Primer probe composition and kit for detecting prawn disease related pathogens and application of primer probe composition and kit

Abstract

The invention discloses a primer probe composition for detecting prawn disease related pathogens, a kit and application of the primer probe composition. The primer probe composition comprises a first primer probe group used for detecting vibrio parahaemolyticus, a second primer probe group used for detecting a tetrapod iridovirus 1, a third primer probe group used for detecting vibrio vitriocultatus and a fourth primer probe group used for detecting enterocytozoon hepatopenaei. The primer probe composition has the characteristics of high sensitivity, good repeatability and strong specificity, and can be used for specifically detecting vibrio parahaemolyticus, tetrapod iridovirus 1, vibrio vitrioculi and enterocytozoon hepatopenaei.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a primer-probe composition, a kit and their applications for detecting pathogens related to shrimp diseases. Background Art

[0002] At present, there are many pathogenic species of 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. Therefore, it is necessary to take measures as early as possible to identify the pathogenic species causing the diseases and take corresponding prevention and control means. Therefore, early detection and early diagnosis are the main key technologies for current prevention and control, and it is of great significance to establish an accurate and rapid differential diagnosis method. Currently, a variety of shrimp disease detection methods have been developed, including section detection, PCR detection, LAMP, immunoassay, etc., but generally can only perform nucleic acid detection or isolation and identification on a single pathogen or a few pathogens, with cumbersome operation steps and long time, which is not conducive to quickly taking countermeasures. Therefore, there is an urgent need for a rapid detection method with not only high accuracy but also wide pathogen coverage. Summary of the Invention

[0003] 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 composition for detecting pathogens related to shrimp diseases.

[0004] The present invention also provides a kit containing the above primer-probe composition.

[0005] The present invention also provides the applications of the above primer-probe composition and kit.

[0006] The present invention also provides a system for detecting Vibrio parahaemolyticus, Decapod iridescent virus 1, Glass shrimp disease vibrio and / or Enterocytozoon hepatopenaei.

[0007] According to the first aspect embodiment of the present invention, there is provided a primer-probe composition for detecting pathogens related to shrimp diseases, the primer-probe composition including a first primer-probe group for detecting Vibrio parahaemolyticus, a second primer-probe group for detecting Decapod iridescent virus 1, a third primer-probe group for detecting Glass shrimp disease vibrio, and a fourth primer-probe group for detecting Enterocytozoon hepatopenaei;

[0008] 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;

[0009] 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;

[0010] 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;

[0011] 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.

[0012] The primer-probe composition for detecting pathogens related to shrimp diseases according to the embodiments of the present invention has at least the following beneficial effects:

[0013] The primer-probe composition for detecting pathogens related to shrimp diseases provided by the present invention has the characteristics of high sensitivity, good repeatability and strong specificity, and can be used for the specific detection of Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio penaeicida and Enterocytozoon hepatopenaei.

[0014] 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.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] According to the embodiments of the second aspect of the present invention, a kit is provided, which includes the primer-probe composition 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 composition of the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments.

[0019] According to some embodiments of the present invention, the kit further includes at least one of a positive control, a negative control, and a PCR premix.

[0020] According to some embodiments of the present invention, the PCR premix includes Tris-HCl, dNTPs, MgCl2, KCl, Triton X-100, DNA polymerase, and UDG enzyme.

[0021] According to some embodiments of the present invention, the PCR premix is 2×Animal Detection U+Probe qPCR Super PreMix.

[0022] According to some embodiments of the present invention, the positive control includes a recombinant vector containing nucleic acid molecules of Vibrio parahaemolyticus, Decapod iridescent virus 1, Glass seedling vibrio, and / or Enterocytozoon hepatopenaei.

[0023] According to some embodiments of the present invention, the negative control includes water.

