6-layer mosquito-borne pathogen nucleic acid detection kit

Abstract

This utility model discloses a six-fold mosquito-borne pathogen nucleic acid detection kit, including a box body with a placement slot on the side wall. The placement slot contains a reagent bottle fixing structure and a reagent bottle. The reagent bottle fixing structure includes: a partition dividing the placement slot into an upper and lower slot with an insertion hole; a hinge post mounted on the partition and extending upwards; a hinge frame including a first frame and a second frame hinged to the hinge post, the first and second frames forming a scissor-like structure with arc-shaped hooks at their outer ends, and upper guide slopes on the left and right sides of the first and second frames; a spring located at the inner end of the first and second frames; and a push frame slidably disposed within the partition, with a pressing head at its bottom located within the insertion hole and a U-shaped push frame at its top. The inner side of the push frame has a lower guide slope that conforms to the upper guide slope. The bottom of the reagent bottle is disposed within the insertion hole and supported by the top of the pressing head, and the side wall of the reagent bottle is held by two hooks. Even if the reagent bottle is overturned or shaken during transportation, there is no risk of it slipping out, and the entire process of removing the reagent bottle can prevent it from tipping over.

Description

Technical Field

[0001] This invention relates to the field of detection technology, and in particular to a nucleic acid detection kit for six mosquito-borne pathogens. Background Technology

[0002] Vector-borne infectious diseases are important zoonotic diseases, posing significant threats to human health and animal husbandry. Mosquitoes and other arthropods are important obligate blood-sucking ectoparasites, widely distributed throughout the world. They can infect various hosts, including birds, reptiles, and mammals, and transmit a wide range of diseases harmful to humans and animals. They carry over 5,000 pathogens, more than half of which are transmitted by mosquitoes. Most of these vector-borne infectious diseases are important zoonotic diseases, and most lack effective vaccines, posing significant threats to human health and animal husbandry.

[0003] To enable rapid and accurate analysis of samples, a high-throughput rapid detection platform for vector-borne (mosquito-borne) infectious diseases based on nucleic acid technology has been established. This platform can simultaneously conduct high-throughput rapid monitoring of vector-borne infectious diseases such as chikungunya virus, Zika virus, dengue virus, West Nile virus, yellow fever virus, and Japanese encephalitis virus. Currently, a combination of fluorescence coding technology and flow cytometry is generally used to achieve rapid detection of these multiple viruses. Detection kits are required during the detection process. Patent application number 202221966835.9 discloses a PCR gene detection kit, including a box body, a lid, a slide groove, a pull rope, a support plate, a support side plate, a support bottom plate, a placement slot, test tubes, and a lid opening adjustment component. The slide groove is located on the inner side wall of the box body, the support plate slides on the slide groove, the support side plate is located below the support plate, the support bottom plate is located below the support side plate, the placement slot is located on the support bottom plate, the test tubes penetrate the support plate and are placed in the placement slot, the lid covers the box body, and one end of the pull rope is located under the lid.

[0004] The above-mentioned reagent kit needs to be pulled up with a rope when in use. Firstly, it is not convenient to observe and then take out the reagent bottle as needed. Secondly, the simple fixation by the socket is not stable. If it is overturned during transportation, it is easy to fall out of the socket and be damaged by impact. Utility Model Content

[0005] Addressing the shortcomings of existing technologies, such as inconvenience in removal as needed and unstable fixation, this invention provides a six-fold nucleic acid detection kit for mosquito-borne pathogens.

[0006] The technical solution adopted by this utility model to solve the above-mentioned technical problems is as follows:

[0007] A six-component mosquito-borne pathogen nucleic acid detection kit includes a box body with a placement slot on the side wall. The placement slot contains a reagent bottle fixing structure and a reagent bottle. The reagent bottle fixing structure includes:

[0008] A partition divides the placement slot into an upper slot and a lower slot, and has insertion holes on it;

[0009] A hinged column, which is mounted on the partition and extends upward;

[0010] The hinge frame includes a first frame and a second frame hinged to the hinge post. The first frame and the second frame form a scissor-like structure and the outer ends of both are provided with arc-shaped hooks. The left and right sides of the first frame and the second frame are provided with upper guide slopes.

