A primer set, microfluidic chip, kit and method for simultaneously detecting four pathogens of prawns

Abstract

The present invention discloses a primer set, a microfluidic chip, a kit and a method for simultaneously detecting four pathogens of shrimp, including a primer set for detecting Enterocytozoon hepatopenaei, a primer set for detecting acute hepatopancreatic necrosis disease, a primer set for detecting decapod iridescent virus, and a primer set for detecting white spot syndrome virus of shrimp. The kit further includes a multi-channel microfluidic chip embedding the primer set, DNA polymerase, 2× reaction buffer, fluorescent dye, mineral oil, standard positive template and negative control. The advantages of the present invention include: it can directly detect four pathogenic microorganisms, namely Enterocytozoon hepatopenaei, acute hepatopancreatic necrosis disease, decapod iridescent virus, and white spot syndrome virus of shrimp simultaneously from complex samples at one time, with the advantages of high throughput, strong specificity, good experimental repeatability, and convenient and rapid operation.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial detection, and particularly to a technique for simultaneously detecting four pathogens of prawns. Background Art

[0002] With the rapid development of the aquaculture industry, the scale of aquaculture has gradually grown, and problems of aquaculture diseases have followed. There are many aquaculture varieties and large yields, resulting in many disease types, complex disease occurrence situations, and easy outbreaks of major epidemic diseases, which are likely to cause devastating blows to the aquaculture industry. At present, China is still in a state of extensive and low-level development. Most aquaculture fishermen focus on treatment rather than prevention, and their concepts of disease prevention and control are backward; our aquatic product pharmaceutical industry is still relatively backward. There are only a very small number of highly effective therapeutic agents for related pathogens on the market, making it difficult to meet the treatment needs of aquatic diseases in China. Therefore, disease prevention and control is particularly important in aquaculture. In the prevention and control of aquaculture diseases, the most basic step is to carry out disease diagnosis, and it is urgent for us to improve the ability of rapid pathogen diagnosis. It is not only limited to single-disease detection, but also the improvement of rapid and efficient diagnostic techniques such as dual or even multiple detections is imminent. Four pathogenic microorganisms, namely Enterocytozoon hepatopenaei (EHP), Acute Hepatopancreatic Necrosis Disease (AHPND), Decapod Iridescent Virus 1 (DIV1), and White Spot Syndrome Virus (WSSV) of prawns, are currently the four major prawn diseases that most trouble the aquaculture industry and pose a potential huge threat to the prawn aquaculture industry in China.

[0003] Loop-mediated isothermal amplification (LAMP) technology is a new type of isothermal nucleic acid amplification method, which is an improvement on the traditional PCR technology. Four or six different specific primers are designed for 6 or 8 sites of the target gene. Under the action of a strand displacement active DNA polymerase (Bst DNA polymerase), rapid amplification of DNA is achieved under constant temperature conditions (about 65°C), and 10 9 ~10 10 copies of the target sequence can be obtained within 1 h. This method is simple to operate, fast in detection, high in sensitivity, and strong in specificity. However, it can only perform single-index single-reaction tests, and it is relatively time-consuming and laborious to perform multi-index tests on the same sample.

[0004] Microfluidics technology integrates the entire reaction and analysis processes of biology, chemistry, and medicine onto a single chip, automatically completing the entire complex experimental process that was previously carried out in a laboratory. This includes basic operation processes such as sample preparation, reaction, separation, and detection. Due to its great potential in the fields of biology, chemistry, medicine, etc., it has developed into a brand-new research field that intersects multiple disciplines such as biology, chemistry, medicine, fluidics, electronics, materials, and machinery. Microdroplet chip technology is developed based on traditional single-phase microfluidics chip technology and is a two-phase system based on the immiscible characteristics of oil and water droplets. Compared with single-phase microfluidic systems, it has more advantages, such as smaller droplets, reaching the sub-microliter level, consuming less sample and reagent, no loss of sample volume, faster mixing speed, less likely to cause cross-contamination, easy to manipulate, enabling the system to have digital characteristics and being easy to detect, etc. Summary of the Invention

