Method and device for simultaneously detecting aroA1, aroA2 and aroA3 and application

Abstract

The invention discloses a method and a device for simultaneously detecting aroA1, aroA2 and aroA3 and application, a sample tube is simultaneously irradiated by a coaxial reflection LED light source outside 808nm, a photosensitizer CsYF4 (at) Yb2O3 provides photoelectrons and heat for LAMP (loop-mediated isothermal amplification) through light triggering, a target gene is used as a template to initiate an amplification reaction, and a large number of double-stranded DNA (deoxyribonucleic acid) products are obtained. A nucleic acid chain is used for modifying CsYF4 (at) Yb2O3 to obtain a signal probe, a photothermal detection test paper detection area captures an amplification product and the signal probe in a sandwich form, a photothermal temperature change signal positively related to the content of the amplification product is given, and the aroA1, aroA2 and aroA3 in a sample are quantified according to the temperature change signal. Photoelectron and photothermal effects are generated by using the photoactivity of the photosensitizer CsYF4 (at) Yb2O3, the temperature is rapidly controlled, the photosensitizer is in direct contact with an amplification system on a molecular level, and then photothermal detection test paper detection is performed, so that the photothermal efficiency is remarkably improved.

Description

Technical Field

[0001] The present invention relates to a method, a device and an application for simultaneously detecting aroA1, aroA2 and aroA3, belonging to the technical field of inspection and detection. Background Art

[0002] Herbicide-resistant genes are genes that enable plants to survive treatment with specific herbicides. These genes are usually introduced into crops through transgenic technology to give them resistance to specific herbicides. This helps to simplify field management and reduce labor costs. Detecting herbicide-resistant genes helps verify whether transgenic crops have successfully acquired herbicide-resistant characteristics, thereby ensuring their effectiveness and safety in field management. In addition, genetic testing can also help breeders optimize breeding strategies and provide regulatory agencies with a scientific basis to evaluate the safety and compliance of transgenic crops.

[0003] The detection methods of herbicide-resistant 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 depends on professionals and equipment, it is not suitable for rapid on-site detection. Loop-mediated isothermal amplification, or LAMP technology, can use strand displacement DNA polymerase (such as Bst DNA polymerase) and a set of specific primers under isothermal conditions (usually 60℃~65℃) to complete the rapid amplification of target nucleic acids through self-circulating strand displacement DNA synthesis. LAMP technology designs four specific primers for six specific regions of the target gene. These four primers are FIP (forward inner primer), BIP (reverse inner primer), F3 (forward outer primer) and B3 (reverse outer primer). These primers play a key role in the amplification reaction and can ensure the specificity and efficiency of amplification. The amplification process of LAMP is mainly divided into two stages: the initial stage: in this stage, both the inner primer and the outer primer participate in the reaction. They bind to the specific region of the target DNA and extend under the action of DNA polymerase to form a double dumbbell structure DNA. This structure provides a template for subsequent cyclic amplification. Cyclic amplification stage: After entering this stage, only the inner primer participates in the reaction. The inner primer continuously produces new stem-loop structure DNA products through strand displacement DNA synthesis reaction. These products can continue to participate in the reaction as templates, thereby achieving exponential amplification of the target sequence.

[0004] Light-triggered amplification, by adding photosensitizers to the amplification system, using light-emitting diodes (LEDs) or lasers as light sources, evenly dispersing photothermal materials in the amplification system, and being excited by LEDs, may provide energy supply for nucleic acid amplification. At present, there are no reports on the use of light-triggered amplification technology to achieve simultaneous LAMP amplification of multiple genes. The challenge of using photosensitizer-mediated, light-triggered LAMP is that photosensitizers with higher activity are required, and the operational difficulty is that the photosensitizer needs to be compatible with the LAMP system and not interfere with the reaction; there needs to be a light source and light triggering conditions that can be precisely controlled to avoid affecting the stability of amplification; and a highly sensitive and specific detection method needs to be developed.

[0005] 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.

[0006] Currently, there is no report on the simultaneous detection of herbicide-resistant genes using external coaxial reflection LED light source excitation-light-triggered amplification combined with photothermal detection test paper technology. Summary of the invention

[0007] Technical issues: Existing methods for loop-mediated isothermal amplification of herbicide-resistant genes have the following limitations: agarose gel electrophoresis cannot be used to distinguish multiple loop-mediated isothermal amplification products; the amplification time is long, and the amplification efficiency needs to be improved. At the same time, light-triggered amplification technology has not yet been applied to the simultaneous amplification of multiple genes; although the external coaxial reflection LED light source has the advantages of light uniformity and reduced shadows, it has not yet been combined with light-triggered amplification and photothermal detection paper technology for the detection of herbicide-resistant genes. The challenges and operational difficulties of using photosensitizer-mediated, light-triggered LAMP are that the photosensitizer must be compatible with the LAMP system and not interfere with the reaction; there must be a precisely controllable light source and light triggering conditions to avoid affecting the stability of the amplification; and a highly sensitive and specific detection method must be developed.

