Dual fluorescent PCR detection of freeze-dried microspheres, and preparation method and application thereof

Abstract

The application relates to the field of microbial detection, in particular to a double fluorescent PCR detection freeze-dried microsphere and a preparation method and application thereof. The double fluorescent PCR detection freeze-dried microsphere is prepared by vacuum freeze-drying a mixed solution containing the following components: a hot-start Taq DNA polymerase, a uracil N-glycosylase (UNG), dNTPs, specific primer pairs and fluorescent probes for detecting mycoplasma gallisepticum (MG), specific primer pairs and fluorescent probes for detecting mycoplasma synoviae (MS), a buffer, potassium chloride, magnesium chloride, a freeze-drying protective agent and an anti-inhibitor. The hot-start Taq DNA polymerase, the UNG enzyme, the dUTP-containing dNTPs, the MG / MS specific primer probes, the buffer salt, the freeze-drying protective agent and the anti-inhibitor are all prepositioned in the same freeze-dried microsphere to realize synergistic action, so that the freeze-dried microsphere can meet the requirements of anti-pollution, anti-inhibition and double-target balanced amplification under extremely simple operation.

Description

Technical Field

[0001] This application relates to the field of microbial detection, and in particular to a dual fluorescence PCR detection method for lyophilized microspheres, its preparation method, and its application. Background Technology

[0002] Mycoplasma avianis (MG) and Mycoplasma synoviae (MS) are two major pathogens in poultry farming. Dual fluorescence PCR technology has become the mainstream method in the industry because it can identify and detect these two pathogens simultaneously.

[0003] Existing detection technologies suffer from a complex and insurmountable drawback in practical applications: on the one hand, the risk of false positives due to aerosol contamination of PCR products remains constant, and conventional UNG enzyme-dUTP anti-contamination systems are difficult to integrate effectively into lyophilized formulations due to the damage to enzyme activity caused by the lyophilization process; on the other hand, characteristic samples for avian mycoplasma detection (such as joint effusion, cyst fluid, etc.) contain high concentrations of amplification inhibitors such as proteins, mucopolysaccharides, and nucleases, and existing products require complex nucleic acid purification before detection, while introducing anti-inhibitor components into the lyophilization system will further exacerbate the compatibility issues between components and the imbalance of target amplification.

[0004] The aforementioned problems are intertwined, making it impossible to guarantee simultaneous achievement of pollution prevention, inhibition resistance, and balanced amplification of dual targets. Summary of the Invention

[0005] To achieve a balance between pollution prevention, inhibition resistance, and dual-target amplification, this application provides a method for preparing and applying lyophilized microspheres for dual-fluorescence PCR detection.

[0006] In the first aspect, this application provides a dual fluorescence PCR detection method for lyophilized microspheres, employing the following technical solution:

[0007] A dual-fluorescence PCR detection lyophilized microsphere is prepared by vacuum freeze-drying of a mixed solution containing the following components: hot-start Taq DNA polymerase, uracil-N-glycosylation enzyme (UNG), dNTPs, specific primer pairs and fluorescent probes for detecting Mycoplasma avianis MG, specific primer pairs and fluorescent probes for detecting Mycoplasma synoviae MS, buffer solution, potassium chloride, magnesium chloride, lyophilization protectant, and inhibitor.

[0008] By employing the above technical solution, all components, including hot-start Taq DNA polymerase, UNG enzyme, dNTPs containing dUTP, MG / MS specific primers and probes, buffer salts, lyophilization protectants, and inhibitors, are pre-contained in the same lyophilized microsphere. First, the lyophilized microsphere morphology allows for long-term stable storage and transportation of reagents at room temperature, completely eliminating reliance on the cold chain. Second, the UNG enzyme and dUTP constitute a built-in anti-contamination system, selectively degrading uracil-containing aerosol products during the initial PCR stage (incubation at 50℃ for 5-10 minutes), reducing the risk of false positives from the source. Third, the inhibitor component endows the microspheres with direct tolerance to samples rich in proteins and polysaccharides, such as synovial fluid and exudate, eliminating the need for complex nucleic acid purification. Finally, the dual primer and probe design enables simultaneous detection of Mycoplasma avian sepsis (MG) and Mycoplasma synoviae (MS) in a single tube. These synergistic features allow the lyophilized microspheres to simultaneously meet the requirements of anti-contamination, anti-inhibition, and balanced amplification of dual targets with extremely simplified operation.

[0009] Furthermore, the freeze-drying protectant includes one or more of trehalose, mannitol, and polyvinylpyrrolidone, and the anti-inhibitor includes one or more of bovine serum albumin (BSA) and betaine.

[0010] By employing the above technical solution, trehalose, mannitol, and polyvinylpyrrolidone were selected as freeze-drying protectants. These three agents work synergistically to provide multiple protective effects: trehalose and mannitol stabilize the native conformation of the enzyme protein through glassy solidification, preventing ice crystal damage; polyvinylpyrrolidone increases the glass transition temperature of the freeze-drying system, resulting in a looser microsphere structure and significantly improved rehydration speed. Simultaneously, bovine serum albumin (BSA) and betaine act as anti-inhibition agents. BSA preferentially binds to impurities such as polysaccharides and humic acids in the sample, while betaine stabilizes polymerase activity and reduces interference from inhibitors on amplification. The combined use of these two agents produces a synergistic anti-inhibition effect, providing crucial assurance for the long-term stability of the microspheres and their compatibility with crude samples.

