CN109679836B - Slide trace detection system applied to prawn pathogen high-flux detection - Google Patents
Slide trace detection system applied to prawn pathogen high-flux detection Download PDFInfo
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Abstract
The invention belongs to the field of microbial detection, and particularly relates to a slide micro-detection system applied to high-throughput detection of prawn pathogens. The system comprises a trace detection slide provided with a detection amplification pool, a trace liquid transfer device and imaging equipment for visual nucleic acid amplification detection; wherein, the detection pool is provided with a pre-coated reaction reagent and a specific primer of a specific pathogen. On the basis of the established new technology for the constant-temperature amplification of the pathogenic nucleic acid of the prawns, the invention develops a glass slide micro-detection system, and has the advantages of low consumption of detection reagents, miniaturization of equipment, convenience and quickness in operation and the like; is a rapid and portable, low-consumption, low-cost, sensitive and specific and high-flux detection and analysis means.
Description
Technical Field
The invention belongs to the field of microbial detection, and particularly relates to a slide micro-detection system applied to high-throughput detection of prawn pathogens.
Background
In 2018, the number of crustacean diseases recorded by World Animal Health Organization (OIE) in the aquatic Animal Health code is 11, 10 or more of other prawn diseases are not recorded, and 2 of newly-discovered prawn diseases. In 2018, this subject group conducted epidemiological investigation of penaeid shrimps in 9 provinces of the major cultivated penaeid shrimps in Guangdong, Guangxi, Hainan, etc., wherein the major positive detection rates of plagues were, in order, Covertortaliment nodavirus (CMNV) (29.15%), Penaeid shrimps (Enteromozon hepatopenaei, EHP) (17.07%), Vibrio parahaemolyticus (Acute hepato necrosea-haemolyticus) (VpahpnD) (13.21%), Infectious hypodermal and Hematopoietic Necrosis Viruses (HNV) (8.70%), White spot syndrome (White spot syndrome virus, syndrovirus) (WSSV 6.82, WSSV 82, Yellow spot syndrome virus (Infectious necrosis syndrome, Infectious necrosis of head of Yellow head of Shrimp, 6.82%), and Infectious necrosis of Yellow spot syndrome virus (Woovirus of Yellow spot virus (Woothecid virus, Woofer necrosis of Yellow spot, Woofer virus, 0, Woofer virus, Woofer, IMNV) (0.0%), the pathogen threatening the prawn culture is many and is mixed infection. Currently, prawn pathogen monitoring mainly adopts technologies such as nucleic acid analysis-based detection in a laboratory after sample collection, Polymerase Chain Reaction (PCR), Real-time quantitative fluorescence PCR (Real-time PCR), and gene chip, and is widely applied due to high sensitivity, rapid detection, and multi-target analysis. However, such analysis requires special instruments and equipment, such as a PCR instrument, a matched electrophoresis instrument, a gel imaging instrument, a real-time fluorescence PCR instrument, a gene chip scanner, etc., which are time-consuming, labor-consuming, expensive in cost, and unsuitable for simple and rapid analysis. Therefore, the development of a novel pathogen detection technology with high throughput, high sensitivity and low cost, which is suitable for on-site real-time analysis, is imperative.
The Nucleic acid detection method based on the Nucleic Acid Isothermal Amplification Technology (NAIAT) can realize rapid, simple and efficient amplification, has low instrument dependence, is very suitable for Point-of-care test (POCT), and is a Nucleic acid analysis means with great application potential. The subject group is dedicated to the research of the prawn pathogen rapid detection technology in recent years, NAIAT is applied to prawn pathogen detection, and a series of on-site rapid and sensitive detection technologies and detection kits such as WSSV, IHHNV, TSV, YHV, SHIV and the like of prawns are successfully developed at present, but in practical application, certain limitations exist: (1) there are limitations to the accuracy of visualization results based on visual interpretation; (2) the detection of a single sample is difficult to realize the simultaneous detection of multiple targets, and the detection cost is too high; (3) the convenience of the operation process needs to be further improved.
