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NucliSens Basic Kit NASBA with real-time detection for accelerated laboratory diagnosis of enterovirus infections



NucliSens Basic Kit NASBA with real-time detection for accelerated laboratory diagnosis of enterovirus infections



Abstracts of the General Meeting of the American Society for Microbiology 101: 264



Background: Enteroviruses are among the most common viral pathogens of humans and are responsible for a wide range of clinical symptoms. While culture is useful in the detection of patients with an enterovirus infection, it is time-consuming and lacks sensitivity. Objective: The aim of this study was to develop a molecular diagnostic assay for the detection of enteroviruses in a variety of clinical specimens using the NASBA amplification technology. Methods: Primers and probes were derived from the 5' non-coding region of the enterovirus genome (pan-enterovirus assay). For nucleic acid extraction and subsequent RNA amplification and detection, reagents from the NucliSens Basic Kit were used. Reference enterovirus strains and clinical samples were analyzed. Results: Initially, the assay utilized degenerate primers and 'end-point' detection of amplification products by electrochemiluminescence. The sensitivity of the pan-enterovirus assay, determined on cultures of a range of enteroviruses, was about 1 TCID50. The specificity was 100%, as determined by examination of enterovirus reference strains and non-enteroviruses including genetically closely related rhinoviruses. Prospectively-analyzed clinical samples (cerebrospinal fluids (CSF), respiratory tract samples, and stool specimens) revealed concordant results for NASBA and an 'in-house' RT-PCR. The assay format was simplified and made more user-friendly by incorporating non-degenerate primers for the amplification and a molecular beacon for 'real time' detection. In addition, an internal control RNA was introduced to monitor isolation, amplification and detection at the individual sample level. None of these changes had a major impact on the sensitivity and specificity of the assay. Conclusions: The pan-enterovirus NASBA, in NucliSens Basic Kit format, is a clinically useful assay applicable to a wide range of clinical specimens. Use of an internal control RNA and 'real time' detection enlarges the utility of the assay in a routine diagnostic setting and enables diagnosis of enterovirus infections within a few hours.

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Accession: 035413812

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