[0011] Furthermore, the concentration of each component in the mixed solution is calculated based on the working concentration after reconstitution of the lyophilized microspheres as follows:

[0012] Hot-start Taq DNA polymerase: 0.05-0.5 U / μL;

[0013] Uracil-N-glycosylation enzyme: 0.01-0.1 U / μL;

[0014] dNTPs include dATP, dCTP, dGTP and dUTP, each at a concentration of 0.2-0.5 mM;

[0015] MG upstream primer: 0.2-1.0 μM, MG downstream primer: 0.2-1.0 μM, MG fluorescent probe: 0.1-0.5 μM;

[0016] MS upstream primer: 0.2-1.0 μM, MS downstream primer: 0.2-1.0 μM, MS fluorescent probe: 0.1-0.5 μM;

[0017] Tris-HCl buffer: 10-50 mM, pH 8.0-8.5;

[0018] KCl: 30-100 mM;

[0019] MgCl2: 2-6 mM;

[0020] Trehalose: 2-10%, w / v;

[0021] Mannitol: 1-5%, w / v;

[0022] Polyvinylpyrrolidone: 0.5-3%, w / v;

[0023] Bovine serum albumin: 0.1-1.0 mg / mL;

[0024] Betaine: 0.2-1.0 M.

[0025] By adopting the above technical solution, hot-start Taq enzyme and UNG enzyme at this concentration can ensure both amplification efficiency and anti-contamination effect, while avoiding non-specific amplification or cost waste caused by excessively high concentrations; each dNTP and Mg 2+ The optimal ratio maintains the polymerase's optimal activity window; the primer and probe concentrations ensure balanced amplification of the dual targets, avoiding competition between them; the lyophilization protectant (trehalose 2-10%, mannitol 1-5%, PVP 0.5-3%) and the inhibitor (BSA 0.1-1.0 mg / mL, betaine 0.2-1.0 M) within this range ensure that the microspheres have a regular appearance, rapid reconstitution, and enzyme activity retention of over 90%, and exhibit significantly better tolerance to common inhibitors (heme, heparin, etc.) than ratios outside this range, thus achieving a balance between detection sensitivity (≤10 copies / reaction) and stability.

[0026] Furthermore, the mixed solution also contains the nonionic surfactant Pluronic F-68, which has a working concentration of g / 100 mL after reconstitution.

[0027] By employing the above technical solution, a nonionic surfactant, Pluronic F-68 (0.05-0.5% w / v), is further introduced into the mixed solution. Pluronic F-68 effectively reduces the nonspecific adsorption of enzyme proteins to the PCR tube wall after reconstitution of lyophilized microspheres, ensuring uniform dispersion of hot-start Taq enzyme and UNG enzyme in the solution, thereby improving the consistency of Ct values ​​among reaction tubes within the same batch (intra-batch CV < 3%). Simultaneously, this surfactant also protects enzyme molecules from gas-liquid interface denaturation during lyophilization, further enhancing enzyme activity retention after lyophilization. This is of great significance for achieving high precision and large-scale batch-to-batch stability of lyophilized microspheres.

[0028] Furthermore, the upstream primer, downstream primer, and fluorescent probe for detecting Mycoplasma avianis MG have the sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the upstream primer, downstream primer, and fluorescent probe for detecting Mycoplasma synoviae MS have the sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

[0029] By employing the above-described technical solution, specific primer and probe sequences (SEQ ID NO: 1-6) for detecting MG and MS were defined. These sequences were designed targeting conserved regions in the MG and MS genomes and have been experimentally verified to exhibit high species specificity and no cross-reactivity with common chicken pathogens (IBV, AIV, NDV, Escherichia coli, Salmonella, etc.). In a dual-fluorescence PCR system, the FAM-labeled MG probe and the VIC-labeled MS probe show no spectral overlap and do not interfere with each other, enabling accurate qualitative and semi-quantitative detection of both targets simultaneously. Based on the validated liquid reagents using this primer and probe set, this application successfully integrated them into lyophilized microspheres, retaining the original sensitivity and specificity, further expanding their potential for field applications.

[0030] Secondly, this application provides a method for preparing lyophilized microspheres for dual fluorescence PCR detection, employing the following technical solution:

[0031] A method for preparing lyophilized microspheres for dual fluorescence PCR detection includes the following steps:

[0032] S1. Dissolve all components except for hot-start Taq DNA polymerase, uracil-N-glycosylation enzyme, primers and probes in nuclease-free water, adjust the pH to 8.0-8.5, filter to sterilize, and obtain the base solution;

[0033] S2. Under ice bath conditions, add hot-start Taq DNA polymerase, uracil-N-glycosylation enzyme, MG primer probe and MS primer probe to the base solution, mix well, and obtain the premix solution;

[0034] S3. Drop the premixed liquid into a mold pre-cooled by liquid nitrogen to form spherical droplets, and freeze-solidify.

[0035] S4. The frozen microspheres are freeze-dried under vacuum until the residual moisture content is less than 2% to obtain freeze-dried microspheres;

[0036] S5. The freeze-dried microspheres are sealed and packaged under inert gas protection.

[0037] By employing the above technical solution, a method for preparing lyophilized microspheres suitable for large-scale production is provided. Key steps include: preparation and filtration sterilization of the basal solution (ensuring sterility and component homogeneity), addition of enzymes and primer probes under ice bath conditions (avoiding high-temperature inactivation), liquid nitrogen drop freezing (instant solidification to form regular spheres without ice crystal damage), stepwise vacuum freeze-drying (pre-freezing at -40℃ → gradually increasing temperature to -20℃ → 0℃ → 25℃ to ensure residual moisture <2% and maintain enzyme activity), and inert gas sealing packaging (preventing moisture absorption). This method exhibits good process reproducibility, minimal batch-to-batch variation, and highly consistent microsphere diameter, reconstitution time, and amplification performance across batches, making it suitable for industrial production of high-quality dual-fluorescence PCR lyophilized detection reagents.

