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. 2016 May 18;7:753. doi: 10.3389/fmicb.2016.00753

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Copyright © 2016 Wang, Wang, Zhang, Liu, Luo, Li, Cao, Liu, Xu and Ye.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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The real-time analysis of multiplex targets in an ET-MCDA reaction. Two sets of ET-MCDA primers targeting invA and ipaH genes were simultaneously added to a reaction tube. (A,B) were simultaneously yielded from Cy5 (labeling Sal-E-CP2 of invA) and Hex (labeling Shi-E-CP1 of ipaH) channels, respectively. The analytical sensitivity of multiplex-ET-MCDA for simultaneously detecting Salmonella (A) and Shigella (B) was analyzed by real-time format, and signals 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, and 13 represent DNA levels of 25 ng, 2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, 25 fg, 12.5 fg, 6.25 fg, 3.125 fg, 1.56 fg, and 0.78 fg per tube and negative control. The LoD of multiplex ET-MCDA assay for Salmonella detection was 6.25 fg per reaction, and the LoD of multiplex ET-MCDA for Shigella detection was 3.125 fg per reaction.