Grouper culture water source pathogen identification primer group, identification method, kit and application

Abstract

The invention discloses a grouper culture water source pathogen identification primer group, an identification method, a kit and application. The identification method comprises two rounds of PCR (Polymerase Chain Reaction) amplification, namely nested PCR amplification, and primers as shown in Seq No.1-6 are used in the first round of PCR; a primer as shown in Seq No.7-12 is used in the second round of PCR (Polymerase Chain Reaction). The method is the first application of the eDNA technology in the monitoring of the pathogen in the water area of the grouper culture water source, and the provided identification method improves the detection sensitivity, realizes diversified detection and improves the detection efficiency.

Description

Technical Field

[0001] The present invention relates to the technical field of pathogenic species identification, in particular to the technical field of molecular biology for identification of pathogenic microorganisms and parasite species in aquaculture, and in particular to a primer set, an identification method, a kit and an application for identification of pathogens in water source of grouper aquaculture. Background Art

[0002] The detection and identification of grouper pathogens is the premise for grouper farming, and it is also the most basic work for the prevention and treatment of grouper diseases. Common methods for detecting and identifying grouper diseases usually collect diseased fish tissues for microscopic observation and molecular identification, but this cannot detect grouper-related pathogens from the source of the water, so it is impossible to identify potential pathogens earlier and take effective preventive measures. It is easy to miss the early window of pathogen prevention, resulting in delayed pathogen identification. With the development and progress of molecular biology technology, the molecular identification method of grouper pathogens based on species-specific gene fragments has become the most commonly used method for grouper pathogen identification. However, this method often requires direct collection of tissue samples of grouper individuals to extract total nucleic acid, which will inevitably cause a lag in the detection of grouper pathogens. In recent years, with the advent and development of eDNA (environmental DNA) technology, we can directly predict the types and abundance of pathogens in the water area by collecting water samples from grouper farming water sources, control the pathogen situation of grouper from the source, and greatly improve the comprehensiveness and efficiency of detection. If this method only uses traditional PCR means and only designs a pair of amplification primers to capture the target pathogen fragment, it will result in high randomness and low sensitivity. If loop-mediated isothermal amplification technology (LAMP technology) is used for amplification, although the sensitivity and specificity are improved, it is still impossible to completely avoid the occurrence of false positives, which will cause certain misjudgments in the results of pathogen investigations in water sources and waters, and bring great misleading to the guidance of disease prevention.

[0003] Environmental DNA technology is a method of extracting DNA directly from environmental samples without separating any species, which can minimize the impact of biological monitoring on the environment. The application scope of this technology is becoming more and more extensive. The following are some specific application cases: 1. Ecological protection and monitoring: Environmental DNA technology can be used to assess the health of ecosystems, including species diversity, community structure and ecosystem functions. In addition, eDNA technology can also be used to monitor and warn of potential biological invasions. 2. Fish monitoring: Environmental DNA technology is also increasingly used in fish monitoring. Compared with traditional biological monitoring methods based on morphological identification, eDNA technology has the advantages of simplicity, efficiency, high sensitivity and environmental friendliness. 3. Cyanobacteria bloom monitoring: The large-scale outbreak of cyanobacteria blooms under global climate change has become an important environmental problem. Accurate, efficient and real-time monitoring of cyanobacteria is the key to the prevention and control of cyanobacteria blooms. In recent years, environmental DNA macrobarcoding technology has begun to be applied to cyanobacteria community monitoring, which makes up for the shortcomings of difficult species identification by microscopy and being greatly affected by subjective experience. These cases show that environmental DNA technology has great potential and application value, and can play an important role in ecological protection, species monitoring and early warning of environmental problems.

[0004] At present, the main strategy for detecting pathogens in grouper is to collect grouper tissues or shrimp pond water. This strategy can often only detect pathogens in shrimp ponds and amplify the target sequences of several pathogens. The detection range and the number of detected pathogens are limited. The traditional sampling and detection method in shrimp ponds requires multiple processing and analysis of samples, and can only detect some pathogens, so its detection throughput is relatively low. This method can only be used for detection when sampling in the breeding pond, and it is impossible to know the potential pathogens in the water source in advance, so it is impossible to timely discover and deal with problems from the upstream of the pathogen source. It only detects a few pathogens, and the effective reference information it can provide is very limited. It can only detect some pathogens and environmental factors, and cannot fully reflect the ecological environment and health status of the entire breeding area. It fails to play an early warning role in disease prevention and early intervention. Summary of the invention

[0005] The main purpose of the present invention is to solve the problem of investigating and identifying potential pathogens in seawater samples of water sources for grouper aquaculture with a high degree of degradation and a low abundance of target pathogen nucleic acid, thereby providing a primer set, an identification method, a kit and an application for identifying pathogens in water sources for grouper aquaculture.