[0038] Furthermore, the freeze-drying procedure for vacuum freeze drying in step S4 is as follows: pre-freeze at -40℃ for 1-3 hours, and then dry at -20℃ for 8-16 hours, 0℃ for 8-12 hours, and 25℃ for 3-6 hours under a vacuum of <10 Pa.

[0039] The specific procedure for vacuum freeze-drying was further optimized by adopting the following technical solution: pre-freezing at -40℃ for 1-3 hours to completely solidify the sample, followed by sequential drying at -20℃ for 8-16 hours under a vacuum of <10 Pa to remove most of the free water, drying at 0℃ for 8-12 hours to remove bound water, and drying at 25℃ for 3-6 hours to thoroughly dry until the residual moisture is <2%. This stepwise temperature increase procedure avoids microsphere collapse or enzyme activity loss caused by excessively rapid temperature rise, while ensuring uniform sublimation of water inside the microspheres, ultimately yielding high-quality microspheres that are plump, porous, and rehydrate rapidly (<30 seconds). After storage at 25℃ for 12 months, the amplification Ct value of freeze-dried microspheres prepared by this procedure increased by no more than 0.5 cycles compared to fresh reagents.

[0040] Thirdly, this application provides an application for dual fluorescence PCR detection of lyophilized microspheres, employing the following technical solution:

[0041] An application of lyophilized microspheres for dual fluorescence PCR detection of Mycoplasma synoviae in avian septicemia / avian synoviposition.

[0042] By adopting the above technical solution, the specific application of the freeze-dried microspheres was clarified for dual-fluorescent PCR detection of avian septicemia / avian synoviposition mycoplasma. Based on the advantages of the aforementioned components and preparation method, these microspheres can be directly applied to the following scenarios: rapid on-site screening in farms (no cold chain or professional laboratory required), disease eradication monitoring (large-scale parallel sample testing), and direct detection of clinical samples (throat swabs, joint effusion, cyst fluid, etc.) (no nucleic acid purification required, only simple lysis and sample loading). In use, only one microsphere and the nucleic acid solution of the sample to be tested (or lysis supernatant, 5 μL is recommended) need to be added to the PCR tube. After reconstitution, the total reaction volume is 25 μL. After reconstitution, amplification is performed, with a total time not exceeding 50 minutes. The detection sensitivity reaches 10 copies / reaction, and the concordance rate with pathogen isolation and identification is ≥99%. This application solution greatly reduces the technical threshold and cost of molecular detection, making it suitable for promotion and use by grassroots veterinary stations and port quarantine departments.

[0043] In summary, this application has the following beneficial effects:

[0044] 1. Room temperature storage and transportation, eliminating reliance on cold chain: In Example 4, after 12 months of storage at 25°C, the Ct value of the lyophilized microspheres increased by only 0.2 cycles (Table 7), while the liquid reagents became ineffective after one week under the same conditions. The combination of lyophilization protectants and the addition of Pluronic F-68 in this application resulted in an enzyme activity retention rate of up to 94.2%, achieving truly stable long-term storage and transportation at room temperature.

[0045] 2. Extremely simple operation, suitable for on-site use: When using, only the sample nucleic acid solution (or lysis supernatant) needs to be added. The reconstitution time is as fast as 14 seconds, and the total detection time is about 50 minutes, which is more than half of that of commercial liquid reagent kits. Moreover, no nucleic acid purification step is required, and the individual packaging avoids cross-contamination.

[0046] 3. Excellent anti-contamination effect and reduced false positive risk: The UNG-dUTP system is pre-placed in the freeze-dried microspheres, which can effectively degrade dU-containing aerosol contamination, ensuring the reliability of test results from the source. With the built-in UNG-dUTP anti-contamination system, the false positive rate is 0 in the simulated aerosol contamination environment, while the false positive rate of UNG without integration is as high as 85%.

[0047] 4. Strong resistance to inhibition and adaptability to complex samples: The BSA + betaine combination maintains excellent activity in the lyophilized microspheres. Under 5 μM heme inhibition, the Ct increase is only 1.2, while the Ct increase with Tween-20 substitution is 5.0. The lyophilized microspheres of this application can directly detect samples such as joint effusion and cyst fluid after rapid lysis treatment, without the need for nucleic acid purification. The detection sensitivity for clinical samples reaches 98.2%, which is highly consistent with the pathogen isolation results.

[0048] 5. Excellent detection performance: The lowest detection limit is 10 copies / reaction, with intra-assay CV ≤ 0.48% and inter-assay CV ≤ 0.60%. The dual-target amplification balance is good (ΔCt is only 0.2), and there is no cross-reaction with common chicken pathogens. Detailed Implementation

[0049] The present application will be further described in detail below with reference to the embodiments.

[0050] Example of raw material and intermediate preparation

[0051] raw material

[0052] It should be noted that: in the following examples, unless otherwise specified, the conditions shall be in accordance with conventional conditions or the manufacturer's recommended conditions; and the raw materials used in the following examples, unless otherwise specified, shall be from commercially available sources.

[0053] Hot-start Taq DNA polymerase, 5 U / μL, catalog number: R007A, purchased from Takara Bio Inc.

