Triple-arthrovirus fluorescent quantitative PCR (polymerase chain reaction) detection kit

Abstract

The utility model discloses a triple-arthrovirus fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit which comprises a square storage seat, square notches are formed in the front side and the rear side of the storage seat, a row of circular storage holes are formed right above each square notch, detection reagent tubes are inserted into the row of storage holes in the rear side, and the detection reagent tubes are inserted into the storage holes in the rear side. Reaction test tubes are inserted into the row of storage holes in the front side. The two rows of storage holes are used for storing detection reagent tubes and reaction test tubes respectively, during detection, liquid relief is convenient, the conditions, such as the adding amount and the like, in the test tubes can be observed in the square notches, multi-tube operation can be performed, repeated taking and placing are not needed, and the fluorescent quantitative detection efficiency of the multiple arthroviruses is improved.

Description

Technical Field

[0001] The utility model relates to a PCR detection kit, in particular to a triple insect-borne virus fluorescence quantitative PCR detection kit. Background Art

[0002] Arboviruses are primarily transmitted through the bites of blood-sucking arthropods and can infect both arthropods and vertebrates. Arboviruses have natural foci and are widely distributed by diverse vectors, primarily mosquitoes, ticks, sand flies, and midges. Predicting epidemic trends is difficult. In my country, the most common viruses are dengue virus, Japanese encephalitis virus, and novel bunyaviruses. Dengue virus (DENV) is primarily transmitted by Aedes aegypti and Aedes albopictus mosquitoes. Infection can cause mild or moderate fever, headache, muscle pain, and joint pain, while severe cases can lead to severe dengue hemorrhagic fever. Studies have shown that from 2005 to 2023, a total of 14,376 imported cases of dengue fever were reported nationwide, indicating an overall upward trend. Provinces with the highest number of reported cases include Yunnan, Guangdong, Fujian, and Zhejiang, posing a significant risk of local re-transmission of the epidemic caused by imported cases. Japanese encephalitis virus (JEV), first discovered in Japan in 1934, causes high fever and viral encephalitis. It is a zoonotic disease with five genotypes (JEV I-V) that are prevalent in Southeast Asia and the Western Pacific. Culex tritaeniorhynchus is the most important vector of JEV in my country. Other mosquito species include Aedes aegypti, Culex pipiens, Culex pipiens pallens, Culex xigensis, Anopheles sinensis, and Gnatia albopictus. In nature, the virus is transmitted between mosquitoes and vertebrate hosts such as bats, waterfowl, and pigs. In the 1960s and 1970s, Japanese encephalitis broke out in China, posing a significant threat to human health. SFTSV (Sphingovirus) is a novel virus of the genus Phlebovirus in the family Bunyaviridae, primarily transmitted by the longicorn tick. Domestic animals such as cattle, goats, and dogs are potential hosts. These three arboviruses primarily cause severe fever with thrombocytopenia syndrome (SFTS). The pathogenesis is not yet fully understood, and clinical symptoms are diverse and lack specificity. Severe cases progress rapidly, with a mortality rate of 30%-60%. These three arboviruses cause similar clinical symptoms such as fever, fatigue, and rash. Rapid and accurate pathogen detection is crucial for treatment strategies and epidemic early warning, surveillance, and prevention. Currently, most arbovirus detection methods use single real-time fluorescence quantitative PCR (qPCR), which can only identify one virus at a time, making it time-consuming and labor-intensive.

[0003] The multiplex qPCR method can detect multiple pathogens simultaneously in the same PCR reaction tube, thereby shortening the operation time and providing timely and accurate diagnostic information for clinical diagnosis and epidemic prevention and control. In the detection process, multiple test tubes are needed, such as detection reagent tubes and reaction test tubes. At present, the test kits for multiplex qPCR detection on the market usually include a box body and a placement table with a socket placed in the box body. During use, the test tubes need to be taken out and added with detection reagents and then put back in place. When multiple tubes need to be operated for multiple detections, frequent taking and placing are required, which is inconvenient to operate. In addition, the situation in the test tubes cannot be observed during the detection process. Utility Model Content

[0004] Based on the shortcomings of the prior art, such as the need to frequently take out and put away test tubes and the inability to observe the conditions inside the test tubes, the utility model provides a triple insect-borne virus fluorescence quantitative PCR detection kit.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a triple insect-borne virus fluorescent quantitative PCR detection kit, including a square storage seat, with square slots on the front and back sides of the storage seat, and a row of circular storage holes directly above the two square slots, wherein the storage holes in the rear row are inserted with detection reagent tubes, and the storage holes in the front row are inserted with reaction tubes.

