Triple natural epidemic disease fluorescent quantitative PCR (polymerase chain reaction) detection kit

Abstract

The utility model discloses a fluorescent quantitative PCR (Polymerase Chain Reaction) detection kit for triple natural epidemic diseases, as shown in Figure 1-Figure 5, the fluorescent quantitative PCR detection kit for triple natural epidemic diseases comprises a cylindrical outer box body and three inner box bodies arranged in the outer box body, the outer box body is provided with a containing cavity, and the three inner box bodies are arranged in the containing cavity. A containing cavity is formed in the outer box body, three annular grooves extending in the circumferential direction are formed in the cavity wall of the containing cavity, vertical sliding grooves are formed in the cavity wall of the containing cavity, the sliding grooves extend to the annular grooves in the bottom from the top of the containing cavity, bosses are arranged on the outer side walls of the three inner box bodies, and the bosses on the outer box body are aligned with the sliding grooves, vertically slide into the containing cavity and rotate so that the bosses can be clamped in the annular grooves. Wherein the bosses of the three inner box bodies are respectively clamped in the three annular grooves, placing plates are arranged in the inner box bodies, a plurality of circular inserting holes are formed in the placing plates, detection reagent tubes or reaction test tubes are inserted in the inserting holes, and three detection reagents aiming at triple natural epidemic diseases are respectively stored in the detection reagent tubes in the three inner box bodies. According to the scheme, the detection kit is provided with the inner box body and the outer box body, internal reagents can be sealed and stored, the effectiveness of the detection reagents can be effectively guaranteed, in addition, different detection reagents are contained in the reagent tubes of the three inner box bodies so as to cope with triple natural epidemic diseases, and the pertinence is high.

Description

Technical Field

[0001] The utility model relates to a PCR detection kit, in particular to a triple natural epidemic disease fluorescence quantitative PCR detection kit. Background Art

[0002] The pathogens of natural epidemic diseases usually spread among animals, mainly infecting animals. Under certain conditions, they can infect humans, causing serious harm [1]. Brucellosis (abbreviated as brucellosis) is a natural epidemic disease caused by Brucella (BS). Cattle, sheep, pigs, and dogs are the sources of animal brucellosis transmission. Humans are generally susceptible to it. Clinically, it is characterized by fever, sweating, fatigue, muscle pain, etc. [2]. The current epidemic form of brucellosis is a multi- and scattered point epidemic [3], but it is on the rise. Leptospirosis caused by pathogenic Leptospira (P. Leptospi ra, LEP) is a statutory infectious disease in my country and one of the most widely distributed zoonotic diseases [4]. Pathogenic Leptospira can infect a variety of animals, including livestock and wild animals, especially rodents [5-7]. Humans are infected through contact with infected animal urine or water and soil contaminated by urine [8]. Leptospirosis is characterized by high fever, fatigue, body pain, and superficial lymphadenopathy. Hemorrhagic fever is a natural epidemic disease caused by Hantavirus (HTNV), with rodents as the main source of infection. Patients present with fever, fatigue, body pain, eyelid pain, and severe bleeding, hypotension, shock, and kidney damage [9]. The reported incidence rate in Shanghai has fluctuated between 0.01 / 100,000 since 2010

[10] . The incidence rate of hemorrhagic fever (HF) shows a trend of "decline-rise-slow decline". BS, LEP, and HTNV pathogens, after infecting humans, can cause similar clinical manifestations such as fever, fatigue, muscle or joint pain, and a history of animal contact, which makes it difficult for clinicians to make early differential diagnosis. In addition, the emergence of co-infection cases poses a huge challenge to clinical diagnosis and treatment, and is very likely to lead to missed diagnosis and misdiagnosis.

