Detection chip, device, primer probe set, kit and method for pathogenic bacteria in aquaculture water body

Abstract

The application discloses a breeding water pathogenic bacteria detection chip, device, primer probe group, kit and method, relates to the technical field of aquatic animal pathogenic bacteria detection, and the detection chip comprises a nucleic acid extraction chamber, which can extract pathogenic bacteria nucleic acid solution in breeding water; a reagent storage chamber is communicated with a detection chamber at one end, can store a reaction solution, and can deliver the reaction solution into the detection chamber; a pipetting module is arranged between the detection chamber and the nucleic acid extraction chamber, can deliver a set amount of pathogenic bacteria nucleic acid solution into the detection chamber; and a fluorescence detection module of the detection device can detect fluorescence signals of mixed pathogenic bacteria nucleic acid solution and reaction solution in the detection chamber. The breeding water pathogenic bacteria detection chip, device, primer probe group, kit and method provided by the application can realize on-site rapid detection of breeding water pathogenic bacteria.

Description

Technical Field

[0001] This invention relates to the field of pathogen detection technology for aquatic animals, and in particular to a chip, device, primer and probe set, reagent kit, and method for detecting pathogens in aquaculture water. Background Technology

[0002] The health of aquaculture water directly affects the yield and quality of aquaculture. However, the presence of pathogens in aquaculture water seriously threatens the survival of aquatic animals, and disease outbreaks often result in huge economic losses for farmers. Therefore, rapid and accurate detection of pathogens in aquaculture water is crucial for timely implementation of prevention and control measures and for ensuring the healthy development of aquaculture.

[0003] Traditional methods for detecting pathogens in aquaculture water involve time-consuming and labor-intensive enrichment cultures, failing to meet the demands for rapid detection in actual aquaculture processes. With continuous advancements in modern analytical techniques, molecular biology detection technologies, due to their high sensitivity, rapid response, and precise identification, are widely used for pathogen detection in aquaculture animals. However, molecular biology detection technologies require specialized equipment and skilled operators, resulting in high costs and limited applicability in on-site testing.

[0004] Therefore, there is an urgent need to design a technical solution that can enable rapid on-site detection of pathogens in aquaculture water. Summary of the Invention

[0005] The purpose of this invention is to provide a chip, device, primer and probe set, reagent kit and method for detecting pathogens in aquaculture water, so as to solve the problems existing in the prior art and realize rapid on-site detection of pathogens in aquaculture water.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a chip for detecting pathogenic bacteria in aquaculture water, comprising:

[0008] The nucleic acid extraction chamber is capable of extracting nucleic acid solutions from pathogenic bacteria in aquaculture water.

[0009] A reagent storage chamber, one end of which is connected to a detection chamber, is capable of storing reaction solutions and delivering reaction solutions to the detection chamber.

[0010] A pipetting module, located between the detection chamber and the nucleic acid extraction chamber, is capable of delivering a set amount of pathogenic bacterial nucleic acid solution into the detection chamber.

[0011] Preferably, it also includes a chip body, wherein the nucleic acid extraction chamber, reagent storage chamber, and pipetting module are all located on the chip body.

[0012] Preferably, the chip body has a first air inlet and a second air inlet. One end of the first air inlet is used to connect to a first driving device, which can drive the DNA template solution in the nucleic acid extraction chamber to flow through the pipetting module and quantitatively transfer it to the detection chamber. One end of the second air inlet is used to connect to a second driving device, which can transfer the MIRA reaction reagent in the reagent storage chamber to the detection chamber to react with the DNA template solution.

[0013] Preferably, the chip body has a first heating device hole and a second heating device hole. The first heating device hole is located near the nucleic acid extraction chamber and is used to install a first heating device to heat the solution in the nucleic acid extraction chamber to a set temperature. The second heating device hole is located near the detection chamber and is used to install a second heating device to heat the mixed solution in the detection chamber to a set temperature. A vibration device hole is provided between the detection chamber and the nucleic acid extraction chamber. The vibration device hole is used to install a vibration drive device to drive the detection chamber and / or the nucleic acid extraction chamber to vibrate for a set time.

[0014] Preferably, the pipetting module includes multiple solution retention structures connected in series, with the multiple solution retention structures connected end to end in sequence; one end of the nucleic acid extraction chamber is connected to the first solution retention structure located on one side, and the detection chamber is connected to the last solution retention structure located on the other side.

[0015] Preferably, the chip body includes a first mounting plate and a second mounting plate arranged symmetrically. The first mounting plate has a first extraction groove, a first detection groove and a first storage groove, and the second mounting plate has a second extraction groove, a second detection groove and a second storage groove. After the first mounting plate and the second mounting plate are fixedly connected, the sealed cavity between the first extraction groove and the second extraction groove forms the nucleic acid extraction chamber, the sealed cavity between the first detection groove and the second detection groove forms the detection chamber, and the sealed cavity between the first storage groove and the second storage groove forms the reagent storage chamber.

[0016] The present invention also provides a pathogen detection device for aquaculture water, comprising a housing, a top cover on the top of the housing, and a display screen mounted on the top cover; a horizontally arranged partition is provided inside the housing, and a power supply and control system are provided below the partition, the power supply and control system being electrically connected to the display screen; a vertically arranged placement seat is provided on the partition, and a placement groove is opened on one side of the placement seat, the placement groove being used to fix the pathogen detection chip for aquaculture water as described above;

[0017] The partition is provided with a fluorescence detection module, a vibration driving device, a first driving device, a second driving device, a first heating device, and a second heating device; the fluorescence detection module is located on the side close to the detection chamber; the fluorescence detection module includes a diode placement slot, in which a photodiode is fixedly installed, and the emitting end of the photodiode is provided with a filter, which is located between the detection chamber and the photodiode.

