CN116694459A

CN116694459A - Portable nucleic acid detection system

Info

Publication number: CN116694459A
Application number: CN202310718989.9A
Authority: CN
Inventors: 李峰; 董天彧
Original assignee: Sichuan University
Current assignee: Sichuan University
Priority date: 2023-06-16
Filing date: 2023-06-16
Publication date: 2023-09-05

Abstract

The invention discloses a portable nucleic acid detection system, which comprises: a nucleic acid amplification reaction tube for performing LAMP reaction on a target nucleic acid; a chip part capable of displaying the movement distance of the amplicon; the portable amplification detection integrated machine provides a reaction environment for LAMP reaction and provides illumination conditions for detection of amplicons. The method adopts a mode of reading the movement distance of the amplicon to carry out qualitative and quantitative detection analysis on the target nucleic acid, and compared with the traditional PCR and qLAMP amplification, the method has the advantages that the detection result is more robust, the operation is simpler and more convenient, and the requirements on technicians and detection equipment are low; in addition, the invention has low manufacturing cost, small volume and light weight, and is more suitable for primary hospitals, clinics and remote areas with limited medical resources.

Description

Portable nucleic acid detection system

Technical Field

The invention belongs to the technical field of gene detection in biomedicine, and particularly relates to a portable nucleic acid detection system.

Background

Nucleic acid detection is a technique for detecting genetic markers from biological or clinical samples. Genetic information of the object to be detected can be extracted and analyzed from a trace sample so as to be applied to the next detection. Nucleic acid detection technology is widely applied to a plurality of fields such as disease detection and prevention, food safety analysis, biological identification and the like, and particularly the nucleic acid detection has an indispensable position in disease detection and prevention. Since the 1985, the Polymerase Chain Reaction (PCR) was invented by Mullis, nucleic acid amplification has been widely used for various nucleic acid detection applications as a detection method excellent in performance, but PCR requires a trained person to perform the operation using a specialized laboratory apparatus, and has a series of problems in that the operation steps are complicated, and a specialized person and a specialized nucleic acid detection laboratory are required to perform the operation, so that conventional nucleic acid detection has been performed in a specialized laboratory of a general or large disease detection center. These problems limit the deployment of nucleic acid assays to the field for testing.

For the need to deploy nucleic acid detection techniques from the laboratory to the site where detection is required, the world health organization sets forth a series of requirements: reasonable price, sensitive measurement, high specificity, easy use, high speed and reliability, no need of extra equipment, and being able to be transported to the hands of the end user (ASSURED standard). However, integrating nucleic acid detection technologies together for field deployment still faces a series of problems, such as complex equipment, high risk of cross contamination in traditional operation procedures, and excessive cost of detection reagents, which are required in fluorescence-based high-precision detection technologies. Therefore, many research results have been developed to combine traditional nucleic acid detection techniques with microfluidic, nanotechnology, and synthetic biology techniques, and many in-situ detection techniques have been developed. However, these methods are complicated in operation, have the risk of cross contamination, require additional auxiliary equipment, and are difficult to apply to Point-of-care testing (POCT) on a large scale.

Thanks to the advantages of high efficiency, controllable reaction, convenient use and the like of the PCR amplification reaction, the PCR amplification reaction is still a gold standard for nucleic acid amplification so far. However, the characteristic of the temperature-changing reaction of the PCR makes the PCR highly dependent on temperature-changing equipment such as a thermal cycler, so that most of the PCR can be deployed in central hospitals and laboratories and is difficult to deploy to resource-limited areas to detect disease transmission. Therefore, a number of isothermal amplification techniques, such as loop-mediated isothermal amplification (LAMP), rolling Circle Amplification (RCA), recombinase Polymerase Amplification (RPA), strand Displacement Amplification (SDA), have been developed to compensate for the drawbacks of PCR temperature swing amplification. Wherein the LAMP reaction uses 2 to 3 pairs of primers to amplify the target at a constant temperature of 65 ℃. Compared with PCR, the LAMP reaction has the advantages of higher selectivity, more amplified DNA orders of magnitude and lower detection line, and is very suitable for detecting low-abundance disease nucleic acid targets.

Paper is considered an excellent raw material for making field-deployable nucleic acid detectors because of its low cost, ready availability and ease of processing. Paper-based microfluidic analytical devices were originally traced to the 2007 whiteside study group, which innovatively combined paper and microfluidic technology, and patterns formed by hydrophobic barriers formed by epoxy-based polymers were fabricated on paper using photolithography, and by partitioning the paper surface, simultaneous detection of multiple targets by manipulation of liquid samples was achieved. The research group of Whitesides in 2015 developed microfluidic devices that fully integrate nucleic acid detection, which successfully integrated sample preparation, nucleic acid amplification and detection on one paper-based device (paper machine). The device has very low detection lines, and can successfully detect the minimum of 5 E.coli added into human serum or as low as1 artificially synthesized DNA chain. In terms of packaging paper chip detection pathogens, the Copper research group developed an integrated paper chip system for detection of malaria infection in human blood based on nucleic acid extraction, amplification and detection of paper chips.

Although many portable nucleic acid detecting apparatuses are reported, it is often difficult to integrate all the functions of nucleic acid detection into one apparatus, and cumbersome operations and additional instrument assistance are required to quantify the concentration of nucleic acid, which undesirably increases the difficulty and cost of nucleic acid detection. In addition, open detection systems are extremely prone to aerosol cross-contamination.

Therefore, the miniaturization of standard nucleic acid detection technology, the development of a portable nucleic acid immediate detection platform which is efficient, low-cost and capable of being deployed on site, has important significance for disease detection and control. The method is not only beneficial to rapid diagnosis at the bedside of patients or doctors' offices, but also can effectively monitor and manage various diseases caused by viruses, bacteria, fungi, parasites and the like in remote areas with limited resources or epidemic situation large-scale outbreak areas.