[0005] The object of the present invention is to provide a primer set, a microfluidic chip, a kit, and a method for simultaneously detecting four pathogens of penaeid shrimp, so as to solve the limitation problems in multiple detection and diagnostic levels in the prior art.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a primer set for simultaneously detecting four pathogens of penaeid shrimp, including:

[0008] Outer primers, inner primers, and loop primers designed according to the 18S rRNA specific gene of Enterocytozoon hepatopenaei:

[0009] The sequence of its outer primers is shown as SEQ ID NO:1~SEQ ID NO:2;

[0010] The sequence of its inner primers is shown as SEQ ID NO:3~SEQ ID NO:4;

[0011] The sequence of its loop primers is shown as SEQ ID NO:5~SEQ ID NO:6.

[0012] Outer primers, inner primers, and loop primers designed according to the pirA specific gene of acute hepatopancreatic necrosis disease:

[0013] The sequence of its outer primers is shown as SEQ ID NO:7~SEQ ID NO:8;

[0014] The sequence of its inner primers is shown as SEQ ID NO:9~SEQ ID NO:10;

[0015] The sequence of its loop primers is shown as SEQ ID NO:11~SEQ ID NO:12.

[0016] Outer primers, inner primers, and loop primers designed based on the ATPase-specific gene of decapod iridovirus:

[0017] The sequences of its outer primers are shown in SEQ ID NO: 13 to SEQ ID NO: 14;

[0018] The sequences of its inner primers are shown in SEQ ID NO: 15 to SEQ ID NO: 16;

[0019] The sequences of its loop primers are shown in SEQ ID NO: 17 to SEQ ID NO: 18.

[0020] Outer primers, inner primers, and loop primers designed based on the specific gene of white spot syndrome virus of shrimp:

[0021] The sequences of its primers are shown in SEQ ID NO: 19 to SEQ ID NO: 20;

[0022] The sequences of its inner primers are shown in SEQ ID NO: 21 to SEQ ID NO: 22;

[0023] The sequences of its loop primers are shown in SEQ ID NO: 23 to SEQ ID NO: 24.

[0024] The present invention also provides a multi-channel microfluidic chip embedded with the primer set.

[0025] Furthermore, the molar ratio of the outer primers, inner primers, and loop primers designed based on the 18S rRNA specific gene of Enterocytozoon hepatopenaei is 1-2:4-8:2-4;

[0026] The molar ratio of the outer primers, inner primers, and loop primers designed based on the pirA specific gene causing acute hepatopancreatic necrosis disease is 1-2:4-8:2-4;

[0027] The molar ratio of the outer primers, inner primers, and loop primers designed based on the ATPase specific gene of decapod iridovirus is 1-2:4-8:2-4;

[0028] The molar ratio of the outer primers, inner primers, and loop primers designed based on the specific gene of white spot syndrome virus of shrimp is 1-2:4-8:2-4.

[0029] Furthermore, in the multi-channel microfluidic chip, the embedding amount of each pair of outer primers for each pathogen is 0.2 μM, the embedding amount of each pair of inner primers is 0.8 μM, and the embedding amount of each pair of loop primers is 0.4 μM.

[0030] The present invention also provides a kit containing the multi-channel microfluidic chip described above. The kit further includes DNA polymerase, 2× reaction buffer, fluorescent dye, sealing liquid, standard positive template and negative control.

[0031] Preferably, the DNA polymerase is Bst DNA polymerase;

[0032] The 2× reaction buffer consists of 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 8 mM MgSO 4 , 10 mM (NH 4 ) 2 SO 4 , 0.1% Tween 20, 1 M betaine, 6 mM MgSO 4 , 1.6 mM dNTP;

[0033] The fluorescent dye is 0.02 mM SYTO-9;

[0034] The sealing liquid is mineral oil;

[0035] The standard positive template is a mixed plasmid DNA containing specific target genes of four pathogenic microorganisms: Enterocytozoon hepatopenaei, Acute Hepatopancreatic Necrosis Disease, Decapod Iridescent Virus, and White Spot Syndrome Virus of Shrimp;

[0036] The negative control is sterilized ultrapure water.

