Detection reagents and kits for identifying sub-salmonid tolerance to bacterial gill disease

Abstract

The present application belongs to the technical field of biological detection, and particularly relates to a detection reagent and a detection kit for identifying sub-dong salmon resistant to bacterial gill disease. The detection reagent for identifying sub-dong salmon resistant to bacterial gill disease is obtained by performing transcriptome sequencing on the head kidney of 6 susceptible and 6 resistant sub-dong salmon, screening out 104 SNP specific sites, performing multiple PCR post-genomic sequencing, and analyzing and population verifying the SNP sites through susceptible and resistant sub-dong salmon samples. Six SNP specific sites are screened out. The six SNP sites are used as detection items of the kit for screening sub-dong salmon resistant to bacterial gill disease. The reagent and the kit have the advantages of short library construction cycle, good repeatability, simple operation, etc. The sub-dong salmon resistant to bacterial gill disease can be quickly screened without damaging the sub-dong salmon, so as to quickly form a sub-dong salmon breeding population resistant to bacterial gill disease.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a detection reagent and a detection kit for identifying the bacterial gill disease tolerance of Salmo trutta. BACKGROUND

[0002] Salmonid fish is one of the three major farmed fish in the world, and its farming yield is only inferior to that of carp and tilapia. The farming areas in China are mainly distributed in Heilongjiang, Jilin, Liaoning and Gansu, and the main farmed species are Cheryn salmon, rainbow trout, white salmon and red-spotted salmon. Salmo trutta, also known as river salmon and catfish, belongs to Salmoniformes, Salmonidae and Salmo, and is a homonym of Salmo trutta. Salmo trutta has established a relatively stable population in the Yading River in Tibet and can grow and breed naturally, and has become a local breed, which belongs to cold-water fish. It is deeply loved by consumers because of its tender meat, no intermuscular spines, rich nutrition and delicious taste.

[0003] Bacterial gill disease (BGD) is a common disease in the farming of Salmo trutta, and if not promptly treated, it will cause serious damage to the development of fisheries. However, there is no good method for detecting the BGD resistance of Salmo trutta at present. SUMMARY

[0004] The present application aims to provide a detection reagent and a detection kit for identifying the bacterial gill disease tolerance of Salmo trutta, and a method of gene sequencing after multiplex PCR by the kit, so as to quickly identify whether Salmo trutta has bacterial gill disease resistance.

[0005] The present application provides a detection reagent for identifying the bacterial gill disease tolerance of Salmo trutta, which comprises specific primer pairs respectively designed for 104 SNP sites;

[0006] The SNP sites are screened by using susceptible bacterial gill disease and Salmo trutta individuals with bacterial gill disease resistance for transcriptome sequencing, respectively;

[0007] The template for the transcriptome sequencing is the RNA of the head kidney tissue.

[0008] Preferably, the SNP sites are as follows:

[0009]

[0010]

[0011]

[0012] The information of the SNP site is based on Salmo_trutta.fSalTru1.1 (http: / / asia.ensembl.org / Salmo_trutta / Info / Index?).

[0013] The application further provides a detection kit for the bacterial gill disease resistant trait of Salmo salar, comprising the detection reagent.

[0014] The application further provides a detection kit for the bacterial gill disease resistant trait of Salmo salar, comprising specific primer pairs respectively designed for the 6 SNP sites.

[0015] The 6 SNP sites include: 16842538-16842787 of chromosome 4, 77517014-77517244 of chromosome 12, 87581588-87581818 of chromosome 13, 18173575-18173832 of chromosome 18, 11029040-11028277 of chromosome 27 and 7014792-7015027 of chromosome 40.

