A composition for rapid detection of grouper iridovirus and vibrio harveyi and application thereof

Abstract

The application provides a composition for rapid detection of grouper iridovirus and Vibrio harveyi and application thereof. The composition comprises a first primer set and a first fluorescent probe for detecting the grouper iridovirus, and a second primer set and a second fluorescent probe for detecting the Vibrio harveyi, wherein the nucleotide sequences of the first primer set and the first fluorescent probe are shown as SEQ ID NO: 1-3, and the nucleotide sequences of the second primer set and the second fluorescent probe are shown as SEQ ID NO: 4-6. The composition provided by the application can realize rapid quantitative detection of SGIV and VH simultaneously through multiplex fluorescent quantitative PCR, and the interference between the primers and the probes is less, and the detection result is not affected, so that the detection result has the characteristics of high specificity, high sensitivity, stability and good repeatability. In addition, the SGIV and the VH can be subjected to nucleic acid amplification in the same reaction system, the detection time is greatly shortened compared with ordinary PCR in which amplification is followed by agarose gel electrophoresis analysis, the detection efficiency is improved, and the detection cost is lower than that of single fluorescent quantitative PCR.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection technology, specifically to a composition for rapid detection of grouper iridovirus and Vibrio harveyi and its application. Background Technology

[0002] Grouper is highly nutritious and has significant medicinal value, making it an important aquaculture species in my country. Currently, the annual domestic production of grouper exceeds 200,000 tons, with a direct industry output value exceeding 10 billion yuan, demonstrating its extremely high economic value. However, with the increasing density and scale of grouper farming, various diseases have frequently broken out, causing substantial economic losses.

[0003] Singapore grouperiridovirus (SGIV), isolated from diseased grouper, is a major viral pathogen causing disease in grouper. It exhibits strong tropism for the epithelial and endothelial tissues of fish, and is particularly destructive to hematopoietic organs and tissues such as the spleen and kidneys, leading to anemia, multiple organ failure, and death. Current diagnostic methods for grouper iridovirus primarily involve molecular biological and immunological detection methods, including PCR, quantitative real-time PCR, and ELISA.

[0004] Vibrio harveyi (VH) is widely found in seawater and seafood. It is one of the common pathogens in aquaculture in coastal areas of my country. It is commonly found in various fish, shrimp, crabs, squid, mud snails, etc., and also in eggs, meat, or vegetables. Clinically, it mainly causes acute gastroenteritis, pneumonia, septicemia, etc.

[0005] SGIV and VH are two significant pathogens in grouper farming. Currently, there are no effective drugs or treatments for SGIV. For VH infection, the main control technique is the use of antibiotics. However, the overuse of antibiotics has caused significant harm to water bodies and the farming environment, posing potential dangers to aquaculture and even human health. SGIV infection can weaken the fish's immune system, making grouper more susceptible to pathogens like VH. Conversely, bacterial infections can exacerbate the symptoms and severity of viral infections. Early diagnosis and interruption of pathogen transmission remain the most effective preventative measures.

[0006] Currently, the main detection methods for SGIV and VH include ELISA, colloidal gold test strips, quantitative real-time PCR, LAMP, and gene sequencing. Each of these methods has its own advantages and disadvantages in practical testing. For example, although ELISA has high sensitivity, the operation steps are cumbersome and the cost is high; PCR requires electrophoresis analysis, which is also time-consuming and has low sensitivity; LAMP is fast and sensitive, but it is prone to false positives; while quantitative real-time PCR has the characteristics of high sensitivity, strong specificity, good repeatability, accurate quantification, and short detection time, and has been widely used in laboratory testing.

[0007] Multiplex quantitative PCR technology is based on single quantitative PCR technology. Different fluorescent groups are labeled on different genes and detected in real time in the same reaction system. It is used to detect multiple pathogens in one sample at the same time. It has the advantages of reducing detection costs, improving work efficiency and simplifying operation.

[0008] SGIV and VH are infectious pathogens that cause serious damage in grouper farming. Epidemiological surveys of farmed species such as grouper, sea bass, and golden pomfret have found that mixed infections of viruses and bacteria are common. At present, laboratories mainly use single fluorescent quantitative PCR to detect SGIV and ordinary PCR to detect VH. The operation is cumbersome and time-consuming, especially when conducting epidemiological surveys of a large number of samples, the detection timeliness is relatively low.

