Mask type respiratory virus real-time nucleic acid detection method based on micro-fluidic chip and smart phone

Abstract

The invention discloses a mask type respiratory virus real-time nucleic acid detection method based on a micro-fluidic chip and a smart phone, and belongs to the field of biological detection. The invention develops a convenient wearable micro-fluidic device which can be integrated in a mask to complete the whole process operation of sample collection, splitting decomposition, amplification and detection of novel coronavirus detection. Besides, through application software developed by a smart phone, collection, statistics and analysis of signals are realized, and real-time, convenient and accurate quantitative detection of target nucleic acid is provided. The invention constructs a novel coronavirus instant nucleic acid detection (POCT) platform. Compared with existing RT-PCR, PCR and other nucleic acid detection methods, the system integrates sample collection and detection processes in the mask, has the advantages of no pollution, simplicity and convenience in operation and high portability, has a wide application prospect, and is particularly suitable for detection of novel coronavirus and other respiratory infectious diseases.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a mask-type respiratory virus instant nucleic acid detection method based on a microfluidic chip and a smart phone, and related products and applications thereof. Background Art

[0002] COVID-19, like severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS), is a highly lethal virus that spreads faster. Its transmission model clearly shows that rapid and accurate identification of COVID-19 can effectively control its pandemic when diagnosed in the early stage. Reverse transcription polymerase chain reaction (RT-PCR) technology is recommended by the World Health Organization as the standard guideline for COVID-19 testing. However, the current RT-PCR test still has shortcomings such as the need for sample collection, RNA extraction, amplification, long processing time, and requirements for equipment and instruments. As a typical respiratory infectious disease, COVID-19 has been confirmed to be transmitted through respiratory aerosols. Studies have shown that patients with early novel coronavirus infection can emit a large amount of aerosols containing novel coronavirus particles through breathing, coughing, talking or sneezing, and masks can be used as a simple and effective tool to avoid human-to-human transmission. For example, N95 masks are widely recommended for preventing inhalation of viral aerosols. These masks use permanently charged electret fibers as filter media, and in vitro experiments have shown that they can collect 95% of the atomized particles in respiratory aerosols. Unlike nasopharyngeal swabs, aerosol sampling does not require trained professional medical personnel and does not produce potentially infectious waste. It can basically be used for COVID-19 testing and screening at any time and anywhere.

[0003] RT-PCR technology is currently the most commonly used method for viral nucleic acid amplification. First, RNA is reverse transcribed into cDNA, and then the target fragment is amplified at an exponential rate through the continuous cycle of the three basic reaction steps of denaturation-annealing-extension. Denaturation is to dissociate the double-stranded DNA into single-stranded DNA by heating it to serve as a template for replication; annealing is to lower the temperature to about 55°C so that the complementary sequence between the primer and the template DNA single strand can pair and bind; extension is to synthesize a new DNA single strand complementary to the template DNA strand at the optimal temperature of DNA polymerase according to the principle of base complementary pairing and semi-conservative replication. After each cycle is completed, the target sequence in the sample will be amplified twice. Therefore, after several cycles, the number of target fragments will show exponential growth. PCR technology provides high sensitivity for current nucleic acid detection. However, their specificity in mutation identification is limited, the precision is affected, the sensitivity of polymerase inhibitors is high, and the target nucleic acid cannot be absolutely quantified. In addition, special supporting instruments are required, which is not suitable for POCT detection.

[0004] Recombinase polymerase amplification (RPA) is a highly sensitive and specific isothermal amplification technology that can amplify as few as 1 to 10 copies of target nucleic acid within 20 minutes at 37°C. It has been used to amplify various target nucleic acids, including RNA, miRNA, ssDNA, and dsDNA. In 2019, ShuQi Wang et al. invented a wearable microfluidic device based on RPA, which was the first wearable microfluidic device to detect nucleic acids. The RPA amplification reaction only requires normal human body temperature, and the target nucleic acid is accurately quantitatively analyzed through a homemade convenient fluorescence exciter, achieving the detection of 100 copies / mL of target nucleic acid in the sample.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a mask-type respiratory virus instant nucleic acid detection method based on a microfluidic chip and a smart phone.

[0007] The present invention is achieved in that:

[0008] In the first aspect, an embodiment of the present invention provides a reagent combination for use in a mask-type respiratory virus instant nucleic acid detection method based on a microfluidic chip and a smart phone, the reagent combination comprising: a solid phase carrier, a nanohydrogel, a capture probe, a dNTP, a reverse transcription reagent and an RPA detection reagent; wherein the dNTP is selected from at least one of dATP, dTTP, dCTP, dGTP and dUTP.

[0009] In a second aspect, an embodiment of the present invention provides a mask-type respiratory virus instant nucleic acid detection method based on a microfluidic chip and a smart phone, which includes using the reagent combination described in the above embodiment to detect the sample to be tested.