Low volume PCR technology has been used for single cell isolation and testing, and NAIAT-based low volume amplification has mainly focused on studying microfluidic chip technology. In recent years, the development of nucleic acid research technology based on microfluidic chips is vigorous, such as the microfluidic chip digital PCR technology and NAIAT-based microfluidic chip systems; however, the amplification detection based on the microfluidic chip technology generally requires high-precision instruments and equipment, and the development cost and the production cost of the chip are expensive, so that the application of the chip in clinical rapid detection is limited.
Disclosure of Invention
The invention aims to solve the technical problem that amplification detection based on a microfluidic chip technology generally requires high-precision instrument equipment, and the research and development cost and the production cost of the chip are very expensive, so that the application of the chip in clinical rapid detection is limited.
Based on the defects, the invention develops a slide micro-detection system on the basis of the established new technology for the constant-temperature amplification of the pathogenic nucleic acid of the prawns, and has the advantages of low consumption of detection reagent, miniaturization of equipment, convenience and quickness in operation and the like; is a rapid and portable, low-consumption, low-cost, sensitive and specific and high-flux detection and analysis means.
In order to achieve the purpose, the invention specifically adopts the following technical scheme:
the slide trace detection system applied to the prawn pathogeny high-flux detection comprises a trace detection slide provided with a detection amplification pool, a trace liquid transfer device and imaging equipment for visual nucleic acid amplification detection; printing a hydrophobic and oleophobic coating on a trace detection slide by a screen printing, reserving a detection amplification pool, carrying out super-hydrophilic modification on the detection amplification pool, arranging a pre-coated reaction reagent and a specific primer of a specific pathogen, and arranging a plugging piece in the detection amplification pool; the micropipettor consists of 2-8 special pipette needles, each pipette needle is a cylinder with the diameter of 1mm, the lateral surface of each pipette needle is coated with a super-hydrophobic and super-oleophobic coating (ZXL-WNS super-hydrophobic and super-oleophobic, Zicily technology), and the bottom surface of each pipette needle is subjected to super-hydrophilic treatment; be equipped with battery, LED constant current drive IC and blue LED in the imaging device, through the line connection between the three, still be equipped with the slide groove in the imaging device, the side opening in slide groove from this puts into the slide, slide groove top is equipped with golden yellow light filter, slide groove both sides equidistant, respectively be provided with 4 blue LED of symmetry, blue light is evenly shone from the both sides of slide, the effective blue and purple stray light of filtering of golden yellow light filter to promote the discernment of exciting light in the detection pond.
Furthermore, 48 groups of identical detection cells are arranged on the trace detection slide, each detection cell is arranged in a4 x 12 mode, and 4 columns are correspondingly arranged in 1 row of each 12 detection cells; further, the micro test slide is rectangular and has a size of 25X 75 mm; the glass slide is made of glass.
Further, the plugging piece is dimethyl silicone oil.
Furthermore, the number of the special pipetting needles is 4, the distance between the needles is 4.10mm (namely, the distance is the same as the longitudinal interval of the detection amplification pool), and the samples are longitudinally spotted along the direction of ABCD during the operation.