[0011] A spring is located at the inner end of the first and second frames;

[0012] The push frame is slidably disposed in the partition, with a pressing head located in the insertion hole at its bottom and a U-shaped push frame at its top. The inner side of the push frame is provided with a lower guide slope that fits against the upper guide slope.

[0013] The bottom of the reagent bottle is positioned inside the socket and supported by the top of the press head, while the side wall of the reagent bottle is held by two hook-like bodies.

[0014] Preferably, a gap is provided between the inner ends of the first and second frames and the side wall of the groove.

[0015] Preferably, in the clamped state, the ends of the two hooks have a gap that is smaller than the diameter of the reagent bottle.

[0016] Preferably, the top of the press head is provided with a soft pad with a hemispherical groove, and the bottom of the reagent bottle is placed in the hemispherical groove.

[0017] Preferably, the soft gasket is an elastic gasket.

[0018] Preferably, there are two placement slots arranged symmetrically at the front and back, and the two placement slots are separate structures;

[0019] Preferably, each placement slot is equipped with six reagent bottle fixing structures and six reagent bottles.

[0020] Preferably, the reagent bottle includes a bottle body and a sealing cap, with the bottle body containing a six-component mosquito-borne pathogen nucleic acid detection reagent.

[0021] Compared with the prior art, the advantages of this utility model are as follows: In this application, the reagent bottle is supported by the top of the pressing head after being placed in the socket. At the same time, the hook-shaped body holds the reagent bottle under the action of the spring, so that even if the reagent bottle is overturned and shaken during transportation, there is no risk of it slipping out. When taking out the reagent bottle, it can be taken out individually as needed. First, press the pressing head upward. As the hook-shaped body is released, the reagent bottle rises. When the opening of the hook-shaped body is larger than the diameter of the reagent bottle, the bottom of the reagent bottle is still in the socket. At this time, the reagent bottle can be pulled out upward to complete the removal. The whole process can avoid the reagent bottle tipping over. Attached Figure Description

[0022] The present invention will be further described in detail below with reference to the accompanying drawings and preferred embodiments. However, those skilled in the art will understand that these drawings are drawn only for the purpose of explaining the preferred embodiments and therefore should not be construed as limiting the scope of the present invention. Furthermore, unless specifically indicated, the drawings are only schematic representations of the composition or structure of the described objects and may contain exaggerated depictions, and the drawings are not necessarily drawn to scale.

[0023] Figure 1 This is a side view of this application;

[0024] Figure 2 This is a perspective view of the present application;

[0025] Figure 3 This is a perspective view of the present application;

[0026] Figure 4 for Figure 3 Enlarged view of point A in the middle;

[0027] Figure 5 A three-dimensional view of the reagent bottle fixing structure;

[0028] In the diagram: 10, box body; 101, placement slot; 102, partition; 1021, insertion hole; 20, reagent bottle; 30, reagent bottle fixing structure; 3011, pressing head; 3012, push frame; 302, hinge column; 304, first frame; 305, second frame; 306, hook-shaped body; 307, spring. Detailed Implementation

[0029] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. Those skilled in the art will appreciate that these descriptions are merely descriptive and exemplary and should not be construed as limiting the scope of protection of the present invention.