[0003] The multiplex fluorescence PCR method has good specificity, sensitivity and repeatability, and has the advantages of being fast and simple. During the detection process, PCR kits are mainly used to detect pathogens and genetic diseases, improve diagnostic accuracy and treatment levels, and are an indispensable part of the detection process. The detection kits on the market are generally packaged in paper boxes, and the reagent tubes inside are fixed by plugs. The protection of the kits is insufficient and they are easily damaged during transportation, resulting in contamination of the detection reagents and affecting the test results. In addition, multiple kits are required for multiple natural epidemic diseases, which has poor targeting. Utility Model Content

[0004] Based on the fact that the protective structure of the test kit in the prior art is insufficient and easily damaged and contaminated by the outside world, thus affecting the test results, and the deficiency that a single test kit cannot detect multiple natural epidemic diseases, the present invention provides a triple natural epidemic disease fluorescence quantitative PCR detection kit.

[0005] The technical solution adopted by the present invention to solve the above-mentioned technical problems is: a triple natural epidemic disease fluorescent quantitative PCR detection kit, comprising a cylindrical outer box body and three inner box bodies arranged in the outer box body, the outer box body having a accommodating cavity, the cavity wall of the accommodating cavity being provided with three circumferentially extending annular grooves and the cavity wall of the accommodating cavity being provided with a vertical slide groove, the slide groove extending from the top of the accommodating cavity to the annular groove at the bottom, the outer side walls of the three inner box bodies being provided with a boss, the boss on the outer box body being aligned with the slide groove and vertically sliding into the accommodating cavity and rotating so that the boss is stuck in the annular groove, wherein the bosses of the three inner box bodies are respectively stuck in the three annular grooves, a placement plate is provided in the inner box body, a plurality of circular jacks are provided on the placement plate, detection reagent tubes or reaction test tubes are inserted in the jacks, and the detection reagent tubes in the three inner box bodies respectively store three detection reagents for triple natural epidemic diseases.

[0006] Preferably, two groups of slide grooves and bosses are provided and are symmetrical about the central axis of the outer box body and the inner box body respectively.

[0007] Preferably, an annular elastic gasket is embedded in the side wall of the insertion hole, and the reagent tube or reaction tube passes through the middle through hole of the elastic gasket and is compressed.

[0008] Preferably, the inner box body includes an inner bottom shell and an inner upper cover, a sealing gasket is embedded in the inner upper cover, and the inner upper cover is threadedly connected to the inner bottom shell.

[0009] Preferably, the placement plate is located inside the bottom shell, a socket is provided at the center of the placement plate, and a plurality of sockets arranged in an array are provided around the center of the placement plate.

[0010] Preferably, a concave groove is provided on the top of the inner cover body, a raised mounting platform is provided in the middle of the groove, and an inner handle is hinged at the mounting platform.

[0011] Preferably, the height of the boss is the same as the width of the annular groove, and the width of the sliding groove is greater than the width of the boss.

[0012] Preferably, when the inner box bodies are installed in the accommodating cavity, adjacent inner box bodies abut against each other in the height direction.

[0013] Preferably, the outer diameter of the inner box body is the same as the inner diameter of the outer box body.

[0014] Preferably, the outer box body includes an outer shell and an outer cover. A sealing gasket is provided on the front of the outer cover. The outer cover is threadedly connected to the outer shell and a sealing structure is formed through the sealing gasket. An outer handle is provided on the outer cover.

[0015] Compared with the existing technology, the advantages of the present invention are: the detection kit in this application has an inner and outer box body, which can seal and store the internal reagents, effectively ensuring the effectiveness of the detection reagents. In addition, the reagent tubes in the three inner boxes are packaged with different detection reagents to deal with three natural epidemic diseases, which is highly targeted. 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 This is an exploded view of the application;

[0019] Figure 3 is a three-dimensional diagram of the outer shell;

[0020] Figure 4 is a cross-sectional view of the outer shell;

[0021] Figure 5-Figure 6 This is the exploded view of the inner box;

[0022] In the figure: 10, outer box body; 101, outer shell; 1011, accommodating cavity; 1012, annular groove; 1013, slide groove; 102, outer cover body; 103, outer handle; 20, inner box body; 201, inner bottom shell; 2011, boss; 2012, placement plate; 202, inner cover body; 203, inner handle; 30, detection reagent tube; 40, reaction tube. DETAILED DESCRIPTION

[0023] 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.

