Method and device for simultaneously detecting qnr1, qnr2 and qnr3 and application

Abstract

The invention discloses a method and device for simultaneously detecting qnr1, qnr2 and qnr3 and application. A sample is irradiated by a coaxial reflection LED light source outside 808nm, a sensitizer Sb2WO6 coated NiS provides photoelectrons and heat for LAMP through light driving, a target gene is used as a template to initiate an amplification reaction, and a large number of double-chain DNALAMP light induction products are obtained. Sb2WO6 (at) NiS is modified by a nucleic acid chain to obtain a signal probe, an LAMP light-induced product and the signal probe are captured by a detection region in a sandwich form, a photo-thermal temperature change signal positively related to the content of the LAMP light-induced product is given, and qnr1, qnr2 and qnr3 in a sample are quantified according to the temperature change signal. Photoelectron and photothermal effects are generated by using the photoinduced activity of the sensitizer Sb2WO6 at NiS, the temperature is rapidly controlled, the sensitizer is in direct contact with an amplification system on a molecular level, then photothermal analysis test paper color development detection is performed, and the photothermal efficiency is remarkably improved.

Description

Technical Field

[0001] The invention relates to a method, a device and an application for simultaneously detecting QNR1, QNR2 and QNR3, belonging to the technical field of analysis and detection. Background Art

[0002] Antibiotic resistance genes are genes that encode a series of proteins or other molecular mechanisms that enable bacteria to become resistant to antibiotics. Their existence and spread pose a serious threat to public health, food safety, and environmental safety. Fluoroquinolones (FQs) are a class of drugs widely used to treat bacterial infections. They are widely used in clinical practice and animal husbandry due to their broad-spectrum antibacterial activity. However, the emergence and spread of fluoroquinolone resistance genes pose a serious threat to public safety and human health.

[0003] Normally, only a small amount of FQs is metabolized or absorbed after entering the body, and most of them are excreted in the form of parent compounds with urine or feces, and eventually enter the sewage treatment plant. Due to the constraints of sewage treatment technology, antibiotics cannot be completely removed and can enter the environmental medium with wastewater discharge. At present, the presence of FQs has been detected in various environmental media such as surface water such as rivers and lakes, groundwater, marine environment, wastewater and sludge, sediment, and soil. FQs in the environment can cause chronic toxicity, affect the reproduction, development and growth of organisms to a certain extent, and also lead to the production of bacterial resistance and resistance genes, posing a huge threat to human health and the stability of the ecosystem. FQs can also interfere with biogeochemical cycles such as nitrogen cycle by affecting microbial communities, inhibit the growth of denitrifying bacteria, change the community structure and functional gene abundance of denitrifying bacteria, and inhibit the denitrification process. In addition, it will increase the accumulation of nitrite and the release of nitrous oxide (N2O).

[0004] Therefore, there is an urgent need to develop on-site rapid detection methods for fluoroquinolone antibiotic resistance genes.

[0005] The detection methods of antibiotic resistance genes are mainly based on nucleic acid amplification, including polymerase chain reaction (PCR) and isothermal amplification technology. Because PCR amplification takes a long time and relies on professionals and equipment, it is not suitable for rapid on-site detection. Among them, loop-mediated isothermal amplification uses a DNA polymerase with chain displacement activity and a set of specially designed primers to amplify the target gene at a constant temperature of 60 to 65°C. It is efficient, sensitive, easy to operate, and widely used in many fields such as food safety and pathogenic microorganism detection.

[0006] LAMP photoinduction, by adding various photothermal nanomaterials or sensitizers into the amplification system, using light-emitting diodes (LEDs) or lasers to excite, provides energy supply for nucleic acid amplification. LAMP photoinduction improves amplification efficiency and has been combined with single-plex PCR, LAMP, and RCA amplification technology with good amplification effects. It is a promising on-site detection platform, but there are no reports on the simultaneous amplification of multiple antibiotic resistance genes using LAMP photoinduction technology.

[0007] There are significant differences between the external coaxial reflective LED light source and other LED light sources in many aspects, mainly reflected in the structural design, light characteristics, application scenarios and optical efficiency. The external coaxial reflective LED light source is mainly composed of a lateral light source and a beam splitter. The light emitted by the light source first passes through the beam splitter and then is reflected parallel to the object to be measured. Other LED light sources such as ring light sources, strip light sources, and linear light sources have different structural designs, but usually do not include a beam splitter component. For example, the ring light source directly illuminates the top of the object to be measured, while the strip light source provides a variety of illumination effects such as direct, oblique and photometric. Due to the use of a beam splitter design, the light of the external coaxial reflective LED light source can be evenly illuminated on the surface of the object after passing through the beam splitter, reducing the interference caused by shadows and reflections. At the same time, this light source usually has high brightness and uniformity, which can highlight the contour features of the object. Although other LED light sources also have their own light characteristics, such as the ring light source can solve the problem of diagonal illumination shadows, and the strip light source can adjust the illumination angle and installation at will, but in comparison, the external coaxial reflective LED light source has more advantages in light uniformity and reducing shadows. The external coaxial reflection LED light source beam splitter design reduces light loss and thus improves optical efficiency.