[0018] The present invention also provides a primer-probe combination for detecting Aeromonas hydrophila, which is used in conjunction with the chip or device described above. The primer-probe combination includes a forward primer F1, a reverse primer R1, and a fluorescent probe.

[0019] The forward primer F1 comprises the nucleotide sequence shown in SEQ ID NO:1;

[0020] The reverse primer R1 comprises a nucleotide sequence as shown in SEQ ID NO:2;

[0021] The fluorescent probe comprises a nucleotide sequence as shown in SEQ ID NO:7; wherein the 3' end of the nucleotide sequence shown in SEQ ID NO:7 is modified with C3Spacer, Y represents tetrahydrofuran, the base at 24 bp in the 5'-3' direction carries a fluorescein group, and the base at 27 bp in the 5'-3' direction carries black hole quencher-1.

[0022] The present invention also provides a kit for detecting Aeromonas hydrophila, the kit comprising a rapid DNA nucleic acid release agent, a multi-enzyme isothermal rapid amplification reagent, and the primer-probe combination described in the above scheme.

[0023] The present invention also provides a method for detecting Aeromonas hydrophila in aquaculture water for non-diagnostic purposes using the above-described chip or device, comprising the following steps:

[0024] The aquaculture water sample to be tested was mixed with a rapid DNA nucleic acid release agent, and genomic DNA was extracted from the aquaculture water sample.

[0025] Using the genomic DNA as a template, multi-enzyme isothermal rapid amplification (MIRA) is performed using the primer and probe set described above. The fluorescence signal intensity of the amplification product is detected to determine whether Aeromonas hydrophila is present in the aquaculture water to be tested.

[0026] The present invention achieves the following technical effects compared to the prior art:

[0027] This invention integrates molecular biology, microfluidics, and electronic information technology. The nucleic acid extraction chamber of the microfluidic chip enables sample collection, mixing, and nucleic acid extraction, while the reagent storage chamber stores MIRA reaction reagents and delivers them to the detection chamber. The DNA template solution in the nucleic acid extraction chamber flows through a pipetting module, allowing a predetermined volume of the DNA template solution to be delivered to the detection chamber for reaction with the MIRA reaction reagents. The fluorescence detection module of the pathogen detection device in aquaculture water detects the fluorescence signal of the reacted solution. This integrated approach of sample introduction, mixing, nucleic acid extraction, and detection achieves rapid, sensitive, and specific automated detection of pathogens in aquaculture water. It effectively solves the problems of complex operation steps, long detection times, and low automation levels faced by existing detection methods, enabling rapid on-site detection of pathogens in aquaculture water.

[0028] This invention designs primer-probe combinations using Aeromonas hydrophila as an example, and screens out primer-probe combinations with superior amplification efficiency. Based on the pathogen detection chip or device for aquaculture water provided by this invention, multi-enzyme isothermal rapid amplification can be performed, and concentrations in aquaculture water ranging from 1×10⁻⁶ can be detected within 40 minutes. 2 ~1×10 7 The detection limit for Aeromonas hydrophila at CFU / mL is 100 CFU / mL. The procedure is simple and the detection time is short.

[0029] The nucleic acid extraction chamber of the chip of this invention can realize sample collection, mixing and nucleic acid extraction, the reagent storage chamber can store the reaction solution and deliver it to the detection chamber; the pipetting module can select a set volume of the extracted nucleic acid solution and deliver it to the detection chamber to react with the reaction solution.

[0030] The detection device of the present invention can install and fix the chip. The fluorescence detection module of the detection device detects the fluorescence signal of the solution after the reaction in the chip. The detection device works with the chip to integrate sample introduction, mixing, nucleic acid extraction and detection, realizing rapid, sensitive and specific automated detection of pathogens in aquaculture water. It effectively solves the problems of complex operation steps, long detection time and low degree of automation faced by existing detection methods, and can realize rapid on-site detection of pathogens in aquaculture water. Attached Figure Description

[0031] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0032] Figure 1This is a schematic diagram of the mold structure in one or more embodiments of the present invention;

[0033] Figure 2 This is a schematic diagram of the pathogen detection chip structure in one or more embodiments of the present invention;

[0034] Figure 3 This is a schematic diagram of the placement base structure in one or more embodiments of the present invention;

[0035] Figure 4 This is a schematic diagram of a fluorescence detection module in one or more embodiments of the present invention;

[0036] Figure 5 The results of multi-enzyme isothermal rapid amplification using different primer-probe combinations of the present invention are shown below; where (A) is the fluorescence signal intensity of the negative control experiment; (B) is the time threshold of the negative control experiment; (C) is the fluorescence signal intensity of Aeromonas hydrophila detected by different primer-probe combinations; and (D) is the time threshold of Aeromonas hydrophila detected by different primer-probe combinations.

[0037] Figure 6 The results of multi-enzyme isothermal rapid amplification after optimization of relevant parameters are shown. Among them, (A) time threshold at different reaction temperatures; (B) time threshold at different primer amounts; (C) time threshold at different probe amounts; (D) fluorescence signal intensity at different probe amounts; (E) time threshold at different DNA template amounts.

[0038] Figure 7 The results represent the rapid, isothermal amplification of different concentrations of Aeromonas hydrophila in aquaculture water using multiple enzymes.

[0039] Figure 8 The results show the optimization of nucleic acid extraction reaction conditions; where A represents nucleic acid extraction under different temperature conditions; B represents nucleic acid extraction under different lysis times; and C represents nucleic acid extraction under different bacterial suspension volumes.

