Multiple Endonuclease Restriction Real-Time Loop-Mediated Isothermal Amplification  Yi Wang, Yan Wang, Ruiting Lan, Huaqing Xu, Aijing Ma, Dongxun Li, Hang Dai, Xuejiao Yuan, Jianguo Xu, Changyun Ye  The Journal of Molecular Diagnostics  Volume 17, Issue 4, Pages 392-401 (July 2015) DOI: 10.1016/j.jmoldx.2015.03.002 Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Outline of multiple endonuclease restriction real-time loop-mediated isothermal amplification (MERT-LAMP) reactions. A: Schematic depiction of a new forward/backward inner primer (EFIP/EBIP). EFIP/EBIP, which was an extension of the forward (F)/backward (B) inner primer (FIP; F1c + F2)/(BIP; B1c + B2) with an endonuclease recognition site (Es, its complementary sequence named CEs) at the 5′ end, was modified with a 5′-fluorophore (F) and a quencher (Q) in the middle. B: Outline of MERT-LAMP reactions, with core MERT-LAMP primers EFIP (Es + F1c + F2), backward (B) inner primer (BIP; B1c + B2), F3, and B3. For clarity, loop forward and loop backward primers are not shown. 1. MERT-LAMP initiates at the F2c sequence of the target, and this new strand is displaced by upstream synthesis from the F3 primer. 2. The BIP anneals to the B2c site in the newly synthesized strand. 3. The primer annealed to the B2c sequence synthesizes the complementary sequence of the new strand, and Nb.BsrDI digests the new double-stranded terminal sequence (Es and CEs). 4. This process releases the quenching, resulting in a gain of signal. The newly synthesized strand is displaced by extension from the B3 primer. 5. The resulting structure undergoes the cycling amplification step in the MERT-LAMP reaction from step 5 to step 9, similar to that in LAMP, and the fluorophores are released at step 9. The products of steps 10 and 11 serve as the template for subsequent elongation and cycling steps, which give rise to additional release of fluorophores, resulting in exponential signal detection. EBIP, new backward interior primer. The Journal of Molecular Diagnostics 2015 17, 392-401DOI: (10.1016/j.jmoldx.2015.03.002) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Location and sequences of Listeria monocytogenes (lmo0733) and Listeria ivanovii (smcL) genes used to design multiple endonuclease restriction real-time loop-mediated isothermal amplification primers. The nucleotide sequences of the sense strands of lmo0733 (A) and smcL (B) are shown. The sequences of the primer sites are underlined. Right arrows and left arrows indicate sense and complementary sequences that are used. The Journal of Molecular Diagnostics 2015 17, 392-401DOI: (10.1016/j.jmoldx.2015.03.002) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Result of the multiple endonuclease restriction real-time loop-mediated isothermal amplification (MERT-LAMP) on detection of Listeria monocytogenes and Listeria ivanovii. A: Amplification products of MERT-LAMP assays are visually detected by observation of the color change: tube 1, positive amplification of L. monocytogenes reference strains (EGD-e); tube 2, negative control; tube 3, positive amplification of L. ivanovii reference strains (ATCCBAA-678); and tube 4, negative control. B: Agarose gel electrophoresis of MERT-LAMP products is shown: lane M, DNA markers DL100; lane 1, MERT-LAMP products of L. monocytogenes; lane 2, negative control; lane 3, MERT-LAMP products of L. ivanovii; and lane 4, negative control. DL, DNA ladder 100; M, marker. The Journal of Molecular Diagnostics 2015 17, 392-401DOI: (10.1016/j.jmoldx.2015.03.002) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 The optimal temperature for the multiple endonuclease restriction real-time loop-mediated isothermal amplification (MERT-LAMP) assay. The MERT-LAMP reactions were monitored by means of real-time detection, and the corresponding curves of DNA concentrations are shown. Signal 1 indicates Listeria ivanovii strains of