WO2019038706A2

WO2019038706A2 - Paper-based molecular diagnostic device and uses thereof

Info

Publication number: WO2019038706A2
Application number: PCT/IB2018/056384
Authority: WO
Inventors: Feng Li; Guan Wang; Tianyu DONG
Original assignee: Brock University
Priority date: 2017-08-24
Filing date: 2018-08-23
Publication date: 2019-02-28
Also published as: WO2019038706A3; CN107384769A; CN107384769B

Abstract

The disclosure provides a paper-based nucleic acid detecting device and measurement method. This device comprises paper layer set with pattern. This paper layer comprises a sample loading zone and a test zone. Herein, the pattern on paper layer indicates the measurement of nucleic acid by the nucleic acid detecting device. This measuring method can perform quantitative analysis of nucleic acid molecules by reading the retention distance of the chromogenic substance, the colored substance or the fluorescent substance. The device and measurement method of this disclosure can achieve rapid and accurate measurement of nucleic acid by reading the test result of the test sample.

Description

PAPER-BASED MOLECULAR DIAGNOSTIC DEVICE AND USES

THEREOF

This application claims priority from China Patent Application No. CN201710737730.3, filed August 24, 2017, the contents of which are hereby incorporated by reference in their entirety for all purposes.

TECHNICAL FIELD

This disclosure relates to the field of biological detection, in particular to a paper-based nucleic acid detecting device for quantitative analysis.

BACKGROUND

Nucleic acid testing using microfluidic paper-based analytical devices (μΡΑϋ) holds great promises for clinical uses at resource-limited and/or decentralized settings. To convert nucleic acid analysis into a paper-based format, many efforts have been made to explore paper as a low-cost and flexible fluidic transportation system that integrates nucleic acid isolation, purification, mixing, and amplification moieties into a single μΡΑϋ. This idea has been successfully exemplified in systems including lateral flow paper strips, slip layer-based "paper machines", two-dimensional paper networks, and three-dimensional paper origami. It is also of critical importance to engineer paper as unique sensing platforms to simplify and/or enhance nucleic acid detection on μΡΑϋ.

Krull et al. (Anal. Chem. 2013, 85, 7502-7511) immobilized quantum dots and nanoparticles into cellulose paper which creates a series of fluorescence resonance energy transfer (FRET)-based μΡΑϋβ for DNA detection. In addition, functional DNA polymers and hydrogels have been incorporated with paper to simplify the on-paper detection/amplification of DNA. Diverse electrochemical and electrophoretic mechanisms have also been integrated with μΡΑΟβ for the concentration and detection of nucleic acids. For example, Gong et al. (J. Am. Chem. Soc. 2015, 137, 13913-13919) has recently demonstrated direct DNA analysis on paper by integrating nanoporous membrane and ion concentration polarization (ICP) mechanism with uPAD. So far, most paper- specific nucleic acid sensing platforms rely on external power supplies or extensive modifications to achieve signal transductions.

US2009215194A1 discloses a microfluidic device provided with a body defining at least one first inlet for loading a fluid for analysis and a masking region in fluid communication with the first inlet. The analysis chamber is in fluid communication with the masking region and the interface cover is fluidly sealed over the microfluidic device. The interface cover is provided with a sealing portion corresponding to the analysis chamber, adapted to present a first configuration that opens the analysis chamber when at rest, and presents a second configuration that closes the same analysis chamber in a fluid-tight manner due to pressure.

CN104004850B discloses a detection method of paper-based microfluidic chip enhanced chemiluminescence for gene hlyA. This method comprises: pressing a screen printing plate on a chromatography paper, waxing, and then heating; the chromatography paper is naturally detached from the screen printing plate and dried; the capture probe and signal probe are designed; the pretreatment of the paper-based microfluidic chip; the DNA sample to be tested is incubated with the capture probe, and the signal probe is added to hybridize with the DNA sample to be tested and then HRP-SA was added for incubation; finally, the base solution was added to trigger enhanced chemiluminescence, and the illuminating signal is imaged and acquired by a CCD digital imaging device.

WO2016064881A1 discloses a paper-based microfluidic device for POC diagnostics. It comprises a first end and a hydrophobic substrate having a second end opposites the first end. The device includes a detection zone in a first surface on a substrate. This detection zone defines a region for sensing an analyte in a sample. This detection zone comprises a first electrode and a second electrode disposed on a first surface of the substrate and a hydrophilic ink layer disposed on the two electrodes and a region between the first and second electrodes. CN104293945B discloses a paper microf uidic comprising a reaction layer made of filter paper and at least one sample loading layer. A sample hole is arranged in the center of the sample loading layer. Several reaction holes are arranged around the sample hole and the orifices are coated with a hydrophobic material. The sample loading layer is placed on top of the reaction layer. The reaction layer is provided with a sample region corresponding to the sample hole, several reaction regions corresponding to the reaction holes, and reaction channel drawn by the hydrophobic material. Reaction zone has the same diameter as reaction hole. The diameter of sample region is smaller than that of sample hole. Reaction zone is connected to the sample region by a reaction channel whose width is gradually reduced from the reaction region to the sample region.

However, nucleic acid quantitative detection devices and methods based on visual measurement have not been disclosed. There is currently no paper chip capable of quantitatively analyzing nucleic acids by distance. Especially for molecular diagnosis of infection or chronic diseases with limited resources, there is urgent need for measuring devices or methods that are fast, accurate, and inexpensive.

SUMMARY

The disclosure provides a paper-based nucleic acid detecting device and measurement method. This device comprises paper layer set with pattern. This paper layer comprises a sample loading zone and a test zone. Herein, the pattern on paper layer indicates the measurement of nucleic acid by the nucleic acid detecting device. This measuring method can perform quantitative analysis of nucleic acid molecules by reading the retention distance of the chromogenic substance, the colored substance or the fluorescent substance. The device and measurement method of this disclosure can achieve rapid and accurate measurement of nucleic acid by reading the test result of the test sample. DESCRIPTION OF DRAWINGS

FIG. 1 is schematic illustration of the paper-based microwell device for quantitative nucleic acid analysis (Scheme SI).

FIG. 2 is quantifying varying concentrations of dsDNA target using SG-1 and paper-based microwells with (A) or without (B) cellulose component.

FIG. 3 is schematic illustrating the design and fabrication of qPDR capable of quantifying genetic STH markers by simply reading the retention distance as readout. (010: Bottom glass slide; 020: Middle layer paraffin film; 030: Top layer wax-patterned paper; 040: Test zone; 050: Sample loading zone.)