[0008] 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 aroA1, aroA2 and aroA3, adopting the photosensitizer CsYF4@Yb2O3 as the photothermal material, utilizing its photoactivity to generate photoelectrons and photothermal effects, and realizing rapid temperature control under the irradiation of 808nm external coaxial reflective LED light source and direct contact with the amplification system at the molecular level, so that the photothermal efficiency is significantly improved, and then the photothermal detection test paper color development detection is carried out, thereby realizing the intensive and efficient amplification and detection of the two links of light-triggered amplification and photothermal detection test paper detection.

[0009] Technical solution: The present invention adopts the following technical solution.

[0010] The first object of the present invention is to provide a method for realizing light-triggered LAMP and detection of herbicide-resistant genes aroA1, aroA2, and aroA3 using the photothermal effect of photosensitizer CsYF4@Yb2O3. The method includes two links: light-triggered amplification and photothermal detection test paper detection. An 808nm external coaxial reflection LED light source is used to irradiate the amplification system, and the photosensitizer CsYF4@Yb2O3 in the amplification system is stimulated to produce photoelectrons and heat, providing energy for LAMP, so that aroA1, aroA2, and aroA3 are used as templates to trigger an amplification reaction, and a large amount of double-stranded DNA amplification products are obtained; then, the amplification products and signal probes are captured in a sandwich form by photothermal detection test paper, and a photothermal temperature change signal positively correlated with the content of the amplification product is given to achieve quantitative detection of the target gene. The isothermal amplification system includes the following components: a primer set, a photosensitizer CsYF4@Yb2O3, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer.

[0011] Specifically, the present invention provides a method for simultaneously detecting aroA1, aroA2, and aroA3, comprising the following steps:

[0012] S1. Preparation of photosensitizer CsYF4@Yb2O3

[0013] S2. Light-triggered LAMP amplification

[0014] Under the mediation of photosensitizer CsYF4@Yb2O3, the sample to be tested was irradiated with an 808nm external coaxial reflection LED light source, and the light-triggered LAMP system was used to amplify the sample to obtain the amplified product, wherein:

[0015] The light-triggered LAMP system includes the following components: a photosensitizer CsYF4@Yb2O3 and a DNA template, wherein the DNA template is herbicide-resistant genes aroA1, aroA2, and aroA3.

[0016] S3, Photothermal test paper detection

[0017] Capture probes were fixed in the T and C regions of the test strip to obtain a multi-target photothermal detection test strip for the light-triggered LAMP products of the target genes aroA1, aroA2, and aroA3

[0018] The photosensitizer CsYF4@Yb2O3 was modified with nucleic acid chains of aroA1, aroA2, and aroA3 to obtain photothermal signal probes;

[0019] The amplification product is detected using the photothermal signal probe and the photothermal detection test paper. The detection area of ​​the photothermal detection test paper captures the amplification product and the photothermal signal probe in a sandwich form, and gives a photothermal temperature change signal positively correlated with the content of the amplification product under the irradiation of an 808nm external coaxial reflection LED light source. AroA1, aroA2, and aroA3 in the sample to be tested are qualitatively and / or quantitatively determined based on the temperature change signal.

[0020] As an optional implementation, in the light-triggered LAMP system, the concentration of each group of DNA templates is 3%-5% v / v.

[0021] As an optional embodiment, the light-triggered LAMP system further includes primers, and 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 loopF and loop B.

[0022] As an optional embodiment, in the light-triggered LAMP system, the concentration of the two external primers F3 and B3 in each set of primers is 4%-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 loopF and loop B is 0%-4% v / v.

[0023] As an optional embodiment, the light-triggered LAMP system further comprises the following components: BSA, dNTPs, betaine, Bst3.0 DNA polymerase, ddH2O and isothermal amplification buffer.

[0024] As an optional embodiment, the isothermal amplification buffer includes Tris-HCl, (NH4)2SO4, KCl, MgSO4, and Tween-20.

[0025] As an optional embodiment, the photothermal signal probe is used independently of the photothermal detection test paper body, or is dropped and dried on the conjugate pad and the conjugate pad is integrated on the photothermal detection test paper.

[0026] As an optional implementation, the irradiation parameters of the 808nm external coaxial reflection LED light source are power 4.5-6W and irradiation time 10-60min.

[0027] As an optional embodiment, the preparation method of the photosensitizer CsYF4@Yb2O3 comprises the following steps:

[0028] (1) Add 140-160 mg of EDTA to 20-30 mL of ultrapure water and stir for 5-10 min to obtain a mixed solution;

[0029] (2) Add Y(NO3)3 and Yb2O3 with a concentration of 0.6-0.8 M to the mixed solution and stir rapidly for 30-40 min;

[0030] (3) Add 0.8-1.2M NH4F and CsF to the mixed solution, stir for 30-40 minutes, and heat in a reactor at 180-220°C for 23-25 ​​hours. After the reaction is completed, the initial product is obtained;

[0031] (4) After cooling, washing and drying, the initial product is obtained as photosensitizer CsYF4@Yb2O3.

[0032] As an optional embodiment, the volume of Y(NO3)3 used in step (2) is 0.8-1.0 mL, and the volume of Yb2O3 used is 0.1-0.3 mL, preferably 1 mL Y(NO3)3 and 0.2 mL Yb2O3.

[0033] As an optional embodiment, the volume of NH4F and CsF used in step (3) is 2-4 mL, preferably 3 mL.