[0016] Preferably, the nucleotide sequences of the specific primer pairs designed for 16842538-16842787 of chromosome 4 are shown as SEQ ID NO. 1 and SEQ ID NO. 2, the nucleotide sequences of the specific primer pairs designed for 77517014-77517244 of chromosome 12 are shown as SEQ ID NO. 3 and SEQ ID NO. 4, the nucleotide sequences of the specific primer pairs designed for 87581588-87581818 of chromosome 13 are shown as SEQ ID NO. 5 and SEQ ID NO. 6, the nucleotide sequences of the specific primer pairs designed for 18173575-18173832 of chromosome 18 are shown as SEQ ID NO. 7 and SEQ ID NO. 8, the nucleotide sequences of the specific primer pairs designed for 11029040-11028277 of chromosome 27 are shown as SEQ ID NO. 9 and SEQ ID NO. 10, and the nucleotide sequences of the specific primer pairs designed for 7014792-7015027 of chromosome 40 are shown as SEQ ID NO. 11 and SEQ ID NO. 12.

[0017] Preferably, the kit further comprises reagents for post-multiplex PCR gene sequencing.

[0018] Preferably, the reagents for post-multiplex PCR gene sequencing comprise independently packaged multiplex amplicon library reagents and multiplex amplicon adapter reagents.

[0019] The application also provides application of the reagent or the kit in creating sub-Donghu salmon germplasm with bacterial gill disease resistance.

[0020] Beneficial effects: the application provides a detection reagent for identifying sub-Donghu salmon resistant to bacterial gill disease, 104 SNP specific sites are screened out by performing transcriptome sequencing on the head kidney of 6 susceptible and resistant sub-Donghu salmons, primers are designed, and the method of multiplex PCR followed by gene sequencing is used, Figure 1 6 SNP specific sites are finally screened out, which can be used as SNP sites for screening sub-Donghu salmon resistant to bacterial gill disease, and a kit is prepared. For sub-Donghu salmon individuals with unknown bacterial gill disease resistance, only about 0.5g of tissue (fat fin, etc.) of the sub-Donghu salmon is needed for DNA extraction, and the kit is used for multiplex PCR followed by gene sequencing, so that whether the sub-Donghu salmon has bacterial gill disease resistance can be quickly identified. The kit uses multiplex PCR to construct a second-generation sequencing library, which is suitable for the Illumina platform, and uses two rounds of PCR reaction to construct the library, which has the advantages of short library construction period, good repeatability, and simple operation. The detection kit described in the application helps researchers to quickly screen sub-Donghu salmon with bacterial gill disease resistance without damaging the sub-Donghu salmon, so as to quickly establish a sub-Donghu salmon breeding population with bacterial gill disease resistance, which is beneficial to the development of sub-Donghu salmon breeding. BRIEF DESCRIPTION OF DRAWINGS

[0021] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings described below are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor.

[0022] Figure 1 The flow chart for library construction capture;

[0023] Figure 2 The kit Library Prep Kit (Module A Universal). DETAILED DESCRIPTION

[0024] The application provides a detection reagent for identifying sub-Donghu salmon resistant to bacterial gill disease, the detection reagent comprising specific primer pairs designed for 104 SNP sites respectively;

[0025] The SNP sites include 104 SNP sites screened after transcriptome sequencing of the head kidney of susceptible bacterial gill disease and A. transmontanus individuals with bacterial gill disease resistance, respectively.

[0026] The present application isolates and purifies a suspected pathogenic bacterium from the rotten gills of A. transmontanus with bacterial gill disease in A. transmontanus breeding population in Yadian County, and identifies the bacterium as Aeromonas salmonicida (ATCC 33658) through 16S rRNA gene sequencing comparison.

[0027] The present application determines whether A. transmontanus has bacterial gill disease resistance by using the above-mentioned Aeromonas salmonicida, preferably including artificial infection experiment based on the use of the Aeromonas salmonicida to infect A. transmontanus, and A. transmontanus with rotten gills or even death is identified as a susceptible individual to bacterial gill disease, and A. transmontanus with no obvious abnormalities in the gill and survival is identified as an individual with bacterial gill disease resistance.

[0028] The present application performs transcriptome sequencing on the head kidney of 6 A. transmontanus individuals with susceptibility and resistance determined by the above-mentioned artificial infection experiment, and screens out 104 SNP specific sites, which are based on Salmo_trutta.fSalTru1.1 (http: / / asia.ensembl.org / Salmo_trutta / Info / Index?), same below.

