Freeze-drying type quadruple detection kit for prebiotic four-item limited amplification and application of quadruple detection kit

Abstract

The invention relates to the technical field of biology, in particular to a freeze-drying type quadruple detection kit for prebiotic four-item limited amplification and application of the freeze-drying type quadruple detection kit. Through optimization of primers, reaction reagents and a reaction system and by means of a hydrogel limiting amplification technology, a limiting amplification reaction system suitable for prebiotic four items, namely toxoplasma gondii, rubella virus, human cytomegalovirus and herpes simplex virus, is developed; the limiting amplification system is used for detecting toxoplasma gondii, rubella virus, human cytomegalovirus and herpes simplex virus, the steps are simple and rapid, the equipment requirement is low, aerosol pollution can be effectively prevented and treated, and the limiting amplification system has high sensitivity and high specificity while having good storage stability.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a lyophilized quadruple detection kit for four limiting amplification parameters for eugenics and its applications. Background Technology

[0002] Toxoplasma gondii (TOX), rubella virus (RV), human cytomegalovirus (HCMV), and herpes simplex virus (HSV) are the core pathogens screened for infertility by TORCH. Infection with these pathogens in pregnant women can lead to serious consequences. Currently, clinical laboratory testing for Toxoplasma gondii, rubella, cytomegalovirus, and herpes simplex virus primarily uses serological tests, such as ELISA (enzyme-linked immunosorbent assay), RIA (radioimmunoassay), and IFA (indirect immunofluorescence assay), to detect specific antibodies in serum. A positive result for viral IgM in serum indicates recent or acute infection. A positive result for viral IgG in serum suggests past infection. Serological tests only detect antibodies and have limitations such as a window period, missed detection in immunocompromised individuals, and the inability to distinguish between recent and past infections; therefore, antigen or nucleic acid testing is necessary.

[0003] Highly sensitive and specific quantitative real-time PCR has also been used for clinical detection of toxoplasmosis, rubella, cytomegalovirus, and herpes simplex virus. However, its reliance on sophisticated instruments (such as thermal cyclers and fluorescence detection modules), complex operation, long processing time (usually 1-2 hours or more), and high cost limit its application in primary healthcare institutions or resource-scarce areas.

[0004] Therefore, it is particularly necessary to develop highly sensitive, rapid, and convenient screening methods for TOX, RV, HCMV, and HSV. Summary of the Invention

[0005] In view of this, the technical problem to be solved by the present invention is to provide a lyophilized quadruple detection kit for four limiting amplifications of reproductive health and its application.

[0006] This invention provides primer combinations for four LAMP amplification parameters for eugenics, including: primer combinations for Toxoplasma gondii detection, primer combinations for rubella virus detection, primer combinations for human cytomegalovirus detection, and primer combinations for herpes simplex virus type I and type II detection.

[0007] The primer combination for Toxoplasma gondii detection includes: the F3 primer with the nucleotide sequence shown in SEQ ID NO:6, the B3 primer with the nucleotide sequence shown in SEQ ID NO:7, the FIP primer with the nucleotide sequence shown in SEQ ID NO:8, the BIP primer with the nucleotide sequence shown in SEQ ID NO:9, the LF primer with the nucleotide sequence shown in SEQ ID NO:10, and the LB primer with the nucleotide sequence shown in SEQ ID NO:11;

[0008] The primer combination for rubella virus detection includes: the F3 primer with the nucleotide sequence shown in SEQ ID NO:18, the B3 primer with the nucleotide sequence shown in SEQ ID NO:19, the FIP primer with the nucleotide sequence shown in SEQ ID NO:20, the BIP primer with the nucleotide sequence shown in SEQ ID NO:21, the LF primer with the nucleotide sequence shown in SEQ ID NO:22, and the LB primer with the nucleotide sequence shown in SEQ ID NO:23;

[0009] The primer combination for human cytomegalovirus detection includes: the F3 primer with the nucleotide sequence shown in SEQ ID NO:39, the B3 primer with the nucleotide sequence shown in SEQ ID NO:40, the FIP primer with the nucleotide sequence shown in SEQ ID NO:41, the BIP primer with the nucleotide sequence shown in SEQ ID NO:42, the LF primer with the nucleotide sequence shown in SEQ ID NO:43, and the LB primer with the nucleotide sequence shown in SEQ ID NO:44;

[0010] The primer combinations for detecting type I and type II herpes simplex virus include: the F3 primer with nucleotide sequences as shown in SEQ ID NO:71 and / or SEQ ID NO:77, the B3 primer with nucleotide sequences as shown in SEQ ID NO:72 and / or SEQ ID NO:78, the FIP primer with nucleotide sequences as shown in SEQ ID NO:73 and / or SEQ ID NO:79, the BIP primer with nucleotide sequences as shown in SEQ ID NO:74 and / or SEQ ID NO:80, the LF primer with nucleotide sequences as shown in SEQ ID NO:75 and / or SEQ ID NO:81, and the LB primer with nucleotide sequences as shown in SEQ ID NO:76 and / or SEQ ID NO:82.

[0011] Furthermore, the primer combination described in this invention also includes a primer combination for internal reference detection, wherein the internal reference is β-actin.

[0012] This invention provides a teratogenic amplification reagent, comprising reagent A and reagent B.

[0013] The reagent A includes: buffer solution, divalent cation, fluorescent dye, dithiol polyethylene glycol, first reducing agent and lyophilization protectant A;

[0014] The reagent B includes: the primer combination described in this invention, dNTPs, Bst DNA polymerase, a second reducing agent, an eight-arm polyethylene glycol acrylate, and a lyophilization protectant B.