ATCCBAA-678, and signal 2 indicates negative control. Six kinetic graphs (A–F) were obtained at different temperature (61°C to 66°C) with an L. ivanovii genomic DNA template at the level of 2.5 pg per reaction. The graphs from C to E show robust amplification. Norm. Fluoro., normal fluorescence signal intensity. The Journal of Molecular Diagnostics 2015 17, 392-401DOI: (10.1016/j.jmoldx.2015.03.002) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 The sensitivity of multiple endonuclease restriction real-time loop-mediated isothermal amplification (MERT-LAMP) assay for detecting a single target. Two sets of MERT-LAMP primer targeting the lmo0733 and smcL genes were used in distinct reactions: Listeria monocytogenes (A) and Listeria ivanovii (B). Signals 1, 2, 3, 4, 5, 6, 7, and 8 represent DNA levels of 2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, 125 fg, and 62.5 fg per reaction and negative control, and the genomic DNA levels of 125 fg and 62.5 fg per reaction and negative control provide the negative signal. The limit of detection of the MERT-LAMP methods for detecting a single target was 250 fg DNA per reaction. Norm. Fluoro., normal fluorescence signal intensity. The Journal of Molecular Diagnostics 2015 17, 392-401DOI: (10.1016/j.jmoldx.2015.03.002) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 6 The multiple endonuclease restriction real-time loop-mediated isothermal amplification (MERT-LAMP) assay for simultaneously detecting two targets. Two sets of MERT-LAMP primer targeting lmo0733 and smcL genes were used in the same reaction. A and B were simultaneously obtained from FAM (labeling EFIP of lmo0733) and HEX (labeling EFIP of smcL) channels, respectively. Signals 1, 2, 3, 4, 5, 6, 7, and 8 represent DNA levels of 2.5 ng, 250 pg, 25 pg, 2.5 pg, 250 fg, 125 fg, and 62.5 fg per reaction and negative control, and the genomic DNA levels of 125 fg and 62.5 fg per reaction and negative control provide the negative signal. The limit of detection of the multiplex MERT-LAMP assay for detecting multiple targets was 250 fg each genomic DNA per reaction. Norm. Fluoro., normal fluorescence signal intensity. The Journal of Molecular Diagnostics 2015 17, 392-401DOI: (10.1016/j.jmoldx.2015.03.002) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 7 Specificity of multiplex multiple endonuclease restriction real-time loop-mediated isothermal amplification (MERT-LAMP) detection for different strains. The multiplex MERT-LAMP amplifications were performed using different genomic DNA templates and were monitored by means of real-time detection. A and B were simultaneously obtained from FAM and HEX channels, respectively. Signals 1–12, Listeria monocytogenes strains of serovar 1/2a (EGD-e), 3a (ICDCLM023), 1/2b (ICDCLM007), 3b (ICDCLM078), 7 (NCTC10890), 1/2c (ICDCLM010), 3c (ICDCLM446), 4a (ATCC19114), 4c (ATCC19116), 4b (ICDC419), 4d (ATCC19117), and 4e (ATCC19118); signals 13 and 14, Listeria ivanovii strains of ATCCBAA-678 and ICDCLIS001; signals 15–18, other Listeria reference strains of Listeria innocua (ATCCBAA-680), Listeria seeligeri (ATCC35967), Listeria welshimeri (ATCC35897), Listeria grayi (ATCC25402); signals 19–35, non-Listeria strains of Enterobacter cloacae, Enterococcus faecalis, enteropathogenic Escherichia coli, enterotoxigenic Escherichia coli, enteroaggregative Escherichia coli, enteroinvasive Escherichia coli, enterohemorrhagic Escherichia coli, Bacillus cereus, Vibrio fluvialis, Vibrio parahaemolyticus, Yersinia enterocolitica, Streptococcus pneumoniae, Streptococcus bovis, Shigella flexneri, Plesiomonas shigelloides, Salmonella enterica, and Klebsiella pneumoniae; and signal 36, negative control. Norm. Fluoro., normal fluorescence signal intensity. The Journal of Molecular Diagnostics 2015 17, 392-401DOI: (10.1016/j.jmoldx.2015.03.002) Copyright © 2015 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions