Primer probe combination for detecting multiple prawn pathogens and application thereof

Abstract

The invention discloses a primer probe combination for detecting various prawn pathogens and application of the primer probe combination. The primer probe combination comprises a first primer probe set for detecting vibrio harveyi, a second primer probe set for detecting infectious subcutaneous and hematopoietic necrosis viruses, a third primer probe set for detecting Taura syndrome viruses and a fourth primer probe set for detecting infectious muscle necrosis viruses. The primer probe combination has the characteristics of high sensitivity, good repeatability and strong specificity, and can be used for specifically detecting vibrio harveyi, infectious subcutaneous and hematopoietic necrosis viruses, Taura syndrome viruses and infectious muscle necrosis viruses.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a primer-probe combination for detecting multiple shrimp pathogens and its application. Background Art

[0002] At present, there are many pathogen species causing shrimp diseases, and the resulting diseases are characterized by strong infectivity, wide spread, high incidence and fatality rates, posing a huge threat to the global shrimp aquaculture industry and causing serious economic losses.

[0003] So far, there are no effective vaccines and therapeutic drugs for most common shrimp diseases. Early detection and elimination of pathogen carriers and cutting off the transmission route are considered the most effective preventive measures. Therefore, early and rapid diagnosis and screening play a decisive role in the prevention and control of shrimp diseases. Currently, a variety of methods for detecting shrimp diseases have been developed, including section detection, PCR detection, LAMP, immunoassay, etc. However, generally, they can only perform nucleic acid detection or isolation and identification on a single pathogen or a few pathogens, with cumbersome operation steps and a long time, which is not conducive to taking rapid countermeasures. Therefore, there is an urgent need for a rapid detection method with high accuracy and wide pathogen coverage. Summary of the Invention

[0004] The present invention aims to at least solve one of the technical problems existing in the prior art. For this purpose, the present invention provides a primer-probe combination for detecting multiple shrimp pathogens.

[0005] The present invention also provides a kit containing the above primer-probe combination.

[0006] The present invention also provides the application of the above primer-probe combination and kit.

[0007] The present invention also provides a system for detecting Vibrio harveyi, Infectious Hypodermal and Hematopoietic Necrosis Virus, Taura Syndrome Virus and / or Infectious Myonecrosis Virus.

[0008] According to the first aspect embodiment of the present invention, a primer-probe combination for detecting multiple shrimp pathogens is provided. The primer-probe combination includes a first primer-probe group for detecting Vibrio harveyi, a second primer-probe group for detecting Infectious Hypodermal and Hematopoietic Necrosis Virus, a third primer-probe group for detecting Taura Syndrome Virus, and a fourth primer-probe group for detecting Infectious Myonecrosis Virus.

[0009] In the first primer-probe group, the primer sequences are as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe sequence is as shown in SEQ ID NO:3.

[0010] In the second primer-probe group, the primer sequences are as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe sequence is as shown in SEQ ID NO:6;

[0011] In the third primer-probe group, the primer sequences are as shown in SEQ ID NO:7 and SEQ ID NO:8, and the probe sequence is as shown in SEQ ID NO:9;

[0012] In the fourth primer-probe group, the primer sequences are as shown in SEQ ID NO:10 and SEQ ID NO:11, and the probe sequence is as shown in SEQ ID NO:12.

[0013] The primer-probe combination for detecting multiple shrimp pathogens according to the embodiments of the present invention has at least the following beneficial effects:

[0014] The primer-probe combination for detecting multiple shrimp pathogens provided by the present invention has the characteristics of high sensitivity, good repeatability and strong specificity, and can be used for specific detection of Vibrio harveyi, Infectious Hypodermal and Hematopoietic Necrosis Virus, Taura Syndrome Virus and Infectious Myonecrosis Virus.

[0015] According to some embodiments of the present invention, both ends of the probe are respectively labeled with a fluorescent reporter group and a fluorescent quenching group.

[0016] According to some embodiments of the present invention, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quenching group.

[0017] According to some embodiments of the present invention, the fluorescent reporter group is selected from FAM, JOE, VIC, HEX, ROX, CY3 or CY5; the fluorescent quenching group is selected from BHQ, TAMRA or MGB.

[0018] According to some embodiments of the present invention, the probes in the first primer-probe group, the second primer-probe group, the third primer-probe group and the fourth primer-probe group have different fluorescent reporter groups.

[0019] According to the embodiments of the second aspect of the present invention, a kit is provided, which includes the primer-probe combination described in the embodiments of the first aspect of the present invention. Since the kit of the embodiments of the present invention adopts all the technical solutions of the primer-probe combination of the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments.

[0020] According to some embodiments of the present invention, the kit further includes at least one of a positive control product, a negative control product, and a PCR premix.

[0021] According to some embodiments of the present invention, the PCR premix includes RT Buffer, Taq / UNG Mix, and reverse transcriptase (RTase).

[0022] According to some embodiments of the present invention, the RT Buffer includes Tris-HCl, dNTPs, MgCl2, KCl, and Triton X-100.

[0023] According to some embodiments of the present invention, the positive control includes a recombinant vector containing nucleic acid molecules of Vibrio harveyi, Infectious Hypodermal and Hematopoietic Necrosis Virus, Taura Syndrome Virus, and / or Infectious Myonecrosis Virus.

[0024] According to some embodiments of the present invention, the negative control includes water.

[0025] According to the embodiments of the third aspect of the present invention, there is provided an application of the above primer-probe combination or kit in any one of the following:

[0026] 1) Detecting or assisting in the detection of Vibrio harveyi for non-diagnostic purposes;

[0027] 2) Preparing a product for detecting or assisting in the detection of Vibrio harveyi;

[0028] 3) Detecting or assisting in the detection of Infectious Hypodermal and Hematopoietic Necrosis Virus for non-diagnostic purposes;

[0029] 4) Preparing a product for detecting or assisting in the detection of Infectious Hypodermal and Hematopoietic Necrosis Virus;

[0030] 5) Detecting or assisting in the detection of Taura Syndrome Virus for non-diagnostic purposes;

[0031] 6) Preparing a product for detecting or assisting in the detection of Taura Syndrome Virus;

[0032] 7) Detecting or assisting in the detection of Infectious Myonecrosis Virus for non-diagnostic purposes;

[0033] 8) Preparing a product for detecting or assisting in the detection of Infectious Myonecrosis Virus.

[0034] According to some embodiments of the present invention, the product is selected from reagents, kits, or chips.

[0035] According to some embodiments of the present invention, the usage method of the product includes the following steps:

[0036] Using the above primer-probe combination or kit to detect the DNA of the sample to be tested.

[0037] According to some embodiments of the present invention, the detection is performed by fluorescence quantitative PCR.

[0038] According to some embodiments of the present invention, the reaction system of the fluorescence quantitative PCR is as follows:

[0039]

[0040]

[0041] According to some embodiments of the present invention, the reaction system of the fluorescence quantitative PCR is as follows:

[0042]

[0043] According to some embodiments of the present invention, the amplification program of the fluorescence quantitative PCR is: 45 - 55°C for 8 - 12 min; 94 - 96°C for 25 - 35 s; then enter the cycling stage: 93 - 96°C for 8 - 12 s, 55 - 65°C for 25 - 35 s, for 40 - 48 cycles, and collect fluorescence data.

[0044] According to some embodiments of the present invention, the amplification program of the fluorescence quantitative PCR is: 50°C for 10 min; 95°C for 30 s; then enter the cycling stage: 95°C for 10 s, 60°C for 30 s, for 45 cycles, and collect fluorescence data.

[0045] In some embodiments of the present invention, after the detection, it further includes the steps of statistically analyzing the Ct value of the sample and interpreting the result; the result interpretation is as follows:

[0046] Positive: The Ct value of the sample test result ≤ 40 or there is an obvious exponential growth phase;

[0047] Negative: The Ct value of the sample test result > 40 or there is no Ct value.

[0048] According to the embodiments of the fourth aspect of the present invention, a method for detecting or assisting in the detection of Vibrio harveyi, infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and / or infectious myonecrosis virus for non-diagnostic purposes is provided, including the following steps:

[0049] Detect the DNA of the sample to be tested using the above primer-probe combination or kit.