[0024] Triple natural epidemic disease fluorescence quantitative PCR detection kit, such as Figure 1-6As shown, it includes a cylindrical outer box body 10 and three inner box bodies 20 arranged in the outer box body 10. The outer box body 10 has a accommodating cavity 1011. The cavity wall of the accommodating cavity 1011 is provided with three circumferentially extending annular grooves 1012 and the cavity wall of the accommodating cavity 1011 is provided with a vertical slide groove 1013. The slide groove 1013 extends from the top of the accommodating cavity 1011 to the annular groove 1012 at the bottom. The outer side walls of the three inner box bodies 20 are provided with bosses 2011. The bosses 2011 on the outer box body 10 are aligned with the slide grooves 1013. The groove 1013 slides vertically into the accommodating cavity 1011 and rotates so that the boss 2011 is stuck in the annular groove 1012, wherein the bosses 2011 of the three inner box bodies 20 are respectively stuck in the three annular grooves 1012. A placement plate 2012 is provided in the inner box body 20, and a plurality of circular jacks are provided on the placement plate 2012. The jacks are provided with detection reagent tubes 30 or reaction tubes 40. The detection reagent tubes 30 in the three inner box bodies 20 respectively store three detection reagents for three natural epidemic diseases. The detection kit in this solution has an inner and outer double box body, which can seal and store the reagents inside, effectively ensuring the effectiveness of the detection reagents. In addition, the reagent tubes in the three inner box bodies 20 are packaged with different detection reagents to deal with three natural epidemic diseases, which is highly targeted.

[0025] As an advantage, there are two sets of slide grooves 1013 and bosses 2011, which are symmetrical about the central axis of the outer box body 10 and the inner box body 20. Two sets can be quickly aligned and can be taken out after rotating a small angle.

[0026] Preferably, an annular elastic gasket is embedded in the side wall of the socket, and the reagent tube or reaction tube 40 is passed through the central through hole of the elastic gasket and compressed. This solution uses the elastic gasket to clamp the reagent tube or test tube in the socket, which can provide a certain degree of protection. In addition, the elastic gasket can also adapt to expand and contract in freezing environments.

[0027] Preferably, the inner box body 20 includes an inner bottom shell 201 and an inner upper cover, a sealing gasket is embedded in the inner upper cover, and the inner upper cover is threadedly connected to the inner bottom shell 201. This solution ensures the sealing of the inner box body 20, which is conducive to ensuring the effectiveness of the internal detection reagent.

[0028] Preferably, the placement plate 2012 is located inside the bottom shell, with a central insertion hole and a plurality of circular insertion holes arranged in an array around the central periphery of the placement plate 2012. This solution allows reagent tubes and test tubes to be placed in separate areas for easier operation and management.

[0029] As an advantage, a concave groove is provided on the top of the inner cover 202, a raised mounting platform is provided in the middle of the groove, and an inner handle 203 is hinged at the mounting platform. The sunken design provides conditions for the two box bodies to abut against each other.

[0030] Preferably, the height of the boss 2011 is the same as the width of the annular groove 1012, and the width of the sliding groove 1013 is greater than the width of the boss 2011. This solution can reduce friction when sliding in vertically and ensure that there will be no vertical shaking after horizontal rotation.

[0031] Preferably, when the inner box bodies 20 are installed in the accommodating cavity 1011 , adjacent inner box bodies 20 abut against each other in the height direction.

[0032] Preferably, the outer diameter of the inner box body 20 is the same as the inner diameter of the outer box body 10. This solution can ensure that the inner and outer box bodies 10 fit together and avoid relative shaking of the inner box body 20 and the outer box body 10 during transportation.