[0054] Uracil-N-glycosylation enzyme, UNG, 5 U / μL, catalog number: M0280S, purchased from New England Biolabs;

[0055] dATP, dCTP, dGTP, dUTP, 100 mM solution, catalog number: R0181, purchased from Thermo Fisher Scientific;

[0056] Primers and probes: purified by HPLC and stored as dry powder at -20℃;

[0057] Trehalose, D-(+)-trehalose dihydrate, purity ≥99%, product number: T9531, purchased from Sigma-Aldrich;

[0058] Mannitol, D-mannitol, purity ≥98%, catalog number: M4125, purchased from Sigma-Aldrich;

[0059] Polyvinylpyrrolidone, PVP K30, average molecular weight 40,000;

[0060] Pluronic F-68, nonionic surfactant, product number: P1300, purchased from Sigma-Aldrich;

[0061] Bovine serum albumin, BSA, molecular biology grade, catalog number: B9200, purchased from New England Biolabs;

[0062] Betaine, anhydrous betaine, purity ≥99%, product number: B0300-5VL, purchased from Sigma-Aldrich;

[0063] Tris-HCl, 1 M buffer, pH 8.0, catalog number: AM9855G, purchased from Thermo Fisher Scientific;

[0064] Nuclease-free water, catalog number: AM9937, purchased from Thermo Fisher Scientific.

[0065] The description of the sequences involved in this application is provided in Table 1:

[0066] Table 1 Sequence Information:

[0067] SEQ ID NO:1 (MG upstream primer, 20 nt in length) 5'-GCT GAA GGA GCT GAA GCT TG-3' SEQ ID NO:2 (MG downstream primer, 20 nt in length) 5'-CGC CAA TCA TCA AAA TCC AG-3' SEQ ID NO:3 (MG probe, 21 nt in length, 5' end labeled with FAM fluorescent group, 3' end labeled with BHQ1 quencher group) 5'-CAA CGC TTG CAG CTG CAA CGT-3' SEQ ID NO:4 (MS upstream primer, 21 nt in length) 5'-CAA GAG ATA CCC ATT GCG ATA-3' SEQ ID NO:5 (MS downstream primer, 20 nt in length) 5'-AAC TCC TTC TGC TGT TCC AG-3' SEQ ID NO:6 (MS probe, 21 nt in length, 5' end labeled with VIC fluorescent group, 3' end labeled with BHQ1 quencher group) 5'-TCA ACG GCT CCT GCT TCA ACT-3'

[0068] Example

[0069] Examples 1-3

[0070] The formulation of the mixed solution (working concentration after reconstitution) for a dual-fluorescence PCR detection of lyophilized microspheres is shown in Table 2:

[0071] Table 2 Formulation of Mixed Solutions in Examples 1-3

[0072] Hot-start Taq DNA polymerase (U / μL) 0.05 0.30 0.50 Uracil-N-glycosylation enzyme (U / μL) 0.10 0.05 0.01 dNTPs (dATP / dCTP / dGTP / dUTP each) (mM) 0.2 0.3 0.5 MG upstream primer (μM) 0.2 0.6 1.0 MG downstream primer (μM) 0.2 0.6 1.0 MG fluorescent probe (FAM labeled) (μM) 0.1 0.3 0.5 MS upstream primer (μM) 0.2 0.6 1.0 MS downstream primer (μM) 0.2 0.6 1.0 MS fluorescent probe (VIC labeled) (μM) 0.1 0.3 0.5 Tris-HCl buffer (mM) 10 30 50 KCl (mM) 100 60 30 [MgCl2 (mM)] 2 4 6 Trehalose (%, w / v) 10 6 2 Mannitol (%, w / v) 5 3 1 Polyvinylpyrrolidone (%, w / v) 0.5 2.0 3.0 Bovine serum albumin (mg / mL) 1.0 0.5 0.1 Betaine (M) 0.2 0.6 1.0

[0073] Preparation method:

[0074] S1. Dissolve all components except hot-start Taq DNA polymerase, uracil-N-glycosylation enzyme, primers and probes in nuclease-free water, adjust the pH to 8.3, and sterilize by 0.22 μm filtration to obtain the base solution;

[0075] S2. Under ice bath (4℃) conditions, add hot-start Taq enzyme, UNG enzyme, MG / MS primers and probes to the base solution, mix gently to obtain the premix solution;

[0076] S3. Use a precision dropper to drop the premixed liquid into a polytetrafluoroethylene mold (3.5mm aperture) pre-cooled by liquid nitrogen to form spherical droplets, and then freeze-solidify it;

[0077] S4. Vacuum freeze drying: Vacuum degree <10 Pa, dry at -20℃ for 12 hours → dry at 0℃ for 10 hours → dry at 25℃ for 4 hours to obtain freeze-dried microspheres;

[0078] S5. Sealed packaging in aluminum foil bags under nitrogen protection.

[0079] Microsphere specifications: Each microsphere contains 25 μL of reaction system components, and the total reaction volume after reconstitution is 25 μL. The diameter is approximately 3.2 mm, and the residual moisture content is 1.5%.

[0080] Example 4

[0081] In Example 4, Pluronic F-68 0.2g / 100 mL was added to the formulation of Example 2, and the remaining components and preparation methods were the same as in Example 2.

[0082] Example 5

[0083] The formula is the same as in Example 2;

[0084] Preparation method: Same as in Example 2, but in step S3, the liquid nitrogen pre-cooling mold is replaced by direct contact quick-freezing with liquid nitrogen at -196℃, and the freeze-drying program in step 4 is adjusted as follows:

[0085] Pre-freeze at -40℃ for 2 hours

[0086] Dry at -30℃ for 4 hours (vacuum degree <5 Pa).

