Method for rapidly detecting pathogen nucleic acid of three imported bee diseases through microchip integration

Abstract

The invention discloses a method for rapidly detecting nucleic acid of three imported bee disease pathogens through microchip integration, and relates to the technical field of pathogen detection. The method comprises the following steps: S1, acquiring DNA of a to-be-detected bee sample; s2, respectively preparing nucleic acid amplification reaction systems of EFB, AFB and CDB pathogens, wherein each system contains an exclusive primer probe and the same basic component; s3, loading the three detection systems, the negative control and the positive control to different reaction units of the same microchip; s4, executing fluorescent quantitative PCR amplification; and S5, judging the existence of pathogens according to the detection signal. According to the invention, the limitation of cross-type pathogen detection of bacteria and fungi is broken through, single synchronous detection is realized, the time is shortened, the cost of reagent consumables is reduced by more than 80% by a 1.2 mu L small system, and the kit is strong in specificity, high in sensitivity and good in repeatability, is adaptive to a conventional PCR instrument, and assists entry quarantine and bee disease prevention and control.

Description

Technical Field

[0001] This invention relates to the field of pathogen detection technology, and in particular to a method for rapid detection of nucleic acids of three imported bee disease pathogens using a microchip integrated system. Background Technology

[0002] Beekeeping is a vital support for the pollination ecology of animal husbandry and agriculture in my country. Three bee diseases—European foulbrood (EFB), American foulbrood (AFB), and chalkbrood (CDB)—are core threats to bee colony survival. All three are highly pathogenic and contagious, leading to larval mortality, decreased colony productivity, and similar morphological appearances that make differentiation difficult. Diagnosis also suffers from significant delays; by the time symptoms are detected, the hive is often already extensively infected, severely impacting beekeepers' economic benefits.

[0003] To address diagnostic challenges, the industry has developed detection technologies such as PCR, real-time fluorescence PCR, ELISA, and high-throughput sequencing. Among these, real-time fluorescence PCR has become the mainstream technology due to its high sensitivity, strong specificity, and real-time monitoring capability. However, existing technologies have key drawbacks: the detection of the three bee diseases requires three independent operations, each involving the preparation of a separate system, the use of an independent reaction unit, and the execution of a separate procedure. The detection time is at least one hour, and it consumes three times the reagent and labor costs, resulting in low efficiency and high cost. This makes it difficult to meet the timeliness requirements of port quarantine and large-scale preventive testing in apiaries, and also increases the risk of operational errors and contamination.

[0004] In summary, there is an urgent need for an integrated detection technology that can simultaneously detect three bee diseases, shorten the detection time, and reduce costs, in order to fill the industry gap and support the early prevention and control of bee diseases. Summary of the Invention

[0005] In order to address the aforementioned defects and shortcomings in the existing technology, this application aims to provide a method for rapid detection of nucleic acids of three imported bee disease pathogens using a microchip integrated system.

[0006] One objective of this invention is to provide a method for rapid detection of nucleic acids of three imported bee disease pathogens using a microchip integrated system, comprising the following steps: S1. Obtain and extract DNA from the bee samples to be tested; S2. Using the DNA of the bee samples to be tested as the template, prepare nucleic acid amplification reaction systems for the European foulbrood EFB pathogen, the American foulbrood AFB pathogen, and the bee chalkbrood CDB pathogen, respectively. S3. Load the negative control system, positive control system, EFB pathogen nucleic acid amplification reaction system, AFB pathogen nucleic acid amplification reaction system and CDB pathogen nucleic acid amplification reaction system into different reaction units of the same microchip respectively. The negative control system is a buffer system that does not contain EFB pathogen nucleic acid, AFB pathogen nucleic acid and CDB pathogen nucleic acid, and the positive control is a system containing EFB pathogen nucleic acid, AFB pathogen nucleic acid and CDB pathogen standard plasmid. S4. Perform real-time PCR amplification on the loaded microchip and collect the detection signals of each reaction unit. S5. Based on the detection signals of each reaction unit, determine whether the bee sample to be tested contains EFB pathogen nucleic acid, AFB pathogen nucleic acid, and CDB pathogen nucleic acid.

[0007] Preferably, the nucleic acid amplification reaction system for the EFB pathogen includes an upstream specific primer for the EFB pathogen, a downstream specific primer for the EFB pathogen, a fluorescent probe for the EFB pathogen, and a first basic component. The nucleic acid amplification reaction system for the AFB pathogen includes upstream specific primers for the AFB pathogen, downstream specific primers for the AFB pathogen, a fluorescent probe for the AFB pathogen, and a second basic component. The nucleic acid amplification reaction system for the CDB pathogen includes upstream specific primers for the CDB pathogen, downstream specific primers for the CDB pathogen, a fluorescent probe for the CDB pathogen, and a third basic component. The first, second, and third basic components have identical composition and content; each of the first, second, and third basic components contains polymerase chain reaction (PCR) buffer, deoxynucleoside triphosphate (dNTP), HS Taq enzyme, and DNA from the bee sample to be tested.

[0008] Preferably, the upstream specific primer sequence of the EFB pathogen is 5′-GACCTGTTTAGCTATTATCACTA-3′; the downstream specific primer sequence of the EFB pathogen is 5′-CACCTACAATGAATGATTCATTC-3′; and the fluorescent probe sequence of the EFB pathogen is 5′-FAM-TCCGCCTAAGCTACCACCTAAGAAC-BHQ1-3′.

