An anti-optical interference multiplexed biosensor based on argonaute and selective optical filtering and applications thereof

Abstract

This invention discloses an anti-optical interference multiplex biosensor (AMDRIL) based on Argonaute and selective optical filtering, and its applications. By using wavelength-selective excitation and optical filtering, it employs a convenient and low-cost solution to avoid optical interference in multiplex detection. AMDRIL is compatible with EHP and Vibrio... AHPND The detection sensitivity is as high as 10 per reaction. 0 Multiple copies are observed in a single tube with good specificity, and the detection process takes only about one hour. By configuring conventional LED lights, a light source filter, and an observation filter in a dark field, multiple fluorescence signals can be observed in the same tube. These signals are clearly visible and can be read with the naked eye, allowing for definitive result interpretation. AMDRIL supports EHP and Vibrio... AHPND This invention enables on-site monitoring and provides an economical and portable technology route suitable for on-the-spot detection, for multiplex nucleic acid biosensing detection with fluorescence readout capability.

Description

Technical Field

[0001] This invention belongs to the technical field, specifically relating to an anti-light interference multichannel biosensor based on Argonaute and selective optical filtering and its application. Background Technology

[0002] Effective pathogen surveillance is crucial for the prevention of infectious diseases. In aquaculture, preventing the invasion of aquatic pathogens is paramount, as outbreaks can cause not only economic losses but also ecological imbalances in aquaculture areas and the spread of infection among wild aquatic organisms. For aquatic pathogen surveillance, detection technologies need to adapt to point-of-care testing (POCT) requirements to meet the need for high-frequency on-site monitoring. This means that detection methods must be accurate and reliable, rely on common tools and personnel with limited training, and the testing process must be concise.

[0003] Nucleic acid sensors, characterized by their sensitivity and accuracy, have become important tools for monitoring aquatic pathogens. Polymerase chain reaction (PCR)-based methods, including quantitative PCR and nested PCR, have been used for the detection of aquatic pathogens. However, they are not suitable for field monitoring because they require complex thermal cyclers and trained personnel. Combining isothermal nucleic acid amplification with programmable nucleases has become a widely used point-of-care testing (POCT) sensor strategy because isothermal amplification avoids the use of thermal cyclers, and the loss of detection accuracy is compensated by the use of programmable nucleases. PfAgo is a programmable nuclease capable of specifically recognizing target sequences under the guidance of 5'-phosphorylated single-stranded guide DNA (gDNA) and cleaving them at specific locations on the target. It has now been combined with recombinase polymerase amplification (RPA) technology to develop highly sensitive sensors suitable for point-of-care detection of aquatic pathogens.

[0004] Furthermore, PfAgo is naturally well-suited for developing multiplex assays because it does not possess trans-cleavage activity like CRISPR nucleases. Successful examples include PfAgo-based multiplex assays for human papillomavirus (HPV) subtypes and several aquatic pathogens. In any case, multiplex assays are crucial because they simplify procedures and reduce costs.

[0005] Another factor to consider when developing field monitoring sensors is the result reading method. Fluorescent reading is a good choice because it can easily generate "specific cleavage" fluorescent signals. By introducing nucleic acid molecular beacons (MBs) with fluorescent and quenching groups into the detection reaction, the presence of a specific cleavage can be converted into a fluorescent signal. Furthermore, in-tube fluorescence reading is simple and clean, superior to other methods such as test strips, because it avoids exposing the detection reaction to the environment. In-tube fluorescence reading has become the primary method for result output in biological assays.

[0006] When applying in-tube fluorescence reading technology to the field monitoring of aquatic pathogens, result interpretation becomes difficult due to interference from other light sources. This includes interference from the emission of other fluorescent components in the multi-detection tube. Visual reading alone is insufficient to distinguish fluorescence signals in multi-detection tubes because the eye has limited wavelength resolution. Ambient light and / or excitation light should also be avoided, as they may mask the fluorescence signal when reflected or refracted by the tube wall or reaction solution. While these light interferences can be overcome using specialized fluorescence detectors, they are difficult to resolve in field monitoring scenarios. Summary of the Invention