Disclosure of Invention

In order to solve the technical problems, the miniaturization of the nucleic acid detection technology is realized, and a portable nucleic acid instant detection platform which is efficient, low in cost and capable of being deployed on site is developed.

In a first aspect, the present invention provides a portable nucleic acid detection system comprising:

a nucleic acid amplification reaction tube for performing loop-mediated isothermal amplification of nucleic acids of a sample to be detected;

the chip component can be detachably connected with the nucleic acid amplification reaction tube, the chip component comprises a paper chip, and a sample adding area and a detection area which are mutually communicated are arranged on the paper chip; fluorescent dye is preset in the sample adding area, and the fluorescent dye is chimeric fluorescent dye which can release fluorescence after being combined with DNA double chains;

after entering a sample adding area from a nucleic acid amplification reaction tube, the amplicon combined with the fluorescent dye permeates into a detection area through capillary action and can flow along the detection area, and under a blue light background, the human eye can directly observe the moving distance of the amplicon in the detection area, so that qualitative/quantitative analysis is carried out on a sample to be detected;

wherein the distance of movement of the amplicon within the detection zone is proportional to the concentration of amplicon.

Preferably, the nucleic acid amplification reaction tube is a PCR tube, and the volume of the PCR tube is selected from any one of 0.1mL, 0.15mL, 0.2mL, and 0.5mL, and more preferably 0.2mL.

Preferably, the chip component comprises a middle plastic package chip and a shell adhered to the upper surface of the plastic package chip. The shell is provided with a pipeline communicated with the nucleic acid amplification reaction tube, and the other end of the pipeline is communicated with a sample adding area on the paper chip.

Preferably, the housing is bonded to the plastic encapsulated chip by, for example, scotch tape to ensure the overall hermeticity of the chip components.

Preferably, the housing is 3D printed from transparent plastic.

Preferably, the plastic package chip comprises a middle paper chip and plastic package films attached to the upper surface and the lower surface of the paper chip. A hydrophobic sealing film is arranged between the paper chip and the lower plastic packaging film. The upper plastic packaging film is provided with a hollowed-out area which is the same as the pattern of the sample adding area and the detection area so as to expose the sample adding area and the detection area of the paper chip (the movement distance of the amplicon combined with the fluorescent dye in the detection area is convenient for sample adding and observation).

Preferably, the paper chip is based on cellulose paper, and further preferably Whatman No.1 cellulose paper is used.

Preferably, on the paper chip, the sample application zone and the detection zone are hydrophilic and the other zone is hydrophobic to define a zone of sample solution diffusion, which can be achieved by partial zone hydrophobization of the base paper, for example by penetration of melted wax into the base paper.

In one embodiment of the invention, the wax pattern is printed on the base paper, heated to melt and infiltrate the wax into the base paper to provide a wax-defined detection zone (sample application zone and detection zone in communication with each other).

Preferably, the thickness of the plastic packaging film is 100-150 μm, more preferably 110-130 μm, for example: 110 μm, 113 μm, 115 μm, 118 μm, 120 μm, 123 μm, 125 μm, 128 μm, 130 μm, more preferably 125 μm.

Preferably, the sealing film is a paraffin film, and the amplicon solution on the sample adding area and the detection area can be prevented from penetrating into the plastic sealing film at the bottom layer through the paraffin film, so that the amplicon sample solution is wasted and the accuracy of the detection result is affected.

Preferably, during sample adding operation, amplicons in the nucleic acid amplification reaction tube sequentially enter a pipeline on the shell of the chip component, a gap between the top plastic packaging film and the shell, a hollowed-out area corresponding to the sample adding area and the sample adding area from the nucleic acid amplification reaction tube.

Preferably, after the loading operation is completed, the amplicon can enter the detection zone along the loading zone.

Preferably, a scale is provided at the edge of the detection zone to allow the operator to read and mark the distance of movement of the amplicon.

Preferably, the inner diameter of the tube on the housing communicating with the nucleic acid amplification reaction tube is 0.5 to 2.5mm, more preferably 0.5 to 2.0mm, for example: 0.5mm, 0.7mm, 1.0mm, 1.2mm, 1.5mm, 1.7mm, 2.0mm, more preferably 1.0mm.

Specifically, the fluorescent dye is SYBR Green I, evaGreen, syto82. Preferably, the fluorescent dye is SYBR Green I.

The principle of quantitative nucleic acid detection on paper chips is based on the interaction between cellulose paper and DNA chimeric dyes (e.g. SYBR GREEN I (SGI)) and DNA. There is a strong interaction between the SGI and the cellulose so that when the SGI is added to the cellulose paper, the SGI does not flow with the solution to the test channel due to capillary action, but is retained in the sample application zone. Meanwhile, the interaction force between the SGI and the cellulose paper is larger than that between the SGI and the amplification primer (single-stranded DNA) but smaller than that between the SGI and the amplicon (double-stranded DNA). Thus, the SGI can be eluted efficiently into the detection channel by the amplicon generated by the target DNA amplification, but in the absence of target DNA, the single-stranded DNA primers in the amplification solution cannot elute the SGI efficiently into the detection channel (see fig. 1). The SGI elution length on paper can be identified by observing its fluorescence length under blue light. Therefore, the inventor skillfully converts the traditional SGI-based nucleic acid detection means into a detection method for observing the flowing distance on paper, the method can read the sample by naked eyes without a precise fluorescence intensity detection instrument, and the method is very suitable for developing POCT (Point-of-care testing) detection technology and deploying the POCT detection technology to the site for detecting diseases.