[0050] According to some embodiments of the present invention, the detection is performed using fluorescence quantitative PCR.

[0051] According to some embodiments of the present invention, the reaction system of the fluorescence quantitative PCR is as follows:

[0052]

[0053]

[0054] According to some embodiments of the present invention, the reaction system of the fluorescence quantitative PCR is as follows:

[0055]

[0056] According to some embodiments of the present invention, the amplification program of the fluorescence quantitative PCR is: 45 - 55°C for 8 - 12 min; 94 - 96°C for 25 - 35 s; then enter the cycling stage: 93 - 96°C for 8 - 12 s, 55 - 65°C for 25 - 35 s, for 40 - 48 cycles, and collect fluorescence data.

[0057] According to some embodiments of the present invention, the amplification program of the fluorescence quantitative PCR is: 50°C for 10 min; 95°C for 30 s; then enter the cycling stage: 95°C for 10 s, 60°C for 30 s, for 45 cycles, and collect fluorescence data.

[0058] In some embodiments of the present invention, after the detection, it further includes the steps of statistically analyzing the Ct value of the sample and interpreting the result; the result interpretation is as follows:

[0059] Positive: The Ct value of the sample test result ≤ 40 or there is an obvious exponential growth phase;

[0060] Negative: The Ct value of the sample test result > 40 or there is no Ct value.

[0061] A method for detecting or assisting in detecting Vibrio harveyi, Infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and / or Infectious myonecrosis virus for non-diagnostic purposes according to an embodiment of the present invention has at least the following beneficial effects:

[0062] The method of the present invention's solution can simultaneously detect Vibrio harveyi (VH), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV), and Infectious myonecrosis virus (IMNV) in a single tube of reaction solution by using the above primer-probe combination for fluorescence quantitative PCR detection. For one specimen, only one nucleic acid extraction and one PCR detection are required simultaneously to obtain 4 test results. Moreover, there is no interference during the PCR process, the test results are accurate, which greatly improves the detection efficiency, shortens the detection cycle, reduces the detection cost, and provides a basis for the rapid diagnosis of shrimp diseases and the exclusion of different pathogen infections. In addition, the detection method in the present invention can integrate PCR, molecular hybridization, and photochemistry based on real-time fluorescence PCR, combine the sensitivity of PCR with the specificity of the probe, greatly change the defects of traditional PCR, shorten the reaction time, simplify the operation steps, and the whole process of PCR amplification and product analysis is carried out under closed conditions, which can monitor the PCR process throughout, avoiding false positives caused by cross-contamination between samples and environmental pollution.

[0063] According to an embodiment of the fifth aspect of the present invention, a system for detecting Vibrio harveyi, Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Taura Syndrome Virus (TSV) and / or Infectious Myonecrosis Virus (IMNV) includes:

[0064] A detection module: for performing fluorescence PCR amplification on the nucleic acid of the sample to be tested by using the above primer-probe combination or kit;

[0065] An analysis module: for evaluating whether the sample to be tested contains Vibrio harveyi, Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV), Taura Syndrome Virus (TSV) and / or Infectious Myonecrosis Virus (IMNV).

[0066] According to some embodiments of the present invention, the system further includes an extraction module for extracting the nucleic acid of the sample to be tested.

[0067] Other features and advantages of the present invention will be described in the following specification, and, in part, will be obvious from the specification, or will be understood by implementing the present invention. Brief Description of the Drawings

[0068] Figure 1 It is a test effect and specific detection effect diagram of the primer-probe combination 1 of the present invention for detecting samples;

[0069] Figure 2 It is a result diagram of the primer-probe combination 2 of the present invention for detecting samples;

[0070] Figure 3 It is a sensitivity detection result diagram of the primer-probe combination 1 of the present invention for detecting Vibrio harveyi (VH);

[0071] Figure 4 It is a sensitivity detection result diagram of the primer-probe combination 1 of the present invention for detecting Infectious Hypodermal and Hematopoietic Necrosis Virus (IHHNV);

[0072] Figure 5 It is a sensitivity detection result diagram of the primer-probe combination 1 of the present invention for detecting Taura Syndrome Virus (TSV);

