Rapid and inexpensive species differentiation using a multiplex real-time polymerase chain reaction high-resolution melt assay

Analytical Biochemistry

Volume 500, 1 May 2016, Pages 15–17

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• Kelly M. Elkins^, ,
• Anjelica C.U. Perez,
• Katherine C. Sweetin

doi:10.1016/j.ab.2016.01.013

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Abstract

We demonstrate a method for developing real-time polymerase chain reaction (PCR) high-resolution melt (HRM) assays to identify multiple species present in a mixture simultaneously using LCGreen Plus and melt temperatures. Highly specific PCR primers are designed to yield amplicons with different melt temperatures for simple routine species identification compared with differentiating melt curve kinetics traces or difference plots. This method is robust and automatable, and it leads to savings in time and reagent costs, is easily modified to probe any species of interest, eliminates the need for post-PCR gel or capillary electrophoresis in routine assays, and requires no expensive dye-labeled primers.

Keywords

• Real-time PCR;
• High-resolution melt;
• SYBR Green I;
• LCGreen plus

Abbreviations used

• PCR, polymerase chain reaction;
• HRM, high-resolution melt;
• T_m, melt temperature;
• SNP, single nucleotide polymorphism;
• CS, Cannabis sativa (marijuana);
• DS, Datura stramonium (jimson weed);
• MT, Merremia tuberosa (Hawaiian woodrose);
• IP, Ipomoea purpurea (morning glory);
• PS, Papaver somniferum (opium poppy);
• SE, Salmonella enterica substrain typhimurium;
• EC, Escherichia coli;
• SF, Shigella flexneri;
• NCBI, National Center for Biotechnology Information;
• ATCC, American Type Culture Collection;
• IPC, internal positive control

Corresponding author.

Copyright © 2016 Elsevier Inc. All rights reserved.