In one embodiment of the present invention, the inventors designed the sample application area to be circular and the detection area to be rectangular. The circular shape is provided with a section of arc notch, the length of the arc corresponding to the chord is smaller than or equal to the width of the rectangle, and more preferably, the length of the chord is equal to the width of the rectangle.

Preferably, the diameter of the circle is 2mm-10mm, further preferably 4-8mm, for example: 4.0mm, 4.4mm, 4.8mm, 5.2mm, 5.6mm, 6.0mm, 6.4mm, 6.8mm, 7.2mm, 7.6mm, 8.0mm. More preferably 5.6mm.

Preferably, the rectangle is 20mm to 60mm long, more preferably 20mm to 50mm long, for example: 20mm, 25mm, 30mm, 35mm, 40mm, 45mm, 50mm, more preferably 35mm.

Preferably, the width of the rectangle is 1.5mm to 3.0mm, more preferably 1.5mm to 2.5mm, for example: 1.5mm, 1.8mm, 2.0mm, 2.2mm, 2.5mm, more preferably 1.8mm.

Because LAMP is more sensitive to cross-contamination than PCR, integrating LAMP into POCT systems requires isolation from cap-open and cross-contamination. LAMP reaction is carried out in a slightly alkaline solution for reaction and amplification, but SGI and amplicon are combined to have good reaction effect under pH slightly acidic condition. In order to realize the completion of two steps of reactions under different reaction conditions without uncovering the chip, the inventor skillfully designs the reaction system of LAMP into 3 layers which are mutually separated.

Preferably, the LAMP reaction system comprises an LAMP reaction reagent, an aqueous spacer, and an acidic buffer. When a sample is detected, LAMP reaction reagents, aqueous isolating agents and acid buffer solutions mixed with target nucleic acids are distributed in the nucleic acid amplification tube from bottom to top in sequence corresponding to the sequence of addition;

specifically, the LAMP reaction reagent contains reagents required for LAMP amplification reaction, such as primers, dNTPs, enzymes, mg ²⁺ (e.g., in the form of magnesium chloride, magnesium sulfate), water, etc.

Preferably, the LAMP reaction reagent has a pH of 7.2 to 8.2, for example: 7.2, 7.6, 8.0, 8.2, more preferably pH 8.0.

Preferably, the pH of the acidic buffer is 3.0-5.0, for example: 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0, more preferably pH 4.0.

Preferably, the acidic buffer is selected from the group consisting of disodium hydrogen phosphate-citric acid buffer, citric acid-sodium hydroxide-hydrochloric acid buffer, acetic acid-sodium acetate buffer, citric acid-sodium citrate buffer, and more preferably citric acid-sodium citrate buffer.

Preferably, the volume of acidic buffer is 5-20. Mu.L, more preferably 5-15. Mu.L, for example: 5. Mu.L, 8. Mu.L, 10. Mu.L, 12. Mu.L, 15. Mu.L, more preferably 8. Mu.L.

In a specific embodiment of the invention, the inventor skillfully arranges a groove matched with the sample adding area and the detection area on the shell at the corresponding position of the sample adding area and the detection area of the paper chip, and arranges an inverted funnel-shaped structure in the groove at the corresponding position of the sample adding area (see fig. 4 and 5). The small opening of the funnel is a pipe orifice communicated with the sample adding area through a pipeline communicated with the PCR pipe, and the large opening of the funnel is buckled in the sample adding area. So that a relatively closed space is formed between the interior of the funnel and the upper surface of the loading zone. The space is communicated with the outside only through the pipe orifice of the pipeline communicated with the nucleic acid amplification reaction pipe. After the LAMP amplification reaction is finished, when a detector holds a chip connected with a nucleic acid amplification tube in hand and throws, the funnel-shaped structure can prevent LAMP amplification reaction liquid and buffer liquid from being thrown into a detection area of the paper chip directly from a sample adding area of the paper chip.

Preferably, the small opening diameter of the funnel is 4.0-6.0mm, for example: 4.0mm, 4.5mm, 5.0mm, 5.5mm, 6.0mm, more preferably 5mm.

Preferably, the funnel has a large opening diameter of 5-8mm, for example: 5.0mm, 5.5mm, 6.0mm, 6.5mm, 7.0mm, 7.5mm, 8.0mm, more preferably 6.0mm.

Preferably, the height of the funnel is 0.4-0.8mm, for example: 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, more preferably 0.6mm.

In one embodiment of the invention, the nucleic acid detection system further comprises a portable amplification detection integrated machineFast,Inexpensive Nucleic acid test with Distance-based Readout, FINDR) which can be connected to the nucleic acid amplification reaction tube and the chip part. The portable amplification detection integrated machine provides a reaction environment for the loop-mediated isothermal amplification reaction and provides illumination conditions for the detection of amplicons.

Preferably, the portable amplification detection integrated machine comprises an isothermal heating module, an illumination module, an LCD display screen and a power supply.

The isothermal heating module comprises a circuit control board, a heating plate, a heating block, a temperature sensor, a digital output converter and a cooling fan. The heating block is positioned above the heating plate, holes for holding the nucleic acid amplification reaction tubes are arranged on the heating block, and the holes are arranged in a matrix on the heating block.

The temperature sensor is connected with the heating plate, preferably the temperature sensor is arranged on the heating plate, and preferably the temperature sensor is a thermocouple.

The illumination module comprises an LED lamp, a chip slot, an optical filter and an observation window which are sequentially arranged from bottom to top.

The power source is a built-in rechargeable battery, preferably a lithium battery.