[0007] Based on the background technology, to address the interference present in reading fluorescence signals in reaction tubes, such as ambient light and / or excitation light potentially masking the signal; different fluorescence signals being indistinguishable from each other; and the fluorescence signal spectrum lacking clear peaks, and the human eye lacking the resolution to extract the signal from the background, this invention proposes a light-interference-resistant multichannel biosensor based on Argonaute and selective optical filtering, and its applications.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] An anti-light-interference multichannel biosensor based on Argonaute and selective optical filtering includes: a) a dual or multiplex recombinase polymerase amplification (RPA) reaction system for simultaneously amplifying multiple target nucleic acids; b) a dual or multiplex PfAgo enzyme digestion reaction system cascaded with the RPA reaction system, the system containing guide DNA (gDNA) specific to each target and fluorescently labeled molecular beacons (MBs), wherein the PfAgo enzyme cleaves specific target sequences under the guidance of the corresponding gDNA, thereby cleaving the corresponding MBs to release fluorescence signals of different wavelengths; c) an optical detection unit, including a dark-field observation environment and optical components independently configured for each fluorescence signal to be measured; each optical component includes: an LED light source emitting excitation light of a specific wavelength, an excitation filter disposed between the LED light source and the reaction container, and an emission filter disposed in the observation direction; wherein the excitation filter is used to limit the wavelength range of the excitation light, enabling it to excite the corresponding fluorescence but effectively separating the spectral range from the fluorescence emission spectrum; the emission filter is used to allow only a narrow band of light near the target fluorescence emission peak to pass through.

[0010] Furthermore, the excitation light source is a 470 nm blue LED, the excitation filter has a diameter of 12.7 mm, a thickness of 2 mm, a center wavelength of 500 nm, a transmission band of 300 - 490 nm, a cutoff band of 515 - 715 nm, an average transmittance > 90%, an effective aperture > 90%, an incident angle of 0°, and a cutoff depth OD≥2. The emission filter has a diameter of 12.7 mm, a thickness of 2 mm, a center wavelength of 525 nm, a bandwidth of 20 nm, a peak transmittance > 85%, and a cutoff depth OD6@200 - 900 nm.

[0011] A light-interference-resistant multichannel biosensor based on Argonaute and selective optical filtering was developed for detecting Enterocytozoa hepatocellular carcinoma (EHP) and Vibrio filamentosa, a pathogenic Vibrio bacterium that causes acute hepatopancreatic necrosis in shrimp. AHPND Application in two aquatic pathogens.

[0012] Furthermore, detection is performed using biosensors, as detailed below:

[0013] S1. Extract DNA from the sample to be tested; S2. Simultaneously add the extracted DNA to a dual or multiple RPA reaction system for isothermal amplification to obtain amplification products; S3. Add the amplification products to a dual or multiple PfAgo enzyme digestion reaction system and perform enzyme digestion at a preset temperature to generate corresponding fluorescence signals in positive samples; S4. Place the reaction vessel that has completed the enzyme digestion reaction in the dark field environment of the optical detection unit, turn on the corresponding LED light source for each fluorescence signal to be tested, and observe through the corresponding excitation filter and emission filter combination. Determine the presence or absence of the corresponding target pathogen based on the presence or absence of a clear fluorescence signal.

[0014] Furthermore, the PfAgo enzyme digestion reaction was carried out at 95°C for 30 minutes, and FAM and HEX fluorescence readings were performed.

[0015] Furthermore, the dual-target RPA-PfAgo detection reaction achieved a sensitivity of 10 for each reaction against EHP and AHPND. 0 One copy.

[0016] Furthermore, the two detection targets, the SWP gene fragment of EHP and the PirB gene fragment of AHPND, are amplified by double RPA. The amplicon is then transferred to the PfAgo reaction and specifically cleaved by PfAgo under the guidance of specially designed gDNA. The cleavage products sequentially guide PfAgo to cleave their respective MBs and release fluorescent signals. FAM is used for EHP and HEX is used for AHPND. Among them, gDNA1 is used for the EHP target and gDNA2 / gDNA3 is used for the AHPND target.