A preparation method of a micro detection slide comprises the following steps:
(1) plate making: film-exposure-hardening-development: firstly, manufacturing a film according to a structural schematic diagram of a trace detection slide; secondly, manufacturing a screen plate by using a 300-mesh silk screen, and drying for later use; thirdly, degreasing and cleaning the blank screen printing plate for later use; fourthly, coating photosensitive glue on the clean screen printing plate, and coating for many times when necessary; drying the glued screen printing plate in a blast drying box, coating for many times and drying for many times; sixthly, placing the film on the plate burning table glass with the photosensitive surface upward, placing the coated screen plate, tightly attaching the film to the screen plate by vacuum air exhaust, and turning on a plate burning lamp to carry out photosensitive plate making; seventhly, developing and developing the photosensitive screen printing plate in water, observing plate making effect, and drying the screen printing plate in a drying oven when the quality requirement is met;
(2) screen printing of hydrophobic and oleophobic coatings: preparing special ink, wherein the product requires hydrophobicity and oleophobicity, and polytetrafluoroethylene special resin, fluorocarbon additive, silicon dioxide and other components are added into the basic ink in different proportions; secondly, the prepared screen printing plate is arranged in a screen printing table for positioning; thirdly, pouring the mixed printing ink into a silk screen, carrying out silk screen printing, and inserting the printed slide into a special alloy frame;
(3) and (3) glass slide solidification: putting the glass slide and the frame into a forced air drying oven, and baking for 90min at 200 ℃ to fully cure the ink;
(4) cleaning treatment of the glass slide: soaking the cured finished product glass slide in a cleaning agent for 80min, then cleaning in a cleaning machine, and inspecting the cleaned glass slide;
(5) super-hydrophilic treatment of a detection pool: uniformly coating or spraying super-hydrophilic ZXL-CQS nano self-cleaning solution (Laiyangzi Xilai environmental protection science and technology Co., Ltd.) in each detection pool, and curing and drying;
(6) pre-coating a detection reagent in a detection pool: the micro detection slide is provided with 48 detection pools with the same holes, and each 8 holes form a group of detection units, and the total number of the detection units is 6; wherein, 4 groups are sample detection units, and the rest 2 groups are respectively negative and positive control detection units; each group of detection units has 8 detection pools for respectively detecting different pathogen nucleic acids;
(7) placing the micro detection slide pre-embedded with the detection reagent in a model LY0-0.5 Dongfulong freeze dryer (Shanghai Dongfulong science and technology Co., Ltd.) for freeze-drying: pre-freezing for 1 hour (the temperature of two probes in the front box is below minus 40 ℃, and the temperature of the rear box is below minus 45 ℃); secondly, vacuumizing the front box and the rear box, wherein the vacuum degree of the front box is displayed to reach the required vacuum degree of 0.08-0.10 mbar; adjusting the manual heating temperature of the interface to-5 ℃, and finishing primary sublimation when the temperature of the product is gradually close to the oil guiding temperature and the vacuum degree curve is obviously bent; thirdly, heating and raising the temperature of the product after the first sublimation is finished; when the temperature of the front box rises to-10 ℃, the temperature starts to rise; the interface is adjusted to manually heat at-5 ℃, and when the temperature of the product rises to-5 ℃, the heat preservation time is 1.5 hours.
(8) And (3) carrying out vacuum packaging on the slide to obtain a qualified product: vacuum packaging the finished product, and storing at a temperature below-20 deg.C.
Further, the micro detection slide is a conventional slide glass in texture and 25 x 75mm in size; the diameter of the detection cells is 2.25mm, and the distance between the detection cells is 3.00 mm.
Further, in the step (2), one or more of polytetrafluoroethylene special resin, fluorocarbon additive and silicon dioxide is/are added into the base ink to meet the hydrophobic and oleophobic characteristics; wherein the proportions of the special polytetrafluoroethylene resin, the fluorocarbon additive and the silicon dioxide are sequentially 2-4% (g/mL), 1-2% (g/mL) and 0.3-0.5% (g/mL).
Further, each detection cell is pre-embedded with a detection reagent: bst 2.0
DNA polymerase,
RTx reverse transcriptase, 10 × Bst buffer, 10mM dNTPs, 5M Betaine, 100mM MgSO4、350mMMnCl2、3.5mM Caclein、ddH2O and specific primers of 8 pathogens of the prawns, wherein the 8 pathogens of the prawns comprise WSSV, IHHNV, SHIV, AHPND, EHP, YHV-8, TSV and CMNV.
The sequences of the primers specific to the above 8 pathogens are shown in the following table:
furthermore, the freeze-drying protective agents of the detection reagent pre-embedded in the detection cell are gelatin, trehalose, Bovine Serum Albumin (BSA) and thiourea, and the proportion is 2% -3% (g/mL), 10% -15% (g/mL), 1% -2% (g/mL) and 1% -2% (g/mL) in sequence.