[0006] In a first aspect, the present invention provides a primer set for identifying pathogens in water source of grouper aquaculture, comprising the following primers:

[0007] The primers used in the first round of PCR were as shown in SeqNo.1-6;

[0008] The primers used in the second round of PCR were shown in Seq No. 7-12.

[0009] Table 1. Primers for the first round of nested PCR

[0010]

[0011]

[0012] Table 2. Primers for the second round of nested PCR

[0013]

[0014] In a second aspect, the present invention provides a method for identifying pathogens in water source of grouper aquaculture using the primer set as described in the first aspect. The method comprises two rounds of PCR amplification, i.e., nested PCR amplification, comprising the following steps:

[0015] Step 1: Using the total DNA of the water body to be tested (i.e., eDNA) as a template, using the primers described in SeqNo.1-6, perform the first round of PCR amplification;

[0016] Step 2: Take the first round PCR amplification product as a template, use the primers described in Seq No.7-12 to perform a second round of PCR amplification under the same conditions as the first round of PCR to obtain the amplified target product;

[0017] Step 3: Perform agarose gel electrophoresis on the target product and identify the pathogen based on the electrophoresis results.

[0018] Preferably, the conditions for the two rounds of PCR amplification are:

[0019] Pre-denaturation at 94-95°C for 5 minutes (for example, it can be 94.1°C, 94.2°C, 94.3°C, 94.4°C, 94.5°C, 94.6°C, 94.7°C, 94.8°C, 94.9°C or all values ​​within the range, which will not be repeated due to space limitations); denaturation at 94-96°C for 30 seconds (for example, it can be 94.1°C, 94.2°C, 94.3°C, 94.4°C, 94.5°C, 94.6°C, 94.7°C, 94.8°C, 94.9°C, 95°C, 95.1°C, 95.2°C, 95.3°C, 95.4°C, 95.5°C, 95.6°C, 95.7°C, 95.8°C, 95.9°C, 96°C or all values ​​within the range, which are not described in detail due to space limitations), annealing at 55-57°C for 30 seconds (for example, it can be 55.1°C, 55.2°C, 55.3°C, 55.4°C, 55.5°C, 55.6°C, 55.7°C, 55.8°C, 55.9°C, 56°C, 56.1°C, 56.2°C, 56.3°C, 56.4°C, 56.5°C, 56.6°C, 56.7°C, 56.8°C, 56.9°C, 57°C or all values ​​within the range, which are not described in detail due to space limitations), and extension at 72-74°C for 30 seconds (for example, it can be 72.1°C, 72.2°C, 72.3°C, 72.4°C, 72.5°C, 72.6°C, 72.7°C, 72.8°C, 72.9°C, 73°C, 73.1°C, 73.2°C, 73.3°C, 73.4°C, 73.5°C, 73.6°C, 73.7°C, 73.8°C, 73.9°C, 74°C or all values ​​within the range, which are not repeated due to space limitations), cycle 35 times; 72-74°C for another 10 minutes (for example, 72.1°C, 72.2°C, 72.3°C, 72.4°C, 72.5°C, 72.6°C, 72.7°C, 72.8°C, 72.9°C, 73°C, 73.1°C, 73.2°C, 73.3°C, 73.4°C, 73.5°C, 73.6°C , 73.7℃, 73.8℃, 73.9℃, 74℃ or all values ​​within the range, which are not described in detail due to space limitations), and finally keep warm at 3-6℃ (for example, it can be 3.1℃, 3.2℃, 3.3℃, ​​3.4℃, 3.5℃, 3.6℃, ​​3.7℃, 3.8℃, 3.9℃, 4℃, 4.1℃, 4.2℃, 4.3℃, 4.4℃, 4.5℃, 4.6℃, 4.7℃, 4.8℃, 4.9℃, 5℃, 5.1℃, 5.2℃, 5.3℃, 5.4℃, 5.5℃, 5.6℃, 5.7℃, 5.8℃, 5.9℃, 6℃ or all values ​​within the range, which are not described in detail due to space limitations).