[0009] Preferably, the upstream specific primer sequence of the AFB pathogen is 5′-TACGCTTTTCGATTCTCTG-3′; the downstream specific primer sequence of the AFB pathogen is 5′-GTCTGTACTGAACCAAGTC-3′; and the fluorescent probe sequence of the AFB pathogen is 5′-VIC-ATCTGCTTCCACTTGTTCACTCACCA-BHQ1-3′.

[0010] Preferably, the upstream specific primer sequence of the CDB pathogen is 5′-ATTAATGTGTGTCTGTGCGGCTA-3′; the downstream specific primer sequence of the CDB pathogen is 5′-GCCGAAGCAACAGGTAAGGT-3′; and the fluorescent probe sequence of the CDB pathogen is 5′-FAM-CCGCGCACTCCCACCC-BHQ1-3′.

[0011] Preferably, the volumes of the nucleic acid amplification reaction systems for EFB pathogen, AFB pathogen, and CDB pathogen in the microchip are all 1.2 μL. The PCR buffer concentration was 1×PCR Buffer, the dNTP concentration was 0.2 mmol / L, and the HS Taq enzyme concentration was 0.01 U / μL.

[0012] Preferably, the concentrations of the upstream specific primer, the downstream specific primer, and the fluorescent probe of the CDB pathogen are all 0.75 μmol / L.

[0013] Preferably, the concentrations of the upstream specific primer of the EFB pathogen, the upstream specific primer of the AFB pathogen, the downstream specific primer of the EFB pathogen, and the downstream specific primer of the AFB pathogen are all 0.4 μmol / L; the concentrations of the fluorescent probe of the EFB pathogen and the fluorescent probe of the AFB pathogen are all 0.1 μmol / L.

[0014] Preferably, the real-time PCR amplification includes a pre-denaturation stage and a cycle amplification stage; The pre-denaturation stage is maintained at a constant temperature of 95°C for 3 minutes. The cyclic amplification phase comprises 45 cycles, each cycle consisting of: The process includes a denaturation step at 95℃ for 10 seconds, an annealing step at 55℃ for 10 seconds, and an extension step at 60℃ for 30 seconds. The collection of detection signals from each reaction unit is performed during the extension phase.

[0015] Preferably, after obtaining the DNA of the bee sample to be tested, the method further includes: dissolving the DNA in DEPC water to obtain a DNA solution of the bee sample to be tested; the added volume of the DNA solution of the bee sample to be tested in the nucleic acid amplification reaction system of EFB pathogen, the nucleic acid amplification reaction system of AFB pathogen, and the nucleic acid amplification reaction system of CDB pathogen is 0.24 μL.

[0016] The beneficial effects of this invention are: This invention leverages the integrated characteristics of microchip carriers, combines dedicated primer and probe designs for three types of imported bee pathogens, and constructs a simultaneous and rapid detection scheme for the nucleic acids of the three bee pathogens through synergistic optimization of the reaction system and amplification conditions. Compared with existing detection technologies, it exhibits significant advantages in many aspects.

[0017] This invention overcomes the limitations of cross-type pathogen detection of bacteria (EFB, AFB) and fungi (CDB). Through the design of dedicated primers and probes and the adjustment of differentiated concentrations, it achieves simultaneous detection of three pathogens in a single test, solving the problems of inefficiency and difficulty in differentiating symptoms in traditional separate testing. Relying on the multi-reaction unit and fast thermal conductivity of the microchip, the detection time is shortened, and the system volume is optimized to 1.2μL, reducing reagent and consumable costs and labor costs, making it feasible for large-scale promotion.

[0018] Meanwhile, this invention ensures accurate detection through specific primers and probes, as well as positive and negative controls, achieving industry-leading sensitivity (EFB 6.5×10⁻⁶). -5 ng / μL, AFB 9.8×10 -5 ng / μL, CDB 4.1×10 -5 (ng / μL), Ct value variation coefficient <1%; the process is clear and easy to operate, compatible with conventional fluorescence quantitative PCR instruments, no need to purchase additional equipment, assisting in import quarantine and bee disease control, and protecting beekeepers' income and agricultural ecological security. Attached Figure Description

[0019] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention, wherein: Figure 1 The results of serial dilutions were analyzed to establish the EFB microchip fluorescent PCR detection method. Figure 2 The standard curve for the EFB microchip fluorescence PCR detection method; Figure 3 The results of serial dilutions were analyzed to establish the AFB microchip fluorescent PCR detection method. Figure 4 The standard curve for the AFB microchip fluorescence PCR detection method; Figure 5 The results of serial dilution detection were used to establish the CDB microchip fluorescent PCR detection method; Figure 6 The standard curve for the CDB microchip fluorescence PCR detection method; Figure 7 This is a result of a specific test. Detailed Implementation

[0020] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. It should also be noted that, for ease of description, only the parts relevant to the invention are shown in the drawings. Unless otherwise specified, the embodiments and features described herein can be combined with each other. The present application will now be described in detail with reference to the accompanying drawings and embodiments.

[0021] According to a first aspect of the present invention, a method for rapid detection of nucleic acids of three imported bee pathogens using a microchip integrated system is provided, comprising the following steps: S1. Obtain DNA from the bee sample to be tested; Select suspected infected bee larvae and remove any beeswax and honey residue from their bodies; Take 1-2 larvae from the pretreated sample, add 200-500 μL of sterile physiological saline (0.9% NaCl solution), place them in a sterile grinding tube, and homogenize them for 3-5 minutes in an ice bath using a tissue homogenizer to prepare a uniform sample homogenate, ensuring that pathogen cells are fully released. Transfer the homogenate to a 1.5 mL sterile centrifuge tube, centrifuge at 5000 rpm for 5-8 min, collect the supernatant, and discard the tissue residue at the bottom to avoid the residue inhibiting enzyme activity.

[0022] Pathogen cell lysis: Take 200 μL of the supernatant of the sample after centrifugation, add 200 μL of lysis buffer, and vortex for 10-15 seconds to mix the liquid thoroughly. Incubate in a 56°C water bath for 10-15 minutes, gently inverting the centrifuge tube every 5 minutes to ensure complete lysis. Proteinase K requires this temperature to activate and thoroughly degrade protein impurities in the sample, preventing them from binding to DNA or inhibiting subsequent HS Taq enzyme activity.

[0023] Nucleic acid purification: Add 200 μL of chloroform, vortex for 30 seconds, and let stand at room temperature for 2 minutes to allow protein impurities to denature fully; Centrifuge at 12,000 rpm for 10 min at 4°C. At this point, the liquid will separate into three layers: the upper layer is an aqueous phase containing DNA, the middle layer is protein impurities, and the lower layer is an organic phase. Carefully aspirate the upper aqueous phase (about 150 μL) with a pipette and transfer it to a new sterile centrifuge tube, avoiding aspiration of the middle layer impurities. Add an equal volume of isopropanol, gently invert the centrifuge tube 5-10 times, and let it stand at room temperature for 5 minutes to allow the DNA to precipitate fully. Centrifuge at 12000 rpm for 10 min at 4℃, discard the supernatant, and white DNA precipitate will be visible at the bottom of the tube; add 500 μL of 75% ethanol, gently invert the centrifuge tube to wash the DNA precipitate, and remove residual salts and organic solvents. Centrifuge at 12,000 rpm for 5 minutes at 4°C, discard the ethanol supernatant, invert the centrifuge tube on sterile filter paper, and air dry at room temperature for 5-10 minutes to avoid ethanol residue affecting subsequent PCR. Do not dry excessively, otherwise the DNA will be difficult to dissolve.

[0024] DNA dissolution and quality verification: Add 20-50 μL of DEPC water to the centrifuge tube. DEPC water can inhibit RNase activity, avoid RNA contamination, and is free of enzymes and impurities, so it will not affect PCR. Gently blow the precipitate at the bottom of the tube to fully dissolve the DNA.

[0025] S2. Using the DNA of the bee samples to be tested as the template, prepare nucleic acid amplification reaction systems for the European foulbrood EFB pathogen, the American foulbrood AFB pathogen, and the bee chalkbrood CDB pathogen, respectively. The nucleic acid amplification reaction system for the EFB pathogen includes upstream specific primers for the EFB pathogen, downstream specific primers for the EFB pathogen, a fluorescent probe for the EFB pathogen, and a first basic component. The nucleic acid amplification reaction system for the AFB pathogen includes upstream specific primers for the AFB pathogen, downstream specific primers for the AFB pathogen, a fluorescent probe for the AFB pathogen, and a second basic component. The nucleic acid amplification reaction system for the CDB pathogen includes upstream specific primers for the CDB pathogen, downstream specific primers for the CDB pathogen, a fluorescent probe for the CDB pathogen, and a third basic component. The first, second, and third basic components have identical composition and content; each of the first, second, and third basic components contains polymerase chain reaction (PCR) buffer, deoxynucleoside triphosphate (dNTP), HS Taq enzyme, and DNA from the bee sample to be tested.

[0026] For identification of European foulbrood of honey bees (EFB), *Melissococcus plutonius* ATCC 35311 strain was selected; for identification of American foulbrood of honey bees (AFB), *Paenibacillus larvae* DSM25430 strain was selected; and for identification of Chalkbrood Disease of honey bees (CDB), conserved 5.8S rRNA was screened from relevant gene sequences of *Gnaphalium affineum*. Based on multiple sequence alignment, specific primers and probes were designed and screened according to relevant genes. The specific primer sequences and fluorescent probe sequences for the three pathogens are shown in Table 1. Table 1 Primer and probe sequences

[0027] The concentrations of the upstream specific primers for EFB pathogen, the upstream specific primers for AFB pathogen, the downstream specific primers for EFB pathogen, and the downstream specific primers for AFB pathogen were all 0.4 μmol / L; the concentrations of the fluorescent probes for EFB pathogen and AFB pathogen were both 0.1 μmol / L.

[0028] The formulations of the nucleic acid amplification reaction systems for the three pathogens are shown in Table 2: Table 2 Reaction system formulation

[0029] dNTPs were purchased from TaKaRa, and primers and probes were synthesized by Shanghai Sangon Biotech Co., Ltd. The 10×PCR Buffer was a commercially available buffer. Taq HS DNA polymerase 5 U / μL, purchased from TaKaRa; DEPC-treated water was prepared by double distillation of purified water, followed by the addition of DEPC to a final concentration of 0.1%, and treated at 37°C with stirring for 12 hours at 15 lbf / in² (1.034 × 10⁻⁶). 5 High-pressure steam sterilization for 15 minutes.