[0033] Preferably, the outer box 10 includes an outer shell 101 and an outer cover 102. A sealing gasket is provided on the front of the outer cover 102. The outer cover 102 is threadedly connected to the outer shell 101 and forms a sealed structure through the sealing gasket. An external handle 103 is provided on the outer cover 102. This solution ensures the sealing of the outer box 10. In addition, the provision of the external handle 103 ensures the portability of the test kit.

[0034] The following is an introduction to the test content:

[0035] 1 Materials and Methods

[0036] 1.1 Materials

[0037] 1.1.1 Sample sources Three plasmid standards (BS, LEP, HTNV) were synthesized; Staphylococcus aureus (ATCC25923), Enterococcus faecalis (ATCC29212), pathogenic Escherichia coli EPEC (CICC24189), enterotoxigenic Escherichia coli ETEC (10667), Salmonella Paratyphi B (CMCC(B)50094), Vibrio parahaemolyticus (ATCC17802), and Yersinia enterocolitica (ATCC23715) were purchased standard strains; norovirus GⅠ (NI), norovirus GⅡ (NVⅡ), dengue virus (DENV), severe fever with thrombocytopenia syndrome virus (SFTSB), influenza A (H1N3), and influenza B (BV) were derived from clinical positive samples.

[0038] 1.1.2 Instruments and Reagents

[0039] Main reagents: primers, probes;

[0040] Main instrument: CFX96 TMFluorescence quantitative PCR instrument (BIO-RAD, USA); Tianlong extractor GeneRotex96; high-speed centrifuge LEGEND MICRO 21

[0041] 1.2. Methods

[0042] 1.2.1 Primers, probes, and positive plasmid sequences

[0043] The BS, LEP, and NTNV gene sequences were downloaded from the NCBI database. Primer and probe sequences were aligned and screened using the bioinformatics software BLAST and Primer Premier 5.0. Upstream and downstream primers were designed from conserved regions upstream and downstream of the target gene. A probe specifically binding to the target gene was then designed between the upstream and downstream primers. The corresponding primer and probe sequences are shown in Table 1.

[0044] Table 1: BS, Lep, HTNV multiplex fluorescence quantitative PCR amplification probes and primers

[0045] Tab le1 Probes and pr imers of tr ip lexrea lt ime PCR for detection of BS / LEp / HTNV

[0046]

[0047]

[0048] 1.2.2 Preparation of plasmid positive template

[0049] 4 μg of each of the three synthetic plasmids BS, LEP, and HTNV were dissolved in 40 μL ddH2O. The plasmid copy number concentrations were calculated according to the formula to be 4.9×10 11 , 6.3×10 11 , 5.1×10 11 . formula:

[0050] Copy number = (6.02 x 10 23 )x(ng / μl x 10 -9 ) / (DNA length x 660)=copies / μl Note: 6.02x 10 23 represents the number of molecular copies of 1 mole of substance; DNA length represents the total length of the plasmid and the target fragment; 660 represents the average molecular weight of double-stranded DNA.

[0051] 1.2.3 Optimization of multiplex fluorescence PCR reaction conditions

[0052] The multiplex PCR was optimized for 25 μL reaction system. 6 A positive plasmid containing 100 copies / μL of DNA was used as a template. Primer and probe concentrations were screened using a matrix-based approach. The optimal annealing temperature, which resulted in high fluorescence intensity, minimum threshold cycle Ct values, maximum amplification efficiency, and no nonspecific signal generation, was determined for reaction optimization. The optimal reaction conditions were defined as those that produced a typical S-shaped amplification curve and a minimum threshold cycle Ct value. The optimal reaction system was confirmed to be: 5 μL reaction solution; 1 μL, 1 μL, and 1 μL of the BS, LEP, and HNTV upstream and downstream primers (10 μmol / L), respectively; and 1 μL, 1 μL, and 1 μL of the probe (10 μmol / L), respectively; 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, followed by 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.