[0017] The above technical solution can achieve the following beneficial effects:

[0018] This invention utilizes a low-cost, rapid, in-tube simultaneous detection sensor or platform for typing two aquatic pathogens, based on a combination of Argonaute enzyme, LED light, light source filter, and dark-field observation filter. AMDRIL is effective for EHP and Vibrio... AHPND The detection sensitivity is as high as 10 per reaction. 0 The assay yields only one copy, exhibits good specificity, and the detection process takes approximately one hour. By configuring conventional LED lights, light source filters, and observation filters in a dark field, multiple fluorescence signals can be observed in the same tube. These signals are clearly visible and can be read with the naked eye, leading to definitive results. This assay also has significant value for the rapid detection of other nucleic acid targets related to disease diagnosis, food safety, environmental protection, and public health. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the AMDRIL principle.

[0020] (A) Detection Principle. The target is amplified via double RPA, and the specific fluorescence signal is generated through a double PfAgo reaction. Visual readout of the signal is crucial to the configuration of FAM and HEX. "+" and "-" indicate the presence or absence of the target. (B) Detailed Configuration for Visual Readout of FAM and HEX Signals. For each signal, the configuration includes an LED, a light source filter, and a filter for observation in the dark field. Reaction components, optical components, and optical paths are represented and labeled with different colors and shapes.

[0021] Figure 2 This establishes the detection reaction of the double RPA-PfAgo.

[0022] (AB) Sensitivity confirmation: Two types of targets, plasmid DNA standard (A) and DNA extracted from infected shrimp (B), were detected. The endpoint fluorescence signal values ​​obtained on the qPCR instrument for different amounts of the target were represented by a bar graph. (CD) Specificity confirmation: A series of pathogens were detected. The endpoint fluorescence signal values ​​of the qPCR instrument were represented by a bar graph for FAM signal (C) and HEX signal (D). NTC: No template control.

[0023] Figure 3 This is an observation of fluorescence signals obtained through wavelength-selective excitation and filtering. (A and C) Emission spectra of FAM (EHP target, A) and HEX (AHPND target, C) were measured using Raman spectroscopy. (B and D) Visualization of fluorescence signals in the EHP (B) and AHPND (D) detection tubes under different filter configurations. These photos were taken with a smartphone camera. NTC: Template-free control

[0024] Figure 4 This is a clinical sample validation of the AMTRIL detection platform. (AB) Spectra of the light emitted by FAM (EHP target) and HEX (AHPND target) tested in the wavelength ranges of 515-535 nm (A) and 550-575 nm (B) using Raman spectroscopy. (C) Visual reading of multiple fluorescence signals in one tube using FAM and HEX configurations. (D) Visual fluorescence detection of a range of pathogens tested by AMTRIL in one tube, confirming good specificity. (EF) Validation of AMTRIL using 10 clinical samples (1-10). The results of EHP (E) and AHPND (F) detection were compared with nested PCR. Agarose gel electrophoresis analysis of PCR products showed that bands at expected sizes indicated the presence of the target. Images observed with the naked eye were taken with a smartphone. "+" and "-" indicate the presence and absence of the target. NTC: No template control. Detailed Implementation

[0025] The following is in conjunction with the appendix Figure 1-4 The present invention will be further illustrated by the following examples:

[0026] Aquaculture is a vital component of the aquatic product supply, contributing to food security by providing high-quality protein. On-site monitoring of aquatic pathogens in aquaculture farms helps prevent outbreaks of aquatic diseases. This requires detection methods suitable for point-of-care testing, meaning these methods should be easy to set up in the field, simple to operate, have short procedures, provide definitive results, and possess sufficient accuracy. The combination of isothermal RPA amplification and the programmable nuclease PfAgo provides a suitable nucleic acid detection method for aquatic pathogens for point-of-care testing. Particularly for the two most serious pathogens in shrimp farming—EHP and AHPND—the RPA-PfAgo combined detection method achieves a good balance between convenience and accuracy.

[0027] To further improve the user experience of detection methods, multiplexing is an important development direction. Multiplexing means processing multiple detection targets in a single reaction tube, thereby generating multiple distinguishable signals. The example utilizes multiplex RPA technology and the specific cleavage properties of PfAgo (PfAgo has no trans-cleavage activity), and successfully established detection methods for EHP and Vibrio. AHPND A dual RPA-PfAgo detection reaction was developed. This dual reaction exhibits good specificity, with a sensitivity of 10 for both targets. 0 Each reaction produces one copy of the sample, and each copy generates a different fluorescence signal.