Preferably, the holes are arranged in a 3×8 matrix, the holes are in one-to-one correspondence with the nucleic acid amplification reaction tubes, the LED lamps are arranged in a matrix, and each row of LED lamps corresponds to one chip slot. The LED lamps are preferably arranged in a 2X 4 matrix

In a second aspect, the present invention provides a method of using a portable nucleic acid detection system, comprising the steps of:

step 1, extracting nucleic acid of a sample to be detected, uniformly mixing the nucleic acid with an LAMP reaction solution, slowly adding the mixture into the bottom of a nucleic acid amplification reaction tube, and sequentially adding silicone oil and an acidic buffer solution.

And 2, hermetically connecting the nucleic acid amplification reaction tube to the chip shell through a pipeline on the chip shell.

And 3, inserting a nucleic acid amplification reaction tube into the hole, starting the portable amplification detection integrated machine, and controlling the temperature of the LAMP reaction through an isothermal heating module to perform the LAMP reaction.

Step 4, an operator holds the chip and shakes the chip to mix the amplified LAMP reaction solution with the acid buffer solution, then sequentially passes through a pipeline connecting the chip and the nucleic acid amplification reaction tube and a funnel-shaped structure to enter a sample adding area on the paper chip, fully combines the amplicon and the fluorescent dye, and horizontally stands for 10min until the solution permeates into a detection area from the sample adding area through capillary action.

And 5, horizontally placing the chip in a chip slot, standing, starting an LED lamp, and observing the moving distance of the amplicon with the fluorescent mark through an observation window.

And 6, analyzing the detection result of the movement distance of the amplicon to obtain the detection result of the detection sample.

Preferably, the temperature of the LAMP reaction in step 3 is 60 ℃ to 65 ℃, for example: 61 ℃, 62 ℃, 63 ℃, 64 ℃, 65 ℃, more preferably 65 ℃.

Preferably, the chip in step 5 is left standing for 5-15min, more preferably 8-12min, for example: 8min, 9min, 10min, 11min, 12min, more preferably 10min.

Preferably, the distance of movement of the amplicon in step 5 can be observed by the naked human eye, and image acquisition can also be performed for recording and subsequent data processing using a photographable electronic device, such as a smart phone.

In order to enable the nucleic acid detecting system of the present invention to be suitable for use in primary hospitals, clinics and remote areas where medical resources are limited, and to be suitable for mass production. The inventor has carefully studied and continuously perfected the design, and the manufacturing cost of the portable amplification detection integrated machine is controlled below 300 RMB, and the manufacturing cost of each chip component is about 2 RMB. Especially, the disposable charging of the portable amplification detection integrated machine can support the LAMP reaction at 65+/-0.5 ℃ for 260min, and can support the completion of 192 groups of LAMP reactions. A portable amplification and detection integrated machine has the size of about 12.8X115.6X13 cm and the weight of about 0.5kg. Whereas the existing ABI 7000qPCR instrument had dimensions of 39X 51X 53cm and a weight of 34kg. In addition, the size of GeneXpert IV was 28X 30.5X 30cm, and the weight was 12kg.

The invention has the beneficial effects that:

1. the method adopts a mode of reading the movement distance of the amplicon to carry out qualitative and quantitative detection analysis on the target nucleic acid, and compared with the qualitative and quantitative analysis of the traditional PCR and qLAMP amplification, the method is simpler and more convenient and has low requirements on technicians and detection equipment, thereby improving the detection efficiency.

2. The FINDR of the present invention is more robust than qLAMP, can detect single copy target DNA from a large mixture of non-target DNA, and can be applied to any disease diagnosis based on nucleic acid detection.

3. The size and the weight of the FINDR all-in-one machine have an order of magnitude difference relative to the volume of a traditional detection instrument, and the combination of the FINDR chip can realize the on-site detection of nucleic acid without uncovering, thereby meeting the on-site detection in a resource limited area.

4. Compared with the existing PCR and qPCR detection equipment, the FINDR integrated machine and the chip have low manufacturing cost, are more suitable for remote areas with limited basic hospitals, clinics and medical resources, and are suitable for mass production.

Drawings

FIG. 1 illustrates the detection principle of the present invention;

FIG. 2 is a schematic flow chart of the detection of target nucleic acid according to the present invention;

FIG. 3 shows a process for fabricating a chip according to the present invention;

FIG. 4 is a schematic diagram showing the structure of a chip housing in the portable FINDR all-in-one machine according to the present invention;

FIG. 5 is a schematic view showing a recess and funnel-like structure of a chip housing of the portable FINDR integrated machine according to the present invention;

FIG. 6 is a schematic diagram of a portable FINDR all-in-one machine according to the present invention;

FIG. 7 is a schematic diagram showing the operation of the portable FINDR all-in-one machine according to the present invention;

FIG. 8 shows the results of experiment 2.1 of the present invention, in which the concentrations of H37Rv genomic DNA in the respective histograms were 10 in order from left to right ³ copies/reaction、10 ² A negative control (N.C) of copies/reaction, 10copies/reaction, 1 copies/reaction;

FIG. 9 shows the amplification results in experiment 2.1 of the present invention versus the negative control (N.C.) flow length (d) _R ) Comparing, wherein the numerical value is 3 groups of repeated mean values plus or minus standard deviation, and the p value is calculated from the t test of the two-tailed students;

FIG. 10 shows the results of the qLAMP reaction in experiment 2.2 of the present invention;

FIG. 11 is a thermal graph comparing FINDR detection results in experiment 2.1 of the present invention with qLAMP detection results in experiment 2.2;

FIG. 12 shows the stability test results of experiment 2.3 of the present invention;

FIG. 13 is a bar graph comparing the moving distance averages of amplicons of experiment 2.3 of the present invention;

FIG. 14 is a schematic diagram showing the flow of DNA detection in sputum samples of tuberculosis patients of experiment 3.1 according to the present invention;

FIG. 15 is a ROC curve showing the results of DNA detection in sputum samples from tuberculosis patients of experiment 3.1 according to the present invention;

FIG. 16 is a box chart showing the results of DNA detection using FINDR in sputum samples from tuberculosis patients of experiment 3.1 according to the present invention, wherein the dotted line represents the detection line separating FINDR positive (blue) from FINDR negative (red);

FIG. 17 is a photograph showing the result of FINDR chip fluorescence of DNA in sputum sample of tuberculosis patient in experiment 3.1 according to the present invention.