[0040] Figure 9 This is a schematic diagram of the structure of a pathogen detection device in aquaculture water in one or more embodiments of the present invention.

[0041] In the diagram: 1-Upper mold base plate, 2-Lower mold base plate, 3-Positioning hole, 4-First extraction tank forming space, 5-First detection tank forming space, 6-First storage tank forming space, 7-Second extraction tank forming space, 8-Second detection tank forming space, 9-Second storage tank forming space, 10-Solution retention structure, 11-Nucleic acid extraction chamber, 12-Reagent storage chamber, 13-Pipeline module, 14-Detection chamber, 15-First air inlet, 16-Second air inlet, 17-First driving device, 18-First heating device, 19-Second driving device, 20-Second heating device, 21-Vibration driving device, 22-Diode placement slot, 23-Fluorescence detection window, 24-Placement seat, 25-Ceramic heating plate placement hole, 26-Miniature vibration motor placement hole, 27-Fluorescence detection module placement slot, 28-Flow channel, 29-Housing shell, 30-Display screen, 31-Partition plate, 32-Power supply, 33-Control system, 34-Fixing plate. Detailed Implementation

[0042] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0043] The purpose of this invention is to provide a chip, device, primer and probe set, reagent kit and method for detecting pathogens in aquaculture water, so as to solve the problems existing in the prior art and realize rapid on-site detection of pathogens in aquaculture water.

[0044] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0045] Traditional methods for detecting pathogens in aquaculture water involve time-consuming and labor-intensive enrichment cultures, failing to meet the demands for rapid detection in actual aquaculture processes. While molecular biology detection techniques offer high sensitivity and rapid response, they require specialized equipment and skilled operators, resulting in high costs and limited applicability in field testing, thus also unsuitable for rapid on-site detection. To address this technical problem, the first objective of this invention is to provide a chip for detecting pathogens in aquaculture water, referencing... Figures 1-4As shown, it includes a nucleic acid extraction chamber 11, which can extract nucleic acid solution of pathogenic bacteria from aquaculture water, the pathogenic bacteria nucleic acid solution being a DNA template solution; a reagent storage chamber 12, one end of which is connected to a detection chamber 14, which can store reaction solution and transport the reaction solution to the detection chamber 14, the reaction solution being a multi-enzyme isothermal nucleic acid amplification reaction system solution; a pipetting module 13 is located between the detection chamber 14 and the nucleic acid extraction chamber 11, and a first driving device can drive the DNA template solution in the nucleic acid extraction chamber 11 to flow through the pipetting module 13 and quantitatively transfer it to the detection chamber 14.

[0046] The nucleic acid extraction chamber 11, reagent storage chamber 12, and pipetting module 13 are all part of the detection chip. In this embodiment, the detection chip is a microfluidic chip, comprising a symmetrically arranged first mounting plate and a second mounting plate. The first mounting plate has a first extraction groove, a first detection groove, and a first storage groove, while the second mounting plate has a second extraction groove, a second detection groove, and a second storage groove. After the first and second mounting plates are fixedly connected, the sealed cavity between the first and second extraction grooves forms the nucleic acid extraction chamber 11; the sealed cavity between the first and second detection grooves forms the detection chamber 14; and the sealed cavity between the first and second storage grooves forms the reagent storage chamber 12. The flow channel formed by the fixed connection between the first and second mounting plates and the solution retention structure within the flow channel constitute the pipetting module 13. The first and second mounting plates can be formed by injection molding or casting.

[0047] In one embodiment, the mold includes an upper mold base plate 1 and a lower mold base plate 2. The upper mold base plate 1 has a first extraction groove forming space 4, a first detection groove forming space 5, a first storage groove forming space 6, and forming spaces for the upper half of each corresponding flow channel 28. A first mounting plate can be formed by injection molding or casting. Similarly, the lower mold base plate 2 has a second extraction groove forming space 7, a second detection groove forming space 8, a second storage groove forming space 9, and forming spaces for the lower half of each corresponding flow channel 28. A second mounting plate can be formed by injection molding or casting. In this embodiment, the mold is used to create the first and second mounting plates, which are then bonded together after plasma treatment to obtain a complete pathogen detection chip. Positioning holes 3 are designed on the first and second mounting plates respectively to facilitate the positioning and bonding of the first and second mounting plates.

[0048] In one embodiment, the detection chip has a first air inlet 15 and a second air inlet 16. One end of the first air inlet 15 is externally connected to a first driving device 17, and the other end is connected to a nucleic acid extraction chamber 11 via a flow channel. The first driving device 17 drives the DNA template solution in the nucleic acid extraction chamber 11 to flow through the pipetting module 13 and quantitatively transfer it into the detection chamber. One end of the second air inlet 16 is externally connected to a second driving device 19, and the other end is connected to a reagent storage chamber 12 via a flow channel. The second driving device 19 transfers the MIRA reaction reagent from the reagent storage chamber 12 to the detection chamber 12 to react with the DNA template solution. The specific structure of the first driving device 17 and the second driving device 19 is not limited. In this embodiment, both the first driving device 17 and the second driving device 19 are miniature air pumps, and the first air inlet 15 and the second air inlet 16 are each connected to a miniature air pump for precise fluid control. The detection chip has a first heating device hole and a second heating device hole. The second heating device hole is located near the detection chamber 14 and contains a second heating device 20, which can heat the mixed solution in the detection chamber 14 to a set temperature. The first heating device hole is located near the nucleic acid extraction chamber 11 and contains a first heating device 18, which can heat the solution in the nucleic acid extraction chamber 11 to a set temperature. The first heating device 18 and the second heating device 20 can be heating wires, heating elements, or thermocouples, etc. In this embodiment, a ceramic heating element is used to provide suitable temperatures for the nucleic acid extraction process and the nucleic acid amplification process, respectively. A PT100 temperature sensor is fixed to the ceramic heating element using high-temperature insulating tape for easy temperature detection and control of the heating process. A vibration device hole is provided between the detection chamber 14 and the nucleic acid extraction chamber 11, and a vibration drive device 21 is provided at the vibration device hole to drive the detection chamber 14 and / or the nucleic acid extraction chamber 11 to vibrate for a set time. In this embodiment, the vibration device uses a miniature vibration motor, which can achieve efficient mixing of reagents in the nucleic acid extraction chamber 11 and the detection chamber 14. A fluorescence detection window 23 is provided at one end of the detection chip, located near the detection chamber 14, for positioning the fluorescence detection module of the detection device.