[0028] Based on the dual RPA-PfAgo detection reaction, different fluorescence signals are generated in the same reaction tube, thus requiring a solution for making a clear interpretation of the results. Reading the fluorescence signals in the reaction tube is not easy because: 1) ambient light and / or excitation light may mask the signal; 2) different fluorescence signals are indistinguishable from each other. The fluorescence signal spectrum is not a clear peak, and the human eye lacks the resolution to extract the signal from the background. By employing wavelength-selective excitation and optical filtering techniques, in AMDRIL, each fluorescence signal is configured with a conventional LED, a source filter, and an observation filter, placed in a dark field, thus achieving extremely clear and definitive result readings. This configuration is designed to block any light that might interfere with signal reading. The source filter limits the wavelength range of the excitation light, ensuring it is within the wavelength range capable of exciting fluorescence but far from the spectral range of the emitted light. Simultaneously, the observation filter only allows a narrow wavelength range of approximately 20 nm around the peak spectrum of the emitted light to pass through. This configuration ensures that only the correct signal can pass through the observation filter. It is worth mentioning that this configuration is suitable not only for reading multiple fluorescence signals, but also for reading a single signal, because even reading a single signal is easily affected by ambient light and / or excitation light.

[0029] The anti-interference optical detection platform AMDRIL can achieve EHP and Vibrio AHPND Multiple detection with good specificity, and a sensitivity of up to 10 per reaction. 0 This theoretically highest sensitivity is achieved with a single copy, and the detection process takes only about one hour. Operation requires only a pipette set, a metal bath, and a mini centrifuge; these devices are easy to assemble and can be taken to the field. Since standard LED lights and light filters are common optical components, configuring a standard LED light, a light source filter, and an observation filter in the dark field for each fluorescence signal does not significantly increase the cost of detection; on the contrary, it improves the clarity of the results. AMDRIL supports detection of aquatic pathogens EHP and Vibrio. AHPND This technology enables on-site detection and provides a low-cost, point-of-care testing (POCT)-compatible technical route for multiplex nucleic acid biosensing. It is also of great value for the rapid detection of other nucleic acid targets related to disease diagnosis, food safety, environmental protection, and public health.

[0030] Based on the above introduction of the anti-interference optical detection platform AMDRIL and sensor information, the following examples illustrate this in detail:

[0031] This embodiment describes an anti-interference multichannel biosensor based on Argonaute and selective optical filtering, namely an Ago enzyme-based multiplex detection platform (anti-interference optical detection platform, abbreviated as AMDRIL), used for on-site detection of two important aquatic pathogens—*Enterocystis hepatis* (EHP) in shrimp and *Vibrio*, a pathogenic Vibrio that causes acute hepatopancreatic necrosis. AHPND Due to their rapid spread and high morbidity, these are two of the most serious pathogens in aquaculture. This embodiment, using wavelength-selective excitation and optical filtering, is convenient and cost-effective, avoiding light interference in multiplex detection. AMDRIL is effective against EHP and Vibrio. AHPND The detection sensitivity is as high as 10 per reaction. 0 The test yields multiple copies with good specificity, and the detection process takes only about one hour. More importantly, by configuring conventional LED lights, light source filters, and observation filters in a dark field, multiple fluorescence signals can be observed in the same tube. These signals are clearly visible and can be read with the naked eye, allowing for definitive result interpretation. AMDRIL supports EHP and Vibrio... AHPND This technology enables on-site monitoring and provides an economical and portable approach suitable for on-the-go testing, enabling multiplex nucleic acid biosensing detection with fluorescence readout capabilities.