[0009] Therefore, it is necessary to establish a multiplex quantitative PCR method that can simultaneously detect SGIV and VH, so as to realize the epidemiological investigation and daily monitoring of aquaculture species such as grouper, bass, and golden pomfret, and provide technical support for the rapid diagnosis and scientific prevention and control of SGIV and VH in aquaculture animals such as grouper and bass. Summary of the Invention

[0010] To address the issues of cumbersome and time-consuming procedures when using single-mode quantitative PCR to detect grouper iridovirus and Vibrio harveyi separately, and to achieve rapid detection of grouper iridovirus and Vibrio harveyi while shortening the detection time and improving detection efficiency, this invention provides a composition for rapid detection of grouper iridovirus and Vibrio harveyi and its application.

[0011] According to a first aspect of the present invention, a composition for rapid detection of grouper iridovirus and Vibrio harveyi is provided, the composition comprising a first primer set and a first fluorescent probe for detecting grouper iridovirus, and a second primer set and a second fluorescent probe for detecting Vibrio harveyi; the first primer set comprises a first forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a first reverse primer with a nucleotide sequence as shown in SEQ ID NO: 2; the second fluorescent probe has a nucleotide sequence as shown in SEQ ID NO: 3; the second primer set comprises a second forward primer with a nucleotide sequence as shown in SEQ ID NO: 4 and a second reverse primer with a nucleotide sequence as shown in SEQ ID NO: 5; the second fluorescent probe has a nucleotide sequence as shown in SEQ ID NO: 6.

[0012] The inventors of this application designed specific primers and TaqMan probes targeting the major capsid protein gene (MCP gene) of grouper iridovirus (SGIV) and the conserved rpoB gene (encoding the β subunit of RNA polymerase) of Vibrio harveyi (VH). After screening, they obtained the first primer set and first fluorescent probe for detecting grouper iridovirus, as shown in SEQ ID NO: 1-6, and the second primer set and second fluorescent probe for detecting Vibrio harveyi, which is the composition for rapid detection of grouper iridovirus and Vibrio harveyi provided by this invention. The composition provided by this invention is applied to the multiplex quantitative PCR detection of grouper iridovirus and Vibrio harveyi. Firstly, it can simultaneously achieve rapid identification, diagnosis and quantification of two pathogens, SGIV and VH. Moreover, the primers and probes obtained through design and screening have less interference and do not affect the detection results, resulting in high specificity, high sensitivity, stability and good repeatability. Secondly, it can simultaneously amplify nucleic acids of SGIV and VH in the same reaction system, saving more than 90 minutes compared to ordinary PCR which involves amplification followed by agarose gel electrophoresis analysis. This can significantly improve work efficiency, especially for large-scale epidemiological surveys. Thirdly, it saves more than 30% of the detection cost compared to single quantitative PCR.

[0013] Preferably, one end of the first fluorescent probe is labeled with the fluorescent group FAM and the other end is labeled with the quenching group TAMRA; one end of the second fluorescent probe is labeled with the fluorescent group HEX and the other end is labeled with the quenching group TAMRA.

[0014] Preferably, the first fluorescent probe is 5'-FAM-CGGTACCGTTTGCGCCGAGCC-TAMRA-3', and the second fluorescent probe is 5'-HEX-TCACCGAAACGCTGACCACCG-TAMRA-3'.

[0015] By labeling the 5' end of the first fluorescent probe used to detect SGIV with the fluorescent group FAM, the 5' end of the second fluorescent probe used to detect VH with the fluorescent group HEX, and the 3' end of the first and second fluorescent probes with the same quencher group TAMRA, it is possible to ensure that the emission peaks of the different fluorescent groups of the first and second fluorescent probes do not overlap, thus avoiding signal crosstalk. This allows for the rapid detection of both grouper iridovirus and Vibrio harveyi, and also enables good differentiation between them.

[0016] According to a second aspect of the invention, the above-described composition for rapid detection of grouper iridovirus and Vibrio harveyi is provided for use in the preparation of a kit for rapid detection of grouper iridovirus and Vibrio harveyi.

[0017] According to a third aspect of the present invention, a kit for rapid detection of grouper iridovirus and Vibrio harveyi is provided, the kit comprising the above-described composition for rapid detection of grouper iridovirus and Vibrio harveyi.

[0018] Preferably, the kit also includes 2×qPCR Mix.