[0008] Currently, there is no report on the simultaneous detection of fluoroquinolone resistance genes using excitation light from an external coaxial reflected LED light source to drive multiple LAMP amplifications combined with photothermal analysis test paper technology. Summary of the invention

[0009] Technical issues: The efficiency of existing LAMP photoinduction sensitizers is not ideal; LAMP photoinduction technology has not yet been applied to the simultaneous amplification of multiple resistance genes; although the external coaxial reflective LED light source has the advantages of light uniformity and reduced shadows, it has not yet been combined with LAMP photoinduction and photothermal analysis test paper technology for the detection of fluoroquinolone resistance genes.

[0010] Purpose of the invention: In order to overcome the above-mentioned deficiencies and defects of the prior art, the present invention provides a method, device and application for simultaneously detecting qnr1, qnr2 and qnr3, adopting the sensitizer Sb2WO6@NiS as the photothermal material, utilizing its photoactivity to generate photoelectrons and photothermal effects, realizing rapid temperature control under the irradiation of 808nm external coaxial reflection LED light source and direct contact with the LAMP photoinduction system at the molecular level, significantly improving the photothermal efficiency, and then performing photothermal analysis test paper colorimetric detection, thereby realizing intensive and efficient amplification and detection of the two links of LAMP photoinduction and photothermal analysis test paper detection.

[0011] Technical solution: The present invention adopts the following technical solution.

[0012] LAMP light-induced product LAMP light-induced product LAMP light-induced product The first object of the present invention is to provide a method for simultaneously detecting qnr1, qnr2 and qnr3, comprising the following steps:

[0013] S1. Preparation of sensitizer Sb2WO6@NiS

[0014] S2, LAMP light induction

[0015] Under the irradiation of 808nm external coaxial reflection LED light source, the sample to be tested undergoes an amplification reaction through the LAMP light-induced system to obtain a LAMP light-induced product, wherein:

[0016] The LAMP light-induced system includes the following components: a primer set, a sensitizer Sb2WO6@NiS, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer, wherein the DNA template is the resistance genes qnr1, qnr2, and qnr3;

[0017] S3, Photothermal analysis test paper detection

[0018] Capture probes were fixed on the T and C regions of the test strip to obtain a multi-target photothermal analysis test strip for the light-driven LAMP products of the target genes qnr1, qnr2, and qnr3;

[0019] The photothermal signal probes were obtained by modifying the sensitizer Sb2WO6@NiS with nucleic acid chains of qnr1, qnr2 and qnr3 respectively;

[0020] The photothermal signal probe and the photothermal analysis test paper are used to detect the LAMP light-induced product. The detection area of ​​the photothermal analysis test paper captures the LAMP light-induced product and the photothermal signal probe in a sandwich form, and gives a photothermal temperature change signal positively correlated with the content of the LAMP light-induced product under the irradiation of an 808nm external coaxial reflection LED light source. The qnr1, qnr2 and qnr3 in the sample to be tested are qualitatively and / or quantitatively determined based on the temperature change signal.

[0021] The present invention utilizes the photothermal effect of the sensitizer Sb2WO6@NiS to realize the light-driven LAMP and detection method of the resistance genes qnr1, qnr2, and qnr3. The method includes two steps: LAMP photoinduction and photothermal analysis test paper detection: first, the LAMP photoinduction system contains a sensitizer Sb2WO6@NiS, and an 808nm external coaxial reflection LED light source is used to irradiate the LAMP photoinduction system to excite the sensitizer Sb2WO6@NiS in the LAMP photoinduction system to generate photoelectrons and heat, provide energy for LAMP, and use qnr1, qnr2 and qnr3 as templates to initiate amplification reactions, thereby obtaining a large number of double-stranded DNA LAMP photoinduction products; secondly, the sensitizer Sb2WO6@NiS is modified with three nucleic acid chains to obtain three photothermal signal probes, and T probes and C probes are fixed in the detection area and quality control area of ​​the photothermal analysis test paper, respectively, to obtain multi-target photothermal analysis test paper for LAMP photoinduction products of target genes qnr1, qnr2, and qnr3; finally, the photothermal analysis test paper captures the LAMP photoinduction products and signal probes in a sandwich form, and gives a photothermal temperature change signal positively correlated with the content of the LAMP photoinduction products, thereby realizing quantitative detection of the target gene.

[0022] As an optional implementation, in step S2, in the LAMP light-inducible system, the concentration of each group of DNA templates is 3%-6% v / v.

[0023] As an optional embodiment, the primer set includes three sets of primers, each set of primers includes two external primers F3 and B3, two internal primers FIP and BIP, and two loop primers loop F and loop B.