[0073] Figure 6 It is a sensitivity detection result diagram of the primer-probe combination 1 of the present invention for detecting Infectious Myonecrosis Virus (IMNV);

[0074] Figure 7 It is a linear relationship and regression equation diagram between the Ct value of the primer-probe combination 1 of the present invention for detecting Vibrio harveyi (VH) and the initial template concentration;

[0075] Figure 8This is a graph showing the linear relationship and regression equation between the Ct value detected by the primer-probe combination 1 of the present invention and the initial template concentration for infectious hypodermal and hematopoietic necrosis virus (IHHNV);

[0076] Figure 9 This is a graph showing the linear relationship and regression equation between the Ct value detected by the primer-probe combination 1 of the present invention and the initial template concentration for Taura syndrome virus (TSV);

[0077] Figure 10 This is a graph showing the linear relationship and regression equation between the Ct value detected by the primer-probe combination 1 of the present invention and the initial template concentration for infectious myonecrosis virus (IMNV). Detailed implementation mode

[0078] The above and / or additional aspects and advantages of the present invention will become obvious and easy to understand from the description of the embodiments in conjunction with the following drawings, where: The concept and technical effects of the present invention will be clearly and completely described below in conjunction with the embodiments to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present invention.

[0079] For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0080] Unless otherwise specified, nucleotide sequences are in the 5' to 3' order.

[0081] Example 1

[0082] This example provides a primer-probe combination for simultaneously detecting Vibrio harveyi, infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and infectious myonecrosis virus. The design and screening steps are as follows:

[0083] (1) Design of the primer-probe combination

[0084] When jointly detecting multiple pathogens, there are many primers and probes, and it is easy for dimers to occur between primers and primers, probes and probes, or primers and probes. To ensure the conservativeness of the design (conservativeness is crucial for the accuracy of detection) and also consider the mutual interference between different primer-probes, it is necessary to carefully design and verify the primer-probes.

[0085] Through a large number of literature retrievals and analyses, and through sequence retrievals and alignment analyses in the GenBank database, the conserved sequences of Vibrio harveyi (VH), Infectious hypodermal and hematopoietic necrosis virus (IHHNV), Taura syndrome virus (TSV), and Infectious myonecrosis virus (IMNV) were selected, a series of primer-probes were designed, and after synthesis, they were screened and verified through experiments. The primer-probe combination 1 (sequences are shown in Table 1) and primer-probe combination 2 (sequences are shown in Table 2) designed for Vibrio harveyi, Infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and Infectious myonecrosis virus were preferably obtained.

[0086] Table 1

[0087]

[0088] Table 2

[0089]

[0090]

[0091] (2) Screening of primer-probe combinations

[0092] Positive control: A cloned plasmid containing the amplified target fragments of Vibrio harveyi, Infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and Infectious myonecrosis virus synthesized by General Biosystems.

[0093] Negative control: Sterilized deionized water.

[0094] The specific detection sample is a cloned plasmid containing the amplified target fragments of Iridovirus decapod 1, Hepatopancreatic parvovirus, Yellow head virus, Acute hepatopancreatic necrosis virus, White spot syndrome virus, Enterocytozoon hepatopenaei, Vibrio parahaemolyticus, Highly pathogenic Vibrio, Vibrio alginolyticus, and Vibrio harveyi synthesized by General Biosystems.

[0095] Reagent preparation:

[0096] According to the amounts of the test samples, positive controls, and negative controls, the corresponding amounts of PCR reaction solution (20 μL / test) were taken, centrifuged at 2000 rpm for 10 s, and then reserved for use.

[0097] Sample processing and loading:

[0098] No more than 30 mg of the specific detection sample, negative control, and positive control were respectively added to 1.5 mL centrifuge tubes, and nucleic acid extraction was performed according to the instructions of the Marine Animal Genomic DNA Extraction Kit from Tiangen to obtain template DNA.