FIG. 4A is schematic illustration of the procedure for sample loading and data collection using qPDR. FIG. 4B is typical fluorescent images of dsDNA/ssDNA SG-I binding complexes captured using a smartphone camera. FIG. 4C is procedure for splitting colour channels and digitizing the image using ImageJ. FIG. 4D is typical chromatograms of DNA-SG-I complexes extracted from the fluorescence images.

FIG. 5 is retention distances of varying concentrations of SG-I from 1.25 μΜ to 40 μΜ on qPDR.

FIG. 6 is retention behavior of RG-labeled dsDNA (500 nM) and ssDNA (500 nM) on qPDR.

FIG. 7 is characterization of the retention behaviours of DNA-SG-I binding complexes on qPDR. Varying concentrations of SG-I from 1.25 μΜ to 40 μΜ were mixed with 500 nM dsDNA (A, B) or 500 nM ssDNA (C, D) and then loaded onto qPDR. Chromatograms (B, D) were extracted using ImageJ with the protocol outlined in Figure 3.

FIG. 8A is the DNA-SG-I binding curves constructed by measuring binding complexes using qPDR. For dsDNA-SG-I binding complexes, the binding curve is a function of maximum normalized fluorescence extracted from Figure 7B as a function of SG-I concentrations. For ssDNA-SG-I binding complexes, the binding curve is a function of maximum normalized fluorescence when dR> 5 mm at SG-I concentrations from 1.25 μΜ to 10 μΜ and dR > 10 mm at SG-I concentrations at 20 μΜ and 40 μΜ. The range for ssDNA-SG-I binding complex was chosen based on the result in Figure 5, where SG-I alone leads to high fluorescence at low dR range. FIG. 8B is DNA-SG-I binding curves constructed using a solution-based fluorescence turn-on assay. Each reaction contains varying concentrations 500 nM dsDNA or ssDNA and varying concentrations of SG-I in pH4 citrate buffer.

FIG. 9 is the effect of polyethylene glycol (PEG) 100,000 on the retention behaviours of DNA-SG-I binding complexes in qPDR. Varying concentrations of PEG from 0 to 10 mg/mL were mixed with 20 μΜ SG-I and 500 nM dsDNA (A, B) or 500 nM ssDNA (C, D), and then loaded into the paper device. Chromatograms (B, D) were extracted using Image J with the protocol outlined in Figure 3.

FIG. 10 is the effect of PEG 100,000 on the retention of RG-labeled dsDNA on qPDR.

FIG. 11 is Concentration dependency of the retention distance dR on the concentration of dsDNA. (A) Representative images of qPDR loaded with 20 μΜ SG-I, 1 mg/mL PEG, and varying concentrations of dsDNA from 4 nM to 250 nM or 500 nM ssDNA. (B) Chromatograms of varying concentrations of dsDNA extracted using ImageJ. (C) Retention distance determined either from chromatograms extracted using ImageJ (blue) by direct reading using naked eyes (red) as a function of dsDNA concentrations. Each error bar represents one standard deviation from triplicate analyses.

FIG. 12 is a photo of portable molecular diagnostic laboratory assembled using a smartphone-controlled thermal cycler (miniPCR, -600 USD), a blue light transluminator (-80 USD), and qPDR (-10 cents).

FIG. 13A is schematic illustrating the amplification of genetic DNA marker using PCR. DNA standards or genomic DNA samples were amplified using standard PCR protocol for 35 cycles and then mixed with 20 μΜ SG-I. The reaction mixture was then loaded onto the paper device for quantitative analyses. FIG. 13B is representative images of paper-based devices loaded with PCR amplicons of varying concentrations of DNA standards from 1 aM to 1 pM. FIG. 13C is chromatograms extracted using ImageJ for the quantitative analysis of PCR amplicons. FIG. 13D is retention distance determined either from chromatograms extracted using ImageJ (blue) by direct reading using naked eyes (red) as a function of log copy numbers of DNA standards in 2 μΐ. sample. Each error bar represents one standard deviation from triplicate analyses.

FIG. 14 is quantitative analysis of genomic DNA samples obtained from 10 Trichuris trichiura (TT) and 2 Ascaris lumbricoides (AL) worms that were collected from infected children in Honduras. A calibration curve was established by measuring varying concentrations of DNA standards from 1 aM to 1 pM (A). Copy numbers of genomic markers (β-tubulin gene) in each TT sample were then calculated using the external calibration (B). Each error bar represents one standard deviation from triplicate analyses.

FIG. 15 is representative images of PCR amplicons measured using qPDR. Samples were amplified from varying concentrations of synthetic DNA standards from 1 aM to 1 pM. The blank contains identical PCR reagent except that there was no DNA standard added.

FIG. 16 is quantitative analysis of 12 genomic samples from STH worms, include 10 TT worms and 2 AL worms (negative controls), using qPDR.

FIG. 17 is analyzing PCR amplicons from DNA standards and STH worm samples using gel electrophoresis. Lane 1: PCR amplicons from AL1; Lane 2: 20-bp DNA ladders; Lane 3-9: PCR amplicons from varying concentrations of DNA standards; Lane 10-19: PCR amplicons from 10 TT samples.

DETAILED DESCRIPTION

The first aspect of the disclosure provides a paper-based nucleic acid detecting device (quantitative paper-based DNA reader, qPDR). The detecting device includes a paper layer with a pattern. This paper layer comprises a sample loading zone and a test zone, wherein the pattern on the paper layer indicates the measurement of the nucleic acid by the nucleic acid detecting device. Preferably, the measurement is analysis of the nucleic acid molecule by reading the retention distance of the chromogenic substance, the colored substance or the fluorescent substance. More preferably, the analysis is quantitative.

In one embodiment, the paper layer of the nucleic acid detecting device of the disclosure contains cellulose. The inventors have surprisingly found that the intercalating dye and the cellulose will fluoresce under the light source as long as the nucleic acid is present in the test zone. After removal of the cellulose component, the fluorescent start-up ability of the intercalating dye for the nucleic acid will be restored. Using the above properties, the inventors firstly realized quantitative detection of nucleic acids by a paper-based nucleic acid detecting device containing cellulose.

In one embodiment, the sample loading zone of the disclosure is circular. Preferably, the inner diameter of the circular sample loading zone is 2-10 mm. More preferably, the inner diameter of the circular sample loading zone is 4-8 mm. Particularly preferably, the inner diameter of the circular sample loading zone is 6 mm.