[0015] The freeze-drying protectant A includes pullulan, PEG20000, PVP, hydroxypropyl-β-cyclodextrin, and glutathione;

[0016] The freeze-drying protectant B includes: mannitol, BSA, trehalose, glycine, hydroxypropyl-β-cyclodextrin, and PVP.

[0017] Furthermore,

[0018] In reagent B, the final concentration of the F3 primer is 0.04~0.06 μM, the concentration ratio of the FIP primer, LF primer and F3 primer is 8:4:1, the concentration of the BIP primer is the same as that of the FIP primer, and the concentration of the LB primer is the same as that of the LF primer.

[0019] The divalent cation includes magnesium ions;

[0020] The first reducing agent includes TCEP;

[0021] The second reducing agent includes DTT.

[0022] Further

[0023] In a 30 μL reaction system, reagent A comprises: 1×Bst Buffer, 4–6 mM magnesium ions, 0.5–1 mM MTCEP, 1× nucleic acid dye, 8–11 mM dithioglycol, 4–6 g / L pullulan, 1.5–2.5 g / L PVP, 4–6 g / L PEG20000, 6–10 g / L hydroxypropyl-β-cyclodextrin, and 0.25–0.75 mM glutathione;

[0024] In a 30 μL reaction system, reagent B comprises: 0.04–0.06 μM F3 primer, 0.04–0.06 μM B3 primer, 0.16–0.02 μM LF primer, 0.16–0.2 μM LB primer, 0.32–0.4 μM FIP primer, 0.32–0.4 μM BIP primer, 0.27–0.37 U / μL LBst enzyme, 2.0–2.75 mM octa-arm polyethylene glycol acrylate, 0.2–0.6 mM DTT, 0.8–1.25 mM dNTP, 1–3 g / L mannitol, 0.8–1.2 g / L BSA, 2–4 g / L trehalose, 14–16 mM glycine, 2.4–2.6 g / L hydroxypropyl-β-cyclodextrin, and 1–3 g / L PVP.

[0025] In a specific embodiment of the present invention, in a 30 μL reaction system, reagent A comprises: 1× Bst Buffer, 5 mM magnesium ions, 1 mM TCEP, 1× nucleic acid dye, 10 mM dithioglycol, 5 g / L pullulan, 2 g / L PVP, 5 g / L PEG20000, 8 g / L hydroxypropyl-β-cyclodextrin, and 0.5 mM glutathione;

[0026] In a specific embodiment of the present invention, in a 30 μL reaction system, reagent B comprises: 0.04 μM F3 primer, 0.04 μM B3 primer, 0.16 μM LF primer, 0.16 μM LB primer, 0.32 μM FIP primer, 0.32 μM BIP primer, 0.32 U / μL LBst enzyme, 2.5 mM polyethylene glycol acrylate, 0.33 mM DTT, 1.25 mM dNTP, 2 g / L mannitol, 1 g / L LBSA, 3 g / L trehalose, 15 mM glycine, 2.5 g / L hydroxypropyl-β-cyclodextrin, and 2 g / L PVP.

[0027] In some other specific embodiments of the present invention, reagent B further includes: reverse transcriptase and RNA protectant.

[0028] The final concentration of the reverse transcriptase is 2 U / μL; the RNA protectant is Murine RNase Inhibitor, with a final concentration of 1.33 U / μL.

[0029] This invention provides the application of at least one of the following (I) to (II) in the preparation of a product for four prenatal screening tests:

[0030] I) The primer combination described in this invention;

[0031] II) The limiting amplification reagent described in this invention.

[0032] This invention provides a kit for four tests for eugenics, which includes: a sample release agent and the limiting amplification reagent described in this invention.

[0033] The conditions for the restricted amplification are 65℃ for 10~30 min; preferably 65℃ for 20 min.

[0034] The sample release agent includes: resin, EDTA, and Triton X-100.

[0035] Furthermore, the sample release agent is: 30g / L~50g / L resin, 1mM~3mM EDTA, 2.5g / L~7.5g / L Triton X-100 and water.

[0036] In a specific embodiment of the present invention, the sample release agent is: 40 g / L resin, 2 mM EDTA, 5 g / L Triton X-100 and water.

[0037] This invention provides a method for detecting four prenatal screening tests for non-diagnostic purposes, which involves testing a sample using at least one of the following methods (A) to (C):

[0038] A) The primer combination described in this invention;

[0039] B) The reagents described in this invention;

[0040] C) The reagent kit described in this invention.

[0041] Furthermore, the detection method of the present invention includes the following steps:

[0042] Step 1: Mix the sample with the sample release agent to obtain the pretreatment solution;

[0043] Step 2: The pretreatment solution is mixed with the limiting amplification reagent according to any one of claims 3 to 5 and then limited amplification is performed;

[0044] Step 3: Determine the positive or negative status of the sample based on the results of the limited amplification.

[0045] The criteria for determining positive and negative are as follows:

[0046] Internal control well: Must show a positive result (fluorescent spot), otherwise the test is invalid and the sample, reagents or procedure need to be checked.

[0047] Target wells: No green fluorescent amplification spots (only uniform weak background fluorescence) indicate a negative result. The presence of one or more discrete green fluorescent amplification spots indicates a positive result. The relative concentration of the corresponding target nucleic acid in the sample can be estimated based on the number of fluorescent spots.

[0048] This invention can simultaneously detect Toxoplasma gondii, rubella virus, human cytomegalovirus and herpes simplex virus. Specifically, the reagents used to detect them can be pre-placed in different reaction pools or reaction tubes.