FIG. 18 is a graph showing the comparison of FINDR detection results in experiment 3.1, qPCR detection results in experiment 3.2, and qLAMP detection results in experiment 3.3 according to the present invention;

FIG. 19 is a confusion matrix of FINDR test results in experiment 3.1 and qPCR test results in experiment 3.2 according to the present invention.

Detailed Description

The invention is further illustrated in the following description, in conjunction with the accompanying drawings and specific embodiments. These examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally performed under conditions conventional in the art or according to manufacturer's recommendations. Unless otherwise specified, the methods are all conventional. Unless defined otherwise, professional and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

Example preparation and use of FINDR nucleic acid detection System

1.1 fabrication of FINDR chip

As shown in FIG. 3, a chip pattern is designed on a computer, which mainly comprises a circular sample adding area with the diameter of 5.6mm, a rectangular detection area with the diameter of 35mm multiplied by 1.8mm and a graduated scale beside the rectangular detection area, then Whatman No.1 cellulose paper is selected as a substrate, the designed pattern is printed on a paper chip by a wax printer (Xerox ColorQube 8580), wherein a section of circular arc notch with the chord length of 35mm is arranged on the circular sample adding area, and corresponds to the width of the rectangular detection area. And heating the cellulose paper chip on a heating plate at 150 ℃ for 40 seconds to melt and infiltrate wax into the paper feeding chip, and forming a circular sample adding area and a detection area with the edges of the rectangular detection area being wax hydrophobic boundaries on the paper chip. And then the heated paper chip is subjected to upper and lower plastic packaging by using a plastic packaging film with the thickness of 125 mu m so as to ensure the overall strength of the FINDR chip.

As shown in fig. 3, a hollowed-out area similar to the pattern of the paper chip is dug out on the upper plastic packaging film, and the detection area of the paper chip is exposed for sample application. And a layer of paraffin film is added between the lower plastic packaging film and the paper chip for bottom sealing.

As shown in FIG. 3, 2. Mu.L of a 300 Xconcentration fluorescent dye SYBR Green I (SGI) dissolved in an acetone solution was added to a circular sample addition zone of a paper chip, and the SGI remained in the cellulose paper gap of the sample addition zone after acetone was volatilized.

As shown in fig. 4, a piece of transparent plastic housing is printed using 3D printing technology. The shell is provided with a groove matched with the paper chip sample adding area and the detection area in position, and an inverted funnel-shaped structure corresponding to the sample adding area is arranged in the groove. The small opening of the funnel is a pipe orifice of a pipeline which is communicated with the PCR pipe and the sample adding area, and the diameter of the small opening is 5mm. The large opening diameter of the funnel was 6mm and the height of the funnel was 0.6mm. The plastic shell is provided with a PCR tube interface and a pipeline with the diameter of 1mm, and is mainly used for connecting a PCR tube containing an amplification reagent with a paper chip.

As shown in fig. 4 and 5, a plastic shell is attached to the upper surface of the plastic package paper chip, so that the groove structure corresponds to the sample adding area and the detection area on the paper chip, and the funnel structure is guaranteed to be reversely buckled on the paraffin hydrophobic edge at the edge of the circular sample adding area. So that a relatively closed space is formed between the interior of the funnel and the upper surface of the circular loading zone. And then the plastic shell and the paper chip are adhered together by adopting the transparent adhesive tape so as to ensure the tightness between the plastic package paper chip and the shell.

1.2 manufacturing of FINDR all-in-one machine

FIG. 6 shows the main structure of the FINDR all-in-one machine of the present invention. The FINDR all-in-one machine is mainly divided into two functional modules: isothermal heating module and illumination observation module. The isothermal heating module uses an electric heating module controlled by an Arduino Nano circuit board, a 24-hole (3×8) heating block is attached to the electric heating plate, and 24 PCR tubes can be inserted for heating at the same time. The temperature of the heating plate is measured by a K-type thermocouple embedded in the heating plate, a thermocouple signal is converted into a digital signal by a thermocouple-to-digital output converter (MAX 31855) and is output to an Arduino plate, the temperature is judged through a preprogrammed code, when the temperature exceeds a threshold value (65 ℃), the Arduino plate closes the heating plate through a relay (SRD-05 VDC-SL-C) and activates two cooling fans to cool the chip, and if the temperature does not reach 65 ℃, the chip is continuously heated until the temperature is heated to 65 ℃ and is in a constant-temperature heating mode. The heating time and temperature are displayed on the LCD display screen in real time, and whether the amplification reaction is executed or not is controlled by the control switch. The illumination observation module consists of an LED matrix and a filter. 8 blue LEDs form a 2X 4 matrix corresponding to the positions of the upper 4 chip slots. The LED light is converted into a uniform area light source through the soft light cover, so that illumination uniformity is ensured. An observation window is arranged right above the chip slot, and an acrylic filter is attached to the observation window so as to reduce background blue light and distinguish SGI green fluorescence from background. After the FINDR chip is inserted into the slot, the LED lamp is turned on, light irradiates from the lower part of the chip, and the moving distance of the amplicon combined with the SGI in the detection area can be observed through the observation window.