[0010] In a third aspect, an embodiment of the present invention provides a mask based on a microfluidic chip and a smart phone that can be used for real-time nucleic acid detection of respiratory viruses, and the kit includes a reagent combination as described in the above embodiments.

[0011] The present invention has the following beneficial effects:

[0012] 1. High sensitivity: The nucleic acid amplicons released in the solution after the reaction are gathered at the modification sites of each capture probe (primer) to form a single fluorescent spot similar to a colony, which reduces the requirements for signal readout equipment and improves the sensitivity of detection to 10-100 copies / mL.

[0013] 2. High portability: The RT-RPA reaction system is prepared in advance as freeze-dried nano-hydrogel particles and integrated into a microfluidic chip. When an amplification reaction is required, the hydrogel particles are renatured into a whole hydrogel film, avoiding the need for professionals to add samples for each reaction. Virus particles can be captured and lysed by wearing a mask with an integrated microfluidic chip, and the chip with captured virus particles can be worn on the face for amplification, without the need for large-scale instruments and equipment and additional professionals for sampling. At the same time, smartphone-based signal detection and analysis software is developed and used to analyze amplification results anytime and anywhere, and accurately realize quantitative analysis of target nucleic acids.

[0014] 3. Low pollution: The project intends to integrate the virus capture microfluidic chip on the mask. The patients can capture and lyse the virus particles by wearing the mask without generating any additional pollutants.

[0015] 4. Low cost: The project adopts microfluidic technology, which has the characteristics of low cost, low sample consumption, automation and easy integration.

[0016] 5. Wide range of applications: It is applicable to most nucleic acid detection systems and has broad application potential in cell biology, molecular biology, clinical medicine and other fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0018] Figure 1 It is the lyophilized RT-RPA reaction principle;

[0019] Figure 2 This is a schematic diagram of the principle of a wearable chip integrated into a mask;

[0020] Figure 3 Optimized for response time;

[0021] Figure 4 For reaction temperature optimization;

[0022] Figure 5 Verify the sensitivity of the detection method provided in Example 1;

[0023] Figure 6 The linear range of the detection method provided in Example 1;

[0024] Figure 7 Specificity verification of the detection method provided in Example 1;

[0025] Figure 8 The detection time of the detection method provided in Example 1 is compared with that of qPCR. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0027] The present invention couples the primers to the nano-hydrogel coating by adding Acrydite-modified primers, reverse transcribes after virus lysis, and captures the reverse transcribed DNA on the nano-hydrogel. The target nucleic acid chain aggregates under the extension of the polymerase, and the result is detected by the mobile phone intelligent software combined with the Image-J software. The nano-hydrogel is the reaction layer, and the PDMS is the support layer. After being made into a simple chip and fixed on the mask, the target nucleic acid chain in the exhaled gas can be captured. The RT-RPA reaction gets rid of the limitation of the need for a matching nucleic acid amplification instrument, and the detection can be completed only by relying on the body's own temperature. It has the advantages of high sensitivity, high accuracy, low cost and easy integration.

[0028] Specifically, an embodiment of the present invention provides a mask-type respiratory virus instant nucleic acid detection method based on a microfluidic chip and a smart phone, wherein the reagent combination includes: a solid phase carrier, a nanohydrogel, a capture primer / probe, a dNTP, a reverse transcription reagent and an RPA detection reagent; wherein the dNTP is selected from at least one of dATP, dTTP, dCTP, dGTP and dUTP.

[0029] In some embodiments, the solid support comprises any one of PDMS, polystyrene, polyvinyl chloride, PVDF membrane, cellulose and gel.

[0030] In some embodiments, the solid phase carrier includes PDMS. PDMS is used as a solid phase carrier as a support layer of a microfluidic chip, and PDMS is the most commonly used material for microfluidic chips. PDMS is low in cost, easy to make, and has adjustable size. Other solid phase carriers can also achieve similar effects.

[0031] In some embodiments, the nano hydrogel includes any one of polyethylene glycol hydrogel, gelatin hydrogel, sodium alginate hydrogel, chitosan hydrogel and polypropylene hydrogel;

[0032] In some embodiments, the capture primer / probe is fixed to the surface of the nano hydrogel carrier. The method of fixing the capture primer to the hydrogel carrier can be implemented based on conventional technical knowledge in the field, for example, the 5-end of the primer sequence can be modified with a (-Acrydite) group in advance, which can be combined with the C=C double bond of the hydrogel carrier itself, and the two are mixed and reacted to obtain a hydrogel carrier with a capture primer fixed on the surface.

[0033] In some embodiments, the probe is capable of specifically binding to a target nucleic acid, which includes any of single-stranded DNA, single-stranded RNA, double-stranded DNA, and double-stranded RNA.