[0087] Dry at -20℃ for 10 hours

[0088] Dry at 0℃ for 10 hours

[0089] Dry at 25℃ for 5 hours.

[0090] Example 6

[0091] The formula is the same as in Example 4, but it does not contain trehalose. The remaining components and preparation methods are the same.

[0092] Example 7

[0093] The formula is the same as in Example 4, but it does not contain mannitol. The remaining components and preparation methods are the same.

[0094] Example 8

[0095] The formulation is the same as in Example 4, but it does not contain polyvinylpyrrolidone. The remaining components and preparation methods are the same.

[0096] Example 9

[0097] The formulation is the same as in Example 4, but the freeze-drying protectant contains only 10% w / v trehalose.

[0098] Example 10

[0099] The formulation is the same as in Example 4, but it does not contain BSA. The remaining components and preparation methods are the same.

[0100] Example 11

[0101] The formula is the same as in Example 4, but it does not contain betaine; the other components and preparation methods are the same.

[0102] Comparative Example

[0103] Comparative Example 1

[0104] The formulation is the same as in Example 1, but the premix does not contain UNG enzyme and dUTP (dNTPs are replaced with dTTP). After the lyophilized microspheres are prepared, the UNG enzyme is packaged separately in liquid form and added on-site when needed.

[0105] Comparative Example 2

[0106] The formulation is the same as in Example 1, but the anti-inhibitor is changed to Tween-20 0.1% w / v.

[0107] Comparative Example 3

[0108] The formulation is the same as in Example 1, but the hot-start Taq DNA polymerase is replaced with a regular Taq DNA polymerase.

[0109] Comparative Example 4

[0110] The formula is the same as in Example 1, but the freeze-drying process is changed to a one-step temperature increase: after pre-freezing at -40°C for 2 hours, the temperature is directly increased to 25°C and dried for 20 hours.

[0111] Performance testing

[0112] 1. Appearance and reconstitution time

[0113] Take 10 lyophilized microspheres from each example and comparative example, observe their appearance, add 25 μL of nuclease-free water, and record the time (seconds) required for complete dissolution. Take 10 microspheres from each group (n=10) for measurement; the "average reconstitution time" is the arithmetic mean of 10 measurements. The "standard deviation (SD)" is calculated using the formula SD= The calculations were used to evaluate the consistency of reconstitution time within the same group of microspheres. A smaller standard deviation indicates better reconstitution uniformity of the microspheres in that group. The results are shown in Table 3.

[0114] Table 3 Appearance and Reconstitution Time

[0115] Example 1 White sphere, smooth surface, no collapse 22 2.1 Example 2 White sphere, smooth surface, no collapse 18 1.8 Example 3 White sphere, smooth surface, no collapse 20 2.0 Example 4 White sphere, smooth surface, no collapse 14 1.5 Example 5 White sphere with slight fine cracks on the surface. 26 2.5 Example 6 Light yellow sphere, rough surface, collapsed edges 58 5.2 Example 7 A white sphere with cracks on the surface and slight collapse. 45 4.1 Example 8 White sphere, smooth but relatively hard surface 52 4.8 Example 9 White sphere with obvious surface depressions 65 6.0 Example 10 White sphere with smooth surface 15 1.6 Example 11 White sphere with a smooth surface 16 1.7 Comparative Example 1 White sphere with a smooth surface 19 1.9 Comparative Example 2 White sphere with a smooth surface 18 1.8 Comparative Example 3 White sphere with a smooth surface 20 2.0 Comparative Example 4 The white sphere has obvious depressions and cracks on its surface. 72 7.5

[0116] Example 4 exhibited the shortest reconstitution time, with an average reconstitution time of only 14 seconds and a small standard deviation (1.5). This indicates that the addition of Pluronic F-68 and the complete protective agents (trehalose + mannitol + PVP) not only resulted in rapid reconstitution but also produced a highly uniform pore structure in the microspheres.

[0117] Examples 6-9 were poor, with long average reconstitution times (>45 seconds) and large standard deviations (>4). This indicates that the absence or alteration of the protective agent components (e.g., absence of trehalose, mannitol, PVP, or only a single component) led to microsphere structural collapse or inhomogeneity, resulting in large interparticle differences.

[0118] Comparative Example 4 was the worst, with the longest average reconstitution time (72 seconds) and the largest standard deviation (7.5). This indicates that an inappropriate freeze-drying procedure (one-step heating) led to extremely unstable microsphere quality.

[0119] 2. Limit of detection (sensitivity)

[0120] 10-fold serial dilutions of MG and MS plasmid standards (10 6 -10 0 Using copies / reactions as templates, 1 μL of sample was added for each reaction, with a total reaction volume of 25 μL. The reagents of each example and comparative example were used for detection. Each concentration was repeated 20 times. The detection limit was determined with a 100% detection rate as the criterion. The results are shown in Table 4.

[0121] Table 4. Limit of Detection (Unit: copies / reaction)

[0122] Example 1 20 20 Example 2 15 15 Example 3 16 15 Example 4 10 10 Example 5 16 15 Example 6 45 43 Example 7 35 34 Example 8 40 38 Example 9 50 48 Example 10 30 29 Example 11 28 27 Comparative Example 1 15 14 Comparative Example 2 50 48 Comparative Example 3 55 52 Comparative Example 4 25 24

[0123] Note: The above-mentioned limit of detection is based on high-purity plasmid standards. The actual limit of detection may be higher for crude clinical samples due to the presence of inhibitors such as proteins and polysaccharides. However, this application utilizes a BSA + betaine anti-inhibition system to achieve direct detection of the lysate supernatant without the need for nucleic acid purification.

