Publicação
Development of a real time PCR Methodology to detect systemic fungal infections
| Resumo: | The work established aimed to adapt a previously developed methodology based on a multiplex PCR assay coupled with GeneScan analysis, to a quantitative PCR methodology (qPCR). This adaptation was related to the diagnostic preferences in hospital settings, since the commercially available qPCR methods are capable of delivering fast and accurate results, with low hands-on time, in an entirely closed system with a reduced likelihood of contamination. With qPCR methods the diagnosis can be attributed as the reaction ends, with no need of samples transfer for PCR products analysis, which takes extra time. This qPCR method uses SYBR Green and melting curve for PCR products analysis. The use of primers designed outside the ITS regions enhances the specificity and reduces cross-amplification of the methodology. Two panels previously designed in our laboratory were evaluated, the Candida Panel, and the Filamentous Fungi Panel. Regarding the adaptation of the Candida Panel, this conversion was not possible since the PCR products of each species had very similar melting temperature values. Regarding the Filamentous Fungi Panel, the melting temperature values obtained for each species were very distinct from each other which allowed to identify and distinguish the most prevalent pathogens associated with invasive aspergillosis (Aspergillus fumigatus, A. niger, A. flavus and A. terreus), and invasive mucormycosis (Rhizopus arrhizus), as well as coinfections in a qPCR multiplex reaction. The qPCR method was also able to detect samples with low amounts of fungal gDNA, explicitly 1.3 pg/μL for A. fumigatus and R. arrhizus, 13 pg/μL for A. flavus and A. niger, and 33 pg/μL for A. terreus. When human plasma was spiked with fungal DNA, the methodology was still able to distinguish and correctly identify the fungal pathogens. No false-positive results were obtained with nontarget species, including bacteria or human DNA. Thus, this work provides evidence for the possibility of the adaptation of the Filamentous Fungi Panel to a qPCR multiplex methodology, as well as the potential diagnostic capability of this method for the identification of the most relevant filamentous fungi involved in invasive fungal infections. |
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| Autores principais: | Mendonça, Paulo Alexandre Silva |
| Assunto: | Molecular diagnosis Multiplex quantitative PCR Invasive fungal infections Invasive aspergillosis Invasive mucormycosis Invasive candidiasis Diagnóstico molecular Multiplex PCR quantitativo Infeções fúngicas invasivas Aspergilose invasiva Mucormicose invasiva Candidíase invasiva |
| Ano: | 2021 |
| País: | Portugal |
| Tipo de documento: | dissertação de mestrado |
| Tipo de acesso: | acesso aberto |
| Instituição associada: | Universidade do Minho |
| Idioma: | inglês |
| Origem: | RepositóriUM - Universidade do Minho |
| Resumo: | The work established aimed to adapt a previously developed methodology based on a multiplex PCR assay coupled with GeneScan analysis, to a quantitative PCR methodology (qPCR). This adaptation was related to the diagnostic preferences in hospital settings, since the commercially available qPCR methods are capable of delivering fast and accurate results, with low hands-on time, in an entirely closed system with a reduced likelihood of contamination. With qPCR methods the diagnosis can be attributed as the reaction ends, with no need of samples transfer for PCR products analysis, which takes extra time. This qPCR method uses SYBR Green and melting curve for PCR products analysis. The use of primers designed outside the ITS regions enhances the specificity and reduces cross-amplification of the methodology. Two panels previously designed in our laboratory were evaluated, the Candida Panel, and the Filamentous Fungi Panel. Regarding the adaptation of the Candida Panel, this conversion was not possible since the PCR products of each species had very similar melting temperature values. Regarding the Filamentous Fungi Panel, the melting temperature values obtained for each species were very distinct from each other which allowed to identify and distinguish the most prevalent pathogens associated with invasive aspergillosis (Aspergillus fumigatus, A. niger, A. flavus and A. terreus), and invasive mucormycosis (Rhizopus arrhizus), as well as coinfections in a qPCR multiplex reaction. The qPCR method was also able to detect samples with low amounts of fungal gDNA, explicitly 1.3 pg/μL for A. fumigatus and R. arrhizus, 13 pg/μL for A. flavus and A. niger, and 33 pg/μL for A. terreus. When human plasma was spiked with fungal DNA, the methodology was still able to distinguish and correctly identify the fungal pathogens. No false-positive results were obtained with nontarget species, including bacteria or human DNA. Thus, this work provides evidence for the possibility of the adaptation of the Filamentous Fungi Panel to a qPCR multiplex methodology, as well as the potential diagnostic capability of this method for the identification of the most relevant filamentous fungi involved in invasive fungal infections. |
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