Real-time quantitative polymerase chain reaction (qPCR) technology is being widely used for high throughput diagnostics of pathogens in the food production chain as an alternative to the more time-consuming and laborious culture-based standard methods. This chapter discusses the basics of qPCR, including data analysis, PCR instruments, and detection chemistries. The importance of choosing appropriate pre-PCR processing strategies - i.e., sampling, sample preparation, and PCR chemistry - to avoid PCR inhibition and achieve correct quantification is furthermore included. Major novel trends such as portable PCR instruments for in-field diagnostics, high-resolution melting curve analysis, and digital PCR are addressed. Finally, some perspectives on future trends are given. © 2015 Elsevier Ltd. All rights reserved.