Further, each of the detection cell pre-embedded detection reagents for detecting 5 DNA pathogens of WSSV, IHHNV, SHIV, AHPND and EHP comprises: bst 2.0
DNA polymerase 0.042. mu.L, 10 × Bst buffer 0.15. mu.L, 25mM dNTPs 0.084. mu.L, 5M beta 0.33. mu.L, 100mM MgSO40.09μL、350mM MnCl20.0018μL、3.5mM Caclein 0.0102μL、ddH2O0.267 mu L, gelatin (g/mL)0.045 mu L, trehalose (g/mL)0.225 mu L, BSA (g/mL)0.03 mu L, thiourea (g/mL)0.015 mu L and corresponding specific primers of 5 DNA pathogens of prawns; the specific primer comprises: 40 μ M FIP0.06 μ L, 40 μ M BIP 0.06 μ L, 40 μ M LF0.03 μ L, 40 μ M LB 0.03 μ L (same amount of ddH used without LB)2O substitution), 20 μ M F30.015 μ L, 20 μ M B30.015 μ L.
Further, each of the detection pool pre-embedded detection reagents for detecting 3 RNA pathogens YHV-8, TSV and CMNV comprises: bst 2.0
DNA polymerase 0.042. mu.L,
RTx reverse transcriptase 0.042. mu.L, 10 × Bst buffer 0.15. mu.L, 25mM dNTPs 0.084. mu.L, 5M Betaine 0.33. mu.L, 100mM MgSO40.09μL、350mM MnCl20.0018μL、3.5mM Caclein 0.0102μL、ddH2O0.225 μ L, gelatin (g/mL)0.045 μ L, trehalose (g/mL)0.225 μ L, BSA (g/mL)0.03 μ L, thiourea (g/mL)0.015 μ L and corresponding prawn 3 RNA pathogen specific primers; the specific primer comprises: 40 μ M FIP0.06 μ L, 40 μ M BIP 0.06 μ L, 40 μ M LF0.03 μ L, 40 μ M LB 0.03 μ L, 20 μ M F30.015 μ L, 20 μ M B30.015 μ L.
Further, the micropipette was pipetted in a volume of 1.525 μ L.
Compared with the prior art, the invention has the following advantages:
the invention designs and manufactures a slide trace detection system, which comprises a trace detection slide, a micropipette and imaging equipment, wherein the trace detection slide is provided with a 48-hole detection pool and is pre-coated with all detection reagents except sample nucleic acid, and the imaging equipment is used for visualizing an amplification detection result. Hydrophobic and oleophobic and super-hydrophilic materials are mainly combined in the manufacturing process of the trace detection slide, so that an independent space for detection is realized, and aerosol pollution is avoided in a water-in-oil mode; the micropipettor is also combined with super-hydrophobic, super-oleophobic and super-hydrophilic materials, and the amount added into the reaction system is accurately calculated, so that the simplicity and convenience of operation and the accuracy of metering are realized; the imaging device is small and exquisite in design and portable, and visual interpretation of detection results can be achieved. The slide micro-detection system can synchronously and immediately detect 4 groups of samples and 8 important pathogens of prawns, and is a low-cost, portable and high-flux nucleic acid (RNA and DNA) detection technology in a real sense.
