This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.
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.
Low-cost, quick, and easy to use systems for the simultaneous detection of multiple nucleic acids are urgently needed in a wide range of fields, such as clinical diagnostics1,2,3, GMO detection4,5,6, microbial monitoring7,8,9, forensic analysis10,11, and especially point-of-care tests (POCTs), where resources are usually limited12,13,14.
Polymerase chain reaction (PCR), including its derivative methods real-time PCR and multiplex PCR, is the most widely applied technique for detection in these fields. However, these methods typically only detect one target in one test15 and they require electricity and sophisticated professional equipment.
Another promising technology for detecting nucleic acids is Loop-mediated isothermal amplification (LAMP), which was first described in 200016. LAMP is a high efficiency DNA detection method. Theoretically, it can amplify from 1 copy to 109 copies of amplicons within one hour, all performed at a constant temperature, (i.e., between 60 – 65 °C). Successful amplification will produce a large amount of the insoluble byproduct pyrophosphate and cause a change in turbidity17, which could be directly observed by the naked eye. A color change can also be observed by the addition of metal ions or fluorescent dyes such as Calcein18, Nucleic acid dye19, and hydroxyl naphthol blue20. Because of the advantages of high sensitivity and convenience of operation, LAMP is being widely applied in nucleic acid detection.
Currently, there are mainly two strategies for multiplex LAMP assays. One is to perform multiple LAMP assays by having multiple LAMP primer sets in one tube21,22,23. However, the multiplicity and the amplification efficiency would be limited by the intrinsic interference and competition among different primer sets. Furthermore, it can be difficult to identify different LAMP products in the same reaction. Another strategy is based on physical isolation. Different primer sets were isolated into individual miniaturized compartments, and multiple LAMP reactions are then performed simultaneously24,25. These approaches, which are generally based on microfluidic chips, provide a potential solution for high-throughput LAMP reactions. However, the manufacture of the chips and the multiplex pre-coating of primer sets is complicated, which may increase costs and decrease reproducibility.
Recently, a few studies have described performing LAMP reactions in capillaries to bypass the complicated fabrication of microfluidic chips and have achieved low-cost detection26,27. However, with regards to high-throughput analysis, these capillaries are similar to miniature versions of PCR strip tubes, because the samples and reaction reagents (including the different primer sets) must be individually prepared and delivered to different reaction units within capillaries. To achieve parallel and multiplex analysis, additional equipment, for example a multichannel syringe pump, is required for parallel loading of samples or reagents.
To overcome the limitations associated with the current methods for multiplex detection of nucleic acids, we have developed a miniaturized platform which combines visual LAMP technology with a capillary array. This platform is multi-target, compact in size, low cost, and easy to operate28. Herein, we describe the details of how to fabricate the capillary array and perform the LAMP reactions in the array. The protocol described here has been standardized using genetically modified organism (GMO) detection as a model. Importantly, this protocol can also be used in high-throughput detection of other nucleic acid targets.
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.
The CALM platform demonstrated here, which combines the LAMP technology with a capillary array, enables the simultaneous detection of multiple GMO-related gene targets in a single, highly effective and easy to operate test.
To successfully perform the multiplex LAMP reactions in the cassette, three critical points need to be noticed. Firstly, achieving the same height for the upper side of the capillaries and the hydrophilic and hydrophobic pattern of the capillary array are critical for simultaneously loading reagents into all the capillaries. The capillaries should be aligned by a plate after initial inserting into the PDMS support to ensure all of them can touch the loaded reaction mixture in the dish of the loading adaptor. When treating the capillary array with the super-hydrophobic coat, do not allow the coat to soak into the inner surface of the capillaries. Treat the outer surface of the capillaries by just loading coat on the top surface of the top PDMS support, allowing the coating to spread to the outer surfaces of the upper parts of the capillaries. If it occasionally happens that not all the capillaries are filled with the reagents, it may be prudent to load reagent directly into a specific capillary.
Secondly, be careful with the preparation of LAMP reagents. The whole process should be gently and quickly carried out on ice, due to the relatively low reaction temperature of LAMP and the fragility of the Bst polymerase. It's advisable to shake the reaction tube with the LAMP mixture gently instead of vortexing the tube violently after adding Bst polymerase. An inappropriate handling may cause failure of the reaction. In this experiment, ADH1(endogenous reference gene of Maize) is set as the positive control and two capillaries without primers are set as blank controls. So, if LAMP mixture is correctly handled, the positive control would show green and the blank control would remain unchanged after the test is performed.
Thirdly, due to the high efficiency of the LAMP reaction, false positive or carryover contamination may happen if there is a trace amount of DNA amplicon in the environment. Therefore, always bear in mind that the operating processes of pre-reaction and post-reaction should be strictly separated in different areas and the LAMP products should be tightly sealed and discarded after analysis. Furthermore, a blank control should be set for indicating an adequate performance of the LAMP.
Intrinsically, CALM is a universal multi-target nucleic acid detection method with good flexibility and expandability. LAMP reactions performed in capillaries can be easily substituted by other types of isothermal amplification methods, such as rolling circle amplification (RCA)29, and recombinase polymerase amplification (RPA)30. It can also be applied in other detection fields, such as pathogen detection27 and disease diagnosis31.
In the current form of this method, although primer sets are pre-fixed with chitosan, the process of LAMP mixture preparation is still needed when the test is performed, which lowers the simplicity of the method. To address this, we would try to preload all the reagents of LAMP in the capillary and then pipette the sample into the cassette. Another limitation for our method may be the lack of nucleic acid extraction, but we are now developing a mobile-based machine which would combine the extraction of nucleic acids, automatic image taking, and data analysis to achieve a real "sample-in and results-out" method.
In summary, we have developed the CALM platform by integrating multiplex LAMP with a capillary array. As a general nucleic acid detection method, CALM may also have potential in a wide range of other nucleic acid analysis applications.
The authors have nothing to disclose.
This study was funded in part by National Natural Science Foundation of China Grants (31370813, 3147670, 31670831 and 31600672,), the National Transgenic Plant Special Fund (2016ZX08012-003, 2016ZX08012-005), Program for New Century Excellent Talents in University, the National Key Research and Development Project of China (2016YFA0500601) and China Postdoctoral Science Foundation (2016M591667).
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 |