[0019] Preferably, the 2×qPCR Mix contains dNTP Mix (a nucleotide mixture containing four dNTPs), dUTP Mix (a nucleotide mixture containing dUTPs), Heat-labile UDG (thermally sensitive uridine-DNA glycosylase, also known as thermosensitive UDG enzyme), and Champagne Taq DNA Polymerase (Taq DNA polymerase).

[0020] Preferably, the concentration of dNTP Mix is ​​10–20 mmol / L, and / or the concentration of dUTP Mix is ​​10–20 mmol / L.

[0021] Preferably, the above-mentioned 2×qPCR Mix also contains a buffer solution, which is prepared by equal volumes of 1300 mmol / L Tris-HCl solution (pH=8.8), 1300 mmol / L potassium chloride solution, 180 mmol / L magnesium chloride solution, 260 mmol / L dithiothreitol solution, and 3.9 wt% Triton X-100 aqueous solution.

[0022] The kit for rapid detection of grouper iridovirus and Vibrio harveyi involved in this protocol uses primers and probes with nucleotide sequences as shown in SEQ ID NO: 1-6, and introduces the above reagents into the kit. Among them, Heat-labile UDG can catalyze the hydrolysis of uracil bases and N-glycosidic bonds of the sugar phosphate backbone of DNA strands containing uracil, releasing free uracil. Compared with ordinary UDG enzymes, the heat-sensitive UDG enzyme avoids the residual activity that may exist after the inactivation of conventional UDG enzymes, which may degrade dU-containing amplification products at room temperature. Champagne Taq DNA Polymerase can improve the detection sensitivity of this kit in the rapid detection of grouper iridovirus and Vibrio harveyi, and the detection sensitivity can reach 0.1 pg total RNA or <10 copies of RNA template. Therefore, the kit for rapid detection of grouper iridovirus and Vibrio harveyi involved in this scheme, through the synergistic effect of its components, enables the rapid detection of grouper iridovirus and Vibrio harveyi and has the advantages of high specificity, high sensitivity, good stability and repeatability.

[0023] According to a fourth aspect of the present invention, a method for rapid detection of grouper iridovirus and Vibrio harveyi is provided, comprising the following steps: mixing the nucleic acid template to be tested, 2×qPCR Mix, and the above-mentioned composition for rapid detection of grouper iridovirus and Vibrio harveyi to prepare a mixed solution, and detecting the fluorescence intensity of the mixed solution using multiplex quantitative PCR; the 2×qPCR Mix contains dNTP Mix, dUTP Mix, buffer, Heat-labile UDG, and Champagne Taq DNA Polymerase; the buffer solution is prepared by equal volumes of 1300 mmol / L Tris-HCl solution (pH=8.8), 1300 mmol / L potassium chloride solution, 180 mmol / L magnesium chloride solution, 260 mmol / L dithiothreitol solution, and 3.9 wt% Triton X-100 aqueous solution.

[0024] The rapid detection method for grouper iridovirus and Vibrio harveyi provided in this scheme uses primers and probes with nucleotide sequences as shown in SEQ ID NO: 1-6, combined with a 2×qPCR Mix containing the above components. Through the synergistic effect of each component, the rapid detection of grouper iridovirus and Vibrio harveyi can be achieved simultaneously, and it has the advantages of high specificity, high sensitivity, good stability and reproducibility.

[0025] Preferably, the reaction procedure for multiplex quantitative PCR is as follows: 37℃ for 2 min to remove contaminants, 95℃ for 30 s to pre-denature, 95℃ for 10 s to denature, 60℃ for 30 s to anneal, (collect fluorescence), 45 cycles. Attached Figure Description

[0026] Figure 1 The results of the SGIV and VH standard curves obtained in Example 4 are shown in the figure.

[0027] Figure 2 The graph shows the sensitive amplification results of SGIV-VH multiplex real-time PCR provided for test example 3.

[0028] Figure 3 The image shows the specific amplification results of SGIV-VH multiplex real-time PCR provided for test example 3.

[0029] Figure 4 The image shows the repeatability amplification results of SGIV-VH multiplex real-time PCR provided for test example 3.

[0030] Figure 5 The image shows the stability amplification results of SGIV-VH multiplex real-time PCR provided for test example 3. Detailed Implementation

[0031] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0032] Example 1: A composition for rapid detection of grouper iridovirus and Vibrio harveyi.