In one embodiment, the sample loading zone of the disclosure includes the intercalating dye, which is selected one or a combination of two or more from SYBRTM Green I (SG-I), PO-PROTM-1, BO-PROTM-1, SYTOTM43, SYTOTM44, SYTOTM45, SYTOXTM Blue, POPOTM-1, POPOTM-3, BOBOTM-1, BOBOTM-3, LO-PROTM-1, JO-PROTM-1, YO-PROTM1, TO-PROTM1, SYTOTM11, SYTOTM13, SYTOTM15, SYTOTM16, SYTOTM20, SYTOTM23, TOTOTM-3, YOYOTM3, GelStar and thiazoleorange. Preferably, the intercalating dye is SYBRTM Green I.

In one embodiment, the test zone of the disclosure is linear. Preferably, the width of the linear test zone is 1-6 mm. Preferably, the width of the linear test zone is 1.5-3 mm. More preferably, the width of the linear test zone is 1.5-2.0 mm. The length of the linear test zone of the disclosure is 10-50 mm. Preferably, the length of the linear test zone of the disclosure is 20-40 mm. More preferably, the length of the linear test zone of the disclosure is 30- 40mm. It will be understood by those skilled in the art that the pattern on the paper layer of the disclosure can be designed as needed, as long as the pattern can indicate the measurement of nucleic acid by the nucleic acid detecting device. For example, the pattern includes one or a combination of two or more of a scale mark, a numerical mark, a shape indication mark, a color indication mark, and so on. Preferably, the measurement of nucleic acids in the disclosure is visual.

In an embodiment of the disclosure, the pattern is scale mark (ie, a ruler). The resolution of the scale mark can be selected according to different samples measured. For example, the resolution may be from 0.5 to 5 mm, preferably, the resolution may be from 1 to 3 mm, and particularly preferably, the resolution may be 2.0 mm. The pattern of the disclosure may be disposed on one side or both sides of the test zone, may also be disposed on the test zone. In one embodiment of the disclosure, the pattern is a graphic mark disposed on the test zone, e.g. a circle, preferably the inner diameter of the circular is 2-8 mm, preferably 4-6 mm.

Understandably, since the disclosure firstly discovers the effect of the presence of a nucleic acid on the luminescent properties of the intercalating dye and cellulose, quantitative analysis of the nucleic acid is achieved by measuring the retention distance of the dye having an amount corresponding to the amount of the nucleic acid molecule in the mixed nucleic acid.

In one embodiment, the nucleic acid detecting device further includes a bottom layer. The bottom layer may be prepared from any material to support the paper layer. For example, the bottom layer is preferably prepared from a cardboard layer, more preferably from a plastic sheet, more preferably from a glass piece, as long as the bottom layer can support the paper layer.

In one embodiment, the nucleic acid detecting device further includes a middle layer. The middle layer is preferably prepared from a paraffin film. The paraffin film of the disclosure can fix the paper chip and the glass sheet. In particular, the paraffin film is hydrophobic, and its fixing effect is superior to that of double-sided tape. In a preferred embodiment of the disclosure, the nucleic acid detecting device comprises a bottom layer, a middle layer and a top layer. The bottom layer is preferably prepared from a glass slide. The middle layer is preferably prepared from a paraffin film. The top layer is preferably prepared from a patterned paper layer. More preferably, the top layer is prepared from a wax patterned paper.

The device of the disclosure can read the test result of the test sample by naked eyes. It achieves fast and accurate measurements of various infections and chronic diseases. The device of the disclosure can be applied to molecular detection based on gene or nucleic acid, such as bacterial detection in environment. The device of the disclosure can be applied to authenticity identification of food or medicine at molecular level, such as the authenticity identification of Chinese herbal medicines and the identification of genetically modified foods, etc. The device of the disclosure can be applied to nucleic acid detection for scientific purposes, such as gene quantitative expression.

The second aspect of the disclosure provides a preparation method of paper-based nucleic acid detecting device. The nucleic acid detecting device is manufactured by a wax printing technique.

In one embodiment, the paper-based nucleic acid detecting device is prepared as followed. The pattern is printed on a paper layer. The paper layer is heated at 80-200°C to obtain a paper layer with a pattern. The pattern indicates the measurement of nucleic acid by the nucleic acid detecting device. Preferably, the heating temperature is 120-180°C. More preferably, the heating temperature is 150°C.

In one embodiment, the paper-based nucleic acid detecting device is prepared as followed. Place and stack the middle layer and the top layer on the bottom layer. They are heated at 80-200 °C for 5-120s.The top layer is a paper layer with a pattern. Preferably, the heating temperature is 80-120 °C. More preferably, the heating temperature is 110 °C. Preferably, the heating time is from 10 to 60 s. More preferably, the heating temperature is from 20 to 40 s, such as 30 s. The third aspect of the disclosure provides an application of a paper layer with a pattern in preparing a nucleic acid detecting device. The paper layer comprises a sample loading area and a test area. The pattern on the paper layer indicates the measurement of the nucleic acid by the nucleic acid detecting device. The nucleic acid detecting device can be used for disease detection, such as the detection of parasitic infection, authenticity identification of Chinese medicine, genetically modified food, and food safety.

In one embodiment, the disease detection of the disclosure is the detection of an infection or chronic disease at point-of-care. Preferably, the disease of the disclosure is soil-transmitted helminth infections. More preferably, the worm is whipworm Trichuristrichiura (TT) and/or roundworm Ascarislumbricoides (AL).

The fourth aspect of the disclosure provides a paper-based nucleic acid detecting method. The method comprises:

(1) A nucleic acid is obtained;

(2) The nucleic acid is contacted with the intercalating dye to obtain a mixture;

(3) The mixture is placed on the sample loading zone of the patterned paper layer and passes through the test zone of the patterned paper layer;

(4) Read the test results.

In one embodiment, the nucleic acid of the disclosure is DNA. More preferably, the nucleic acid is double- stranded DNA (dsDNA).

It will be understood by those skilled in the art that the way to obtain nucleic acid in the step (1) of the disclosure is conventional. For example, it can be completed by using a commercially available DNA extraction kit or PCR amplification. Thus, in one embodiment of the disclosure, the step (1) includes obtaining an amplicon by PCR amplification.