The distance of nucleic acid movement may be observed by the naked human eye or image acquisition may be performed using a photographable electronic device, such as a smart phone, for recording and subsequent data processing. All components are packaged by a 3D printing shell to be manufactured into a portable integrated machine, and a built-in rechargeable lithium battery is used for supplying power. FINDR all-in-one machine was 12.8X15.6X13 cm in size and 0.5kg in weight. The FINDR integrated machine and the FINDR chip can be combined to realize the on-site detection of nucleic acid without uncovering, thereby meeting the on-site detection in a resource limited area.

1.3 methods of Using FINDR nucleic acid detection systems

As shown in FIG. 7, the FINDR nucleic acid detection system operates as follows:

step 1, extracting nucleic acid of a sample to be detected, uniformly mixing the nucleic acid with the LAMP reaction solution, slowly adding the mixture into the bottom of a PCR tube, and sequentially adding silicone oil and citric acid/sodium citrate buffer solution with pH of 4.0. The total reaction liquid volume was adjusted to 10. Mu.L.

And 2, hermetically connecting the PCR tube to the chip shell through a pipeline on the FINDR chip shell.

And 3, inserting the PCR tube into a hole of a heating block in the FINDR all-in-one machine, starting the FINDR all-in-one machine, controlling the reaction temperature of the LAMP to be 65 ℃ through an isothermal heating module, and performing LAMP reaction.

Step 4, an operator holds the FINDR chip with the LAMP reaction completed, shakes the chip to mix LAMP reaction liquid with citric acid/sodium citrate buffer solution, then sequentially passes through a pipeline connecting the chip and a PCR tube and a funnel-shaped structure to enter a circular sample adding area on the FINDR chip, fully combines an amplicon and fluorescent dye SGI, horizontally stands for 10min, and is subjected to capillary action to permeate into a detection area from the sample adding area.

And 5, horizontally placing the chip in a slot of a chip frame, standing, starting an LED lamp, and observing the moving distance of the amplicon with the fluorescent mark through an observation window.

Example detection of Mycobacterium tuberculosis Strain H37Rv genome

2.1 detection of H37Rv genome by Portable nucleic acid detection System

(1) Construction of LAMP reaction System preparation

The LAMP reaction system (2. Mu.L) was prepared as follows:

LAMP mix 2x(NEB)1μL；

primer mixture: 0.2 μl;

h37Rv genomic DNA: 0.4. Mu.L;

ddH ₂ O：0.4μL；

wherein, the final concentration of each primer (see table 1) in the LAMP reaction system in the primer mixture liquid is respectively as follows: FIP 1.2. Mu. M, BIP 1.2.2. Mu. M, LF 0.4. Mu. M, LB 0.4.4. Mu. M, F30.4.4. Mu. M, B30.4.4. Mu.M.

The final concentration of H37Rv in the LAMP reaction system was 10 ³ The copies/reaction is from 1 copy/reaction.

TABLE 1 primer information in LAMP amplification reactions

(2) LAMP reaction

The LAMP amplification reaction solution in (1) was added to a 200. Mu.LPCR tube, followed by sequentially adding 5. Mu.L of AS100 silicone oil (Merck) and 8. Mu.L of buffer (pH 4.0 citric acid buffer, containing 0.1% (v/v) Tween-20), thereby forming a water-oil-water layered structure in the PCR tube. The PCR tube was then mounted on a FINDR chip, and the PCR tube was inserted into a heating block of a FINDR instrument and heated at 65℃for 25min. The reaction system of the negative control adopts deionized water with the same volume as the H37Rv genome DNA to replace the H37Rv genome DNA, and the rest reaction conditions are the LAMP reaction with the H37Rv genome DNA.

(3) Fluorescence detection

The FINDR chip after the LAMP amplification reaction is immediately taken down from the instrument, the chip is pinched by hand, the PCR tube is directed against the palm, the chip is swung 5 times to mix the solutions in the PCR tube and flows into the sample loading area of the paper-based chip in the FINDR chip through the pipeline on the outer shell of the FINDR chip. And then horizontally standing the FINDR chip for 10min, enabling the LAMP amplicon combined with the fluorescent dye to flow along a detection area of a paper-based chip in the FINDR chip, inserting the FINDR chip into the detection area of a FINDR instrument, turning on an LED LAMP, and visually reading the fluorescent length on the FINDR chip through an observation window on the instrument, or performing image data acquisition by using an intelligent device with a camera, and calculating detection data by using a two-tailed student t test, wherein the result is shown in fig. 8 and 9.

As can be seen from FIGS. 8 and 9, the H37Rv genomic DNA concentrations were 10, respectively ³ copies/reaction、10 ² copies/reaction、10 ¹ When the copies/reactions are performed, the p value of the detection result of the H37Rv genome and the p value of the negative control (N.C) are less than 0.0001, and the difference is very obvious; when the concentration of the H37Rv genomic DNA was 1copies/reaction, the difference was not significant between the detection result of the H37Rv genome and the p-value= 0.4985 of the negative control (N.C). The detection concentration of H37Rv genomic DNA using the portable nucleic acid detection system of the present invention can be as low as10 ¹ The copies/reactions. The detection time is short, and the detection technology is stable and reliable.

