biotecnologia. Curitiba, PR, Brasil / Fundação Oswaldo Cruz. Instituto Carlos Chagas. Curitiba, PR, Brasil.Instituto de Biologia Molecular do Paraná. Curitiba, PR, Brasil.Arboviruses have been emerging and reemerging worldwide, predominantly in tropical and subtropical areas. As many arbovirus infections, including dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV), have similar signs and symptoms, clinical diagnosis of arbovirus infections is challenging. Therefore, reliable laboratory tests are necessary to improve the clinical management of patients with suspected arbovirus infections. Real-time reverse-transcription PCR (RT-qPCR) is among the more effective methods to distinguish these viruses. The aim of this study was to construct a unique positive external control derived from a unique plasmid using genetic engineering for specific use in RT-qPCR assays to detect Zika, dengue (1-4), and chikungunya. An external control derived from the MS2 bacteriophage was constructed using sequences from arbovirus and human genomes. Laboratories were asked to test the control in the ZDC Biomol kit, a RT-qPCR kit which is able to detect Zika, dengue serotypes 1-4, chikungunya, and an internal human control. RNA extracted from the external control was able to be amplified and detected in RT-qPCR assays for each virus detected by using the ZDC Biomol kit. The external control, samples from viral culture, and infected patient samples display similar amplification using this assay. The pET47b(+)MS2-ZDC vector is a viable expression system for the production of external control viral-like particles (MS2-ZDC). The RNA from the recombinant particles can be easily extracted and can function as a tool to validate all steps of process from the extraction to the amplification of all targets in specific reaction. Thus, the MS2-ZDC particles are laboratory-safe in order to avoid risk for operators, and the phages are effective as positive control for use in the ZDC Biomol kit amplifying all kit targets making them effective for commercial profile

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses

Epidemiological profile of Zika, Dengue and Chikungunya virus infections identified by medical and molecular evaluations in Rondonia, Brazil

ABSTRACT Several arboviruses have emerged and/or re-emerged in North, Central and South-American countries. Viruses from some regions of Africa and Asia, such as the Zika and Chikungunya virus have been introduced in new continents causing major public health problems. The aim of this study was to investigate the presence of RNA from Zika, Dengue and Chikungunya viruses in symptomatic patients from Rondonia, where the epidemiological profile is still little known, by one-step real-time RT-PCR. The main clinical signs and symtoms were fever (51.2%), headache (78%), chills (6.1%), pruritus (12.2%), exanthema (20.1%), arthralgia (35.3%), myalgia (26.8%) and retro-orbital pain (19.5%). Serum from 164 symptomatic patients were collected and tested for RNA of Zika, Dengue types 1 to 4 and Chikungunya viruses, in addition to antibodies against Dengue NS1 antigen. Direct microscopy for Malaria was also performed. Only ZIKV RNA was detected in 4.3% of the patients, and in the remaining 95.7% of the patients RNA for Zika, Dengue and Chikungunya viruses were not detected. This finding is intriguing as the region has been endemic for Dengue for a long time and more recently for Chikungunya virus as well. The results indicated that medical and molecular parameters obtained were suitable to describe the first report of symptomatic Zika infections in this region. Furthermore, the low rate of detection, compared to clinical signs and symptoms as the solely diagnosis criteria, suggests that molecular assays for detection of viruses or other pathogens that cause similar symptoms should be used and the corresponding diseases could be included in the compulsory notification list