[0034] As an optional embodiment, the drying comprises drying in an oven at 65-75° C. for 2-3 h, preferably drying at 70° C. for 2 h.

[0035] The second object of the present invention is to provide a device for simultaneously detecting aroA1, aroA2, and aroA3, which is used to realize the combined and convenient operation of the above method. Specifically, the device includes an 808nm external coaxial reflection LED light source, a light-triggered LAMP device, and a photothermal detection test paper assembly, wherein:

[0036] The light-triggered LAMP device comprises: a light-proof and heat-insulating shell, and a light-triggered LAMP component arranged inside the light-proof and heat-insulating shell, wherein:

[0037] The light-triggered LAMP component 8 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 light-triggered LAMP system, and the light-triggered LAMP system contains a primer set, a photosensitizer CsYF4@Yb2O3, a DNA template, Bst3.0 DNA polymerase and an isothermal amplification buffer, and the DNA template is the herbicide-resistant genes aroA1, aroA2, and aroA3;

[0038] The front part of the light-proof and heat-insulating shell has a transversely arranged sample channel, and the light-triggered LAMP 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 light-proof and heat-insulating shell, and is arranged toward the light-triggered LAMP component and the photothermal detection test paper component;

[0039] The photothermal detection test strip assembly includes a photothermal detection test strip arranged vertically or horizontally, and the photothermal signal probe is obtained by modifying the photosensitizer CsYF4@Yb2O3 with nucleic acid chains of aroA1, aroA2, and aroA3, respectively, wherein:

[0040] When the photothermal test paper is arranged vertically, specifically, a vertical test paper groove is arranged at the inner bottom of the front side of the light-proof and heat-insulating shell, and the photothermal test paper is vertically inserted and fixed in the test paper groove;

[0041] When the photothermal detection test paper is arranged horizontally, specifically, the photothermal detection test paper assembly includes a test paper holder and the photothermal detection 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 detection test paper is fixed in the rectangular groove.

[0042] 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 light-proof and heat-insulating outer shell 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.

[0043] As an optional implementation, the wavelength band of the LED light source is 800-810nm.

[0044] As an optional implementation, the irradiation parameters of the LED light source are power 4.5-6W and irradiation time 10-60min.

[0045] As an optional implementation, two sides of the light-proof and heat-insulating housing are provided with beam splitter grooves and plano-convex cylindrical lens grooves for inserting beam splitters and plano-convex cylindrical lenses, respectively.

[0046] As an optional implementation, an observation channel is provided on the top of the light-proof and heat-insulating housing, located on the front side of the plane of the plano-convex cylindrical lens, for observing the light-triggered LAMP component and / or the photothermal detection test paper.

[0047] Beneficial Effects

[0048] The amplification method provided by the present invention uses the photoactive photosensitizer CsYF4@Yb2O3 to generate photoelectrons and photothermal effects, which can quickly control the temperature and directly contact the amplification system at the molecular level, and the heating speed is faster than that of traditional water baths and metal baths. At the same time, the photosensitizer CsYF4@Yb2O3 is used as a signal probe for photothermal detection test paper, providing a sensitive and quantifiable signal. The whole set of methods can quickly achieve efficient amplification and detection of multiple target DNAs. 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 the device of the present invention;

[0051] Figure 3 Schematic diagram of the self-assembly device of the present invention.

[0052] Figure 4 The photothermal temperature rise of the photothermal material of the present invention;

[0053] Figure 5 It is the validation of the light-triggered multiple LAMP primers of the present invention;

[0054] Figure 6 The electrophoresis photos, color development and photothermal detection test paper photos of the light-triggered single and multiple LAMP products of the present invention;

[0055] Figure 7 The light-triggered LAMP of the present invention is combined with lateral flow chromatography test paper for photothermal quantification;

[0056] Figure 8 The electrophoretic diagrams of the products of light-triggered LAMP of aroA using the device and scattered light LED, respectively;

[0057] Fig. 9 The present invention uses gel electrophoresis of light-triggered multiplex LAMP and water-bath LAMP amplification products. 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] The challenges and operational difficulties of using photosensitizer-mediated, light-triggered LAMP are that the photosensitizer must be compatible with the LAMP system and not interfere with the reaction; there must be a light source and light triggering conditions that can be precisely controlled to avoid affecting the stability of amplification; and a high-sensitivity, high-specificity detection method that matches light-triggered multiple LAMP must be developed. Based on this technical difficulty, the present invention discloses an analysis method, device and application of light-triggered loop-mediated isothermal amplification, i.e., LAMP combined with photothermal detection test paper, for simultaneous detection of herbicide-resistant genes aroA1, aroA2, and aroA3. This method includes two steps: light-triggered LAMP and photothermal detection test paper detection. N sample tubes are simultaneously irradiated with an 808nm external coaxial reflection LED light source, and the photosensitizer CsYF4@Yb2O3 therein provides photoelectrons and heat for LAMP through light triggering. The target gene in the positive sample is used as a template to trigger the amplification reaction, and a large amount of double-stranded DNA amplification products are obtained. CsYF4@Yb2O3 is modified with the three nucleic acid chains aroA1, aroA2, and aroA3 to obtain three corresponding signal probes. The detection areas of the three photothermal detection test strips capture the amplification products and signal probes in a sandwich form, and give a photothermal temperature change signal that is positively correlated with the content of the amplification products. AroA1, aroA2, and aroA3 in the original sample are further quantified according to the temperature change signal. The present invention uses the photoactivity of the photosensitizer CsYF4@Yb2O3 to generate photoelectrons and photothermal effects, quickly control the temperature, and directly contact the light-triggered LAMP system at the molecular level, and then perform color development detection with the photothermal detection test strip, and the photothermal efficiency is significantly improved.

