Method for detecting beta-glucuronidase to evaluate ability of prawns to resist tetrapod iridovirus type 1

Abstract

The invention discloses a method for detecting beta-glucuronidase to evaluate the ability of shrimps to resist a decapod iridovirus type 1, and belongs to the technical field of biological breeding of aquatic animals. The method comprises the following steps: detecting beta-glucuronidase in a sample by qPCR (quantitative polymerase chain reaction) to obtain a 2-delta delta Ct value of the beta-glucuronidase, and evaluating the anti-decapod iridovirus type 1 capability of the shrimps according to the 2-delta delta Ct value, when the 2-delta delta Ct is less than or equal to 0.15, the resistance is evaluated to be high, when the 2-delta delta Ct is more than 0.15 and less than 0.45, the resistance is evaluated to be medium, and when the 2-delta delta Ct is more than or equal to 0.45, the resistance is evaluated to be susceptible. And through experimental verification, the prediction accuracy of unknown sample verification is more than 86.7%, and the AUC area of the ROC curve is 0.8864. In conclusion, the invention provides a standardized and operable evaluation method for macrobrachium rosenbergii disease-resistant molecular breeding.

Description

Technical Field

[0001] This invention relates to the field of aquatic animal breeding technology, and in particular to a method for assessing the ability of shrimp to resist Decapoda iridovirus type 1 by detecting β-glucuronidase. Background Technology

[0002] Decapodiform iridovirus type 1 (DIV1) is a pathogen that seriously harms crustacean aquaculture. It can infect a variety of important economic shrimp species, such as giant freshwater prawns and Litopenaeus vannamei, and has a wide range of transmission and is prone to causing high mortality. Traditional disease resistance breeding relies on phenotypic screening based on survival rate after artificial infection, which is time-consuming (7-10 days), costly, inefficient, and cannot be screened in the early stages.

[0003] Proteomics technology provides a high-throughput screening method for discovering disease-resistance-related biomarkers. Data-independent acquisition (DIA) combined with parallel reaction monitoring (PRM) targeted validation enables highly accurate and reproducible quantitative protein analysis. However, mass spectrometry equipment is expensive and complex to operate, making it unsuitable for large-scale application in grassroots breeding units. Therefore, it is necessary to establish a complete technology transfer system from mass spectrometry discovery to routine qPCR detection, and to establish operable judgment thresholds to realize the industrial application of molecular biomarkers. Summary of the Invention

[0004] The purpose of this invention is to provide a method for assessing the ability of shrimp to resist Decapoda iridovirus type 1 by detecting β-glucuronidase, in order to solve the problems existing in the prior art. This invention establishes a complete technical chain from mass spectrometry discovery to routine qPCR detection, providing a standardized and operable assessment method for molecular breeding of disease resistance in giant freshwater prawns.

[0005] To achieve the above objectives, the present invention provides the following solution: This invention provides a method for assessing the ability of shrimp to resist Decapoda iridovirus type 1 by detecting β-glucuronidase, comprising detecting β-glucuronidase in a sample by qPCR and obtaining its 2 -ΔΔCt Value, based on 2 -ΔΔCt Steps for assessing the ability of shrimp to resist Decapodiform iridovirus type 1; The 2 -ΔΔCt When the value is ≤0.15, it is assessed as high resistance, the 2 -ΔΔCt In 0.15 < 2 -ΔΔCt When the value is <0.45, it is assessed as moderate resistance, the 2 -ΔΔCt A value ≥0.45 indicates susceptibility.

[0006] This invention also provides a method for breeding shrimp highly resistant to Decapoda iridovirus type 1, comprising detecting β-glucuronidase in samples by qPCR to obtain its 2 -ΔΔCt Value, keep 2-ΔΔCt Steps for shrimp with a value ≤ 0.15.

[0007] Optionally, the primers for the qPCR are a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.2.

[0008] Optionally, the qPCR reaction system is SYBR. ® Premix Ex Taq™ II 10 μL, forward primer 0.8 μL, reverse primer 0.8 μL, cDNA template 2 μL and sterile water 6.4 μL.

[0009] Optionally, the qPCR reaction program is 95℃ pre-denaturation for 30 s; 95℃ for 5 s, 60℃ for 30 s, for a total of 40 cycles.

[0010] Optionally, the shrimp includes giant freshwater prawns.

[0011] The present invention also provides the use of primers for detecting β-glucuronidase in any of the following: (1) To assess the shrimp’s resistance to Decapoda iridovirus type 1; (2) Prepare products to evaluate the ability of shrimp to resist Decapoda iridovirus type 1; (3) Select and breed shrimp that are highly resistant to Decapoda iridovirus type 1.

