Simultaneous detection of multiple pathogens with the TaqMan Array Card

Quantitative polymerase chain reaction (qPCR) is a gold standard method for the detection and quantification of pathogenic organisms. Standard qPCR is inexpensive, sensitive and highly specific to the pathogen of interest. While qPCR assays can be multiplexed to allow the detection of multiple organisms in one reaction, it is prohibitively labour intensive to screen large numbers of samples for several pathogens at the same time. The TaqMan Array Card (TAC) is a cost-effective and accurate technique that expands the number of assays that can be simultaneously performed on a sample, with no increase in set-up time and only small reductions in sensitivity. This approach is highly beneficial in settings where there is a need to monitor a large panel of pathogens. We illustrate the application of TAC to the monitoring of gastrointestinal pathogens, which span viral, bacterial, protist and helminth taxa. This protocol outlines the laboratory set-up of a TaqMan Array Card, and some recommended data processing steps to aid in accurate interpretation of the results. A video protocol is additionally provided to assist in the use of the technique. • The TAC is designed primarily for gene expression assays, but has recently been utilised in several studies for pathogen detection in human clinical samples. • We expand the use of TAC for pathogen detection across human, animal and environmental sample types, and have developed a protocol and guidelines for the processing and interpretation of results that circumvents issues with the automated outputs. • This technique is applicable to pathogen or organism detection in any context, if quality nucleic acid extracts can be obtained from the sample type of interest.