[0033] This embodiment provides a composition for rapid detection of grouper iridovirus and Vibrio harveyi. The composition includes a first primer set and a first fluorescent probe for detecting grouper iridovirus (SGIV), and a second primer set and a second fluorescent probe for detecting Vibrio harveyi (VH). The first primer set includes a first forward primer and a first reverse primer, and the second primer set includes a second forward primer and a second reverse primer. Specific nucleotide sequences and the lengths of the amplification products obtained by amplifying SGIV and VH using the above primers are shown in Table 1. Primer Premier 5.0 software was used to target the major capsid protein gene of SGIV (MCP gene, whose nucleotide sequence is shown in SEQ ID NO: 7) and the conserved gene rpoB of VH (the gene encoding the β subunit of RNA polymerase, whose nucleotide sequence is shown in SEQ ID NO: 7). As shown in NO:8, multiple sets of specific primers and TaqMan probes were designed and synthesized by Wuhan Aoke Biotechnology Co., Ltd., resulting in the first forward primer (SGIV_F1), the first reverse primer (SGIV_R1), the first fluorescent probe (SGIV_P1), the second forward primer (VH_F1), the second reverse primer (VH_R1), and the second fluorescent probe (VH_P1) in the composition provided in this embodiment.

[0034] SEQ ID NO: 7

[0035]

[0036]

[0037] SEQ ID NO: 8

[0038]

[0039]

[0040]

[0041] Table 1. Composition for rapid detection of grouper iridovirus and Vibrio harveyi provided in Example 1.

[0042]

[0043]

[0044] Example 2: Kit for rapid detection of grouper iridovirus and Vibrio harveyi

[0045] This embodiment provides a kit for rapid detection of grouper iridovirus and Vibrio harveyi. The kit includes the rapid detection composition (SGIV_F1, SGIV_R1, SGIV_P1, VH_F1, VH_R1, VH_P1) provided in Example 1 for grouper iridovirus and Vibrio harveyi and 2×qPCR Mix. The 2×qPCR Mix contains 10-20 mmol / L dNTP Mix, 10-20 mmol / L dUTP Mix, buffer (prepared by equal volumes of 1300 mmol / L Tris-HCl solution at pH 8.8, 1300 mmol / L potassium chloride solution, 180 mmol / L magnesium chloride solution, 260 mmol / L dithiothreitol solution, and 3.9 wt% Triton X-100 aqueous solution), Heat-labile UDG, and Champagne Taq DNA Polymerase.

[0046] Example 3: A method for rapid detection of grouper iridovirus and Vibrio harveyi.

[0047] This embodiment provides a method for rapid detection of grouper iridovirus and Vibrio harveyi. The method includes the following steps: mixing the nucleic acid template to be tested, 2×qPCR Mix, and the composition for rapid detection of grouper iridovirus and Vibrio harveyi provided in Example 1 evenly to prepare a mixed solution, and detecting the fluorescence intensity of the mixed solution using multiplex quantitative PCR.

[0048] The 2×qPCR Mix contains 10–20 mmol / L dNTP Mix, 10–20 mmol / L dUTP Mix, buffer (prepared by equal volumes of 1300 mmol / L Tris-HCl solution at pH 8.8, 1300 mmol / L potassium chloride solution, 180 mmol / L magnesium chloride solution, 260 mmol / L dithiothreitol solution, and 3.9 wt% Triton X-100 aqueous solution), Heat-labile UDG, and Champagne Taq DNA Polymerase.

[0049] The above mixture includes the following components: 10 μL of 2×qPCR Mix, 0.4 μL of SGIV_F1, 0.4 μL of SGIV_R1, 0.2 μL of SGIV_P1, 0.4 μL of VH_F1, 0.4 μL of VH_R1, 0.2 μL of VH_P1, 5 μL of the nucleic acid template to be tested, and ultrapure water to a final volume of 20 μL.

[0050] The reaction procedure for multiplex quantitative PCR is as follows: 37℃ for 2 min to remove contaminants, 95℃ for 30 s for pre-denaturation, 95℃ for 10 s for denaturation, 60℃ for 30 s for annealing (for fluorescence collection), 45 cycles.