[0024] As an optional embodiment, in the LAMP light-inducible system, the concentration of the two external primers F3 and B3 in each set of primers is 2%-6% v / v, the concentration of the two internal primers FIP and BIP is 2%-6% v / v, and the concentration of the two loop primers loop F and loop B is 0-4% v / v.

[0025] As an optional embodiment, the LAMP light-inducible system further comprises the following components: BSA, dNTPs, betaine and ddH2O.

[0026] As an optional embodiment, the LAMP isothermal amplification buffer includes Tris-HCl, (NH4)2SO4, KCl, MgSO4, and Tween-20.

[0027] As an optional embodiment, the photothermal signal probe is used independently of the test paper body, or is dropped and dried on the conjugate pad and the conjugate pad is integrated on the test paper.

[0028] As an optional implementation, the irradiation parameters of the 808nm external coaxial reflection LED light source are power 4.5-6W and irradiation time 10-30min.

[0029] As an optional implementation, in step S1, the preparation method of the sensitizer Sb2WO6@NiS comprises the following steps:

[0030] (1) dissolving nickel nitrate and CH4N2S in water at a molar ratio of 1:4-1:5, stirring for 1-2 hours to obtain a mixed solution;

[0031] (2) injecting the mixed solution into a Sb2WO6 aqueous solution, wherein the molar ratio of Sb2WO6 to nickel nitrate is 1:2-1:4, and heating in a reactor at 160-180° C. for 1-2 h. After the reaction is completed, a primary product is obtained;

[0032] (3) After centrifugation, washing and drying, the initial product was obtained as the sensitizer Sb2WO6@NiS.

[0033] As an optional embodiment, in step (2), the concentration of Sb2WO6 dissolved in water is 0.1-0.25 mM, preferably 0.2 mM.

[0034] As an optional embodiment, in step (3), the centrifugation includes centrifugation at 10000-12000 rpm for 10-15 min, preferably centrifugation at 10000 rpm for 15 min.

[0035] As an optional embodiment, the drying includes drying in an oven at 50-55° C. for 20-24 hours, preferably drying in an oven at 50° C. for 24 hours.

[0036] The second object of the present invention is to provide a device for LAMP light induction-detection of qnr1, qnr2, and qnr3, the device comprising an 808nm external coaxial reflection LED light source, a LAMP light induction device, and a test paper detection component, wherein:

[0037] The LAMP light induction device comprises a housing, the housing is light-proof, and a LAMP light induction component is arranged inside the housing, wherein:

[0038] The LAMP light-induced component includes a sample holder, which is provided with N PCR tube channels for placing PCR tubes, wherein N≥1; the PCR tubes are used to hold the LAMP light-induced system, and the LAMP light-induced system contains a primer set, a sensitizer Sb2MoO6@NiFeS2, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer, wherein the DNA template is resistance genes qnr1, qnr2, and qnr3;

[0039] The front part of the housing has a transversely arranged sample channel, and the LAMP light induction component is inserted and fixed in the sample channel; the 808nm external coaxial reflection LED light source is located at the rear part of the housing, and is arranged toward the LAMP light induction component and the test paper detection component;

[0040] The test strip detection assembly includes a photothermal analysis test strip and a photothermal signal probe, wherein the photothermal signal probe is obtained by modifying the photoactivator Sb2MoO6@NiFeS2 with the nucleic acid chains of qnr1, qnr2, and qnr3, respectively. The photothermal analysis test strip is arranged vertically or horizontally, wherein:

[0041] When the photothermal analysis test paper is arranged vertically, specifically, a vertical test paper groove is arranged at the inner bottom of the front side of the housing, and the photothermal analysis test paper is vertically inserted and fixed in the test paper groove;

[0042] When the photothermal analysis test paper is arranged horizontally, specifically, the test paper detection assembly includes a test paper holder and a photothermal analysis test paper, the test paper holder is inserted into and fixed in the sample channel, the test paper holder contains a rectangular groove, and the photothermal analysis test paper is fixed in the rectangular groove.

[0043] As an optional embodiment, the 808nm external coaxial reflection LED light source includes an LED light source, a beam splitter and a plano-convex cylindrical lens. The plano-convex cylindrical lens is vertically arranged inside the housing with its flat side facing forward. The LED light source is horizontally placed at the bottom position of the rear side of the plano-convex cylindrical lens. The beam splitter is arranged at a 45° angle between the plano-convex cylindrical lens and the LED light source. After passing through the beam splitter and the plano-convex cylindrical lens, the LED light source is gathered into a linear light spot to constitute an external coaxial reflection LED light source.

[0044] As an optional implementation, the wavelength band of the LED light source is 800-810nm.

[0045] As an optional implementation, the irradiation parameters of the LED light source are power 4.5-6W and irradiation time 10-30min.

[0046] As an optional implementation, two sides of the housing are provided with a beam splitter groove and a plano-convex cylindrical lens groove, which are used to insert the beam splitter and the plano-convex cylindrical lens respectively.