[0024] According to the embodiments of the third aspect of the present invention, there is provided an application of the above primer-probe composition or kit in any one of the following:

[0025] 1) Detection for non-diagnostic purposes or auxiliary detection of Vibrio parahaemolyticus;

[0026] 2) Preparation of a product for detecting or assisting in the detection of Vibrio parahaemolyticus;

[0027] 3) Detection for non-diagnostic purposes or auxiliary detection of Decapod iridescent virus 1;

[0028] 4) Preparation of a product for detecting or assisting in the detection of Decapod iridescent virus 1;

[0029] 5) Detection for non-diagnostic purposes or auxiliary detection of Glass seedling vibrio;

[0030] 6) Preparation of a product for detecting or assisting in the detection of Glass seedling vibrio;

[0031] 7) Detection for non-diagnostic purposes or auxiliary detection of Enterocytozoon hepatopenaei;

[0032] 8) Preparation of a product for detecting or assisting in the detection of Enterocytozoon hepatopenaei.

[0033] According to some embodiments of the present invention, the product is selected from reagents, kits, or chips.

[0034] According to the embodiments of the fourth aspect of the present invention, there is provided a method for detecting or assisting in the detection of Vibrio parahaemolyticus, Decapod iridescent virus 1, Glass seedling vibrio, and / or Enterocytozoon hepatopenaei for non-diagnostic purposes, comprising the following steps:

[0035] Detecting the DNA of the sample to be tested using the above primer-probe composition or kit.

[0036] According to some embodiments of the present invention, the detection is performed by fluorescence quantitative PCR.

[0037] According to some embodiments of the present invention, the reaction system of the fluorescence quantitative PCR is as follows:

[0038] Animal Detection U+Probe qPCR Super PreMix 1×;

[0039] The forward primer sequence in the first primer-probe group is 0.1 - 0.2 μM;

[0040] The reverse primer sequence in the first primer-probe group is 0.1 - 0.2 μM;

[0041] The probe sequence in the first primer-probe group is 0.05 - 0.15 μM;

[0042] The forward primer sequence in the second primer-probe group is 0.1 - 0.3 μM;

[0043] The reverse primer sequence in the second primer-probe group is 0.1 - 0.3 μM;

[0044] The probe sequence in the second primer-probe group is 0.05 - 0.15 μM;

[0045] The forward primer sequence in the third primer-probe group is 0.05 - 0.2 μM;

[0046] The reverse primer sequence in the third primer-probe group is 0.05 - 0.2 μM;

[0047] The probe sequence in the third primer-probe group is 0.01 - 0.1 μM;

[0048] The forward primer sequence in the fourth primer-probe group is 0.1 - 0.2 μM;

[0049] The reverse primer sequence in the fourth primer-probe group is 0.1 - 0.2 μM;

[0050] The probe sequence in the fourth primer-probe group is 0.05 - 0.15 μM.

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

[0052] Animal Detection U+Probe qPCR Super PreMix 1×;

[0053] The forward primer sequence in the first primer-probe group is 0.16 μM;

[0054] The reverse primer sequence in the first primer-probe group is 0.16 μM;

[0055] The probe sequence in the first primer-probe group is 0.08 μM;

[0056] The forward primer sequence in the second primer-probe group is 0.2 μM;

[0057] The reverse primer sequence in the second primer-probe group is 0.2 μM;

[0058] The probe sequence in the second primer-probe group is 0.1 μM;

[0059] The forward primer sequence in the third primer-probe group is 0.12 μM;

[0060] The reverse primer sequence in the third primer-probe group is 0.12 μM;

[0061] The probe sequence in the third primer-probe group is 0.06 μM;

[0062] The forward primer sequence in the fourth primer-probe group is 0.16 μM;

[0063] The reverse primer sequence in the fourth primer-probe group is 0.16 μM;

[0064] The probe sequence in the fourth primer-probe group is 0.08 μM.

[0065] According to some embodiments of the present invention, the reaction system of the fluorescence quantitative PCR further includes water.

[0066] According to some embodiments of the present invention, the amplification program of the fluorescence quantitative PCR is: 35 - 39 °C for 1 - 3 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.

[0067] According to some embodiments of the present invention, the amplification program of the fluorescence quantitative PCR is: 37 °C for 2 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.

[0068] 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:

[0069] Positive: The Ct value of the sample test result is ≤ 40 or there is an obvious exponential growth phase;

[0070] Negative: The Ct value of the sample test result is > 40 or there is no Ct value.