[0037] The present invention also provides the application of a kit containing a multi-channel microfluidic chip in detecting Enterocytozoon hepatopenaei, Acute Hepatopancreatic Necrosis Disease, Decapod Iridescent Virus, and White Spot Syndrome Virus of Shrimp.

[0038] The present invention also provides a reaction system using the multi-channel microfluidic chip described above, which further includes reaction reagents. The reaction reagents are: 12.5 μL of 2× reaction solution, 8 U of DNA polymerase, 0.5 μL of 0.02 mM SYTO-9, 2 μL of the sample to be tested, and add sterilized ultrapure water to make up to 25 μL;

[0039] The reaction conditions are 63°C for 15 s, 60°C for 45 s, for 30 cycles.

[0040] More preferably, the 2× reaction buffer consists of 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 8 mM MgSO 4 , 10 mM (NH 4 ) 2 SO 4 , 0.1% Tween 20, 1 M betaine, 6 mM MgSO 4 , 1.6 mM dNTP.

[0041] The present invention also provides an application of the primer set in a kit.

[0042] The present invention determines the test result according to the amplification curve. According to the curve color of the amplification index displayed by the instrument, if the amplification curve is in an "S" shape, the test result is positive, that is, the test sample contains the pathogen corresponding to the index; if no "S" - shaped amplification curve appears, the test result is negative, that is, the test sample does not contain the pathogen corresponding to the index.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] (1) Good specificity: The target genes used for primer design in the present invention are specific fragments of each pathogen. Six specific primers are designed for amplification of the target fragment, with strong specificity. There is no cross - detection among the primer sets of the four pathogens, and there is no amplification for other common shrimp viruses.

[0045] (2) High - throughput: Only one test is required to quickly obtain the positive and negative results of four pathogenic microorganisms, namely Enterocytozoon hepatopenaei (EHP), Acute hepatopancreatic necrosis disease (AHPND), Decapod iridescent virus 1 (DIV1), and White spot syndrome virus (WSSV) in shrimp at the same time.

[0046] (3) High sensitivity: For the mixed positive plasmid containing the detection target genes of these four pathogenic microorganisms, the lowest detection limit can reach 10 2 copies / μL;

[0047] (4) Simple operation and direct and objective results: The operation steps are simple. It can directly detect Enterocytozoon hepatopenaei (EHP), Acute hepatopancreatic necrosis disease (AHPND), Decapod iridescent virus 1 (DIV1), and White spot syndrome virus (WSSV) from complex samples. By observing the amplification curve, the positive and negative can be directly judged. The results are direct and objective, without the need for cumbersome electrophoresis and other analysis steps, and have low requirements for the experience of operators, being suitable for rapid and accurate on - site detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] The drawings described herein are used to provide a further understanding of the present invention, form a part of this application, and do not constitute an improper limitation to the present invention. In the drawings:

[0049] Figure 1 It is a schematic diagram of the results of the sample for detecting Enterocytozoon hepatopenaei (EHP) by the isothermal amplification microfluidic chip method in Example 1.

[0050] Figure 2 It is a schematic diagram of the results of the sample for detecting Acute hepatopancreatic necrosis disease (AHPND) by the isothermal amplification microfluidic chip method in Example 1.

[0051] Figure 3Schematic diagram of the results of detecting samples of Decapod iridovirus (DIV1) by the isothermal amplification microfluidic chip method in Example 1.

[0052] Figure 4 Schematic diagram of the results of detecting samples of white spot syndrome virus (WSSV) of shrimp by the isothermal amplification microfluidic chip method in Example 1.

[0053] Figure 5 For the results of detecting the positive control of the mixed plasmid DNA of four pathogens with a concentration of 10 4 copies / μL by the isothermal amplification microfluidic chip method in Example 2.

[0054] Figure 6 For the results of detecting the positive control of the mixed plasmid DNA of four pathogens with a concentration of 10 3 copies / μL by the isothermal amplification microfluidic chip method in Example 2.