[0029] The present application also provides a detection kit for bacterial gill disease resistance of A. transmontanus, which comprises the above-mentioned detection reagent.

[0030] The detection kit of the present application preferably uses multiplex PCR followed by gene sequencing to detect the tolerance of A. transmontanus to bacterial gill disease, and the specific process is as shown in Figure 1 Based on the specific site information of the above-mentioned 104 SNP sites, the present application designs primers, analyzes and population verifies the 104 SNP sites by multiplex PCR followed by gene sequencing, and supplements the susceptible and resistant A. transmontanus samples (30 individuals in each group) obtained by artificial infection experiment, and finally screens out 6 SNP specific sites, which can be used as SNP sites for screening bacterial gill disease resistance of A. transmontanus, and are prepared into commercial kits.

[0031] The present application also provides a detection kit for bacterial gill disease resistance of A. transmontanus, which comprises specific primer pairs designed for 6 SNP sites, respectively.

[0032] The six SNP loci include: chromosome 4 16842538-16842787, chromosome 12 77517014-77517244, chromosome 13 87581588-87581818, chromosome 18 18173575-18173832, chromosome 27 11029040-11028277, and chromosome 40 7014792-7015027.

[0033] Table 1 Primer information for the detection kit

[0034]

[0035]

[0036] The detection kit of the present invention preferably comprises three main parts in separate packages, namely, multiplex amplicon library preparation reagent (… Library Prep Kit), Multiplex Amplicon Primer Set ( PrimerPool) and multiplex amplicon adapter reagents ( Connector kit).

[0037] In this invention, Library Prep Kit Figure 2 It is divided into two sub-modules, Module A and Module B. The Library Prep Kit (Module A Universal) includes the reagents shown in Table 2, and the recommended storage temperature is -20℃±5℃. The recommended storage conditions for Enhancer Buffer M (2M betaine) are 2℃~8℃, or it can be stored at -20℃±5℃. Enhancer buffer NB: 0.1mg / ml BSA.

[0038] Table 2 Library Prep Kit(Module A Universal)

[0039]

[0040] The present invention The Library Prep Kit (Module B) 960rxn can be packaged and split into Module B1 and Module B2, where Module B1 contains the IGT. TMEM808 Polymerase Mixture and Enhancer Buffer M, Module B2 contains YF Buffer B. The recommended storage condition of Enhancer Buffer M and YF Buffer B is 2℃-8℃, which can also be stored at -20℃±5℃.

[0041] Table 3 Library Prep Kit (Module B)

[0042]

[0043]

[0044] The present application The composition of the Primer Pool is shown in Table 4, wherein N is a tube identifier, and according to the tube design, The Primer Pool includes T1, T1-T2, T1-T3, etc. The total amount is the total amount of each tube-specific primer pool.

[0045] Table 4 Primer Pool

[0046]

[0047] The present application The adapter kit can be selected according to experimental requirements CDI Primer or Dual-Indexed Primer; and in Table 5 and Table 6, N is divided into four combinations of 1-96, 97-192, 193-288, and 289-384 according to the kit.

[0048] Table 5 CDI Primer

[0049]

[0050] Table 6 Dual-Indexed Primer

[0051]

[0052] The detection kit described in the present application is suitable for Custom Panel (A412XV1), Illumina sequencing platform, specifically: The Custom Panel (A412XV1, for Illumina) enables next-generation sequencing library construction via multiplex PCR and is compatible with the Illumina platform. This kit utilizes a multi-factor algorithm to design primers for the genome of the target region of interest, synthesizing effective and specific primers to amplify the target sequence. This significantly reduces the amount of sequencing data, requiring only sequencing and analysis of key regions of interest. It has enormous application potential in clinical diagnostics and drug development, and also holds broad application prospects in related research in the fields of plants and animals. The kit employs two rounds of PCR for library construction, offering advantages such as short construction cycle, high reproducibility, and ease of operation.

[0053] The present invention also provides the application of the above-described reagents or kits in the creation of Asian salmon germplasm resistant to bacterial gill disease.