[0047] As an optional embodiment, an observation channel is provided on the top of the housing, located on the front side of the plane of the plano-convex cylindrical lens, for observing the LAMP light-induced component and / or the photothermal analysis test paper.

[0048] Beneficial effects: The method, device and application for simultaneously detecting qnr1, qnr2 and qnr3 provided by the present invention utilize the photoactivity of the sensitizer Sb2WO6@NiS to generate photoelectrons and photothermal effects, which can quickly control the temperature and directly contact the LAMP photoinduction system at the molecular level, which is significantly more efficient than traditional water baths and metal baths. At the same time, the sensitizer is used as a signal probe for photothermal analysis test paper, providing a sensitive and quantifiable signal. The 808nm external coaxial reflection LED light source has more advantages in light uniformity and shadow reduction, and has greater advantages for multiple light-controlled reactions LAMP photoinduction and photothermal analysis test paper detection. The whole set of methods can quickly achieve efficient amplification and detection of qnr1, qnr2, and qnr3 target DNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 It is a schematic diagram of the working process of the present invention;

[0050] Figure 2 It is a schematic diagram of a device for simultaneously detecting qnr1, qnr2, and qnr3 of the present invention;

[0051] Figure 3 It is a schematic diagram of an integrated device for simultaneously detecting qnr1, qnr2, and qnr3 of the present invention;

[0052] Figure 4 The scanning electron microscope, transmission electron microscope and XRD spectrum of the sensitizer Sb2WO6@NiS of the present invention;

[0053] Figure 5 The photothermal temperature rise of the sensitizer of the present invention;

[0054] Figure 6 Verification of the qnr1, qnr2 and qnr3 multiple LAMP light-induced primers of the present invention;

[0055] Figure 7 The electrophoresis photos, color development and photothermal analysis test paper photos of the single and multiple LAMP light-induced products of qnr1, qnr2 and qnr3 of the present invention;

[0056] Figure 8 The qnr1, qnr2 and qnr3 LAMP light induction of the present invention is combined with the photothermal analysis test paper for photothermal quantification;

[0057] Fig. 9 The electrophoresis diagram of the products of LAMP light induction of qnr1 using the device of the present invention and using scattered light LED respectively; Fig.10 Gel electrophoresis of the products of qnr1, qnr2 and qnr3 amplified using multiplex LAMP light-induced and water-bath LAMP for the present invention. DETAILED DESCRIPTION

[0058] The present invention can be better understood according to the following examples. However, the specific material ratios, process conditions and results described in the examples are only used to illustrate the present invention, and should not and will not limit the present invention described in detail in the claims.

[0059] Embodiment 1:

[0060] A method for simultaneously detecting qnr1, qnr2 and qnr3, specifically a method for realizing multiple LAMP light induction and photothermal analysis detection of qnr1, qnr2 and qnr3 by using the photothermal effect of the sensitizer Sb2WO6@NiS, the method comprising the following steps:

[0061] (1) Preparation of sensitizer Sb2WO6@NiS:

[0062] Nickel nitrate and CH4N2S were dissolved in deionized water at a molar ratio of 1:4 and stirred for 1 hour. Then, it was injected into an aqueous solution of Sb2WO6 with a molar concentration of 1 / 4 of nickel and heated at 180°C in a reactor for 1 hour. After the reaction, the product was centrifuged at 10,000 rpm for 15 minutes and washed with deionized water. After centrifugal washing three times, it was dried in an oven at 50°C for 24 hours to obtain the sensitizer Sb2WO6@NiS. Its scanning electron microscope, transmission electron microscope and XRD patterns are shown in Figure 4 .

[0063] The sensitizer Sb2WO6@NiS solution was irradiated with an 808nm external coaxial reflection LED light source and compared with the solutions of Sb2WO6 and NiS with the same concentration. The photothermal heating curves of the three are as follows: Figure 5 As shown, it can be seen that the heating efficiency of the Sb2WO6@NiS composite is significantly improved compared with that of a single substance, which is beneficial to improving its efficiency as a sensitizer.

[0064] (2) Primer sequence design:

[0065] According to the principle of LAMP light induction, amplification primer sets were designed for six different regions of the target gene qnr1, qnr2 and qnr3 sequences. The primer sets included two external primers (F3 and B3), two internal primers (FIP and BIP) and two loop primers (loop F and loop B). The primer sequences are shown in Table 1:

[0066] Table 1 Primer sequences of the LAMP light-induced system of the present invention

[0067]

[0068]

[0069] (3) Plasmid DNA extraction:

[0070] The preserved bacteria containing the target genes (qnr1, qnr2 and qnr3) were cultured in LB medium for 24 hours, and plasmid DNA was extracted. The specific method was: plasmid DNA was extracted using a DNA extraction kit, and the kit included RNase A, Buffer S, Solution I, Solution II, Solution III, Wash Solution, Elution Buffer, EZ-10 Column, and Collection Tube.

