RESUMO
A new mechanism is described for DNA amplification using nucleic acid sequence-based amplification (NASBA) including a restriction enzyme digestion and P1 primer binding directly upstream of the digestion. For hepatitis B virus (HBV), herpes simplex virus (HSV) and methicillin resistant Staphylococcus aureus (MRSA) DNA, which all show very poor amplification with normal NASBA, assay sensitivity was improved by a factor 100-1000 when restriction enzyme digestion was performed prior to amplification. For the quantitative HBV assay, in combination with the NucliSENS Extractor (bioMérieux, Boxtel, The Netherlands), a 95% target detection rate of 242 WHO IU/ml and 50% detection rate of 35 WHO IU/ml was achieved. The lowest detectable HBV concentration was 10 WHO IU/ml. HBV DNA could be quantified with an algorithm comparable to that used for RNA quantitation and by using a two step approach a dynamic range of 10(2)-10(9)WHO IU/ml (>6 log) was shown to be quantifiable. For the qualitative HSV assay, in combination with the NucliSENS miniMAG (bioMérieux, Boxtel, The Netherlands), the 95% detection rate was determined to be 84 and 138 copies/isolation for HSV 1 and HSV 2, respectively, which corresponds to approximately 10 copies per amplification for both targets. For MRSA, the limit of detection was <10 equivalent CFU per amplification.