[0034] In some embodiments, the length of the capture probe is greater than the length of the target nucleic acid.

[0035] In some embodiments, the capture probe is 10 to 80 bp long. Within this length range, the effects described herein can be achieved. The longer the probe, the higher the specificity. Specifically, it can be any one of 10bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp or a range between any two of them.

[0036] In some embodiments, the product comprises a reagent or a kit.

[0037] In some embodiments, the RT-RPA detection reagent includes: 4 dNTPs, Mg2+, reverse transcriptase, Oligo dT, specific primers, DNA polymerase, recombinase and RPA reaction buffer, and virus lysis solution.

[0038] In some embodiments, the virus lysis solution includes any one or a range between any two of 0.01-5% SDS, 1-100 mmol / L Tris pH 7.4, 0.1-20 mmol / L EDTA and 0.1-20 mg / mL proteinase K.

[0039] In some embodiments, the instant nucleic acid detection method for mask-type respiratory viruses based on microfluidic chips and smartphones can be defined in the practical instructions and included in the kit as one of the components in the reagent combination.

[0040] In some embodiments, the reaction temperature is 35-40°C, specifically any one of 35°C, 36°C, 37°C, 38°C, 39°C, 40°C or a range between any two thereof.

[0041] In some embodiments, the mixing reaction time is 5 min to 2 h, specifically can be any one of 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 60 min, 65 min, 70 min, 75 min, 80 min, 85 min, 90 min, 95 min, 100 min, 105 min, 110 min, 115 min, 120 min or a range between any two of them. In some embodiments, the concentration of the primer modified with Acrydite is 0.02 μM to 2 mM, specifically 0.02 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.3 μM, 0.4 μM, 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1 μM, 10 μM, 50 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM. , 350μM, 400μM, 450μM, 500μM, 550μM, 600μM, 650μM, 700μM, 750μM, 800μM, 850μM, 900μM, 950μM, 1mM, 1.1mM, 1.2mM, 1.3mM, 1.4mM, 1.5mM, 1.6mM, 1.7mM, 1.8mM, 1.9mM, 2mM, any one or a range between any two of them.

[0042] An embodiment of the present invention also provides a mask-type respiratory virus instant nucleic acid detection method based on a microfluidic chip and a smart phone, which includes using a reagent combination as described in any of the aforementioned embodiments to detect a sample to be tested.

[0043] In some embodiments, the detection step includes the mask-type respiratory virus instant nucleic acid detection method based on a microfluidic chip and a smartphone as described in any of the preceding embodiments.

[0044] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0045] Example 1

[0046] A mask-type respiratory virus instant nucleic acid detection method based on microfluidic chips and smartphones. The reaction principle diagram is as follows Figure 1 As shown, it includes the following steps.

[0047] (1) Design a specific target nucleic acid probe for the target nucleic acid to be detected, and then extend the target nucleic acid chain. At the same time, modify the 5-end of the forward primer with an (-Acrydite) group. The sequence information of the primers, probes and target nucleic acid used is shown in Table 1.

[0048] Table 1 Sequence information of capture primers, probes and target nucleic acids

[0049]

[0050] (2) dissolving the primer obtained in step (1) in ultrapure water according to the required working concentration, incubating with the nano-hydrogel coating, adding 1% APS and 1% TEMED as initiators, and reacting for 10 minutes to obtain solution I;

[0051] (3) adding an appropriate amount of target nucleic acid, 10 U of reverse transcriptase and reaction buffer to solution I to obtain solution II;

[0052] (4) Using RPA reagent, add appropriate amount of primers and probes to RPA reaction buffer, add to solution II, and react at 37°C for 15-30 minutes;

[0053] (5) After the reaction is completed, a smartphone with image processing software is used to collect the fluorescence signal and perform statistical analysis.

[0054] Example 2

[0055] Based on the method provided in Example 1, the reaction (step (4)) time was used as a single factor variable, and multiple experimental groups were set to verify the effect of different reaction times on the detection effect. The detection results are shown in Tables 2 and Figure 3 .

[0056] Table 2 Test results

[0057]

[0058]

[0059] Note: copy / mL is the concentration of target nucleic acid; cluster number is the digital signal of high fluorescence clusters.

[0060] Example 3

[0061] Based on the method provided in Example 1, the reaction temperature in the reaction (step (4)) was set as a single factor variable, and multiple experimental groups were set to verify the effect of the reaction temperature on the detection effect. The detection results are shown in Tables 3 and Figure 4 .

[0062] Table 3 Test results

[0063]

[0064] Example 4

[0065] The sensitivity of the detection method provided in Example 1 was verified, and the detection results are shown in Tables 4 and Figure 5 .