[0124] Example 4 exhibited the highest sensitivity, with a detection limit of 10 copies / reaction. Comparative Examples 2 and 3 showed significantly decreased sensitivity. Examples 6-9 suffered from reduced sensitivity due to impaired enzyme activity caused by incomplete lyophilization protectant.

[0125] 3. Specificity

[0126] Using MG genomic DNA, MS genomic DNA, Mycoplasma gallinarum, Mycoplasma anatis, Infectious bronchitis virus (IBV), H9 subtype avian influenza virus (AIV), Newcastle disease virus (NDV), Escherichia coli, and Salmonella as templates, the freeze-dried microspheres of Example 4 were used for detection, and the results are shown in Table 5.

[0127] Table 5. Specific test results (Ct value, >40 or no Ct is considered negative).

[0128] MG genomic DNA 24.3 >40 MS genomic DNA >40 24.1 M. gallinarum >40 >40 M. anatis >40 >40 IBV >40 >40 H9 AIV >40 >40 NDV >40 >40 Chicken Escherichia coli >40 >40 Salmonella in chickens >40 >40 negative control >40 >40

[0129] Only the MG and MS templates showed amplification in their respective channels, with no cross-reaction, indicating that the lyophilized microspheres of this application have high specificity.

[0130] 4. Precision

[0131] Take 20 lyophilized microspheres from the same batch (Example 4) and test them for MG and MS positive standards (10). 3 (Copies / reaction), calculate the intra-batch standard deviation. Three additional batches of lyophilized microspheres from Example 4, prepared consecutively (20 microspheres per batch), were tested with the same standard, and the inter-batch standard deviation was calculated. The results are shown in Table 6.

[0132] Table 6 Precision Results

[0133] Within a batch (n=20) 24.8±0.12 0.12 24.6±0.11 0.11 Between batches (3 batches, n=60) 24.8±0.15 0.15 24.6±0.14 0.14

[0134] The small intra-batch and inter-batch standard deviations indicate that the freeze-dried microspheres of this application have good precision and high inter-batch consistency.

[0135] 5. Stability during room temperature storage

[0136] The lyophilized microspheres of each embodiment and comparative example (representative samples) were stored at 25°C, and samples were taken at 0, 1, 3, 6, and 12 months to detect MG standard (10 3 (Copies / reaction), record the Ct value. A Ct value increase ≤ 1.0 is considered a pass criterion for stability; results are shown in Table 7.

[0137] Table 7. Storage stability at 25℃ (MG Ct value)

[0138] Example 1 25.6 25.7 25.8 26.0 26.3 0.7 Example 2 24.9 24.9 25.0 25.2 25.4 0.5 Example 3 25.2 25.3 25.5 25.7 26.0 0.8 Example 4 24.8 24.8 24.9 24.9 25.0 0.2 Example 5 25.0 25.1 25.3 25.7 26.2 1.2 Example 6 29.5 30.2 31.8 34.0 38.5 9.0 Example 7 27.8 28.3 29.5 31.2 34.0 6.2 Example 8 28.2 28.8 30.0 32.0 35.5 7.3 Example 9 30.5 31.5 33.2 36.0 >40 >10 Comparative Example 1 25.8 26.0 26.3 26.8 27.5 1.7 Comparative Example 2 28.5 29.5 31.0 33.5 37.0 8.5 Comparative Example 3 29.0 30.5 32.5 35.5 39.0 10.0 Comparative Example 4 26.0 27.0 28.5 30.5 34.0 8.0

[0139] Example 4 exhibited the best stability, with a Ct increase of only 0.2 after 12 months of storage at 25°C, meeting the acceptable standard. Example 5 showed a Ct increase of 1.2, exceeding the acceptable standard (not greater than 1.0), indicating that the freeze-drying procedure (adding a -30°C step and extending the drying time) failed to improve stability and may have even led to changes in microsphere structure or loss of enzyme activity due to excessive drying time. Examples 6-9 showed significantly decreased stability due to the lack of a protective agent component. Comparative Example 4 also exhibited poor stability. This demonstrates that the optimized protective agent combination and freeze-drying procedure of this application are crucial for long-term room temperature storage.

[0140] 6. Anti-pollution performance

[0141] The lyophilized microspheres from Example 1 and Comparative Example 1 were used to conduct simulated aerosol contamination tests: first, aerosols were generated by amplifying the same target PCR product (containing dU) and then the caps were opened; then, a new batch of 20 negative samples were tested, and the false positive rate (the proportion of negative samples that were mistakenly judged as positive) of the two groups under contaminated conditions was compared. The results are shown in Table 8.

[0142] Table 8 Anti-pollution performance

[0143] Example 1 0%（0 / 20） 0%（0 / 20） Comparative Example 1 85%（17 / 20） 0%（0 / 20）

[0144] Example 1 effectively degrades dU-containing aerosol contamination with a false positive rate of 0. Comparative Example 1 (UNG-dUTP not integrated into lyophilized microspheres, added separately in liquid form) showed poor anti-contamination performance due to delayed addition or uneven mixing during operation. This demonstrates the importance and advantages of pre-integrating the UNG-dUTP system into lyophilized microspheres.

