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Efficient amplification with NASBA of hepatitis B virus, herpes simplex virus and methicillin resistant Staphylococcus aureus DNA.
- Source :
-
Journal of virological methods [J Virol Methods] 2008 Aug; Vol. 151 (2), pp. 283-293. Date of Electronic Publication: 2008 Jun 02. - Publication Year :
- 2008
-
Abstract
- 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.
Details
- Language :
- English
- ISSN :
- 0166-0934
- Volume :
- 151
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Journal of virological methods
- Publication Type :
- Academic Journal
- Accession number :
- 18514336
- Full Text :
- https://doi.org/10.1016/j.jviromet.2008.04.009