[0030] It should be noted that similar labels in the following figures indicate similar items; therefore, once an item is defined in one figure, it may not be further defined and explained in subsequent figures. Example

[0031] This embodiment mainly describes the title of the 6-fold mosquito-borne pathogen nucleic acid detection kit, as follows:

[0032] See attached document Figure 1-5 A six-component mosquito-borne pathogen nucleic acid detection kit includes a box body 10, a placement slot 101 on the side wall of the box body 10, a reagent bottle fixing structure 30 and a reagent bottle 20 within the placement slot 101, and the reagent bottle fixing structure 30 includes:

[0033] The partition 102 divides the placement slot 101 into an upper slot and a lower slot, and has an insertion hole 1021 on it;

[0034] A hinged post 302 is disposed on the partition 102 and extends upward;

[0035] The hinge frame includes two first frame bodies 304 and second frame bodies 305 hinged to the hinge column 302. The first frame bodies 304 and second frame bodies 305 form a scissor-like structure and the outer ends of both are provided with arc-shaped hook-like bodies 306. The left and right sides of the first frame bodies 304 and second frame bodies 305 are provided with upper guide slopes.

[0036] Spring 307 is disposed at the inner end of the first frame 304 and the second frame 305;

[0037] The pusher 3012 is slidably disposed in the partition 102. Its bottom has a pressing head 3011 located in the insertion hole 1021, and its top has a U-shaped pusher 3012. The inner side of the pusher 3012 is provided with a lower guide slope that fits against the upper guide slope.

[0038] The bottom of the reagent bottle 20 is positioned within the socket 1021 and supported by the top of the pressing head 3011. The sidewalls of the reagent bottle 20 are held by two hooks 306. In this design, after the reagent bottle 20 is placed within the socket 1021, it is supported by the top of the pressing head 3011. Simultaneously, under the action of the spring 307, the hooks 306 clamp the reagent bottle 20, thus preventing it from slipping out even if it is tilted or shaken during transportation. To remove the reagent bottle 20, it can be taken out individually as needed. First, press the pressing head 3011 upwards. As the hooks 306 release, the reagent bottle 20 rises. When the opening of the hooks 306 is larger than the diameter of the reagent bottle 20, the bottom of the reagent bottle 20 remains within the socket 1021. At this point, the reagent bottle 20 can be pulled out upwards to complete the removal. The entire process prevents the reagent bottle 20 from tipping over.

[0039] Preferably, a gap is provided between the inner ends of the first frame 304 and the second frame 305 and the side wall of the groove. This gap is used to prevent interference between the first frame 304 and the second frame 305 and the wall of the placement groove 101 when they rotate.

[0040] Preferably, in the clamped state, the ends of the two hooks 306 have a gap smaller than the diameter of the reagent bottle 20. This gap is used to reduce the opening angle of the first frame 304 and the second frame 305. The inner walls of the hooks 306 may also be padded for cushioning.

[0041] Preferably, the top of the pressing head 3011 is provided with a soft pad with a hemispherical groove, and the bottom of the reagent bottle 20 is placed in the hemispherical groove. Preferably, the soft pad is an elastic pad. This structure not only plays a buffering role, but also, when pressing upwards, if the clamping force at the hook 306 is too large, the soft pad will deform first, avoiding excessive force on the bottom of the reagent bottle 20.

[0042] Preferably, two placement slots 101 are symmetrically arranged front and back, and the two placement slots 101 are separate structures. The placement slots 101 allow for side viewing of the labels on the reagent bottles 20, and having two slots increases the amount of reagents that can be stored.

[0043] Preferably, each placement slot 101 is provided with six reagent bottle fixing structures 30 and six reagent bottles 20.

[0044] Preferably, the reagent bottle 20 includes a bottle body and a sealing cap, and the bottle body contains a six-fold nucleic acid detection reagent for mosquito-borne pathogens.

[0045] Additionally, it should be noted that the test kit described in this application is placed inside the packaging box during packaging, and its shape matches the cavity of the packaging box.