2.2qLAMP detection of H37Rv genome

(1) Preparation of qLAMP reaction System

The qLAMP reaction system (25. Mu.L) was prepared as follows:

Isothermal Mastermix(OptiGene,UK)15μL；

5 mu L of primer mixture;

H37Rv(10 ⁰ ng/μL-10 ^-6 ng/. Mu.L) 2. Mu.L (1. Mu.L 1 ng/. Mu.LH 37Rv contains 21 ten thousand copies of DNA, i.e.1 ng/. Mu.L equals 2.1X10) ⁵ copies/μL)；

ddH ₂ O 3μL。

Wherein, the final concentration of each primer in the primer mixture in the LAMP reaction system is as follows:

the final concentration of each primer in the qLAMP reaction system is as follows: FIP 1.6. Mu. M, BIP 1.6.6. Mu. M, LF 0.6. Mu. M, LB 0.6.6. Mu. M, F30.2.2. Mu. M, B30.2.2. Mu.M.

(2) qLAMP reaction

The qLAMP amplification reaction solution of (1) was added to a 200. Mu. LPCR tube, and then amplified and detected using a CFX96 fluorescent quantitative PCR instrument (BioRad, USA). The qLAMP reaction conditions were: 95 ℃ for 30s; 40s at 60 ℃ for 40 cycles; 95℃for 15s. The reaction was then completed by raising the temperature to 0.5℃to 95℃every 5s after lowering the temperature to 60 ℃. The experimental results are shown in FIG. 10.

As can be seen from FIG. 10, qLAMP can detect 10 at the lowest ^-5 ng/. Mu.L (2.1 copies/reaction) of H37Rv genomic DNA. In addition, due to the N.C. curve and 10 ^-6 ng/. Mu.L overlap each other and are not shown.

The results of experiments 2.1 and 2.2 were compared using a heat map, and the results are shown in fig. 11.

As can be seen from FIG. 11, FINDR paper chips can detect up to 1copies/reaction, and are superior to qLAMP detection results on the lowest detection line.

2.3 Portable nucleic acid detection System stability test

The experiment was repeated 10 times with the H37Rv genome (1 ng/. Mu.L) of experiment 2.1 and the negative control as target nucleic acid for 2.1, and the experimental results are shown in FIGS. 12 and 13.

As can be seen from FIGS. 12 and 13, the difference between the detection distances of 10 shifts of the H37Rv genomic DNA is small, the repeatability of the detection result is high, and the portable nucleic acid detection system of the present invention operates stably.

Example three Portable nucleic acid detection System detection of sputum samples from tuberculosis patients

3.1FINDR System detection of DNA from sputum samples of tuberculosis patients

The specific experimental procedure is shown in fig. 14.

(1) Pretreatment of nucleic acid samples

DNA was extracted from 80 sputum samples (59 tuberculosis patients and 21 healthy) and then the total nucleic acid amount was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, US) and then diluted uniformly to a concentration of 10 ng/. Mu.L using deionized water.

(2) Construction of LAMP reaction System preparation

The LAMP reaction system was identical to the reaction system in experiment 2.1, except that H37Rv was replaced with sputum DNA to extract the sample. Of these, 59 cases of tuberculosis patients had DNA in sputum and 21 healthy persons had DNA in sputum (10 ng/. Mu.L).

(3) LAMP reaction and fluorescence detection

The LAMP amplification reaction solution in (2) was added to a 200. Mu. LPCR tube, and the remaining detection steps were the same as those of experiment 2.1. The results are shown in FIGS. 15, 16 and 17.

As can be seen from FIG. 15, the length of movement of the nucleic acid amplicon on the chip in all 80 sputum samples (59 infections and 21 healthy) was used as the receiver operating characteristic curve (ROC), with an area under the curve of 0.99, demonstrating the high resolution of the FINDR nucleic acid detection system. Using ROC curve calculations, the minimum on-chip shift length at 100% specificity was 11.8mm, thus setting 11.8mm as the threshold for detection positive on the fidr chip.

As can be seen from FIGS. 16 and 17, of the 59 sputum samples from tuberculosis patients, 57 samples had a nucleic acid amplicon moving distance greater than the detection line, and only 2 samples (Nos. 23 and 47) had a nucleic acid amplicon moving distance less than the detection line. The FINDR monitoring system can be used for detecting nucleic acid samples with the concentration of 10 ng/. Mu.L, and has high detection sensitivity.

3.2qPCR detection of DNA from sputum samples of tuberculosis patients

(1) Pretreatment of nucleic acid samples

Nucleic acid samples from sputum of 59 tuberculosis patients in experiment 3.1 were each quantified for total nucleic acid amount using NanoDrop spectrophotometer (Thermo Fisher Scientific, US) and diluted to a concentration of 1 ng/. Mu.l.

(2) Preparation of qPCR reaction System

The reaction system (20. Mu.L) was prepared as follows:

ChamQ SYBR Color qPCRMaster Mix 2x 10μL；

forward primer F3.4 μl;

negative primer B3, 0.4. Mu.L;

ddH ₂ O 7.2μL；

sputum DNA (1 ng/. Mu.L) from tuberculosis patients was 2. Mu.L.

Wherein, the final concentration of F3 and B3 in the reaction system is 0.2 mu M.

(2) qPCR reaction

The qPCR amplification reaction solution in (2) was added to a 200. Mu. LPCR tube, and then amplified and detected using a CFX96 fluorescent quantitative PCR instrument (BioRad, USA). The qPCR reaction conditions were: 95 ℃ for 30s;95 ℃ for 10s,60 ℃ for 30s,40 cycles; 95℃for 15s. The reaction was then completed by raising the temperature to 0.5℃to 95℃every 5s after lowering the temperature to 60 ℃.

3.3qLAMP detection of sputum from tuberculosis patients

(1) Pretreatment of nucleic acid samples

The specific procedure was the same as in experiment 2.2.