Drawings
FIG. 1 is a schematic view of the structure of a micro test slide; detection cells A1, A3, A5, A7, A9 and A11 are coated with reagents required by WSSV detection; detection cells B1, B3, B5, B7, B9 and B11 are coated with reagents required for detecting IHHNV; the detection cells C1, C3, C5, C7, C9 and C11 are coated with reagents required for detecting SHIV; detection pools D1, D3, D5, D7, D9 and D11 are coated with reagents required for detecting AHPND; the detection pools A2, A4, A6, A8, A10 and A12 are all coated with reagents required for detecting EHP; detection cells B2, B4, B6, B8, B10 and B12 are all coated with reagents required for YHV-8 detection; the detection pools C2, C4, C6, C8, C10 and C12 are all coated with reagents required for detecting TSV; the detection pools D2, D4, D6, D8, D10 and D12 are coated with reagents required for detecting CMNV;
fig. 2 is a schematic diagram of a micropipette structure;
fig. 3 is a schematic diagram of the operation of a micropipette;
fig. 4 is a schematic structural view of the image forming apparatus;
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the embodiments described are only a few embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1:
the slide trace detection system applied to the prawn pathogeny high-flux detection comprises a trace detection slide 1 provided with a detection amplification pool 11, a trace pipettor 2 and an imaging device 3 for visual nucleic acid amplification detection; coating a hydrophobic and oleophobic coating on the trace detection slide 1, reserving a detection amplification pool 11, modifying the detection amplification pool 11 by super hydrophilicity, arranging a pre-coated reaction reagent and a specific primer of a specific pathogen, and arranging a plugging piece in the detection amplification pool 11 during detection; the micropipettor 2 consists of 2-8 special pipette needles, the pipette needles are cylindrical and have the diameter of 1mm, the lateral surfaces of the pipette needles are coated with super-hydrophobic coatings, and the bottom surfaces of the pipette needles are subjected to super-hydrophilic treatment; be equipped with battery 31, LED constant current drive IC32 and blue LED33 in the imaging device 3, pass through the line connection between the three, be located the left side, imaging device 3 right side still is equipped with the slide groove, and its right side opening is put into the slide from this, is equipped with golden yellow light filter 34 on the slide groove, and slide groove bilateral symmetry, equidistant respectively are equipped with 4 blue LED 33.
The micro detection slide is provided with 48 groups of same detection pools, each detection pool is arranged in a4 multiplied by 12 mode, and 4 columns are correspondingly arranged in 1 row of each 12 detection pools; further, the micro test slide is rectangular and has a size of 25X 75 mm; the glass slide is made of glass.
The plugging piece is dimethyl silicone oil.
The number of the special liquid transferring needles is 4.
A preparation method of a micro detection slide comprises the following steps:
(1) plate making: film-exposure-hardening-development: firstly, manufacturing a film according to a structural schematic diagram of a trace detection slide; secondly, manufacturing a screen plate by using a 300-mesh silk screen, and drying for later use; thirdly, degreasing and cleaning the blank screen printing plate for later use; fourthly, coating photosensitive glue on the clean screen printing plate, and coating for many times when necessary; drying the glued screen printing plate in a blast drying box, coating for many times and drying for many times; sixthly, placing the film on the plate burning table glass with the photosensitive surface upward, placing the coated screen plate, tightly attaching the film to the screen plate by vacuum air exhaust, and turning on a plate burning lamp to carry out photosensitive plate making; seventhly, developing and developing the photosensitive screen printing plate in water, observing plate making effect, and drying the screen printing plate in a drying oven when the quality requirement is met;
(2) screen printing of hydrophobic and oleophobic coatings: preparing special ink, wherein the product requires hydrophobicity and oleophobicity, and polytetrafluoroethylene special resin, fluorocarbon additive, silicon dioxide and other components are added into the basic ink in different proportions; secondly, the prepared screen printing plate is arranged in a screen printing table for positioning; thirdly, pouring the mixed printing ink into a silk screen, carrying out silk screen printing, and inserting the printed slide into a special alloy frame;
(3) and (3) glass slide solidification: putting the glass slide and the frame into a forced air drying oven, and baking for 90min at 200 ℃ to fully cure the ink;
(4) cleaning treatment of the glass slide: soaking the cured finished product glass slide in a cleaning agent for 80min, then cleaning in a cleaning machine, and inspecting the cleaned glass slide;
(5) super-hydrophilic treatment of a detection pool: uniformly coating or spraying super-hydrophilic ZXL-CQS nano self-cleaning solution (Laiyangzi Xilai environmental protection science and technology Co., Ltd.) in each detection pool, and curing and drying;
(6) pre-coating a detection reagent in a detection pool: the micro detection slide is provided with 48 detection pools with the same holes, and each 8 holes form a group of detection units, and the total number of the detection units is 6; wherein, 4 groups are sample detection units, and the rest 2 groups are respectively negative and positive control detection units; each group of detection units has 8 detection pools for respectively detecting different pathogen nucleic acids; specifically, the detection pool is respectively embedded with 5 DNA pathogen reagents (1.5 muL system) for detecting WSSV, IHHNV, SHIV, AHPND, EHP and the like, comprising: bst 2.0
DNA polymerase 0.042. mu.L, 10 × Bst buffer 0.15. mu.L, 25mM dNTPs 0.084. mu.L, 5M beta 0.33. mu.L, 100mM MgSO40.09μL、350mM MnCl20.0018μL、3.5mM Caclein 0.0102μL、ddH2O0.267 mu L, gelatin (g/mL)0.045 mu L, trehalose (g/mL)0.225 mu L, BSA (g/mL)0.03 mu L, thiourea (g/mL)0.015 mu L, and corresponding specific primers of 5 DNA pathogens of prawns comprise: 40 μ M FIP0.06 μ L, 40 μ M BIP 0.06 μ L, 40 μ M LF0.03 μ L, 40 μ M LB 0.03 μ L (same amount of ddH used without LB)2O substitution), 20 μ M F30.015 μ L, 20 μ M B30.015 μ L; the embedded reagents (1.5 μ L system) for detecting 3 RNA pathogens including YHV-8, TSV and CMNV respectively comprise: bst 2.0
DNA polymerase 0.042. mu.L,
RTx reverse transcriptase 0.042. mu.L, 10 × Bst buffer 0.15. mu.L, 25mM dNTPs 0.084. mu.L, 5M Betaine 0.33. mu.L, 100mM MgSO40.09μL、350mM MnCl20.0018μL、3.5mM Caclein 0.0102μL、ddH2O0.225. mu.L, gelatin (g/mL) 0.045. mu.L, trehalose (g/mL) 0.225. mu. L, BSA (g/mL) 0.03. mu.L, thiourea (g/mL) 0.015. mu.L and the corresponding specific primers of the prawn 3 RNA pathogens include: 40 μ M FIP0.06 μ L, 40 μ M BIP 0.06 μ L, 40 μ M LF0.03 μ L, 40 μ M LB 0.03 μ L, 20 μ M F30.015 μ L, 20 μ M B30.015 μ L; in addition, the detection cells with the same 48 holes of the micro detection slide can be freely coated with 1, 2, 4, 6, 8 and 12 pathogens for synchronous real-time detection, and the pathogens can be replaced by any other suitable pathogens which can be detected by constant-temperature amplification;
(7) placing the micro detection slide pre-embedded with the detection reagent in a LY0-0.5 type Dongfulong freeze dryer (Shanghai Dongfulong science and technology Co., Ltd.) for freeze-drying treatment, and comprises the following steps:
step one, pre-freezing: firstly, clicking to start manually (turning green), opening a compressor COM 15 s, then opening a circulating pump CP130s, then opening a cold mixing valve SV1, and cooling a board layer; when the temperature of the entering box is reduced to-16 ℃, closing a cold mixing valve SV1, opening a rear box refrigerating valve SV21, manually clicking an interface, clicking heating control, and opening the heating control (the heating control is displayed as green); keeping the temperature and timing for 30 min; beginning to regulate the temperature and continuously pulling the temperature of the product downwards; the click interface is manual, and the click heating temperature is set to be-45 ℃; clicking heating control, repeating the switch for 2 times (the heating control shows green), keeping the temperature and timing for 1 hour (the two probes in the front box are below-40 ℃, and the probes in the rear box are below-45 ℃);
step two, sublimation for the first time: opening vacuum pumps VP1 and FV32 in sequence, opening a septum valve FV1 after 1min, and vacuumizing a front box and a rear box; secondly, when the vacuum degree of the current box is displayed to reach the required vacuum degree of 0.08-0.10 mbar; adjusting the manual heating temperature of the interface to be-5 ℃; clicking the heating control to repeat the switch for 2 times (the heating control displays green); during sublimation, observing the shape of the product, and observing whether the temperature of the product and a sublimation interface are clear or not, wherein the temperature of the product is controlled not to exceed the eutectic point so as to prevent the product from melting due to overhigh temperature; thirdly, when the temperature of the product is gradually close to the oil guiding temperature, the vacuum degree curve is obviously bent, and the one-time sublimation is finished;
step three, analyzing stage: firstly, finishing one-time sublimation, and heating and raising the temperature of a product according to the freeze-drying process requirement of the product; when the temperature of the front box rises to-10 ℃, the temperature starts to rise; adjusting the manual heating temperature of the interface to be-5 ℃; clicking the heating control to repeat the switch for 2 times (the heating control displays green); secondly, when the temperature of the product rises to-5 ℃, heat preservation is needed; adjusting the manual heating temperature of the interface to be-5 ℃; clicking the heating control to repeat the switch for 2 times (the heating control displays green); keeping the temperature and timing for 1.5 hours; and (5) finishing the freeze-drying.