[0046] The following describes the testing steps:

[0047] GenBank database was searched, and bioinformatics analysis was used to perform sequence alignment and conserved sequence analysis on mosquito-borne pathogens such as chikungunya virus, Zika virus, dengue virus, West Nile virus, yellow fever virus, and Japanese encephalitis virus. Interspecific variation regions and intraspecific conserved regions were selected as detection targets. After comparative analysis of the target sequences using NCBI, the sequences of the top 100 closely related species were downloaded and compared again. The following three criteria were repeatedly tested: ① Interspecific specificity analysis of the target (passed); ② Intraspecific conservation analysis of the target (passed); ③ Target sequence structure analysis (passed).

[0048] Specific primers were designed using PrimerPlex software, with each primer pair labeled with biotin at the 5' end. Probes were designed within the primer amplification regions. Probes were designed using PrimerSelect, with a size of approximately 20-35 bases, GC content of 40-45%, and annealing temperature of 55-60℃. All probe sequences meeting the criteria were screened, and probe specificity was determined by BLAST analysis. A specific tag sequence (TAG) was added to the 5' end of each probe to form a dual-binding probe; the TGA sequence was complementary to the microsphere coupling sequence. Simultaneously, complementary probe sequences were synthesized and biotin-labeled at the 5' end, serving as quality control reference sequences for the suspension chip. Through repeated primer and probe screening, the final primer and probe sequences for each pathogen were selected.

[0049] 2. Plasmid standards containing pathogen target gene sequences

[0050] The overlap extension method was used to construct plasmids containing pathogen target gene sequences. The target gene sequence was divided into 50-70 nt fragments according to its length (ensuring that the 3' end fragments were numbered evenly). The fragments were numbered sequentially from the 5' end to the 3' end. Odd-numbered gene fragments were selected from the forward sequence, and even-numbered gene fragments were selected from the reverse sequence. Adjacent gene fragments had to overlap by 20 nt. PCR was then performed to construct the plasmids, which were then sequenced for later use.

[0051] 3. Establishment and optimization of multiplex PCR

[0052] Dilute the template to 1000 copies / μL, 100 copies / μL, and 10 copies / μL in a fume hood for later use. Prepare a multiplex PCR reaction system: 6 μL of 5 × One Step U+ Mix, 1.5 μL of One Step U+ Enzyme Mix, 0.3 × 7 μL of Primer F (10 μM) and 0.3 × 7 μL of Primer R (10 μM) and 0.3 × 7 μL of Primer R, 1 μL of DNA template, and 16 μL of RNase-free ddH2O. The system preparation can be performed in a clean bench. All operations involving template, primers, and probes should be performed in a fume hood or biosafety cabinet. After sample loading, cycle 35 times: 95℃ for 30 seconds; 95℃ for 10 seconds; 57~60℃ (preliminary experiment) for 60 seconds. Store at 4~8℃. After PCR amplification, confirm the target product by 2% agarose gel electrophoresis. After multiple rounds of optimization, using plasmid standards as templates, the PCR system with an annealing temperature of 60℃ was determined to be the optimal solution. Under conditions of 60~120 copies / μL (500 CFU / mL~1000 CFU / mL), the 7-fold target was successfully amplified, and hybridization detection was scheduled.

[0053] 4. Selection of microspheres, coupling of microspheres with anti-tag sequences, counting and quality control of coupled microspheres.

[0054] 4.1 Selection of Fluorescent Encoded Microspheres: To meet the detection requirements of 10-30 molecular targets, low-adsorption fluorescent encoded microspheres (7-fold, second-generation spheres - COOH) will be selected. Based on the requirements of liquid phase chip molecular detection products, second-generation -COOH microspheres will be evaluated, and fluorescent encoded microspheres suitable for multiplex nucleic acid detection will be screened. CY3-labeled target testing data for the second-generation spheres -COOH show good microsphere clustering, good thermal stability, and high microsphere coupling efficiency (MFI of 350,000-450,000). The signal-to-noise ratio (SNR) for low-copy (0.02 pM) target hybridization is 50-80, and the SNR for high-copy (0.2 pM) target hybridization is close to 100, meeting the molecular detection requirements! This type of microsphere is highly recommended.

