Review © Future Drugs Ltd. All rights reserved. ISSN 1473-7159 219 CONTENTS Real-time capillary thermal cycler Detection strategies offered by the LightCycler Specimen processing for the LightCycler Optimization of PCR parameters for the LightCycler Multiplex & nested PCR platforms on the LightCycler Remote viewing & control of the LightCycler Investigation of virus outbreaks Conclusion Expert opinion Five-year view Key issues References Affiliations www.future-drugs.com Diagnostic value of real- time capillary thermal cycler in virus detection Boon-Huan Tan † , Elizabeth Ai- Sim Lim, Jasper Chin- Wen Liaw, Shirley Gek- Kheng Seah and Eric Peng- Huat Yap Molecular-based detection methods such as PCR techniques have had a significant impact on the diagnosis of viral infections because of their superior sensitivity and rapid turnaround time. This review describes the use of real-time PCR on the capillary thermal cycler, the Roche LightCycler™, for early disease detection in diagnostic virology. The advantages of using the LightCycler, the detection processes using SYBR ® Green I and different hybridization strategies will be discussed in detail, with specific examples drawn from our in-house viral assays. The use of the LightCycler for the investigation of two recent viral outbreaks in Singapore will also be briefly described. Expert Rev. Mol. Diagn. 4(2), 219–230 (2004) † Author for correspondence Virology Group, Defence Medical and Environmental Research Institute, DMERI@DSO(KENT RIDGE), 27 Medical Drive 13-00, Singapore 117510 Tel.: +65 6 485 7240 Fax: +65 6 485 7262 tboonhua@dso.org.sg KEYWORDS: capillary thermal cycler, FRET, hybridization probes, LightCycler™ , melting curve analysis, real-time PCR, SYBR ® Green I, TaqMan probes Diseases caused by emerging and re-emerging infections represent a global health threat for the 21st century [1]. Recent examples of emerg- ing and re-emerging diseases are caused by RNA viruses. T hese RNA viruses could quickly adapt and exploit varying conditions using viral-encoded replication machinery to multiply their viral genomes at high error rates. As a result, new genetic variants are constantly derived through processes of muta- tion, recombination and assortment. The abundance and distribution of host or vectors for these RNA viruses, if present, could also be modified by environmental factors, such as the changing weather patterns or tropical deforest- ation for urbanization. The explosion in human populations and urbanization, together with advances in the speed and volume of global transportation, has further increased opportunities for emergence and re-emergence of RNA viral diseases [2]. In Southeast Asia, emerging viral diseases have been described as zoonotic and vectorborne [3]. Singaporeans are also vulnerable to being exposed to exotic infectious agents as leisure pursuits bring them to foreign countries. T here is also an increase in travel to or outside regional areas due to occupational needs of many workers residing in Singapore. Diseases may also be introduced unknowingly into Singapore by immigrants and visitors from foreign countries. As infectious organisms evolved, the US Centers for Disease Control and Prevention (CDC) identified nine target areas in public health on which to focus in order to control human diseases: antimicrobial resistance, food- borne, waterborne and bloodborne diseases, chronic diseases caused by infectious agents, diseases affecting immunocompromised people, diseases of pregnant women and pediatric patients, and diseases associated with travelers, immigrants and refugees [4]. Four strategies were then identified to prevent and control these dis- eases [5]: to strengthen surveillance and response capacity for the identification of unusual occurrences that may indicate a new disease or a bioterrorism incident; to be able to prioritize applied research to improve diagnostic tools and to understand the disease by the identifi- cation of risk factors; to maintain certain core capacities in the routine laboratories so that initial response could be conducted; and to have access to special capabilities that the normal routine laboratories do not have. Our research institute responded by devel- oping a panel of molecular assays using the capillary thermal cycler, the LightCycler™ (Roche Diagnostics, NJ, USA), to provide For reprint orders, please contact reprints@future-drugs.com