[0012] Optionally, the primers are forward primers with nucleotide sequences as shown in SEQ ID NO.1 and reverse primers with nucleotide sequences as shown in SEQ ID NO.2.

[0013] The present invention also provides a product for evaluating the ability of shrimp to resist Decapoda iridovirus type 1, said product comprising primers for detecting β-glucuronidase; The primers are a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.2.

[0014] Optionally, the shrimp includes giant freshwater prawns.

[0015] The present invention discloses the following technical effects: This invention discloses a method for evaluating the resistance of Macrobrachium rosenbergii to DIV1 virus using β-glucuronidase as a biomarker. DIA-PRM proteomics screening revealed that this enzyme was significantly downregulated in the high-resistance group (DIA: 0.383-fold, P=0.000; PRM: 0.277-fold, P=0.008). The determination threshold was established by qPCR: 2. -ΔΔCt≤0.15 indicates high resistance, ≥0.45 indicates susceptibility, and <0.15 indicates low susceptibility. -ΔΔCt A value <0.45 indicates moderate resistance. Experimental verification showed a prediction accuracy of over 86.7% for unknown samples, with an AUC area of ​​0.8864 and a 95% confidence interval of 0.7216–1.000, indicating significant differences. In summary, this invention establishes a complete technical chain from mass spectrometry discovery to routine qPCR detection, providing a standardized and operable evaluation method for molecular breeding of disease resistance in giant freshwater prawns. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0017] Figure 1 The relative expression levels of the β-glucuronidase gene in different resistant populations (qPCR results); C: control group; S: susceptible group; HR: highly resistant group; Figure 2 The working characteristic curve for the subjects. Detailed Implementation

[0018] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0019] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0020] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0021] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0022] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0023] Example 1: DIA-PRM screening revealed β-glucuronidase. 1. Animal infection and grouping Sixty giant freshwater prawns (12.8 ± 0.5 cm) were temporarily held for 14 days and then randomly divided into an infection group (n=30, injected with DIV1 virus 3.23 × 10⁻⁶). 5 copies·μL -1 The study group consisted of two groups: a control group (n=30, injected with the same volume of PBS). Patients were observed for 7 days post-infection and divided into the following three groups: High resistance group (HR): Survival > 7 days (n=10); Susceptible group (S): 72-120 h near death (n=10); Control group (C): PBS treatment (n=10).

[0024] 2. Plasma collection and protein extraction Blood was collected from the base of the foot 72 h post-infection (3.2% sodium citrate anticoagulation, volume ratio 8:2), centrifuged at 4000 rpm for 10 min at 4°C, and plasma was collected.

[0025] Collect plasma and add lysis buffer (containing protease inhibitor, lysis buffer:inhibitor = 50:1), vortex to mix, and quantify using the BCA method. Take 15 μg for SDS-PAGE quality control, and store the remainder at -80℃.

[0026] 3. DIA mass spectrometry analysis Enzymatic hydrolysis: Take an appropriate amount of protein, 100 mmol·L -1 Add ammonium bicarbonate to a final volume of 100 μL, then add TCEP (final concentration 10 mmol·L⁻¹). -1 ) and IAA (final concentration 40 mmol·L -1 Vortex for 30 seconds, incubate at 95°C and 600 rpm for 5 minutes. Add trypsin, and incubate overnight at 37°C and 600 rpm with shaking. Add 10% formic acid to terminate the process, desalt using a C18 desalting pipette tip, concentrate to dryness under vacuum, and store at -80°C.

[0027] Chromatographic conditions: Vanquish Neo UHPLC system, EASY-Spray C18 column (2 μm, 150 μm × 15 cm, catalog number ES906), column temperature 55℃, autosampler temperature 5℃.

[0028] Mass spectrometry conditions: Orbitrap Astral mass spectrometer, primary scan range 380-980 m / z, resolution 240,000 (m / z 200), AGC target value 500%, maximum IT 5 ms; DIA mode 300 scan windows, isolation window 2 m / z, HCD collision energy 25 eV, AGC target value 500%, maximum IT 3 ms.

[0029] Data analysis: Spectronaut software (version 19.4, Biognosys) for protein identification and quantification. Differentially expressed proteins (DEPs) screening criteria: HR vs. S, fold change > 2 and P < 0.05.

[0030] Results: β-glucuronidase (GenBank accession number: XP_066938200.1, protein name: SK128_002912) was significantly downregulated by 0.383-fold in the HR group (P=0.000, Table 1), and was identified as a candidate biomarker.