[0055] 4.2.1 Take out the original microsphere tube (10000 / ul), vortex for 2 min to mix thoroughly, and immediately aspirate 10ul into the PCR tube. Add 90ul of H2O to the PCR tube, vortex for 2 min to mix thoroughly, and then aspirate 1ul for instrument detection. The diluted microsphere concentration is 1000 / ul.

[0056] 4.2.2 After thoroughly mixing the PCR tube (99 μL, ~100,000 microspheres) by vortexing for 2 min, immediately place it on a magnetic rack and magnetically attach it for 5 min. After the magnetic microspheres are attached to the PCR tube wall, gently remove the supernatant (be careful not to disturb the microspheres!).

[0057] 4.2.3 Remove the PCR tube from the magnetic rack, add 50 μL of coupling buffer, vortex for 2 min to mix thoroughly, and immediately place it on the magnetic rack for 5 min to magnetically adhere the microspheres to the PCR tube wall. Then, gently aspirate the supernatant (be careful not to disturb the microspheres!).

[0058] 4.2.4 Repeat step 4.2.3 once.

[0059] 4.2.5 Prepare a 10 mg / mL EDC solution using coupling buffer (weigh 1 mg EDC into a 1.5 mL centrifuge tube and dissolve it in 100 μL of coupling buffer), and use it immediately after preparation.

[0060] 4.2.6 Add 50 μL of coupling buffer to the PCR tube containing microspheres, vortex for 2 min to mix thoroughly, then add 5 μL of amino-modified nucleic acid probe (20 pmol / μL), add 5 μL of 10 mg / mL EDC solution, vortex again for 2 min to mix thoroughly, and immediately place on a constant temperature shaker to incubate in the dark for 30 min (25℃, 1300 rpm), vortexing for 2 min every 10 min to mix thoroughly.

[0061] 4.2.7 Prepare a 10 mg / mL EDC solution using coupling buffer, following the same method as in step 4.2.5, and use immediately after preparation. 4.2.8 Add 5 μL of the 10 mg / mL EDC solution to the reaction tube from step 4.2.6, vortex for 2 min to mix thoroughly, and immediately place on a constant temperature shaker to incubate in the dark for 45 min (25℃, 1300 rpm), vortexing for 2 min every 10 min to mix.

[0062] 4.2.9 After incubation, remove the PCR tube, vortex for 2 minutes to mix thoroughly, and immediately place it on a magnetic rack for 5 minutes to magnetically adhere the microspheres to the PCR tube wall. Then, gently remove the supernatant (be careful not to disturb the microspheres!).

[0063] 4.2.10 Remove the PCR tube from the magnetic rack, add 50 μL of microsphere washing solution 1, vortex for 2 min to mix thoroughly, and immediately place it on the magnetic rack for 5 min to magnetically adhere to the PCR tube wall. After the microspheres are adsorbed, gently aspirate the supernatant (be careful not to disturb the microspheres).

[0064] 4.2.11 Remove the PCR tube from the magnetic rack, add 50 μL of microsphere washing solution 2, vortex for 2 min to mix thoroughly, and immediately place it on the magnetic rack for 5 min to magnetically adhere to the PCR tube wall. After the microspheres are adsorbed, gently aspirate the supernatant (be careful not to disturb the microspheres).

[0065] 4.2.12 Remove the PCR tube from the magnetic rack, add 50 μL of microsphere blocking solution to the PCR tube, vortex for 2 min to mix thoroughly, and immediately place it on a constant temperature shaker to incubate in the dark for 1 h (25℃, 1300 rpm) or overnight at 4℃.

[0066] 4.2.13 After incubation, remove the PCR tube, vortex for 2 minutes to mix thoroughly, and immediately place it on a magnetic rack for 5 minutes to magnetically adhere the microspheres to the PCR tube wall. Then, gently remove the supernatant (be careful not to disturb the microspheres).