[0032] 1. Strains and shrimp samples

[0033] Standard strains, including *Vibrio harveyi* (ATCC 43516), *Vibrio vulnificus* (ATCC 27562), *Vibrio alginolyticus* (ATCC 17749), *Vibrio mimicus* (MCCC 1A02602), *Vibrio cholerae* (ATCC 14100), and *Vibrio parahaemolyticus* (non-AHPND type, ATCC 17802), were obtained from preservation facilities. An environmentally isolated *Vibrio parahaemolyticus* strain containing the pVA1 plasmid was also included. AHPND Shrimp samples infected with white spot disease virus (WSSV), shrimp hemocyte iridovirus (SHIV), and shrimp enterocytozoon hepaticus (EHP) were provided by the Jiangsu Provincial Marine Fisheries Research Institute. Clinical shrimp samples were collected from shrimp farms in different regions of Jiangsu Province. Healthy shrimp were purchased from the local market in Lianyungang. Bacterial or shrimp DNA was extracted using the Tiangen Bacterial DNA Extraction Kit or the Tiangen Genomic DNA Extraction Kit, respectively, and subsequently quantified using a micro-spectrophotometer.

[0034] 2. DNA standards

[0035] The EHP DNA standard is constructed using a standard molecular cloning method using a 3206-bp plasmid containing a 514-bp swp gene fragment against a pMD18T background (GenBank ID: AF173993).AHPND DNA standards were obtained from Vibrio parahaemolyticus. AHPND The virulence plasmid pVA1 (GenBank ID: NC_025152.1) was extracted from environmental isolates.

[0036] 3. RPA reaction

[0037] The RPA reaction was performed according to the instructions of the RAA Nucleic Acid Amplification Kit (Hangzhou Zhongce Biotechnology Co., Ltd., Hangzhou, China). One lyophilized particle was used to prepare a 50 µl reaction mixture containing 25 µl of buffer A, 2 µl of each RPA primer (10 µM), 2–5 µl of template, an appropriate amount of deionized water, and 2.5 µl of buffer B. The reaction was carried out at 37°C for 30 minutes. If necessary, the RPA amplification products could be analyzed using non-denaturing PAGE (15%, 0.5×TBE). The oligonucleotide sequences (primers, gDNA, and MB) are shown in Table 1.

[0038] 4. PfAgo enzyme digestion reaction

[0039] PfAgo was prepared in our laboratory. A 20 µl PfAgo cleavage reaction mixture contained 5 µl of column-purified RPA amplification product, 2.5 µM PfAgo, gDNA (0.5 µM gDNA1-EHP, 0.25 µM gDNA2-AHPND, and 0.25 µM MgDNA3-AHPND), molecular beacons (1 µM MB-EHP and 1 µM MB-AHPND), and 2 µl of 10× reaction buffer (200 mM HEPES, pH 7.5, 2.5 M NaCl, and 5 mM MnCl2). The reaction was performed on a Roche LightCycler 480 II qPCR system (Basel, Switzerland) at 95°C for 30 minutes, with FAM and HEX fluorescence readouts.

[0040] 5. Nested PCR

[0041] Nested PCR experiments were performed as previously reported (see Table 1). For EHP, primers SWP-1F and SWP-1R were used in the first round of PCR, and primers SWP-2F and SWP-2R were used in the second round. The amplified fragment sizes in the first and second rounds were 514 bp and 147 bp, respectively. For AHPND, primers AP4-1F and AP4-1R were used in the first round of PCR, and primers AP4-2F and AP4-2R were used in the second round. The amplified fragment sizes in the first and second rounds were 1269 bp and 230 bp, respectively. PCR products were analyzed by 1.5% agarose gel electrophoresis.

[0042] 6. Fluorescence spectroscopy recording

[0043] Fluorescence spectra of FAM and HEX were recorded using an inVia microconfocal Raman spectrometer from Renishaw. 10 µl of the PfAgo reaction mixture was coated onto a glass substrate. For the FAM fluorescence signal, the excitation wavelength was set to 470 nm, and the excitation power was 25 mW. Spectral data were recorded in the wavelength range of 515 nm to 535 nm, with an integration time of 10 s. For the HEX fluorescence signal, the excitation wavelength was set to 525 nm, and spectral data were recorded in the wavelength range of 550 nm to 575 nm.

[0044] 7. Observing fluorescence signals using wavelength-selective excitation and optical filtering techniques.