[0051] Example 4: SGIV standard curve and VH standard curve and their test result judgment criteria

[0052] This embodiment aims to use the composition provided in Example 1 and the kit provided in Example 2, and refer to the method provided in Example 3, to detect the positive standard (SGIV-VH-pUC-57 positive plasmid) to plot the SGIV standard curve and the VH standard curve. The specific operation steps are as follows:

[0053] 1. Preparation of positive plasmids

[0054] The gene fragments containing the SGIV and VH primers were synthesized by Wuhan Aoke Biotechnology Co., Ltd., and then cloned into the pUC-57 plasmid vector to obtain the SGIV-VH-pUC-57 positive plasmid. The copy number was calculated and diluted to a concentration of 10⁻⁶. 9 The positive standard was obtained by dispersing copies / μL and stored at -20℃ for later use.

[0055] 2. Plot the SGIV and VH standard curves.

[0056] A serial dilution of 10-fold was performed to obtain a concentration of 10. 5 10 4 10 3 10 2 10 1 10 0 Positive standards of copies / μL were analyzed using the composition provided in Example 1 and the kit provided in Example 2, following the method provided in Example 3. The serially diluted positive standards were then tested. SGIV and VH standard curves were plotted with template (positive standard) concentration as the x-axis and average CT value as the y-axis. The results are as follows: Figure 1 As shown.

[0057] Depend on Figure 1 The formula for the SGIV standard curve is as follows: y = -1.055ln(x) + 30.88, and the correlation coefficient R0 is... 2 =0.9992; The VH standard curve formula is as follows: y = -1.044ln(x) + 30.324, correlation coefficient R 2 =0.9979.

[0058] 3. Criteria for Judging Test Results

[0059] Using the composition provided in Example 1 and the kit provided in Example 2, and referring to the method provided in Example 3, the nucleic acid template to be tested was detected based on the SGIV standard curve and VH standard curve obtained above. The detection results were judged by the Cq value (the number of cycles in which the sample reaction exceeds the fluorescence threshold, also known as the cycle threshold) of the FAM and HEX channels and the amplification curve. The specific judgment criteria are as follows:

[0060] (1) The amplification results of the positive control are: the Cq values ​​of the FAM and HEX channels are all less than or equal to 35 and both have typical “S” type amplification curves. The negative control has no Cq value and no amplification curve. The experiment is valid.

[0061] (2) If the nucleic acid template to be tested has a Cq value of less than or equal to 35 in both FAM and HEX channels and has a typical “S” type amplification curve, then the nucleic acid template to be tested is positive for both SGIV and VH.

[0062] (3) If the nucleic acid template to be tested has a Cq value in the FAM channel that is less than or equal to 35 and has a typical “S”-shaped amplification curve, but has no Cq value and no amplification curve in the HEX channel, then the nucleic acid template to be tested is positive for SGIV and negative for VH.

[0063] (4) If the nucleic acid template to be tested has a Cq value in the HEX channel that is less than or equal to 35 and has a typical “S”-shaped amplification curve, while the FAM channel has no Cq value and no amplification curve, then the sample to be tested is positive for VH and negative for SGIV.

[0064] (5) If the nucleic acid template to be tested has no Cq value and no amplification curve in both FAM and HEX channels, then the nucleic acid template to be tested is determined to be negative for both SGIV and VH.

[0065] (6) If the Cq value of the nucleic acid template to be tested is greater than 35 in both the FAM and HEX channels and there is or is not a typical “S” type amplification curve, it is judged as a suspicious or uncertain sample and needs to be re-extracted for nucleic acid retesting. If the retest results are the same, it is judged that both SGIV and VH are positive. If the retest results have no Cq value, it is judged that both SGIV and VH are negative. If either the FAM or HEX channel has a Cq value and a corresponding amplification curve in the retest, the corresponding gene is judged as positive. Otherwise, it is judged as negative.

[0066] Comparative Example 1

[0067] This comparative example provides a composition for rapid detection of grouper iridovirus and Vibrio harveyi. The composition includes a third primer set and a third fluorescent probe for detecting grouper iridovirus (SGIV), and a fourth primer set and a fourth fluorescent probe for detecting Vibrio harveyi (VH). The third primer set includes a third forward primer and a third reverse primer, and the fourth primer set includes a fourth forward primer and a fourth reverse primer. Primer Premier 5.0 software is used to target the major capsid protein gene (MCP gene, whose nucleotide sequence is shown in SEQ ID NO: 7) of SGIV and the conserved gene rpoB gene (encoding the RNA polymerase β subunit, whose nucleotide sequence is shown in SEQ ID NO: 7) of VH. (NO: 8) Multiple sets of specific primers and TaqMan probes were designed and synthesized by Wuhan Aoke Biotechnology Co., Ltd., resulting in the third forward primer (SGIV_F2), the third reverse primer (SGIV_R2), the third fluorescent probe (SGIV_P2), the fourth forward primer (VH_F2), the fourth reverse primer (VH_R2), and the fourth fluorescent probe (VH_P2) in the composition provided in this comparative example. The specific nucleotide sequences are shown in Table 2.