[0055] Figure 7 For the results of detecting the positive control of the mixed plasmid DNA of four pathogens with a concentration of 10 2 copies / μL by the isothermal amplification microfluidic chip method.

[0056] Figure 8 Schematic diagram of the results of detecting samples of infectious hypodermal and hematopoietic necrosis virus of shrimp by the isothermal amplification microfluidic chip method.

[0057] Figure 9 Schematic diagram of the results of detecting samples of baculovirus of shrimp by the isothermal amplification microfluidic chip method.

[0058] Figure 10 Schematic diagram of the results of detecting samples of acute hepatopancreatic necrosis virus of shrimp by the isothermal amplification microfluidic chip method.

[0059] Figure 11 Schematic diagram of the results of detecting samples of Vibrio parahaemolyticus by the isothermal amplification microfluidic chip method.

[0060] Figure 12 Schematic diagram of the results of detecting samples of Vibrio harveyi by the isothermal amplification microfluidic chip method.

[0061] Figure 13 Schematic diagram of a multi-channel microfluidic chip. Detailed implementation manners

[0062] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. Here, the illustrative embodiments and descriptions of the present invention are used to explain the present invention, but not to limit the present invention.

[0063] Example 1

[0064] I. Composition of the kit:

[0065] It includes a set of LAMP primer sets designed according to the specific gene of Enterocytozoon hepatopenaei (EHP) 18S rRNA, a set of LAMP primer sets designed according to the specific gene of pirA of Acute Hepatopancreatic Necrosis Disease (AHPND), a set of LAMP primer sets designed according to the specific gene of ATPase of Decapod iridescent virus (DIV1), a set of LAMP primer sets for detecting the specific gene of White Spot Syndrome Virus (WSSV) of shrimp, a multi-channel microfluidic chip, DNA polymerase, 2× reaction buffer, fluorescent dye, mineral oil, standard positive template and negative control.

[0066] Among them, for the LAMP primer set for detecting Enterocytozoon hepatopenaei (EHP), the sequences of the outer primers are as shown in SEQ ID NO:1 to SEQ ID NO:2, the sequences of the inner primers are as shown in SEQ ID NO:3 to SEQ ID NO:4, and the sequences of the loop primers are as shown in SEQ ID NO:5 to SEQ ID NO:6. For the primer set for detecting Acute Hepatopancreatic Necrosis Disease, the sequences of the outer primers are as shown in SEQ ID NO:7 to SEQ ID NO:8, the sequences of the inner primers are as shown in SEQ ID NO:9 to SEQ ID NO:10, and the sequences of the loop primers are as shown in SEQ ID NO:11 to SEQ ID NO:12. For the primer set for detecting Decapod iridescent virus, the sequences of the outer primers are as shown in SEQ ID NO:13 to SEQ ID NO:14, the sequences of the inner primers are as shown in SEQ ID NO:15 to SEQ ID NO:16, and the sequences of the loop primers are as shown in SEQ ID NO:17 to SEQ ID NO:18. For the primer set for detecting White Spot Syndrome Virus of shrimp, the sequences of the outer primers are as shown in SEQ ID NO:19 to SEQ ID NO:20, the sequences of the inner primers are as shown in SEQ ID NO:21 to SEQ ID NO:22, and the sequences of the loop primers are as shown in SEQ ID NO:23 to SEQ ID NO:24. Synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0067] LAMP primer set for detecting Enterocytozoon hepatopenaei (EHP):

[0068] Outer primer OF (SEQ ID NO:1): 5’-TCCGTAGTCGTGGATGC-3’;

[0069] Outer primer OB (SEQ ID NO:2): 5’-CCAATCTACGATAGACTTGACC-3’;

[0070] Inner primer IF (SEQ ID NO:3): 5’-GAACTACAGCGGTGTCTAATCACTTACCTGGACCAACGGAG-3’;

[0071] Inner primer IB (SEQ ID NO:4):

[0072] 5’-AGAGCGATGCTTGGTGTGGACTCCTGGTAGTGTCCTTC-3’;