[0071] In other optional embodiments, the plasmid DNA extraction method may also be any one of the adsorption column method, magnetic bead method, CTAB method, etc.

[0072] (4) Construction of multiple LAMP light-induced systems:

[0073] A multiple LAMP light-inducible system was constructed using qnr1, qnr2, and qnr3 in the extracted plasmid DNA as DNA templates, respectively. The multiple LAMP light-inducible system included the following components: a primer set, a sensitizer Sb2WO6@NiS (referring to the sensitizer Sb2WO6@NiS solution prepared in step (1)), BSA, MgSO4, an isothermal amplification buffer (Tris-HCl, (NH4)2SO4, KCl, MgSO4, Tween-20), dNTPs, betaine, Bst 3.0 DNA polymerase, ddH2O, and a DNA template.

[0074] Under the premise of maintaining the final volume at 25 μL, the concentrations of the components and the reaction volumes of the multiple LAMP light-inducible system are shown in Table 2 below.

[0075] Table 2 Concentrations of components and reaction volumes of the multiple LAMP light-induced system of this example

[0076]

[0077]

[0078] (5)LED light source excitation:

[0079] The multiple LAMP light-inducing system was irradiated with an 808 nm external coaxial reflection LED to excite the sensitizer in the multiple LAMP light-inducing system to provide the energy required for multiple loop-mediated isothermal amplification. The excitation power was 4.5 W and the excitation time was 20 min.

[0080] In this embodiment, an integrated LAMP light induction-detection device for qnr1, qnr2, and qnr3 is also provided, which integrates LAMP light induction and photothermal analysis test paper detection in a portable device to achieve the purpose of portable and rapid detection. Specifically, the integrated device includes an 808nm external coaxial reflection LED light source, a LAMP light induction device, and a test paper detection component 9, wherein,

[0081] The LAMP light-inducing device includes a shell 3, which is light-proof. A LAMP light-inducing component 8 is arranged inside the shell 3, wherein the LAMP light-inducing component 8 includes a sample holder, on which N PCR tube channels are provided for placing PCR tubes, wherein N≥1; the PCR tubes are used to hold the LAMP light-inducing system, and the LAMP light-inducing system contains a primer set, a sensitizer Sb2MoO6@NiFeS2, a DNA template, Bst3.0 DNA polymerase and an isothermal amplification buffer, and the DNA template is resistance genes qnr1, qnr2, and qnr3.

[0082] The front of the shell 3 has a transversely arranged sample channel 1, and the LAMP light induction component 8 is inserted and fixed in the sample channel 1; the 808nm external coaxial reflection LED light source is located at the rear of the shell 3, and is arranged toward the LAMP light induction component 8 and the test paper detection component 9.

[0083] The test paper detection component 9 includes a photothermal analysis test paper 12 and a photothermal signal probe. The photothermal signal probe is obtained by modifying the photoactivator Sb2MoO6@NiFeS2 with the nucleic acid chains of qnr1, qnr2, and qnr3 respectively. The photothermal analysis test paper 12 is arranged vertically or horizontally. In some examples, the photothermal analysis test paper 12 is arranged vertically, specifically, a vertical test paper groove 5 is provided on the inner bottom of the front side of the shell 3, and the photothermal analysis test paper 12 is vertically inserted and fixed in the test paper groove 5; in other examples, the photothermal analysis test paper 12 is arranged horizontally, specifically, the test paper detection component 9 includes a test paper holder and a photothermal analysis test paper 12, the test paper holder is inserted and fixed in the sample channel 1, the test paper holder contains a rectangular groove, and the photothermal analysis test paper 12 is fixed in the rectangular groove.

[0084] In the present invention, the 808nm external coaxial reflection LED light source can be realized by selecting existing equipment, such as Figure 2 As shown, it is not limited in the present invention.

[0085] Furthermore, the embodiment of the present invention also provides a combined 808nm external coaxial reflection LED light source. Specifically, the 808nm external coaxial reflection LED light source includes an LED light source 7, a beam splitter 11 and a plano-convex cylindrical lens 10. The plano-convex cylindrical lens 10 is vertically arranged inside the housing 3, with its flat side facing forward. The LED light source 7 is horizontally arranged at the bottom position of the rear side of the plano-convex cylindrical lens 10. The beam splitter 11 is arranged between the plano-convex cylindrical lens 10 and the LED light source 7 at an angle of 45°. After passing through the beam splitter 11 and the plano-convex cylindrical lens 10, the LED light source 7 is gathered into a linear light spot to form an external coaxial reflection LED light source 13. The two sides of the housing 3 are relatively provided with a beam splitter groove 6 and a plano-convex cylindrical lens groove 4, which are used to insert the beam splitter 11 and the plano-convex cylindrical lens 10 respectively. The material of the beam splitter is AR glass, and the size is length×width×thickness=141mm×104mm×2mm. The material of the plano-convex cylindrical lens is N-SF11, the size is length×width×center thickness=100mm×104mm×50mm, the focal length is 60mm, and the edge thickness is 0mm. The distances between the bottom end of the spectroscope and the bottom ends of the LED light source and the plano-convex cylindrical lens are 1cm and 15cm respectively. An observation channel 2 is provided at the top of the housing 3, located on the front side of the plane of the plano-convex cylindrical lens 10, for observing the LAMP light induction component 8 and / or the photothermal analysis test paper 12.