[0045] All optical components were housed in a 3D-printed box to provide a dark environment for observation. A 470 nm blue LED was used as the excitation source for observing the FAM fluorescence signal. The light first passed through an excitation filter (filter 1A, diameter 12.7 mm, thickness 2 mm, center wavelength 500 nm, transmission band 300-490 nm, cutoff band 515-715 nm, average transmittance > 90%, effective aperture > 90%, incident angle 0°, cutoff depth OD≥2) and then was perpendicularly incident onto the PfAgo reaction mixture in the test tube. The fluorescence signal in the test tube was observed through an emission filter (filter 1B, diameter 12.7 mm, thickness 2 mm, center wavelength 525 nm, bandwidth 20 nm, peak transmittance > 85%, cutoff depth OD6@200-900 nm), which was set at a 90° angle to the excitation light. A 525 nm green LED was used as the excitation source for observing the HEX fluorescence signal. Light was first passed through an excitation filter (filter 2A, diameter 12.7 mm, thickness 2 mm, center wavelength 525 nm, bandwidth 20 nm, peak transmittance > 85%, cutoff depth OD6@200 - 900 nm) and then perpendicularly incident on the reaction mixture. Fluorescence signals were observed through a filter (filter 2B, diameter 12.7 mm, thickness 2 mm, center wavelength 564 nm, bandwidth 20 nm, peak transmittance > 85%, cutoff depth OD6@200-900 nm). The optical components were purchased from United Optics Technology (Beijing) Co., Ltd.

[0046] 8. Experimental Procedure

[0047] (1) Detection principle of AMTRIL platform

[0048] AMDRIL for EHP and AHPND (Vibrio) AHPND The detection reaction includes a dual RPA reaction that specifically amplifies the detection target and a dual PfAgo reaction that generates a fluorescent signal. Figure 1 Two detection targets, the SWP gene fragment of EHP and the PirB gene fragment of AHPND, were amplified via double RPA. The amplicons were then transferred to a PfAgo reaction and specifically cleaved by PfAgo under the guidance of specially designed gDNAs (gDNA1 for the EHP target, gDNA2 / gDNA3 for the AHPND target). The cleavage products sequentially guided PfAgo to cleave their respective MBs and release fluorescent signals: FAM for EHP and HEX for AHPND. Through specific photoexcitation and filtering, the signals were clearly distinguishable from each other and from negative results. Figure 1 A). Specific photoexcitation and filtering were achieved in a dark field provided by the 3D printing chamber. To observe the FAM signal, light emitted from a 470 nm blue LED was passed through filter 1A onto the reaction mixture, and the emitted fluorescence was observed through filter 1B on the chamber wall at a 90° angle to the excitation light. Similarly, to observe the HEX signal, excitation light emitted from a 525 nm green LED was filtered through filter 2A, and the emitted fluorescence was observed through filter 2B. Figure 1 B). Due to the use of a dark field and the combination of light source and filter, interference from ambient / excitation light and other fluorescence emissions is eliminated, making the observation results of fluorescence signals within a single tube clearer.

[0049] (2) Establish a dual RPA-PfAgo reaction for detecting EHP and AHPND.

[0050] The dual RPA-PfAgo detection reaction comprises dual RPA amplification and dual PfAgo cleavage. “Dual” means that the reaction can respond to both targets with equal efficiency and specificity. We used single-target RPA primer sequences and gDNA sequences (Table 1) identified in previous studies for developing a combined RPA and PfAgo detection method for EHP and AHPND.

[0051] Table 1 Nucleic Acid Sequences

[0052]

[0053] The sensitivity and specificity of the dual RPA-PfAgo detection reaction were confirmed. To confirm sensitivity, two types of targets were used: plasmid DNA standards and DNA extracted from infected shrimp. The two targets were mixed in a 1:1 molar ratio in the reaction, with target amounts ranging from 10... 0 Up to 10 3The number of copies varies. Fluorescent signals indicate that, regardless of the type of DNA used, the sensitivity of the dual-target RPA-PfAgo detection reaction to both EHP and AHPND is 10 per reaction. 0 One copy ( Figure 2 AB). To confirm specificity, tests were performed on a range of Vibrio species and some common aquatic pathogens. Figure 2 (CD). Fluorescence signals showed that only EHP and AHPND targets were positive, while all other samples were negative, indicating that the dual-target RPA-PfAgo detection reaction has good specificity.

