Visual establishment method for Panonymus alatus RPA-SG

Abstract

The invention relates to the technical field of stored grain pest detection, and discloses a Pesportunus trifoliatus RPA-SG visualization establishment method, the method comprises a DNA rapid extraction module, an RPA amplification module, a visualization interpretation module and a result output module, by optimizing primer concentration and reaction time parameters, an efficient and specific RPA isothermal amplification system is constructed, and the RPA-SG visualization establishment method for Pesportunus trifoliatus RPA-SG visualization is realized. Specific primers are designed on the basis of a gene conserved region of the mitochondrial COI of the prymaria rugosa, the sequences of the specific primers are as shown in SEQ ID NO: 1 and SEQ ID NO: 2, and it is ensured that only target species are accurately recognized; nucleic acid amplification is completed within 10 minutes at the constant temperature of 37 DEG C by combining an SYBR Green I fluorescent dye labeling technology, and field visual interpretation without complicated instruments is realized after purification treatment. By applying the technology, the whole process from sample processing to result output can be completed within 40 minutes, the prevention and control efficiency of prymaria rugosoannulata in granaries, customs and other scenes is improved, the single detection cost is reduced, and reliable technical support is provided for intelligent monitoring of stored grain pests.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of stored grain pest detection, in particular to a RPA-SG visualization method for Tribolium castaneum. BACKGROUND

[0002] The stored grain pest detection technology refers to a technical system for real-time monitoring of grain pest activities through intelligent equipment and innovative methods to achieve early warning and precise prevention and control. The RPA-SG visualization method for Tribolium castaneum is a complex method integrating various advanced technologies, aiming to improve the efficiency and accuracy of Tribolium castaneum detection, especially in the field of instant diagnosis in the field or warehouse. It is one of the core components of the method, responsible for rapid genomic DNA extraction, RPA isothermal amplification, and fluorescence signal visualization. The DNA extraction module can efficiently lyse samples and purify nucleic acids to ensure high integrity in subsequent amplification. The RPA amplification module can quickly complete gene amplification under isothermal conditions, and the SYBR Green I dye is used for fluorescence labeling. Combined with the visualization interpretation module, the blue-green fluorescence signal is excited by a 395 nm ultraviolet light source to ensure that the presence of Tribolium castaneum can be clearly identified during detection, and the RPA-SG visualization detection effect of Tribolium castaneum is guaranteed.

[0003] Currently, due to the existence of various process restrictions in the detection process of Tribolium castaneum, the RPA amplification system relied on during the field detection of Tribolium castaneum samples may not be able to monitor the stability of the amplification temperature in real time. If there is temperature fluctuation or deviation, it may cause non-specific amplification or background noise, affecting the reliability of the amplification product. At the same time, during fluorescence interpretation, it is not possible to correct environmental light interference or signal bleaching in real time, which may lead to false positive or false negative results, and the detection position is easily affected by external factors during field operation and cannot be calibrated in real time. During the specific detection of Tribolium castaneum, due to the high gene homology of the related species in the family of Tribolium castaneum, during sample cross-validation, it is not possible to achieve simultaneous analysis of multiple waveband spectra, which may ignore background impurities or non-target species interference during detection, further affecting detection accuracy and diagnostic efficiency.

[0004] Therefore, the present application provides a RPA-SG visualization method for Tribolium castaneum to solve the above problems. SUMMARY

[0005] (I) Technical problems solved

[0006] In view of the deficiencies of the prior art, the present application provides a RPA-SG visualization method for Tribolium castaneum to solve the problems raised in the background art.

[0007] (II) Technical solutions

[0008] In order to achieve the above object, the present application provides the following technical scheme to achieve it: The rust flat valley thief RPA-SG visualization establishment method, the method comprises the following steps:

[0009] S1, extract the genomic DNA of the sample to be tested, adopt CTAB method for efficient lysis and purification, ensure the DNA integrity and concentration suitable, suitable for subsequent amplification reaction;

[0010] S2, construct RPA amplification system, the system is carefully designed to optimize the reaction efficiency and specificity, including RPA reaction buffer, rust flat valley thief specific primer pair, SYBR Green I fluorescent dye, DNA template, MgOAc and ddH2O, wherein the primer pair is designed based on the conserved region of mitochondrial COI gene, the sequence is shown as SEQ ID NO: 1 and SEQ ID NO: 2, ensure that only the target gene of rust flat valley thief is amplified with high selectivity;