[0031] Table 1. Changes in β-glucuronidase expression in differential proteomics analysis of HR vs S using DIA. 4. PRM Target Validation Method establishment: Specific peptides suitable for PRM were screened for β-glucuronidase and imported into Xcalibur software (Thermo Scientific) to construct a PRM acquisition method.

[0032] Chromatographic conditions: Mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid-80% acetonitrile aqueous solution. The column was equilibrated with 96% solution A, and the flow rate was 2.5 μL·min. -1 Gradient elution program: 0-0.9 min, solution B 4%→8.5%; 0.9-13.9 min, solution B 8.5%→22.5%; 13.9-21.8 min, solution B 22.5%→35%; 21.8-22.3 min, solution B 35%→99%; 22.3-24 min, solution B maintained at 99%.

[0033] Mass spectrometry parameters: analysis time 24 min, positive ion detection mode; primary scan range 100-1000 m / z, resolution 45,000 (m / z 200), AGC target value Standard, maximum IT 50 ms; secondary mass spectrometry acquires tMS2 according to the inclusion list, isolation window 1.6 Th, normalized AGC target value 100%, maximum IT 50 ms, HCD collision energy 25%.

[0034] Data analysis: Quantitative analysis was performed using Skyline software (version 25.1.0).

[0035] Results: PRM confirmed that β-glucuronidase was downregulated by 0.277-fold in the HR group (P=0.008, Table 2), consistent with the trend of DIA data, and was confirmed as a reliable biomarker.

[0036] Table 2. Changes in β-glucuronidase expression verified by PRM. Example 2: qPCR Validation and Threshold Establishment 1. Sample collection and RNA extraction Same as in Example 1, blood was collected 72 h post-infection: the joint membrane at the base of the second step foot was cut open, and hemolymph was dripped into a pre-cooled anticoagulant tube (3.2% sodium citrate, hemolymph: anticoagulant = 8:2), centrifuged at 4°C and 4000 rpm for 10 min, and the blue supernatant plasma was collected and stored at -80°C.

[0037] Total RNA was extracted from plasma using the TRIzol method. Genomic DNA was removed by DNase I treatment. First-strand cDNA was synthesized via reverse transcription using the PrimeScript™ RTreagent Kit with gDNA Eraser (TaKaRa). RNA concentration and purity were determined using NanoDrop. 260 / A 280 =1.9-2.1).

[0038] 2. qPCR detection Primer design: Specific primers were designed based on the β-glucuronidase gene sequence. The primer sequences are as follows.

[0039] Forward primer: 5'-GATGCGAAGGCGTCATCAAC-3', SEQ ID NO.1; Reverse primer: 5'-CCCGGGTCATCCGACATTAG-3', SEQ ID NO.2.

[0040] The extension factor 1α (EF1α) was used as an internal reference gene, and its primer sequence is as follows.

[0041] Forward primer: 5'-TGCGCTGTGTTGATTGTAGC-3', SEQ ID NO.3; Reverse primer: 5'-ACAATGAGCTGCTTGACACC-3', SEQ ID NO.4.

[0042] Reaction system (20 μL): SYBR ® Premix Ex Taq™ II (TaKaRa) 10 μL, forward primer 0.8 μL (10 μmol·L⁻¹) -1 0.8 μL of reverse primer (10 μmol·L⁻¹) -1 ), 2 μL of cDNA template and 6.4 μL of sterile water.

[0043] Reaction procedure: 95℃ pre-denaturation for 30 s; 95℃ for 5 s, 60℃ for 30 s, for a total of 40 cycles. Three technical replicates were set up for each sample.

[0044] Quantitative methods: 2 -ΔΔCt The relative expression level of the target gene is calculated using the following method: ΔCt = Ct(GUSB) - Ct(EF1α), ΔΔCt = ΔCt(sample) - ΔCt(C group reference).

[0045] 3. Experimental Results and Statistical Analysis All experimental data are expressed as mean ± standard deviation (Mean ± SD). Statistical analysis was performed using GraphPad Prism 10.0 software. One-way ANOVA combined with Tukey's multiple comparison test was used for comparisons between groups. P < 0.05 was considered statistically significant, and P < 0.001 was considered highly statistically significant.

[0046] Results: Significant differences were found among the three groups. Figure 1 The expression gradient was obvious: C group > S group > HR group. The difference between HR group and C group was extremely significant (P<0.001), the difference between S group and C group was significant (P<0.05), and the difference between HR group and S group was significant (P<0.01).

