کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
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3407562 | 1223631 | 2008 | 11 صفحه PDF | دانلود رایگان |

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 102–109 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.
Journal: Journal of Virological Methods - Volume 151, Issue 2, August 2008, Pages 283–293