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).

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
<ol>
	<li>Clean the stainless-steel mold.
	<ol>
		<li>Wash the stainless-steel mold by d...

<ol>
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D., Kessel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+Ebola+tells+us+about+outbreak+diagnostic+readiness.">What Ebola tells us about outbreak diagnostic readiness.</a> Nat Biotechnol. 33 (5), 464-469 (2015).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+universal+multiplex+PCR+strategy+for+100-plex+amplification+using+a+hydrophobically+patterned+microarray.">A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray.</a> Lab Chip. 11 (21), 3609-3618 (2011).</li><li>Notomi, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+of+DNA.">Loop-mediated isothermal amplification of DNA.</a> Nucleic Acids Res. 28 (12), (2000).</li><li>Mori, Y., Nagamine, K., Tomita, N., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+loop-mediated+isothermal+amplification+reaction+by+turbidity+derived+from+magnesium+pyrophosphate+formation.">Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation.</a> Biochem Biophys Res Commun. 289 (1), 150-154 (2001).</li><li>Tomita, N., Mori, Y., Kanda, H., Notomi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+(LAMP)+of+gene+sequences+and+simple+visual+detection+of+products.">Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products.</a> Nat Protoc. 3 (5), 877-882 (2008).</li><li>Iwamoto, T., Sonobe, T., Hayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loop-mediated+isothermal+amplification+for+direct+detection+of+Mycobacterium+tuberculosis+complex,+M-avium,+and+M-intracellulare+in+sputum+samples.">Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M-avium, and M-intracellulare in sputum samples.</a> J Clin Microbiol. 41 (6), 2616-2622 (2003).</li><li>Goto, M., Honda, E., Ogura, A., Nomoto, A., Hanaki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorimetric+detection+of+loop-mediated+isothermal+amplification+reaction+by+using+hydroxy+naphthol+blue.">Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue.</a> Biotechniques. 46 (3), 167-172 (2009).</li><li>Iseki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+a+multiplex+loop-mediated+isothermal+amplification+(mLAMP)+method+for+the+simultaneous+detection+of+bovine+Babesia+parasites.">Development of a multiplex loop-mediated isothermal amplification (mLAMP) method for the simultaneous detection of bovine Babesia parasites.</a> J Microbiol Methods. 71 (3), 281-287 (2007).</li><li>Liang, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+loop-mediated+isothermal+amplification+detection+by+sequence-based+barcodes+coupled+with+nicking+endonuclease-mediated+pyrosequencing.">Multiplex loop-mediated isothermal amplification detection by sequence-based barcodes coupled with nicking endonuclease-mediated pyrosequencing.</a> Anal Chem. 84 (8), 3758-3763 (2012).</li><li>Shao, Y., Zhu, S., Jin, C., Chen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+multiplex+loop-mediated+isothermal+amplification-RFLP+(mLAMP-RFLP)+to+detect+Salmonella+spp.+and+Shigella+spp.+in+milk.">Development of multiplex loop-mediated isothermal amplification-RFLP (mLAMP-RFLP) to detect Salmonella spp. and Shigella spp. in milk.</a> Int J Food Microbiol. 148 (2), 75-79 (2011).</li><li>Fang, X., Chen, H., Yu, S., Jiang, X., Kong, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+viruses+accurately+by+a+multiplex+microfluidic+loop-mediated+isothermal+amplification+chip.">Predicting viruses accurately by a multiplex microfluidic loop-mediated isothermal amplification chip.</a> Anal Chem. 83 (3), 690-695 (2011).</li><li>Stedtfeld, R. 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The authors have nothing to disclose.

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 simul...

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 &#176;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.