[0020] Preferably, the method for identifying pathogens in grouper farming water includes the following steps:

[0021] Step 1: Using the total DNA of the water body to be tested as a template, use the primers described in Seq No.1-6 to perform the first round of PCR amplification;

[0022] Step 2: Take the first round PCR amplification product as a template, use the primers described in Seq No.7-12 to perform a second round of PCR amplification under the same conditions as the first round of PCR to obtain the amplified target product;

[0023] Step 3: subject the target product to agarose gel electrophoresis and identify the pathogen based on the electrophoresis results;

[0024] Wherein, the conditions for the two rounds of PCR amplification are:

[0025] Pre-denaturation at 94-95°C for 5 minutes; denaturation at 94-96°C for 30 seconds, annealing at 55-57°C for 30 seconds, extension at 72-74°C for 30 seconds, cycle 35 times; extension at 72-74°C for another 10 minutes, and finally incubation at 3-6°C.

[0026] In a third aspect, the present invention provides a grouper pathogen identification kit, the kit comprising at least 6 pairs of primer sequences as described in SeqNo.1-12.

[0027] In a fourth aspect, the present invention provides the application of the method for identifying pathogens in grouper aquaculture water as described in the second aspect in the field of grouper pathogen identification.

[0028] Compared with the prior art, the present invention has at least the following beneficial effects:

[0029] The present invention can make up for the shortcomings of traditional grouper pathogen detection technology by adopting a method combining nested PCR and high-throughput sequencing technology. Under the premise of not approaching, touching, or interfering with grouper farming, seawater samples from the water source sea area for grouper farming are collected, and a set of nested PCR amplification primers for multiple grouper pathogens are designed. Nested PCR is performed on the target pathogen-specific sequence region, and eDNA is sequenced using a high-throughput sequencing method to supplement the limitations of nested PCR, and finally the type of pathogen species is analyzed and determined by means of data assembly, comparison, etc., so as to achieve a comprehensive and accurate identification of potential pathogens in the water source water area of ​​the farming water. The present invention solves the problem of investigating and identifying potential pathogens in seawater samples of grouper farming water sources with a high degree of degradation and a low abundance of target pathogen nucleic acids.

[0030] Compared with the traditional method of only sampling and detecting pathogens of grouper in fish ponds, the identification method provided by the present invention has at least the following advantages:

[0031] 1. Non-destructive sampling: Water source environmental DNA testing can obtain the DNA of grouper pathogens from upstream source waters without the need to catch or sample groupers, thus avoiding damage and loss to groupers.

[0032] 2. Improve detection sensitivity: Water source environmental DNA testing can detect pathogens inside and outside the shrimp pond, including potential pathogens and pathogens that do not show symptoms. In contrast, traditional sampling and testing only in shrimp ponds may miss some potential pathogens.

[0033] 3. Diversity detection: Water source environmental DNA can detect multiple pathogens at the same time through high-throughput sequencing analysis, while traditional sampling and identification in shrimp ponds can usually only detect specific pathogens. This can provide a more comprehensive understanding of the pathogen composition and distribution of the shrimp pond.

[0034] 4. Improve detection efficiency: After high-throughput sequencing of water source environmental DNA, a comprehensive and accurate pathogen database is established for comparison and identification, which can quickly and accurately detect pathogens in a large number of samples. In contrast, the traditional sampling and testing in shrimp ponds requires processing and testing of each sample one by one, which is less efficient.

[0035] 5. Currently, eDNA technology is mainly used in ecological protection and monitoring of fish and cyanobacteria. There is no application of pathogen monitoring in grouper aquaculture water sources based on eDNA technology, nor is there any case related to the identification of potential pathogens in grouper aquaculture water sources using a combination of eDNA and nested PCR technology. The present invention is the first application of eDNA technology in pathogen monitoring in grouper aquaculture water sources. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0037] Figure 1 The agarose gel electrophoresis result of Example 2 is shown in FIG.

[0038] Figure 2 The agarose gel electrophoresis result of Comparative Example 1 is shown in FIG.

[0039] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] It should be noted that all directional indications in the embodiments of the present invention (such as up, down, left, right, front, back, etc.) are only used to explain the relative position relationship, movement status, etc. between the components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indication will also change accordingly.

[0042] In addition, the descriptions of "first", "second", etc. in the present invention are only used for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in the field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0043] In a first aspect, the present invention provides a primer set for identifying pathogens in water source of grouper aquaculture, comprising the following primers:

[0044] The primers used in the first round of PCR were as shown in SeqNo.1-6;

[0045] The primers used in the second round of PCR were shown in Seq No. 7-12.