[0011] S3, carry out RPA amplification reaction under the condition of 37 DEG C constant temperature for 10 min, the temperature and time parameters are strictly optimized, avoid non-specific amplification and background noise, at the same time ensure the abundance of amplification product;

[0012] S4, after the termination of the reaction, the amplification product is purified, the nucleic acid purification kit is used to remove impurities and unreacted components, and the signal-to-noise ratio of fluorescence detection is improved;

[0013] S5, the purified product is mixed with SYBR Green I dye, and the fluorescence signal is directly observed under the excitation of 395 nm ultraviolet light, and this step adopts portable equipment to realize on-site instant interpretation;

[0014] S6, according to the fluorescence signal determination result: blue-green fluorescence appears positive, indicating that rust flat valley thief exists, and no fluorescence is negative.

[0015] Preferably, the extraction of genomic DNA in step S1 adopts CTAB method, which specifically comprises:

[0016] Sample lysis, protease digestion, organic solvent extraction and ethanol precipitation;

[0017] The lysis solution contains CTAB and β-mercaptoethanol, which destroys the cell wall and membrane structure, releases high-quality DNA, protease K digestion is carried out at 56 DEG C for 1 h, and the protein impurities are completely degraded, the ratio of chloroform-isoamyl alcohol extraction is 24:1, the lipid and pigment are removed, after ethanol precipitation, the salt is removed by washing twice with ethanol, finally dissolved in TE buffer, the pH value is adjusted to 8.0, ensure the stability and amplification compatibility of DNA.

[0018] Preferably, the design of the specific primer pair of Cryptolestes ferrugineus is based on the conserved region of the mitochondrial COI gene, and the primer sequences are as follows:

[0019] Upstream primer Cf-F10: 5'-TTTGGTCAACTAATCATAAAGATATTGG-3';

[0020] Downstream primer Cf-R10: 5'-TATACTTCAGGATGTCCAAAAAATCA-3';

[0021] The primer is verified by BLAST, only matches the COI gene of Cryptolestes ferrugineus, and has relatively low homology with related species in the family Cryptophagidae, thereby ensuring interspecific specificity; the primer length and Tm value are optimized to 28 bp and 60°C, avoiding the formation of primer dimers and improving the amplification efficiency.

[0022] Preferably, the purification treatment in step S4 adopts a nucleic acid purification kit, and the specific operation includes:

[0023] Add 3 times the volume of binding buffer to the amplification product to promote DNA adsorption, transfer to a silica gel membrane adsorption column, centrifuge and discard the filtrate, remove the enzyme and dNTP residues, wash twice with rinsing solution containing ethanol, centrifuge and dry for 5 min to remove organic solvents, add 30 μL eluent, pH 8.0 TE buffer, dissolve the DNA, and ensure that the purified product does not interfere with fluorescence detection.

[0024] Preferably, in step S5, a portable 395 nm ultraviolet flashlight is used for fluorescence interpretation, the observation distance is controlled to be 8 cm to optimize the signal intensity, and the observation time is not more than 30 s to prevent photobleaching. At the same time, a background correction area is set, a negative control sample is used as a reference to reduce environmental light interference, and when interpreting, the sample is placed on a black background plate to enhance the contrast of blue-green fluorescence.

[0025] Preferably, the method further comprises setting positive and negative controls: the positive control is pure DNA of Cryptolestes ferrugineus with a concentration greater than 1 ng / μL to ensure amplification effectiveness, and the negative control is ddH2O or non-target insect DNA to verify no cross-reaction. The control samples are processed in parallel with the samples to be tested to improve the reliability of the results.

[0026] Preferably, the detection sensitivity of the method is 10 -3 ng / μL DNA template, and Cryptolestes ferrugineus adults, larvae and fragments can be detected. The sensitivity is defined as the minimum detectable concentration , which is determined by a series of dilution experiments, and the formula is as follows:

[0027]

[0028] wherein, The minimum DNA concentration for reliably generating a fluorescent signal at a 95% confidence level, verified by triplicate experiments, ensures a stable detection limit.

[0029] Preferably, the method cross-reactivity verification for closely related species of flat grain weevils includes:

[0030] The test samples are selected from 12 species of flat grain weevils, including Tribolium castaneum and Tribolium turcicum. No blue-green fluorescent signal is observed in the amplification results of all non-target samples, confirming the specificity. During verification, the DNA template concentration is uniformly 1 ng / μL to avoid concentration bias.