(8) And (3) carrying out vacuum packaging on the slide to obtain a qualified product: vacuum packaging the finished product, and storing at a temperature below-20 deg.C.
The micro detection slide is a conventional slide glass in texture and 25 x 75mm in size; the diameter of the detection cells is 2.25mm, and the distance between the detection cells is 3.00 mm.
The freeze-drying protective agents of the detection cell pre-embedded detection reagent are gelatin, trehalose, Bovine Serum Albumin (BSA) and thiourea, and the proportion is 2% -3% (g/mL), 10% -15% (g/mL), 1% -2% (g/mL) and 1% -2% (g/mL) in sequence;
the volume of the micropipette to be removed is 1.525 mu L, and the measuring and calculating method comprises the following steps:
step one, establishing a standard curve: preparing standard solution by using equal volume of a Carkoverlu fluorescent whitening agent and 10% KOH (potassium hydroxide), accurately taking 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 mu L of the standard solution, reading a fluorescence value at 355nm by using a microplate reader, and taking the fluorescence value as an X axis and the concentration as a Y axis to obtain a standard curve as follows: y is 44.702 x-0.0081; r2＝0.9978。
And step two, measuring and calculating the moving volume of the micropipettor: the fluorescence value for each pipette removal of 0.0368 minus the residual fluorescence value of 0.0025 equals 0.0343 and the fluorescence value is assigned to the standard curve to give a volume of 1.525. mu.L removed.
The embodiment of the invention provides a micro-system slide detection system, and the detection method using the system has the characteristics of simplicity, convenience, low cost, no need of special instruments, high flux and the like, and can be applied to the field instant detection of various pathogen nucleic acids by adopting constant-temperature amplification detection.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Sequence listing
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Claims (8)
1. Be applied to slide trace detecting system of shrimp pathogeny high flux detection which characterized in that: comprises a trace detection slide provided with a detection amplification pool, a trace liquid transfer device and imaging equipment for visual nucleic acid amplification detection; printing a hydrophobic and oleophobic coating on a trace detection slide by a screen printing, reserving a detection amplification pool, carrying out super-hydrophilic modification on the detection amplification pool, arranging a pre-coated reaction reagent and a specific primer of a specific pathogen, and arranging a plugging piece in the detection amplification pool; the micropipettor consists of 2-8 special liquid transferring needles, the liquid transferring needles are cylinders, the diameter of each liquid transferring needle is 1mm, the side surfaces of the liquid transferring needles are coated with super-hydrophobic and oleophobic coatings, and the bottom surfaces of the liquid transferring needles are subjected to super-hydrophilic treatment; be equipped with battery, LED constant current drive IC and blue LED in the imaging device, pass through the line connection between the three, still be equipped with the slide groove in the imaging device, slide groove top is equipped with golden yellow light filter, slide groove both sides equidistant, symmetrical respectively is provided with 4 blue LED.
2. The slide micro-detection system applied to the high-throughput detection of prawn pathogens according to claim 1, characterized in that: the micro detection slide is provided with 48 groups of same detection pools, each detection pool is arranged in a4 multiplied by 12 mode, 1 row of each 12 detection pools is provided with 4 columns correspondingly.
3. The slide micro-detection system applied to the high-throughput detection of prawn pathogens according to claim 1, characterized in that: the plugging piece is dimethyl silicone oil.