(2) Preparation of qLAMP reaction System

The reaction system (25. Mu.L) was prepared according to the following procedure:

Isothermal Mastermix(OptiGene,UK)15μL；

5 mu L of primer mixture;

sputum DNA (1 ng/. Mu.L) of tuberculosis patient is 2. Mu.L;

ddH ₂ O 3μL。

wherein, the final concentration of each primer in the primer mixture in the qLAMP reaction system is as follows: FIP 1.6. Mu. M, BIP 1.6.6. Mu. M, LF 0.6. Mu. M, LB 0.6.6. Mu. M, F30.2.2. Mu. M, B30.2.2. Mu.M.

(3) qLAMP reaction

The qLAMP amplification reaction solution of (2) was added to a 200. Mu. LPCR tube, and then amplified and detected using a CFX96 fluorescent quantitative PCR instrument (BioRad, USA). The qLAMP reaction conditions were: 95 ℃ for 30s; 40s at 60 ℃ for 40 cycles; 95℃for 15s. The reaction was then completed by raising the temperature to 0.5℃to 95℃every 5s after lowering the temperature to 60 ℃.

The results of the nucleic acid sample detection of experiment 3.1, experiment 3.2 and experiment 3.3 were compared using a heat map, and the results are shown in FIG. 18.

As can be seen from FIG. 18, qPCR was used as a gold standard method to detect nucleic acid samples in sputum of tuberculosis patients, and all 59 tuberculosis samples could be detected. The FINDR detection result is similar to the qPCR detection result. However, 23 tuberculosis samples in the qLAMP detection result are detected as false negative. The qLAMP detection effect was inferior to the FINDR detection result.

The detection line obtained by using the ROC curve in FIG. 15 was used to compare the detection results of nucleic acids in sputum of 59 tuberculosis patients in experiment 3.1 and experiment 3.2 using a confusion matrix, and the results are shown in FIG. 19.

As can be seen from FIG. 19, the sensitivity of the FINDR detection method was 96.6% and the specificity was 100% compared to the qPCR gold standard detection results. The portable nucleic acid detecting system of the present invention can be used for detecting a nucleic acid sample having a concentration of 10 ng/. Mu.L, and has high detection sensitivity.

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solution of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.

Claims

1. A portable nucleic acid detection system, characterized by: the nucleic acid detection system includes:

the chip component can be detachably connected with the nucleic acid amplification reaction tube, the chip component comprises a paper chip, and a sample adding area and a detection area which are mutually communicated are arranged on the paper chip; fluorescent dye is preset in the sample adding area, and the fluorescent dye is chimeric fluorescent dye.

2. The portable nucleic acid detection system of claim 1, wherein: the edge of the detection area is provided with a graduated scale for marking the moving distance of the amplicon.

3. The portable nucleic acid detection system of claim 1, wherein: the chip component comprises a plastic package chip in the middle and a shell adhered to the upper surface of the plastic package chip;

the plastic package chip comprises the paper chip in the middle and plastic package films attached to the upper surface and the lower surface of the paper chip;

one end of the shell, which is adjacent to the sample adding area, is provided with a pipeline which is communicated with the nucleic acid amplification reaction tube, and the other end of the pipeline is communicated with the sample adding area.

4. The portable nucleic acid detection system of claim 3, wherein: a hydrophobic sealing film is arranged between the paper chip and the lower plastic packaging film;

and a hollowed-out area with the same pattern as the sample adding area and the detection area is arranged on the upper layer of the plastic packaging film.

5. The portable nucleic acid detection system of claim 3, wherein: the shell is provided with grooves matched with the sample adding area and the detection area at the corresponding positions of the sample adding area and the detection area;

an inverted funnel-shaped structure is arranged in the groove, a small opening of the funnel is a pipe orifice of the pipeline communicated with the sample adding area, and a large opening of the funnel is buckled in the sample adding area.

6. The portable nucleic acid detection system of any one of claims 1 to 4, wherein: the sample adding area is round, and the detection area is rectangular;

a section of arc notch is arranged in the circle, and the length of the arc corresponding to the chord is smaller than or equal to the width of the rectangle.

7. The portable nucleic acid detection system of any one of claims 1 to 4, wherein: the fluorescent dye is selected from SYBRGreenI, evaGreen or Syto82.

8. The portable nucleic acid detection system of any one of claims 1 to 4, wherein: the nucleic acid detection system further comprises an LAMP reaction reagent, an aqueous isolating agent and an acidic buffer solution;

when a sample is detected, the LAMP reaction reagent, the water-based isolating agent and the acid buffer solution which are mixed with target nucleic acid are distributed in the nucleic acid amplification tube from bottom to top in sequence corresponding to the sequence of addition;

the pH value of the acidic buffer solution is 3.0-5.0, and the pH value of the LAMP reaction reagent is 7.2-8.2.

9. The portable nucleic acid detection system of any one of claims 1 to8, wherein: the nucleic acid detection system further comprises a portable amplification detection integrated machine which can be connected with the nucleic acid amplification reaction tube and the chip component;

the portable amplification detection integrated machine provides a reaction environment for the loop-mediated isothermal amplification reaction and provides illumination conditions for detection of the amplicon.

10. The portable nucleic acid detection system of claim 9, wherein: the portable amplification detection integrated machine comprises an isothermal heating module, an illumination module, an LCD display screen and a power supply;

the isothermal heating module comprises a circuit control board, a heating plate, a heating block, a temperature sensor, a digital output converter and a cooling fan;

the heating block is positioned above the heating plate, holes for containing the nucleic acid amplification reaction tubes are arranged on the heating block, and the holes are arranged in a matrix on the heating block;

the temperature sensor is connected with the heating plate, preferably the temperature sensor is arranged on the heating plate, and the temperature sensor is preferably a thermocouple;

the illumination module comprises an LED lamp, a chip slot, an optical filter and an observation window which are sequentially arranged from bottom to top;

the power supply is a built-in rechargeable battery.