[0066] Table 4 Test results

[0067]

[0068] Example 5

[0069] Verify the linear range of the detection method provided in Example 1. The results are shown in Table 5 and Figure 6 shown.

[0070] Table 5 Detection results of linear range

[0071]

[0072]

[0073] Example 6

[0074] The specificity of Example 1 was verified.

[0075] The sequence information of the probe, target nucleic acid and mutant nucleic acid added to the test sample used in this example is shown in Table 6.

[0076] Table 6 Sequence information

[0077]

[0078]

[0079] Note: WT is the target nucleic acid, and the bold / underlined bases are the mutation sites in the mutant nucleic acid.

[0080] The test results are shown in Table 7 and Figure 7 .

[0081] Table 7 Test results

[0082]

[0083] Example 7

[0084] Verify the detection time of the method of the present invention.

[0085] The samples were tested using the detection method of Example 1 of the present invention. At the same time, a control group was set based on qPCR, and the target nucleic acid concentration of the two groups was 6000 copy / mL. The qPCR reaction system was 1x qPCR mix, 0.1 μM primer F, 0.1 μM primer R and target nucleic acid, and the thermal cycle temperature was shown in Table 8.

[0086] Table 8 qPCR thermal cycle temperature

[0087]

[0088] The time required for the two methods to complete the detection is shown in Table 9 and Figure 8 shown.

[0089] Table 9 Detection time

[0090]

[0091]

[0092] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A mask-type respiratory virus instant nucleic acid detection method based on a microfluidic chip and a smart phone, characterized in that: The detection method is based on the coordinated application of a microfluidic chip and a smart phone. The reagent combination includes: a solid phase carrier, a nanohydrogel, a capture primer / probe, a deoxynucleoside triphosphate (dNTP), a reverse transcription reagent, and a recombinase polymerase amplification (RPA) detection reagent; wherein the dNTP is selected from at least one of dATP, dTTP, dCTP, dGTP, and dUTP.

2. The use according to claim 1, characterized in that: The capture primer / probe is fixed on the surface of the nano hydrogel; Preferably, the nano hydrogel comprises any one of polyethylene glycol hydrogel, gelatin hydrogel, sodium alginate hydrogel, chitosan hydrogel or polyacrylamide hydrogel; Preferably, the solid phase carrier comprises any one of polydimethylsiloxane (PDMS), polystyrene, polyvinyl chloride, polyvinylidene fluoride (PVDF) membrane, cellulose or gel.

3. The use according to claim 1, characterized in that: The dNTP is selected from any one of dATP, dTTP, dCTP, dGTP and dUTP.

4. The use according to any one of claims 1 to 3, characterized in that: The capture probe can specifically bind to a target nucleic acid, and the target nucleic acid includes any one of single-stranded DNA, single-stranded RNA, double-stranded DNA or double-stranded RNA; Preferably, the sequence length of the capture probe is greater than the length of the target nucleic acid; Preferably, the capture probe is 10 to 80 bp in length; Preferably, the product comprises a reagent or a kit; Preferably, the RT-RPA detection reagent includes: four dNTPs, magnesium ions (Mg 2 +), reverse transcriptase, Oligo dT, specific primers, DNA polymerase, recombinase and RPA reaction buffer.

5. The use according to claim 4, characterized in that: The product's mask-type respiratory virus instant nucleic acid detection method based on microfluidic chips and smartphones includes: By adding Acrydite-modified primers, the primers are coupled to the nanohydrogel coating, and the virus is lysed and reverse transcribed. The reverse transcribed cDNA is captured on the nanohydrogel, and the target nucleic acid chain is continuously extended and aggregated under the action of the polymerase; The nano-hydrogel is used as a reaction layer and PDMS is used as a support layer. After being made into a simple chip, it is fixed on the mask and can capture the target nucleic acid chain in the exhaled gas; Signal detection and result analysis were performed by a smartphone combined with Image-J software.

6. The use according to claim 5, characterized in that: The reaction temperature is 36 to 40°C; Preferably, the mixing reaction time is from 10 minutes to 2 hours.

7. The use according to claim 5, characterized in that: The working concentration of the Acrydite modified primers is 0.02 μM to 2 mM.

8. A mask-type respiratory virus instant nucleic acid detection method based on a microfluidic chip and a smart phone, characterized in that: The method comprises using the reagent combination as described in any one of claims 1 to 7 to detect a sample to be tested.

9. The detection method according to claim 8, characterized in that: The detection step includes the mask-type respiratory virus instant nucleic acid detection method based on a microfluidic chip and a smart phone as described in any one of claims 5 to 7.

10. A mask-type respiratory virus instant nucleic acid detection method based on a microfluidic chip and a smart phone, characterized in that: The kit comprises the reagent combination according to any one of claims 1 to 7.

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