Efficient amplification with NASBA of hepatitis B virus, herpes simplex virus and methicillin resistant Staphylococcus aureus DNA.
J Virol Methods
; 151(2): 283-293, 2008 Aug.
Article
em En
| MEDLINE
| ID: mdl-18514336
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Staphylococcus aureus
/
Vírus da Hepatite B
/
Simplexvirus
/
Replicação de Sequência Autossustentável
Tipo de estudo:
Diagnostic_studies
/
Qualitative_research
Idioma:
En
Revista:
J Virol Methods
Ano de publicação:
2008
Tipo de documento:
Article
País de afiliação:
Holanda