<table>
	<tbody>
		<tr>
			<td>UltraEverDry(super-hydrophobic coat)</td>
			<td>UltraTech</td>
			<td>4001</td>
			<td>supplier:Exiron chemistry(CHINA) CO.,LTD.</td>
		</tr>
		<tr>
			<td>PDMS</td>
			<td>Dow Corning</td>
			<td>8332557</td>
			<td></td>
		</tr>
		<tr>
			<td>Bst polymerase</td>
			<td>New England BioLabs</td>
			<td>M0275L</td>
			<td></td>
		</tr>
		<tr>
			<td>betain</td>
			<td>Sigma-Aldrich</td>
			<td>B0300-1VL</td>
			<td></td>
		</tr>
		<tr>
			<td>calcein</td>
			<td>Sigma-Aldrich</td>
			<td>C0875-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>MnCl2</td>
			<td>Sigma-Aldrich</td>
			<td>MKBP0495V</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>New England BioLabs</td>
			<td>B1003S</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Shanghai Sangon</td>
			<td>B804BA0022</td>
			<td></td>
		</tr>
		<tr>
			<td>chitosan</td>
			<td>Shanghai Sangon</td>
			<td>LJ0805S309J</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop 7.0 software</td>
			<td>Adobe Systems Inc., CA, USA</td>
			<td></td>
			<td>Image analysis</td>
		</tr>
		<tr>
			<td>GenePix Pro 6.1</td>
			<td>Molecular Devices, CA, USA</td>
			<td></td>
			<td>microarray analysis software</td>
		</tr>
		<tr>
			<td>AutoCAD</td>
			<td>Adobe Systems Inc.</td>
			<td></td>
			<td>3D construction software</td>
		</tr>
		<tr>
			<td>UV filter (ZWB2)</td>
			<td>YXSensing</td>
			<td></td>
			<td>supplier : taobao</td>
		</tr>
	</tbody>
</table>

visual detection of multiple nucleic acids in a capillary array

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.

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 &#34;T&#34; and &#34;U&#34;, as illustrated in Figure 3a. As...

Watch this Scientific Journal Video about Visual Detection of Multiple Nucleic Acids in a Capillary Array at JoVE.com

Visual Detection of Multiple Nucleic Acids in a Capillary Array

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 ...

