Efficient amplification with NASBA® of hepatitis B virus, herpes simplex virus and methicillin resistant Staphylococcus aureus DNA
2008; Elsevier BV; Volume: 151; Issue: 2 Linguagem: Inglês
10.1016/j.jviromet.2008.04.009
ISSN1879-0984
AutoresBirgit Deiman, Corinne Jay, Carine Zintilini, Saskia Vermeer, Dianne van Strijp, Fokke Venema, Paul van de Wiel,
Tópico(s)Bacteriophages and microbial interactions
ResumoA 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.
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