[0068] Table 2 shows the compositions provided in Comparative Example 1 for the rapid detection of grouper iridovirus and Vibrio harveyi.

[0069] SGIV_F2 SEQ ID NO:9 5'-TAGAGCACGCTTCTCTCACC-3' SGIV_R2 SEQ ID NO:10 5'-ATGCGTTTACCTTCGCACAT-3' SGIV_P2 SEQ ID NO:11 5'-FAM-ACGACATTTGCGCGCAGCAGT-TAMRA-3' VH_F2 SEQ ID NO:12 5'-GTATTGACCGCCGTCGTAAG-3' VH_R2 SEQ ID NO:13 5'-TCACCACGTAGACGATCAGG-3' VH_P2 SEQ ID NO:14 5'-HEX-ACCAGCTTCGATCATCCTTCGTGCA-TAMRA-3'

[0070] Test Example 1

[0071] This test example aims to compare the amplification effects of the two sets of primers and probes provided in Example 1 (SGIV_F1, SGIV_R1, SGIV_P1, VH_F1, VH_R1, VH_P1) and Comparative Example 1 (SGIV_F2, SGIV_R2, SGIV_P2, VH_F2, VH_R2, VH_P2) on grouper iridovirus and Vibrio harveyi, referring to the rapid detection method of the composition in Example 3. The results are shown in Table 3. The CT value represents the Cycle Threshold, which indicates the number of amplification cycles corresponding to when the fluorescence signal of the amplified product reaches the set fluorescence threshold. Simply put, the CT value represents the number of cycles when the initial template amplification reaches a certain amount of product.

[0072] Table 3 Comparison of amplification effects between the two sets of primers and probes

[0073]

[0074]

[0075] According to Table 3, which shows the amplification effects of the primers and probes provided in Example 1 and Comparative Example 1 on grouper iridovirus and Vibrio harveyi, when using SGIV_F1, SGIV_R1, and SGIV_P1 provided in Example 1 to detect grouper iridovirus, the detection rate is higher than that of SGIV_F2, SGIV_R2, and SGIV_P2 provided in Comparative Example 1 when the template concentration is low. Similarly, when using VH_F1, VH_R1, and VH_P1 provided in Example 1 to detect Vibrio harveyi, the detection rate is higher than that of VH_F2, VH_R2, and VH_P2 provided in Comparative Example 1 when the template concentration is low. The above results prove that the composition provided in Example 1 has better amplification and detection effects than the composition provided in Comparative Example 1 when jointly detecting grouper iridovirus and Vibrio harveyi. That is, the detection sensitivity of the composition provided in Example 1 when jointly detecting grouper iridovirus and Vibrio harveyi is higher than that of the composition provided in Comparative Example 1.

[0076] Test Example 2

[0077] This test case aims to investigate whether the primers and probes provided in Example 1 (SGIV_F1, SGIV_R1, SGIV_P1, VH_F1, VH_R1, VH_P1) interfere with each other during the joint detection of SGIV and VH, referring to the rapid detection method for grouper iridovirus (SGIV) and Vibrio harveyi (VH) provided in Example 3. SGIV was detected individually using SGIV_F1, SGIV_R1, and SGIV_P1, and VH was detected individually using VH_F1, VH_R1, and VH_P1. The combination provided in Example 1 was also used to jointly detect SGIV and VH. The results of individual detection and duplex qPCR detection using the combination provided in Example 1 are compared in Table 4. The approximate pathogen copy number in the sample can be calculated by substituting the obtained CT values ​​into the standard curve formula.