[0049] This invention improves the specificity and accuracy of detecting Toxoplasma gondii, rubella virus, human cytomegalovirus, and herpes simplex virus types I and II through primer optimization; further improves reagent stability through lyophilization protectant optimization, ensuring good accuracy even after long-term storage; and further enhances accuracy through optimization of magnesium ions, etc. Based on this, the invention optimizes the hydrogel system using a hydrogel limiting system, further improving detection sensitivity, accuracy, and specificity; and makes the detection results visual and easier to observe. Therefore, the various parameters in this invention interact to influence the accuracy, specificity, and reagent stability of the detection results for Toxoplasma gondii, rubella virus, human cytomegalovirus, and herpes simplex virus, and should be protected as a whole.

[0050] The beneficial effects of this invention are:

[0051] (1) High sensitivity and specificity: The hydrogel limiting effect effectively reduces the non-specific amplification background, improves the signal-to-noise ratio, and the detection limit can reach about 1000 copies / mL. It also has no cross-reaction with common pathogens.

[0052] (2) Fast and efficient: The entire detection process (from sample processing to result interpretation) takes ≤25 minutes, of which the core reaction takes only 20 minutes.

[0053] (3) Simple operation and low equipment dependence: Only a pipette, a common heating block (or the constant temperature module of a common PCR instrument) and a simple light source (such as a blue light flashlight) are needed to complete the operation. No complex and precise instruments (such as a fluorescence quantitative PCR instrument or a microfluidic control system) are required.

[0054] (4) Strong anti-interference ability and simple sample processing: The nanopores of the hydrogel can effectively isolate the inhibitors in the sample matrix, allowing the direct heating lysis method (sample release agent) to quickly process the sample. The results are comparable to the gold standard method such as magnetic bead purification, which greatly simplifies the pretreatment steps.

[0055] (5) Excellent stability and portability: The key reagents are freeze-dried using an optimized freeze-drying protectant formulation. The freeze-dried products can be stably stored at room temperature (e.g., 15~30°C) for ≥12 months, which is convenient for room temperature transportation and storage, and is especially suitable for rapid on-site testing in areas with limited resources.

[0056] (6) Low cost: Compared with microfluidic digital PCR and qPCR, it saves the cost of expensive chips, precision instruments and consumables; the price of raw materials such as hydrogel monomers is relatively low.

[0057] (7) Visualized results, relative quantification: The results can be interpreted by observing the fluorescent spots with the naked eye or with simple equipment, which is intuitive and clear. Relative quantitative analysis similar to digital PCR can be achieved by counting the fluorescent spots;

[0058] (8) High signal-to-noise ratio: It restricts the diffusion of amplification products (DNA-dye complex) to form fluorescent spots, which can ignore the influence of background fluorescence, improve the signal-to-noise ratio, and further improve the detection sensitivity (about 1000 copies / mL) and specificity. Attached Figure Description

[0059] Figure 1 The diagram illustrates the interpretation of positive and negative results, where A represents a positive result and B represents a negative result.

[0060] Figure 2 This demonstrates the optimization of hydrogel monomer screening;

[0061] Figure 3 This demonstrates the optimization of hydrogel concentration screening;

[0062] Figure 4 Performance test of lyophilization protectant;

[0063] Figure 5 Cross-testing of freeze-drying protectants. Detailed Implementation

[0064] This invention provides a lyophilized quadruple detection kit for four phenotypic locomotor amplifications and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0065] Overview of the technical solution of this invention

[0066] This invention provides a lyophilized quadruple detection kit based on hydrogel-restricted loop-mediated isothermal amplification (LAMP).

[0067] Confinement mechanism: The hydrogel is formed in situ in the reaction system by the Michael addition reaction of gel monomer A (such as bis-mercaptopolyethylene glycol, SH-PEG-SH) and gel monomer B (such as octahedral polyethylene glycol acrylate, 8-Arm-PEG-AC). Its nanoscale pore size can effectively restrict the free diffusion of amplification products (DNA-dye complex).

[0068] Signal generation: The amplified DNA fragments aggregate in situ, forming discrete, countable fluorescent signal spots (called "amplification spots"). Each fluorescent spot represents an amplification event.

[0069] The lyophilized quadruple detection kit of this invention, through screening of lyophilized additives, can be stored at room temperature for 15 months without affecting detection performance, solving the problem that hydrogel reagents cannot be stored at room temperature for a long time, and is beneficial for long-distance transportation of reagents.

[0070] This invention can simultaneously detect four core pathogens with a detection sensitivity of 1000 copies / mL. When compared with clinical samples using the gold standard qPCR method for molecular diagnostics, it shows high consistency and shorter detection time. This is the first kit to combine hydrogel nanopores and loop-mediated isothermal amplification for the detection of four core pathogens for reproductive health.

[0071] This kit allows for direct processing of clinical samples using a sample release agent; the nanopores formed by the hydrogel effectively isolate macromolecular impurities in the sample, simplifying the sample extraction process. In addition to the eight-tube reaction vessel commonly used in the examples, this kit can also utilize a self-made reaction vessel (not shown); detection results can be directly read visually using a UV-blue light flashlight, or using a fluorescence microscope or a matching simple fluorescence observation device (not shown).

[0072] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0073] Example 1: Primer combination for tert-limited amplification of reproductive tract infection pathogens

[0074] The primer selection for LAMP detection of Toxoplasma gondii, rubella, cytomegalovirus, and herpes simplex virus is illustrated below as an example to illustrate primer optimization. Due to space limitations, other inferior primer combinations (primer groups with different numbers from those in the table below) are not shown:

[0075] Table 1. Primers for pathogens causing reproductive tract infections

[0076]

[0077]

[0078] To verify the amplification efficiency and sensitivity of LAMP primers for four pathogens, qPCR was used to perform limited amplification on samples of the four pathogens after they were identified, with concentrations of 1E6 copies / mL, 1E5 copies / mL, 1E4 copies / mL, 1000 copies / mL, and 500 copies / mL, respectively.