[0071] A method for detecting or assisting in detecting Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio vitreoscilla, and / or Enterocytozoon hepatonaei for non-diagnostic purposes according to an embodiment of the present invention has at least the following beneficial effects:

[0072] The method of the solution of the present invention can simultaneously detect Vibrio parahaemolyticus (VP), Decapod iridescent virus 1 (DIV1), Vibrio tapetis (VTPD), and Enterocytozoon hepatopenaei (EHP) in a single reaction tube by using the above-mentioned primer-probe combination for fluorescence quantitative PCR detection. For one specimen, only one nucleic acid extraction and one PCR detection need to be performed simultaneously to obtain 4 detection results. Moreover, there is no interference in the PCR process, and the detection 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, and avoid false positives caused by cross-contamination between samples and environmental pollution.

[0073] According to an embodiment of the fifth aspect of the present invention, a system for detecting Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio tapetis, and / or Enterocytozoon hepatopenaei includes:

[0074] A detection module: for performing fluorescence PCR amplification on the nucleic acid of the sample to be tested by using the above-mentioned primer-probe composition or kit; An analysis module: for evaluating whether the sample to be tested contains Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio tapetis, and / or Enterocytozoon hepatopenaei.

[0075] 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.

[0076] Other features and advantages of the present invention will be described in the following specification, and some of them will become obvious from the specification or be understood by implementing the present invention. Description of the Drawings

[0077] Figure 1 It is the test effect and specific detection effect diagram of the sample detected by the primer-probe combination 1 of the present invention;

[0078] Figure 2 It is the result diagram of the sample detected by the primer-probe combination 2 of the present invention;

[0079] Figure 3 It is the linear relationship and regression equation between the Ct value of Vibrio parahaemolyticus (VP) in the sample detected by the primer-probe combination 1 of the present invention and the initial template concentration;

[0080] Figure 4The linear relationship and regression equation between the Ct value and the initial template concentration of the primer-probe combination 1 of the present invention for detecting the decapod iridovirus 1 (DIV1) in samples;

[0081] Figure 5 The linear relationship and regression equation between the Ct value and the initial template concentration of the primer-probe combination 1 of the present invention for detecting Vibrio tubiashii (VTPD) in samples;

[0082] Figure 6 The linear relationship and regression equation between the Ct value and the initial template concentration of the primer-probe combination 1 of the present invention for detecting Enterocytozoon hepatopenaei (EHP) in samples;

[0083] Figure 7 The sensitivity test result graph of the primer-probe combination 1 of the present invention for detecting Vibrio parahaemolyticus (VP) in samples. Among them, the curves are 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×10 0 copies / μL;

[0084] Figure 8 The sensitivity test result graph of the primer-probe combination 1 of the present invention for detecting the decapod iridovirus 1 (DIV1) in samples. Among them, the curves are 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×10 0 copies / μL;

[0085] Figure 9 The sensitivity test result graph of the primer-probe combination 1 of the present invention for detecting Vibrio tubiashii (VTPD) in samples. Among them, the curves are 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×10 0 copies / μL;

[0086] Figure 10 This is the sensitivity test result graph of the primer-probe combination 1 of the present invention for detecting Enterocytozoon hepatopenaei (EHP) in samples. Among them, the curves from left to right are 1×10 5 copies / μL, 1×10 4 copies / μL, 1×10 3 copies / μL, 1×10 2 copies / μL, 1×10 1 copies / μL, 1×10 0 copies / μL;

[0087] Figure 11 This is the repeatability test result graph of the primer-probe combination 1 of the present invention for detecting Vibrio parahaemolyticus (VP) at a concentration of 1×10 0 copies / μL;

[0088] Figure 12 This is the repeatability test result graph of the primer-probe combination 1 of the present invention for detecting Decapod iridescent virus 1 (DIV1) at a concentration of 1×10 0 copies / μL;

[0089] Figure 13 This is the repeatability test result graph of the primer-probe combination 1 of the present invention for detecting Vibrio tubiashii (VTPD) at a concentration of 1×10 0 copies / μL;

[0090] Figure 14 This is the repeatability test result graph of the primer-probe combination 1 of the present invention for detecting Enterocytozoon hepatopenaei (EHP) at a concentration of 1×10 0 copies / μL. Detailed implementation manners

[0091] 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.

[0092] For those not specified in the embodiments, they are carried out under conventional conditions or conditions recommended by the manufacturer. For reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0093] Unless otherwise specified, nucleotide sequences are in the order of 5' to 3'.