[0006] Preferably, the storage holes are arranged at equal intervals.

[0007] Preferably, the detection reagent tube includes a first tube body, a first connecting belt and a first cover body provided on the first tube body, the first connecting belt is rotatably connected to the first cover body, and the first cover body covers the top end of the first tube body.

[0008] Preferably, the first tube body is a cylindrical structure, and the first cover body and the first tube body are sealed together.

[0009] Preferably, a first sealing gasket is embedded in the first cover body, and when the first cover body is covered on the first tube body, the first sealing gasket forms a seal with the top wall of the first tube body.

[0010] Preferably, an annular sleeve is provided at one end of the first connecting belt, and an annular groove for the annular sleeve to be sleeved is provided on the side wall of the first cover body, and the annular sleeve is movably sleeved in the annular groove.

[0011] Preferably, the first cover body is threadedly connected to the first tube body.

[0012] Preferably, the reaction tube includes a second tube body, a second connecting band arranged on the second tube body and a second cover body, one end of the second connecting band is provided with an annular sleeve, the side wall of the second cover body is provided with an annular groove for the annular sleeve to be mounted, the annular sleeve is movably mounted in the annular groove, and the second cover body covers the top end of the second tube body.

[0013] Preferably, the bottom of the second cover body is provided with a lower cover groove with a sealing gasket, the side wall of the lower cover groove is provided with a horizontally extending boss, the side wall of the second tube body is provided with a limiting groove formed by two spaced circumferentially extending ribs, and the upper rib is provided with a vertical notch. When the second cover body is closed, the boss enters the limiting groove through the notch and rotates until it is stuck in the limiting groove to lock the second cover body. When the second cover body is locked, the sealing gasket in the second cover body fits tightly against the top wall of the second tube body to form a seal.

[0014] Preferably, a plurality of snap-fit ​​grooves / holes are provided in the middle of the storage seat. When adding the detection reagent, the second cover body is turned over and snap-fitted into the snap-fit ​​grooves / holes.

[0015] Compared with the existing technology, the advantages of the present invention are as follows: the two rows of storage holes in the present application are used to store detection reagent tubes and reaction test tubes respectively. During detection, pipetting is convenient, and the situation in the test tube, such as the amount of addition, can be observed in the square groove. Multi-tube operations can be performed without repeated taking and placing, thereby improving the efficiency of fluorescent quantitative detection of multiple insect-borne viruses. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The present invention will be described in further detail below with reference to the accompanying drawings and preferred embodiments. However, those skilled in the art will appreciate that these drawings are drawn only for the purpose of explaining the preferred embodiments and should not be construed as limiting the scope of the present invention. Furthermore, unless otherwise specified, the drawings are merely schematic representations of the composition or structure of the depicted objects and may contain exaggerated representations. Furthermore, the drawings are not necessarily drawn to scale.

[0017] Figure 1 A perspective view of this application;

[0018] Figure 2 A perspective view of this application;

[0019] Figure 3 This is an exploded view of the application;

[0020] Figure 4 is a stereogram of a reaction tube;

[0021] In the figure: 10, storage seat; 100, square notch; 101, storage hole; 102, snap-fit ​​groove / hole; 20, reaction tube; 201, second tube body; 2011, limiting groove; 2012, notch; 202, second connecting belt; 2021, annular sleeve; 203, second cover body; 2031, boss; 30, detection reagent tube 301, first tube body; 302, first connecting belt; 303, first cover body. DETAILED DESCRIPTION

[0022] 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 illustrative and exemplary and should not be construed as limiting the scope of protection of the present invention.