[0049] The pipetting module 13 includes multiple serially connected solution retention structures 10, which are connected end to end in sequence. Each solution retention structure 10 has a 5mm high right-angled triangular receiving groove with a hydrophilic layer on the side wall to form a pipetting area. A portion of the solution can be retained in the receiving groove, which can replace the operation of a pipette. By adjusting the power of the micro air pump, the solution can flow through the solution retention structure 10 at a set flow rate and flow rate. Within an acceptable error range, the solution can be driven to flow through the solution retention structure 10 to obtain a fixed volume of DNA template solution for subsequent amplification experiments. After the fixed volume of DNA template solution is driven into the detection chamber 14 by the micro air pump, the multi-enzyme isothermal nucleic acid amplification reaction system in the reagent storage chamber 12 is driven into the detection chamber 14 by the micro air pump. One end of the nucleic acid extraction chamber 11 is connected to the first solution retention structure 10 located on one side, and the detection chamber 14 is connected to the last solution retention structure 10 located on the other side.

[0050] A second objective of this invention is to provide a device for detecting pathogenic bacteria in aquaculture water, which is used for placing a pathogenic bacteria detection chip and controlling the detection process; such as Figure 9 As shown, its structure includes a base plate, on which a housing 29 is fixedly mounted. A top cover is provided on the top of the housing 29, and a display screen 30 is mounted on the top cover. A horizontally arranged partition 31 is provided inside the housing 29. A power supply 32 and a control system 33 are located below the partition 31. The power supply 32 can be a battery. The power supply 32 and the control system 33 are electrically connected to the display screen 30. In one embodiment, the fluorescence detection module includes a diode placement slot 22, in which a photodiode is fixedly mounted. A filter is provided at the emitting end of the photodiode, located between the detection chamber 14 and the photodiode. The main controller of the control system 33 is an STM32F103 microcontroller. The microcontroller is used to connect to the photodiode to acquire fluorescence data, to connect to a micro air pump to achieve precise control of the solution within the pathogen detection device, to connect to a micro vibration motor to achieve efficient mixing of the solution within the chamber, to connect to a ceramic heating element to achieve temperature regulation, and to connect to the display screen to achieve human-computer interaction. The power supply 32 includes a switching power supply and a voltage regulation module. The switching power supply rectifies 220V AC power into usable DC power, and the voltage regulation module regulates the voltage output by the switching power supply to the required different output voltages.

[0051] In this embodiment, the partition 31 has wire holes and slots, and is tightly connected to a fixing plate 34. The fixing plate 34 is used to fix a micro air pump. A vertically arranged placement seat 24 is provided on the partition 31 near the fixing plate 34. A placement groove is provided on one side of the placement seat 24, which is used to fix the pathogen detection chip in the aquaculture water as described above. A 470nm wavelength LED is fixed on the placement seat 24, located directly below the chip detection chamber 14, to provide excitation light for fluorescence detection. The placement seat 24 also includes a ceramic heating element placement hole 25 and a micro vibration motor placement hole 26, located directly below the corresponding placement holes of the pathogen detection device, to facilitate the installation of the ceramic heating element and the micro vibration motor. The placement seat 24 also has a fluorescence detection module placement groove 27, which facilitates the tight fit between the filter and photodiode after assembly and the fluorescence detection window 23.

[0052] In this embodiment, the control system of the pathogen detection device in aquaculture water has built-in detection software. This software is developed using C language based on an STM32F103 microcontroller. The software can control the pumping system, temperature control system, vibration mixing system, fluorescence detection system, and human-machine interface system. The pumping system uses the microcontroller's timer to generate PWM output to regulate the gas flow of the miniature air pump, and controls the operating status of a single miniature air pump by driving a relay module through the microcontroller's GPIO pins. The vibration mixing system uses the microcontroller's GPIO pins to drive a relay module to control the operating status of the miniature vibration motor. The temperature control system uses the microcontroller's timer to generate PWM output, combined with a PID control algorithm to achieve precise temperature control of the ceramic heating element. The fluorescence detection system uses the microcontroller's ADC to collect the photodiode fluorescence signal every 30 seconds, and analyzes the sample concentration by calculating the time taken to reach the fluorescence threshold. The human-machine interface system uses the microcontroller's serial communication to interact with the Taojingchi serial touchscreen, displaying the detection time and results, amplification curves, and allowing adjustment of the detection device's operating parameters. In addition, to prevent software systems from crashing under certain circumstances, watchdog timers are used in software systems to improve system reliability.