[0094] Example 1

[0095] This example provides a primer-probe composition for detecting Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio tubiashii, and Enterocytozoon hepatopenaei. The design and screening steps are as follows:

[0096] (1) Design of primer-probe combinations

[0097] When detecting multiple pathogens jointly, there are numerous 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.

[0098] Through a large number of literature retrievals and analyses, and simultaneously through sequence retrievals and alignment analyses in the GenBank database, this invention selects the conserved sequences of Vibrio parahaemolyticus (VP), Decapod iridescent virus 1 (DIV1), Vibrio tubiashii (VTPD), and Enterocytozoon hepatopenaei (EHP), designs a series of primer-probes, and after synthesis, screens and verifies them through experiments. Primer-probe combination 1 designed for Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio tubiashii, and Enterocytozoon hepatopenaei (the sequence is shown in Table 1) and primer-probe combination 2 designed for Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio tubiashii, and Enterocytozoon hepatopenaei (the sequence is shown in Table 2) are preferably obtained.

[0099] Table 1

[0100]

[0101]

[0102] Table 2

[0103]

[0104]

[0105] (2) Screening of primer-probe combinations

[0106] Positive control: A cloned plasmid containing the amplified target fragments of Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio tubiashii, and Enterocytozoon hepatopenaei synthesized by General Biosystems.

[0107] Negative control: Sterilized deionized water.

[0108] The specific detection samples are diseased shrimp samples cultured in a shrimp farming pond and detected to contain shrimp hemocyte iridovirus, Taura syndrome virus, hepatopancreatic parvovirus, yellow head virus, acute hepatopancreatic necrosis virus, infectious myonecrosis virus, white spot syndrome virus, infectious hypodermal and hematopoietic necrosis virus, Vibrio alginolyticus, Vibrio harveyi, and Vibrio splendidus, which are pathogens of shrimp diseases.

[0109] Take no more than 30 mg of the specific detection sample, negative control, and positive control and add them separately to 1.5 mL centrifuge tubes. Use the Marine Animal Genomic DNA Extraction Kit from Tiangen to extract nucleic acids according to the instructions of the kit to obtain template DNA.

[0110] Table 3

[0111]

[0112]

[0113] Prepare the fluorescence quantitative PCR amplification system according to the system in Table 3 for primer-probe combination 1 (sequences shown in Table 1) and primer-probe combination 2 (sequences shown in Table 2) (in primer-probe combination 2, the template DNA of the specific detection sample was not used for amplification). Place the prepared fluorescence quantitative PCR amplification system in a PCR instrument such as the Hongshi SLAN-96S fully automatic medical PCR analysis system and perform fluorescence quantitative PCR amplification according to the procedure shown in Table 4.

[0114] Table 4

[0115]

[0116] The detection results are as Figure 1-2 shown. It can be seen from Figure 1 that primer-probe combination 1 can accurately detect Vibrio parahaemolyticus, Iridovirus decapod 1, Vibrio mytili, and Enterocytozoon hepatopenaei pathogens, and no detection was found in the negative control and other shrimp diseases. It can be seen from Figure 2 that for primer-probe combination 2, only partial amplification curves appeared in the detection, and the amplification curve had a low increase and poor repeatability. Even no amplification curve for the target was observed, and the combined detection of Vibrio parahaemolyticus, Iridovirus decapod 1, Vibrio mytili, and Enterocytozoon hepatopenaei pathogens could not be achieved. Therefore, primer-probe combination 1 (sequences shown in Table 1) was screened for subsequent experimental verification.

[0117] Example 2

[0118] This example provides a kit for detecting Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio penaeicida, and Enterocytozoon hepatopenaei, which consists of 2×Animal Detection U+Probe qPCR Super PreMix, a primer-probe set designed for the target VP (sequences shown in SEQ ID NO: 1-3), a primer-probe set designed for the target DIV1 (sequences shown in SEQ ID NO: 4-6), a primer-probe set designed for the target VTPD (sequences shown in SEQ ID NO: 7-9), a primer-probe set designed for the target EHP (sequences shown in SEQ ID NO: 10-12), a positive control, and a negative control.

[0119] Among them, the positive control: a cloned plasmid synthesized by General Biosystems containing the amplified target fragments of Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio penaeicida, and Enterocytozoon hepatopenaei.