[0046] In a second aspect, the present invention provides a method for identifying pathogens in water source of grouper aquaculture using the primer set as described in the first aspect, comprising the following steps:

[0047] Step 1: Using the total DNA of the water body to be tested (i.e., eDNA) as a template, using the primers described in SeqNo.1-6, perform the first round of PCR amplification;

[0048] Step 2: Take the first round PCR amplification product as a template, use the primers described in Seq No.7-12 to perform a second round of PCR amplification under the same conditions as the first round of PCR to obtain the amplified target product;

[0049] Step 3: Perform agarose gel electrophoresis on the target product and identify the pathogen based on the electrophoresis results.

[0050] As a preferred embodiment, the conditions for the two rounds of PCR amplification are:

[0051] Pre-denaturation at 94-95°C for 5 minutes (for example, it can be 94.1°C, 94.2°C, 94.3°C, 94.4°C, 94.5°C, 94.6°C, 94.7°C, 94.8°C, 94.9°C or all values ​​within the range, which will not be repeated due to space limitations); denaturation at 94-96°C for 30 seconds (for example, it can be 94.1°C, 94.2°C, 94.3°C, 94.4°C, 94.5°C, 94.6°C, 94.7°C, 94.8°C, 94.9°C, 95°C, 95.1°C, 95.2°C, 95.3°C, 95.4°C, 95.5°C, 95.6°C, 95.7°C, 95.8°C, 95.9°C, 96°C or all values ​​within the range, which are not described in detail due to space limitations), annealing at 55-57°C for 30 seconds (for example, it can be 55.1°C, 55.2°C, 55.3°C, 55.4°C, 55.5°C, 55.6°C, 55.7°C, 55.8°C, 55.9°C, 56°C, 56.1°C, 56.2°C, 56.3°C, 56.4°C, 56.5°C, 56.6°C, 56.7°C, 56.8°C, 56.9°C, 57°C or all values ​​within the range, which are not described in detail due to space limitations), and extension at 72-74°C for 30 seconds (for example, it can be 72.1°C, 72.2°C, 72.3°C, 72.4°C, 72.5°C, 72.6°C, 72.7°C, 72.8°C, 72.9°C, 73°C, 73.1°C, 73.2°C, 73.3°C, 73.4°C, 73.5°C, 73.6°C, 73.7°C, 73.8°C, 73.9°C, 74°C or all values ​​within the range, which are not repeated due to space limitations), cycle 35 times; 72-74°C for another 10 minutes (for example, 72.1°C, 72.2°C, 72.3°C, 72.4°C, 72.5°C, 72.6°C, 72.7°C, 72.8°C, 72.9°C, 73°C, 73.1°C, 73.2°C, 73.3°C, 73.4°C, 73.5°C, 73.6°C , 73.7℃, 73.8℃, 73.9℃, 74℃ or all values ​​within the range, which are not described in detail due to space limitations), and finally keep warm at 3-6℃ (for example, it can be 3.1℃, 3.2℃, 3.3℃, ​​3.4℃, 3.5℃, 3.6℃, ​​3.7℃, 3.8℃, 3.9℃, 4℃, 4.1℃, 4.2℃, 4.3℃, 4.4℃, 4.5℃, 4.6℃, 4.7℃, 4.8℃, 4.9℃, 5℃, 5.1℃, 5.2℃, 5.3℃, 5.4℃, 5.5℃, 5.6℃, 5.7℃, 5.8℃, 5.9℃, 6℃ or all values ​​within the range, which are not described in detail due to space limitations).

[0052] As a preferred embodiment, the method for identifying pathogens in grouper farming water includes the following steps:

[0053] Step 1: Using the total DNA of the water body to be tested as a template, use the primers described in Seq No.1-6 to perform the first round of PCR amplification;

[0054] Step 2: Take the first round PCR amplification product as a template, use the primers described in Seq No.7-12 to perform a second round of PCR amplification under the same conditions as the first round of PCR to obtain the amplified target product;

[0055] Step 3: subject the target product to agarose gel electrophoresis and identify the pathogen based on the electrophoresis results;

[0056] Wherein, the conditions for the two rounds of PCR amplification are:

[0057] Pre-denaturation at 94-95°C for 5 minutes; denaturation at 94-96°C for 30 seconds, annealing at 55-57°C for 30 seconds, extension at 72-74°C for 30 seconds, cycle 35 times; extension at 72-74°C for another 10 minutes, and finally incubation at 3-6°C.