[0053] In operation, the detection chip is controlled via a display screen to initiate the detection process. First, the main control circuit controls a micro-air pump at the first air inlet 15 to pump the sample and rapid nucleic acid release agent (180 μL total) into the nucleic acid extraction chamber 11. Then, a micro-vibration motor is activated to vibrate and mix the solution in the nucleic acid extraction chamber 11 for 10 seconds. Subsequently, the ceramic heating element at the first heating device 18 is heated to 40°C for 5 minutes. After completion, the micro-air pump at the first air inlet 15 drives the DNA template solution to the pipetting area. Under gravity, the DNA template solution, after passing through the solution retention structure, is pumped into the detection chamber 14 in a volume of 5 μL. Then, the micro-air pump at the first air inlet 15 is stopped, and the micro-air pump at the second air inlet 16 is activated to pump the multi-enzyme isothermal nucleic acid amplification reaction system solution pre-reserved in the reagent storage chamber 12 into the detection chamber 14. Finally, the vibration motor is activated again to efficiently mix the solution in the detection chamber 14 for 10 seconds. Simultaneously, the ceramic heating element at the second heating device 20 is rapidly heated to 39°C. Furthermore, fluorescence signal detection is performed upon reaching 39°C, with detection occurring every 30 seconds. The concentration of the sample is analyzed by calculating the time required to reach the fluorescence threshold. Finally, the main control circuit displays the detection results and amplification curve on the screen.

[0054] A third objective of this invention is to provide a primer-probe combination for detecting Aeromonas hydrophila, used in conjunction with the chip or device described above. The primer-probe combination includes a forward primer F1, a reverse primer R1, and a fluorescent probe. The forward primer F1 includes the nucleotide sequence shown in SEQ ID NO:1; the reverse primer R1 includes the nucleotide sequence shown in SEQ ID NO:2; and the fluorescent probe includes the nucleotide sequence shown in SEQ ID NO:7. Specifically, the 3' end of the nucleotide sequence shown in SEQ ID NO:7 is modified with C3Spacer, Y represents tetrahydrofuran, the base at 24 bp in the 5'-3' direction carries a fluorescein group, and the base at 27 bp in the 5'-3' direction carries a black hole quencher-1.

[0055] This invention designs and evaluates primer-probe combinations based on the aerolysin gene (aerA) of Aeromonas hydrophila. The results show that the combination of the forward primer F1 with the nucleotide sequence shown in SEQ ID NO:1, the reverse primer R1 with the nucleotide sequence shown in SEQ ID NO:2, and the probe with the nucleotide sequence shown in SEQ ID NO:7 has high specificity, reaches the fluorescence threshold at 7.63 min, and shows the highest amplification efficiency.

[0056] The present invention also provides a kit for detecting Aeromonas hydrophila, the kit comprising a rapid DNA nucleic acid release agent, a multi-enzyme isothermal rapid amplification reagent, and the primer-probe combination described in the above scheme.

[0057] In one embodiment, the *Aeromonas hydrophila* is a type of *Aeromonas hydrophila* found in aquaculture water. In one embodiment, the concentration of *Aeromonas hydrophila* in the aquaculture water is 1 × 10⁻⁶. 2 ~1×10 7 CFU / mL.

[0058] In one embodiment, the rapid nucleic acid release agent and the multi-enzyme isothermal rapid amplification reagent were purchased from Amp Future; the rapid nucleic acid release agent was a rapid nucleic acid release agent (DNA type)-II; and the multi-enzyme isothermal rapid amplification reagent was a DNA isothermal rapid amplification kit (fluorescent type).

[0059] The present invention also provides a method for detecting Aeromonas hydrophila in aquaculture water for non-diagnostic purposes using the chip or device described above, comprising the following steps:

[0060] The aquaculture water sample to be tested was mixed with a rapid DNA nucleic acid release agent, and genomic DNA was extracted from the aquaculture water sample.

[0061] Using the genomic DNA as a template, multi-enzyme isothermal rapid amplification was performed using the primer and probe set described above. The fluorescence signal intensity of the amplification product was detected to determine the content of Aeromonas hydrophila in the aquaculture water to be tested.

[0062] The present invention first mixes the aquaculture water sample to be tested with a rapid DNA nucleic acid release agent to extract genomic DNA from the aquaculture water sample.

[0063] In one embodiment, the extraction temperature is 40°C and the extraction time is 5 minutes.

[0064] In one embodiment, the present invention uses the main control circuit of the pathogen detection device in aquaculture water or the pathogen detection system in aquaculture water described in the above technical solution to control a micro air pump at the first air inlet 15 to pump the aquaculture water sample to be tested and a rapid DNA nucleic acid release agent (total 180 μL) into the nucleic acid extraction chamber 11. Then, a micro vibration motor is started to vibrate and mix the solution in the nucleic acid extraction chamber 11 for 10 seconds. Subsequently, the ceramic heating plate at the first heating device 18 is controlled to heat the solution at 40°C for 5 minutes to obtain genomic DNA.

[0065] After obtaining the genomic DNA of the aquaculture water to be tested, the present invention uses the genomic DNA as a template and performs multi-enzyme isothermal rapid amplification using the primer and probe set described in the above scheme, detects the fluorescence signal intensity of the amplification product, and determines the content of Aeromonas hydrophila in the aquaculture water to be tested, thereby realizing the quantitative detection of Aeromonas hydrophila.

[0066] In one embodiment, the temperature for the isothermal rapid amplification of the multi-enzyme is 39°C.

[0067] In one embodiment, the isothermal rapid amplification time of the multi-enzyme is 20 minutes.

