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Nucleic acid sequence-based amplification, NASBA®, is an isothermal amplification technique for nucleic acids and was used for typing a collection of 24Mycoplasma pneumoniaestrains. A set of primers was chosen from the 16S rRNA sequence alignment ofMycoplasmaspecies. The nucleotide sequences of the (−)RNA amplicons were determined forM. pneumoniaestrains M15/83 (type 1) and FH (type 2), and revealed a one-point difference at the 16S rRNA level between the two types. Based on this result, two type-specific probes were constructed. The probes were hybridized in solution with the amplified nucleic acids of 24M. pneumoniaestrains in an enzyme-linked gel assay (ELGA). The results obtained by NASBA-based typing are in agreement with the classification of the 24M. pneumoniaestrains into two types by other typing methods, confirming the reliability of this technique.

A new method to quantify two individual mRNAs in a single NASBA reaction is described. In this study, tissue factor and CD14 mRNAs were used as a model system. RNA ratios of -4 to +4 log units were determined with good precision (within 0.3 log) and accuracy (within 0.2 log). By measuring both mRNAs in human monocytes that were stimulated with LPS, the multiplex Q-NASBA proved to be a successful tool to monitor the expression levels of two individual mRNAs in a single-tube amplification system. The method has potential in all fields in which quantitative information is needed on two individual RNAs.