[0047] Table 3 qPCR quantification results for each group 4. Threshold Determination and Verification The threshold determination criteria are as follows: The high resistance threshold must ensure that it includes the vast majority of highly resistant individuals while avoiding misjudgment: HR group mean + 1SD = 0.12 + 0.03 = 0.15. This threshold covers 90% of the individuals in the HR group (9 / 10) and is lower than the lowest value of the S group (0.32). The susceptibility threshold must effectively distinguish between susceptible and resistant individuals while taking into account the breeding error tolerance requirements: S group mean ± SD range: 0.34-0.50. Considering the lower limit of the C group (0.68) and the need for conservative culling in actual breeding, it is set to 0.45. Therefore, the judgment criteria are shown in Table 4.

[0048] Table 4 Judgment Criteria Example 3: Application of Unknown Sample Evaluation 1. Sample testing Thirty giant freshwater prawns were collected for testing. Plasma was collected 72 h after infection with DIV1 and qPCR was performed according to the method in Example 2.

[0049] 2. Results Analysis The results are shown in Table 5. The high resistance rate was 87.5% (7 / 8), the susceptibility rate was 91.7% (11 / 12), and the overall compliance rate was 86.7% (26 / 30). The survival rate of the medium resistance group (0.21-0.38) was 50% (5 / 10), which is consistent with the expected intermediate state. This embodiment verifies the rationality of the three-level classification.

[0050] Table 5 Results Simultaneously, the assessment efficacy of β-glucuronidase was analyzed using receiver operating characteristic (ROC) curves. For example... Figure 2 As shown in Table 6, the area under the receiver operating characteristic curve (AUC) for β-glucuronidase was 0.8864, with a 95% confidence interval of 0.7216–1.000, indicating a significant difference (P = 0.0017). This demonstrates that β-glucuronidase has strong efficacy in assessing the resistance of Macrobrachium rosenbergii to DIV1. These results further confirm the feasibility of the assessment model of this invention and provide strong evidence supporting the use of β-glucuronidase in assessing the resistance of Macrobrachium rosenbergii to DIV1 virus.

[0051] Table 6 ROC Results The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for assessing the ability of shrimp to resist Decapoda iridovirus type 1 by detecting β-glucuronidase, characterized in that, This includes detecting β-glucuronidase in samples using qPCR to obtain its 2 -ΔΔCt Value, based on 2 -ΔΔCt Steps for assessing the ability of shrimp to resist Decapodiform iridovirus type 1; The 2 -ΔΔCt When the value is ≤0.15, it is assessed as high resistance, the 2 -ΔΔCt In 0.15 < 2 -ΔΔCt When the value is <0.45, it is assessed as moderate resistance, the 2 -ΔΔCt A value ≥0.45 indicates susceptibility.

2. A method for breeding shrimp highly resistant to Decapoda iridovirus type 1, characterized in that, This includes detecting β-glucuronidase in samples using qPCR to obtain its 2 -ΔΔCt Value, keep 2 -ΔΔCt Steps for shrimp with a value ≤ 0.

15.

3. The method as described in claim 1 or 2, characterized in that, The primers for the qPCR are the forward primer with the nucleotide sequence shown in SEQ ID NO.1 and the reverse primer with the nucleotide sequence shown in SEQ ID NO.

2.

4. The method as described in claim 1 or 2, characterized in that, The qPCR reaction system was SYBR. ® Premix ExTaq™ II 10 μL, forward primer 0.8 μL, reverse primer 0.8 μL, cDNA template 2 μL and sterile water 6.4 μL.

5. The method as described in claim 1 or 2, characterized in that, The qPCR reaction program was 95℃ pre-denaturation for 30 s; 95℃ for 5 s, 60℃ for 30 s, for a total of 40 cycles.

6. The method as described in claim 1 or 2, characterized in that, The shrimp mentioned include giant freshwater prawns.

7. The application of primers for detecting β-glucuronidase in any of the following: (1) To assess the shrimp’s resistance to Decapoda iridovirus type 1; (2) Prepare products to evaluate the ability of shrimp to resist Decapoda iridovirus type 1; (3) Select and breed shrimp that are highly resistant to Decapoda iridovirus type 1.

8. The application as described in claim 7, characterized in that, The primers are a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.

2.

9. A product for evaluating the ability of shrimp to resist Decapoda iridovirus type 1, characterized in that, The product contains primers for detecting β-glucuronidase; The primers are a forward primer with a nucleotide sequence as shown in SEQ ID NO.1 and a reverse primer with a nucleotide sequence as shown in SEQ ID NO.

2.

10. The product as described in claim 9, characterized in that, The shrimp mentioned include giant freshwater prawns.

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