Efficient amplification with NASBA of hepatitis B virus, herpes simplex virus and methicillin resistant Staphylococcus aureus DNA.
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.
bioMérieux bv, Boseind 15, P.O. Box 84, 5280 AB Boxtel, The Netherlands.
Birgit
B
Deiman
bioMérieux Molecular Biology Center, Christophe Mérieux Center, 5 Rue des Berges, 38 024 Grenoble Cedex 01, France.
Corinne
C
Jay
bioMérieux SA, Chemin de l'Orme, 69280 Marcy l'Étoile, France.
Carine
C
Zintilini
bioMérieux bv, Boseind 15, P.O. Box 84, 5280 AB Boxtel, The Netherlands.
Saskia
S
Vermeer
bioMérieux bv, Boseind 15, P.O. Box 84, 5280 AB Boxtel, The Netherlands.
Dianne
D
van Strijp
bioMérieux bv, Boseind 15, P.O. Box 84, 5280 AB Boxtel, The Netherlands.
Fokke
F
Venema
bioMérieux Molecular Biology Center, Christophe Mérieux Center, 5 Rue des Berges, 38 024 Grenoble Cedex 01, France.
Paul
P
van de Wiel
D017931:DNA Primers; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D006515:Hepatitis B virus / Q000235:genetics*; D016106:Methicillin Resistance; D021141:Nucleic Acid Amplification Techniques; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D012680:Sensitivity and Specificity; D018139:Simplexvirus / Q000235:genetics*; D013211:Staphylococcus aureus / Q000235:genetics*
D017931:DNA Primers; D004279:DNA, Viral