[0067] 4.2.14 Remove the PCR tube from the magnetic rack, add 50 μL of storage buffer, vortex for 2 min to mix thoroughly, and immediately place it on the magnetic rack for 5 min to magnetically adhere the microspheres to the PCR tube wall. Then gently aspirate the supernatant (be careful not to disturb the microspheres).

[0068] 4.2.15 Repeat step 4.2.14 once.

[0069] 4.2.16 Remove the PCR tube from the magnetic rack, add 100 μL of storage buffer, vortex for 2 min to mix, and store in the dark at 2-8℃.

[0070] 4.3 Counting of Coupled Microspheres: Take 1 μl of the coupled microsphere solution, add 49 μl of pH 8.0 TE buffer, dilute and mix thoroughly, then add to a 96-well plate. Place the plate in a suspension chip instrument for detection. When setting the detection program, set the number of coupled microspheres to be much higher than the actual number of microspheres. For detection, aspirate one-third of the liquid from the suspension chip. After detection, multiply the number of microspheres detected by the instrument by 3 to obtain the number of microspheres contained in 1 μl of the microsphere solution. After calculating the microsphere concentration using the suspension chip instrument, store at 4℃ protected from light.

[0071] 4.4 Microsphere Coupling Quality Control: Using a solution of biotin-labeled Anti-Tag-conjugated microspheres, prepare the reaction system as follows: 45 μl of detection buffer, 5 μl of biotin-labeled microspheres, and 5 μl of streptavidin-phycoerythrin (SA-PE) diluted 50-fold in each reaction tube. Shake thoroughly and incubate at 50°C for 5 min in a PCR instrument. Place the system solution in a suspension chip detection instrument and detect the fluorescence value of 100 microspheres to obtain the median fluorescence index (MFI). The MFI value is used to determine the coupling status between the probe and the microspheres.

[0072] 4.5 Hybridization Quality Control of Coupled Microspheres: Select the coupled microspheres, prepare the following mixture: 45 μl of detection buffer, 5 μl of coupled microspheres, and 1 μl of hybridization quality control probe (biotin-labeled X-Tag sequence) (set up two parallel reactions with control probe concentrations of 0.2 pmol / μL and 0.02 pmol / μL, respectively). Incubate in a PCR instrument at 95℃ for 5 min and 50℃ for 15 min. After incubation, add 5 μL of streptavidin-phycoerythrin (SA-PE) diluted 50 times to each reaction tube, shake thoroughly, and incubate in a PCR instrument at 50℃ for 5 min. Place the system solution in a suspension chip detection instrument and detect the fluorescence value of the microspheres to obtain the median fluorescence value (MFI). Based on the MFI value, determine the hybridization quality control result. Low copy number (0.02 pM) target hybridization signal is 60,000-120,000, and high copy number (0.2 pM) target hybridization signal is 300,000-400,000, which meets the requirements of molecular detection.

[0073] 5.1 Single Microsphere + Single PCR Validation: A single-microsphere, a corresponding dual-binding probe, and a target analyte were added to each reaction well to verify the efficient binding of the designed probe and PCR product. The results were then analyzed. Test data showed that the average hybridization signal of the singleton PCR product was over 100,000, and all hybridization signals were >50,000, indicating efficient binding and meeting the target detection requirements.

[0074] 5.2 Multiplex Microsphere + Multiplex PCR Validation: To determine whether there is cross-reactivity between the designed probes, multiple conjugated microspheres, corresponding bimolecular probes, and multiplex PCR products (single template) were added to each reaction well. This verified whether the binding of the designed probes and PCR products would affect other target results (generating non-specific hybridization signals), i.e., the probe specificity, and was then detected. Test data showed that the multiplex PCR product hybridization signal was >50,000, indicating good hybridization specificity and meeting the target detection requirements.