[0068] In one embodiment, a micro-air pump at the first air inlet 15 drives the genomic DNA solution to the pipetting area. Under gravity, after 175 μL of the genomic DNA solution is retained in the solution retention structure, 5 μL of the genomic DNA solution is pumped into the detection chamber 14. Then, the micro-air pump at the first air inlet 15 is stopped, and the micro-air pump at the second air inlet 16 is turned on, pumping the multi-enzyme isothermal rapid amplification reaction system solution pre-reserved in the reagent storage chamber 12 into the detection chamber 14. Subsequently, the vibration motor is restarted to efficiently mix the solution in the detection chamber 14 for 10 seconds. Simultaneously, the ceramic heating element at the second heating device 20 is rapidly heated to the incubation temperature. Furthermore, fluorescence signal detection is performed when the temperature reaches the incubation temperature, with detection every 30 seconds. The concentration of the sample is analyzed by calculating the time taken to reach the fluorescence threshold. Finally, the main control circuit controls the display screen to show the detection results and amplification curve.

[0069] In one embodiment, the volume of the DNA template solution pumped into the detection chamber 14 is 5 μL. In one embodiment, the multi-enzyme isothermal rapid amplification reaction system, in 50 μL volume, comprises the following components: 29.4 μL A buffer, 2 μL forward primer, 2 μL reverse primer, 0.6 μL fluorescent probe, 5 μL DNA template solution, and 8.5 μL sterile double-distilled water; the concentrations of the forward primer, reverse primer, and fluorescent probe are all 10 μM.

[0070] The method provided by this invention can determine whether Aeromonas hydrophila is present in the aquaculture water sample to be tested. Furthermore, by establishing a linear relationship between the time threshold and the logarithm of the Aeromonas hydrophila concentration, the concentration of Aeromonas hydrophila in the aquaculture water sample can be detected. This method is highly specific and sensitive.

[0071] Example 1

[0072] Taking the detection of Aeromonas hydrophila in aquaculture water as an example, the specific detection process is as follows:

[0073] 1. Primer design and screening

[0074] 1.1 A set of primers and probes for multi-enzyme isothermal rapid amplification detection of the aerolysin gene (aerA) was designed using the EZassay online design software (https: / / ezassay.com / primer), including 3 forward primers, 3 reverse primers and 1 fluorescent probe, as shown in Table 1.

[0075] Table 1 Primer and probe sequences for multi-enzyme isothermal rapid amplification detection.

[0076]

[0077] Note: The base T (uppercase bold) at 24 bp in the 5'-3' direction of the fluorescent probe carries the fluorescein group (FAM); the Y at 25 bp in the 5'-3' direction of the fluorescent probe represents tetrahydrofuran (THF); the base t (lowercase bold) at 27 bp in the 5'-3' direction of the fluorescent probe carries black hole quencher-1 (BHQ1); the 3' end of the fluorescent probe is modified with C3Spacer.

[0078] 1.2 The primers and probes were evaluated to ensure the efficiency and specificity of the multi-enzyme isothermal rapid amplification reaction.

[0079] (1) Three primer-probe combinations (forward primer + reverse primer + fluorescent probe) were randomly selected for a negative control experiment: using sterile double-distilled water as a template, a multi-enzyme isothermal rapid amplification reaction was performed using the primer-probe combinations. The results showed that none of the combinations produced a fluorescent signal, indicating that the designed primers and probes did not interact nonspecifically with non-target sequences. Figure 5 (A) and (B) confirmed the high specificity of the primers and probes.

[0080] (2) Use the same concentration of DNA template (1×10⁻⁶) 5 CFU·mL -1 All primer-probe combinations were subjected to multi-enzyme isothermal rapid amplification reactions. The results showed that the F1-R1-Probe combination reached the fluorescence threshold at 7.63 minutes, demonstrating superior amplification efficiency. Figure 5 (C) and (D)). Therefore, the F1-R1-Probe combination was selected as the optimal primer-probe combination for subsequent multi-enzyme isothermal rapid amplification experiments;

[0081] The system for the multi-enzyme isothermal rapid amplification reaction consisted of: 29.4 μL A buffer; 2 μL forward primer (10 μM); 2 μL reverse primer (10 μM); 0.6 μL fluorescent probe (10 μM); 5 μL DNA template solution; and 8.5 μL sterile double-distilled water, for a total of 50 μL.

[0082] The temperature for the multi-enzyme isothermal rapid amplification reaction is 39℃.

[0083] 2. When extracting bacterial nucleic acid, the Aeromonas hydrophila suspension is thoroughly mixed with rapid nucleic acid release agents (including LD-1 and LD-2) at a volume ratio of 25:4:1. The mixture is then incubated under optimal reaction conditions to produce a bacterial DNA template solution.

[0084] The efficiency of nucleic acid extraction is crucial to the sensitivity of subsequent nucleic acid detection. The rapid nucleic acid release reagent employs a high-salt lysis mechanism, and the extraction efficiency mainly depends on the lysis temperature, incubation time, and bacterial suspension volume. To ensure the reliability of the optimized experimental data, an in-tube MIRA reaction was performed using a real-time PCR system to determine the optimal nucleic acid extraction reaction conditions.

[0085] To determine the optimal lysis temperature for nucleic acid extraction, 100 μL of 1×10⁻⁶ lysate was lysed at different lysis temperatures (25℃, 30℃, 35℃, 40℃, 45℃, 50℃, 55℃, and 60℃). 5 CFU·mL -1 The Aeromonas hydrophila suspension was incubated for 5 minutes to obtain the DNA template solution for the subsequent MIRA reaction. For example... Figure 8 As shown in Figure A, increasing the temperature from 25°C to 40°C improves the performance of MIRA assays. However, temperatures above 40°C may induce DNA degradation and trigger denaturation and precipitation of proteins, polysaccharides, and other impurities in a high-salt environment, ultimately inhibiting subsequent MIRA amplification. Furthermore, given MIRA's high sensitivity to inhibitory substances, the accumulation of these impurities significantly reduces amplification efficiency. Therefore, 40°C was chosen as the optimal lysis temperature for the nucleic acid extraction reaction.

