Significant Expansion of Real-Time PCR Multiplexing with Traditional Chemistries using Amplitude Modulation

Aditya Rajagopal,Scott Fraser,Claudia Shin,Thomas A Tombrello,Lucien Jacky,Dominic Yurk,Karen Menge,Gregory J Tsongalis

doi:10.1038/s41598-018-37732-y

Abstract

The real time polymerase chain reaction (rtPCR) is an essential method for detecting nucleic acids that has a wide range of clinical and research applications. Current multiplexed rtPCR is capable of detecting four to six nucleic acid targets in a single sample. However, advances in clinical medicine are driving the need to measure many more targets at once. We demonstrate a novel method which significantly increases the multiplexing capability of any existing rtPCR instrument without new hardware, software, or chemistry. The technique works by varying the relative TaqMan probe concentrations amongst targets that are measured in a single fluorometric channel. Our fluorescent amplitude modulation method generates a unique rtPCR signature for every combination of targets present in a reaction. We demonstrate this technique by measuring nine different targets across three color channels with TaqMan reporting probes, yielding a detection accuracy of 98.9% across all combinations of targets. In principle this method could be extended to measure 6 or more targets per color channel across any number of color channels without loss in specificity.

Highlights

Regression of endpoint intensity levels generated by various probe concentrations
All of the probe concentrations used in later experiments were well above 600 pM, in the regime where we can rely on a linear relationship between probe concentration and fluorescent intensity
All PCR reactions where performed in an Applied Biosciences (ABI) 7500 thermocycler with 10 minutes of initial denaturation at 95 °C followed by 50 cycles with 15 s at 95 °C, 30 s at 58 °C, and 60 s at 75 °C

Summary

Introduction

Regression of endpoint intensity levels generated by various probe concentrations. Twelve replicates were performed at each probe concentration, which were used to generate the means and 1-σ error bars shown. 5′ GTA GGG ATA GAC CCT TTC AAA CTG CTT CAA AAC AGC CAA GTA TAC AGC CTA ATC AGA CCG AAT GAG AAT CCA GCA CAC AAG AGT C 3′ To address this gap in rtPCR capability we demonstrate a highly reproducible, maximally-information dense technique for multiplexing rtPCR reactions by adjusting primer and probe levels. This technique improves upon prior work[17] by improving signal processing techniques, expanding to measure targets across multiple color channels, and demonstrating viability on multiple different rtPCR platforms The advantage of this method is that it is run on existing rtPCR hardware without any new software, protocols, design processes, or chemistries. The coding technique used in the method is extensible to an arbitrary number of targets per channel, potentially allowing this bandwidth gain to increase to 6x or higher

Methods

Results

Conclusion