4. A method for preparing a slide for trace detection according to claim 1, comprising the steps of:
(1) plate making: film-exposure-hardening-development;
(2) screen printing a hydrophobic and oleophobic coating; step (2) preparing special ink, wherein the special ink is required to be hydrophobic and oleophobic, and one or more than one of special polytetrafluoroethylene resin, fluorocarbon additive and silicon dioxide is added into the basic ink; secondly, the prepared screen printing plate is arranged in a screen printing table for positioning; thirdly, pouring the mixed printing ink into a silk screen, carrying out silk screen printing, and inserting the printed slide into a special alloy frame;
(3) solidifying the glass slide;
(4) cleaning the glass slide;
(5) performing super-hydrophilic treatment on the detection pool;
(6) the detection pool is pre-coated with a detection reagent;
(7) pre-embedding a detection reagent in a slide detection pool, and freeze-drying; the freeze-drying protective agents of the detection reagent pre-embedded in the detection pool in the step (7) are gelatin, trehalose, bovine serum albumin BSA and thiourea respectively, and the mass volume ratios are 2% -3%, 10% -15%, 1% -2% and 1% -2% in sequence;
(8) and (5) carrying out vacuum packaging on the slide to obtain a qualified product.
5. The method for preparing a slide for microscopic examination according to claim 4, wherein: the micro detection slide is a conventional slide glass in the texture and the size of 25 multiplied by 75 mm; the diameter of the detection cells is 2.25mm, and the distance between the detection cells is 3.00 mm.
6. The method for preparing a slide for microscopic examination according to claim 4, wherein: step (1), manufacturing a film according to a structural schematic diagram of a trace detection slide; secondly, manufacturing a screen plate by using a 300-mesh silk screen, and drying for later use; thirdly, degreasing and cleaning the blank screen printing plate for later use; fourthly, coating photosensitive glue on the clean screen printing plate, and coating for many times when necessary; drying the glued screen printing plate in a blast drying box, coating for many times and drying for many times; sixthly, placing the film on the plate burning table glass with the photosensitive surface upward, placing the coated screen plate, tightly attaching the film to the screen plate by vacuum air exhaust, and turning on a plate burning lamp to carry out photosensitive plate making; and seventhly, developing and developing the photosensitive screen printing plate in water, observing the plate making effect, and drying the screen printing plate in a drying oven when the quality requirement is met.
7. The method for preparing a slide for microscopic examination according to claim 4, wherein: the step (3) is that the glass slide and the frame are put into a blast drying oven and baked for 90min at 200 ℃ to fully cure the printing ink; soaking the cured finished product glass slide in a cleaning agent for 80min, then carrying out subsequent cleaning in a cleaning machine, and inspecting the cleaned glass slide; and (5) uniformly smearing or spraying the super-hydrophilic ZXL-CQS nano self-cleaning solution in each detection pool, and curing and drying.
8. The method for preparing a slide for microscopic examination according to claim 4, wherein: and (3) pre-embedding detection reagents in the detection pools in the step (6): bst 2.0Warm
DNA polymerase, Warm
RTx reverse transcriptase, 10 × Bstbuffer, 10mM dNTPs, 5M Betaine, 100mM MgSO4、350mM MnCl2、3.5mM Caclein、ddH2O and specific primers of 8 pathogens of the prawns, wherein the 8 pathogens of the prawns comprise WSSV, IHHNV, SHIV, AHPND, EHP and Y of the prawnsHV-8、TSV、CMNV。
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| CN110144417A (en) * | 2019-05-21 | 2019-08-20 | 中国水产科学研究院黄海水产研究所 | A kind of primer and kit and method of the EHP cause of disease detecting litopenaeus vannamei |
| CN113913550A (en) * | 2020-07-08 | 2022-01-11 | 宁波大学 | Method for detecting prawn iridovirus by micro-fluidic chip technology |
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| JP5097737B2 (en) * | 2009-03-27 | 2012-12-12 | 株式会社日立ハイテクノロジーズ | Automatic analyzer and sample dispensing nozzle |
| CN102740978A (en) * | 2009-12-18 | 2012-10-17 | 扎芬纳股份公司 | Micropipette |
| CN207276595U (en) * | 2017-09-12 | 2018-04-27 | 深圳市尚维高科有限公司 | Micro-fluidic pcr chip |
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