[0031] Preferably, the specific execution steps are as follows:

[0032] S11, sample automatic coding recognition: read the two-dimensional code on the sample container through a scanning gun, associate the sample information to the method database, and generate a unique identification code:

[0033]

[0034] wherein, is the detection date, is the serial number of the day;

[0035] S12, parallel amplification control: the RPA amplification module has four independent temperature control units, supporting simultaneous processing of 1-4 groups of samples, with a temperature fluctuation of less than 0.3℃, and dynamic calibration of reaction efficiency through the following formula:

[0036]

[0037] wherein, is the amplification efficiency, is the slope of the standard curve, and when 0.9≤ ≤1.1, the reaction system is determined to be effective;

[0038] S13, result automatic association: after the visual interpretation module captures the fluorescence image, the value is bound and stored with the corresponding to generate an encrypted detection report.

[0039] Preferably, the method integrates an ambient light interference compensation mechanism, specifically including:

[0040] S21, real-time background acquisition: before fluorescence interpretation, the CMOS sensor automatically captures the ambient light intensity for 3 times, and calculates the mean ;

[0041] S22, dynamic calibration of fluorescence signal: the sample fluorescence intensity is compensated and corrected, with the formula being:

[0042]

[0043] wherein, is the corrected fluorescence intensity, is the ambient light attenuation coefficient, the preset value is 0.85, and when ≥50lux, the compensation is automatically activated;

[0044] S23, multispectral verification: after ultraviolet excitation, the 515nm and 625nm dual-band data are synchronously collected, and the following formula is used to eliminate non-specific interference:

[0045]

[0046] wherein, is the fluorescence intensity ratio, is the fluorescence intensity at 530nm waveband, is the fluorescence intensity at 650nm waveband, when ≥2.0 and ≥0.5, it is determined as positive.

[0047] (Three) beneficial effects

[0048] Compared with the prior art, the present application provides a rust red flat valley thief RPA-SG visualization establishment method, which has the following beneficial effects:

[0049] 1. By setting the RPA amplification optimization module, when the rust red flat valley thief sample is detected on site, the primer concentration and reaction time parameters are optimized, the non-specific amplification and background noise caused by temperature fluctuation are eliminated, the high abundance and high specificity of the amplification product are ensured, and the accuracy and reliability of the detection are improved, thereby ensuring the accuracy of the rapid diagnosis of the rust red flat valley thief.

[0050] 2. By setting the environmental interference correction module, when the fluorescence signal is interpreted, the ambient light intensity is monitored in real time and the dynamic compensation mechanism is used to avoid signal bleaching or misjudgment, ensure the credibility of the detection result under complex light conditions in the field, further reduce the incidence of false positive or false negative results, and improve the stability and convenience of the on-site operation.

[0051] 3. By setting the multispectral verification module, when the flat valley thief relative species cross detection is performed, the dual-band fluorescence data are synchronously collected and the ratio analysis is performed to distinguish the target signal from the background impurity interference, ensure the high specificity of the detection, avoid misdiagnosis of the relative species, and improve the overall diagnostic efficiency and applicability. BRIEF DESCRIPTION OF DRAWINGS

[0052] Figure 1 is the flowchart of the rust red flat valley thief RPA-SG visualization establishment method of the present application. DETAILED DESCRIPTION

[0053] The technical solutions in the embodiments of the present application will be clearly and completely described with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the scope of the present application.

[0054] Please refer to Figure 1 The rust red flat valley thief RPA-SG visualization establishment method comprises the following steps:

[0055] S1, extract the genomic DNA of the sample to be tested, and use the CTAB method for efficient lysis and purification to ensure the integrity and concentration of the DNA, which is suitable for subsequent amplification reaction;

[0056] S2, construct an RPA amplification system, which is carefully designed to optimize the reaction efficiency and specificity, and contains RPA reaction buffer, rust red flat valley thief specific primer pair, SYBR Green I fluorescent dye, DNA template, MgOAc and ddH2O, wherein the primer pair is designed based on the conserved region of mitochondrial COI gene, and the sequence is shown as SEQ ID NO: 1 and SEQ ID NO: 2, which ensures high selectivity amplification of the target gene of the rust red flat valley thief;

[0057] S3, carry out RPA amplification reaction at 37℃ constant temperature for 10 min, and the temperature and time parameters are strictly optimized to avoid non-specific amplification and background noise, while ensuring the abundance of the amplification product;

[0058] S4, after the reaction is terminated, the amplification product is purified and treated, and the nucleic acid purification kit is used to remove impurities and unreacted components to improve the signal-to-noise ratio of fluorescence detection;