[0030] S3. Load the negative control system, positive control system, EFB pathogen nucleic acid amplification reaction system, AFB pathogen nucleic acid amplification reaction system, and CDB pathogen nucleic acid amplification reaction system into different reaction units of the same microchip; the volume of each nucleic acid amplification reaction system is 1.2 μL; The negative control system was prepared as a 5% liquid using 0.01 mol / L pH 7.2 PBS buffer, which did not contain EFB pathogen nucleic acid, AFB pathogen nucleic acid, or CDB pathogen nucleic acid; the positive control system contained EFB pathogen nucleic acid, AFB pathogen nucleic acid, and CDB pathogen standard plasmid. Negative controls can eliminate false positives caused by reagent contamination or operational contamination; positive controls can verify whether the PCR system is working properly and eliminate false negatives. Loading the control system and the detection system onto the same chip ensures that the control and detection are carried out under the same temperature control and fluorescence acquisition conditions, avoiding the impact of environmental differences on the reliability of the results.

[0031] S4. Perform real-time PCR amplification on the loaded microchip and collect the detection signals from each reaction unit: Take the microchip after the system loading is completed and observe whether there is liquid leakage or residual air bubbles in each reaction unit. If air bubbles are found, gently blow the reaction unit with a sterile pipette to remove the air bubbles and avoid the air bubbles affecting the temperature control uniformity and fluorescence signal acquisition. If leakage is present, replace the microchip and reload the system according to step S3 to ensure that the microchip is well sealed and meets the closed reaction requirements of real-time PCR. Place the qualified microchip into the appropriate chip slot of the real-time PCR instrument, close the instrument door, select the microchip detection mode through the instrument operation interface, and confirm that the microchip is accurately positioned to avoid temperature control deviation or signal acquisition error due to loading misalignment.

[0032] Configure the corresponding fluorescence detection channel on the instrument interface: EFB pathogen and CDB pathogen: The fluorescent probes are both 5′-FAM labeled, the FAM channel is selected, the excitation wavelength is about 494nm, and the emission wavelength is about 518nm; AFB pathogen: The fluorescent probe is labeled with 5′-VIC, the VIC channel is selected, the excitation wavelength is about 538nm, and the emission wavelength is about 554nm; At the same time, a reference fluorescence channel (ROX channel) is set up to correct signal errors caused by fluctuations in the instrument's light source, ensuring the stability of fluorescence signal acquisition.

[0033] Enter the amplification program parameters, strictly following the pre-denaturation and cyclic amplification two-stage program parameters shown in Table 3: Table 3 Amplification program parameters and operating requirements

[0034] Start the PCR instrument and first run the instrument warm-up program to 95°C, which takes about 2-3 minutes. After the instrument temperature stabilizes, click "Start Run". The instrument will automatically enter the pre-denaturation stage: 95°C for 3 minutes. In this stage, the DNA template to be tested is completely denatured by high temperature, breaking the double-stranded structure and activating the HS Taq enzyme in the system, laying the foundation for subsequent cyclic amplification.

[0035] After pre-denaturation, the instrument automatically enters a 45-cycle amplification process, with each cycle performing denaturation-annealing-extension in sequence: Denaturation step (95℃ / 10s): High temperature causes the DNA double strand to unwind into a single strand, ensuring that the primer can bind to the target. During this stage, the instrument temperature control rate must be ≥2℃ / s to quickly heat up to the set temperature and avoid excessive loss of HS Taq enzyme activity. Annealing step (55℃ / 10s): The temperature is reduced to near the primer Tm value (Melting Temperature). 55℃ is the suitable annealing temperature for the three pathogen primers, so that the specific primers of EFB / AFB / CDB can bind precisely to the corresponding target single-stranded DNA. During this stage, the temperature control accuracy needs to be maintained at ±0.3℃ to ensure primer binding specificity. Extension step (60℃ / 30s): The temperature is raised to the optimal activity temperature of HS Taq enzyme, 60℃. The enzyme catalyzes the synthesis of progeny DNA by dNTPs in the direction of primer extension. At the same time, the instrument collects the fluorescence signal of each reaction unit in the preset FAM / VIC channel to monitor the amplification process of the three pathogen target DNAs in real time.

[0036] Ten seconds after the start of each extension step, the instrument's fluorescence detector is activated, and three signal acquisitions are performed on each reaction unit. The average value is taken as the fluorescence signal value for that cycle to avoid random errors from a single acquisition. After the acquisition is completed, the instrument automatically enters the denaturation step of the next cycle until all 45 cycles are completed.

[0037] After the amplification process is completed, the instrument operation interface automatically generates raw data files of fluorescence signal values ​​and cycle numbers for each reaction unit, including automatically calculated Ct values. Ct values ​​represent the number of cycles at which the fluorescence signal reaches a preset threshold. At the same time, an amplification curve is generated, with fluorescence intensity on the vertical axis and cycle number on the horizontal axis, providing raw data support for the result determination of the subsequent S5 step.

[0038] S5. Based on the detection signals of each reaction unit, determine whether the bee sample to be tested contains EFB pathogen nucleic acid, AFB pathogen nucleic acid, and CDB pathogen nucleic acid.

[0039] First, confirm that the signals of the negative and positive control systems meet expectations to rule out any abnormalities in the detection system. The verification criteria are as follows: The negative control system did not exhibit a typical "S-shaped" amplification curve; the Ct value showed "Undetected" with no specific numerical value. The positive control systems EFB, AFB, and CDB all showed typical "S-shaped" amplification curves in their corresponding reaction units; the Ct values ​​of all three were ≤35 (ensuring the effectiveness of the amplification system).

[0040] If the control system does not meet the standard (e.g., the negative control shows a Ct value, or the positive control shows no amplification), the judgment must be terminated, and steps S1-S4 must be repeated to check for problems (e.g., changing reagents, reloading the microchip). Only when the control is qualified can the judgment of the sample to be tested proceed.