[0054] In this invention, bacterial gill disease resistance testing is performed based on 104 screened SNP sites, particularly the polymorphism information of these SNP sites. Some of the polymorphism information is shown in Table 7. In this invention, the primers for the 104 SNP sites were obtained through a website (…). https: / / m.primer.igenetech.com / The design is based on the instructions on the website. After logging in, enter the target area in BED format, select the species as East Asian salmon, the number of tubes as the number of SNPs, leave other parameters as default, and after submitting, select the top-ranked primer sequences.

[0055] Table 7. Information on differentially expressed SNP sites identified by head kidney transcriptome sequencing of the Yadong salmon (partial list).

[0056]

[0057]

[0058] The application is preferred to use DNA extracted from the tissue of sub-eastern salmon as a template when detecting the bacterial gill disease tolerance of the created sub-eastern salmon germplasm, and the tissue of the sub-eastern salmon is preferably about 0.5 g of adipose fin, etc. First, the template is used to construct a first round of multiplex PCR reaction system, which is preferably 30 μl and includes: Enhancer buffer NB (1N) 3.5 μl, Enhancer buffer M 2.5 μl, Primer pool 5 μl, template 40 ng, IGT-EM808 polymerase mixture 10 μl, and the balance of ddH2O. The application uses the constructed first round of multiplex PCR reaction system to perform a first round of multiplex PCR reaction, which preferably includes 105℃ heat lid (Heat lid); 95℃ 3min30s; 98℃ 20s, 60℃ 4min, 18 cycles; 72℃ 5min.

[0059] The application performs magnetic bead purification on the product of the first round of multiplex PCR reaction to obtain purified first round product, and then uses the purified first round product to perform a second round of adapter sequence PCR. The system of the second round of adapter sequence PCR is preferably 30 μl and includes: purified first round product 13.5 μl, Enhancer buffer M 2.5 μl, CDI Primer (pre-mixed adapter primer, concentration 5 μM each) 2 μl, IGT-EM808 polymerase mixture 10 μl, and the balance of ddH2O. The program of the second round of adapter sequence PCR of the application preferably includes 105℃ heat lid (Heat lid); 95℃ 3min30s; 98℃ 20s, 58℃ 1min, 72℃ 30s, 9 cycles; 72℃ 5min. The application performs a second round of magnetic bead purification on the product of the second round of adapter sequence PCR, and then performs library concentration and quality inspection on the machine. The adapter primer of the application is a universal adapter, which is obtained and downloaded from the official website of illumina and can be synthesized.

[0060] The method of two rounds of magnetic bead purification is the same in the preferred embodiment of the application TMPure Beads or Agencourt AMPure XP, the purified magnetic beads are stored at 4℃, and after being taken out, the magnetic beads are first vortexed and resuspended, then the magnetic beads are balanced at room temperature for 25-30 min, and before use, the magnetic beads are vortexed and resuspended to ensure uniformity and consistency of the concentration. The operation of the magnetic bead purification in the application preferably comprises: 1) Prepare 80% ethanol with anhydrous ethanol and Nuclease-Free Water in advance and place it at room temperature for standby. 2) Take the purified magnetic beads out of the 4℃ refrigerator in advance, mix well and balance at room temperature for 30 min; the purified magnetic beads that have been balanced to room temperature are vortexed and mixed well for standby. 3) Add 0.9 times the volume of magnetic beads (27 μL) to the product to be purified, mix well by pipetting or vortexing, and stand at room temperature for 5 min. 4) Centrifuge for a moment, place the PCR tube on the magnetic stand for 3 min, and wait for the solution to clarify. 5) Remove the supernatant completely, take the PCR tube off the magnetic stand, add 50 μL YF buffer B to the tube, mix well by pipetting, and stand at room temperature for 5 min. 6) Centrifuge for a moment, place the PCR tube on the DynaMag-96 Side magnetic stand for 3 min. 7) Keep the PCR tube on the magnetic stand, carefully remove the supernatant, add 180 μL of 80% ethanol solution to the PCR tube, and stand for 30 s. 8) Keep the PCR tube on the magnetic stand, remove the supernatant, and add 180 μL of 80% ethanol solution to the PCR tube again, stand for 30 s, and remove the supernatant. 9) Cover the tube cap, centrifuge for a moment, centrifuge the residual ethanol to the bottom of the tube, carefully remove the residual ethanol at the bottom of the PCR tube with a 10 μL pipette, and make sure not to suck the magnetic beads. 10) Keep the PCR tube on the magnetic stand, stand at room temperature for 3-5 min, and dry the magnetic beads to make the residual ethanol evaporate completely. 11) Add 24 μL of Nuclease-Free Water, take the PCR tube off the magnetic stand, mix well by pipetting or vortexing, and stand at room temperature for 2 min. 12) Centrifuge for a moment, place the PCR tube on the magnetic stand for 2 min, and wait for the solution to clarify. 13) Use a pipette to suck 13.5 μL of the supernatant and transfer it to a new PCR tube, and the supernatant in the tube is the purified multiplex PCR product, which is labeled.

