RESUMO
Here we present an alternative polymerase chain reaction (PCR) approach using 18S rDNA to identify apicomplexan parasites. A new primer set was designed and evaluated in silico and in vitro. This new PCR could detect some apicomplexan genera based on amplicon size and identify at least 45 species after sequencing.
Assuntos
Primers do DNA , Parasitos/isolamento & purificação , Doenças Parasitárias/diagnóstico , Reação em Cadeia da Polimerase/métodos , Animais , DNA de Protozoário/genética , Humanos , Camundongos , Células RAW 264.7 , RNA Ribossômico 18S/genéticaRESUMO
Toxoplasma gondii is a widespread parasite able to infect virtually any nucleated cells of warm-blooded hosts. In some cases, T. gondii detection using already developed PCR primers can be inefficient in routine laboratory tests, especially to detect atypical strains. Here we report a new nested-PCR protocol able to detect virtually all T. gondii isolates. Analyzing 685 sequences available in GenBank, we determine that GRA7 is one of the most conserved genes of T. gondii genome. Based on an alignment of 85 GRA7 sequences new primer sets that anneal in the highly conserved regions of this gene were designed. The new GRA7 nested-PCR assay providing sensitivity and specificity equal to or greater than the gold standard PCR assays for T. gondii detection, that amplify the B1 sequence or the repetitive 529bp element.