In one embodiment, the intercalating dye in the step (2) of the disclosure is selected one or a combination of two or more from SYBRTM Green I (SG-I), PO-PROTM-1, BO-PROTM-1, SYTOTM43, SYTOTM44, SYTOTM45, SYTOXTM Blue, POPOTM-1, POPOTM-3, BOBOTM-1, BOBOTM-3, LO-PROTM-1, JO-PROTM-1, YO-PROTM1, TO-PROTM1, SYTOTM11, SYTOTM13, SYTOTM15, SYTOTM16, SYTOTM20, SYTOTM23, TOTOTM-3, YOYOTM3, GelStar and thiazoleorange. Preferably, the intercalating dye is SYBRTM Green I. The concentration of the intercalating dye in the mixture can be adjusted according to the test result. Preferably, the concentration of the intercalating dye in the mixture is from 1 to 100 μΜ. More preferably, the concentration of the intercalating dye in the mixture is from 10 to 50 μΜ. Particularly preferably, the concentration of the intercalating dye in the mixture is from 15 to 20 μΜ.

In one embodiment, the nucleic acid and the intercalating dye are mixed in a buffer and the nucleic acid is contacted with the intercalating dye to obtain a mixture in the step (2). Alternatively, the buffer containing the nucleic acid is added to the sample loading zone with the patterned paper layer in the step (2). In this way, the nucleic acid is contacted with the intercalating dye to obtain a mixture. The sample loading zone contains the intercalating dye.

In one embodiment, the nucleic acid and the intercalating dye are contacted with the signal enhancer to obtain a mixture in the step (2). Preferably the nucleic acid, the intercalating dye and the signal enhancer are mixed in the buffer in the step (2). In this way, the nucleic acid is contacted with the intercalating dye to obtain a mixture. Or the buffer containing the nucleic acid is added to the sample loading zone of the patterned paper layer in the step (2). In this way, the nucleic acid, the intercalating dye and the signal enhancer are mixed to obtain a mixture. The sample loading zone contains the intercalating dye and the signal enhancer. Or the buffer containing the nucleic acid and the signal enhancer is added to the sample loading zone of the patterned paper layer in the step (2). In this way, the nucleic acid, the intercalating dye and the signal enhancer are mixed to obtain a mixture. The sample loading zone contains the intercalating dye. Preferably, the signal enhancer is a surfactant, such as polyethylene glycol (PEG), Tween (preferably Tween 20), SDS or a combination thereof. More preferably, the signal enhancer is PEG100000. The concentration of the signal enhancer in the mixture is from 0.1 mg/mL to 10 mg/mL. More preferably, the concentration of the signal enhancer is 1, 2, 3, 4 or 5 mg/niL. The most preferably, the concentration of the signal enhancer is 1 mg/mL.

In one embodiment, the patterned paper layer in the step (3) of the disclosure comprises the sample loading zone and the test zone. The pattern on the paper layer indicates the measurement of the nucleic acid by the nucleic acid detecting device. It will be understood by those skilled in the art that the time during which the mixture in the step (3)passes through the test zone of the patterned paper layer is set according to different nucleic acids. Preferably, after the mixture in the step (3) is added to the sample loading zone of the patterned paper layer, it is allowed to stand for 5 to 60 minutes, preferably 10 to 30 minutes, and particularly preferably 15 minutes.

In one embodiment, the reading of the test results in the step (4) of the disclosure comprises measurement of the nucleic acid by the pattern on the paper layer. For example, the test results are read under ultraviolet or visible light. Preferably, the test results are read under a 400-480 nm light source.

In a specific embodiment of the disclosure, the retention distance (di is read by a light source of 400-480 nm in the step (4).

Preferably, the paper-based nucleic acid detection method of the disclosure is a quantitative analysis. That is, the above object is achieved by reading the retention distance (di of a chromogenic substance, a colored substance or a fluorescent substance under a light source of 400-480 nm.

In one embodiment of the disclosure, the test result can also be read by the imaging device in the step (4). For example, take pictures of the test zone with a camera, cell phone, or other digital device and the obtained images are converted into chromatogram. Preferably, the obtained images are converted into chromatogram by dividing the color channel, digitizing into a plurality of pixels, and converting into chromatogram.

The purpose of paper-based nucleic acid detection method of the disclosure may be the disease or non-disease diagnosis. The fifth aspect of the disclosure provides a molecular diagnostic system comprised a PCR instrument, a paper-based nucleic acid detecting device, and a light source. The paper-based nucleic acid detecting device includes a patterned paper layer. The patterned paper layer comprises the sample loading zone and the test zone. Therein the pattern on the patterned paper layer indicates the measurement of nucleic acid by the nucleic acid detecting device.

In one embodiment, the molecular diagnostic system is portable.

In one embodiment, the PCR instrument is a miniPCR instrument.

In one embodiment, the light source is a blue light transilluminator or a UV lamp.

In one embodiment, the molecular diagnostic system further includes a control device. The control device is a portable computer or a smart phone.

The sixth aspect of the disclosure provides a nucleic acid detection method from soil-transmitted helminth. The method is implemented as followed:

(1) A β-tubulin genome sequence of a soil-transmitted helminthis amplified to obtain nucleic acid;

(2) The nucleic acid and the signal enhancer are contacted with the intercalating dye to obtain a mixture;

(4) Read the test results.

In one embodiment, the soil-transmitted helminth of the disclosure is the whipworm Trichuristrichiura (TT) and/or the roundworm Ascarislumbricoides (AL).

EXAMPLES

The technical solution of the disclosure will be further described in detail by mode of execution. The disclosure is not limited to the specific methods and solutions described in this article as they may vary. In addition, the terminology used herein is only for describing specific embodiments and is not intended to limit the scope of the disclosure. All technical and scientific terms and any abbreviations, except as otherwise defined, shall have the same meaning as that generally understood by the ordinary technician in the field of the disclosure. Although the methods and materials used in the embodiments of the disclosure can be similar or equivalent to those described in this article, illustrative methods, devices and materials are described here.

Unless otherwise specially stated, the terms of the disclosure have the meaning commonly used in the field.

In the disclosure, the inventors describe a new paper-based nucleic acid sensing mechanism that leverages the intrinsic chromatographic property of cellulose paper and thus eliminates the need for external energy or modifications. This sensing mechanism uniquely translates the conventionally fluorescence-based nucleic acid quantification into the visual measurement of distance as readout. Using this principle, the inventors design and fabricate a quantitative paper-based DNA reader (qPDR) for the molecular diagnosis of soil-transmitted helminth (STH) infection at resource-limited settings.