[0079] Table 2. Detection sensitivity of different pathogens

[0080]

[0081] Conclusion: The LAMP primers can accurately detect different pathogens at a sample concentration of 1000 copies / mL while maintaining both sensitivity and specificity. Detection is unstable below 1000 copies / mL, indicating that the designed primer sensitivity is 1000 copies / mL. Sensitivity tests were conducted on herpes simplex virus types 1 and 2 separately. The optimal primer sequences are: the second set of primers for Toxoplasma gondii, the first set of primers for rubella virus, the second set of primers for cytomegalovirus, and the third set of primers for herpes simplex virus types 1 and 2.

[0082] Example 2: Preparation and Detection Method of a Lyophilized Quadruple Reproductive Health Kit with Four Limiting Amplification Spectra

[0083] I. Preparation of a lyophilized limit amplification quadruple detection kit

[0084] Based on the selection of the optimal primer set, a lyophilized quadruple detection kit for restriction amplification was prepared. The dNTPs and primers were obtained from Shanghai Sangon Biotech Co., Ltd. The Bst polymerase in the LAMP reaction mixture was purchased from Xinhai Gene, the eight-arm polyethylene glycol acrylate was purchased from Shanghai Tuoyang, and the dithiol polyethylene glycol was purchased from Xi'an Kaixin.

[0085] 1. Preparation of lyophilized components A / B

[0086] The kit contains reagent components A and B in lyophilized powder form. Lyophilized component A contains 10×Bst Buffer, TCEP, magnesium ions, nucleic acid dye, dithiol polyethylene glycol (SH-PEG-SH, Mw=4000), and lyophilization protectant A;

[0087] The final concentrations of Bst Buffer (1×), TCEP (1 mM), magnesium ions (5 mM), nucleic acid dye (1×), and dithiol polyethylene glycol (10 mM) were all set at 2.5 μL.

[0088] Lyophilization protectant A (final concentration): 5 g / L pullulan, 2 g / L PVP, 5 g / L PEG20000, 8 g / L hydroxypropyl-β-cyclodextrin and 0.5 mM glutathione;

[0089] Mix the components according to the volumes shown in Table 3, and mix thoroughly. Dispense into suitable containers (such as centrifuge tubes) and freeze-dry to obtain freeze-dried component A.

[0090] 2. Preparation of freeze-dried components B1-B5 (according to the general formula in Table 4, B2 is special).

[0091] Lyophilized component B contains Bst enzyme, eight-arm polyethylene glycol acrylate (8Arm-PEG-AC, Mw=20000), DTT, dNTP, primers and lyophilization protectant B;

[0092] The final concentrations of Bst enzyme were 0.32 U / μL, octagonal polyethylene glycol acrylate was 2.5 mM, DTT was 1 mM, dNTPs were 1.25 mM, primer set was 0.4×, and lyophilization protectant B was added in an amount of 2.5 μL.

[0093] Lyophilization protectant B (final concentration): 2 g / L mannitol, 1 g / L BSA, 3 g / L trehalose, 15 mM glycine, 2.5 g / L hydroxypropyl-β-cyclodextrin and 2 g / L PVP.

[0094] Mix all components except primer Mix (Bst enzyme, dNTP, DTT, 8-Arm-PEG-AC, lyophilization protectant B) according to the general formula shown in Table 3. Then add the corresponding 10× primer Mix (prepared according to the proportions in Table 4).

[0095] For B2 (rubella virus detection): Add an additional 2 U / μL of reverse transcriptase and 1.33 U / μL of RNA protectant (Murine RNase Inhibitor). Mix thoroughly. Aliquot into reactors and freeze-dry to obtain reactors containing freeze-dried components B1 (TOX), B2 (RV), B3 (HCMV), B4 (HSV), and B5 (internal control), respectively.

[0096] Note: In this patent, "final concentration" is calculated based on the (reaction) concentration in a 30μL reaction system.

[0097] Table 3. Reaction system preparation (based on 1 part of lyophilized component, the volume of the mixed liquid before lyophilization is 10 μL)

[0098]

[0099] 10×Bst Buffer is composed of betaine, KCl, Tris-HCl, and (NH4)2SO4 in a molar ratio of 4~6:5~8:2~5:1, with Tris-HCl having a pH of 7.5~8.8 and a concentration of 40mM; more specifically, the molar ratio of betaine, KCl, Tris-HCl, and (NH4)2SO4 is 5:6:4:1, and the buffer solution consists of 50mM betaine, 60mM KCl, 40mM Tris-HCl, and 10mM (NH4)2SO4.

[0100] The 100× nucleic acid dye was obtained by diluting the commercially available 10000× nucleic acid dye by 100 times.

[0101] 0.5 mg / μL of octahedral polyethylene glycol acrylate (8Arm-PEG-AC, Mw = 20000) and 0.5 mg / μL of dithiol polyethylene glycol (HS-PEG-SH, Mw = 4000) were obtained by dissolving solid powder in DEPC water.

[0102] The primer mixture contains FIP, BIP, LF, LB, F3, and B3; FIP, LF, and F3 are mixed in a molar ratio of 8:4:1. The concentration of primer BIP is the same as that of FIP, primer LB is the same as that of LF, and primer F3 is the same as that of B3. When using, the six primers are prepared into a mixture in different proportions. Some primer pairs contain only one loop primer, with the other replaced by water. Specific preparation ratios are shown in Table 4 (when the concentration of herpes simplex virus primers is doubled to 100 μM, the amount of primers added is halved, while the total volume remains the same).