[0086] In some examples, the wavelength of the LED light source 7 is 800-810 nm, and the irradiation parameters of the LED light source 7 are power 4.5-6 W and irradiation time 10-30 min.

[0087] (6) Analysis of amplification results:

[0088] Three photothermal signal probes were obtained by modifying the sensitizer Sb2WO6@NiS with three nucleic acid chains. The specific method was as follows: 20 μL of three nucleic acid chains were added to 2 mL of sensitizer Sb2WO6@NiS respectively, 50 μL of sodium citrate buffer was added after oscillation and mixing, and the mixture was incubated at 37°C for 30 min. Then, 100 μL of HEPES buffer was added and the mixture was incubated at 37°C for 30 min. The mixture was centrifuged and 150 μL of photothermal reconstitution solution was added to dissolve the mixture, thereby obtaining three photothermal signal probes of sensitizer Sb2WO6@NiS modified with three nucleic acid chains.

[0089] T probe and C probe were fixed in the detection area and quality control area of ​​the test paper respectively to obtain a multi-target photothermal analysis test paper for the light-driven LAMP products of the target genes qnr1, qnr2, and qnr3. The probe sequences of the photothermal analysis test paper for detecting the light-induced LAMP products are shown in Table 3 below.

[0090] Table 3 Probe sequences of the photothermal analysis test paper for detecting the light-induced products of LAMP in this embodiment

[0091]

[0092] The LAMP light-induced product obtained in the above steps is detected using the above integrated device. The specific method is as follows: In this embodiment, the test paper detection component 9 includes a test paper holder and a photothermal analysis test paper 12. The test paper holder is inserted into and fixed in the sample channel 1. The test paper holder contains a rectangular groove. The photothermal analysis test paper 12 is fixed in the rectangular groove. Figure 3 f; turn on the LED light source 7, the LED light source is gathered into a linear light spot after passing through the beam splitter 11 and the plano-convex cylindrical lens 10, and constitutes an external coaxial reflective LED light source 13, which is irradiated onto the photothermal analysis test paper 12 in front, i.e., the multi-target photothermal analysis test paper constructed as described above, to provide it with photothermal heat, thereby stimulating the photothermal effect of the sensitizer Sb2WO6@NiS in the photothermal signal probe, and realizing the detection efficacy of the photothermal analysis test paper; and read the photothermal signal of the T zone on the photothermal analysis test paper through a smart phone equipped with an infrared thermal imaging accessory, judge the presence or absence of the target object according to the change phenomenon of the photothermal signal, and perform a qualitative test; and further perform a quantitative test according to the intensity of the temperature change signal, wherein the standard curve used in the quantitative test can be characterized and drawn according to the concentration gradient method.

[0093] The principle of the present invention is as follows Figure 1 The present invention can realize efficient on-site amplification of qnr1, qnr2, and qnr3, and uses the photothermal effect of 808nm external coaxial reflection LED to excite the sensitizer Sb2WO6@NiS to drive the amplification, which has the characteristics of high efficiency, convenience, and accuracy.

[0094] (7) Specificity verification

[0095] After adding all reagents except primers and templates to 6 light-driven LAMP test tubes, add

[0096] ①qnr1 template, qnr2 primer, qnr3 primer;

[0097] ②qnr1 template, qnr1 primer, qnr2 primer, qnr3 primer;

[0098] ③qnr2 template, qnr3 primer;

[0099] ④qnr2 template, qnr2 primer, qnr3 primer;

[0100] ⑤qnr3 template, qnr2 primer;

[0101] ⑥qnr3 template, qnr2 primer, qnr3 primer.

[0102] Figure 6 The electrophoresis photograph shows that only when the primers and templates of the same gene coexist, the gel electrophoresis image shows diffuse bands, indicating that the method has good selectivity for the three target genes qnr1, qnr2 and qnr3.

[0103] Figure 7 The electrophoresis photograph shows that the coexisting primers and templates do not interfere with the amplification of other target genes, and the test paper photograph shows that the test paper can specifically detect the LAMP photoinduced products of specific target genes in the multiple LAMP photoinduced system in colorimetric and photothermal modes, respectively.

[0104] Figure 8 This is a concentration gradient detection experiment for the three target genes qnr1, qnr2 and qnr3, and a quantitative curve is drawn for the three target genes based on the photothermal signal of the test paper. The above results verify that the present invention can successfully amplify and quantitatively detect the three target genes qnr1, qnr2 and qnr3. After experimental verification, the detection limit of the three target genes qnr1, qnr2 and qnr3 reached 10 copies / μL.