All paper-based devices in the disclosure were fabricated using the wax-printing technology. For example, Y. Lu, et al, Electrophoresis 2009, 30, 1497-1500, and E. Carrilho et al, Anal. Chem. 2009, 81, 7091-7095. Design parameters and fabrication procedures are detailed in the part of the embodiments of the disclosure. The inventors first designed a microwell device (Scheme S I, Figure 1), where varying concentrations of a model double- stranded DNA (dsDNA, 44bp) and the intercalating dye SYBR Green I (SG-I) were loaded for a fluorescence turn-on measurement. To the inventors surprise, all microwells emitted bright fluorescence under a UV lamp regardless the concentrations of dsDNA (Figure 2A). The inventors then removed the cellulose paper component, which restored fluorescence turn-on capability of SG-I for dsDNA quantification (Figure 2B). Because the fluorescence restoration of SG-I is a direct result of the limited intramolecular motion and reduced self-quenching, the observation of the inventors suggests that there might be a strong molecular interaction between SG-I and cellulose on paper. The inventors designed a thermometer- like qPDR (Scheme 1, Figure 3) to explore the chromatographic behaviors of double- stranded DNA (dsDNA), single- stranded DNA (ssDNA), and SG-I on cellulose paper. Figure 4A shows a typical procedure for sample measurement and data analysis. After the addition of a drop of sample (10 pL) containing SG-1 and/or DNA into the sample loading zone (Scheme 1), the solution was allowed to completely wick through the test zone. The device was then positioned on a handheld blue-light transilluminator (470 nm), and the retention distance (dR, defined as migration distance at 15% normalized fluorescence threshold) of the fluorescent components (SG-I or fluorophore-labeled DNA) can then be determined either by naked-eye or imaged using a smartphone camera (Figure 4B). The obtained image can further be converted into a chromatogram by first splitting color channels to enhance sensitivity (Figure 4C) and then digitalizing the test zone (1.5 mm x 30.0 mm) into 18,000 pixels with a resolution of 50 μιη per pixel (Figure 4D).

As shown in Figure 5, SG-I was strongly retained on qPDR with a maximum dR of 6.5 mm at a concentration of 40 μΜ. The fluorophore (rhodamine green) -labelled dsDNA and ssDNA show near identical retention profile on qPDR with dR of 21.0 mm and 18.5 mm, respectively (Figure 6). The inventors then compared the retention behaviours of dsDNA-SG-I and ssDNA-SG-I binding complexes (Figure 7). dsDNA was found to significantly reduce the retention of SG-I on qPDR, evidenced by the dramatic increases of dR upon mixing varying concentrations of SG-I (from 1.25 μΜ to 40 μΜ) with 500 nM unlabelled dsDNA (Figure 7A and 6B). Meanwhile, ssDNA had almost no effect on the retention of SG-I (Figure 7C and 7D). To quantitatively understand this observation, the inventors established a binding curve by plotting maximum fluorescence obtained on qPDR as a function of the concentrations of SG-I (Figure 8A) and compared to that obtained in free solution (Figure 8B). Near identical binding curves were obtained, suggesting that the retention behaviour of dsDNA-SG-I binding complexes is dominated by the affinity between the two species. It is also interesting to notice that the binding curves of ssDNA-SG-I complexes obtained using qPDR (no binding) differs significantly from that obtained in free solution (weak binding), indicating that the affinity between SG-I and ssDNA is even weaker than that between SG-I and cellulose (Figure 8).

The ability of qPDR to quantitatively discriminate dsDNA from ssDNA by simply reading the retention distance on paper makes it an ideal reader to quantify dsDNA products that are generated using polymerase-driven nucleic acid amplification techniques, such as PCR.

To fulfill the analytical potential of qPDR, the inventors first set out to maximize its ability to differentiate dsDNA from ssDNA by systematically optimizing various assay conditions. Through this process, the inventors found that polyethylene glycol (PEG) with molecular weight of 100 kDa could significantly reduce the retention of dsDNA-SG-I complex with increases in dR by as much as 1.7 fold (when [PEG] = 1 mg/mL, Figure 9A). Meanwhile, PEG also enhanced the retention of ssDNA-SG-I with a reduced d_R by 5/7 at 1 mg/mL PEG (Figure 9C). Collectively, the overall improvement of signal-to-background ratio for the detection of dsDNA from ssDNA was near 2.5 times at the optimal PEG concentration (1 mg/mL). Moreover, the use of PEG significantly altered the flow profiles of dsDNA (Figure 10) and dsDNA-SG-I binding complexes (Figure 9B), resulting in much sharper colour transitions that ensures the accurate determination of dR by naked eyes (Figure 9A).

Having identified PEG as a key signal enhancement addictive, the inventors then characterize the analytical performance of qPDR by challenging it with varying concentrations of the model dsDNA (44 bp). Figure 11 reveals a concentration dependency between the observed retention distance dR and the concentration of dsDNA. In this proof-of-principle assay, the dynamic range is 4-250 nM dsDNA and the detection limit is 2 nM. Importantly, the assay is highly specific to dsDNA and dR for 500 nM ssDNA is at the level of a blank.

Based on the promising results of qPDR for quantifying dsDNA, the inventors next explored it as a diagnostic tool for STH through the integration with PCR. To be compatible with the resource-limited settings, PCR was carried out using a smartphone-controlled handheld thermal cycler (miniPCR). A smartphone-mediated portable molecular diagnostic system that can be fit into a single backpack was thus built by assembling the miniPCR, a blue-light transluminator (or a UV lamp), and qPDR (Figure 12).

All worm samples, including Trichuris trichiura (TT) and Ascaris lumbricoides (AL), were collected on-site at the rural areas of La Hicaca, Olanchito, Honduras, where STH prevalence in children is over 50%. Genomic DNA samples were then isolated from TT and AL worms that were expelled from school age children who had received chemotherapy. A pair of primers was designed to specifically amplify a 164-bp gene fragment on β-tubulin gene of TT. This gene fragment containing codon 200 has been well validated for genotyping and drug resistance tests. Figure 13 shows a typical result of quantifying PCR amplicons of varying concentrations of 164-bp synthetic DNA standard from 1 aM (-10 copies in 2 sample) to 1 pM using qPDR. At primer concentrations of 500 nM each, a near linear standard curve was obtained when plotting dR as a function of log copy numbers of DNA standard (Figure 13D). The dynamic range is from 1 aM (-10 copies) to 1 fM (-104 copies) and the detection limit is 1 aM.

A wider linear range from 10 aM (-102 copies) to 100 fM (-106 copies) was obtained by reducing the primer concentrations to 250 nM each (Figure 14A and 15). Using this calibration curve, we quantified 10 TT genomic samples (Figure 14B and 16). The inventors also included 2 AL genomic samples as negative controls. All 10 TT samples were found to be positive with varying levels of β-tubulin marker and the 2 AL samples were both negative. These results were further validated by carrying out a head-to-head comparison with gel electrophoresis (Figure 16). Despite the consistent results with gel electrophoresis, the inventors device enables accurate visual quantification and is much faster and cheaper, which are critical for POC diagnosis at resource-limited settings.

In conclusion, the inventors have successfully developed a paper-based quantitative device and assembled a portable molecular diagnostic system towards the on-site diagnosis and management of STH at resource-limited settings. The working principle that harnesses the differential chromatographic behaviours of SG-I and DNA-SG-I binding complexes on cellulose paper is novel and universal. Therefore, qPDR is not limited to STH and can in principle be applied as a simple molecular diagnostic tool to any disease of interest by simply switching a set of PCR primers. In addition to PCR, qPDR that quantitatively differentiates dsDNA from ssDNA is also fully compatible with any other isothermal nucleic acid amplification reaction that generates new DNA strands, such as loop-mediated isothermal amplification (LAMP) and rolling circle amplification (RCA). Collectively, qPDR is affordable (10 cent per device) (A), sensitive (S), specific (S), user-friendly (sample-in answer-out format) (U), rapid (turn-around time within minutes) (R), equipment-free (capable of distance-based quantification) (E), and deliverable to users (portable laboratory fitting into a single backpack) (D). By meeting such ASSURED criteria, the inventors believe that qPDR will find wide applications to the diagnosis of diverse infectious and chronic diseases at point-of-care (POC).

Example 1.

Materials and reagents. Whatman qualitative filter paper (Grade 1), glass slides, paraffin film, sodium citrate (NasCeHsC ), Citrate acid solution, hydrochloric acid (HC1), sodium hydroxide (NaOH), lOxPBS buffer, TWEEN 20 solution, Polyethylene glycol (PEG) 100,000, and 10,000 x SYBR Green I (SG-I) dye were purchased from Sigma (Oakville, ON, Canada). Taq 2 x PCR Master Mix, Ammonium persulfate (APS), N, N, N', N' tetramethyllethylenediamine (TEMED), 40% acrylamide/bis-acrylamide solution, and DNA loading buffer were purchased from Bio-Rad Laboratories, Inc. (Mississauga, ON, Canada). NANOpure H₂0 (> 18.0 ΜΩ), purified using an Ultrapure Milli-Q water system, was used for all experiments. All synthetic DNA samples (Table S I) were purchased from Integrated DNA Technologies (Coralville, IA) and purified using standard desalting.

Table 1. Synthetic DNA sequences and modifications.

Name Sequence 5'-AA ATT CGC AGT CCC CAA CCT CC A

Sense

ATC ACT CAC CAA CCT CCT GTC-3'

44-bp model Rhodamine Green (RG) 5'-RG-AA ATT CGC AGT CCC CAA CCT DNA -labeled Sense CC A ATC ACT CAC CAA CCT CCT GTC-3'

5'-GAC AGG AGG TTG GTG AGT GAT TGG

Antisense

AGG TTG GGG ACT GCG AAT TT-3'

F-Primer 5'-AGG TTT CAG ATA CAG TTG TAG-3'

R-Primer 5' -CAA ATG ATT TAA GTC TCC G-3'

5'-AGG TTT CAG ATA CAG TTG TAG A AC CAT ATA ATG CAA CTC TGT CAG TCC

ΤΤ-ΌΝΑ ACC AGT TGG TAG AGA ACA CGG ACG

DNA Standard AAA CAT TCT GCA TAG ATA ATG AAG

CGC TTT ACG ATA TTT GTT TCC GAA CTT TGA AGT TAA CAA CAC CAA CTT ACG GAG ACT TAA ATC ATT TG-3'

Genomic DNA samples of soil-transmitted helminth (STH). STH worm samples were recovered from eight school-age children infected with Trichuris trichiura in the rural region of La Hicaca located in the northwestern area of Honduras. Ethical approval was obtained by the National Autonomous University of Honduras and by Brock University. The eight participants received a treatment scheme based on pyrantel pamoate and oxantel pamoate (Conmtel™) during the first 3 days and Albendazole during a fourth day. The adult worms expelled in faeces were washed with saline solution and stored in 70% ethanol. Following the recovery of specimens, DNA was extracted using the Automate Express™ DNA Extraction System (Thermo Fisher Scientific Inc.) with the commercial kit PrepFiler Express BTA™, according to the manufacturer's protocol.

Buffer conditions. DNA stock solutions were prepared by dissolving oligonucleotides using deionized water and then stored at -20 °C. Concentrations of DNA stock solutions were determined by measurement of absorbance at 260 nm using a Thermo Spectronic Unicam UV-visible spectrophotometer. Unless indicated otherwise, 100 mM citrate buffer at a pH of 4 was used as the assay buffer.

Device design and fabrication. All paper-based devices were first designed using graphic design software CorelDraw x8. Each quantitative paper-based DNA reader (qPDR) was designed to have thermometer-like format (Scheme- 1) containing a circular sample loading zone (6 mm inner diameter) and a linear testing zone (2.0 mm in width x 36 mm in length). A ruler with a resolution of 2.0 mm was marked along the testing zone to facilitate the naked-eye measurements of the retention distance. Once designed, the patterns were first printed onto cellulose papers using a XEROX ColorQube 8580 solid ink printer and then heating on a hotplate at 150 °C for 40 seconds. The device was then fabricated by stacking the patterned paper and a layer of paraffin film on a glass side (Scheme 1). This sandwiched device was then bonded by heating on the hotplate at 110 °C for 30 seconds. After the fabrication process, the final width of the testing channel was determined to be 1.5 mm.

Nucleic acid quantification using qPDR. A typical reaction mixture (10 μΐ,) containing varying concentrations of target nucleic acids, 20 μΜ SG-I, and 1 mg/mL PEG 100,000 in citrate buffer was first incubated at room temperature for 5 min and then added into the sample loading zone of qPDR. After sample loading, qPDR was left at room temperature for 15 min to allow the reaction mixture to completely wick through the testing zone. The device was then placed on the top of a blue -light transluminator for visual measurements or photography. Digital photos taken by smartphone (Nexus 6P) camera were then transferred to computer for quantitative data analysis using ImageJ.

Because SG-I emits bright green fluorescence, the inventors split each image into three individual color channels and then picked up the green channel image to enhance signal-to-background ratio. This image was further digitalized into 30 x 600 pixels at a resolution of 50 μιη per pixel and the fluorescence for each pixel was measured in the form of greyscale intensity. The image was further converted into a chromatogram, which is the normalized fluorescence as a function of migration distance d. The fluorescence at each d was determined by first averaging 30 pixels in width and then normalized the fluorescence against the maximum grayscale value (set to 100) and background grayscale value (set to 0). The background grayscale values were obtained from the empty channel adjacent to the end of the flow (typically between dR= 25 mm to 30 mm). To determine the retention distance dR for each sample, we set a normalized fluorescence value of 15 as a threshold, so that each dR corresponds to the migration distance of the fluorescence species when its normalized fluorescence value equals to 15. This threshold was determined by recruiting 59 student volunteers to read 3 dsDNA samples and 3 ssDNA samples (Table 2), from which the inventors found the dR determined by the threshold of 15 was closest to those determined by naked eyes.

Table 1. Retention distances of 3 dsDNA samples (Sl-3) and 3 ssDNA samples

(Bl-3) on qPDR read by 59 student volunteers.

20.1 20.4 22.1 5.7 6.1 6.3

20.0 22.0 22.0 6.0 6.0 8.0

20.0 22.0 23.0 6.0 6.0 8.0

19.0 22.0 24.5 7.0 6.0 8.0

19.0 22.0 22.0 5.0 5.0 6.5

20.0 22.0 21.0 7.0 6.0 7.0

19.0 21.0 23.0 6.0 6.0 7.0

20.0 22.0 23.0 6.0 6.0 8.0

20.0 22.0 22.0 5.0 6.0 7.0

20.0 22.0 22.0 6.0 6.0 8.0

18.0 22.0 22.0 6.0 6.0 6.0

18.0 20.0 22.0 6.0 6.0 8.0

20.0 22.0 23.0 6.0 6.0 7.0

18.9 21.9 22.5 4.1 4.9 6.2

13.0 16.1 18.3 4.5 5.2 6.4

20.0 20.1 21.0 5.8 6.0 7.6

20.0 22.0 24.0 6.0 6.0 8.0

18.0 20.0 22.0 4.0 6.0 8.0

20.0 24.0 26.0 6.0 6.0 8.0

18.5 22.5 23.5 5.9 6.1 7.0

20.0 24.0 26.0 7.0 8.0 8.0

20.0 23.0 23.0 5.0 6.0 7.0

20.0 22.0 24.0 6.0 6.0 7.0

20.0 23.0 23.5 6.0 6.0 7.5

16.2 22.0 22.8 7.5 6.4 7.4

20.1 24.5 23.9 6.2 5.7 8.3

18.5 22.0 22.5 5.9 6.3 6.9

19.0 22.0 22.0 5.0 5.0 6.5 49 18.9 22.0 22.5 5.0 5.8 6.5

50 18.8 23.0 22.0 6.0 5.9 7.0

51 16.0 20.0 22.0 6.0 6.0 7.0

52 18.3 22.0 23.9 6.1 6.3 8.1

53 16.0 21.5 22.0 4.5 5.0 6.0

54 19.8 21.2 21.5 6.7 7.8 7.5

55 20.0 22.0 22.0 5.0 6.0 8.0

56 18.1 22.3 23.5 5.6 6.1 7.9

57 20.0 22.0 22.0 6.0 6.0 8.0

58 20.0 23.0 24.0 6.0 6.0 7.0

59 20.0 22.0 23.0 6.0 6.0 8.0

Average 18.7 21.5 22.9 6.0 6.1 7.5

Normalized

Fluorescence 13.9 17.3 14.3 12.8 17.3 9.7 at d_R

Average 15.18 13.30

Quantification of STH samples and PCR amplicons using qPDR. qPDR was validated and optimized for the quantification of soil-transmitted helminths (STH) infection. A fragment of 164 bp of β-tubulin genomic sequence from whipworm, Trichuris trichiura (TT) was amplified using a smartphone-controlled portable thermal cycler (MiniPCR™). The PCR mixture contained ~2 ng of TT genomic DNA, primers at a final concentration of 0.25 μΜ each and lx Taq master mix. The thermal cycles included an initial incubation at 94 °C for 3 min, followed by 35 cycles of (denaturation at 94 °C, annealing at 42 °C and extension at 72 °C for 30 seconds each) and a final extension at 72 °C for 5 min. A standard curve was generated by measuring varying concentrations of a 164-bp synthetic β-tubulin DNA standard from 1 aM to 1 pM using the same PCR protocol. Two Ascaris lumbricoides (AL) genomic samples were also included as negative controls. After PCR, each amplicon was mixed with SG-I at a final concentration of 20 μΜ and PEG 100,000 at a final concentration of 1 mg/mL and incubated at room temperature for 5 min. The PCR amplicon was then loaded and quantified using qPDR using the protocol outlined above. For STH samples, the total genomic DNA was first quantified using a SG-I-mediated fluorescence turn-on assay and external calibration. Specifically, varying concentrations of synthetic dsDNA standard and 2 μΐ, genomic DNA sample were each mixed with 2 μΜ SG-I in 1 x PBS buffer. The reaction mixtures were then transferred into a 96-well microplate and a multi-mode microplate reader (SpectraMax i3, Molecular Devices) was used to measure fluorescence for each sample or standard. A calibration curve was constructed using the DNA standard and the sample was quantified using the external calibration.

It will be apparent to those skilled in the art that although this article describes the specific embodiments of the disclosure for illustration, while various modifications could be made without departing from the spirit and scope of the disclosure. Therefore, the specific embodiments of the disclosure should not limit the scope of the disclosure. The disclosure is only limited by the appended claim.

REFERENCES

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3. J. T. Connelly, J. P. Rolland, G. M. Whitesides, Anal. Chem. 2015, 87, 7595-7601.

4. L. K. Lafleur, J. D. Bishop, E. K. Heiniger, et al. Lab Chip 2016, 16, 3777-3787.

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6. a) M. O. Noor, U. J. Krull, Anal. Chem. 2013, 85, 7502-7511; b) M. O. Noor, U. J. Krull, Anal. Chem. 2014, 86, 10331-10339; c) S. Doughan, U. Uddayasankar, A. Peri, U. J. Krull, Anal. Chim.Acta 2017, 962, 88-96.

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8. a) X. Li, K. Scida, R. M. Crooks, Anal. Chem. 2015, 87, 9009-9015; b) J. C. Cunningham, N. J. Brenes, R. M. Crooks, Anal. Chem. 2014, 86, 6166-6170; c) X. Li, L. Luo, R. M. Crooks, Lab Chip 2015, 15, 4090-4098.

9. M. M. Gong, R. Nosrati, M. C. San Gabriel, A. Zini, D. Sinton, J. Am. Chem. Soc. 2015, 137, 13913-13919.

10. J. Bethony, S. Brooker, M. Albonico, S. M. Geiger, A. Loukas, D. Diemert, P. J. Hotez, The Lancet 2006, 367, 1521-1532.

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SEQUENCE LISTING

< 110> Brock University

<120> Paper-based molecular diagnostic device and uses thereof <130> 20180820

<160> 5

< 170> Patentin version 3.5

<210> 1

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 1

aaattcgcag tccccaacct ccaatcactc accaacctcc tgtc

<210> 2

<211> 44

<212> DNA

<213> Artificial Sequence

<400> 2

gacaggaggt tggtgagtga ttggaggttg gggactgcga attt

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence <400> 3

aggtttcaga tacagttgta g 21

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 4

caaatgattt aagtctccg

19

<210> 5

<211> 164

<212> DNA

<213> Artificial Sequence

<400> 5

aggtttcaga tacagttgta gaaccatata atgcaactct gtcagtccac cagttggtag 60 agaacacgga cgaaacattc tgcatagata atgaagcgct ttacgatatt tgtttccgaa 120 ctttgaagtt aacaacacca acttacggag acttaaatca tttg 164

Claims

1. A paper-based nucleic acid detecting device comprises a patterned paper layer, wherein the patterned paper layer comprises a sample loading zone and a test zone, wherein the pattern on the paper layer indicates the measurement of nucleic acid by the nucleic acid detecting device, and the patterned paper layer contains cellulose.

2. The paper-based nucleic acid detecting device of claim 1, wherein the pattern comprises one or more marks, wherein the mark is selected from a scale mark, a numerical mark, a shape indication mark and a color indication mark.

3. The paper-based nucleic acid detecting device of claim 2, wherein the pattern is a scale mark that has a resolution of 0.5-5 mm.

4. The paper-based nucleic acid detecting device of claim 1, wherein the sample loading zone is circular, preferably, the inner diameter of the circular sample loading zone is 2-10 mm.

5. The paper-based nucleic acid detecting device of claim 1, wherein the sample loading zone includes one or more intercalating dyes, wherein the intercalating dye is selected from the group consisting of SYBRTM Green I (SG-I), PO-PROTM-1, BO-PROTM-1, SYTOTM43, SYTOTM44, SYTOTM45, SYTOXTM Blue, POPOTM-1, POPOTM-3, BOBOTM-1, BOBOTM-3, LO-PROTM-1, JO-PROTM-1, YO-PROTM1, TO-PROTM1, SYTOTM11, SYTOTM13, SYTOTM15, SYTOTM16, SYTOTM20, SYTOTM23, TOTOTM-3, YOYOTM3, GelStar and thiazoleorange.

6. The paper-based nucleic acid detecting device of claim 1, wherein the test zone is a linear test zone, preferably, the width of linear test zone is 1-6 mm.

7. The paper-based nucleic acid detecting device of claim 1, wherein the nucleic acid detecting device further includes a bottom layer.

8. The paper-based nucleic acid detecting device of claim 7, wherein the bottom layer is cardboard layer, plastic sheet or glass sheet.

9. The paper-based nucleic acid detecting device of claim 1, wherein the nucleic acid detecting device further includes a middle layer.

10. The paper-based nucleic acid detecting device of claim 1, wherein the middle layer is paraffin film.

11. The paper-based nucleic acid detecting device of claim 1, wherein the nucleic acid detecting device comprises a bottom layer, a middle layer and a top layer, wherein the bottom layer is a slide glass, a cardboard layer, a plastic sheet or a glass sheet, and the middle layer is a double- sided tape or a paraffin film, wherein the top layer is a patterned paper layer.

12. The method of preparation the paper-based nucleic acid detecting device of claim 1, wherein the preparation comprises: printing the pattern on a paper layer, heating at 80-200 °C, and obtaining a pattern paper layer.

13. The method of claim 12, wherein the preparation comprises placing the middle layer and the top layer on the bottom layer and heating at 80-200 °C for 5-120 s, wherein the top layer is a patterned paper layer.

14. A use of the patterned paper layer for preparing a nucleic acid detecting device, wherein the patterned paper layer comprises a sample loading zone and a test zone, wherein the pattern on the paper layer indicates measurement of nucleic acid by the nucleic acid detecting device, wherein the nucleic acid detecting device is used for disease detection, authenticity identification of Chinese medicine, detection of genetically modified foods or food safety.

15. The use of claim 14, wherein the detection of disease is the detection of an infection or a chronic disease at point-of-care.

16. The use of claim 15, wherein the detection of disease is a soil-transmitted helminth infection.

17. A paper-based nucleic acid detecting method, wherein the method comprises:

(1) a nucleic acid is obtained;

(4) the test results are read.

18. The paper-based nucleic acid detecting method of claim 17, wherein the nucleic acid is dsDNA.

19. The paper-based nucleic acid detecting method of claim 17, wherein the step (2) includes contacting the nucleic acid, the intercalating dye, and the signal enhancer.

20. The paper-based nucleic acid detecting method of claim 19, wherein the signal enhancer is a surfactant.

21. The paper-based nucleic acid detecting method of claim 20, wherein the signal enhancer is polyethylene glycol, Tween, SDS or a combination thereof.

22. A molecular diagnostic system comprises a PCR instrument, a paper-based nucleic acid detecting device, and a light source, wherein the paper-based nucleic acid detecting device includes a pattern paper layer, where the pattern paper layer comprises the sample loading zone and the test zone, wherein the pattern on the paper layer indicates the measurement of nucleic acid by the nucleic acid detection device.