[0059] S5, mix the purified product with SYBR Green I dye, and directly observe the fluorescence signal under 395 nm ultraviolet light excitation. This step uses a portable device to realize on-site real-time interpretation;

[0060] S6, according to the fluorescence signal determination result: blue-green fluorescence appears positive, indicating the presence of rust red flat valley thief, and no fluorescence is negative;

[0061] The extraction of genomic DNA in step S1 uses the CTAB method, which specifically includes:

[0062] Sample lysis, protease digestion, organic solvent extraction and ethanol precipitation;

[0063] The lysis solution contains CTAB and β-mercaptoethanol, which destroys the cell wall and membrane structure and releases high-quality DNA. Proteinase K digestion is carried out at 56°C for 1 hour to completely degrade protein impurities. The chloroform-isoamyl alcohol extraction ratio is 24:1 to remove lipids and pigments. After anhydrous ethanol precipitation, the salt is removed by washing twice with ethanol. Finally, the DNA is dissolved in TE buffer, the pH value is adjusted to 8.0 to ensure the stability and amplification compatibility of the DNA.

[0064] The specific primer pair of the rust red flat grain beetle is designed based on the conserved region of the mitochondrial COI gene, and the primer sequence is as follows:

[0065] The upstream primer Cf-F10 is 5'-TTTGGTCAACTAATCATAAAGATATTGG-3';

[0066] The downstream primer Cf-R10 is 5'-TATACTTCAGGATGTCCAAAAAATCA-3';

[0067] The primer is verified by BLAST, which only matches the COI gene of the rust red flat grain beetle, and the homology with the related species of the flat grain beetle family is relatively low, thereby ensuring the interspecific specificity. The primer length and Tm value are optimized to 28bp and 60°C to avoid primer dimer formation and improve amplification efficiency.

[0068] The purification treatment in step S4 uses a nucleic acid purification kit, and the specific operation includes:

[0069] Add 3 times the volume of binding buffer to the amplification product to promote DNA adsorption. Transfer to a silica gel membrane adsorption column and centrifuge to discard the filtrate. Remove enzyme and dNTP residues. Wash twice with rinse solution containing ethanol. Centrifuge and dry for 5 minutes to remove organic solvents. Add 30μL eluent, pH 8.0 TE buffer, to dissolve the DNA and ensure that the purified product is free of inhibitors that interfere with fluorescence detection.

[0070] The fluorescence interpretation in step S5 uses a portable 395nm ultraviolet flashlight, and the observation distance is controlled at 8cm to optimize signal intensity. The observation time is not more than 30s to prevent light bleaching. At the same time, a background correction area is set, and a negative control sample is used as a reference to reduce environmental light interference. When interpreting, the sample is placed on a black background plate to enhance the contrast of blue-green fluorescence.

[0071] The method also includes the setting of positive and negative controls. The positive control is pure DNA of the rust red flat grain beetle with a concentration greater than 1ng / μL to ensure amplification effectiveness. The negative control is ddH2O or non-target insect DNA to verify no cross-reaction. The control samples are processed in parallel with the samples to be tested to improve the reliability of the results.

[0072] The detection sensitivity of the method is 10 -31 ng / μL DNA template, the adult, larva and fragment of C. ferruginea can be detected; wherein, the sensitivity is defined as the minimum detectable concentration Through a series of dilution experiments, the formula is:

[0073]

[0074] Among them, is the minimum DNA concentration that can reliably produce a fluorescent signal at 95% confidence, which is verified by three repeated experiments to ensure the stability of the detection limit;

[0075] The cross-reaction verification of the method for closely related species of flat grain beetle includes:

[0076] The test samples are selected from 12 kinds of flat grain beetle insects such as C. longiceps and C. turcicus. The amplification results of all non-target samples have no blue-green fluorescent signal, which confirms the specificity. During verification, the DNA template concentration is uniform at 1 ng / μL to avoid the influence of concentration deviation;

[0077] The specific execution steps are as follows:

[0078] S11, sample automatic coding identification: the two-dimensional code on the sample container is read by a scanning gun, the sample information is associated to the method database, and a unique identification code is generated:

[0079]

[0080] Among them, is the detection date, is the serial number of the day;

[0081] S12, parallel amplification control: the RPA amplification module has four independent temperature control units, which can support simultaneous processing of 1-4 groups of samples, and the temperature fluctuation of each unit is less than 0.3℃. The reaction efficiency is dynamically calibrated by the following formula:

[0082]