[0073] Loop primer LF (SEQ ID NO:5): 5’-CTCTAGCCTTCGTCCTTGATC-3’;

[0074] Loop primer LB (SEQ ID NO:6): 5’-GGATAGTACGCTCGCAAGG-3’;

[0075] LAMP primer set for detecting acute hepatopancreatic necrosis disease:

[0076] Outer primer OF (SEQ ID NO:7): 5’-ACAGAAGTAGACAGCAAACA-3’;

[0077] Outer primer IB (SEQ ID NO:8): 5’-GCATTATCAGGGCGTTGT-3’;

[0078] Inner primer IF (SEQ ID NO:9): 5’-TGGTATTGAATGGTAAGCTCCCCCACCTATCATCCCGGAAGT-3’;

[0079] Inner primer OB (SEQ ID NO:10):

[0080] 5’-AATGGGGTGCGCCATTTATGGAAGTTTCATCACGTTGTACC-3’;

[0081] Loop primer LF (SEQ ID NO:11): 5’-TCTCAATGTCTACACTACGACCG-3’;

[0082] Loop primer LB (SEQ ID NO:12): 5’-TGGCGGCTGGAAAGTGG-3’;

[0083] LAMP primer set for detecting decapod iridovirus (DIV1):

[0084] Outer primer OF (SEQ ID NO:13): 5’-GGCTTGGTATCTTATTCAGAGAT-3’;

[0085] Outer primer OB (SEQ ID NO:14): 5’-ATTCACAACATCGTCACCAT-3’;

[0086] Inner primer IF (SEQ ID NO:15): 5’-CTCTTGATGGATACACTGATCTTCGTTGTAGAGCCAGAGATTGTAAC-3’;

[0087] Inner primer IB (SEQ ID NO:16):

[0088] 5’-ATTCAGTATTCAAGGATTGGTTCAAAAGTTCTTCCATCTACCTCTC-3’;

[0089] Loop primer LF (SEQ ID NO:17): 5’-TTCGGTACGAAGATGTAGC-3’;

[0090] Loop primer LB (SEQ ID NO:18): 5’-GAAGAGTATCCTAATATGACCATCC-3’;

[0091] LAMP primer set for detecting white spot syndrome virus (WSSV) of penaeid shrimp:

[0092] Outer primer OF (SEQ ID NO:19): 5’-TGATTCAGATGGCATGGATACTT-3’;

[0093] Outer primer OB (SEQ ID NO:20): 5’-CCGATACTGCCATTGAAAGC-3’;

[0094] Inner primer IF (SEQ ID NO:21): 5’-TGTTATGGTAGTGAACCCCTTTGCACGACTTATCATTCAAGACATCAAT-3’;

[0095] Inner primer IB (SEQ ID NO:22):

[0096] 5’-GGAAGAAAGATACAAGCCCATTGGCGCTCCCTTACCACCTTCCTTAATC-3’;

[0097] Loop primer LF (SEQ ID NO:23): 5’-GATCGTTAACAACAACAATACTGGA-3’;

[0098] Loop primer LB (SEQ ID NO:24): 5’-GCCATTGAAGCAGTGTTGGGAT-3’;

[0099] The multi-channel microfluidic chip is embedded with dry powder of primers for the above four pathogens. By molar ratio, the proportion of dry powder of primers for each pathogen is outer primer: inner primer: loop primer = 1:4:2. The embedded amount of a pair of outer primers is 0.2 μM, the embedded amount of a pair of inner primers is 0.8 μM, and the embedded amount of a pair of loop primers is 0.4 μM.

[0100] The microfluidic chip includes a cover plate and a substrate. The substrate is provided with an electrical connection port, an electrode assembly, and electrode leads. The electrical connection port sends a voltage signal to the electrode assembly through the electrode leads. The cover plate is arranged above the substrate. The cover plate and the substrate are hermetically connected and form a cavity. A droplet barrier is arranged between the cover plate and the substrate. The cover plate is provided with an oil inlet and more than two sample inlets.