[0120] The negative control: sterilized deionized water.

[0121] Example 3

[0122] This example provides a method for detecting Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio penaeicida, and Enterocytozoon hepatopenaei, and the steps are as follows:

[0123] Extract the nucleic acid sample of the test sample as a template, use the kit prepared in Example 2 to prepare the fluorescence quantitative PCR amplification system as shown in Table 3, and place the prepared fluorescence quantitative PCR amplification system in a PCR instrument such as the Hongshi full-automatic medical PCR analysis system SLAN-96S, and perform fluorescence quantitative PCR amplification according to the program shown in Table 4.

[0124] The result judgment is shown in Table 5. If there is an obvious S-shaped amplification curve in the FAM, HEX (VIC), ROX, and CY5 channels of the sample and the Ct value ≤ 40, it is judged as positive; if there is no amplification curve (No Ct) or the Ct value > 40 in the FAM, HEX (VIC), ROX, and CY5 channels of the sample, it is judged as negative.

[0125] Table 5

[0126]

[0127]

[0128] Example 4 Sensitivity experiment

[0129] The master solutions of cloning plasmids containing the amplified target fragments of Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio vitreoscilla, and Enterocytozoon hepatopenaei, respectively, synthesized by General Biosynthesis, were measured for concentration on a Qubit fluorometer. The ng concentration measured was calculated to obtain the copies / μL concentration. The high-concentration plasmid was diluted to 1.00×10 5 copies / μL using TE buffer, and then serially diluted 10-fold to 1×10 0 copies / μL, with a total of 6 gradients. The nucleic acids of the samples were extracted as detection samples, and the prepared kit in Example 2 and the detection method provided in Example 3 were used to prepare corresponding amounts of detection reagents for testing. Three technical replicates were set for each gradient concentration of the plasmid. Meanwhile, the regression equation between the amplified Ct value and the initial template concentration was calculated.

[0130] Experimental results: The results are as Figures 3-10 shown. It can be seen from the figure that within the range of the initial template concentration of 1.00×10 5 copies / μL, the regression equations between the amplified Ct values and the initial template concentrations of Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio vitreoscilla, and Enterocytozoon hepatopenaei showed good linear relationships, and the correlation coefficients R 2 were all > 0.99. The amplification efficiencies Eff% were all between 90% and 110%. The sensitivities of Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio vitreoscilla, and Enterocytozoon hepatopenaei were 1×10 0 copies / μL.

[0131] Example 5 Repeatability experiment

[0132] The master solutions of cloning plasmids containing the amplified target fragments of Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio vitreoscilla, and Enterocytozoon hepatopenaei, respectively, synthesized by General Biosynthesis, were measured for concentration on a Qubit fluorometer. The ng concentration measured was calculated to obtain the copies / μL concentration. The high-concentration plasmid was diluted to 1.00×10 0 copies / μL as the detection sample, and the nucleic acids of the sample were extracted. The prepared kit in Example 2 and the detection method provided in Example 3 were used to prepare corresponding amounts of detection reagents for testing. Twenty technical replicates were set for the experiment.

[0133] The results are as Figures 11-14 shown. It can be seen from the figure that plasmids with a concentration as low as 1.00×10 0 copies / μL could achieve 100% stable detection, and the detection repeatability was good.

[0134] Example 6 Specificity experiment

[0135] Select diseased shrimp samples cultured in a shrimp pond that are detected to contain shrimp hemocyte iridovirus, Taura syndrome virus, hepatopancreatic parvovirus, yellow head virus, acute hepatopancreatic necrosis virus, infectious myonecrosis virus, white spot syndrome virus, infectious hypodermal and hematopoietic necrosis virus, Vibrio alginolyticus, Vibrio harveyi, Vibrio splendidus, and other shrimp disease pathogens, as well as positive plasmid spiked samples of Vibrio parahaemolyticus, decapod iridovirus 1, glass shrimp disease-causing Vibrio, and Enterocytozoon hepatonaei as test samples. Use the kit prepared in Example 2 and the detection method provided in Example 3 for detection.

[0136] The detection result graph is the same as Figure 1 the same. As can be seen from the graph, only Vibrio parahaemolyticus, decapod iridovirus 1, glass shrimp disease-causing Vibrio, and Enterocytozoon hepatonaei show an S curve, and other results are all negative. The results indicate that the method of the present invention has no cross-reaction with the above-mentioned shrimp disease pathogens.

