Development and evaluation of nucleic acid sequence based amplification (NASBA) for diagnosis of enterovirus infections using the NucliSens Basic Kit.
BACKGROUND
Molecular methods based on RNA amplification are needed for sensitive detection of enteroviruses in clinical samples. Many 'in house' methods based on reverse-transcribed PCR (RT-PCR) could be difficult to use in the routine diagnostic laboratory since they tend to be time-consuming, use reagents from many different suppliers and include non-routine procedures.


OBJECTIVE
The aim of this study was to develop and evaluate methods based on nucleic acid sequence based amplification (NASBA) for detection of enterovirus sequences.


METHODS
'In house' prepared and commercially available reagents were utilised to develop enterovirus-specific NASBA assays. Optimised methods were evaluated using clinical samples (cerebrospinal fluid, respiratory and stool samples), titred virus controls and in vitro produced synthetic RNA. Results for NASBA were compared with RT-PCR and virus culture.


RESULTS
Kit-based reagents gave an equivalent sensitivity to the more laborious 'in house' molecular assays (NASBA and RT-PCR) on clinical material and controls. All molecular methods picked up enterovirus positive clinical samples that were not identified by culture. End point detection sensitivity for the NASBA assay based on the NucliSens Basic Kit was <or=1 tissue culture infective dose 50% of a range of enteroviruses or <100 copies RNA input. The assay was specific for enteroviruses and did not pick up high titre rhinovirus preparations. Enterovirus Basic Kit NASBA results for clinical samples were easily obtained within a single working day.


CONCLUSIONS
NASBA is a suitable alternative to RT-PCR for sensitive amplification and detection of enterovirus sequences in a range of clinical specimens. The use of kit-based reagents will enable a wide range of laboratories to undertake molecular-based diagnostic procedures for RNA viruses and provide results within a time frame relevant to patient management.
Fox
Julie D
JD
Department of Medical Microbiology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, UK. foxjd@cardiff.ac.uk
Han
Song
S
Samuelson
Agneta
A
Zhang
Yadan
Y
Neale
M Lynne
ML
Westmoreland
Diana
D
D004770:Enterovirus / Q000235:genetics / Q000302:isolation & purification; D004769:Enterovirus Infections / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D012367:RNA, Viral / Q000032:analysis; D011933:Reagent Kits, Diagnostic; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic