Device for reliably and quickly responding to coding card to realize multiple virus detection

Abstract

The invention discloses a device for realizing multiple virus detection by a reliable and quick response coding card. The device comprises the quick response coding card and an intelligent photographing and result reading system, when the virus RNA does not exist in the sample, the hydrogel seals the paper-based pores in the paper-based pores, so that the color developing solution stops flowing and cannot reach the color developing layer, and the result is blue; on the contrary, in the presence of a target, the hydrogel is dissociated due to complementation of virus RNA and a gel cross-linking sequence, free fluid is allowed to flow to carry a color developing solution to a color developing layer, and the result is yellow; different codes are combined according to color results generated by negative and positive sample reactions, and a text result report is calculated and output by a built-in algorithm, that is, naked eye judgment is replaced by computer vision, and the detection accuracy is improved, so that the method can be used for detecting various targets.

Description

Technical Field

[0001] The invention relates to the field of biological detection, and in particular to a device for realizing multiple virus detection by reliably and quickly responding to a coding card. Background Art

[0002] The visual colorimetric detection technology in POCT (Point-of-Care Testing) uses color changes as detection signals to analyze the concentration or presence of target substances in samples visually or with the assistance of instruments. This detection method is often based on color changes caused by chemical or biological reactions, such as enzyme-catalyzed reactions or colorimetric reactions of nanomaterials. Its advantages are simple operation, low cost, no need for complex equipment, and it is suitable for environments with limited resources. Colorimetric detection is often combined with platforms such as test strips and microfluidic chips to achieve qualitative or semi-quantitative analysis through immune response or nucleic acid detection. This technology has been widely used in fields such as blood glucose monitoring, infectious disease screening, and environmental monitoring, but its accuracy is limited by the detection environment and sample processing conditions, and colorimetric detection relying on the naked eye still has subjective bias, which reduces the accuracy of detection.

[0003] The development of high-throughput POCT (Point-of-Care Testing) detection technology aims to solve the problems of low throughput and limited detection speed of traditional POCT detection, especially in response to large-scale screening, disease monitoring and public health emergencies. In recent years, researchers have been committed to combining technologies such as microfluidic chips, multi-channel colorimetric detection, nanomaterials and biosensors to improve the throughput and sensitivity of POCT detection.

[0004] Microfluidic chip technology is the core of current high-throughput POCT research. By integrating multiple channels on a single chip, it can realize the simultaneous detection of multiple samples and improve detection efficiency. The introduction of nanomaterials, such as gold nanoparticles and carbon nanotubes, can enhance colorimetric, fluorescent and electrochemical signals, further improving detection sensitivity and specificity. In addition, the combination of automated instruments and mobile devices (such as smartphones) enables POCT testing to have stronger processing capabilities and data reading functions in complex sample processing.

[0005] High-throughput POCT has shown broad prospects in the rapid detection of infectious pathogens, cancer marker screening and environmental pollution monitoring. Future research will focus on developing a higher-throughput detection platform to achieve simultaneous detection of multiple biomarkers, further shorten detection time and reduce costs to meet the diverse needs of clinical and public health. Summary of the invention

[0006] In view of this, an object of the present invention is to provide a device that can reliably and quickly respond to coded cards to achieve multiple virus detection.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] 1. A device that can realize multiple virus detection by reliable and fast response coding cards, including fast response coding cards and intelligent camera reading result system;

[0009] The rapid response coding card consists of four layers from top to bottom, namely the top PVC hollow sample well layer with sample area, reaction area and channel, the second layer corresponding to the PVC hollow sample well layer with DNA hydrogel loaded on the paper base, the third layer is the paper-based color development layer, and the bottom is the PVC coding color card with hollowed-out PVC corresponding to the top sample area and reaction area.

[0010] The intelligent photographing and reading result system comprises a photographing system and a result calculating system. The system recognizes the actual RGB value of the color block according to the picture obtained by the photographing system, performs color correction on the picture according to the actual RGB value and the standard RGB value of the calibration block, and obtains the corrected picture. The RGB value of the specified range of the detection hole is extracted from the color-corrected picture, and then the color of the detection hole is judged to be positive or negative in turn.

[0011] Preferably, the upper end of the PVC coding color card is designed with five standard color blocks, namely yellow, blue, red, green and black.

[0012] Preferably, the paper-based color developing layer is a glucose color developing test paper.

[0013] Preferably, the reaction paper base of the DNA hydrogel adopts Whatman filter paper, the hydrophilic area corresponds to the sample area, reaction area and channel hollow area at the top; the hydrophobic area is processed by a Phaser 8560 wax printer, the reaction area is pre-loaded with chromogenic glucose, and then loaded with DNA hydrogel.

[0014] Preferably, the response sequence of the DNA hydrogel is designed to use 20nt target short RNAs in the constant coding regions of four viruses, H1N1H1N1, Influenza BV, Rhinovirus, and RSV-A, as virus trigger sequences.

[0015] Preferably, the specific process of the judgment is as follows:

[0016] (1) pre-define the standard negative and standard positive RGB values ​​according to the two RGB values ​​with the highest accuracy in the recognition process of the actual reaction color;

[0017] (2) The RGB data of the predefined standard negative, standard positive and detection wells are converted into LAB space data through nonlinear substitution;

[0018] (3) Calculate the extreme color difference value in the LAB color space. Select the two extreme colors of black and white in a broad sense for calculation. Their RGB values ​​are a(0, 0, 0) and b(255, 255, 255) respectively. After converting them into LAB space data, a(L, A, B) is a(0, 0, 0) and b(L, A, B) is b(100, 0, 0). According to the formula d=√((Lb-La)2+(Ab-Aa)2+(Bb-Ba)2), the color difference value of the two colors is calculated, which is defined as the extreme color difference value d max ; Where Lb, Ab, Bb are the values ​​of L, A, B corresponding to a(L, A, B), La, Aa, Ba are the values ​​of L, A, B corresponding to b(L, A, B);

[0019] (4) The test hole in LAB space is defined as c(L, A, B), the standard positive is 1(97, -21, 94), and the standard negative is 2(86, -14, -8). According to the formula d1 = √((Lc-L1)2 + (By-A1)2 + (Bz-B1)2) or d2 = √((Lc-L2)2 + (By-A2)2 + (Bz-B2)2), the color difference d1 between the test hole and the standard negative and the color difference d2 between the test hole and the standard positive are calculated respectively. The data values ​​converted into LAB space according to the standard negative, standard positive and detection wells, where Lc, By and Bz are L, A and B in c(L, A, B), L1, A1 and B1 are 97, -21 and 94 corresponding to the standard positive 1 (97, -21, 94), and L2, A2 and B2 are 86, -14 and -8 corresponding to the color difference d2 (86, -14, -8) of the standard positive;

[0020] (5) Calculate the similarity with the negative color block and the positive color block respectively by the color difference value with the standard negative and standard positive. The formula is S1 = (dmax-d1) / dmax; S2 = (dmax-d2) / dmax. Obtain the negative similarity S1 and the positive similarity S2, compare S1 and S2, and take the larger value as the reaction result color of the detection well. Finally, combine them into a coded color development result and output a text report.

[0021] The beneficial effects of the present invention are as follows: the present invention provides a device for realizing multiple virus detection by reliably and rapidly responding to coding cards, and proposes a unique rapid response coding card, which realizes reliable visual detection of multiple pathogens by encoding different color development results combined with computer vision correction judgment. By using a smartphone to record the color changes on the back of the paper to construct a coding card, accurate detection of multiple viral RNA molecules can be achieved. Traditional QR code paper microfluidic detection often destroys the QR code structure, resulting in impaired built-in error correction capabilities, while our design utilizes simplified QR code rules, which can potentially avoid the risk of false negative or false positive results. Since the target-responsive hydrogel uses nucleic acid sequences as signal recognition elements, this method is generally applicable to a variety of targets; this method provides a new strategy for simple, reliable, and instant detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration:

[0023] Figure 1 Encoding card structures for rapid response;

[0024] Figure 2 Schematic and physical pictures of the response cards;

[0025] Figure 3 This is an illustration of combining computer vision recognition and judgment;

[0026] Figure 4 A practical diagram of implementing virus detection for the response card. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0028] In the present invention, DNA and RNA primers were purchased from Sangon Biotech (Shanghai) Co., Ltd.; Duplex DNase and RNase H were purchased from New England Biolabs (Beijing) LTD (Number: M7635S), and the colorimetric solution standard glucose solution was purchased from Shanghai Yuanye Biotechnology Co., Ltd. (Number: B24407).

[0029] Example 1: Building a Quick Response Coded Card

[0030] Quick response coding card structure such as Figure 1As shown, it includes a sample area, a reaction area, and a channel connecting the sample area and the reaction area, wherein the reaction area is located in the middle and the sample area is located around the reaction area. The quick response coding card has four layers from top to bottom, namely, the top PVC hollow sample well layer for setting the sample area, reaction area and channel, the second layer corresponding to the PVC hollow sample well layer for loading DNA hydrogel, the third layer is the paper-based color development layer, and the bottom is the PVC coding color card corresponding to the top sample area and reaction area.

[0031] The paper base loaded with DNA hydrogel uses Whatman filter paper, and the hydrophilic channel and hydrophobic area are designed. The hydrophilic area corresponds to the sample area, reaction area and hollow area of ​​the channel at the top; the hydrophobic area is processed by Phaser 8560 wax printer, and the reaction area is pre-loaded with chromogen (glucose), and then loaded with DNA hydrogel, wherein the response sequence of DNA hydrogel is L, connecting X and Y on the gel skeleton to form a DNA cross-linking network. The 20nt target short RNA in the constant coding region of four viruses H1N1H1N1, Influenza BV, Rhinovirus, and RSV-A is designed as the virus trigger sequence, and the DNA hydrogel response probe R is pre-combined with the short chain S, and the target RNA is completely matched with S. Under the action of DSN enzyme, the response probe R is released by continuous chain displacement, and R further dissociates L to achieve the dissociation of DNA hydrogel, so as to produce different color results for coding analysis.

[0032] The specific sequence is as follows:

[0033] H1N1 X: 5'-TTGAAGAGGTGCTG-3' (SEQ ID NO. 1);

[0034] H1N1 Y: 5'-CACTCAGCGAACAA-3' (SEQ ID NO. 2);

[0035] H1N1 L: 5'-CGATAGTCAGCACTCTTCAATTGTTCGCTGAGTG-3' (SEQ ID NO.3);

[0036] H1N1 R: 5'-GGGAATCACTCAGCGAACAATTGAAGAGTGCTGACTATCG-3' (SEQ ID NO.4);

[0037] H1N1 S: 5'-TTGTTCGGTCCCATTCCC-3' (SEQ ID NO.5);

[0038] Influenza BV X: 5'-TTGAAGAGGTGCTG-3' (SEQ ID NO. 6);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0039] <h2 style=";text-align:left;direction:ltr"> Influenza BV Y:5'-CACTCAGGACAA-3'(SEQ ID NO.7);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0040] <h2 style=";text-align:left;direction:ltr"> Influenza BV L:5'-GTGAGTCCTGTTAACTTCTCACGACTGATAGC-3'(SEQ ID NO.8);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0041] <h2 style=";text-align:left;direction:ltr"> Influenza BV R:5'-AAGCAC TACTCAC GAGACAA TTGAAGAGTGCTGACTATCG-3'(SEQID NO.9);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0042] <h2 style=";text-align:left;direction:ltr"> Influenza BV S:5'-TTGTCTCCATTAGGGTGCTT-3'(SEQ ID NO.10);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0043] <h2 style=";text-align:left;direction:ltr"> RSV RNA<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0044] <h2 style=";text-align:left;direction:ltr"> RSV RNA Y:5'-CACTCAGACATCCC-3'(SEQ ID NO.12);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0045] <h2 style=";text-align:left;direction:ltr"> RSV RNA L:5'-GTGAGTTCTGTAGGGAACTTCTCACGACTGATAGC-3(SEQ ID NO.13);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0046] <h2 style=";text-align:left;direction:ltr"> RSV RNA R:5'-TTGGAACAATTGGACATCCCTTGAAGAGTGCTGACTATCG-3'<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0047] <h2 style=";text-align:left;direction:ltr"> (SEQ ID NO.14);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0048] <h2 style=";text-align:left;direction:ltr"> RSV RNA S:5'-GGGATGTGTGTAATTTCCAA-3'(SEQ ID NO.15);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0049] <h2 style=";text-align:left;direction:ltr"> Rhinovirus<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0050] <h2 style=";text-align:left;direction:ltr"> Rhinovirus Y:5'-CACTCAGGAATGTG-3'(SEQ ID NO.17);<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0051] Rhinovirus L: 5'-GTGAGTCCTTACACAACTTCTCACGACTGATAGC-3' (SEQ IDNO.18);

[0052] Rhinovirus R: 5'-TCCTCCCAATTAGGAATGTGTTGAAGAGTGCTGACTATCG-3' (SEQ IDNO.19);

[0053] Rhinovirus S: 5'-AGGAGGCCGGGGACTTACAC-3' (SEQ ID NO. 20).

[0054] The loading method is as follows: prepare a 10-fold concentrated hydrogel buffer (TAE-Mg 2+ ), the steps are as follows: 20 mL of 50× TAE buffer, 12.5 mL of magnesium acetate, and 67.5 mL of nuclease-free water for a total of 100 mL. For a typical preparation of gel precursor chains X and Y, 180 μL reactions were performed in 1.5 ml microcentrifuge tubes. For a 4% (wt / vol) acrylamide gel, one gel precursor for chain X (PA-X) and one gel precursor for chain Y (PA-Y) were prepared. The following reagents were added in the following order. 10× TAE-Mg 2+ Buffer 18 μL; 3 mM X or Y 60.0 μL; 40% (wt / vol) acrylamide 18.0 μL Nuclease-free water 37.5 μL Total 85.5 μL. Briefly spin the microcentrifuge tube at 2000 g (room temperature) for 3 seconds to collect its contents. Place the opened tube under a vacuum oven for 10 minutes to degas the solution. During the incubation period, prepare a fresh stock of 2% (wt / vol) APS in 1 ml nuclease-free water and a fresh stock of 20% (vol / vol) TEMED in 0.5 mL nuclease-free water. Add 4.5 μL of APS stock solution to the polymerization reaction centrifuge tube (PA-X, PA-Y), invert the tube several times to mix the reagents, and then briefly spin (2000 g, 20-23°C, 5s) to collect the tube contents. Add 4.5 μL of TEMED stock solution to the polymerization reaction centrifuge tube (PA-X, PA-Y), turn the test tube over several times to mix the reagents, and then briefly spin (2000g, 20-23°C, 5s) to collect the contents of the test tube to obtain gel precursors PA-X and PA-Y, fill 3 μL of a mixture containing PA-X, PA-Y and L (1:1:1 (vol / vol)) onto the paper substrate loaded with the chromogen, and dry at room temperature for 30 min to allow the reagents to precipitate on the paper substrate.

[0055] The paper-based color development layer selects glucose color development test paper, and the glucose color development test paper is aligned with the five sample area holes to ensure that the reaction hole is consistent with the color development hole.

[0056] Make the real thing Figure 2 shown.

[0057] Example 2: Encoded card for rapid detection of multiple pathogens

[0058] During the detection process, the reaction liquid is introduced from the top sample loading hole, and the paper base provides lateral flow force to the circular DNA hydrogel reaction area on the edge. When the viral RNA reacts, the hydrogel response probe is released, and then the DNA hydrogel is dissociated, the paper base molecular pores are unblocked to release the chromogen, and after the color development liquid is introduced, the color development layer can achieve a blue to yellow color change, forming different color combinations on the back of the card. The results can be read by taking a photo with a smartphone and a text report can be output ( Figure 3 ). The specific method is as follows:

[0059] Use a smartphone to identify the actual RGB values ​​of the six color blocks, perform color correction on the image based on the actual RGB values ​​of the calibration blocks and the standard RGB values, and obtain the corrected image; extract the RGB values ​​of the specified range of the five detection holes from the color-corrected image, and then judge the colors of the detection holes as positive or negative in turn. The judgment process is as follows:

[0060] (1) The predefined standard negative and standard positive RGB values ​​are defined based on the two RGB values ​​with the highest accuracy in the recognition process according to the actual reaction color.

[0061] (2) The predefined negative, positive, and detection hole RGB data are converted from RGB to LAB space data through nonlinear substitution, which are L, A, and B respectively. The specific conversion formula uses the CV2.cvtcolor() function.

[0062] (3) Calculate the extreme color difference value in the LAB color space. Select the two extreme colors of black and white in a broad sense for calculation. Their RGB values ​​are a(0, 0, 0) and b(255, 255, 255) respectively. After converting them into LAB space data, a(L, A, B) is a(0, 0, 0) and b(L, A, B) is b(100, 0, 0). According to the formula d=√((Lb-La)2+(Ab-Aa)2+(Bb-Ba)2), the color difference value of the two colors is calculated, which is defined as the extreme color difference value d max ; Among them, Lb, Ab, Bb are the corresponding values ​​of L, A, B in a(L, A, B), and La, Aa, Ba are the corresponding values ​​of L, A, B in b(L, A, B).

[0063] (4) The test hole in LAB space is defined as c(L, A, B), the standard positive is 1(97, -21, 94), and the standard negative is 2(86, -14, -8). According to the formula d1 = √((Lc-L1)2 + (By-A1)2 + (Bz-B1)2) or d2 = √((Lc-L2)2 + (By-A2)2 + (Bz-B2)2), the color difference d1 between the test hole and the standard negative and the color difference d2 between the test hole and the standard positive are calculated respectively. The data values ​​converted into LAB space are obtained according to the standard negative, standard positive and detection wells, where Lc, Ay and Bz are L, A and B in c(L, A, B), respectively; L1, A1 and B1 are 97, -21 and 94 corresponding to the standard positive 1 (97, -21, 94); L2, A2 and B2 are 86, -14 and -8 corresponding to the color difference d2 (86, -14, -8) of the standard positive.

[0064] (5) Calculate the similarity with the negative color block and the positive color block respectively by the color difference value with the standard negative and standard positive. The formula is S1 = (dmax-d1) / dmax; S2 = (dmax-d2) / dmax. Obtain the negative similarity S1 and the positive similarity S2, compare S1 and S2, and take the larger value as the reaction result color of the detection well. Finally, combine them into a coded color development result and output a text report.

[0065] This example detects H1N1 infected samples in mixed samples, and the results are as follows Figure 4 The results show that the virus can be accurately detected.

