Multiplex PCR Primer Design for Simultaneous Detection of Multiple Pathogens

Multiplex PCR provides a powerful tool for simultaneous detection and discrimination of multiple pathogens or different subtypes of a causative agent from humans, animals, and plants in a single reaction, and saves time and cost in the clinical diagnostic laboratory. Here, we describe the specific protocol of multiplex PCR primer design for simultaneous identification of more than one target from a same specimen. Different sizes of amplicons and similar T m values of primer sets are essential to successfully develop a feasible multiplex PCR assay.