[0041] For the three reaction units to be tested on the microchip—EFB, AFB, and CDB—the presence of corresponding pathogen nucleic acids is determined one by one based on the signal characteristics of their fluorescence channels (FAM channels for EFB / CDB and VIC channels for AFB). The specific criteria are as follows: 1. Nucleic acid determination of EFB pathogen A positive result is determined when all three conditions are met: The FAM channel detected a typical "S-shaped" amplification curve, which was smooth and without abnormal fluctuations. Ct value ≤ 35 (compare with the Ct value of the positive control EFB unit to ensure effective amplification and exclude false positives caused by inefficient amplification).

[0042] A negative result is determined if any one of the following conditions is met: There is no typical "S-shaped" amplification curve, such as only baseline noise and no exponential growth period; Ct value > 35 (below the lower limit of detection sensitivity); The Ct value shows "Undetected".

[0043] 2. Nucleic acid determination of AFB pathogen A positive result is determined when all three conditions are met: A typical "S-shaped" amplification curve was detected in the VIC channel; Ct value ≤ 35 (compare with the Ct value of the positive control AFB unit to ensure effective amplification and prevent false positives.) A negative result is determined if any one of the following conditions is met: No typical "S-shaped" amplification curve was observed. Ct value > 35; The Ct value displays "Undetected".

[0044] 3. CDB pathogen nucleic acid identification A positive result is determined when all three conditions are met: A typical "S-shaped" amplification curve was detected in the FAM channel (although it is in the same channel as EFB, there is no signal crosstalk due to the specificity of the primers and probes). Ct value ≤ 35 (to prevent false positives). A negative result is determined if any one of the following conditions is met: No typical "S-shaped" amplification curve was observed. Ct value > 35; The Ct value displays "Undetected".

[0045] If the test reaction unit shows a Ct value of 35-40 or an atypical S-shaped amplification curve, it needs to be verified by repeating the experiment. Take the DNA solution from the same sample and re-prepare the amplification system for the corresponding pathogen according to step S2; Reload the microchip and perform PCR amplification following steps S3-S4; Secondary determination: If the Ct value is ≤40 and a typical S-shaped curve appears after repeated testing, it is determined to be positive; if the Ct value is still >40 or the curve is abnormal, it is determined to be negative, in order to avoid false positives caused by low-concentration non-specific amplification.

[0046] This invention uses a microchip as the PCR reaction carrier. Its high thermal conductivity material allows for rapid response to temperature control commands, significantly shortening the heating and cooling time during the quantitative PCR amplification process. Simultaneously, a customized amplification program adapted for three pathogens is employed: a 3-minute pre-denaturation at 95°C to activate enzyme activity and thoroughly denature the DNA; and subsequent 45 cycles of 10-second denaturation at 95°C, 10-second annealing at 55°C, and 30-second extension at 60°C. Fluorescence signals from each reaction unit are collected simultaneously during the extension phase, enabling simultaneous amplification and signal acquisition. Compared to traditional real-time PCR, which requires over 1 hour for single pathogen detection and over 3 hours for three pathogens, this invention can complete the detection of three pathogens in a single experiment, reducing the time to less than 3 / 4 of traditional techniques. The results are output instantly, efficiently meeting the time-sensitive needs of scenarios such as rapid screening of batches of samples at ports of entry and preventative testing during the peak spring disease season in apiaries.

[0047] In this invention, EFB / AFB primers and probes are designed for bacterial specific genes, and CDB is designed for the conserved 5.8S rRNA gene of fungi. The primer and probe sequences, fluorescent labels and binding affinity of the three are completely different and need to be configured separately as specific components to ensure that the primers and probes of each pathogen bind only to the corresponding target and avoid cross-interference. PCR buffer, dNTPs, and HSTaq enzyme are universally essential components for PCR amplification. PCR buffer provides a stable pH environment and Mg²⁺. + (Activating enzyme activity), dNTPs provide raw materials for amplification, HS Taq enzyme catalyzes DNA synthesis. The roles of the three are independent of the pathogen type, and the amplification of the three pathogens requires the same concentration. Therefore, they are classified as common basic components, which can avoid operational redundancy and concentration errors caused by repeated configuration.

[0048] The three bee disease pathogens exhibit significant differences in their biological properties. EFB and AFB are bacteria, while CDB is a fungus. These pathogens differ fundamentally in their nucleic acid types, extraction difficulty, and PCR amplification efficiency. This need for detecting mixed bacterial and fungal pathogens presents unique challenges to simultaneous detection technologies. The industry has developed detection technologies such as PCR, real-time fluorescence PCR, ELISA, and high-throughput sequencing. Among these, real-time fluorescence PCR has become the mainstream technology due to its high sensitivity, strong specificity, and real-time monitoring capability. While microchip integrated detection technology can achieve parallel detection of multiple pathogens, existing solutions have significant limitations. Most microchip multi-pathogen detection technologies only target similar pathogens, such as all being viruses or all being bacteria. While the system formulation and amplification procedures are generally applicable, integrated detection solutions for mixed bacterial and fungal pathogens are extremely rare and difficult to adapt to the detection needs of these three bee diseases.