[0066] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A device for realizing multiple virus detection by reliable and rapid response coding card, characterized in that: Includes quick response coded cards and intelligent camera reading result system; The rapid response coding card consists of four layers from top to bottom, namely the top PVC hollow sample well layer with sample area, reaction area and channel, the second layer corresponding to the PVC hollow sample well layer with DNA hydrogel loaded on the paper base, the third layer is the paper-based color development layer, and the bottom is the PVC coding color card with hollowed-out PVC corresponding to the top sample area and reaction area. The intelligent photographing and reading result system comprises a photographing system and a result calculating system. The system recognizes the actual RGB value of the color block according to the picture obtained by the photographing system, performs color correction on the picture according to the actual RGB value and the standard RGB value of the calibration block, and obtains the corrected picture. The RGB value of the specified range of the detection hole is extracted from the color-corrected picture, and then the color of the detection hole is judged to be positive or negative in turn.

2. The device for realizing multiple virus detection by reliable and fast response coding card according to claim 1, characterized in that: The upper end of the PVC coding color card is designed with five standard color blocks, namely yellow, blue, red, green and black.

3. The device for realizing multiple virus detection by reliable and fast response coding card according to claim 1, characterized in that: The paper-based color development layer is a glucose color development test paper.

4. The device for realizing multiple virus detection by reliable and fast response coding card according to claim 1, characterized in that: The reaction paper base of the DNA hydrogel adopts Whatman filter paper, the hydrophilic area corresponds to the sample area, reaction area and channel hollow area at the top; the hydrophobic area is processed by Phaser 8560 wax printer, the reaction area is pre-loaded with chromogenic glucose, and then loaded with DNA hydrogel.

5. The device for realizing multiple virus detection by reliable and fast response coding card according to claim 4, characterized in that: The response sequence of the DNA hydrogel is designed with 20nt target short RNA in the constant coding region of four viruses: H1N1H1N1, Influenza BV, Rhinovirus, and RSV-A as the virus trigger sequence.

6. The device for realizing multiple virus detection by reliable and fast response coding card according to claim 1, characterized in that: The specific process of the judgment is as follows: (1) pre-define the standard negative and standard positive RGB values ​​according to the two RGB values ​​with the highest accuracy in the recognition process of the actual reaction color; (2) The RGB data of the predefined standard negative, standard positive and detection wells are converted into LAB space data through nonlinear substitution; (3) Calculate the extreme color difference value in the LAB color space. Select the two extreme colors of black and white in a broad sense for calculation. Their RGB values ​​are a(0, 0, 0) and b(255, 255, 255) respectively. After converting them into LAB space data, a(L, A, B) is a(0, 0, 0) and b(L, A, B) is b(100, 0, 0). According to the formula d=√((Lb-La)2+(Ab-Aa)2+(Bb-Ba)2), the color difference value of the two colors is calculated, which is defined as the extreme color difference value d max ; Where Lb, Ab, Bb are the values ​​of L, A, B corresponding to a(L, A, B), La, Aa, Ba are the values ​​of L, A, B corresponding to b(L, A, B); (4) The test hole in LAB space is defined as c(L, A, B), the standard positive is 1(97, -21, 94), and the standard negative is 2(86, -14, -8). According to the formula d1 = √((Lc-L1)2 + (By-A1)2 + (Bz-B1)2) or d2 = √((Lc-L2)2 + (By-A2)2 + (Bz-B2)2), the color difference d1 between the test hole and the standard negative and the color difference d2 between the test hole and the standard positive are calculated respectively. The data values ​​converted into LAB space according to the standard negative, standard positive and detection wells, where Lc, By and Bz are L, A and B in c(L, A, B), L1, A1 and B1 are 97, -21 and 94 corresponding to the standard positive 1 (97, -21, 94), and L2, A2 and B2 are 86, -14 and -8 corresponding to the color difference d2 (86, -14, -8) of the standard positive; (5) Calculate the similarity with the negative color block and the positive color block respectively by the color difference value with the standard negative and standard positive. The formula is S1 = (dmax-d1) / dmax; S2 = (dmax-d2) / dmax. Obtain the negative similarity S1 and the positive similarity S2, compare S1 and S2, and take the larger value as the reaction result color of the detection well. Finally, combine them into a coded color development result and output a text report.

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