[0137] Example 7 Stability Study

[0138] The kit prepared in Example 2 was stored at the actual storage conditions (-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.

[0139] The above has described the embodiments of the present invention in detail, but the present invention is not limited to the above embodiments. Within the knowledge scope of those of ordinary skill in the art, various changes can be made without departing from the purpose of the present invention.

Claims

1. A primer-probe composition for detecting pathogens related to shrimp diseases, characterized in that, The primer-probe composition includes a first primer-probe group for detecting Vibrio parahaemolyticus, a second primer-probe group for detecting Decapod iridescent virus 1, a third primer-probe group for detecting Vibrio mytili, and a fourth primer-probe group for detecting Enterocytozoon hepatopenaei; 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 composition 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 composition 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.

5. The kit according to claim 4, characterized in that, The positive control includes a recombinant vector containing nucleic acid molecules of Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio mytili, and / or Enterocytozoon hepatopenaei.

6. Use of the primer-probe composition according to any one of claims 1-2 or the kit according to any one of claims 3-5 in any of the following: 1) Detection or auxiliary detection of Vibrio parahaemolyticus for non-diagnostic purposes; 2) Preparation of a product for detecting or auxiliary detecting Vibrio parahaemolyticus; 3) Detection or auxiliary detection of Decapod iridescent virus 1 for non-diagnostic purposes; 4) Preparation of a product for detecting or auxiliary detecting Decapod iridescent virus 1; 5) Detection or auxiliary detection of Vibrio mytili for non-diagnostic purposes; 6) Preparation of a product for detecting or auxiliary detecting Vibrio mytili; 7) Detection or auxiliary detection of Enterocytozoon hepatopenaei for non-diagnostic purposes; 8) Preparation of a product for detecting or auxiliary detecting Enterocytozoon hepatopenaei.

7. A method for detecting or assisting in the detection of Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio hyalinus, and / or Enterocytozoon hepatonaei for non-diagnostic purposes, characterized in that, Including the following steps: Detecting the DNA of the sample to be tested using the primer-probe composition according to any one of claims 1-2 or the kit according to any one of claims 3-5.

8. The method according to claim 7, wherein The amplification is fluorescence quantitative PCR amplification; Preferably, the reaction system of the fluorescence quantitative PCR is: Animal Detection U+Probe qPCR Super PreMix 1×; The forward primer sequence in the first primer-probe group is 0.1 - 0.2 μM; The reverse primer sequence in the first primer-probe group is 0.1 - 0.2 μM; The probe sequence in the first primer-probe group is 0.05 - 0.15 μM; The forward primer sequence in the second primer-probe group is 0.1 - 0.3 μM; The reverse primer sequence in the second primer-probe group is 0.1 - 0.3 μM; The probe sequence in the second primer-probe group is 0.05 - 0.15 μM; The forward primer sequence in the third primer-probe group is 0.05 - 0.2 μM; The reverse primer sequence in the third primer-probe group is 0.05 - 0.2 μM; The probe sequence in the third primer-probe group is 0.01 - 0.1 μM; The forward primer sequence in the fourth primer-probe group is 0.1 - 0.2 μM; The reverse primer sequence in the fourth primer-probe group is 0.1 - 0.2 μM; The probe sequence in the fourth primer-probe group is 0.05 - 0.15 μM; Preferably, the amplification program of the fluorescence quantitative PCR is: 35 - 39 °C for 1 - 3 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 includes the steps of statistically analyzing the Ct value of the sample and interpreting the results; the result interpretation is: Positive: The Ct value of the sample test result is ≤ 40 or there is an obvious exponential growth phase; Negative: The Ct value of the sample test result is > 40 or there is no Ct value.

10. A system for detecting Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio tubiashii, and / or Enterocytozoon hepatopenaei, characterized in that, It includes: Detection module: used to perform fluorescence PCR amplification on the nucleic acid of the sample to be tested using the above primer-probe composition or kit; Analysis module: used to evaluate whether the sample to be tested contains Vibrio parahaemolyticus, Decapod iridescent virus 1, Vibrio vitreous, and / or Enterocytozoon hepatonaei.

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