[0145] 7. Resistance to inhibition

[0146] Using the reagents from Example 1, Comparative Example 2, and Comparative Example 1 (liquid reagent form, not lyophilized but containing the same primers, probes, and UNG-dUTP, freshly prepared for use), MG standard (10 3 The Ct values ​​(copies / reactions) were calculated in the presence of different concentrations of inhibitor. The Ct increment (ΔCt) was calculated based on the Ct value without inhibitor, and the results are shown in Table 9.

[0147] Table 9. Anti-inhibition performance (ΔCt)

[0148] Heme 2 μM +0.3 +2.5 +1.5 Heme 5 μM +1.2 +5.0 +4.0 Heme 10 μM +2.5 +9.0 +7.5 heparin 0.1 U / mL +0.4 +2.8 +1.8 heparin 0.5 U / mL +1.5 +6.0 +5.5 heparin 1.0 U / mL +3.0 +10.5 +9.5 Feather extract 0.1% +0.2 +2.2 +1.0 Feather extract 0.5% +1.0 +4.8 +3.8 Feather extract 1.0% +2.2 +8.5 +7.0

[0149] In Example 1, the ΔCt at all inhibitor concentrations was significantly lower than that of Comparative Example 2 and the liquid reagent, indicating that the BSA+betaine combination of this application can still exert excellent anti-inhibition effects in lyophilized microspheres, and the lyophilization process may enable the anti-inhibitor to form a more stable complex with the enzyme, which is better than the liquid reagent with the same formulation.

[0150] 8. Dual-target amplification balance

[0151] Using the lyophilized microspheres from Example 4, mixed samples containing both MG and MS standards (10³ copies / reaction) were tested, repeated 20 times, and the difference in Ct values ​​between the FAM and VIC channels was compared (ΔCt = |Ct-MG-Ct-MS|). Samples containing only a single target were also tested to confirm no cross-interference. The results are shown in Table 10.

[0152] Table 10 Dual-target amplification balance

[0153] Double positive for MG and MS (n=20) 24.8±0.12 24.6±0.11 0.2±0.08 MG positive only 24.7±0.10 >40 — MS positive only >40 24.5±0.09 —

[0154] In double-positive samples, the average difference in Ct values ​​between MG and MS was only 0.2 cycles, indicating good balance in dual-target amplification with no significant competitive inhibition. In single-target samples, the other channel showed no signal, indicating no cross-interference.

[0155] 9. Enzyme activity retention rate after lyophilization

[0156] The activities of hot-start Taq enzyme and UNG enzyme in the premixed solution before lyophilization and the microsphere reconstituted solution after lyophilization were measured, respectively.

[0157] The activities of heat-started Taq enzyme and UNG enzyme in the premixed solution before lyophilization and the reconstituted solution of microspheres after lyophilization were determined, respectively. The enzyme activity assay method is as follows:

[0158] Taq enzyme activity assay: A quantitative real-time assay was performed using λDNA of known concentration as a template under standard PCR conditions. Relative activity was calculated by comparing the difference in the number of cycles (Ct value) required to reach the same fluorescence threshold before and after lyophilization. Specifically, activity retention rate = 2. -ΔCt × 100%, where ΔCt = Ct value after freeze-drying - Ct value before freeze-drying.

[0159] UNG enzyme activity assay: The uracil-DNA degradation method was used, with oligonucleotides containing dU as substrates. After incubation at 37°C, the degree of substrate degradation was detected by electrophoresis or fluorescence method. The relative activity was calculated by comparing the difference in time required to degrade the same amount of substrate before and after freeze-drying.

[0160] The activity retention rate after freeze-drying was calculated with the activity before freeze-drying as 100%, and the results are shown in Table 11.

[0161] Table 11 Enzyme activity retention rate

[0162] Example 4 94.2 90.5 Example 5 88.5 85.0 Example 6 68.0 62.0 Example 7 72.5 68.0 Example 8 75.0 70.0 Example 9 65.0 58.0 Comparative Example 4 70.0 65.0

[0163] Example 4 showed the highest enzyme activity retention rate, while Examples 6-9 showed a significant decrease due to the absence of the protectant. Example 9 (containing only trehalose) exhibited the lowest enzyme activity retention rate, indicating that a single protectant cannot provide sufficient protection, and the combination of protectants (trehalose + mannitol + PVP) has a synergistic protective effect. This demonstrates that the preferred protectant combination and lyophilization procedure of this application can maximize the protection of enzyme activity.

[0164] 10. Clinical sample comparison and validation

[0165] Fifty throat swab samples and 30 joint fluid samples from chickens clinically suspected of avian mycoplasma infection were collected and tested using: (1) the freeze-dried microsphere method of Example 4 (5 μL of supernatant was added directly after the sample was treated with rapid lysis buffer for 5 minutes); (2) a commercial liquid PCR kit (BioWalt BVP5091A, operated according to the instructions); and (3) pathogen isolation and culture method (as the gold standard). The sensitivity and specificity of each method were calculated, and the results are shown in Table 12.

[0166] Table 12 Comparison of Clinical Sample Test Results

[0167] Example 4 19 12 98.2（55 / 56） 100（24 / 24） Approximately 50 Commercial liquid reagent kits 19 12 98.2（55 / 56） 100（24 / 24） Approximately 120 Pathogen isolation 19 (MG), 12 (MS) — benchmark benchmark 5-7 days

[0168] Note: A total of 80 samples were collected, of which 19 were positive for MG and 12 were positive for MS (including 3 double positive samples). There were 28 actual positive cases and 52 negative cases. A total of 31 positive pathogen isolations were recorded.

