NOTE: This protocol assumes that the stainless steel mold bearing the shape for the desired micro-channels and the loading adaptor have already been made (3D files are provided as Supplemental Files 1 and 2). This protocol also assumes that plant DNA isolation has already been carried out.
1. Fabrication of the Capillary Array-based Ready-to-use Cassette
LAMP primer fixing mix components (initial concentration) | Volume (μL) |
ddH2O | 17.0 |
Chitosan (1.3%) | 1.0 |
FIP/BIP primer (20 μM) | 2.0/2.0 |
LoopF/LoopB primer (20 μM) | 1.0/1.0 |
F3/B3 primer (20 μM) | 0.5/0.5 |
Total volume | 25.0 |
Table 1: The components of LAMP primer fixing reagents. The components of LAMP primer fixing mix are listed in the left column of the table, and the volume of each component is listed in the right column.
Figure 1: The cassette fabrication and assembly. (a) Stainless mold and the PDMS support. The mold consists of 3 parts: cylinder, dam board, and pillar plate. (b) The schematic of PDMS support fabrication and assembly of the capillary cassette. The whole process contains 5 steps: 1. PDMS pouring, 2. Mold removing, 3. Capillary inserting and surface coating, 4. primer fixing and 5. cassette anchoring. 1. Pour PDMS into cylinder of the mold; 2. Push out the dam board to remove the mold from PDMS support; 3. Coat the down surface of PDMS support and then insert the capillaries into the PDMS support, lastly coat the upper surface of PDMS support and exposed capillaries. The thick blue line indicates super-hydrophobic coat; 4. Load primer set into individual capillaries; 5. Anchor the capillary array in a single 96-well plate and install a sample loading adaptor onto it. The details have been described in protocol steps 1.1 – 1.9. Please click here to view a larger version of this figure.
2. Performance of LAMP Reaction in Capillary Array
LAMP components (initial concentration) | Volume (μL) |
ddH2O | 11.6 |
MgSO4 (100 mM) | 2.0 |
dNTPs (25 mM) | 1.4 |
Betaine (5 M) | 4.0 |
Buffer (10x) | 2.5 |
Calcein (1.25 mM) | 0.5 |
MnCl2 (25 mM) | 0.5 |
Bst polymerase (8 U/μL) | 1.5 |
plant DNA (10 ng/μL) | 1.0 |
Total volume | 25.0 |
Table 2: The reaction system of the capillary array-LAMP. The components of the reaction system of capillary array LAMP components are listed in left column, and the volume of each component is listed in the right column.
Figure 2: Diagram of sample-loading using the loading adaptor. The picture shows the loading process employing blue solution as an example. Insert the tip into the inlet and inject the sample into the adaptor slowly, and then remove the adaptor with locked tip. Please click here to view a larger version of this figure.
3. Results Readout and Data Analysis
In this method, it is important to prevent cross-contamination among different capillaries during the sample loading. For this purpose, chitosan was introduced, which could retain the primers in individual capillaries. To test whether it worked or not, we pre-fixed the ADH1 (endogenous reference gene of maize) primer set in the capillary cassette with the pattern of "T" and "U", as illustrated in Figure 3a. As expected, only the capillaries contained primer sets showing positive signals (Figure 3b).
Figure 3: Examples of capillary result. (a) The layout of the capillary array. The green spots indicate that ADH-1 primer sets are pre-fixed in capillaries. (b) Fluorescent photographs of the two capillary arrays after LAMP. The green color presented the positive LAMP amplification. The test was performed in duplicate. Successful amplification only presented in primer-fixed capillaries and without contamination among blank capillaries. Please click here to view a larger version of this figure.
To further evaluate the ability to monitor GMO, we chose seven frequently-used transgenic elements which cover ~75% of the commercialized GMO events (i.e., P-CaMV35S, bar, cp4 epsps, P-FMV35S, pat, T-nos and nptII) (Figure 4, layout) which correspond to the capillaries number 1 – 7, and one endogenous reference gene for maize (ADH1). To show the specificity of this method, three GM events (MON863, MON89034, and 59122) were selected and applied to CALM. To analyze the results, fluorescence images were taken by camera and analyzed as described in the protocol steps. Expected results were obtained for all the tests (Figure 4). For example, for the GM maize MON863, positive signals were obtained for capillaries 1, 6, 7, and 8, which correspond to targets P-CaMV35S, T-nos, nptII, and the endogenous reference gene ADH1, respectively.
Figure 4: The specificity for monitoring GMOs. The detection of different DNA samples, i.e., MON863, MON89034, 59122, mix of all the above three GM events (GMO mix), Non-GM maize and pure water (no template control, NTC). 1 – 8: Capillaries pre-fixed with LAMP primer sets of P-CaMV35S, bar, cp4 epsps, P-FMV35S, pat, T-nos, nptII, and ADH-1, individually. 9 – 10, two no-primer controls. The test was performed in duplicate and we found that all the results were consistent with expectations. See Supplementary Table 2 for data analysis Please click here to view a larger version of this figure.
To test the performance of our method in a real world application, two practical maize samples were selected for analysis by CALM. Then the results were compared with that of real-time PCR and results were consistent (Figure 5, Supplementary Table 2)
Figure 5: The results of testing two maize samples. 1 – 8: Capillaries pre-fixed with the LAMP primer set of P-CaMV35S, bar, cp4 epsps, P-FMV35S, pat, T-nos, nptII, and ADH-1, individually. 9 – 10, two no-primer controls. The test was performed in duplicate and then the results were compared with that of real-time PCR and the results were consistent. See Supplementary Table 3 for comparison results. See Supplementary Table 2 for data analysis. Please click here to view a larger version of this figure.
Supplementary Table 1: List of LAMP primer sequences. Please click here to download this file.
Supplementary Table 2: Data analysis of the LAMP array experiments. The green color coded cells in the table indicate the positive result. Please click here to download this file.
Supplementary Table 3: The real-time PCR results reporting form. Please click here to download this file.
UltraEverDry(super-hydrophobic coat) | UltraTech | 4001 | supplier:Exiron chemistry(CHINA) CO.,LTD. |
PDMS | Dow Corning | 8332557 | |
Bst polymerase | New England BioLabs | M0275L | |
betain | Sigma-Aldrich | B0300-1VL | |
calcein | Sigma-Aldrich | C0875-5G | |
MnCl2 | Sigma-Aldrich | MKBP0495V | |
MgSO4 | New England BioLabs | B1003S | |
dNTPs | Shanghai Sangon | B804BA0022 | |
chitosan | Shanghai Sangon | LJ0805S309J | |
Photoshop 7.0 software | Adobe Systems Inc., CA, USA | Image analysis | |
GenePix Pro 6.1 | Molecular Devices, CA, USA | microarray analysis software | |
AutoCAD | Adobe Systems Inc. | 3D construction software | |
UV filter (ZWB2) | YXSensing | supplier : taobao |
Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.
Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.
Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.