[0101] For the microfluidic chip, one side of the substrate where the electrode assembly is arranged is coated with an insulating coating. The electrode assembly includes more than two electrode units arranged at intervals. One electrode unit includes a main infusion electrode channel, a liquid storage electrode, and a detection electrode group. One sample inlet is located directly above one liquid storage electrode. Primer dry powder is pre-stored on the detection electrode group. A main infusion electrode channel is connected between one liquid storage electrode and one detection electrode group.

[0102] The DNA polymerase is Bst DNA polymerase.

[0103] The 2× reaction buffer is composed of 20 mM Tris-HCl (PH8.8), 10 mM KCl, 8 mM MgSO 4 , 10 mM (NH 4 ) 2 SO 4 , 0.1% Tween 20, 1 M betaine, 6 mM MgSO 4 , and 1.6 mM dNTP.

[0104] The fluorescent dye is 0.02 mM SYTO-9.

[0105] The standard positive template is a mixed plasmid DNA containing specific target genes of four pathogenic microorganisms, namely Enterocytozoon hepatopenaei (EHP), Acute Hepatopancreatic Necrosis Disease (AHPND), Decapod Iridescent Virus 1 (DIV1), and White Spot Syndrome Virus (WSSV).

[0106] The negative control is sterilized ultrapure water.

[0107] II. Detection method

[0108] 1. Grind the sample to a uniform state, add 400 μL of lysis buffer to a centrifuge tube, and mix well by shaking for 30 s. Let it stand at room temperature for 5 - 10 min, and centrifuge at 10000 rpm for 3 min to remove impurities.

[0109] 2. Take out the nucleic acid adsorption column sleeve in the kit, transfer as much supernatant obtained in step 1 as possible to the nucleic acid adsorption column, and centrifuge at 10000 rpm for 1 min. Discard the filtrate, then put the nucleic acid adsorption column back into the collection tube, add 500 μL of washing buffer to the nucleic acid adsorption column, and centrifuge at 10000 rpm for 1 min. Then add 400 μL of washing buffer, centrifuge at 10000 rpm for 1 min, discard the filtrate, centrifuge at 10000 rpm for 3 min without adding anything, transfer the nucleic acid adsorption column to a new 1.5 mL centrifuge tube, and add 100 μL of elution buffer to the center of the membrane of the nucleic acid adsorption column. Let it stand at room temperature for 1 min, and centrifuge at 10000 rpm for 1 min.

[0110] 3. Discard the nucleic acid adsorption column to complete the extraction. The nucleic acid extract can be stored at -20 °C for standby or stored at -80 °C for long term.

[0111] 4. Perform PCR reaction

[0112] (1) Prepare the reaction reagents: 12.5 μL of 2× reaction buffer, 8 U of DNA polymerase, 0.5 μL of 0.02 mM SYTO-90, 2 μL of the sample to be tested, and make up to 25 μL with sterilized ultrapure water;

[0113] (2) Tear open the vacuum package of the microfluidic chip along the packaging notch to take it out, and put the taken-out microfluidic chip into the supporting metal holder with the glass side facing up;

[0114] (3) Use a pipette with a capacity of 1000 μL to aspirate about 500 μL of mineral oil, and slowly add it from the oil inlet of the microfluidic chip with the pipette tip tilted at 45°;

[0115] (4) Use a pipette to aspirate 20 μL of the reaction reagents prepared in step (1), and slowly add it from the arc-shaped sample inlet of the microfluidic chip with the pipette tip tilted at 45°;

[0116] (5) Perform on-machine detection: Insert the chip with samples added together with the holder into the card slot of the detection device. Note that the device will prompt that the chip is successfully connected through a beep and a warning light. Then enter the sample name and the name of the tester, select the reaction program corresponding to the chip kit, and start detecting the extracted DNA by clicking the <Start Detection> button on the software page.

[0117] The reaction program is: 63 °C for 15 s, 60 °C for 45 s, for 30 cycles.

