Mid-level Multiplex PCR

Fri, 10/07/2011 - 11:03am
Carolyn Reifsnyder, Marketing Director, QPCR, PCR, and Bioreagents; Agilent Technologies Inc., La Jolla, Calif.

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Figure 1. Sample results using Agilent’s MassCode software to analyze PCR data. (Source: Agilent)

Real-time PCR has revolutionized life science research over the last decade, due to the sensitivity and precision provided by the monitoring of PCR products in real time. Its effectiveness at amplification and quantification of low levels of nucleic acids has driven the emergence of numerous applications, including biomarker discovery and validation, cancer risk assessment, and development of diagnostic tests for infectious diseases.

Despite its great power, real-time PCR has one significant drawback. Current real-time PCR systems cannot detect and measure more than five nucleic acid targets per PCR experiment. This limitation presents a real roadblock to studies that need to analyze large numbers of targets in the same sample, especially for large numbers of samples. Researchers often need to interrogate a single sample for more than 20 infectious agents to determine the etiology of a clinical syndrome. Analysis of such samples with real-time PCR requires multiple PCR experiments for each sample, increasing the amount of work required to get to a result while decreasing throughput and the reliability of the results.

Agilent Technologies’ MassCode PCR technology provides the sensitivity of endpoint PCR, while enabling 30 to 40 targets to be analyzed in a sample, using a single multiplex PCR experiment. It takes advantage of the ability of a mass spectrometer (MS) to differentiate 60 to 80 different MassCode tags simultaneously, with nanomolar sensitivity. Replacing fluorescent detection with MS provides the mid-level multiplexing capability that real-time PCR lacks. Each forward and reverse primer is conjugated with a small molecule tag of a specific mass to provide a dual signal for a given target sequence. After cDNA synthesis and PCR with enzymes designed specifically for the technology, excess primers and reaction by-products are removed and the 96-well plate is loaded into a liquid chromatography autosampler for automated UV processing to cleave the small molecule tags from the amplified targets (Figure 1). Flow injection into the single-quadrupole MS equipped with an atmospheric pressure chemical ionization (APCI) source enables detection of the respective tags. MassCode PCR software automatically analyzes the data and reports the result for each sample and target of interest. MassCode PCR is a one-day process, just like real-time PCR, but the number of targets analyzed per unit time is much higher, and the cost per target per sample is lower for larger sample numbers.

Typical results for a multi-target analysis using MassCode are shown above, illustrating simultaneous analysis of multiple viral pathogens. This technology has been used to detect multiple respiratory pathogens,1 viral hemorrhagic fever agents, and tick-borne pathogens2 in clinical research. MassCode PCR is a valuable research tool to elucidate the role of multiple co-infecting agents in causation of disease, to prioritize targets for vaccine and drug development.

1. Lamson D, et al. MassTag Polymerase-Chain-Reaction Detection of Respiratory Pathogens, Including a New Rhinovirus Genotype, That Caused Influenza-Like Illness in New York State during 2004-2005. J Infect Dis. 2006;194:1398-02.

2. Tokarz R, et al. Detection of Tick-Borne Pathogens by MassTag Polymerase Chain Reaction. Vector Borne Zoonotic Dis. 2008;2:147-52.



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