[0049] This invention overcomes the limitations of cross-pathogen detection. Among the three target bee disease pathogens, EFB and AFB are bacteria, while CDB is a fungus. These two pathogens differ fundamentally in nucleic acid type, genomic structure, and PCR amplification efficiency. Existing microchip-based multi-pathogen detection technologies are mostly only applicable to pathogens of the same type. This invention addresses this characteristic by designing specific primers and probes targeting the conserved 5.8S rRNA gene of CDB, and designing specific gene sequences for EFB and AFB pathogens targeting bacterial genes. Simultaneously, the primer and probe concentrations in the reaction system are differentiated: the CDB primer and probe concentration is set at 0.75 μmol / L, while the EFB and AFB primer concentrations are set at 0.4 μmol / L and probe concentrations at 0.1 μmol / L. This precisely adapts to the amplification requirements of different pathogen types, enabling simultaneous detection of all three pathogens in a single run without the need for batch operations. This completely solves the industry pain points of traditional detection methods, such as difficulty in distinguishing similar symptoms and low efficiency due to three separate tests, providing crucial technical support for early and accurate diagnosis of bee diseases.

[0050] This invention optimizes the nucleic acid amplification reaction system volume for three pathogens to 1.2 μL, which is much smaller than the 5-10 μL system commonly used in existing microchip detection, significantly reducing reagent consumption. At the same time, a single microchip can integrate multiple reaction units, eliminating the need for three times the consumables required for single pathogen detection as in traditional detection methods, reducing reagent and consumable costs by more than 80%. Labor costs are also significantly reduced because a single operation replaces three independent operations, significantly reducing the testing burden on grassroots apiaries and quarantine agencies, and possessing economic feasibility for large-scale promotion nationwide.

[0051] This invention ensures detection accuracy and stability through multiple design features. First, the proprietary primer and probe sequences exhibit high specificity, and experimental verification has shown no cross-reactivity with other bee pathogens such as yeast, honeybee spore-forming organisms, and Varroa mites, precisely identifying only EFB, AFB, and CDB target pathogens. Second, negative and positive controls are included to effectively eliminate false positive or false negative results caused by reagent contamination and operational errors. Third, the detection sensitivity reaches industry-leading levels, with an EFB detection sensitivity of approximately 6.5 × 10⁻⁶. -5 ng / μL, AFB approximately 9.8 × 10⁻⁶ -5 ng / μL, CDB approximately 4.1 × 10⁻⁶ -5 The concentration of ng / μL can detect early low-concentration pathogens; fourth, it has excellent repeatability, with the coefficient of variation of Ct values ​​for positive samples at different dilutions all less than 1%, and the results are stable and reliable, fully meeting the accuracy requirements for quarantine of imported bee products and long-term monitoring of diseases in bee farms.

[0052] The detection process of this invention is clear, requiring only sample DNA extraction, reaction system preparation, microchip loading, PCR amplification, and result determination. No complex professional operations are needed, and staff can master it after simple training. At the same time, the microchip is compatible with conventional real-time PCR instruments. As long as the instrument is compatible with the microchip's temperature control and multi-channel fluorescence detection functions, existing quarantine agencies and laboratories do not need to purchase additional specialized large-scale equipment. They can carry out detection based on existing instruments, which greatly reduces the equipment investment threshold for technology promotion. This is conducive to rapid implementation and application nationwide and has important practical value for improving my country's quarantine capabilities for imported bees, preventing the cross-regional spread of bee diseases, and ensuring the economic benefits of beekeepers and the ecological security of agricultural pollination.

[0053] Example 1 Standard curve establishment and method amplification efficiency evaluation 1. Preparation of positive control 1.1 Preparation of nucleic acid for EFB and AFB pathogens: Whole genome nucleic acid was extracted from EFB and AFB pathogens. The initial concentration of the extracted nucleic acid was used as the highest detection concentration. The specific concentration was calculated by measuring its A260 absorbance value and used as the stock solution for EFB and AFB positive controls.

[0054] 1.2 Preparation of CDB standard plasmid: A CDB standard plasmid with a concentration of 41 ng / μL was dissolved in a suitable solvent (enzyme-free water) to a final volume of 100 μL to obtain a CDB positive control stock solution. Subsequently, the above EFB and AFB pathogen nucleic acid stock solutions and the CDB standard plasmid stock solution were serially diluted 10-fold (i.e., 10... -1 10 -2 10 -3 10 -4 10 -5 10 -6(Dilution), prepare a series of positive control samples at different concentrations.

[0055] 2. Microchip fluorescent PCR detection and standard curve establishment According to the microchip fluorescent PCR reaction system formula in Table 2, reaction systems were prepared for the above-mentioned EFB, AFB, and CDB positive control samples at various dilutions, and loaded into the corresponding reaction units of the microchip. The fluorescent quantitative PCR amplification program set in Table 3 was executed (pre-denaturation at 95℃ for 3 min, 45 cycles of denaturation at 95℃ / 10 s, annealing at 55℃ / 10 s, extension at 60℃ / 30 s, with fluorescence signals collected during the extension phase).

[0056] Standard curves for EFB, AFB, and CDB were established with the logarithm of the concentration of each positive control sample as the x-axis and the corresponding Ct value as the y-axis. At the same time, the amplification efficiency E=[10^(-1 / k)-1]×100% (K is the slope) and the correlation coefficient R² were calculated.

[0057] 3. Results Analysis 3.1EFB: In 10 -1 -10 -6 Within the dilution range, the logarithm of concentration showed a good linear correlation with the Ct value; the standard curve slope was -3.457, the intercept was 20.483, the amplification efficiency reached 94.5%, and the correlation coefficient R² was 0.9996 (close to 1, indicating excellent linear correlation). The corresponding amplification curves are shown below. Figure 1 Standard curves are shown below. Figure 2 .