[0058] In a third aspect, the present invention provides a grouper pathogen identification kit, the kit comprising at least 6 pairs of primer sequences as described in SeqNo.1-12.

[0059] In a fourth aspect, the present invention provides the application of the method for identifying pathogens in grouper aquaculture water as described in the second aspect in the field of grouper pathogen identification.

[0060] Example 1

[0061] (1) Water sample collection

[0062] In the Huangbu waters of Huidong County, Huizhou City, Guangdong Province, sterilized sampling bottles were used to collect water samples from 4 water sources in the waters three times, and a total of 12 seawater samples were collected, each sample was 5L;

[0063] (2) Water sample filtration

[0064] The collected water samples were filtered and enriched using a sterilized water sample filtration device, and the organisms, tissue fragments and free DNA in the water were enriched on the microporous filter membrane, which was then frozen and stored at -80°C.

[0065] (3) Extraction of total nucleic acid from water

[0066] The enriched microporous filter membrane was taken out, placed in a centrifuge tube and cut into pieces, and the total nucleic acid in the water was extracted from the filter membrane fragments using a total nucleic acid extraction kit. The concentration and quality of the total nucleic acid sample were detected by gel electrophoresis and an ultra-micro spectrophotometer, and the RNA in the total nucleic acid was reverse transcribed into cDNA using a reverse transcription kit.

[0067] Example 2

[0068] The nested PCR amplification reaction used the total water DNA in Example 1 as a template and the sequences listed in Tables 1 and 2 as primers to construct a 25 μl reaction system. The first round of PCR was amplified using external primers, and the PCR cycle was: 94°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 55°C annealing for 30 seconds, 72°C extension for 30 seconds, 35 cycles; 72°C extension for 10 minutes, and finally 4°C incubation. In the second round of PCR, 2ul of the first round amplification product was used as a template and amplified using internal primers under the same PCR cycle conditions.

[0069] The product was identified by electrophoresis in 2% agarose gel, and the size of the band was used to determine whether the target pathogen band was amplified. The amplified target band was sent for further sequence comparison.

[0070] The remaining eDNA was used to construct a DNA sequencing library, and high-throughput sequencing was performed using the MGISEQ-T7 sequencer to obtain high-quality offline data. The original offline data was filtered using cutadapter software, and the filtered data was aligned to the constructed pathogenic species database using SOAP software. The aligned sequences were spliced ​​using pear software, and the spliced ​​sequences were assembled into contigs using SPAdes to further confirm the detected pathogens.

[0071] Example 3

[0072] The steps are the same as those in Example 2, except that the conditions for PCR amplification are changed, specifically:

[0073] Pre-denaturation at 95°C for 5 minutes; denaturation at 96°C for 30 seconds, annealing at 57°C for 30 seconds, extension at 74°C for 30 seconds, cycle 35 times; extension at 74°C for another 10 minutes, and finally incubation at 3°C.

[0074] The product was identified by electrophoresis in 2% agarose gel, and the size of the band was used to determine whether the target pathogen band was amplified. The amplified target band was sent for further sequence comparison.

[0075] The remaining eDNA was used to construct a DNA sequencing library, and high-throughput sequencing was performed using the MGISEQ-T7 sequencer to obtain high-quality offline data. The original offline data was filtered using cutadapter software, and the filtered data was aligned to the constructed pathogenic species database using SOAP software. The aligned sequences were spliced ​​using pear software, and the spliced ​​sequences were assembled into contigs using SPAdes to further confirm the detected pathogens.

[0076] Example 4

[0077] The steps are the same as those in Example 2, except that the conditions for PCR amplification are changed, specifically:

[0078] Pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 73°C for 30 seconds, cycle 35 times; extension at 73°C for another 10 minutes, and finally incubation at 6°C.

[0079] The product was identified by electrophoresis in 2% agarose gel, and the size of the band was used to determine whether the target pathogen band was amplified. The amplified target band was sent for further sequence comparison.

[0080] The remaining eDNA was used to construct a DNA sequencing library, and high-throughput sequencing was performed using the MGISEQ-T7 sequencer to obtain high-quality offline data. The original offline data was filtered using cutadapter software, and the filtered data was aligned to the constructed pathogenic species database using SOAP software. The aligned sequences were spliced ​​using pear software, and the spliced ​​sequences were assembled into contigs using SPAdes to further confirm the detected pathogens.

