Otimização da nested-PCR em turbo unico na detecção do RNA-HCV

AUTOR(ES)
DATA DE PUBLICAÇÃO

1999

RESUMO

In the present study a methodology was optimized to a nested PCR in single tube for the detection of RNA-HCV. The sequences of primers were chosen allowing different optimum annealing temperature between the first and second PCR reaction in single tube. PCR conditions were established using an control produced by amplification of 5 NCR from genome that is highly conserved deleting 72 nucleotides with the enzyme PpuM I and cloned into pBluescript KS vector. The amplification of a single fragment at the end of a single tube reaction in the nested-PCR was reached when concentrations of 2 mM of `MgCI. Ind 2´, 5 nM and 200 nM of external and internal prímers, were used respectively. The temperature of annealing used was 72°C and 46°C for the first and second stage of the nested PCR reaction, with 20 and 35 cycle of al!lplification respectively. Sensibility tests, using different numbers of copies of the control (pB-HCV-2) were able to detect about 900 copies using our method. The conventional nested-PCR showed sensibility 10 times higher, detecting 90 copies. The difference in the sensibility observed between the two methodologies didn t interfere in the detection of RNA HCV when biological samples were tested. Our data indicated that the nested PCR in single tube developed can be used for the detection of RNA-HCV as an alternative method for the detection of RNA-HCV in clinical samples

ASSUNTO(S)

reação em cadeia de polimerase otimização hepatite por virus

ACESSO AO ARTIGO

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