[0061] In order to further illustrate the present application, the identification reagent and kit for identifying the bacteria-resistant gill disease of sub-dong salmon provided by the present application are described in detail below in combination with the drawings and examples, but they cannot be understood as limiting the protection scope of the present application.

[0062] Example 1

[0063] 1. Materials and methods

[0064] 1.1 Experimental materials

[0065] The sub-eastern salmon used in the experiment were from the breeding base of the Institute of Aquaculture Science of the Tibet Academy of Agricultural and Pastoral Sciences, and 24 healthy adult female sub-eastern salmon (body weight 360.00±56.78 g, total length 30.17±1.72 cm, body length 27.33±1.63 cm) were randomly selected for artificial infection experiment of Aeromonas sobiae (preliminary experiments showed that Aeromonas sobiae is the pathogenic bacteria of bacterial gill disease of sub-eastern salmon, which has been published in the journal of Aquaculture Science. Sun Shuaijie, Zhou Jianzhi, Wang Wanli, et al. Isolation and identification of pathogenic bacteria of bacterial gill disease of sub-eastern salmon [J / OL]. Aquaculture Science: 1-14 [2022-09-30]. DOI: 10.16378 / j.cnki.1003-1111.21052.). The sub-eastern salmon showing bacterial gill disease and asymptomatic were selected 6 each to form the susceptible group and the resistant group, and the head kidney tissues of the two groups of sub-eastern salmon were collected. After the sample collection, it was placed in liquid nitrogen for freezing, and after 30 min, it was transferred to a-80℃ refrigerator for storage.

[0066] 1.2 Experimental method

[0067] The above experimental materials were sent to Shanghai Pisenlo Biotechnology Co., Ltd. for transcriptome sequencing, and the SNP sites related to bacterial gill disease resistance were screened from the obtained differential genes.

[0068] 2 Results

[0069] 2.1 Differential gene results

[0070] Compared with the resistant group, 1851 differential genes were screened, including 1026 up-regulated genes and 825 down-regulated genes.

[0071] 2.2 SNP site information

[0072] The SNP sites of each sub-eastern salmon were statistically analyzed, and among the above 1851 differential genes, 104 SNP sites with different genotypes between the susceptible group and the resistant group were finally obtained.

[0073] The information of the 104 SNP sites screened by the above method (part of Table 7) was used to design corresponding primers using the website ( https: / / m.primer.igenetech.com / ) to verify and screen the SNP of the above 6 samples of the susceptible and resistant groups by multiplex PCR method, and 6 SNP sites (Table 1) were screened, which had different genotypes between the susceptible group and the resistant group. Thus, it is preliminarily determined that the 6 SNP sites can be used as SNP sites for screening the bacterial gill disease resistance of sub-eastern salmon.

[0074] Example 2

[0075] Six SNP sites screened out from Example 1 were used as detection basis, and the genotypes of 6 SNP sites of 60 samples were detected by multiplex PCR method, of which 30 were susceptible and 30 were resistant. The method is as follows: 1. The first round of multiplex PCR reaction

[0076] According to the previously provided instructions, the reagent ratio is shown in the following table:

[0077] Reagent Volume(μl) ddH2O 9-x Enhancer buffer NB(1N) 3.5 Enhancer buffer M 2.5 Primer pool 5 sample [1] ]] X IGT-EM808polymerase mixture 10 Total 30

[0078] [1] The initial amount is 40 ng per reaction tube; the concentration of DNA is the quantitative result of Qubit (Thermo Fisher).