[0086] To optimize the lysis time, 100 μL of 1×10⁻⁶ solution was added before MIRA amplification. 5 CFU·mL -1 Aeromonas hydrophila suspension was incubated in lysis buffer at 40°C for different times (3 min, 5 min, 7 min, 9 min, and 11 min). Increasing the lysis time from 3 min to 5 min significantly improved the MIRA detection performance. Figure 8 (B in the original text). However, further extending the lysis time beyond 5 minutes may lead to DNA breakage, especially under high-salt conditions, or increase the release of cell debris and inhibitory substances, thereby cumulatively impairing MIRA performance and reducing detection sensitivity. Therefore, the optimal incubation time for the nucleic acid extraction reaction is 5 minutes.

[0087] Finally, at the optimal lysis temperature and time, 1×10⁻⁶ ions were extracted from different volumes (50 μL, 100 μL, 150 μL, and 200 μL). 5 CFU·mL -1Nucleic acid was extracted from Aeromonas hydrophila suspension and then subjected to MIRA detection. The amplification efficiency of MIRA gradually increased with increasing suspension volume. Figure 8 (C in the text). Considering detection costs and chip size, 150 μL was ultimately chosen as the optimal volume for the bacterial suspension.

[0088] 3. To further improve the efficiency of multi-enzyme isothermal rapid amplification and ensure high sensitivity and reliability of molecular detection results, relevant parameters were optimized.

[0089] (1) Temperature is a key factor affecting enzyme activity, which in turn affects reaction kinetics and overall efficiency. Following step 1.2, a multi-enzyme isothermal rapid amplification reaction was conducted using the F1-R1-Probe combination to investigate the amplification effect at different temperatures (27℃, 39℃, 41℃, 43℃, and 45℃). The results showed that the reaction reached the fluorescence threshold in 9.88 min at 39℃, and the amplification efficiency was highest at this temperature. Therefore, 39℃ is the optimal reaction temperature. Figure 6 (A)

[0090] (2) To ensure effective binding of primers to the target DNA and improve amplification efficiency and reaction specificity, the primer dosage was optimized. Following step 1.2, a multi-enzyme isothermal rapid amplification reaction was performed using the F1-R1-Probe combination to investigate the amplification effect at different primer dosages (1.0 μL, 1.5 μL, 2.0 μL, 2.5 μL, and 3.0 μL). The results showed that with increasing primer dosage, the multi-enzyme isothermal rapid amplification reaction reached the fluorescence threshold in the shortest time (9.81 min) with 2 μL of primer. However, when the primer dosage increased from 2 μL to 3 μL, the time threshold did not decrease significantly. Therefore, 2 μL was selected as the optimal primer dosage. Figure 6 (B)

[0091] (3) To optimize fluorescence signal intensity and reaction efficiency, the amplification effect under different fluorescent probe amounts (0.4 μL, 0.6 μL, 0.8 μL, and 1.0 μL) was investigated under optimal reaction temperature and primer dosage conditions. The results showed that when the fluorescent probe amount increased from 0.4 μL to 0.8 μL, the time threshold gradually decreased, reaching 9.63 min. However, with the continuous increase of probe amount, the time threshold became increasingly larger. Furthermore, with the increase of fluorescent probe amount, the background fluorescence signal gradually increased. Therefore, considering all factors, 0.6 μL was selected as the optimal probe amount (…). Figure 6 (C) and (D)).

[0092] (4) The amplification effect was investigated at different DNA template amounts (3 μL, 5 μL, 7 μL, 9 μL, and 10 μL) under optimal reaction temperature, primer dosage, and fluorescent probe dosage. The results showed that when the DNA template amount increased from 3 μL to 5 μL, the time threshold decreased significantly, reaching 9.73 min. However, further increasing the DNA template amount to 7 μL led to an increase in the time threshold. Therefore, 5 μL was selected as the optimal DNA template amount.

[0093] 4. After establishing and optimizing the above detection method, conduct on-chip detection experiments under optimal conditions, i.e., the detection process of the detection device: The detection device is started via touchscreen control. First, the main control circuit controls the micro-air pump at the first air inlet to pump the sample to be tested and the rapid nucleic acid release agent (180 μL in total) into the nucleic acid extraction chamber. Then, the micro-vibration motor is started to vibrate and mix the solution in the nucleic acid extraction chamber for 10 seconds. Subsequently, the ceramic heating plate at the first heating device is controlled to heat at 40℃ for 5 minutes. After completion, the DNA template solution is driven to the pipetting area by the micro-air pump at the first air inlet. Under gravity, after 175 μL of the DNA template solution is retained in the solution retention structure, 5 μL of the DNA template solution is pumped into the detection chamber. Then, the micro-air pump at the first air inlet is stopped, and the micro-air pump at the second air inlet is turned on to pump the multi-enzyme isothermal rapid amplification reaction system solution reserved in the reagent storage chamber into the detection chamber. Subsequently, the vibration motor was restarted to efficiently mix the solution in the detection chamber for 10 seconds. Simultaneously, the ceramic heating element at the second heating device was rapidly heated to 39°C. Furthermore, fluorescence signal detection was initiated upon reaching 39°C, with detection performed every 30 seconds. The time taken to reach the fluorescence threshold was calculated to analyze the sample concentration. Finally, the main control circuit displayed the detection results and amplification curve on the screen, as shown below. Figure 7 As shown. According to Figure 7 As can be seen, according to the method of this invention, a concentration in the aquaculture water range of 1×10⁻⁶ can be detected within 40 minutes. 2 ~1×10 7 The detection limit for Aeromonas hydrophila at CFU / mL is 100 CFU / mL.