[0103] Table 4.10 Primer Mix Configuration

[0104]

[0105] In the freeze-dried component A of this invention, there are no bioactive components. The thiol-polyethylene glycol-thiol group, besides forming a gel network, also acts as a plasticizer during the freeze-drying process. TCEP in freeze-dried component A effectively reduces and stabilizes the thiol (-SH) groups, preventing their oxidation into disulfide bonds (-SS-). Pullulan in freeze-drying protectant A forms a stable protective film during freeze-drying, preventing oxygen and other oxidants from contacting the thiol-polyethylene glycol-thiol group, thereby reducing oxidation and degradation. PVP and TECP work together to slow down the photobleaching of fluorescent dyes.

[0106] In some embodiments of the present invention, the freeze-drying protectants A and B are respectively applicable to components A and B, and the amount added per person is 2.5 μL.

[0107] II. Four Limitation Amplification Methods for Eugenics

[0108] 1. Sample preparation: Take out the sample to be tested and vortex to mix well (frozen samples should be fully thawed at room temperature before use). Take 200 μL of the sample to be tested, add 200 μL of sample release agent (40 g / L resin, 2 mM EDTA, 5 g / L Triton X-100 and water), vortex to mix well, heat at 95℃ for 5 min, cool to room temperature, and use the sample supernatant for detection.

[0109] 2. Reagent preparation: Take 5 lyophilized components A and add 20 μL of DEPC water to each, shake to dissolve and mix well to form complex solution A;

[0110] 3. Sample addition: Take 5 reactors containing lyophilized components B1 (TOX), B2 (RV), B3 (HCMV), B4 (HSV), and B5 (internal control), respectively. Add 10 μL of the sample supernatant prepared in step 1 to each reactor.

[0111] 4. Start the reaction: Add 20 μL of reconstitution solution A to each reactor. Immediately mix with a pipette several times (at least 3 times). Cover the reactor and briefly centrifuge to ensure the mixture is completely in the reaction chamber.

[0112] 5. Isothermal amplification: Place the reactor in an isothermal heating block preheated to 65°C and incubate for 20 minutes.

[0113] 6. Result Detection: After amplification, remove the reactor. In a dark or light-protected environment, use a portable fluorescence imager (wavelength 470±10nm) to scan the reaction chamber and automatically count the fluorescent spots using AI recognition software.

[0114] 7. Result Interpretation: Internal control (β-actin) well (B5): ≥1 fluorescent spot / field of view; otherwise, the test is invalid, and the sample, reagents, or procedure must be checked. Target wells (B1-B4): ≥1 fluorescent spot / field of view is positive. The relative concentration of the corresponding target nucleic acid in the sample can be estimated based on the number of fluorescent spots.

[0115] Example 3: Concordance rate test of lyophilized tert-dependent amplification quadruple detection kit with commercially available qPCR detection kit in clinical samples.

[0116] One hundred clinical samples were collected, and the extracted DNA was detected using a commercially available qPCR kit and the lyophilized, limited-amplification quadruple detection kit of this invention. The results are as follows:

[0117] Table 5. Concordance rate of clinical samples between the lyophilized quadruple detection kit for limited teratogenic amplification and the qPCR detection kit.

[0118]

[0119]

[0120]

[0121]

[0122] Note: More than 20 fluorescent dots are marked as "more".

[0123] The statistical table of clinical sample test results is shown in Table 6.

[0124] Table 6. Comparison between the lyophilized restriction amplification quadruple detection kit and the commercially available prenatal four-item qPCR detection kit.

[0125]

[0126] The positive concordance rate of the lyophilized limit amplification quadrivalent detection kit and the commercially available 4-item qPCR detection kit for reproductive health in clinical samples was 100%, the negative concordance rate was 98.55%, the overall concordance rate was 99.00%, and the Kappa value was 0.977, demonstrating that the detection results of the two kits are highly consistent.

[0127] Example 4: Screening and Concentration Optimization of Hydrogel Components

[0128] This embodiment aims to systematically evaluate the comprehensive impact of the type and concentration of hydrogel monomers on the limiting effect, detection sensitivity, signal-to-noise ratio, and anti-interference ability of LAMP amplification, thereby determining the optimal hydrogel formulation.

[0129] PEG hydrogels are a type of water-insoluble gel material formed by the crosslinking of hydrophilic polymers. After crosslinking, the polymer aqueous solution forms a hydrogel with a porous structure. By adjusting the polymer parameters, the pore size and physicochemical properties of the gel can be controlled.

[0130] I. Screening of hydrogel monomers

[0131] The combined effect of linear, four-armed, six-armed, and eight-armed polyethylene glycol acrylate hydrogel monomers on the regulation of network pore size and diffusion coefficient on the confinement effect of LAMP amplification was evaluated to determine the optimal hydrogel formulation. Four hydrogel monomers, linear polyethylene glycol diacrylate (Linear-PEG-AC, Mw=5,000), four-armed polyethylene glycol acrylate (4Arm-PEG-AC, Mw=10,000), six-armed polyethylene glycol acrylate (6Arm-PEG-AC, Mw=15,000), and eight-armed polyethylene glycol acrylate (8Arm-PEG-A, Mw=20,000), were selected and formulated with dithiol polyethylene glycol (SH-PEG-SH, Mw=4,000) to form hydrogel-confined LAMP amplification systems. The concentration of dithiol polyethylene glycol (SH-PEG-SH, Mw=4,000) was 10 mM, and the molar ratio of the four polyethylene glycol acrylates to the dithiol polyethylene glycol functional groups was 1:1 (acrylate groups: thiol groups = 1:1). The primers, enzymes, dNTPs, and other components of the amplification system were consistent with those in Example 2 (using Toxoplasma gondii as an example).