[0099] Table 3

[0100] Component Volume / Concentration in Each Reaction 5×RT Buffer 5 μL 12.5×Taq / UNG Mix (with dNTPs) 2 μL 50×RTase 0.5 μL Forward Primer Designed for Target VH (50 pmol / μL) 0.08 μL Reverse Primer Designed for Target VH (50 pmol / μL) 0.08 μL Forward Primer Designed for Target IHHNV (50 pmol / μL) 0.08 μL Reverse Primer Designed for Target IHHNV (50 pmol / μL) 0.08 μL Forward Primer Designed for Target TSV (50 pmol / μL) 0.06 μL Reverse Primer Designed for Target TSV (50 pmol / μL) 0.06 μL Forward Primer Designed for Target IMNV (50 pmol / μL) 0.08 μL Reverse Primer Designed for Target IMNV (50 pmol / μL) 0.08 μL Probe Designed for Target VH (50 pmol / μL) 0.04 μL Probe Designed for Target IHHNV (50 pmol / μL) 0.04 μL Probe Designed for Target TSV (50 pmol / μL) 0.03 μL Probe Designed for Target IMNV (50 pmol / μL) 0.04 μL Sterilized Purified Water 11.75 μL DNA 5 μL total 25 μL

[0101] The primer-probe combination 1 (sequences shown in Table 1) and the primer-probe combination 2 (sequences shown in Table 2) (specific detection of the sample template DNA was not used for amplification in the primer-probe combination 2) were respectively prepared into fluorescence quantitative PCR amplification systems according to the system in Table 3. The prepared fluorescence quantitative PCR amplification systems were placed in PCR instruments such as the Hongshi automatic medical PCR analysis system SLAN-96S, and fluorescence quantitative PCR amplification was carried out according to the program shown in Table 4.

[0102] Table 4

[0103]

[0104]

[0105] The detection results are as Figure 1-2 shown. It can be seen from Figure 1 that the primer-probe combination 1 can accurately detect Vibrio harveyi, infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and infectious myonecrosis virus, and no detection was found in the negative control and other shrimp diseases. It can be seen from Figure 2 that only partial amplification curves appeared in the detection of the primer-probe combination 2, and the amplification curves had a low increase rate and poor repeatability, and even no amplification curves for the target were observed. Therefore, the overall detection effect was poor, and the combined detection of Vibrio harveyi, infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and infectious myonecrosis virus could not be achieved. Therefore, the primer-probe combination 1 (sequences shown in Table 1) was screened for subsequent experimental verification.

[0106] Example 2

[0107] This example provides a kit for Vibrio harveyi, infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and infectious myonecrosis virus, which consists of 5×RT Buffer (purchased from Baorui Biology), Taq / UNG Mix (purchased from Baorui Biology), 50×Rtase (purchased from Baorui Biology), primer-probe groups designed for the target VH (sequences shown in SEQ ID NO: 1-3), primer-probe groups designed for the target IHHNV (sequences shown in SEQ ID NO: 4-6), primer-probe groups designed for the target TSV (sequences shown in SEQ ID NO: 7-9), primer-probe groups designed for the target IMNV (sequences shown in SEQ ID NO: 10-12), positive control products, and negative control products;

[0108] Among them, the positive control: a cloned plasmid containing the amplified target fragments of Vibrio harveyi, infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and infectious myonecrosis virus synthesized by General Biology.

[0109] Negative control: Sterilized deionized water.

[0110] Example 3

[0111] This example provides a method for detecting Vibrio harveyi, Infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and Infectious myonecrosis virus. The steps are as follows:

[0112] Extract the nucleic acid of the sample to be tested as a template. Using the kit prepared in Example 2, prepare a fluorescence quantitative PCR amplification system as shown in Table 3 of Example 1. Place the prepared fluorescence quantitative PCR amplification system in a PCR instrument such as the Hongshi automatic medical PCR analysis system SLAN-96S, and perform fluorescence quantitative PCR amplification according to the program shown in Table 4.

[0113] The result judgment is shown in Table 5. If the sample has obvious S-shaped amplification curves in the FAM, HEX (VIC), ROX, and CY5 channels, and the Ct value ≤ 40, it is judged as positive; if the sample has no amplification curve (No Ct) in the FAM, HEX (VIC), ROX, and CY5 channels, it is judged as negative.