[0105] In addition, the scattered light LED and the external coaxial reflection LED were used as the light-induced light source of the light-controlled LAMP to compare their amplification effects. Fig. 9 As shown in the figure, only the external coaxial reflection LED light source can give a bright diffuse strip, that is, a high-efficiency amplification, while the scattered light LED light source does not induce effective amplification. The reason may be that when the scattered light LED light source is working, it not only generates light energy but also heat energy. Part of the heat energy may be absorbed by the scattering material or structure and converted into other forms of energy (such as internal energy), thereby reducing the heat dissipation efficiency; while the external coaxial reflection LED mainly converts electrical energy into light energy, generates relatively less heat energy, and can be dissipated more efficiently.

[0106] Comparative Example 1: Water Bath LAMP

[0107] In this example, the method of realizing multiple LAMP light induction of qnr1, qnr2 and qnr3 by using the photothermal effect of the sensitizer Sb2WO6@NiS is compared with the traditional water bath LAMP effect to verify the advancement of the method of the present invention in terms of amplification effect.

[0108] The water bath LAMP method includes the following steps:

[0109] (1) Primer sequence design:

[0110] Same as Example 1

[0111] (2) Plasmid DNA extraction:

[0112] Same as Example 1

[0113] (3) Construction of isothermal amplification system:

[0114] The water bath LAMP system was constructed. The water bath loop-mediated amplification system included the following components: primer set, MgSO4, isothermal amplification buffer (Tris-HCl, (NH4)2SO4, KCl, MgSO4, Tween-20), dNTPs, betaine, Bst 3.0 DNA polymerase, ddH2O and DNA template.

[0115] Under the premise of maintaining the final volume at 25 μL, the concentrations of the components and the reaction volumes of the water bath ring-mediated isothermal amplification system are shown in Table 4 below:

[0116] Table 4 Concentrations and reaction volumes of components in water bath ring-mediated isothermal amplification system

[0117]

[0118] (4) Amplification process:

[0119] Add the components of the water bath LAMP system into a 1.5 mL centrifuge tube and place it in a 60 °C water bath for 60 min.

[0120] (5) Amplification product analysis:

[0121] The amplified products were analyzed by agarose gel electrophoresis.

[0122] Gel electrophoresis images such as Fig.10 As shown, the water bath LAMP group of comparative example 1 showed bright diffuse bands after 60 minutes of amplification, while the light-controlled LAMP group showed clear and bright diffuse bands at 20 minutes, indicating that the amplification efficiency of the light-controlled LAMP group was better than that of the water bath LAMP group.

[0123] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneously detecting qnr1, qnr2, and qnr3, characterized in that: The method comprises the following steps: S1. Preparation of sensitizer Sb2WO6@NiS S2, LAMP light induction Under the irradiation of 808nm external coaxial reflection LED light source, the sample to be tested undergoes amplification reaction through the LAMP light induction system to obtain LAMP light induction products, among which, The LAMP light-induced system includes the following components: a primer set, a sensitizer Sb2WO6@NiS, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer, wherein the DNA template is the resistance genes qnr1, qnr2, and qnr3; S3, Photothermal analysis test paper detection Capture probes were fixed on the T and C regions of the test strip to obtain a multi-target photothermal analysis test strip for the light-driven LAMP products of the target genes qnr1, qnr2, and qnr3; The photothermal signal probes were obtained by modifying the sensitizer Sb2WO6@NiS with nucleic acid chains of qnr1, qnr2, and qnr3 respectively; The photothermal signal probe and the photothermal analysis test paper are used to detect the LAMP light-induced product. The detection area of ​​the photothermal analysis test paper captures the LAMP light-induced product and the photothermal signal probe in a sandwich form, and gives a photothermal temperature change signal positively correlated with the content of the LAMP light-induced product under the irradiation of an 808nm external coaxial reflection LED light source. The qnr1, qnr2, and qnr3 in the sample to be tested are qualitatively and / or quantitatively determined based on the temperature change signal.

2. The method according to claim 1, characterized in that In step S2, in the LAMP light-induced system, the concentration of each group of DNA templates is 4%-8% v / v; Preferably, the primer set includes three sets of primers, each set of primers includes two external primers F3 and B3, two internal primers FIP and BIP, and two loop primers loop F and loop B; Preferably, in the LAMP light-inducible system, the concentration of the two external primers F3 and B3 in each set of primers is 2%-6% v / v, the concentration of the two internal primers FIP and BIP is 2%-6% v / v, and the concentration of the two loop primers loop F and loop B is 0%-4% v / v; Preferably, the LAMP light-inducible system further comprises the following components: BSA, dNTPs, betaine and ddH2O; Preferably, the LAMP light-induced buffer comprises Tris-HCl, (NH4)2SO4, KCl, MgSO4, and Tween-20.