[0132] Table 7. Comparison of hydrogel properties of PEG monomers with different structures

[0133]

[0134] A homemade reaction tube was used as the reaction vessel, and a simple fluorescence observation device was used to read the experimental results. For example... Figure 2 As shown in Table 7, the linear structure (Group A) had a larger amplification point size; the four-arm, six-arm, and eight-arm structures could all form a uniform three-dimensional network, which could effectively confine large DNA molecules (reducing the diffusion coefficient to 1 / 100 of that in free solution) and ensure the mass transfer efficiency of the reactants. With the increase of the number of arms of the polyethylene glycol acrylate hydrogel monomer, the crosslinking speed increased, and 8Arm-PEG-AC was the optimal hydrogel monomer.

[0135] II. Optimization of Hydrogel Concentration

[0136] Hydrogel networks are the core of confined amplification (LAMP) technology. Their three-dimensional structure confines the LAMP amplification reaction within tiny "reaction chambers," effectively preventing the diffusion of amplification products, thereby enhancing signal aggregation, reducing the risk of aerosol contamination, and simplifying result interpretation. The concentration of hydrogel monomers directly determines the pore size and density of the network structure, affecting DNA molecule diffusion and fluorescence signal formation. Too low a concentration may result in a loose network structure, failing to effectively confine the amplification reaction; too high a concentration may inhibit mass transfer of reactants, hindering the amplification reaction. This embodiment aims to optimize the optimal working concentrations of eight-arm polyethylene glycol acrylate (8Arm-PEG-AC, Mw=20,000) and dithiol polyethylene glycol (HS-PEG-SH, Mw=4,000). While keeping the concentrations of other components unchanged as described in Example 1's optimal formulation, the molar ratio of the two monomers was fixed at 1:4 (dithiol polyethylene glycol: octaarmer polyethylene glycol acrylate). The final concentration of 8Arm-PEG-AC-20K was set to five gradients: 1.8mM, 2.0mM, 2.5mM, 2.75mM, and 3.0mM (corresponding to 4 times the concentration of HS-PEG-SH-4k), and hydrogel-limited amplification detection was performed at each gradient. The optimization effect of the hydrogel concentration was evaluated by observing the clarity, size, and diffusion of the fluorescent amplification spots formed at different concentrations (using Toxoplasma gondii as an example).

[0137] Table 8. Optimization of hydrogel concentration

[0138]

[0139] A homemade reaction tube was used as the reaction vessel, and a simple fluorescence observation device was used to read the experimental results. The results showed that the concentration of the hydrogel monomer significantly affected the site-limited amplification effect. Figure 3As shown, when the concentration of 8Arm-PEG-AC-20K is 2.5 mM and the concentration of HS-PEG-SH-4k is 10.0 mM (molar ratio 1:4), a three-dimensional gel network with optimal morphology can be formed. Under these conditions, the generated fluorescent amplification spots are clear, of moderate size, and without diffusion, making them most suitable for visual or photographic interpretation using a simple fluorescence observation device or UV lamp illumination. Therefore, this concentration was determined to be the optimal concentration for constructing the hydrogel network.

[0140] Example 5: Optimization of Magnesium Ion Concentration

[0141] Magnesium ions are an essential cofactor for Bst nucleic acid polymerase, crucial for its activity and stability. Both excessively high and low magnesium ion concentrations can affect the efficiency and specificity of the LAMP reaction. Excessively high magnesium ion concentrations may lead to nonspecific amplification, while excessively low concentrations may result in poor reaction initiation or low amplification efficiency. Therefore, optimizing the magnesium ion concentration is key to ensuring efficient amplification and reducing background noise.

[0142] To investigate the effect of magnesium ion concentration in the system on amplified clinical detection limit samples and negative samples, the final magnesium ion concentrations were set to 2mM, 3mM, 4mM, 5mM, and 6mM. The concentrations of other components were as described in Example 2. The results are shown in Table 9.

[0143] Table 9. Detection results at different magnesium ion concentrations

[0144]

[0145] Conclusion: Under the premise of ensuring the accuracy of the detection results, the optimal Mg 2+ The final concentration is 5mM. Too low a concentration (2-4mM) will result in false negatives for HCMV borderline samples; too high a concentration (6mM) will result in false positives.

[0146] Example 6: Optimization of dNTP Concentration

[0147] dNTPs provide essential nucleotide triphosphates for nucleic acid polymerases and are fundamental to nucleic acid synthesis. The concentration of dNTPs must be sufficient to support large-scale nucleic acid synthesis without depleting resources, but also not so high as to inhibit enzyme activity. Inappropriate dNTP concentrations may lead to reduced amplification efficiency or the production of erroneous amplification products. By optimizing the dNTP concentration, the specificity of amplification and the yield of products can be improved.

[0148] To investigate the impact of dNTP concentrations within the system on amplification of clinically detectable samples and negative samples, the effects of final dNTP concentrations (0.6, 0.8, 1.0, 1.25, 1.4 mM) on the detection results of 1000 copies / mL borderline positive and negative samples were tested. The concentrations of other components were referenced in Example 2, and the results are shown in Table 10.

[0149] Table 10. Detection results of different dNTP concentrations

[0150]

[0151] Conclusion: The optimal final concentration of dNTPs is 1.25 mM. Too low a concentration (0.6 mM) will result in false positives; too high a concentration (1.4 mM) will inhibit the PCR reaction and lead to false negatives in borderline samples (TOX, HCMV, HSV).