[0079] Among them, Enhancer Buffer NB (1N), Enhancer Buffer M and IGT TM EM808 Polymerase Mixture are all from the kit Library Prep Kit (Module A Universal). Primer Pool is from the kit Primer Pool is a mixture of 6 selected SNP primers.

[0080] 2. The first round of magnetic bead purification

[0081] According to the previously provided instructions.

[0082] 3. The second round of adapter sequence PCR reaction

[0083] According to the previously provided instructions, the reagent ratio is shown in the following table:

[0084]

[0085] [1] PCR product mixture is the multiplex PCR product after purification in the previous step.

[0086] [2] CDI Primer is a premixed adapter primer with a concentration of 5 μM each

[0087] Among them, IGT TM EM808 Polymerase Mixture and Enhancer Buffer M are from the kit Library Prep Kit (Module A Universal). CDI Primer is from the kit CDI Primer, because there are only 6 SNP sites primer sequence when validation, so N choose 1-96 combination can.

[0088] 4. 2nd round of magnetic bead purification

[0089] According to the previous provided instructions can be done.

[0090] 5. Concentration measurement and quality inspection

[0091] According to the previous provided instructions can be done.

[0092] 6. Sequencing

[0093] Obtain PCR product sequence, analyze SNP site information, and reverse alignment.

[0094] The results show that: 30 susceptible group samples, 26 completely consistent with 6 SNP site susceptible genotype combination, the accuracy rate is 86.67%; 30 tail resistance group samples, 27 completely consistent with 6 SNP site resistance genotype combination, the accuracy rate is 90.00%. The remaining 7 tail does not completely consistent with any one genotype combination, using this method, the bacterial gill disease resistance is not determined.

[0095] Although the above examples make a detailed description of the present application, it is only a part of the embodiments of the present application, not all the embodiments, people can also obtain other embodiments according to the present embodiment without creativity, these embodiments all belong to the protection scope of the present application.

Claims

1. A test kit for the detection of the trait of Atlantic salmon tolerance to bacterial gill disease, characterized in that, The specific primer pairs are respectively designed for 6 SNP sites; The 6 SNP sites include: 16842638 site on chromosome 4, 77517128 site on chromosome 12, 87581629 site on chromosome 13, 18173793 site on chromosome 18, 11029129 site on chromosome 27 and 7014825 site on chromosome 40; and information of the SNP sites is based on Salmo_trutta.fSalTru1.

1.

2. The test kit according to claim 1, characterized in that, The nucleotide sequences of the specific primer pairs designed for the 16842638 site on chromosome 4 are shown as SEQ ID NO. 1 and SEQ ID NO. 2, the nucleotide sequences of the specific primer pairs designed for the 77517128 site on chromosome 12 are shown as SEQ ID NO. 3 and SEQ ID NO. 4, the nucleotide sequences of the specific primer pairs designed for the 87581629 site on chromosome 13 are shown as SEQ ID NO. 5 and SEQ ID NO. 6, the nucleotide sequences of the specific primer pairs designed for the 18173793 site on chromosome 18 are shown as SEQ ID NO. 7 and SEQ ID NO. 8, the nucleotide sequences of the specific primer pairs designed for the 11029129 site on chromosome 27 are shown as SEQ ID NO. 9 and SEQ ID NO. 10, and the nucleotide sequences of the specific primer pairs designed for the 7014825 site on chromosome 40 are shown as SEQ ID NO. 11 and SEQ ID NO.

12.

3. The test kit according to claim 1 or 2, characterized in that, The reagents for post-multiplex PCR gene sequencing are also included.

4. The test kit according to claim 3, characterized in that, The reagents for post-multiplex PCR gene sequencing include independently packaged multiplex amplicon library reagents and multiplex amplicon adapter reagents.

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