Multiplex rtPCR in Microbiology
Nick M. Cirino, Norma P. Tavakoli, Susan Madison-Antenucci and Christina Egan
from: Real-Time PCR in Microbiology: From Diagnosis to Characterization (Edited by: Ian M. Mackay). Caister Academic Press, U.K. (2007)
Real-time PCR, or rtPCR, with its ability to detect and identify microorganisms is contributing to improvements in public health, and is facilitating more rapid attribution of disease-causing agents, whether they are old foes or newly emerging pathogens. Capitalizing on the strengths of rtPCR, we can further expand the capabilities of the methodology, so as to detect multiple target nucleic acid sequences in a single reaction. We refer to this type of an assay as a multiplex diagnostic assay, capable of detecting two (i.e., duplex) or more target signatures simultaneously. While multiplexing of rtPCR assays is achieved on a limited basis, typically two to four target sequences, there are examples of highly multiplexed assays such as DNA arrays or gene chips that can interrogate >10,000 oligonucleotide sequences in a single sample. This chapter will focus on the multiplex ability of rtPCR assays. Through integration of multiple assays into a single reaction, information about assay quality (e.g., internal positive or inhibition controls) can be simultaneously generated, and additional target pathogen sequences queried. Multiplexing therefore reduces analytical costs, improves turnaround time, expands testing capability and capacity, and adds data richness to analyses. In this chapter, we will describe the current applications of multiplex rtPCR to clinical diagnostics and public health, and we will review current applications of rtPCR to various classes of pathogenic microorganisms including viruses, bacteria, fungi, and parasites. Given a basic understanding of multiplexing concepts and critical parameters, it is fairly simple to convert conventional or rtPCR assays to multiplex formats read more ...