Low-Density TaqMan® Array Cards for the Detection of Pathogens

Jude Heaney; Kathryn Rolfe; Nicholas S Gleadall; Jane S Greatorex; Martin D Curran

doi:10.1016/bs.mim.2015.06.002

Low-Density TaqMan® Array Cards for the Detection of Pathogens

Methods Microbiol. 2015:42:199-218. doi: 10.1016/bs.mim.2015.06.002. Epub 2015 Aug 3.

Authors

Jude Heaney^{1

2}, Kathryn Rolfe³, Nicholas S Gleadall³, Jane S Greatorex³, Martin D Curran³

Affiliations

¹ Department of Medicine, University of Cambridge, Cambridge, United Kingdom.
² Institute of Hepatology, Foundation for Liver Research, London, United Kingdom.
³ Public Health England, Cambridge University Hospitals NHS, Cambridge, United Kingdom.

Abstract

Real-time PCR assays have revolutionised diagnostic microbiology over the past 15 years or more. Adaptations and improvements over that time frame have led to the development of multiplex assays. However, limitations in terms of available fluorophores has meant the number of assays which can be combined has remained in single figures. This latter limitation has led to the focus tending to be on individual pathogens and their detection. This chapter describes the development of TaqMan® Array Cards (TACs), technology which allows the detection of multiple pathogens (up to 48 targets) from a single nucleic acid extract, utilising small volumes and real-time PCR. This in turn lends itself to a syndromic approach to infectious disease diagnosis. Using the examples of TACs we have developed in our own laboratory, as well as others, we explain the design, optimisation and use of TACs for respiratory, gastrointestinal and liver infections. Refinement of individual assays is discussed as well as the incorporation of appropriate internal and process controls onto the array cards. Finally, specific examples are given of instances where the assays have had a direct, positive impact on patient care.

Keywords: Array; ECMO; Gastrointestinal; Jaundice; PCR; Pathogen detection; Respiratory; Syndromic; TAC; TaqMan.