Desenvolvimento de primers para identificação e diferenciação de espécies de Candida em secreção vaginal por PCR em tempo real (qPCR)

Abstract

RESUMOInfecções fúngicas são cada vez mais frequentes, principalmente relacionadas ao sistema geniturinário e causadas por Candida spp. A PCR mostra-se promissora para o diagnóstico e para sua aplicação faz-se necessário o desenvolvimento de primers. Para isso obteve-se sequências da região ITS do rDNA 18S de 5 espécies de Candida, alinhou-se e verificou-se os tamanhos dos amplicons e as Tms teóricas. Cepas padrão e cepas cultivadas foram utilizadas como referência. Além disso, 67 amostras de DNA de secreção vaginal foram submetidas a qPCR e os resultados foram confirmados em gel de agarose 2%. Os testes in silico demonstraram a capacidade do par de primers em diferenciar as cinco espécies, o que foi confirmado após a amplificação das cepas padrão e de amostras de secreção vaginal, identificadas em 20 amostras. O limite de detecção foi de 58 células/mL para as cepas padrão de C. albicans e C. tropicalis e 29 células/mL para C. parapsilosis. A sensibilidade da qPCR frente ao teste de crescimento microbiológico foi de 68%, com especificidade de 90%. A técnica possui potencial de diagnóstico, porém precisa ser otimizada para testes quantitativos.Palavras-chave: Infecção geniturinária. qPCR. Candida spp. Desing de primers. ABSTRACTFungal infections are increasingly frequent, mainly related to the genitourinary system and caused by Candida spp. The PCR is promising for the diagnosis and for its application it is necessary the development of primers. For this purpose 18S rDNA ITS region sequences were obtained from 5 Candida species, aligned and the sizes of the amplicons and the theoretical Tms were verified. Standard and cultivated strains were used as reference. In addition, 67 samples of vaginal secretion DNA were subjected to qPCR and the results were confirmed on 2% agarose gel. In silico tests demonstrated the ability of the pair of primers to differentiate the five species, which was confirmed after amplification of standard samples and vaginal secretion (it was possible to identify the species in 20 samples). The detection limit was 58 cells/mL for the standard C. albicans and C. tropicalis strains and 29 cells/mL for C. parapsilosis. The sensitivity of qPCR to the microbiological growth test was 68%, with specificity of 90%. The technique has diagnostic potential, but needs to be optimized for quantitative tests.

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“…The use of the in silico method allows preparation for experimentation in prediction, diagnosis and modeling, since the in silico studies reproduce real conditions and enable the creation of primers that can be used in specific situations without the need of using biological samples. One study used in silico primers to identify and differentiate Candida species in vaginal secretion and validate the primers used the in silico test and real-time PCR (qPCR), demonstrating the ability of the primers to differentiate between 5 different Candida species with specificity of 90% [18].…”

“…It has several species that can be grouped into five genera (alpha, beta, gamma, mu, and nu), corresponding to the letters of the Greek alphabet [2]. Human pathogen species are classified into two groups, high-risk HPV types, closely related to cervical cancer, such as HPV 16,18,31,33,35,39,45,51,52,56,58,59 and 68, and low-risk HPV types, mainly associated with genital warts (condyloma acuminate), such as HPV 2, 3, 10 and 57 [3].…”