[0054] (3) Observation of FAM signals under wavelength selective excitation and filtering

[0055] FAM fluorescence (associated with EHP targets) is generated by excitation with light at a wavelength of 470 nm, followed by the emission of light at a wavelength of 525 nm. Theoretically, illuminating the reaction mixture with 470 nm light and observing the emitted 525 nm light would allow for the reading of the FAM fluorescence signal. However, in practice, it is not so simple. We use a Raman spectrometer to measure the spectrum of the emitted light (…). Figure 3 A). The results showed that the wavelength range was between 515 and 535 nm, indicating that the FAM fluorescence signal was a smooth waveform rather than a sharp peak. Ordinary LED lamps (i.e., non-high-cost ones) typically have a broad spectrum. If used as an excitation source, their spectrum can easily overlap with the emitted light, interfering with signal observation. As we observed in our experiment, directly illuminating the reaction mixture with ordinary 470 nm LED light failed to produce a positive signal that was significantly different from the control group. When we used a light source filter (filter 1A, transmission band 300-490 nm), the excitation light reflected or refracted by the tube wall or reaction liquid still masked the emitted signal, even though the spectrum of the "filtered" excitation light did not overlap with the emitted light. This indicates that the wavelength resolution of our naked eye is insufficient to extract the 515-535 nm signal from the background of the excitation light. When an observation filter (filter 1B, center wavelength 525 nm, bandwidth 20 nm) was added to block the reflected or refracted excitation light, the emitted signal became clearly visible. Figure 3 B). The above results show that, in a dark field, the combination of a standard 470 nm LED light source, a light source filter (filter 1A), and an observation filter (filter 1B) can effectively eliminate light interference when reading FAM signals.

[0056] (4) Observation of HEX signals with wavelength selective excitation and filtering

[0057] Similar optical interference problems were encountered during the observation of the HEX signal (correlated with the AHPND target) in the reaction tube. The HEX fluorescence is generated by excitation with light at a wavelength of 525 nm and emits light at a wavelength of 564 nm. In fact, according to the Raman spectrometer, the emitted light wavelength ranges from 550 to 575 nm. Figure 3 C). When using a standard 525 nm LED light as the excitation light, no significant positive signal is observed without a filter, meaning the emitted HEX signal is masked by the background light. Similarly, using only a source filter (filter 2A, center wavelength 525 nm, bandwidth 20 nm) cannot solve the background light problem, while using both a source filter and an observation filter (filter 2B, center wavelength 564 nm, bandwidth 20 nm) simultaneously produces a distinguishable signal. Figure 3 D). Observations of HEX signals indicate that the configuration required for good visualization is the same, including a combination of a standard LED light in dark conditions, a light source filter, and an observation filter.

[0058] (5) Observe multiple fluorescence signals in one tube.

[0059] Once a feasible configuration for reading a specific fluorescence signal is determined, it becomes easy to simultaneously observe multiple fluorescence signals in a single test tube, as this configuration eliminates any light interference from other fluorescence signals, provided that the spectra of the other signals do not overlap with the signal to be read. In fact, as Raman spectroscopy demonstrates, the emission wavelengths of FAM fluorescence and HEX fluorescence do not overlap within their respective observation windows. Figure 4 (AB). This means that the configuration for reading the FAM signal can block the HEX signal from the same reaction tube and give a FAM-specific positive or negative result, and vice versa. This was confirmed by visualizing EHP-positive, AHPND-positive, double-positive, and double-negative test tubes. Figure 4 C). To date, we have successfully achieved single-tube observation of FAM and HEX fluorescence signals for the detection of EHP and AHPND, and established the detection platform AMDRIL. AMDRIL does not affect the sensitivity of the dual RPA-PfAgo reaction because the target amount we used during development was just at the limit (10). 0 (Copy each reaction). The specificity of AMTRIL was further confirmed by testing on a range of Vibrio species and some common aquatic pathogens. Only the positive controls (samples containing EHP and AHPND targets) showed their respective fluorescence signals, indicating that AMTRIL has good specificity. Figure 4 D).

[0060] (6) Clinical sample validation of the AMDRIL detection platform

[0061] The AMDRIL testing platform was validated using 10 clinical samples. Results showed that 5 samples were positive for both EHP and AHPND (double positive), while the other three samples were positive for AHPND only. The AMDRIL detection results were completely consistent with industry-standard testing methods using nested PCR. Figure 4 (EF). Through visual observation, the difference between positive and negative samples is very obvious, indicating that any user using AMDRIL can obtain a clear test result without hesitation.