[0114] Table 5

[0115]

[0116] Sensitivity experiment of Example 4

[0117] Measure the concentration of the clone plasmid mother solution containing the amplification target fragments of Vibrio harveyi, Infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and Infectious myonecrosis virus synthesized by General Biosystems on a qubit fluorescence quantifier. Calculate the copies / μL concentration from the measured ng concentration. Dilute the high-concentration plasmid to 1×10 6 copies / μL using TE buffer, and then perform 10-fold serial dilution to 1×10 0 copies / μL, with a total of 7 gradients. Extract the sample nucleic acid, use the kit prepared in Example 2, and the detection method provided in Example 3 to prepare corresponding amounts of detection reagents for testing. Set 3 technical replicates for each gradient concentration of the plasmid. At the same time, calculate the regression equation between the amplification Ct value and the initial template concentration.

[0118] Experimental results: The results are as Figure 3-10 shown. It can be seen from the figure that the 100% detection limit of the kit of the present invention's solution for Vibrio harveyi, Infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and Infectious myonecrosis virus is 1×10 0 copies / μL.

[0119] Meanwhile, within the starting template concentration range of 1.00×10 6 copies / μL, the regression equations of the amplification Ct values of Vibrio harveyi, Infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and Infectious myonecrosis virus with the starting template concentration showed a good linear relationship, and the correlation coefficient R 2 was all > 0.99, and the amplification efficiency Eff% was all between 90% and 110%.

[0120] In addition, the kit prepared in Example 2 was used to simultaneously detect the cloned plasmids containing the amplification target fragments of Vibrio harveyi, Infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and Infectious myonecrosis virus. The concentration of the cloned plasmid was 1×10 0 copies / μL, and accurate detection could also be achieved. The 100% detection limit could be as low as 1×10 0 copies / μL.

[0121] Example 5 Specificity Experiment

[0122] Detection template: Cloned plasmids containing the amplification target fragments of Decapod iridovirus 1, Hepatopancreatic parvovirus, Yellow head virus, Acute hepatopancreatic necrosis virus, White spot syndrome virus, Enterocytozoon hepatopenaei, Vibrio parahaemolyticus, Highly pathogenic Vibrio, Vibrio alginolyticus, Vibrio harveyi synthesized by General Biosystems, as well as the positive plasmid spiked samples of Vibrio harveyi, Infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and Infectious myonecrosis virus were used as detection samples. The kit prepared in Example 2 and the detection method provided in Example 3 were used for detection.

[0123] The detection result graph is the same as Figure 1 consistent. It can be seen from the graph that only Vibrio harveyi, Infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and Infectious myonecrosis virus showed S curves, and other results were all negative. The results indicate that the method of the present invention has no cross-reaction with the above-mentioned shrimp disease pathogens.

[0124] Example 6 Stability Study

[0125] The kit prepared in Example 2 was stored at the actual storage condition (-20±5°C) for 11 months and then detected, and its performance was stable. The results of the accelerated stability experiment at 37°C showed that the kit was stored in an incubator at 37°C for 24 hours, and the results met the quality requirements. The freeze-thaw stability test showed that at the actual storage temperature, after one freeze-thaw at each detection time point and continuous detection for 3 times, the results all met the quality requirements.

[0126] The above has described the embodiments of the present invention in detail. However, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the scope of knowledge possessed by those of ordinary skill in the relevant technical field.

Claims

1. A primer-probe combination for detecting multiple shrimp pathogens, characterized in that, The primer-probe combination includes a first primer-probe group for detecting Vibrio harveyi, a second primer-probe group for detecting infectious hypodermal and hematopoietic necrosis virus, a third primer-probe group for detecting Taura syndrome virus, and a fourth primer-probe group for detecting infectious myonecrosis virus; In the first primer-probe group, the primer sequences are as shown in SEQ ID NO:1 and SEQ ID NO:2, and the probe sequence is as shown in SEQ ID NO:3; In the second primer-probe group, the primer sequences are as shown in SEQ ID NO:4 and SEQ ID NO:5, and the probe sequence is as shown in SEQ ID NO:6; In the third primer-probe group, the primer sequences are as shown in SEQ ID NO:7 and SEQ ID NO:8, and the probe sequence is as shown in SEQ ID NO:9; In the fourth primer-probe group, the primer sequences are as shown in SEQ ID NO:10 and SEQ ID NO:11, and the probe sequence is as shown in SEQ ID NO:

12.