[0053] 1.2.4 Optimization of amplification procedures

[0054] Three groups of annealing temperatures were set for the multiplex fluorescence quantitative PCR amplification program, which were set to 54°C, 55°C, and 56°C respectively. The concentration was selected as 10 6 The amplification protocol was optimized using plasmid standards containing BS, LEP, and HNTV at a low copy number / μL as templates. The reaction conditions included reverse transcription at 50°C for 15 minutes, pre-denaturation at 95°C for 3 minutes, denaturation at 95°C for 5 seconds, and 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. The results are shown in Table 2. Different annealing temperatures affected the amplification performance of multiplex fluorescence quantitative PCR. An annealing temperature of 55°C produced the optimal amplification curves and cycle threshold (Ct) values ​​for BS, LEP, and HNTV.

[0055] Table 2: Effect of annealing temperature on multiplex fluorescence quantitative PCR amplification

[0056]

[0057] 1.2.5 Multiplex fluorescence PCR specificity test The 7 bacterial samples and 6 virus-positive samples in 1.1.1 were used as reaction templates to verify the specificity of the established triple fluorescence quantitative PCR method.

[0058] 1.2.6 Multiplex fluorescence PCR sensitivity test selected a concentration gradient of 10 2 copies /

[0059] μL-10 8Multiple sensitivity tests were performed using a standard plasmid with 100 copies / μL as a positive template and ddH2O as a negative control template.

[0060] 1.2.7 Multiplex fluorescence PCR repeatability test results were selected from 10 5 copies / μL-10 3 The standard template with 100 copies / μL was used for repeated experiments within and between groups.

[0061] 1.2.8 Preliminary application of multiplex PCR: 27 blood samples were collected from hospitalized patients with brucellosis, 50 blood samples from patients with a history of contact with cattle, sheep, pigs, and dogs, and symptoms such as fever and pain, as well as 101 mouse viscera (liver, lung, and kidney). After extracting the nucleic acid from the samples, a triple fluorescence PCR was established for detection, and the results were compared with the single fluorescence PCR results of the reagent manufacturer.

[0062] 2. Results

[0063] 2.1 Specificity test results

[0064] Use the bacterial and viral samples described in 1.1.1 to verify the specificity of the established triplex fluorescence quantitative PCR method.

[0065] 2.2 Establishment of standard curve and sensitivity test results

[0066] The standards constructed by BS, LEP and HTNV were mixed and diluted with ddH2O water to a concentration gradient of 10 8 copies / μL-10 2 Multiplex fluorescence PCR amplification was performed with 100 copies / μL standard template, and each concentration was repeated 3 times. The average Ct value of the experimental results was taken. The data of 8 concentrations were selected, and the Ct value was used as the vertical axis (Y) and the logarithm of the copy number of the standard was used as the horizontal axis (X). Excel logarithm was used for analysis to establish a standard curve.

[0067] Note: 8 bits 1*10 8 copies / μL; 7 is 1*10 7 copies / μL; 1*6 is 10 6 copies / μL; 1*5 is 10 5 copies / μL; 1*4 is 1*10 4 copies / μL; 1*3 is 10 3 copies / μL; 2 is 5*10 2 copies / μL; 1 is 2*10 2 copies / μL

[0068] 2.3 Repeatability test results

[0069] Select 10 5 copies / μL-10 3 The results of intra-group and inter-group repeat tests (Table 3) showed that the coefficients of variation within and between groups were all below 3%, indicating that the method has good reproducibility.