[0075] 6.1 For RNA target testing, a reverse transcription step was incorporated to achieve co-detection of DNA and RNA targets. This step (one-step RT-PCR) was added to PCR amplification, focusing on testing the impact of the reverse transcription system and procedure on PCR efficiency and detection sensitivity. Target detection data are marked in yellow. Test data show that the signal-to-noise ratio for target detection is 40–10, meeting the requirements for RNA target detection.

[0076] 6.2 Comparative Testing of Nucleic Acid Extraction Kits The quality of the nucleic acid extraction kit determines the product's detection performance. RNA reverse transcription is an essential step in RNA detection, and validating product performance with real samples is crucial. Selecting a nucleic acid extraction kit requires considering factors such as sample type (blood, swabs, sputum, bronchoalveolar lavage fluid, etc.), extraction method, extraction efficiency, extraction cost, and extraction target type (DNA + RNA). Based on these factors, three extraction kits—Novizan, Kangwei Century, and Aibotek—were selected for comparative testing to verify extraction performance. Test data showed that for blood sample extraction, the Novizan (RM601) extraction kit achieved the best extraction effect (high nucleic acid yield and good reproducibility).

[0077] 6.3 Validation using simulated real samples

[0078] To verify the performance of the test product in actual detection, simulating a positive "real sample" for mosquito-borne pathogen nucleic acid detection, we compared it with qPCR or NGS testing platforms to verify and evaluate the product's detection sensitivity, specificity, and stability parameters. Here, we used standard plasmid DNA mixed with host DNA (Human gDNA) to simulate a "real sample" for product performance verification. The test protocol is as follows: 60 copies of plasmid DNA were mixed with 10 ng / 50 ng of host DNA (Human gDNA) to simulate a "real sample" for product performance verification.

[0079] 6.3.1 Simulated Sample PCR Amplification and PCR Protocol Optimization: A simulated "real sample" was created by incorporating 60 copies of standard plasmid DNA into host DNA (Human gDNA) for product performance verification. PCR amplification was performed using 10 / 50 ng of host DNA. PCR electrophoresis showed successful target amplification, but the proportion of non-specific PCR amplification increased with the increase of host DNA. This was presumably due to host DNA interfering with target amplification. Therefore, we optimized the PCR system and procedure to improve target amplification yield and reduce non-specific amplification.

[0080] 6.3.2 Using 60 copies of plasmid DNA mixed with 10 ng / 50 ng of host DNA (Human gDNA) to simulate "real samples" for product performance verification, the target detection data has been marked in yellow. The test data shows that the target detection signal-to-noise ratio is 50~5, the hybridization specificity is good, and it meets the sample detection requirements.

[0081] 6.3.3 Product performance verification was carried out by incorporating 60 copies of plasmid DNA into swabs, mosquitoes, etc. Target detection data has been marked in yellow. Test data shows that the signal-to-noise ratio of target detection is 50~5, the hybridization specificity is good, and it meets the sample detection requirements.

[0082] 6.4 Method Evaluation

[0083] Under the same conditions, the established suspension chip method was used to perform three detections before and after to verify the repeatability and stability of the method. The coefficient of variation (CV) of the detection fluorescence values ​​of each pathogen sample was less than 10%.

[0084] 6.5 Real Sample Detection

[0085] To verify the performance of the mosquito vector development reagent, we conducted a "real positive sample" test at the Zhejiang Provincial Center for Disease Control and Prevention. We used 6 positive samples (5ul each) provided by the Provincial Center for Disease Control and Prevention to perform "6-fold mosquito vector pathogen nucleic acid detection".

[0086] The concordance rate with qPCR test results was 100%.

[0087] 7. Conclusions and Recommendations

[0088] 7.1 Primers and specific detection probes targeting mosquito-borne pathogens were successfully designed.

[0089] Bioinformatics analysis was used to perform sequence alignment and conserved sequence analysis on the genes of each pathogen. Primers and species-specific probes were designed for each mosquito-borne pathogen. The interspecificity and intraspecific conservation of the targets were verified. Target sequence structure analysis showed that the designed primers and probes not only had good species specificity, but were also validated in subsequent analysis and detection.