[0023] Triple Arbovirus Fluorescent Quantitative PCR Detection Kit, reference Figures 1-4 The apparatus comprises a square storage seat 10 with square notches 100 on both the front and rear sides. A row of circular storage holes 101 is formed directly above each of the two square notches 100. The rear row of storage holes 101 houses test reagent tubes 30, while the front row of storage holes 101 houses reaction tubes 20. In this embodiment, the two rows of storage holes 101 are used to store test reagent tubes 30 and reaction tubes 20, respectively. During testing, pipetting is convenient, and the contents of the test tubes, such as the amount added, can be observed within the square notches 100. Multiple tube operations can be performed without repeated placement and removal, thereby improving the efficiency of fluorescent quantitative testing for multiple insect-borne viruses.

[0024] As an advantage, the storage holes 101 are arranged at equal intervals. Since the test tubes of the same type are of the same size, arranging the storage holes 101 at equal intervals is not only aesthetically pleasing, but also convenient for operation.

[0025] Preferably, the detection reagent tube 30 includes a first tube body 301201, a first connecting belt 302 and a first cover body 303 arranged on the first tube body 301201, the first connecting belt 302 is rotatably connected to the first cover body 303, and the first cover body 303 covers the top of the first tube body 301201.

[0026] Preferably, the first tube body 301201 is a cylindrical structure, and the first cover body 303 and the first tube body 301201 are sealed and covered.

[0027] Preferably, a first sealing gasket is embedded in the first cover 303 , and when the first cover 303 covers the first tube 301201 , the first sealing gasket forms a seal with the top wall of the first tube 301201 .

[0028] Preferably, an annular sleeve 2021 is provided at one end of the first connecting band 302. The sidewall of the first cover 303 is provided with an annular groove for the sleeve 2021 to fit within. The sleeve 2021 is movably positioned within the annular groove. The annular groove can be directly formed or formed by two circumferential annular protrusions provided on the outer sidewall of the first cover 303. The sleeve 2021 has a certain degree of elasticity to facilitate its installation within the annular groove.

[0029] Preferably, the first cover 303 is threadedly connected to the first tube 301201. When tightened, the first sealing gasket is tightly fitted to the top wall of the detection reagent tube 30 to form a seal.

[0030] Preferably, the reaction tube 20 includes a second tube body, a second connecting band 202 arranged on the second tube body, and a second cover body 203. One end of the second connecting band 202 is provided with an annular sleeve 2021, and the side wall of the second cover body 203 is provided with an annular groove for the annular sleeve 2021 to be mounted. The annular sleeve 2021 is movably mounted in the annular groove, and the second cover body 203 covers the top end of the second tube body.

[0031] Preferably, a lower covering groove with a second sealing gasket is provided at the bottom of the second cover body 203, and a horizontally extending boss 2031 is provided on the side wall of the lower covering groove. The side wall of the second tube body is provided with a limiting groove 2011 formed by two spaced circumferentially extending ribs, and a vertical notch 2012 is provided on the upper rib. When the second cover body 203 is closed, the boss 2031 enters the limiting groove 2011 through the notch 2012 and rotates until it is stuck in the limiting groove 2011 to lock the second cover body 203. When the second cover body 203 is locked, the second sealing gasket in the second cover body 203 fits tightly against the top wall of the second tube body to form a seal. After the pipetting is completed, the boss 2031 on the second cover body 203 is pressed into the notch 2012 and the second cover body 203 is rotated so that the boss 2031 is stuck in the limiting groove 2011 and the second sealing gasket is compressed to achieve sealing. When pipetting is required, the second cover body 203 is rotated to align the boss 2031 with the notch 2012 and then the second cover body 203 can be opened. Compared with the threaded connection method, its opening and closing is faster.

[0032] Preferably, a plurality of snap-fitting grooves / holes 102 are provided in the middle of the storage seat 10 . When adding the detection reagent, the second cover 203 is turned over and snap-fitted into the snap-fitting grooves / holes 102 .

[0033] In addition, the reagent kit is further provided with a packaging box body, and the outer side walls of the two test tubes are provided with circumferential limiting rings for locking them in the storage hole 101 and preventing them from falling.

[0034] The following is the detection part:

[0035] Sample source and nucleic acid extraction:

[0036] The plasmid was synthesized manually and cloned into a plasmid. The plasmid was synthesized by Shanghai Sangon Biotechnology Co., Ltd. The concentration was converted to copy number according to the plasmid copy number concentration formula: copies / μL = (6.02 x 1023) x (ng / μL x 10-9) / (DNA length x 660). A serial dilution was performed to obtain a standard concentration for later use. (Note: 6.02 x 1023 represents the number of molecular copies per mole of the substance; DNA length represents the total length of the plasmid and target fragment; 660 represents the average molecular weight of double-stranded DNA. The copy number concentrations of the dengue virus (DENV), novel bunyavirus (SFTSV), and Japanese encephalitis virus (JEV)-positive plasmids were 6.7 x 1011 copies / μL, 5.4 x 1011 copies / μL, and 5.0 x 1011 copies / μL, respectively.)

[0037] Other sample sources:

[0038] 50 blood samples from patients suspected of arbovirus infection in the past two years, 300 tick samples collected in Jinhua City, and 1,000 mosquito samples; clinically positive samples for influenza A (H1N3), influenza B (BV), and Brucella (BS); Staphylococcus aureus (ATCC 25923) and Salmonella paratyphi B (CMCC (B) 50094) as standard strains; nucleic acid was extracted from all samples according to the instructions of commercial extraction kits. Instruments and reagents:

[0039] The detection kits in this application are used for several purposes, such as animal tissue genomic DNA extraction, nucleic acid extraction and bacterial genomic DNA extraction, CFX96TM fluorescence quantitative PCR instrument; Tianlong extractor GeneRotex 96; high-speed centrifuge.

[0040] Primer and probe design:

[0041] The complete gene sequences of DENV, SFTSV, and JEV were downloaded from the NCBI (National Center of Biotechnology Information) database. Primer and probe sequences were aligned and screened using the bioinformatics software BLAST and Primer Premier 5.0. Primers were designed in conserved regions upstream and downstream of the target gene. Probes that specifically bind to the target gene were then designed between the upstream and downstream primers.

[0042] Optimization of reaction system:

[0043] Using a positive plasmid at a concentration of 107 copies / μL as template, primer and probe concentrations were screened using a matrix-based approach. The optimal reaction conditions were those that produced typical "S"-shaped amplification curves and the highest fluorescence signal intensity for all three arboviruses. The optimal reaction system was confirmed to be: a 5 μL reaction solution; 1 μL, 0.6 μL, and 0.6 μL of upstream and downstream primers (20 μmol / L) for DENV, SFTSV, and JEV, respectively; and 1 μL, 0.8 μL, and 0.8 μL of probes (20 μmol / L), respectively; and 5 μL of template, diluted to 25 μL. Amplification conditions included reverse transcription at 50°C for 15 min, pre-denaturation at 95°C for 3 min, denaturation at 95°C for 5 s, and annealing and extension at 55°C for 30 s for 45 cycles. Fluorescence in the FAM, HEX, and CY5 channels was collected during the extension phase of each cycle.

[0044] Optimization of amplification procedures:

[0045] The multiplex qPCR amplification program was optimized using three annealing temperature settings: 54°C, 55°C, and 56°C. Standard plasmids of DENV, SFTSV, and JEV at a concentration of 107 copies / μL were used as templates. The following reaction conditions were used: reverse transcription at 50°C for 15 minutes; pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 5 seconds, followed by annealing and extension at the annealing temperature for 30 seconds for 45 cycles. Fluorescence in the FAM, HEX, and CY5 channels was collected during the extension phase of each cycle.

[0046] Specificity test:

[0047] Using an established multiplex qPCR assay, DENV, SFTSV, and JEV were tested to verify cross-reactivity between primers and probes for the three pathogens. The specificity of the assay was also verified by testing extracted nucleic acids from influenza A (H1N3), influenza B (BV), Brucella (BS), Staphylococcus aureus (ATCC 25923), and Salmonella Paratyphi B (CMCC(B)50094).

[0048] Establishment of standard curve and sensitivity test:

[0049] The three pathogen standards were mixed and diluted in a 10-fold gradient to 1.0×10₂ to 1.0×10₂ copies / μL. The assay was performed using a constructed multiplex qPCR assay, with three replicates performed at each concentration. The Ct values ​​were averaged. Data from eight concentrations were analyzed using Excel to construct a standard curve, with the Ct value plotted on the y-axis and the logarithm of the standard copy number plotted on the x-axis. The sensitivity of the assay was determined by testing the standards with copy numbers ranging from 1.0×10₂ to 1.0×10₂ copies / μL using the constructed multiplex qPCR assay.