[0070] Table 3 Repeatability test results

[0071]

[0072] 2.4 Preliminary application of clinical samples and mouse visceral samples

[0073] Fifty blood specimens were collected from patients with a history of exposure to cattle, sheep, pigs, and dogs, and who presented with symptoms such as fever and pain. Using a newly developed multiplex fluorescent PCR assay, 5 samples tested positive for BS, while LEP and HTNV were negative, for a positive rate of 10.0% (5 / 50). Multiplex fluorescent PCR assays were also performed on 101 murine visceral specimens, detecting pathogenic Leptospira in 7 samples, for a positive rate of 6.93%. Neither Brucella nor Hantavirus were detected. Comparison was also performed with the manufacturer's single fluorescent PCR results, which showed a 100% concordance rate.

[0074] The above describes the triple-fold natural epidemic disease fluorescence 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 concept. 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 also fall within the scope of protection of the claims of the present invention.

Claims

1. 3 Fluorescence quantitative PCR detection kit for severe natural epidemic diseases, characterized in that: It includes a cylindrical outer box body and three inner box bodies arranged in the outer box body. The outer box body has a accommodating cavity. The cavity wall of the accommodating cavity is provided with three circumferentially extending annular grooves and the cavity wall of the accommodating cavity is provided with vertical slide grooves. The slide grooves extend from the top of the accommodating cavity to the annular grooves at the bottom. The outer side walls of the three inner box bodies are provided with bosses. The bosses on the outer box body are aligned with the slide grooves and slide vertically into the accommodating cavity and rotated so that the bosses are stuck in the annular grooves, wherein the bosses of the three inner box bodies are respectively stuck in the three annular grooves. A placement plate is provided in the inner box body. A number of circular jacks are provided on the placement plate. Detection reagent tubes or reaction tubes are inserted in the jacks. The detection reagent tubes in the three inner box bodies respectively store three detection reagents for three natural epidemic diseases.

2. The triple natural epidemic disease fluorescence quantitative PCR detection kit according to claim 1, characterized in that: There are two groups of sliding grooves and bosses, which are symmetrical about the central axis of the outer box body and the inner box body respectively.

3. The triple natural epidemic disease fluorescence quantitative PCR detection kit according to claim 1, characterized in that: An annular elastic gasket is embedded on the side wall of the insertion hole, and the reagent tube or the reaction tube passes through the middle through hole of the elastic gasket and is pressed tightly.

4. The triple natural epidemic disease fluorescence quantitative PCR detection kit according to claim 1, characterized in that: The inner box body comprises an inner bottom shell and an inner upper cover, a sealing gasket is embedded in the inner upper cover, and the inner upper cover is threadedly connected to the inner bottom shell.

5. The triple natural epidemic disease fluorescence quantitative PCR detection kit according to claim 4, characterized in that: The placement plate is located in the bottom shell, a socket is provided at the center of the placement plate, and a plurality of sockets arranged in an array are provided on the periphery of the center of the placement plate.

6. The triple natural epidemic disease fluorescence quantitative PCR detection kit according to claim 5, characterized in that: A concave groove is provided on the top of the inner cover body, a raised mounting platform is provided in the middle of the groove, and an inner handle is hinged at the mounting platform.

7. The triple natural epidemic disease fluorescence quantitative PCR detection kit according to claim 1, characterized in that: The height of the boss is the same as the width of the annular groove, and the width of the sliding groove is greater than the width of the boss.

8. The triple natural epidemic disease fluorescence quantitative PCR detection kit according to claim 7, characterized in that: When the inner box bodies are installed in the accommodating cavity, adjacent inner box bodies abut against each other in the height direction.

9. The triple natural epidemic disease fluorescence quantitative PCR detection kit according to claim 8, characterized in that: The outer diameter of the inner box body is the same as the inner diameter of the outer box body.

10. The triple natural epidemic disease fluorescence quantitative PCR detection kit according to claim 1, characterized in that: The outer box body includes an outer shell and an outer cover. A sealing gasket is provided on the front of the outer cover. The outer cover is threadedly connected to the outer shell and a sealing structure is formed through the sealing gasket. An outer handle is provided on the outer cover.

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