[0062] The above descriptions are all preferred embodiments of the present invention. For those skilled in the art, any modifications to the present invention in various equivalent forms without departing from the principle of the present invention shall fall within the protection scope of the appended claims.

Claims

1. A light-interference-resistant multichannel biosensor based on Argonaute and selective optical filtering, characterized in that: include: a) A dual or multiple recombinase polymerase amplification RPA reaction system for simultaneously amplifying multiple target nucleic acids; b) A dual or multiple PfAgo enzyme digestion reaction system cascaded with the RPA reaction system, the system containing guide DNA (gDNA) corresponding to each target specificity and fluorescently labeled molecular beacons (MBs), wherein the PfAgo enzyme cleaves the specific target sequence under the guidance of the corresponding gDNA, and then cleaves the corresponding MB to release fluorescent signals of different wavelengths; c) An optical detection unit comprising a dark-field observation environment and optical components independently configured for each fluorescence signal to be measured; each optical component comprising: an LED light source emitting excitation light of a specific wavelength, an excitation filter disposed between the LED light source and the reaction vessel, and an emission filter disposed in the observation direction; wherein, the excitation filter is used to limit the wavelength range of the excitation light so that it can excite the corresponding fluorescence but the spectral range is effectively separated from the fluorescence emission spectrum; the emission filter is used to allow only a narrow band of light near the target fluorescence emission peak to pass through.

2. The light interference resistant multichannel biosensor based on Argonaute and selective optical filtering according to claim 1, characterized in that: The excitation source is a 470 nm blue LED. The excitation filter has a diameter of 12.7 mm, a thickness of 2 mm, a center wavelength of 500 nm, a transmission band of 300 - 490 nm, a cutoff band of 515 - 715 nm, an average transmittance of > 90%, an effective aperture of > 90%, an incident angle of 0°, and a cutoff depth OD≥2. The emission filter has a diameter of 12.7 mm, a thickness of 2 mm, a center wavelength of 525 nm, a bandwidth of 20 nm, a peak transmittance of > 85%, and a cutoff depth OD6@200 - 900 nm.

3. A light-interference-resistant multichannel biosensor based on Argonaute and selective optical filtering for detecting Enterocytozoa hepatisimilariae (EHP) and Vibrio filamentosa, a pathogenic Vibrio bacterium causing acute hepatopancreatic necrosis in shrimp. AHPND Application in two aquatic pathogens.

4. The application according to claim 3, characterized in that: The application uses the biosensor described in claim 1 for detection, and the specific method is as follows: S1. Extract DNA from the sample to be tested; S2. Add the extracted DNA to a dual or multiple RPA reaction system and amplify at an isothermal temperature to obtain the amplification product; S3. Add the amplification product to a dual or multiple PfAgo enzyme digestion reaction system and perform the enzyme digestion reaction at a preset temperature to generate the corresponding fluorescent signal in the positive sample; S4. Place the reaction vessel that has completed the enzyme digestion reaction in the dark field environment of the optical detection unit. For each fluorescence signal to be tested, turn on the corresponding LED light source and observe it through the corresponding excitation filter and emission filter combination. Determine the presence or absence of the corresponding target pathogen based on whether there is a clear fluorescence signal.

5. The application according to claim 4, characterized in that: The PfAgo enzyme digestion reaction was carried out at 95°C for 30 minutes, and FAM and HEX fluorescence readings were performed.

6. The application according to claim 1, characterized in that: The dual-target RPA-PfAgo detection reaction achieves a sensitivity of 10 per reaction for both EHP and AHPND. 0 One copy.

7. The application according to claim 6 or 4, characterized in that: The two detection targets, the SWP gene fragment of EHP and the PirB gene fragment of AHPND, were amplified by double RPA. The amplicon was then transferred to the PfAgo reaction and specifically cleaved by PfAgo under the guidance of specially designed gDNA. The cleavage products sequentially guided PfAgo to cleave their respective MBs and release fluorescent signals. FAM was used for EHP and HEX was used for AHPND. Among them, gDNA1 was used for the EHP target and gDNA2 / gDNA3 was used for the AHPND target.

Images