[0152] Example 7: Bst enzyme concentration optimization experiment

[0153] Bst nucleic acid polymerase is the specific enzyme used in LAMP reactions, enabling rapid nucleic acid amplification under isothermal conditions. The enzyme concentration is crucial to the reaction rate and efficiency. Insufficient enzyme concentration may lead to slow or incomplete reactions, while excessive enzyme may increase non-specific amplification, affecting result interpretation. Optimizing the concentration of Bst nucleic acid polymerase ensures efficient nucleic acid synthesis while reducing the risk of erroneous amplification.

[0154] To investigate the effect of Bst enzyme concentration on the amplification of clinically detectable samples and negative samples, the effect of Bst enzyme dosage (0.21, 0.27, 0.32, 0.37, 0.43 U / μL) on the detection results of 1000 copies / mL borderline positive and negative samples was tested. The concentrations of other components were as described in Example 2, and the results are shown in Table 11.

[0155] Table 11. Detection results at different Bst concentrations

[0156]

[0157] Conclusion: The optimal concentration of Bst enzyme is 0.27~0.37 U / μL per reaction; considering both cost and amplification performance, the optimal concentration is 0.32 U / μL. Too low a concentration of Bst enzyme will cause false negatives, while too high a concentration of Bst enzyme will cause false positives.

[0158] Example 8 Primer Mix Concentration Optimization

[0159] The amount of primers in the amplification system affects the amplification effect. Too low a primer amount reduces the amplification product, potentially leading to false negatives; too high a primer concentration promotes non-specific binding and primer dimer formation, resulting in false positives. This invention determines the optimal primer amount by adjusting the ratio of different primer concentrations in the reaction system. The test samples were clinical samples that had undergone fluorescence qPCR assays, with a positive sample concentration of 10... 3 copies / mL. The concentration of primer Mix was adjusted to 0.2×, 0.4×, 0.6×, and 0.8×, while the concentrations of other components were as described in Example 2. The results are shown in Table 12.

[0160] Table 12. Detection results at different primer concentrations

[0161]

[0162] The test results show that samples with too low a primer concentration will have false negative results, while samples with too high a primer concentration will have false positive results. Primer concentrations of 0.4× to 0.6× are all acceptable, and the optimal primer concentration is determined to be 0.4×.

[0163] Example 9 Sample Pretreatment

[0164] Sample release agents: 40 g / L resin, 2 mM EDTA, 5 g / L Triton X-100, and water. Clinical borderline positive samples (1000 copies / mL) and negative samples were treated using the release agent method and the magnetic bead method (detailed procedures refer to the instruction manual), and the detection results of the two methods were compared. All clinical samples were subjected to fluorescence qPCR detection and the results are shown in Table 13.

[0165] Table 13. Detection results of different pathogen pretreatment methods

[0166]

[0167] Conclusion: For samples with detection limits, there is no significant difference between the magnetic bead method and the sample release agent method; both can accurately distinguish sample types. The release agent method is simpler to operate and takes less time. When combined with the limit amplification system, detection can be completed within 25 minutes.

[0168] Example 10 Optimization of Lyophilization Protectant

[0169] This embodiment uses Toxoplasma gondii for testing. 2.5 μL of different formulations of lyophilization protectant A were added to each sample A, and the samples were placed in a lyophilizer for lyophilization. The lyophilized morphology was observed. Similarly, 2.5 μL of different formulations of lyophilization protectant B were added to each sample B, and the lyophilized morphology of the different protectants was observed. The lyophilization procedure is shown in Table 14, and the components of the lyophilization protectants are shown in Table 15 (the concentrations mentioned in the tables are the final concentrations in a 30 μL reaction system).

[0170] Table 14. Freeze-drying process

[0171]

[0172] Table 15. Formulation of freeze-drying protectants

[0173]

[0174] Based on Table 13, different lyophilization protectant formulations were designed and programmed lyophilization was performed, such as... Figure 4 As shown, freeze-drying protectants A1, A2, and A3 all showed good freeze-drying properties; freeze-drying protectants B1 and B2 could not be molded after freeze-drying, while freeze-drying protectant B3 showed the best results. It is recommended to use freeze-drying protectant B3 in combination with A1, A2, and A3 for testing.

[0175] After reconstitution of lyophilization protectant B3 with the corresponding reagents of lyophilization protectants A1, A2, and A3, their amplification efficiency was tested. According to... Figure 5 The fluorescence results of lyophilized and reconstituted Toxoplasma gondii samples at 2000 copies / mL show that the amplification spots formed after reconstitution of lyophilized protectant B3 and its corresponding reagents to lyophilized protectant A1 exhibited diffusion and failed to form. No amplification spots were formed after reconstitution of lyophilized protectant B3 and its corresponding reagents to lyophilized protectant A2. The amplification spots formed after reconstitution of lyophilized protectant B3 and its corresponding reagents to lyophilized protectant A3 were normal, indicating the highest amplification efficiency. This combination was selected as the optimal protectant combination.

[0176] Lyophilization protectants A3 and B3 were selected, and components A and B were lyophilized and stored at room temperature for different periods of time. The storage stability of the lyophilized reagents was tested, and the results are shown in Table 16: After 15 months of storage at room temperature, the lyophilized reagents were still stably detected using a sample of 1000 copies / mL as the detection limit.