[0058] 3.2AFB: In 10 -1 -10 -6 Within the dilution range, the logarithm of concentration was linearly correlated with the Ct value; the standard curve slope was -3.258, the intercept was 24.335, the amplification efficiency reached 103%, and the correlation coefficient R² was 0.9871 (good linear correlation). The corresponding amplification curves are shown below. Figure 3 Standard curves are shown below. Figure 4 .

[0059] 3.3CDB: in 10 -1 -10 -6 Within the dilution range, the logarithm of concentration was linearly correlated with the Ct value; the standard curve slope was -3.2831, the intercept was 20.565, the amplification efficiency reached 102%, and the correlation coefficient R² was 0.9904 (good linear correlation). The corresponding amplification curves are shown below. Figure 5 Standard curves are shown below. Figure 6 .

[0060] An amplification efficiency of 90%-110% is ideal. The amplification efficiencies of EFB, AFB, and CDB mentioned above are all within this range, indicating that the microchip fluorescent PCR detection method of the present invention has stable amplification performance and can be used for subsequent pathogen nucleic acid detection.

[0061] Example 2 Specificity test 1. Preparation of experimental materials The pathogen nucleic acid samples used in this experiment were all from our laboratory. The specific sample information is shown in Table 4, covering both the target pathogen and non-target bee disease-related pathogens, in order to verify the method's ability to specifically identify the target pathogen.

[0062] Table 4. Pathogen nucleic acids used in specific experimental studies.

[0063] 2. Test Methods The three microchip-integrated fluorescent PCR detection methods for imported bee diseases of this invention were used for detection, and the specific operation is as follows: Extract DNA from the above 9 pathogen nucleic acid samples according to step S1. The CDB pathogen plasmid was directly dissolved in DEPC water to the appropriate concentration without additional extraction. Following step S2, prepare corresponding nucleic acid amplification reaction systems for the three target pathogens: EFB, AFB, and CDB. Following step S3, the detection system, negative control system, and positive control system corresponding to each pathogen nucleic acid are loaded into different reaction units of the same microchip. Following step S4, place the microchip into the real-time PCR instrument and execute the preset amplification program: pre-denaturation at 95℃ for 3 min, followed by 45 cycles of denaturation at 95℃ / 10 s, annealing at 55℃ / 10 s, and extension at 60℃ / 30 s. During the extension phase, collect the fluorescence signals from the FAM / VIC channels.

[0064] 3. Experimental Results and Analysis Specific test results such as Figure 7 As shown, only the reaction units corresponding to the three target pathogens EFB, AFB, and CDB showed typical "S-shaped" amplification curves, and the Ct value was ≤35, indicating that the method can effectively identify the target pathogens. The reaction units corresponding to the nucleic acids of six non-target pathogens, including yeast, honeybee spore disease, honeycomb beetle, heat mite, Varroa mite, and Varroa mite, did not have typical amplification curves, and the Ct values ​​showed "Undetected", which was consistent with the results of the negative control.

[0065] The above results demonstrate that the three microchip fluorescent PCR detection methods for bee diseases established in this invention specifically amplify only the target pathogens EFB, AFB, and CDB, and do not cross-react with other non-target pathogens related to bee diseases. The methods are highly specific and can meet the needs of accurate detection.

[0066] Example 3 Repeatable experiments 1. Preparation of experimental materials Positive control samples of the detection targets (EFB, AFB, CDB) of this invention were selected and three gradient dilutions were set: 10-1 -3 Dilution, 10 -4 Dilution, 10 -5 Dilution is used to verify the consistency of test results at different concentrations.

[0067] 2. Test Methods The method for rapid detection of nucleic acids of three imported bee disease pathogens using the microchip integrated of this invention was used to conduct repeatability tests. The specific operation is as follows: For each of the above-mentioned dilutions of EFB, AFB, and CDB positive samples, a nucleic acid amplification reaction system was prepared, with a single system volume of 1.2 μL and a test DNA volume of 0.24 μL. The primer and probe concentrations were set as follows: EFB / AFB primer 0.4 μmol / L, probe 0.1 μmol / L, and CDB primer and probe 0.75 μmol / L. For each pathogen sample at each dilution, three parallel replicate tests were performed, meaning that the same sample was prepared into three identical reaction systems and loaded into different reaction units of the same batch of microchips. Pre-denaturation at 95℃ for 3 min, followed by 45 cycles of denaturation at 95℃ / 10s, annealing at 55℃ / 10s, and extension at 60℃ / 30s. Fluorescence signals were collected during the extension phase for quantitative real-time PCR amplification, and the Ct values ​​for each repeated test were recorded.

[0068] 3. Experimental Results and Analysis The repeatability test results are shown in Table 5. At the same dilution, the Ct values ​​of each pathogen in three repeated tests showed very little difference and no significant fluctuation; the coefficient of variation (CV) for each dilution was <1%. The above results indicate that the microchip fluorescent PCR detection method established in this invention has high consistency and small fluctuation range in multiple repeated tests of positive samples at different concentrations. The method has good repeatability and stability, which can ensure the reliability of batch sample detection results.