[0083] Among them, is the amplification efficiency, is the slope of the standard curve, and when 0.9≤ ≤1.1, it is determined that the reaction system is effective;

[0084] S13, result automatic association: after the visual interpretation module captures the fluorescence image, the value is bound and stored with the corresponding to generate an encrypted detection report;

[0085] The method integrates an ambient light interference compensation mechanism, which specifically includes:

[0086] S21, Real-time background acquisition: Before fluorescence interpretation, the CMOS sensor automatically captures 3 times of ambient light intensity , calculate the mean ;

[0087] S22, Dynamic calibration of fluorescence signal: The sample fluorescence intensity is compensated and corrected, and the formula is:

[0088]

[0089] Wherein, is the corrected fluorescence intensity, is the ambient light attenuation coefficient, the preset value is 0.85, and the compensation is automatically activated when ≥50lux;

[0090] S23, Multi-spectral verification: After ultraviolet excitation, the 515nm and 625nm dual-band data are synchronously collected, and the non-specific interference is eliminated by the following formula:

[0091]

[0092] Wherein, is the fluorescence intensity ratio, is the fluorescence intensity at 530nm waveband, is the fluorescence intensity at 650nm waveband, when ≥2.0 and ≥0.5, it is determined as positive.

[0093] A rust flat valley beetle RPA-SG visual detection method, comprising: DNA rapid extraction module, RPA amplification module, visual interpretation module and result output module, wherein:

[0094] The DNA rapid extraction module integrates a lysis chamber, a centrifugal unit and a buffer storage bin to realize one-key sample processing;

[0095] The RPA amplification module includes a constant temperature reaction chamber, a reagent preloaded tube and a timing controller, and automatically completes the amplification;

[0096] The visual interpretation module is built-in 395nm LED excitation light source, optical filter and fluorescence acquisition sensor, which supports real-time fluorescence capture;

[0097] The result output module is equipped with colorimetric card control interface and wireless data transmission unit, which realizes the result remote sharing. Specific embodiments

[0098] Example one, field rapid detection of rust flat valley beetle

[0099] In the on-site detection scene of grain reserves, the operator first takes a 10g sample of the wheat to be tested, places it in a sterile grinding tube, adds a pre-configured CTAB lysis solution containing CTAB surfactant and β-mercaptoethanol reducing agent, breaks the sample to release the cell contents through an electric grinding device, then adds proteinase K and digests at 56°C in a constant temperature shaker for 1 hour, completely degrading protein impurities. After digestion, extract twice with a chloroform-isoamyl alcohol mixture at a ratio of 24:1 to separate lipids and pigment interference. After centrifugation, add pre-cooled anhydrous ethanol to the supernatant to precipitate the DNA. The precipitate is washed twice with ethanol to remove residual salt ions. Finally, dissolve in TE buffer at pH 8.0 to obtain high-purity genomic DNA. Then construct the RPA amplification system, which contains 29.4 microliters of RPA reaction buffer, 1.8 microliters of specific upstream primer Cf-F10, 1.8 microliters of downstream primer Cf-R10, 5 microliters of DNA template, 2.5 microliters of 280 millimolar magnesium acetate solution, and 9.5 microliters of enzyme-free water. The primer sequence is verified by NCBI database to be completely matched with the mitochondrial COI gene of the rust red flat grain beetle. Place the reaction tube in a portable constant temperature amplifier and set the temperature to 37°C for 10 minutes. After the reaction is completed, use a silica gel membrane nucleic acid purification kit to process the amplification product. Add three volumes of binding buffer to the product, transfer it to the adsorption column and centrifuge to remove the filtrate. After two ethanol rinses, dry for 5 minutes. Finally, elute the DNA with 30 microliters of TE buffer. Mix 20 microliters of purified product with 2 microliters of 100-fold concentrated SYBR Green I dye, place it on a black background plate, and use a portable 395-nanometer ultraviolet flashlight at a vertical distance of 8 centimeters. The operator observes blue-green fluorescence within 30 seconds. Set up positive and negative controls simultaneously. The positive control is known rust red flat grain beetle DNA at a concentration of 1 nanogram per microliter. The negative control is long-headed flat grain beetle DNA. The results show that the fluorescence intensity of the positive sample is more than 5 times that of the negative sample, confirming that the sample is infected with rust red flat grain beetle. The entire process from sample processing to result interpretation takes only 40 minutes.