[0060] Example

[0061] The following embodiment of the present application discloses a method for realizing a light-triggered multiple LAMP system of resistance genes aroA1, aroA2, and aroA3 by utilizing the photothermal effect of photosensitizer CsYF4@Yb2O3, comprising the following steps:

[0062] (1) Primer sequence design:

[0063] According to the LAMP principle, taking a target gene sequence as an example, an amplification primer set was designed for six different regions of the target gene sequence, and the primer set included two outer primers (F3 and B3), two inner primers (FIP and BIP) and two loop primers (loop F and loop B).

[0064] (2) Plasmid DNA extraction:

[0065] The microorganisms collected from the sample or the preserved strains containing the target genes (aroA1, aroA2, aroA3) 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.

[0066] As an optional embodiment, the plasmid DNA extraction method in step (2) can also be any one of the adsorption column method, magnetic bead method, CTAB method, etc.

[0067] (3) Preparation of photosensitizer CsYF4@Yb2O3:

[0068] Add 140-160mg of EDTA to a 50mL beaker, add 25mL of ultrapure water and stir rapidly for 5 minutes. Then add 0.8-1.0mL of Y(NO3)3 with a molar concentration of 0.6-0.8mol / L, 0.1-0.3mL of Yb2O3 with a molar concentration of 0.6-0.8mol / L and stir rapidly for 30min. Subsequently, add 2-4mL of NH4F with a molar concentration of 0.8-1.2mol / L and 2-4mL of CsF with a molar concentration of 0.8-1.2mol / L, and stir continuously for 30 minutes until a milky white solution is produced. After transferring the solution to a 200℃ autoclave, react for 24h, cool to room temperature, wash alternately with ultrapure water and alcohol 2-3 times, and dry at 70℃ for 2h to obtain CsYF4@Yb2O3 nanocomposite material.

[0069] (4) Construction of light-triggered multiple LAMP system:

[0070] A light-triggered multiple LAMP system was constructed using aroA1, aroA2, and aroA3 in the extracted plasmid DNA as templates, respectively. The amplification system included the following components: a primer set, a photosensitizer CsYF4@Yb2O3 (i.e., the photosensitizer CsYF4@Yb2O3 solution prepared in step (3)), 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.

[0071] (5) External coaxial reflection LED light source excitation:

[0072] The light-triggered LAMP system is illuminated by an 808nm external coaxial reflection LED to excite the photosensitizer in the light-triggered LAMP system to provide the energy required for multiple LAMPs.

[0073] In an embodiment of the present invention, a device for simultaneously detecting aroA1, aroA2, and aroA3 is also provided, which integrates the two links of light-triggered LAMP and photothermal detection test strip detection together with the light source into a combined device, further enhancing the portability of the detection system of the present invention. Figure 2 As shown, the device includes an 808nm external coaxial reflection LED light source, a light-triggered LAMP device and a photothermal detection test paper assembly 9, wherein:

[0074] The light-triggered LAMP device comprises: a light-proof and heat-insulating shell 3, and a light-triggered LAMP component 8 arranged inside the light-proof and heat-insulating shell 3, wherein:

[0075] The light-triggered LAMP component 8 includes a sample holder, which is provided with N through-holes for PCR tubes for placing PCR tubes, wherein N≥1; the PCR tubes are used to hold the light-triggered LAMP system, and the light-triggered LAMP system contains a primer set, a photosensitizer CsYF4@Yb2O3, a DNA template, a Bst3.0 DNA polymerase, and an isothermal amplification buffer;

[0076] The front part of the light-proof heat-insulating housing 3 has a transversely arranged sample channel 1, and the light-triggered LAMP component 8 is inserted and fixed in the sample channel 1; the 808nm external coaxial reflection LED light source is located at the rear part of the light-proof heat-insulating housing 3, and is arranged toward the light-triggered LAMP component 8 and the photothermal detection test paper component 9 to provide a light source for them;

[0077] The photothermal detection test paper assembly 9 includes a photothermal detection test paper 12 arranged vertically or horizontally, wherein:

[0078] When the photothermal test paper 12 is arranged vertically, specifically, a vertical test paper groove 5 is provided at the inner bottom of the front side of the light-proof and heat-insulating shell 3, and the photothermal test paper 12 is vertically inserted and fixed in the test paper groove 5;

[0079] When the photothermal detection test paper 12 is arranged horizontally, specifically, the photothermal detection test paper assembly 9 includes a test paper holder and the photothermal detection 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, and the photothermal detection test paper 12 is fixed in the rectangular groove.

[0080] In one example, if Figure 2 As shown in d, the 808nm external coaxial reflection LED light source is provided by the existing external coaxial reflection LED light source 14 with a wavelength of 808nm, which is not specifically limited in the present invention and all belong to the protection scope of the present invention.

[0081] In one example, the present invention also provides a self-assembled 808nm external coaxial reflection LED light source, such as Figure 3 As shown, the self-assembled 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 light-proof and heat-insulating shell 3, with its flat side facing forward, the LED light source 7 is horizontally placed at the bottom position of the rear side of the plano-convex cylindrical lens 10, the beam splitter 11 is arranged at a 45° angle between the plano-convex cylindrical lens 10 and the LED light source 7, and 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, forming an external coaxial reflection LED light source 13.

[0082] The diffuse light emitted by the LED light source is filtered by a beam splitter to obtain uniform parallel light. The parallel light is gathered into a horizontal linear light spot after passing through a plano-convex cylindrical mirror. The power of the LED light source can be adjusted to meet the energy required for light-triggered LAMP and photothermal detection test strip detection under different room temperature conditions. The size of the light spot in the amplification container (0.2mL PCR tube) and the test strip can be adjusted by adjusting the distance between the light-triggered LAMP component, the photothermal detection test strip component and the plano-convex cylindrical mirror to cope with light-triggered LAMP systems of different volumes and different T zones on the test strip.

[0083] In one example, the wavelength band of the LED light source 7 is 800-810 nm.

[0084] In one example, the irradiation parameters of the LED light source 7 are power 4.5-6W and irradiation time 10-60min.

[0085] In one example, two sides of the light-shielding and heat-insulating housing 3 are oppositely provided with a beam splitter groove 6 and a plano-convex cylindrical lens groove 4 for inserting a beam splitter 11 and a plano-convex cylindrical lens 10, respectively.

[0086] In one example, an observation channel 2 is provided on the top of the light-proof and heat-insulating housing 3 , which is located on the front side of the plane of the plano-convex cylindrical lens 10 and is used to observe the light-triggered LAMP component 8 and / or the photothermal detection test paper 12 .

[0087] In one example, the diameter of the PCR tube channel is 5.38 mm, and the center distance between adjacent PCR tube channels is 9 mm.

[0088] In one example, among the N penetrating PCR tube channels, N≥2.

[0089] In one example, the PCR tube is a 200 μL PCR single tube and / or a 200 μL eight-tube strip.

[0090] In one example, the distances between the bottom end of the beam splitter and the bottom ends of the LED light source and the plano-convex cylindrical lens are 1 cm and 15 cm respectively.

[0091] In one example, the beam splitter is made of AR glass, and has a size of length×width×thickness=141 mm×104 mm×2 mm.

[0092] In one example, the material of the plano-convex cylindrical lens is N-SF11, the dimensions are length×width×center thickness=100mm×104mm×50mm, the focal length is 60mm, and the edge thickness is 0mm.

[0093] In one example, the sample channel is 45 mm away from the plane of the plano-convex cylindrical lens and 60 mm away from the inner bottom of the light-proof and heat-insulating shell.

[0094] In one example, the depth of the rectangular groove on the test paper holder is 3 mm.

[0095] (6) Photothermal test paper detection:

[0096] The photosensitizer CsYF4@Yb2O3 was modified with three nucleic acid chains to obtain three photothermal signal probes. The capture probes were fixed in the T and C regions of the test paper to obtain a multi-target photothermal detection test paper for the light-triggered LAMP products of the target genes aroA1, aroA2, and aroA3. The amplified products were detected using the photothermal detection test paper, and the temperature signal was read by a smartphone for quantification.

[0097] The principle of the present invention is as follows Figure 1As shown. The method is efficient, convenient and accurate, and is suitable for the detection of aroA1, aroA2 and aroA3. The present invention can realize efficient on-site amplification of aroA1, aroA2 and aroA3, and light-triggered amplification of photosensitizer CsYF4@Yb2O3 is excited by an 808nm external coaxial reflection LED.

[0098] The following examples use aroA1, aroA2 and aroA3 as examples of resistance genes to fully illustrate the entire amplification method.

[0099] Embodiment 1:

[0100] A method for driving multiple LAMPs using the photothermal effect of photosensitizer CsYF4@Yb2O3, the method comprising the following steps:

[0101] (1) Primer sequence design:

[0102] According to the principle of loop-mediated isothermal amplification, amplification primer sets were designed for six different regions of the target gene sequence. 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 sequences of the external primers are shown in Table 1:

[0103] Table 1 Primer sequences of the light-triggered multiple LAMP system of the present invention

[0104]

[0105] (2) Plasmid DNA extraction:

[0106] The preserved bacteria containing the target genes (aroA1, aroA2, aroA3) 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.

[0107] (3) Preparation of photosensitizer CsYF4@Yb2O3:

[0108] Add 140mg of EDTA to a 50mL beaker, add 25mL of ultrapure water and stir rapidly for 5 minutes. Then add 0.8mL of Y(NO3)3 with a molar concentration of 0.6mol / L and 0.1mL of Yb2O3 with a molar concentration of 0.6mol / L and stir rapidly for 30min. Subsequently, add 2mL of NH4F with a molar concentration of 0.8mol / L and 2mL of CsF with a molar concentration of 0.8mol / L, and stir continuously for 30 minutes until a milky white solution is produced. After transferring the solution to a 200℃ autoclave, react for 24h, cool to room temperature, wash it alternately with ultrapure water and alcohol 3 times, and dry it at 70℃ for 2h to obtain CsYF4@Yb2O3 nanocomposite material.

[0109] The photosensitizer CsYF4@Yb2O3 solution was irradiated with an 808nm external coaxial reflection LED light source in the combined device and compared with the solutions of CsYF4 and Yb2O3 with the same concentration. The photothermal heating curves of the three are shown in Figure 2. Figure 4 As shown, it can be seen that the heating efficiency of the CsYF4@Yb2O3 complex is significantly improved compared with that of a single substance, which is beneficial to improving its efficiency as a photosensitizer.

[0110] (4) Construction of light-triggered multiple LAMP system:

[0111] The light-triggered multiple LAMP system includes the following components: a primer set, a photosensitizer CsYF4@Yb2O3 (referring to the photosensitizer CsYF4@Yb2O3 solution prepared in step (3)), 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.

[0112] Under the premise of maintaining the final volume at 25 μL, the concentrations of the components and the reaction volumes of the light-triggered multiple LAMP system are shown in Table 2 below.

[0113] Table 2 Concentrations of components and reaction volumes of the light-triggered multiple LAMP system in this example

[0114]

[0115]

[0116] (5)LED light source excitation:

[0117] In this embodiment, the two links of light-triggered multiple LAMP and photothermal detection test strips are integrated into a combined device together with the light source to study the detection effect of the combined device on the three target genes aroA1, aroA2 and aroA3. Figure 2, 3 As shown, the device includes an 808nm external coaxial reflection LED light source, a light-triggered LAMP device and a photothermal detection test paper assembly 9, wherein:

[0118] The light-triggered amplification device comprises: a light-proof and heat-insulating housing 3, and a light-triggered LAMP component 8 arranged inside the light-proof and heat-insulating housing 3, wherein:

[0119] The light-triggered LAMP assembly 8 includes a sample holder, on which 10 PCR tube channels are provided for placing PCR tubes, the PCR is a 200 μL PCR single tube, the diameter of the tube channel is 5.38 mm, and the center distance between adjacent PCR tube channels is 9 mm;

[0120] The PCR tube is used to hold the light-triggered LAMP system. As described in the above steps, the light-triggered LAMP system contains a primer set, a photosensitizer CsYF4@Yb2O3, a DNA template, Bst3.0 DNA polymerase, and an isothermal amplification buffer;

[0121] The front part of the light-proof and heat-insulating housing 3 has a transversely arranged sample channel 1, and the light-triggered LAMP component 8 is inserted and fixed in the sample channel 1; the 808nm external coaxial reflection LED light source is located at the rear part of the light-proof and heat-insulating housing 3, and is arranged toward the light-triggered LAMP component 8 and the photothermal detection test paper component 9 to provide light source for them;

[0122] The photothermal detection test paper assembly 9 includes a photothermal detection test paper 12 arranged vertically or horizontally. An observation channel 2 is provided on the top of the light-proof and heat-insulating shell 3, which is located on the front side of the plane of the plano-convex cylindrical lens 10 and is used to observe the light-triggered LAMP assembly 8 and / or the photothermal detection test paper 12.

[0123] In one example, when the photothermal test paper 12 is arranged vertically, specifically, a vertical test paper groove 5 is arranged at the inner bottom of the front side of the light-proof and heat-insulating shell 3, and the photothermal test paper 12 is vertically inserted and fixed in the test paper groove 5;

[0124] In another example, when the photothermal detection test paper 12 is arranged horizontally, specifically, the photothermal detection test paper assembly 9 includes a test paper holder and the photothermal detection test paper 12, the test paper holder is inserted into and fixed in the sample channel 1, and the test paper holder contains a rectangular groove with a depth of 3 mm, and the photothermal detection test paper 12 is fixed in the rectangular groove.

[0125] like Figure 3As shown, the self-assembled 808nm external coaxial reflection LED light source of this embodiment 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 light-proof heat-insulating shell 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 a 45° inclination. 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 light-proof heat-insulating shell 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 beam splitter and the bottom ends of the LED light source and the plano-convex cylindrical lens are 1cm and 15cm respectively.

[0126] One of the challenges and operational difficulties of using photosensitizer-mediated, light-triggered multiple LAMP is that a precisely controllable light source and light triggering conditions are required to avoid affecting the stability of amplification. In order to solve this technical problem, the present invention uses this self-assembled device to cleverly filter the diffuse light emitted by the LED light source through a spectroscope to obtain uniform parallel light, and the parallel light is gathered into a horizontal linear light spot after passing through a plano-convex cylindrical mirror. By adjusting the power of the LED light source, the energy required for light-triggered LAMP and photothermal detection test paper detection under different room temperature conditions can be met. By adjusting the distance between the light-triggered LAMP component, the photothermal detection test paper component and the plano-convex cylindrical mirror, the size of the light spot on the amplification container (0.2mL PCR tube) and the test paper can be adjusted to cope with light-triggered LAMP systems of different volumes and different T zones on the test paper.

[0127] The 808nm external coaxial reflection LED in the combined device is used to illuminate the light-triggered LAMP system, and the photothermal material in the light-triggered LAMP system is excited to provide the energy required for multiple LAMPs, thereby achieving amplification and obtaining amplified products. In this embodiment, the excitation power of the 808nm external coaxial reflection LED is 4.5W, and the excitation time is 15min.

[0128] (6) Analysis of light-triggered LAMP results:

[0129] Three photothermal signal probes were obtained by modifying the photosensitizer CsYF4@Yb2O3 with three nucleic acid chains. The specific method is as follows: 20 μL of three nucleic acid chains, aroA1, aroA2, and aroA3, were added to 2 mL of photosensitizer CsYF4@Yb2O3 respectively, and 50 μL of sodium citrate buffer was added after oscillation and mixing. The mixture was incubated at 37°C for 30 min, and then 100 μL of HEPES buffer was added. The mixture was incubated at 37°C for 30 min, centrifuged, and 150 μL of photothermal reconstitution solution was added to dissolve the mixture. Thus, three photothermal signal probes of the photosensitizer CsYF4@Yb2O3 modified with three nucleic acid chains were obtained.

[0130] Capture probes were fixed in the T and C regions of the test strip to obtain a multi-target photothermal detection test strip for the light-triggered LAMP products of the target genes aroA1, aroA2, and aroA3.

[0131] The probe sequences of the amplified products detected by the photothermal detection test strip are shown in Table 3 below.

[0132] Table 3 Probe sequences of amplified products detected by the photothermal test strip in this embodiment

[0133]

[0134] The photothermal detection test paper assembly 9 of the combined device is used to detect the amplification product obtained in the above steps. The specific method is as follows: In this embodiment, the photothermal detection test paper 12 is horizontally inserted and fixed in the sample channel 1, such as 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 detection test paper 12 in front to provide it with photothermal heat, thereby stimulating the photothermal effect of the photosensitizer CsYF4@Yb2O3 in the photothermal signal probe, and realizing the detection efficacy of the photothermal detection test paper; and read the photothermal signal of the T zone on the photothermal detection test paper 12 through a smart phone equipped with an infrared thermal imaging accessory, and judge whether the target object exists 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.

[0135] (7) Specificity verification

[0136] After adding all reagents except primers and templates to 6 light-triggered LAMP test tubes, add

[0137] ① aroA1 template, aroA2 primer, aroA3 primer;

[0138] ② aroA1 template, aroA1 primer, aroA2 primer, aroA3 primer;

[0139] ③ aroA2 template, aroA3 primer;

[0140] ④ aroA2 template, aroA2 primer, aroA3 primer;

[0141] ⑤ aroA3 template, aroA2 primer;

[0142] ⑥ aroA3 template, aroA2 primer, aroA3 primer;

[0143] Figure 5 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 aroA1, aroA2, and aroA3.

[0144] Figure 6 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 amplification products of specific target genes in the multiple LAMP system in colorimetric and photothermal modes, respectively.

[0145] Figure 7 This is a concentration gradient detection experiment for the three target genes aroA1, aroA2 and aroA3, 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 aroA1, aroA2 and aroA3. After experimental verification, the detection limit of the three target genes sul1, sul2 and sul3 reached 10 copies / μL.

[0146] In addition, the scattered light LED and the external coaxial reflection LED were used as the light-triggered LAMP light source to compare their amplification effects. Figure 8 As shown in the figure, only the external coaxial reflection LED can give a bright diffuse strip, that is, a high-efficiency amplification, while the scattered light LED does not induce effective amplification. The reason may be that when the scattered light LED 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.

[0147] Comparative Example 1: Water Bath LAMP

[0148] This example compares the method of realizing light-triggered multiple LAMP using the photothermal effect of the photosensitizer CsYF4@Yb2O3 with the effect of traditional water bath LAMP to verify the advancement of the method of the present invention in terms of amplification effect.

[0149] The water bath ring-mediated isothermal amplification method comprises the following steps:

[0150] (1) Primer sequence design:

[0151] Same as Example 1

[0152] (2) Plasmid DNA extraction:

[0153] Same as Example 1

[0154] (3) Construction of isothermal amplification system:

[0155] 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.

[0156] Under the premise of maintaining the final volume at 25 μL, the concentrations of the components and the reaction volume of the water bath LAMP system are shown in Table 4 below:

[0157] Table 4 Concentrations of components and reaction volumes of water bath LAMP system

[0158]

[0159] (4) Amplification process:

[0160] Add the components of the water bath LAMP system into a 1.5 mL centrifuge tube and place it in a 65 °C water bath for 60 min.

[0161] (5) Amplification product analysis:

[0162] The amplified products were analyzed by agarose gel electrophoresis.

[0163] Gel electrophoresis images such as Fig. 9 As shown, the negative group image without template DNA only shows primers and no diffuse bands, while the image of the positive group with template DNA successfully amplified shows diffuse bands. At the same time, the water bath LAMP group of comparative example 1 only shows bright diffuse bands after 60 minutes of amplification, while the light-triggered LAMP group shows clear and bright diffuse bands at 20 minutes, indicating that the amplification efficiency of the light-triggered LAMP group is better than that of the water bath LAMP group.

[0164] 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 aroA1, aroA2, and aroA3, characterized in that: The following steps are involved: S1. Preparation of photosensitizer CsYF4@Yb2O3 S2. Light-triggered LAMP amplification Under the mediation of photosensitizer CsYF4@Yb2O3, the sample to be tested was irradiated with an 808nm external coaxial reflection LED light source, and the light-triggered LAMP system was used to amplify the sample to obtain the amplified product, wherein: The light-triggered LAMP system includes a photosensitizer CsYF4@Yb2O3 and a DNA template, wherein the DNA template is herbicide-resistant genes aroA1, aroA2, and aroA3; S3, Photothermal test paper detection Capture probes were fixed on the T and C regions of the test strip to obtain a multi-target photothermal detection test strip for the light-triggered LAMP products of the target genes aroA1, aroA2, and aroA3; The photosensitizer CsYF4@Yb2O3 was modified with nucleic acid chains of aroA1, aroA2, and aroA3 to obtain photothermal signal probes; The amplification product is detected using the photothermal signal probe and the photothermal detection test paper. The detection area of ​​the photothermal detection test paper captures the amplification product and the photothermal signal probe in a sandwich form, and gives a photothermal temperature change signal positively correlated with the content of the amplification product under the irradiation of an 808nm external coaxial reflection LED light source. AroA1, aroA2, and aroA3 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 the light-triggered LAMP system, the concentration of each group of DNA templates is 3%-5% v / v; Preferably, the light-triggered LAMP system further comprises primers, wherein the concentration of the two external primers F3 and B3 in each set of primers is 4%-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 light-triggered LAMP system further comprises the following components: BSA, dNTPs, betaine, Bst3.0 DNA polymerase, ddH2O and isothermal amplification buffer; Preferably, the isothermal amplification buffer comprises Tris-HCl, (NH4)2SO4, KCl, MgSO4, Tween-20).

3. The method according to claim 1, characterized in that: The irradiation parameters of the external coaxial reflection LED light source are power 4.5-6W and irradiation time 10-60min.

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

5. The method according to claim 1, characterized in that The preparation method of the photosensitizer CsYF4@Yb2O3 comprises the following steps: (1) Add 140-160 mg of EDTA to 20-30 mL of ultrapure water and stir for 5-10 min to obtain a mixed solution; (2) Add Y(NO3)3 and Yb2O3 with a concentration of 0.6-0.8M to the mixed solution and stir rapidly for 30-40 minutes; (3) Add 0.8-1.2M NH4F and CsF to the mixed solution, stir for 30-40 minutes, and heat in a reactor at 180-220°C for 23-25 ​​hours. After the reaction is completed, the initial product is obtained; (4) After cooling, washing and drying, the initial product is obtained as photosensitizer CsYF4@Yb2O3.

6. The method according to claim 5, characterized in that The volume of Y(NO3)3 used in step (2) is 0.8-1.0 mL, and the volume of Yb2O3 used is 0.1-0.3 mL.

7. The method according to claim 5, characterized in that The volume of NH4F and CsF used in step (3) is 2-4 mL.

8. The method according to claim 5, characterized in that The drying in step (4) includes drying in an oven at 65-75° C. for 2-3 hours.

9. A device for simultaneously detecting aroA1, aroA2, and aroA3, characterized in that: It comprises an 808nm external coaxial reflection LED light source, a light-triggered LAMP device and a photothermal detection test paper assembly (9), wherein: The light-triggered LAMP device comprises: a light-proof and heat-insulating outer shell (3), and a light-triggered LAMP component (8) arranged inside the light-proof and heat-insulating outer shell (3), wherein: The light-triggered LAMP 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 light-triggered LAMP system, the light-triggered LAMP system contains a primer set, a photosensitizer CsYF4@Yb2O3, a DNA template, Bst3.0 DNA polymerase and an isothermal amplification buffer, wherein the DNA template is the herbicide-resistant genes aroA1, aroA2 and aroA3; The front part of the light-proof heat-insulating shell (3) has a transversely arranged sample channel (1), and the light-triggered LAMP 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 light-proof heat-insulating shell (3), and is arranged toward the light-triggered LAMP component (8) and the photothermal detection test paper component (9); The photothermal detection test paper assembly (9) comprises a photothermal detection test paper (12) arranged vertically or horizontally, wherein the photothermal signal probe is obtained by modifying the photosensitizer CsYF4@Yb2O3 with nucleic acid chains of aroA1, aroA2, and aroA3, respectively, wherein: When the photothermal detection test paper (12) is arranged vertically, it specifically includes that a vertical test paper groove (5) is arranged on the inner bottom of the front side of the light-proof and heat-insulating shell (3), and the photothermal detection test paper (12) is vertically inserted and fixed in the test paper groove (5); When the photothermal detection test paper (12) is arranged horizontally, the photothermal detection test paper assembly (9) comprises a test paper holder and the photothermal detection 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 detection 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 light-shielding and heat-insulating shell (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 10-60min; and / or Preferably, the light-shielding and heat-insulating housing (3) has a beam splitter groove (6) and a plano-convex cylindrical lens groove (4) on two sides thereof, for inserting a beam splitter (11) and a plano-convex cylindrical lens (10) respectively; and / or Preferably, an observation channel (2) is provided on the top of the light-proof and heat-insulating housing (3), located on the front side of the plane of the plano-convex cylindrical lens (10), for observing the light-triggered LAMP component (8) and / or the photothermal detection test paper (12).

Images