[0118] III. Result Judgment

[0119] The actual samples of Enterocytozoon hepatopenaei (EHP) were detected by the above method, and the results are as Figure 1 shown. The samples showed typical "S"-shaped curve amplification, which was a positive result, proving that Enterocytozoon hepatopenaei (EHP) was detected in the samples. Conversely, the result was negative.

[0120] The actual samples of Acute Hepatopancreatic Necrosis Disease (AHPND) were detected by the above method, and the results are as Figure 2 shown. The samples showed typical "S"-shaped curve amplification, which was a positive result, proving that Acute Hepatopancreatic Necrosis Disease (AHPND) was detected in the samples. Conversely, the result was negative.

[0121] The actual samples of Decapod Iridescent Virus 1 (DIV1) were detected by the above method, and the results are as Figure 3 shown. The samples showed typical "S"-shaped curve amplification, which was a positive result, proving that Decapod Iridescent Virus 1 (DIV1) was detected in the samples. Conversely, the result was negative.

[0122] The actual samples of White Spot Syndrome Virus (WSSV) of penaeid shrimp were detected by the above kit, and the results are as Figure 4 shown. The samples showed typical "S"-shaped curve amplification, which was a positive result, proving that White Spot Syndrome Virus (WSSV) of penaeid shrimp was detected in the samples. Conversely, the result was negative.

[0123] Example 2 Sensitivity Experiment

[0124] The standard positive template (mixed plasmid DNA containing specific target genes of four pathogenic microorganisms, namely Enterocytozoon hepatopenaei (EHP), Acute Hepatopancreatic Necrosis Disease (AHPND), Decapod Iridescent Virus 1 (DIV1), and White Spot Syndrome Virus (WSSV) of penaeid shrimp) was diluted by 10-fold gradient, and DNA at three gradient concentrations of 10 4 copies / μL, 10 3 copies / μL, and 10 2 copies / μL were used as templates, and the detection was carried out with reference to the method of Example 1.

[0125] As Figures 5 to 7 shown, in the detection tests of each gradient concentration, the specific target genes of each pathogenic microorganism could all show typical "S"-shaped curve amplification. The results showed that when the mixed plasmid DNA with an initial concentration of 10 5 copies / μL was diluted by gradient, the detection limit of the microfluidic detection kit for detecting four pathogens of penaeid shrimp established in Example 1 could reach a concentration of 10 2 copies / μL.

[0126] Example 3 Specificity Experiment

[0127] Refer to the method of Example 1 to detect the DNA of samples of Taura syndrome virus, Baculovirus penaei, Penaeus vannamei nodavirus, Vibrio parahaemolyticus, and Vibrio harveyi respectively.

[0128] As Figures 8 to 12 shown, no amplification occurred in the samples, indicating that the established microfluidic detection kit for detecting four pathogens of shrimp has good specificity.

[0129] The technical solutions provided by the embodiments of the present invention have been introduced in detail above. Specific examples are used in this article to elaborate on the principles and implementation manners of the embodiments of the present invention. The descriptions of the above embodiments are only applicable to helping understand the principles of the embodiments of the present invention; at the same time, for those of ordinary skill in the art, according to the embodiments of the present invention, there will be changes in the specific implementation manners and application scopes. In summary, the content of this specification should not be construed as a limitation to the present invention.