[0100] Example Two, Multi-channel Batch Sample High-throughput Screening

[0101] In the application of customs quarantine laboratory, the method needs to process 50 samples of grain species at a time. The operator pre-pastes a two-dimensional code label on the sample container, and the scanning gun automatically reads the date number and serial number to generate a unique identification code. In the amplification stage, a four-channel independent temperature control unit of RPA module is started. Four groups of samples are loaded into freeze-dried microsphere tubes preloaded with reaction solution. The reaction is carried out at a constant temperature of 37 degrees Celsius for 10 minutes. The method calibrates the amplification efficiency in real time through the slope of the standard curve to avoid false negatives caused by temperature fluctuations. After purification, the amplification product is transferred to the visual interpretation module. The CMOS sensor captures the fluorescence images of the four groups of samples in turn, automatically calculates the R value of each sample, which is the ratio of the sample fluorescence intensity after deducting the background to the negative control intensity. When the R value is greater than 0.5, the associated identification code generates a positive report. The detection results show that the R values of three corn samples are 0.8, 1.2 and 0.9 respectively. The method automatically marks the rust flat grain beetle as positive, and uploads the encrypted report to the central database. The whole process of processing 50 samples in this batch takes less than two hours, and the efficiency is four times higher than that of traditional single-channel.

[0102] Example three, accurate interpretation under strong light environment interference

[0103] In the open-air warehouse inspection scene, the environmental light intensity reaches ten thousand lumens. The operator detects the suspected rust flat grain beetle infected rice sample. After mixing the amplification product with SYBR Green I, it is placed on the detection table. The visual interpretation module first starts the environmental light compensation mechanism. The CMOS sensor automatically collects the environmental light intensity three times and calculates the average value as eight hundred lumens. Because it exceeds the fifty lumens threshold, the dynamic calibration algorithm is activated. The original fluorescence intensity of the sample is input into the formula, multiplied by the preset decay coefficient of zero point eight five to generate the corrected fluorescence intensity, eliminating the interference of strong light. Then the ultraviolet light source excites the sample, and the sensor synchronously captures the fluorescence data of the target waveband of five hundred fifteen nanometers and the background waveband of six hundred twenty five nanometers. By calculating the ratio of the two wavebands of five hundred thirty nanometers and six hundred fifty nanometers, if the target signal intensity is more than twice the background and the corrected R value is greater than zero point five, it is determined to be positive. The current sample has a fluorescence intensity of six hundred fifty units at five hundred fifteen nanometers and three hundred units at six hundred twenty five nanometers. The ratio of the two wavebands is two point one seven, and the corrected R value is zero point nine, which meets the positive determination standard. The method outputs the detection result in real time and triggers an alarm. The double waveband ratio of the non-target sample in the control group is only zero point six, avoiding false positives. This process still maintains a low false positive rate under strong light in the wild, verifying the effectiveness of the environmental light compensation and multi-spectral verification mechanism.

[0104] Example four, detection of larval fragments in storage environment

[0105] In the daily monitoring scene of large grain warehouse, the operator found that there were suspicious insect fragments in the wheat storage area, and then started the RPA-SG rapid detection process. First, 0.1 g of sample containing larva fragments was selected and placed in a grinding tube preloaded with lysis solution, β-mercaptoethanol was added to enhance cell membrane permeability, and a portable oscillator was used for homogenization at a speed of 300 rpm for 3 minutes to release nucleic acid substances. Then, proteinase K solution was added, and the sample was digested in a constant temperature metal bath at 56°C for 45 minutes to completely decompose protein impurities. After digestion, the sample was extracted by chloroform-isoamyl alcohol mixed solution, and the lipid interference was removed by 24:1 volume ratio layering. The supernatant was taken after centrifugation, and pre-cooled anhydrous ethanol was added to precipitate DNA. The precipitate was dissolved in TE buffer solution with pH 8.0 after two ethanol rinses, and high-purity DNA template was obtained. Then, the RPA amplification reaction system was configured, and 29.4 μL of reaction buffer, 1.8 μL of specific upstream primer Cf-F10, 1.8 μL of downstream primer Cf-R10, 5 μL of DNA extraction solution, 2.5 μL of 280 mM magnesium acetate solution and 9.5 μL of nuclease-free water were accurately added. The mixture was transferred to a freeze-dried microsphere preloaded tube and placed in a palm-sized thermostat at 37°C for 10 minutes. After amplification, the product was treated with a silica gel membrane purification column, three volumes of binding buffer were added to bind DNA, and impurities were removed by two ethanol rinses. Finally, 30 μL of TE buffer solution was used to elute the target fragment, 25 μL of purified product was mixed with 2.5 μL of 100×SYBR Green I dye, and was spread in a black detection card slot. In the relatively dark environment of the grain warehouse, a 395 nm ultraviolet flashlight was used for vertical irradiation at a distance of 8 cm, and a clear blue-green fluorescence signal was observed within 20 seconds. The positive control showed the same intensity of fluorescence, and the negative control of cryptolestes turcicus sample showed no fluorescence, confirming that the insect fragments were R. dominica larva residues. The method automatically recorded the detection results and uploaded them to the central monitoring platform, triggering the grain warehouse fumigation warning. The whole process took 35 minutes, which was 6 hours faster than traditional morphological identification. The detection sensitivity of the fragments reached 0.001 ng / μL, blocking the risk of pest spread.