[0081] Example 5

[0082] The steps are the same as those in Example 2, except that the conditions for PCR amplification are changed, specifically:

[0083] Pre-denaturation at 94°C for 5 minutes; denaturation at 94°C for 30 seconds, annealing at 55°C for 30 seconds, extension at 72°C for 30 seconds, cycle 35 times; extension at 74°C for another 10 minutes, and finally incubation at 5°C.

[0084] The product was identified by electrophoresis in 2% agarose gel, and the size of the band was used to determine whether the target pathogen band was amplified. The amplified target band was sent for further sequence comparison.

[0085] The remaining eDNA was used to construct a DNA sequencing library, and high-throughput sequencing was performed using the MGISEQ-T7 sequencer to obtain high-quality offline data. The original offline data was filtered using cutadapter software, and the filtered data was aligned to the constructed pathogenic species database using SOAP software. The aligned sequences were spliced ​​using pear software, and the spliced ​​sequences were assembled into contigs using SPAdes to further confirm the detected pathogens.

[0086] Comparative Example 1

[0087] The total water DNA in Example 1 was used as a template and amplified using conventional grouper pathogen detection PCR primers in a patent. The amplification conditions were the same as in Example 2, and other treatments were the same.

[0088] Performance Verification

[0089] The agarose gel electrophoresis test results of Example 2 are as follows Figure 1As shown, combined with DNA sequencing to further confirm the detected pathogens, the results show that the pathogens detected in Example 2 include: 1. Viral neuronecrosis virus, 2. Lymphocystis virus, 3. Grouper iridovirus, 4. Negative control.

[0090] The results of Examples 3-5 are similar to those of Example 2 and will not be described in detail.

[0091] The agarose gel electrophoresis test results of Comparative Example 1 are as follows Figure 2 As shown, combined with DNA sequencing to further confirm the detected pathogens, the results show that Comparative Example 1 did not detect any pathogens detected in Example 2 (viral neuronecrosis virus, lymphocystis virus, grouper iridovirus). This shows that the detection effect of Examples 2-5 is significantly better than that of Comparative Example 1, and the primer sequence and detection method provided by the present invention have good effects.

[0092] In summary, the present invention can make up for the shortcomings of traditional grouper pathogen detection technology by adopting a method combining nested PCR and high-throughput sequencing technology. Under the premise of not approaching, touching, or interfering with grouper farming, seawater samples from the water source sea area for grouper farming are collected, and a set of nested PCR amplification primers for multiple grouper pathogens are designed. Nested PCR is performed on the target pathogen-specific sequence region, and eDNA is sequenced using a high-throughput sequencing method to supplement the limitations of nested PCR, and finally the type of pathogen species is analyzed and judged by means of data assembly, comparison, etc., so as to achieve a comprehensive and accurate identification of potential pathogens in the water source water area for this piece of farming. The present invention solves the problem of investigating and identifying potential pathogens in seawater samples of grouper farming water sources with a high degree of degradation and a low abundance of target pathogen nucleic acids.

[0093] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification and drawings under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A primer set for identifying pathogens in water source of grouper aquaculture, characterized in that: The primers included are: The primers used in the first round of PCR were as shown in SeqNo.1-6; The primers used in the second round of PCR were shown in Seq No. 7-12.

2. A method for identifying pathogens in water source of grouper aquaculture using the primer set as claimed in claim 1, characterized in that: The steps include: Step 1: Using the total DNA of the water body to be tested as a template, use the primers described in Seq No.1-6 to perform the first round of PCR amplification; Step 2: Take the first round PCR amplification product as a template, use the primers described in SeqNo.7-12 to perform a second round of PCR amplification under the same conditions as the first round of PCR to obtain the amplified target product; Step 3: Perform agarose gel electrophoresis on the target product and identify the pathogen based on the electrophoresis results.

3. The method for identifying pathogens in water source for grouper breeding as claimed in claim 2, characterized in that: The conditions for the two rounds of PCR amplification are: Pre-denaturation at 94-95°C for 5 minutes; denaturation at 94-96°C for 30 seconds, annealing at 55-57°C for 30 seconds, extension at 72-74°C for 30 seconds, cycle 35 times; extension at 72-74°C for another 10 minutes, and finally incubation at 3-6°C.

4. A grouper pathogen identification kit, characterized in that: The kit at least includes 6 pairs of primer sequences as described in SeqNo.1-12.

5. Application of the method for identifying pathogens in water sources for grouper breeding as claimed in claim 2 or 3 in the field of pathogen identification of grouper.

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