[0094] Specific examples have been used to illustrate the principles and implementation methods of this invention. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this invention. Furthermore, those skilled in the art will recognize that, based on the ideas of this invention, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of this invention.

Claims

1. A chip for detecting pathogenic bacteria in aquaculture water, characterized in that: include: The nucleic acid extraction chamber is capable of extracting nucleic acid solutions from pathogenic bacteria in aquaculture water. A reagent storage chamber, one end of which is connected to a detection chamber, is capable of storing reaction solutions and delivering reaction solutions to the detection chamber. A pipetting module, located between the detection chamber and the nucleic acid extraction chamber, is capable of delivering a set amount of pathogenic bacterial nucleic acid solution into the detection chamber. The pipetting module includes multiple solution retention structures connected in series, with the multiple solution retention structures connected end to end in sequence. One end of the nucleic acid extraction chamber is connected to the first solution retention structure located on one side, and the detection chamber is connected to the last solution retention structure located on the other side. The solution retention structure has a 5mm high right-angled triangular receiving groove, and the side wall of the receiving groove is provided with a hydrophilic layer to form a pipetting area. A portion of the solution can be retained in the receiving groove to replace the operation of a pipette.

2. The pathogen detection chip in aquaculture water according to claim 1, characterized in that: It also includes the chip body, on which the nucleic acid extraction chamber, reagent storage chamber, and pipetting module are all located.

3. The pathogen detection chip in aquaculture water according to claim 2, characterized in that: The chip body has a first air inlet and a second air inlet. One end of the first air inlet is used to connect to a first driving device, which can drive the DNA template solution in the nucleic acid extraction chamber to flow through the pipetting module and quantitatively transfer it to the detection chamber. One end of the second air inlet is used to connect to a second driving device, which can transfer the MIRA reaction reagent in the reagent storage chamber to the detection chamber to react with the DNA template solution.

4. The pathogen detection chip in aquaculture water according to claim 2, characterized in that: The chip body has a first heating device hole and a second heating device hole. The first heating device hole is located near the nucleic acid extraction chamber and is used to install a first heating device to heat the solution in the nucleic acid extraction chamber to a set temperature. The second heating device hole is located near the detection chamber and is used to install a second heating device to heat the mixed solution in the detection chamber to a set temperature. A vibration device hole is provided between the detection chamber and the nucleic acid extraction chamber. The vibration device hole is used to install a vibration drive device to drive the detection chamber and / or the nucleic acid extraction chamber to vibrate for a set time.

5. The pathogen detection chip in aquaculture water according to claim 2, characterized in that: The chip body includes a first mounting plate and a second mounting plate arranged symmetrically. The first mounting plate has a first extraction groove, a first detection groove and a first storage groove, and the second mounting plate has a second extraction groove, a second detection groove and a second storage groove. After the first mounting plate and the second mounting plate are fixedly connected, the sealed cavity between the first extraction groove and the second extraction groove forms the nucleic acid extraction chamber, the sealed cavity between the first detection groove and the second detection groove forms the detection chamber, and the sealed cavity between the first storage groove and the second storage groove forms the reagent storage chamber.

6. A device for detecting pathogenic bacteria in aquaculture water, characterized in that: The device includes a housing, a top cover on the top of the housing, and a display screen mounted on the top cover; a horizontally arranged partition is provided inside the housing, and a power supply and control system is provided below the partition, the power supply and control system being electrically connected to the display screen; a vertically arranged placement seat is provided on the partition, and a placement groove is provided on one side of the placement seat, the placement groove being used to fix the pathogen detection chip in aquaculture water as described in any one of claims 1 to 5; The partition is provided with a fluorescence detection module, a vibration driving device, a first driving device, a second driving device, a first heating device, and a second heating device; the fluorescence detection module is located on the side close to the detection chamber; the fluorescence detection module includes a diode placement slot, in which a photodiode is fixedly installed, and the emitting end of the photodiode is provided with a filter, which is located between the detection chamber and the photodiode.

7. A primer-probe combination for detecting Aeromonas hydrophila, used in conjunction with the chip according to any one of claims 1 to 5 or the device according to claim 6, characterized in that: The primer-probe combination includes a forward primer F1, a reverse primer R1, and a fluorescent probe; The forward primer F1 has the nucleotide sequence shown in SEQ ID NO:1; The reverse primer R1 is the nucleotide sequence shown in SEQ ID NO:2; The fluorescent probe is the nucleotide sequence shown in SEQ ID NO:7; wherein, the 3' end of the nucleotide sequence shown in SEQ ID NO:7 is modified with C3Spacer, Y represents tetrahydrofuran, the base at 24 bp in the 5'-3' direction carries a fluorescein group, and the base at 27 bp in the 5'-3' direction carries black hole quencher-1.

8. A kit for detecting Aeromonas hydrophila, characterized in that: The kit includes a rapid DNA nucleic acid release agent, a multi-enzyme isothermal rapid amplification reagent, and the primer-probe combination as described in claim 7.

9. A method for detecting Aeromonas hydrophila in aquaculture water for non-diagnostic purposes using the chip according to any one of claims 1 to 5 or the device according to claim 6, characterized in that, Includes the following steps: The aquaculture water sample to be tested was mixed with a rapid DNA nucleic acid release agent, and genomic DNA was extracted from the aquaculture water sample. Using the genomic DNA as a template, multi-enzyme isothermal rapid amplification is performed using the primer and probe set described in claim 8. The fluorescence signal intensity of the amplification product is detected to determine the content of Aeromonas hydrophila in the aquaculture water to be tested.

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