The overall goal of this procedure is to fabricate a small, ready to use cassette that can be applied for visual detection of multiple nucleic acids in a single, easy to operate test. This method can help answer key questions in the multiplex nucleic acid detection field, such as genetically modified organisms, or GMO detection. To clean the capillaries, don a lab uniform, safety goggles, and chemical protective gloves.Place capillaries into a beaker. Clean the capillaries with 30 milliliters of acetone for five minutes to remove organic matter. And then, wash the capillaries with deionized water.Next, prepare the piranha solution by pouring about ten milliliters of hydrogen peroxide into the beaker. Then, add 30 milliliters of sulfuric acid into the hydrogen peroxide with slow shaking to prevent overheating. Make sure that the solution completely covers the capillaries for at least 30 minutes.Then, wash away the piranha solution with a large amount of water. Wash the capillaries with ethanol and deionized water for five minutes each. Dry the capillaries in a drying oven.Next, weigh PDMS base and gearing reagents with a ratio of one-to-one in a 50 milliliter tube. Typically, mix five grams of elastomer base with five grams of elastomer curing agent. Stir the mixture thoroughly for about five minutes with a glass rod.Place the beaker with the PDMS in a vacuum bell jar for thirty minutes for degassing. Then, pour the PDMS slowly into the cylinder of the stainless steel mold. Allow the PDMS to cure for three hours in a drying oven at 60 degrees Celsius.After curing the PDMS remove the mold by pulling out the mold with cylindar. Remove the PDMS from the cylinder by cutting the margin of the mold with a scalpel. Wash PDMS three times with ethanol and deionized water.Then, dry the PDMS support with nitrogen. Treat the lower surface of the PDMS support to be hydrophobic by soaking the PDMS support in a super hydrophobic coat for one second. Then, dry it for ten minutes at room temperature.Next, insert the cleaned four millimeter capillaries into the holes of the PDMS support, leaving zero point five millimeters of the capillaries outside the top surface of the PDMS support. Make sure the ends of the capillaries are on the same level. To treat the outer surface of the capillaries and the top surface of the PDMS support to be hydrophobic, add 15 microliters of the super hydrophobic coat on top of the surface of the PDMS support.Air dry the super hydrophobic modified capillary array. Next, add one point six microliters of the primer fixing mixture of one set of primers to fill one corresponding capillary of the capillary array according to a pre-designed order. Anchor the array in a transparent well of a standard flat bottom 96-well plate and dry the array at 60 degrees Celsius for at least two hours.To assemble the cassette, place a loading adapter onto the top of the anchored array. Make sure the dish of the adapter just covers the exposed parts of all ten capillaries. To prepare the LAMP reaction mix, add reagents to the mixture according to the text protocol and vortex the reaction mixture for five seconds after adding calcine.Gently invert the tube twenty times after Bst polymerase is added. Trace DNA that exists in the environment can be eliminated by cleaning earlier with one mole per liter of hydrochloric acid before handing the LAMP mix. Aspirate 20 microliters of the LAMP reaction mixture with a standard 100 microliter tip.Insert the tip into the inlet of the loading adapter to lock it. Gently inject the reaction mixture into the dish of adapter. The reaction mixture will quickly fill the dish, and then load the capillaries automatically through capillary force.Remove the adapter with the lock tip and seal the well by a PCR compatible transparent sealing film. Then, incubate the capillary array in an incubator at 63 degrees Celsius for one hour. To acquire images of the fluorescence emission, use a UV flashlight to excite the dissociated calcine to emit fluorescence.Capture images from the top of the capillary array by either a digital camera or a smart phone. When taking an image, ensure that the camera is zoomed in on the area of the capillaries as much as possible to get high quality, clear images. Next, open the image by image analysis software.And then select image, mold, grayscale, yes and image, mold, 16-bit, yes. Select file, save as, format, TIFF, to convert the image to 16-bit TIFF format. To extract the value of fluorescence intensity, open the microarray analysis software.Drag the 16-bit TIFF format image into the interface of the software, and then select the wavelength of 532 nanometers and a color of green to display the image. Then, create a new block to locate the fluorescence signal from the capillaries. Select tools, new blocks, and then put the number of columns and rows as one-to-one and two-to-five in the blocks and features interfaces separately.Right click and select the features model. Then, adjust the location and diameter to fit the fluorescence area of the capillaries. Select analyze to extract the value of fluorescence intensity.Finally, select file, save settings as, to save the block's documents. Then, select, file, save results as to save the fluorescence intensity results. Shown here is the layout of the capillary array.Capillaries one through eight, prefixed with LAMP primer sets. Capillaries nine and ten represent two, no primer controls. Shown here is the fluorescent photograph for the detection of genetically modified maize, MON863, where the green color represents the positive LAMP amplification.Successful amplification, only presented in expected capillaries one, six, seven and eight, without contamination among blank capillaries. This fluorescent photograph is for the detection of the GMO mix. The green color presented the positive LAMP amplification.Successful amplification only presented in expected capillaries one, four, five, six, seven and eight without contamination among blank capillaries. The fluorescent photograph shown here is for the detection of non-genetically modified maize, with the green color presenting the positive LAMP amplification. Successful amplification only presented in the expected capillary eight, and without contamination among blank capillaries.Once mastered, this detection procedure can be done in one point five hours if it is performed properly. After watching this video, you should have a good understanding of how to fabricate capillary-based cassette to perform multiple nucleic acid detection.

visual-detection-of-multiple-nucleic-acids-in-a-capillary-array

Research

JoVE Journal

Bioengineering

7.2K Görüntüleme.  Shanghai Jiao Tong University. The overall goal of this procedure is to fabricate a small, ready to use cassette that can be applied for visual detection of multiple nucleic acids in a single, easy to operate test. This method can help answer key questions in the multiplex nucleic acid detection field, such as genetically modified organisms, or GMO detection. To clean the capillaries, don a lab uniform, safety goggles, and chemical protective gloves.Place capillaries into a beaker. Clean the capillaries with 30 mill...