[0078] Table 4 Comparison of results between standalone and dual qPCR detection

[0079]

[0080] The results of individual and dual qPCR detection are shown in Table 4. The ΔCT values ​​for individual and dual qPCR detection of SGIV and VH were calculated based on ΔCT = (average CT value of SGIV in the SGIV / VH combination) - (average CT value of SGIV) and ΔCT = (average CT value of VH in the SGIV / VH combination) - (average CT value of VH). The calculated ΔCT value for individual SGIV detection combined with dual qPCR detection of SGIV was 0.563, and the ΔCT value for individual VH detection combined with dual qPCR detection of VH was 0.796. Both average ΔCT values ​​were less than 0.8. This demonstrates that applying the composition provided in Example 1 to the combined detection of grouper iridovirus (SGIV) and Vibrio harveyi (VH) can simultaneously achieve differential diagnosis and quantification of both pathogens. Furthermore, there is minimal interference between the multiple primers and probes, which does not affect the detection results of clinical samples, enabling simultaneous specific quantitative detection of SGIV and VH.

[0081] Test Example 3

[0082] This test case aims to verify the sensitivity, specificity, repeatability, and stability of the rapid detection composition for grouper iridovirus and Vibrio harveyi provided in Example 1 and the rapid detection method for grouper iridovirus and Vibrio harveyi using this composition in Example 3 for detecting SGIV and VH.

[0083] 1. Sensitivity test of multiplex quantitative PCR

[0084] The SGIV-VH-pUC-57 positive plasmid prepared in Example 4 was diluted to a concentration of 10. 3 ~10 0 The sensitivity of multiplex quantitative PCR was tested using copies / μL of the diluted SGIV-VH-pUC-57 positive plasmid as a template, following the reaction system (mixture) and procedure provided in Example 3. The results are as follows: Figure 2 As shown, the curves from left to right correspond to SGIV-VH-pUC-57 positive plasmid template concentrations of 10... 3 10 2 10 1 10 0 copies / μL.

[0085] Depend on Figure 2 It can be seen that the detection limits of SGIV (blue curve) and VH (green curve) of the established SGIV-VH multiplex real-time PCR were both 1 copies / μL.

[0086] 2. Specificity assays for multiplex quantitative PCR

[0087] (1) Four common viruses and four common pathogens were selected: viral nervous necrosis virus (NNV), rhabdovirus, large mouth bassranavirus, grass carp hemorrhagic disease virus, Vibrio parahaemolyticus (VP), Vibrio alginolyticus, Vibrio Edwardsiella, and Aeromonas hydrophila.

[0088] (2) Nucleic acid (DNA / RNA) was extracted from each virus or pathogen. Using the SGIV-VH-pUC-57 positive plasmid standard as a positive control and sterile, enzyme-free water or ultrapure water as a negative control, multiplex quantitative PCR was performed to determine the specificity of the established multiplex quantitative PCR detection. The results are as follows: Figure 3 As shown.

[0089] Depend on Figure 3 The specificity detection results of the multiplex quantitative PCR showed that only SGIV (blue curve) and VH (green curve) could be amplified (with typical "S"-shaped amplification curves), while other pathogens were not amplified. These results demonstrate that the established multiplex quantitative PCR detection system has good specificity.

[0090] 3. Repeatability experiments of multiplex quantitative PCR

[0091] Take a dilution range of 10 4 ~10 0 Using SGIV-VH-pUC-57 positive plasmid standard (copies / μL) as a template, a repeatability experiment for multiplex quantitative PCR was performed according to the reaction system (mixture) and procedure provided in Example 3. Four replicates were set up for each dilution gradient. The repeatability of this method was evaluated by the overlap of the amplification curves of the four replicates at each dilution gradient. The results are as follows: Figure 4 As shown, the curves from left to right correspond to SGIV-VH-pUC-57 positive plasmid standard concentrations of 10. 3 10 2 10 1 10 0 copies / μL.

[0092] Depend on Figure 4The repeatability test results show that the amplification curves corresponding to SGIV (blue curve) and VH (green curve) in the five repeat experiments at each dilution largely overlap. These results demonstrate that the established multiplex quantitative PCR detection method exhibits good repeatability.

[0093] 4. Stability test of multiplex quantitative PCR

[0094] The prepared reaction system was placed in a constant temperature environment of 37℃. Samples were taken on days 0, 1, 3, 5, and 7 and stored at -20℃. Using the same concentration of SGIV-VH-pUC-57 positive plasmid standard as a template, stability experiments for multiplex quantitative PCR detection were performed according to the reaction system (mixture) and reaction procedure provided in Example 3. The stability of this method was evaluated by the degree of overlap of the amplification curves. The results are as follows: Figure 5 As shown.