Claims

1. A primer set for simultaneously detecting four pathogens of penaeid shrimp, characterized in that, it includes: Outer primers, inner primers, and loop primers designed according to the 18S rRNA specific gene of Enterocytozoon hepatopenaei: The sequences of its outer primers are shown as SEQ ID NO:1 to SEQ ID NO:2; The sequences of its inner primers are shown as SEQ ID NO:3 to SEQ ID NO:4; The sequences of its loop primers are shown as SEQ ID NO:5 to SEQ ID NO:6; Outer primers, inner primers, and loop primers designed according to the pirA specific gene causing acute hepatopancreatic necrosis disease: The sequences of its outer primers are shown as SEQ ID NO:7 to SEQ ID NO:8; The sequences of its inner primers are shown as SEQ ID NO:9 to SEQ ID NO:10; The sequences of its loop primers are shown as SEQ ID NO:11 to SEQ ID NO:12; Outer primers, inner primers, and loop primers designed according to the ATPase specific gene of decapod iridovirus: The sequences of its outer primers are shown as SEQ ID NO:13 to SEQ ID NO:14; The sequences of its inner primers are shown as SEQ ID NO:15 to SEQ ID NO:16; The sequences of its loop primers are shown as SEQ ID NO:17 to SEQ ID NO:18; Outer primers, inner primers, and loop primers designed according to the specific gene of white spot syndrome virus of penaeid shrimp: The sequences of its outer primers are shown as SEQ ID NO:19 to SEQ ID NO:20; The sequences of its inner primers are shown as SEQ ID NO:21 to SEQ ID NO:22; The sequences of its loop primers are shown as SEQ ID NO:23 to SEQ ID NO:

24.

2. A multi-channel microfluidic chip embedding the primer set described in claim 1.

3. The multi-channel microfluidic chip according to claim 2, characterized in that: The molar ratio of the outer primers, inner primers, and loop primers designed according to the 18S rRNA specific gene of Enterocytozoon hepatopenaei is 1 - 2:4 - 8:2 - 4; The molar ratio of the outer primers, inner primers, and loop primers designed according to the pirA specific gene causing acute hepatopancreatic necrosis disease is 1 - 2:4 - 8:2 - 4; The molar ratio of the outer primers, inner primers, and loop primers designed according to the ATPase specific gene of decapod iridovirus is 1 - 2:4 - 8:2 - 4; The molar ratio of the outer primers, inner primers, and loop primers designed according to the specific gene of white spot syndrome virus of penaeid shrimp is 1 - 2:4 - 8:2 - 4.

4. The multi-channel microfluidic chip according to claim 3, characterized in that: For each pathogen in the multi-channel microfluidic chip, the embedding amount of a pair of outer primers is 0.2 μM, the embedding amount of a pair of inner primers is 0.8 μM, and the embedding amount of a pair of loop primers is 0.4 μM.

5. A kit containing the multi-channel microfluidic chip according to any one of claims 2 - 4, characterized in that: The kit further includes DNA polymerase, 2× reaction buffer, fluorescent dye, sealing liquid, standard positive template and negative control.

6. A kit containing a multi-channel microfluidic chip according to claim 5, characterized in that: the DNA polymerase is Bst DNA polymerase; The 2× reaction buffer consists of 20 mM Tris-HCl pH 8.8, 10 mM KCl, 8 mM MgSO 4 , 10 mM (NH 4 ) 2 SO 4 , 0.1% Tween 20, 1 M betaine, 6 mM MgSO 4 , and 1.6 mM dNTPs; the fluorescent dye is 0.02 mM SYTO-9; the sealing liquid is mineral oil; the standard positive template is a mixed plasmid DNA containing specific target genes of four pathogenic microorganisms, namely Enterocytozoon hepatopenaei, Acute Hepatopancreatic Necrosis Disease, Decapod iridescent virus, and White Spot Syndrome Virus of shrimp; the negative control is sterilized ultrapure water.

7. A reaction system using the multi-channel microfluidic chip according to any one of claims 2 to 4, characterized in that: it further includes reaction reagents, and the reaction reagents are: 12.5 μL of 2× reaction solution, 8 U of DNA polymerase, 0.5 μL of 0.02 mM SYTO-9, 2 μL of the sample to be tested, and add sterilized ultrapure water to 25 μL; the reaction conditions are 63°C for 15 s, 60°C for 45 s, for 30 cycles.

8. The reaction system using the multi-channel microfluidic chip according to claim 7, characterized in that: The 2× reaction buffer consists of 20 mM Tris-HCl pH 8.8, 10 mM KCl, 8 mM MgSO 4 , 10 mM (NH 4 ) 2 SO 4 , 0.1% Tween 20, 1 M betaine, 6 mM MgSO 4 , and 1.6 mM dNTPs.

9. Use of the primer set according to claim 1 in the preparation of a kit.

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