[0106] It should be noted that, in the present document, relational terms such as first and second and the like can be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can include other elements not expressly listed or inherent to such process, method, article, or apparatus. An element proceeded by "comprises... a" does not, without more constraints, exclude the presence of additional identical elements in the process, method, article, or apparatus that comprises the element.

[0107] While embodiments of the application have been shown and described, it is to be understood that the application is not limited to the details of the embodiments described, since numerous changes, modifications, substitutions and variations can be made thereto without departing from the spirit and scope of the application as defined by the appended claims and their equivalents.

Claims

1. A visualization method for RPA-SG of the Rusty Red Flat Rice Robber, characterized by: The method includes the following steps: S1. Extract genomic DNA from the sample to be tested, and perform efficient lysis and purification using the CTAB method to ensure DNA integrity and appropriate concentration, suitable for subsequent amplification reactions; S2. Construct an RPA amplification system. This system is carefully designed to optimize reaction efficiency and specificity. It includes RPA reaction buffer, specific primer pairs for *Berberis rubrum*, SYBR Green I fluorescent dye, DNA template, MgOAc, and ddH2O. The primer pairs are designed based on the conserved region of the mitochondrial COI gene, and the sequences are shown in SEQ ID NO:1 and SEQ ID NO:2, to ensure highly selective amplification of the *Berberis rubrum* target gene. S3. Perform RPA amplification reaction at a constant temperature of 37℃ for 10 min. These temperature and time parameters have been strictly optimized to avoid non-specific amplification and background noise, while ensuring the abundance of amplification products. S4. After terminating the reaction, the amplification product is purified. Impurities and unreacted components are removed using a nucleic acid purification kit to improve the signal-to-noise ratio of fluorescence detection. S5. Mix the purified product with SYBR Green I dye and observe the fluorescence signal directly under 395nm ultraviolet light excitation. This step uses a portable device to achieve on-site real-time interpretation. S6. Determine the result based on the fluorescence signal: the presence of blue-green fluorescence indicates a positive result, indicating the presence of *Berberis rubrum*, while the absence of fluorescence indicates a negative result.

2. The RPA-SG visualization establishment method for the Rusty Red Flat Rice Robber according to claim 1, characterized in that: The extraction of genomic DNA in step S1 uses the CTAB method, specifically including: Sample lysis, protease digestion, organic solvent extraction, and ethanol precipitation; The lysis buffer contains CTAB and β-mercaptoethanol to disrupt cell walls and membrane structures, releasing high-quality DNA. Proteinase K digestion is performed at 56°C for 1 hour to thoroughly degrade protein impurities. Chloroform-isoamyl alcohol extraction at a ratio of 24:1 removes lipids and pigments. After precipitation with anhydrous ethanol, the DNA is washed twice with ethanol to remove salts and finally dissolved in TE buffer. The pH is adjusted to 8.0 to ensure DNA stability and amplification compatibility.

3. The RPA-SG visualization establishment method for the Rusty Red Flat Rice Robber according to claim 1, characterized in that: The specific primer pair for *Bombyx mori* was designed based on the conserved region of the mitochondrial COI gene, and its primer sequence is as follows: Upstream primer Cf-F10: 5'-TTTGGTCAACTAATCATAAAGATATTGG-3'; Downstream primer Cf-R10: 5'-TATACTTCAGGATGTCCAAAAAATCA-3'; The primers were verified by BLAST and matched only with the COI gene of the rusty red flat beetle. They showed low homology with closely related species of the flat beetle family, thus ensuring interspecificity. Primer length and Tm value were optimized to 28 bp and 60℃ to avoid primer dimer formation and improve amplification efficiency.