3. The method according to claim 1, characterized in that The irradiation parameters of the 808nm external coaxial reflection LED light source are power 4.5-6W and irradiation time 10-30min.

4. The method according to claim 1, characterized in that: In step S1, the preparation method of the sensitizer Sb2WO6@NiS comprises the following steps: (1) dissolving nickel nitrate and CH4N2S in water at a molar ratio of 1:4-1:5, stirring for 1-2 hours to obtain a mixed solution; (2) injecting the mixed solution into a Sb2WO6 aqueous solution, wherein the molar ratio of Sb2WO6 to nickel nitrate is 1:2-1:4, and heating in a reactor at 160-180° C. for 1-2 h. After the reaction is completed, a primary product is obtained; (3) After centrifugation, washing and drying, the initial product was obtained as the sensitizer Sb2WO6@NiS.

5. The method according to claim 4, characterized in that In step (1), the concentration of nickel nitrate dissolved in water is 0.4-0.5 mM.

6. The method according to claim 4, characterized in that In step (2), the concentration of Sb2WO6 dissolved in water is 0.1-0.25 mM.

7. The method according to claim 4, characterized in that In step (3), the centrifugation includes centrifugation at 10000-12000 rpm for 10-15 min; Preferably, the drying comprises drying in an oven at 50-55° C. for 20-24 hours.

8. The method according to claim 1, characterized in that: The photothermal signal probe is used independently of the test paper body, or is dropped and dried on a conjugate pad and the conjugate pad is integrated on the test paper.

9. A device for LAMP light-induced detection of qnr1, qnr2, and qnr3, characterized in that: It includes an 808nm external coaxial reflection LED light source, a LAMP light induction device and a test paper detection component (9), wherein: The LAMP light induction device comprises a housing (3), the housing (3) is light-proof, and a LAMP light induction component (8) is arranged inside the housing (3), wherein: The LAMP light-induced component (8) comprises a sample holder, on which N PCR tube channels are provided for placing PCR tubes, wherein N≥1; the PCR tubes are used to hold the LAMP light-induced system, wherein the LAMP light-induced system contains a primer set, a sensitizer Sb2MoO6@NiFeS2, a DNA template, a Bst3.0 DNA polymerase and an isothermal amplification buffer, wherein the DNA template is resistance genes qnr1, qnr2, and qnr3; The front part of the housing (3) has a transversely arranged sample channel (1), and the LAMP light induction component (8) is inserted into and fixed in the sample channel (1); the 808nm external coaxial reflection LED light source is located at the rear part of the housing (3), and is arranged toward the LAMP light induction component (8) and the test paper detection component (9); The test paper detection assembly (9) comprises a photothermal analysis test paper (12) and a photothermal signal probe, wherein the photothermal signal probe is obtained by modifying the photoactivator Sb2MoO6@NiFeS2 with the nucleic acid chains of qnr1, qnr2 and qnr3 respectively, and the photothermal analysis test paper (12) is arranged vertically or horizontally, wherein: When the photothermal analysis test paper (12) is arranged vertically, it specifically comprises that a vertical test paper groove (5) is arranged at the inner bottom of the front side of the housing (3), and the photothermal analysis test paper (12) is vertically inserted into and fixed in the test paper groove (5); When the photothermal analysis test paper (12) is arranged horizontally, the test paper detection assembly (9) comprises a test paper holder and a photothermal analysis test paper (12), the test paper holder is inserted into and fixed in the sample channel (1), the test paper holder comprises a rectangular groove, and the photothermal analysis test paper (12) is fixed in the rectangular groove.

10. The device according to claim 9, characterized in that The 808nm external coaxial reflection LED light source comprises an LED light source (7), a beam splitter (11) and a plano-convex cylindrical lens (10); the plano-convex cylindrical lens (10) is vertically arranged inside the housing (3) with its flat side facing forward; the LED light source (7) is horizontally arranged at the bottom position of the rear side of the plano-convex cylindrical lens (10); the beam splitter (11) is arranged between the plano-convex cylindrical lens (10) and the LED light source (7) at an angle of 45°; the LED light source (7) is gathered into a linear light spot after passing through the beam splitter (11) and the plano-convex cylindrical lens (10), thereby forming an external coaxial reflection LED light source (13); Preferably, the wavelength of the LED light source (7) is between 800-810 nm; and / or Preferably, the irradiation parameters of the LED light source (7) are power 4.5-6W, irradiation time 10-30min; and / or Preferably, the housing (3) has a beam splitter groove (6) and a plano-convex cylindrical lens groove (4) on two opposite sides thereof, for inserting the beam splitter (11) and the plano-convex cylindrical lens (10) respectively; and / or Preferably, an observation channel (2) is provided on the top of the housing (3), located on the front side of the plane of the plano-convex cylindrical lens (10), for observing the LAMP light-induced component (8) and / or the photothermal analysis test paper (12).

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