[0050] Repeatability test results:

[0051] Three replicates were performed on each of the 105 and 107 copies / μL standards to verify intra-group reproducibility. Three replicates were performed on each of the 104 and 106 copies / μL standards to verify inter-group reproducibility. Data were analyzed using Excel, and the mean, standard deviation, and coefficient of variation (CV) of the Ct values ​​were calculated.

[0052] Initial verification:

[0053] Fifty blood samples from patients suspected of being infected with arboviruses in the past two years, 300 hard tick samples and 1000 mosquito samples collected in the field were collected. The three pathogens were detected using the established multiplex qPCR method, and the results were analyzed.

[0054] The above describes the triple-fold arbovirus fluorescent quantitative PCR detection kit provided by the present invention. Specific examples are used herein to illustrate the principles and implementation methods of the present invention. The description of the above examples is intended only to facilitate understanding of the present invention and its core concepts. It should be noted that those skilled in the art may make various improvements and modifications to the present invention without departing from the principles of the present invention, and such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. 3-fold insect-borne virus fluorescence quantitative PCR detection kit, characterized in that: It includes a square storage seat with square slots on both the front and back sides. There is a row of round storage holes just above the two square slots. The detection reagent tubes are inserted into the storage holes in the rear row, and the reaction tubes are inserted into the storage holes in the front row.

2. The triple insect-borne virus fluorescence quantitative PCR detection kit according to claim 1, characterized in that: The storage holes are set at equal intervals.

3. The triple insect-borne virus fluorescence quantitative PCR detection kit according to claim 1, characterized in that: The detection reagent tube includes a first tube body, a first connecting belt and a first cover body provided on the first tube body. The first connecting belt is rotatably connected to the first cover body, and the first cover body covers the top end of the first tube body.

4. The triple insect-borne virus fluorescence quantitative PCR detection kit according to claim 3, characterized in that: The first tube body is a cylindrical structure, and the first cover body and the first tube body are sealed and covered.

5. The triple insect-borne virus fluorescence quantitative PCR detection kit according to claim 4, characterized in that: A first sealing gasket is embedded in the first cover. When the first cover is covered on the first tube, the first sealing gasket forms a seal with the top wall of the first tube.

6. The triple insect-borne virus fluorescence quantitative PCR detection kit according to claim 3, characterized in that: An annular sleeve is provided at one end of the first connecting belt, and an annular groove for the annular sleeve to be sleeved is provided on the side wall of the first cover body, and the annular sleeve is movably sleeved in the annular groove.

7. The triple insect-borne virus fluorescence quantitative PCR detection kit according to claim 3, characterized in that: The first cover body is threadedly connected to the first tube body.

8. The triple insect-borne virus fluorescence quantitative PCR detection kit according to claim 1, characterized in that: The reaction tube includes a second tube body, a second connecting band arranged on the second tube body and a second cover body. One end of the second connecting band is provided with an annular sleeve. The side wall of the second cover body is provided with an annular groove for the annular sleeve to be arranged. The annular sleeve is movably sleeved in the annular groove. The second cover body covers the top end of the second tube body.

9. The triple insect-borne virus fluorescence quantitative PCR detection kit according to claim 8, characterized in that: The bottom of the second cover body is provided with a lower covering groove with a second sealing gasket, and the side wall of the lower covering groove is provided with a horizontally extending boss, and the side wall of the second tube body is provided with a limiting groove formed by two spaced circumferentially extending ribs, and the upper rib is provided with a vertical notch. When the second cover body is closed, the boss enters the limiting groove through the notch and rotates until it is stuck in the limiting groove to lock the second cover body. When the second cover body is locked, the second sealing gasket in the second cover body is tightly fitted with the top wall of the second tube body to form a seal.

10. The triple insect-borne virus fluorescence quantitative PCR detection kit according to claim 9, characterized in that: A plurality of buckling grooves / holes are provided in the middle of the storage seat. When adding the detection reagent, the second cover body is turned over and buckled into the buckling grooves / holes.

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