[0069] Table 5 Repeatability Test Results

Claims

1. A method for rapid detection of nucleic acids of three imported bee pathogens using a microchip integrated system, characterized in that, Includes the following steps: S1. Obtain DNA from the bee sample to be tested; S2. Using the DNA of the bee samples to be tested as the template, prepare nucleic acid amplification reaction systems for the European foulbrood EFB pathogen, the American foulbrood AFB pathogen, and the bee chalkbrood CDB pathogen, respectively. S3. Load the negative control system, positive control system, EFB pathogen nucleic acid amplification reaction system, AFB pathogen nucleic acid amplification reaction system and CDB pathogen nucleic acid amplification reaction system into different reaction units of the same microchip respectively. The negative control system is a buffer system that does not contain EFB pathogen nucleic acid, AFB pathogen nucleic acid and CDB pathogen nucleic acid, and the positive control is a system containing EFB pathogen nucleic acid, AFB pathogen nucleic acid and CDB pathogen standard plasmid. S4. Perform real-time PCR amplification on the loaded microchip and collect the detection signals of each reaction unit. S5. Based on the detection signals of each reaction unit, determine whether the bee sample to be tested contains EFB pathogen nucleic acid, AFB pathogen nucleic acid, and CDB pathogen nucleic acid.

2. The method for rapid detection of three imported bee disease pathogens using a microchip integrated as described in claim 1, characterized in that, The nucleic acid amplification reaction system for the EFB pathogen includes upstream specific primers for the EFB pathogen, downstream specific primers for the EFB pathogen, a fluorescent probe for the EFB pathogen, and a first basic component. The nucleic acid amplification reaction system for the AFB pathogen includes upstream specific primers for the AFB pathogen, downstream specific primers for the AFB pathogen, a fluorescent probe for the AFB pathogen, and a second basic component. The nucleic acid amplification reaction system for the CDB pathogen includes upstream specific primers for the CDB pathogen, downstream specific primers for the CDB pathogen, a fluorescent probe for the CDB pathogen, and a third basic component. The first, second, and third basic components have identical composition and content; each of the first, second, and third basic components contains polymerase chain reaction (PCR) buffer, deoxynucleoside triphosphate (dNTP), HS Taq enzyme, and DNA from the bee sample to be tested.

3. The method for rapid detection of three imported bee pathogens using a microchip integrated as described in claim 2, characterized in that, The upstream specific primer sequence for the EFB pathogen is 5′-GACCTGTTTAGCTATTATCACTA-3′; the downstream specific primer sequence for the EFB pathogen is 5′-CACCTACAATGAATGATTCATTC-3′; and the fluorescent probe sequence for the EFB pathogen is 5′-FAM-TCCGCCTAAGCTACCACCTAAGAAC-BHQ1-3′.

4. The method for rapid detection of three imported bee disease pathogens using a microchip integrated as described in claim 2, characterized in that, The upstream specific primer sequence for the AFB pathogen is 5′-TACGCTTTTCGATTCTCTG-3′; the downstream specific primer sequence for the AFB pathogen is 5′-GTCTGTACTGAACCAAGTC-3′; and the fluorescent probe sequence for the AFB pathogen is 5′-VIC-ATCTGCTTCCACTTGTTCACTCACCA-BHQ1-3′.

5. The method for rapid detection of three imported bee pathogens using a microchip integrated as described in claim 2, characterized in that, The upstream specific primer sequence for the CDB pathogen is 5′-ATTAATGTGTGTCTGTGCGGCTA-3′; the downstream specific primer sequence for the CDB pathogen is 5′-GCCGAAGCAACAGGTAAGGT-3′; and the fluorescent probe sequence for the CDB pathogen is 5′-FAM-CCGCGCACTCCCACCC-BHQ1-3′.

6. The method for rapid detection of three imported bee disease pathogens using a microchip integrated as described in claim 2, characterized in that, The volumes of the nucleic acid amplification reaction systems for EFB pathogen, AFB pathogen, and CDB pathogen in the microchip are all 1.2 μL. The PCR buffer concentration was 1×PCR Buffer, the dNTP concentration was 0.2 mmol / L, and the HS Taq enzyme concentration was 0.01 U / μL.

7. The method for rapid detection of three imported bee disease pathogens using a microchip integrated as described in claim 2, characterized in that, The concentrations of the upstream specific primer, the downstream specific primer, and the fluorescent probe of the CDB pathogen were all 0.75 μmol / L.

8. The method for rapid detection of three imported bee disease pathogens using a microchip integrated as described in claim 2, characterized in that, The concentrations of the upstream specific primers for the EFB pathogen, the downstream specific primers for the EFB pathogen, and the downstream specific primers for the AFB pathogen are all 0.4 μmol / L; the concentrations of the fluorescent probes for the EFB pathogen and the fluorescent probes for the AFB pathogen are all 0.1 μmol / L.

9. The method for rapid detection of three imported bee disease pathogens using a microchip integrated as described in claim 2, characterized in that, The quantitative real-time PCR amplification includes a pre-denaturation stage and a cycle amplification stage; The pre-denaturation stage is maintained at a constant temperature of 95°C for 3 minutes. The cyclic amplification phase comprises 45 cycles, each cycle consisting of: The process includes a denaturation step at 95℃ for 10 seconds, an annealing step at 55℃ for 10 seconds, and an extension step at 60℃ for 30 seconds. The collection of detection signals from each reaction unit is performed during the extension phase.

10. The method for rapid detection of three imported bee disease pathogens using a microchip integrated as described in claim 1, characterized in that, After obtaining the DNA of the bee sample to be tested, the method further includes: dissolving the DNA in DEPC water to obtain a DNA solution of the bee sample to be tested; the added volume of the DNA solution of the bee sample to be tested in the nucleic acid amplification reaction system of EFB pathogen, the nucleic acid amplification reaction system of AFB pathogen, and the nucleic acid amplification reaction system of CDB pathogen is 0.24 μL.

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