[0090] 7.2 A suspension chip method for detecting multiple mosquito-borne pathogens was successfully established.

[0091] The developed high-throughput detection platform for mosquito-borne diseases can simultaneously conduct high-throughput rapid monitoring of emerging or important mosquito-borne diseases such as chikungunya virus, Zika virus, dengue virus, West Nile virus, yellow fever virus, and Japanese encephalitis virus. It can quickly and accurately identify samples, and there is no cross-reaction between the probes. In addition, the coefficient of variation (CV) of fluorescence intensity values ​​between batches is within 10%, indicating that the method has good repeatability and stability.

[0092] It is of great significance in the epidemiological investigation, clinical diagnosis, and disease prevention and control of infectious diseases, and can provide a basis for clinical diagnosis and government risk assessment of tick-borne diseases and formulation of prevention and control strategies.

[0093] The title of the six-fold mosquito-borne pathogen nucleic acid detection kit provided by this utility model has been described in detail above. Specific examples have been used to illustrate the principle and implementation of this utility model. The above description of the embodiments is only for the purpose of helping to understand this utility model and its core ideas. It should be noted that for those skilled in the art, several improvements and modifications can be made to this utility model without departing from the principle of this utility model, and these improvements and modifications also fall within the protection scope of the claims of this utility model.

Claims

1. 6 Nucleic acid detection kit for mosquito-borne pathogens, comprising a box body, wherein a placement slot is provided on the side wall of the box body, characterized in that, The placement slot is equipped with a reagent bottle fixing structure and a reagent bottle. The reagent bottle fixing structure includes: A partition divides the placement slot into an upper slot and a lower slot, and has insertion holes on it; A hinged column, which is mounted on the partition and extends upward; The hinge frame includes a first frame and a second frame hinged to the hinge post. The first frame and the second frame form a scissor-like structure and the outer ends of both are provided with arc-shaped hooks. The left and right sides of the first frame and the second frame are provided with upper guide slopes. A spring is located at the inner end of the first and second frames; The push frame is slidably disposed within the partition, with a pressing head located in the insertion hole at its bottom and a U-shaped push frame at its top. The inner side of the push frame is provided with a lower guide slope that fits against the upper guide slope. The bottom of the reagent bottle is positioned inside the socket and supported by the top of the press head, while the side wall of the reagent bottle is held by two hook-like bodies.

2. The six-fold mosquito-borne pathogen nucleic acid detection kit according to claim 1, characterized in that, There is a gap between the inner ends of the first and second frames and the side wall of the groove.

3. The six-fold mosquito-borne pathogen nucleic acid detection kit according to claim 1, characterized in that, When clamped, the ends of the two hooks have a gap that is smaller than the diameter of the reagent bottle.

4. The six-fold mosquito-borne pathogen nucleic acid detection kit according to claim 1, characterized in that, The top of the press head is equipped with a soft pad with a hemispherical groove, and the bottom of the reagent bottle is placed in the hemispherical groove.

5. The six-fold mosquito-borne pathogen nucleic acid detection kit according to claim 4, characterized in that, The soft gasket is an elastic gasket.

6. The six-fold mosquito-borne pathogen nucleic acid detection kit according to claim 1, characterized in that, There are two placement slots arranged symmetrically at the front and back, and the two placement slots are separate structures.

7. The six-fold mosquito-borne pathogen nucleic acid detection kit according to claim 6, characterized in that, Each placement slot contains six reagent bottle fixing structures and six reagent bottles.

8. The six-fold mosquito-borne pathogen nucleic acid detection kit according to claim 1, characterized in that, The reagent bottle consists of a bottle body and a sealing cap, and the bottle body contains a nucleic acid detection reagent for six mosquito-borne pathogens.

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