Fig 1.
Schematic presentation of the mechanism of mLAMP.
Table 1.
The fluorophores and detection channels.
Table 2.
Primer and FD probe sequences for the mLAMP reaction.
Fig 2.
The effect of the ratio of FD-FIP composite probe to unlabeled primer on FMDV amplification.
The mLAMP reaction was optimized by using FMDV-specific primers and an FD-FIP composite probe (labeled with FAM and BHQ1). The products were imaged by using the 520 nm channel (A), and the turbidity curve was generated by a real-time turbidimeter (B). The total amount of FIP in each reaction was maintained at 0.8 μM and composed of various ratios of FD-FIP to unlabeled FIP as follows: 1: FD-FIP: unlabeled FIP = 0; 3: 25%; 5: 50%; 7: 75%; 9: 100%, 2, 4, 6, 8, 10 were the negative controls for the corresponding proportions. With the increase in the FD-FIP ratio, the inhibition became more severe with a slower amplification time. At FD-FIP concentrations equal to or higher than 75%, the turbidity curve and fluorescence increase were not observed, suggesting that the reaction was completely inhibited at these concentrations. An equimolar ratio (50%) was used to balance the fluorescence signal and amplification rate for FMDV amplification in the mLAMP reaction.
Fig 3.
The effect of the labeling position of the fluorophore on the mLAMP reaction.
The mLAMP products amplified by using FMDV-specific composite probes with fluorophores/quenchers labeling different positions were imaged using a 520 nm channel (A), and the turbidity curve was monitored by a real-time turbidimeter (B). 1: The FD-FITC/FIP-BHQ1 composite probe is composed of FD labeled with FITC at the 3’ end and FIP labeled with BHQ1 at the 5’ end, and the amplification product showed a robust fluorescence signal with a shorter initial reaction time. 2: For the FD-BHQ1/FIP-FITC composite probe with the quencher and fluorophore position reversed, the initial reaction time was slightly longer than that of composite probe 1. 3: The FD-FAM/FIP-BHQ1 composite probe had a similar fluorescence signal and initial reaction time as composite probe 1. 4: BD-FAM/BIP-BHQ1 composite probe labeled with fluorophore at the BIP terminus. 5: Both the FIP and BIP termini were labeled with fluorophores (FD-FAM/FIP-BHQ1, BD-FAM/BIP-BHQ1). Composite probes 4 and 5 inhibited mLAMP with a weak fluorescence signal compared to that of 1, 2 and 3.
Fig 4.
The effect of FD length on the mLAMP reaction.
The mLAMP products amplified by using FMDV-specific composite probes with different lengths of FD were imaged using a 520 nm channel (A), and the turbidity curve was monitored by a real-time turbidimeter (B). 1: FD1 consists of 22 bases and is completely complementary to FMDV-F1c with a TM value of 64.2°C, and the initial reaction time is 57.3 min; 2: FD2 consists of removing 3 bases from the 5’ end of FMDV-FD1 with a TM value of 57.1°C, and the initial reaction time is 37.2 min; 3: FD3 consists of removing 6 bases from the 5’ end of FD1 with a TM value of 50.9°C, and the initial reaction time is 34.2 min; 4: FMDV-FD4 consists of removing 9 bases from the 5’ end of FMDV-FD1 with a TM value of 39.4°C, and the initial reaction time is 28.3 min; 5–8: negative controls of FD1-4.
Fig 5.
The effect of different strand-displacing enzymes on the mLAMP reaction.
The fluorescent mLAMP products were imaged separately using multiple channels (A), and the turbidity was monitored by a real-time turbidimeter (B). Amplification time dependence upon different strand-displacing enzymes with different working amounts. Bst 2.0 WarmStart showed shorter amplification times than Bst 3.0 in the mLAMP reaction. 1: Without addition of enzyme, the reaction was completely inhibited without any amplification; 2: each reaction with 8 U Bst 2.0 WarmStart had an initial amplification time of 52 minutes, 3: each reaction with 16U Bst 2.0 WarmStart had an initial amplification time of approximately 36 minutes, 4: each reaction with 3U Bst 3.0 had an initial amplification time of 55 minutes, 5: each reaction with 16U Bst 3.0 had an initial amplification time of 42 minutes, 6: negative control.
Fig 6.
The fluorescent mLAMP products were imaged separately using multiple channels (A). The turbidity curve was generated by a real-time turbidimeter to interpret the process of amplification (B). Green fluorescence (FAM) indicates FMDV-positive amplification, red fluorescence (Cy5) indicates VSV-positive amplification, and blue fluorescence (Cy3) indicates BTV-positive amplification. Overlapping fluorescence indicates multiple positive amplifications. Lane 1: FMDV A, lane 2: FMDV O, lane 3: FMDV Asia I, lane 4: VSV IND, lane 5: VSV ND, lane 6: BTV 1, lane 7: BTV 2, lanes 8: FMDV A+ VSV IND + BTV1; 9–15: PPRV, EHDV, SVDV, BVDV, MB, IBRV, negative control.
Table 3.
Probit analysis to determine the detection limit of mLAMP.
Table 4.
Performance of the mLAMP assay for detection in clinical samples.