[0177] Table 16. Results of the storage stability of lyophilized reagents

[0178]

[0179] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Primer combinations for LAMP amplification of four genetic predispositions, characterized in that, include: Primer combinations for Toxoplasma gondii detection, primer combinations for rubella virus detection, primer combinations for human cytomegalovirus detection, and primer combinations for herpes simplex virus type I and type II detection. The primer combination for Toxoplasma gondii detection includes: the F3 primer with the nucleotide sequence shown in SEQ ID NO:6, the B3 primer with the nucleotide sequence shown in SEQ ID NO:7, the FIP primer with the nucleotide sequence shown in SEQ ID NO:8, the BIP primer with the nucleotide sequence shown in SEQ ID NO:9, the LF primer with the nucleotide sequence shown in SEQ ID NO:10, and the LB primer with the nucleotide sequence shown in SEQ ID NO:11; The primer combination for rubella virus detection includes: the F3 primer with the nucleotide sequence shown in SEQ ID NO:18, the B3 primer with the nucleotide sequence shown in SEQ ID NO:19, the FIP primer with the nucleotide sequence shown in SEQ ID NO:20, the BIP primer with the nucleotide sequence shown in SEQ ID NO:21, the LF primer with the nucleotide sequence shown in SEQ ID NO:22, and the LB primer with the nucleotide sequence shown in SEQ ID NO:23; The primer combination for human cytomegalovirus detection includes: the F3 primer with the nucleotide sequence shown in SEQ ID NO:39, the B3 primer with the nucleotide sequence shown in SEQ ID NO:40, the FIP primer with the nucleotide sequence shown in SEQ ID NO:41, the BIP primer with the nucleotide sequence shown in SEQ ID NO:42, the LF primer with the nucleotide sequence shown in SEQ ID NO:43, and the LB primer with the nucleotide sequence shown in SEQ ID NO:44; The primer combination for detecting type I and type II herpes simplex virus includes: the F3 primer with nucleotide sequences as shown in SEQ ID NO:71 and / or SEQ ID NO:77, the B3 primer with nucleotide sequences as shown in SEQ ID NO:72 and / or SEQ ID NO:78, the FIP primer with nucleotide sequences as shown in SEQ ID NO:73 and / or SEQ ID NO:79, the BIP primer with nucleotide sequences as shown in SEQ ID NO:74 and / or SEQ ID NO:80, the LF primer with nucleotide sequences as shown in SEQ ID NO:75 and / or SEQ ID NO:81, and the LB primer with nucleotide sequences as shown in SEQ ID NO:76 and / or SEQ ID NO:

82.

2. A limiting amplification reagent, characterized in that, Including reagent A and reagent B, The reagent A includes: buffer solution, divalent cation, fluorescent dye, dithiol polyethylene glycol, first reducing agent and lyophilization protectant A; The reagent B comprises: the primer combination as described in claim 1 or 2, dNTP, Bst DNA polymerase, a second reducing agent, an octagonal polyethylene glycol acrylate, and a lyophilization protectant B. The freeze-drying protectant A includes pullulan, PVP, PEG20000, hydroxypropyl-β-cyclodextrin, and glutathione; The freeze-drying protectant B includes: mannitol, BSA, trehalose, glycine, hydroxypropyl-β-cyclodextrin, and PVP.

3. The limiting amplification reagent according to claim 2, characterized in that, In reagent B, the final concentration of the F3 primer is 0.04~0.08 μM, the concentration ratio of the FIP primer, LF primer and F3 primer is 8:4:1, the concentration of the BIP primer is the same as that of the FIP primer, and the concentration of the LB primer is the same as that of the LF primer. The divalent cation includes magnesium ions; The first reducing agent includes TCEP; The second reducing agent includes DTT.

4. The limiting amplification reagent according to claim 3, characterized in that, Reagent A comprises: 1×Bst Buffer, 4-6 mM magnesium ions, 0.5-1 mM TCEP, 1× nucleic acid dye, 8-11 mM dithioglycol, 4-6 g / L pullulan, 1.5-2.5 g / L PVP, 4-6 g / L PEG20000, 6-10 g / L hydroxypropyl-β-cyclodextrin, and 0.25-0.75 mM glutathione; The reagent B comprises: 0.04–0.06 μM F3 primer, 0.04–0.06 μM B3 primer, 0.16–0.02 μM LF primer, 0.16–0.2 μM LB primer, 0.32–0.4 μM FIP primer, 0.32–0.4 μM BIP primer, 0.27–0.37 U / μL LBst enzyme, 2.0–2.75 mM octa-arm polyethylene glycol acrylate, 0.2–0.6 mM DTT, 0.8–1.25 mM dNTP, 1–3 g / L mannitol, 0.8–1.2 g / L BSA, 2–4 g / L trehalose, 14–16 mM glycine, 2.4–2.6 g / L hydroxypropyl-β-cyclodextrin, and 1–3 g / L LPVP.

5. The limiting amplification reagent according to claim 4, characterized in that, Reagent B also includes: reverse transcriptase and RNA protectant.

6. The application of at least one of the following (I) to (II) in the preparation of products for four prenatal screening tests: I) The primer combination as described in claim 1; II) The limiting amplification reagent according to any one of claims 2 to 5.

7. A kit for four prenatal screening tests, characterized in that, include: The sample release agent and the limiting amplification reagent according to any one of claims 3 to 5.

8. The reagent kit according to claim 7, characterized in that, The sample release agent includes: resin, EDTA, and Triton X-100.

9. A method for detecting four prenatal screening tests for non-diagnostic purposes, characterized in that, To detect the sample using at least one of the following methods A) to C): A) The primer combination as described in claim 1 or 2; B) The limiting amplification reagent according to any one of claims 3 to 5; C) The kit according to claim 7 or 8.

10. The detection method according to claim 9, characterized in that, Includes the following steps: Step 1: Mix the sample with the sample release agent to obtain the pretreatment solution; Step 2: The pretreatment solution is mixed with the limiting amplification reagent according to any one of claims 3 to 5 and then limited amplification is performed; Step 3: Determine the positive or negative status of the sample based on the results of the limited amplification.

Images