2. The primer-probe combination according to claim 1, wherein Both ends of the probe are respectively labeled with a fluorescent reporter group and a fluorescent quenching group; Preferably, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quenching group; Preferably, the fluorescent reporter group is selected from FAM, JOE, VIC, HEX, ROX, CY3 or CY5; the fluorescent quenching group is selected from BHQ, TAMRA or MGB; Preferably, the probes in the first primer-probe group, the second primer-probe group, the third primer-probe group, and the fourth primer-probe group have different fluorescent reporter groups.

3. A kit, characterized in that, The kit contains the primer-probe combination according to any one of claims 1-2.

4. The kit according to claim 3, characterized in that, The kit further includes at least one of a positive control, a negative control, and a PCR premix; Preferably, the positive control includes a recombinant vector containing nucleic acid molecules of Vibrio harveyi, infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus, and / or infectious myonecrosis virus; Preferably, the PCR premix includes RT Buffer, Taq / UNG Mix, and reverse transcriptase; More preferably, the RT Buffer includes Tris-HCl, dNTPs, MgCl2, KCl, and Triton X-100.

5. Use of the primer-probe combination according to any one of claims 1-2 or the kit according to any one of claims 3-4 in any of the following: 1) Detection for non-diagnostic purposes or auxiliary detection of Vibrio harveyi; 2) Preparation of a product for detecting or auxiliary detecting Vibrio harveyi; 3) Detection for non-diagnostic purposes or auxiliary detection of infectious hypodermal and hematopoietic necrosis virus; 4) Preparation of a product for detecting or auxiliary detecting infectious hypodermal and hematopoietic necrosis virus; 5) Detection for non-diagnostic purposes or auxiliary detection of Taura syndrome virus; 6) Preparation of a product for detecting or auxiliary detecting Taura syndrome virus; 7) Detection for non-diagnostic purposes or auxiliary detection of infectious myonecrosis virus; 8) Preparation of a product for detecting or auxiliary detecting infectious myonecrosis virus.

6. The application according to claim 5, characterized in that, The product is selected from a reagent, a kit or a chip; and / or, the method for using the product comprises the following steps: Detecting the DNA of a sample to be tested by using the primer-probe combination according to any one of claims 1-2 or the kit according to any one of claims 3-4.

7. A method for detecting or assisting in the detection of Vibrio harveyi, Infectious Hypodermal and Hematopoietic Necrosis Virus, Taura Syndrome Virus, and / or Infectious Myonecrosis Virus for non-diagnostic purposes, characterized in that, Comprising the following steps: Detecting the DNA of a sample to be tested by using the primer-probe combination according to any one of claims 1-2 or the kit according to any one of claims 3-4.

8. The method according to claim 7, wherein The amplification is real-time fluorescence quantitative PCR amplification; Preferably, the reaction system of the real-time fluorescence quantitative PCR is: Preferably, the amplification program of the real-time fluorescence quantitative PCR is: 45-55°C for 8-12 min; 94-96°C for 25-35 s; then enter the cycling stage: 93-96°C for 8-12 s, 55-65°C for 25-35 s, for 40-48 cycles, and collect fluorescence data.

9. The method according to claim 7, wherein After the detection, it further comprises the steps of statistically analyzing the Ct value of the sample and interpreting the result; the result interpretation is: Positive: the Ct value of the sample detection result is ≤40 or there is an obvious exponential growth phase; Negative: the Ct value of the sample detection result is >40 or there is no Ct value.

10. A system for detecting Vibrio harveyi, Infectious Hypodermal and Hematopoietic Necrosis Virus, Taura Syndrome Virus, and / or Infectious Myonecrosis Virus, characterized in that, Including: Detection module: for performing fluorescence PCR amplification on the nucleic acid of a sample to be tested by using the primer-probe combination according to any one of claims 1-2 or the kit according to any one of claims 3-4; Analysis module: for evaluating whether the sample to be tested contains Vibrio harveyi, infectious hypodermal and hematopoietic necrosis virus, Taura syndrome virus and / or infectious myonecrosis virus.

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