[0169] Sensitivity calculation explanation: Based on 28 positive cases, each case was tested for 2 targets (MG and MS), for a total of 56 positive detection opportunities (both targets from double-positive cases were included). Example 4 and the commercial liquid kit both correctly detected 55 targets, with 1 missed target (one target from one double-positive sample was not detected), resulting in a sensitivity of 55 / 56 = 98.2%.

[0170] Example 4: The lyophilized microsphere method yielded completely consistent results with the commercial liquid reagent kit, with a sensitivity of 98.2% and a specificity of 100%, but the time was reduced by more than half, and no nucleic acid purification step was required, making the operation simpler.

[0171] This application integrates a hot-start Taq enzyme, an UNG-dUTP anti-contamination system, a BSA+betaine anti-inhibitor system, an optimized lyophilization protectant (trehalose+mannitol+PVP), and a nonionic surfactant Pluronic F-68 into a single lyophilized microsphere. Combined with an optimized step-by-step lyophilization process, it achieves a unified approach to room-temperature storage and transportation, simplified operation, contamination prevention, anti-inhibition, and balanced amplification of dual targets for the dual fluorescent PCR detection reagent for avian septicemia / avian mycoplasma synoviae. This makes it suitable for rapid on-site detection applications in grassroots farms, veterinary stations, and port quarantine departments.

[0172] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.

Claims

1. A dual-fluorescence PCR detection lyophilized microsphere, characterized in that, The lyophilized microspheres are prepared by vacuum freeze-drying of a mixed solution containing the following components: hot-start Taq DNA polymerase, uracil-N-glycosylation enzyme (UNG), dNTPs, specific primer pairs and fluorescent probes for detecting Mycoplasma septicemia (MG) in avian septicemia, specific primer pairs and fluorescent probes for detecting Mycoplasma synoviae (MS) in avian synoviae, buffer solution, potassium chloride, magnesium chloride, lyophilization protectant, and inhibitor.

2. The dual fluorescence PCR detection lyophilized microspheres according to claim 1, characterized in that, The freeze-drying protectant includes one or more of trehalose, mannitol, and polyvinylpyrrolidone, and the anti-inhibitor includes one or more of bovine serum albumin (BSA) and betaine.

3. The lyophilized microspheres for dual fluorescence PCR detection according to claim 1, characterized in that, The concentrations of each component in the mixed solution are calculated based on the working concentration after reconstitution of the lyophilized microspheres: Hot-start Taq DNA polymerase: 0.05-0.5 U / μL; Uracil-N-glycosylation enzyme: 0.01-0.1 U / μL; dNTPs include dATP, dCTP, dGTP and dUTP, each at a concentration of 0.2-0.5 mM; MG upstream primer: 0.2-1.0 μM, MG downstream primer: 0.2-1.0 μM, MG fluorescent probe: 0.1-0.5 μM; MS upstream primer: 0.2-1.0 μM, MS downstream primer: 0.2-1.0 μM, MS fluorescent probe: 0.1-0.5 μM; Tris-HCl buffer: 10-50 mM, pH 8.0-8.5; KCl: 30-100 mM; MgCl2: 2-6 mM; Trehalose: 2-10%, w / v; Mannitol: 1-5%, w / v; Polyvinylpyrrolidone: 0.5-3%, w / v; Bovine serum albumin: 0.1-1.0 mg / mL; Betaine: 0.2-1.0 M.

4. The lyophilized microspheres for dual fluorescence PCR detection according to claim 1, characterized in that, The mixed solution also contains the nonionic surfactant Pluronic F-68, which has a working concentration of 0.05-0.5 g / 100 mL after reconstitution.

5. The dual fluorescence PCR detection lyophilized microspheres according to claim 1, characterized in that, The upstream primer, downstream primer, and fluorescent probe for detecting Mycoplasma avianis MG have the sequences shown in SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively, and the upstream primer, downstream primer, and fluorescent probe for detecting Mycoplasma synoviae MS have the sequences shown in SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6, respectively.

6. A method for preparing lyophilized microspheres for dual fluorescence PCR detection as described in any one of claims 1-5, characterized in that, Includes the following steps: S1. Dissolve all components except for hot-start Taq DNA polymerase, uracil-N-glycosylation enzyme, primers and probes in nuclease-free water, adjust the pH to 8.0-8.5, filter to sterilize, and obtain the base solution; S2. Under ice bath conditions, add hot-start Taq DNA polymerase, uracil-N-glycosylation enzyme, MG primer probe and MS primer probe to the base solution, mix well, and obtain the premix solution; S3. Drop the premixed liquid into a mold pre-cooled by liquid nitrogen to form spherical droplets, and freeze-solidify. S4. The frozen microspheres are freeze-dried under vacuum until the residual moisture content is less than 2% to obtain freeze-dried microspheres; S5. The freeze-dried microspheres are sealed and packaged under inert gas protection.

7. The method for preparing lyophilized microspheres for dual fluorescence PCR detection according to claim 6, characterized in that, The freeze-drying procedure in step S4 is as follows: pre-freeze at -40℃ for 1-3 hours, then dry at -20℃ for 8-16 hours, at 0℃ for 8-12 hours, and at 25℃ for 3-6 hours under a vacuum of <10 Pa.

8. An application of the dual fluorescence PCR detection of lyophilized microspheres as described in any one of claims 1-5, characterized in that, For dual fluorescent PCR detection of avian septicemia / avian synoviocyte mycoplasma.