[0095] Depend on Figure 5 The stability test results showed that the amplification curves corresponding to SGIV (blue curve) and VH (green curve) largely overlapped at different number of days. These results indicate that the established multiplex quantitative PCR detection method has good stability.

[0096] Test Example 4: Clinical Application of the Multiplex Real-Time PCR Kit

[0097] This test case aims to clinically apply the kit provided in Example 2 for the rapid detection of grouper iridovirus and Vibrio harveyi to further verify the detection efficacy of the kit. The specific experimental steps are as follows:

[0098] 1. Clinical sample preparation: Three clinical samples of SGIV and VH that have been isolated, sequenced and identified as positive were stored in the laboratory. After grinding, the samples were combined to obtain clinical samples that are positive for SGIV, VH and SGIV-VH, respectively, which were used as samples to be tested.

[0099] 2. Nucleic acid (DNA / RNA) was extracted from each sample to be tested. The SGIV-VH-pUC-57 positive plasmid standard was used as a positive control, and sterile enzyme-free water or ultrapure water was used as a negative control. Real-time PCR amplification was performed to determine the accuracy of the established multiplex real-time PCR in clinical applications in fish samples. The detection results are shown in Table 5.

[0100] Table 5. Results of SGIV-VH Multiplex Quantitative PCR Detection in Clinical Samples

[0101] SGIV 3 0 / 3 100 VH 3 0 / 3 100 SGIV-VH 3 0 / 3 100

[0102] As shown in Table 5, the detection rate of the multiplex real-time PCR detection system established using the rapid detection kit for grouper iridovirus and Vibrio harveyi provided in Example 2 was 100%. This indicates that the composition for rapid detection of grouper iridovirus and Vibrio harveyi provided by the present invention and the kit using it have high accuracy in clinical testing of fish samples.

[0103] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A composition for rapid detection of grouper iridovirus and Vibrio harveyi, characterized in that: The composition includes a first primer set and a first fluorescent probe for detecting grouper iridovirus, and a second primer set and a second fluorescent probe for detecting Vibrio harveyi. The first primer set includes a first forward primer with a nucleotide sequence as shown in SEQ ID NO: 1 and a first reverse primer with a nucleotide sequence as shown in SEQ ID NO: 2; The nucleotide sequence of the first fluorescent probe is shown in SEQ ID NO: 3; The second primer set includes a second forward primer with a nucleotide sequence as shown in SEQ ID NO: 4 and a second reverse primer with a nucleotide sequence as shown in SEQ ID NO: 5; The nucleotide sequence of the second fluorescent probe is shown in SEQ ID NO:

6.

2. The composition for rapid detection of grouper iridovirus and Vibrio harveyi as described in claim 1, characterized in that: One end of the first fluorescent probe is labeled with the fluorescent group FAM, and the other end is labeled with the quenching group TAMRA; The second fluorescent probe is labeled with the fluorescent group HEX at one end and the quenching group TAMRA at the other end.

3. The use of the composition for rapid detection of grouper iridovirus and Vibrio harveyi as described in claim 1 or 2 in the preparation of a kit for rapid detection of grouper iridovirus and Vibrio harveyi.

4. A kit for rapid detection of grouper iridovirus and Vibrio harveyi, characterized in that: The kit includes the composition for rapid detection of grouper iridovirus and Vibrio harveyi as described in claim 1 or 2.

5. The kit for rapid detection of grouper iridovirus and Vibrio harveyi as described in claim 4, characterized in that: The kit also includes 2×qPCR Mix.

6. The kit for rapid detection of grouper iridovirus and Vibrio harveyi as described in claim 5, characterized in that: The 2×qPCR Mix contains dNTP Mix, dUTP Mix, Heat-labile UDG, and Champagne Taq DNA Polymerase.

7. The kit for rapid detection of grouper iridovirus and Vibrio harveyi as described in claim 6, characterized in that: The concentration of the dNTP Mix is ​​10-20 mmol / L, and / or the concentration of the dUTP Mix is ​​10-20 mmol / L.

8. The kit for rapid detection of grouper iridovirus and Vibrio harveyi as described in claim 6, characterized in that: The 2×qPCR Mix also contains a buffer solution, which is prepared by equal volumes of 1300 mmol / L Tris-HCl solution (pH=8.8), 1300 mmol / L potassium chloride solution, 180 mmol / L magnesium chloride solution, 260 mmol / L dithiothreitol solution, and 3.9 wt% Triton X-100 aqueous solution.

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