4. The RPA-SG visualization establishment method for the Rusty Red Flat Rice Robber according to claim 1, characterized in that: The purification process in step S4 uses a nucleic acid purification kit, and the specific operations include: Add 3 times the volume of binding buffer to the amplification product to promote DNA adsorption. Transfer to a silica membrane adsorption column, centrifuge and discard the filtrate to remove enzyme and dNTP residues. Wash twice with rinsing buffer containing ethanol, centrifuge and dry for 5 min to remove organic solvents. Add 30 μL of elution buffer and pH 8.0 TE buffer to dissolve the DNA, ensuring that the purified product is free of inhibitors that interfere with fluorescence detection.

5. The RPA-SG visualization establishment method for the Rusty Red Flat Rice Robber according to claim 1, characterized in that: In step S5, fluorescence interpretation is performed using a portable 395nm ultraviolet flashlight. The observation distance is controlled at 8cm to optimize signal intensity, and the observation time is not more than 30s to prevent photobleaching. At the same time, a background correction area is set up, and a negative control sample is used as a benchmark to reduce ambient light interference. During interpretation, the sample is placed on a black background plate to enhance the contrast of blue-green fluorescence.

6. The RPA-SG visualization establishment method for the Rusty Red Flat Rice Robber according to claim 1, characterized in that: The method also includes the setting of positive and negative controls: the positive control is pure DNA of the rusty red flat rice beetle at a concentration greater than 1 ng / μL to ensure amplification effectiveness; the negative control is ddH2O or DNA from non-target insects to verify no cross-reaction. The control samples and the test samples are processed in parallel to improve the reliability of the results.

7. The RPA-SG visualization establishment method for the Rusty Red Flat Rice Robber according to claim 1, characterized in that: The detection sensitivity of the method is 10. -3 A DNA template of ng / μL can detect adult, larval, and fragmentary *Russula rubescens*; sensitivity is defined as the lowest detectable concentration. The formula was determined through a series of dilution experiments: ； in, The minimum DNA concentration required to reliably generate a fluorescent signal at a 95% confidence level was verified through triple-replication experiments to ensure the stability of the detection limit.

8. The RPA-SG visualization establishment method for the Rusty Red Flat Rice Robber according to claim 1, characterized in that: The method for cross-response verification of closely related species in the family Coprinus includes: The test samples were selected from 12 species of flat meal beetles, including the long-headed flat meal beetle and the Turkish flat meal beetle. The amplification results of all non-target samples showed no blue-green fluorescence signal, confirming the specificity. During the verification, the DNA template concentration was uniformly 1 ng / μL to avoid the influence of concentration deviation.

9. The RPA-SG visualization establishment method for the Rusty Red Flat Rice Robber according to claim 1, characterized in that: The method also includes a batch sample processing procedure, the specific execution steps of which are as follows: S11. Automatic Sample Encoding and Identification: The QR code on the sample container is read using a scanner, the sample information is linked to the method database, and a unique identifier is generated. ； in, For the date of testing, This is the serial number for the day; S12. Parallel Amplification Control: The RPA amplification module has four built-in independent temperature control units, supporting simultaneous processing of 1-4 sets of samples. The temperature fluctuation of each unit is less than 0.3℃, and the reaction efficiency is dynamically calibrated using the following formula: ； in, For amplification efficiency, The slope of the standard curve, when 0.9 ≤ The reaction system is considered effective when the value is ≤1.

1. S13. Automatic Result Association: After the visualization and interpretation module captures the fluorescence image, it will automatically associate the results with the fluorescence image. Value and Correspondence Bind storage and generate an encrypted detection report.

10. The RPA-SG visualization establishment method for the Rusty Red Flat Rice Robber according to claim 1, characterized in that: The method integrates an ambient light interference compensation mechanism, specifically including: S21. Real-time background acquisition: Before fluorescence interpretation, the CMOS sensor automatically captures the ambient light intensity three times. Calculate the mean ; S22. Dynamic calibration of fluorescence signal: This involves calibrating the sample fluorescence intensity. The compensation and correction are performed using the following formula: ； in, The corrected fluorescence intensity, This is the ambient light attenuation coefficient, with a preset value of 0.

85. Compensation is automatically activated when the value is ≥50 lux. S23. Multispectral Verification: After UV excitation, data from both 515nm and 625nm bands are acquired simultaneously. Non-specific interference is eliminated using the following formula: ； in, The ratio of fluorescence intensity. The fluorescence intensity at 530 nm. For the fluorescence intensity in the 650nm band, when ≥2.0 and A value ≥0.5 is considered positive.

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