A primer pair and kit for quantitatively detecting american eel adenovirus

Abstract

The application discloses a primer pair and a kit for quantitatively detecting American eel adenoma virus, and belongs to the technical field of molecular biology. The primer pair is designed based on the superfamily 3 helicases (S3H) gene of the American eel adenoma virus. The American eel adenoma virus quantitative detection kit provided by the application can realize rapid and accurate quantitative detection of suspected American eel adenoma virus samples, has the characteristics of high sensitivity, strong specificity and good repeatability, and is of great significance for improving the detection rate of American eel adenoma virus in asymptomatic eels and preventing and controlling the "hemorrhagic and rotten gill disease" of American eels in production.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to a primer pair and a kit for quantitatively detecting American eel adenomas virus. Background Art

[0002] China boasts the world's largest eel aquaculture industry, with Fujian Province being the primary eel production area. Since 2015, the American eel has become the primary species farmed in Fujian. However, in recent years, a highly contagious disease has emerged in two-year-old American eels farmed in Fujian Province, with a long duration (3-6 months) and a high cumulative mortality rate (20%-50%). While symptoms are mild, the primary pathological change is the dense distribution of pinpoint hemorrhages on the gill filaments, hence the name "hemorrhagic gill rot." In previous research, we isolated a new virus, named American Eel Adenomas (AEAdoV), from eels with "hemorrhagic gill rot" using eel ovary (EO) cells. This is the first time an adenomas virus has been isolated from an American eel.

[0003] Studies have shown that AEAdoV is a non-enveloped dsDNA virus with an icosahedral structure and a diameter of 75nm to 85nm, and has homology with the relevant regions of Marbled Eel Adomavirus (MEAdoV). In order to study the relationship between AEAdoV and "hemorrhagic gill rot" and understand the prevalence of AEAdoV in American eels, it is necessary to establish a sensitive, effective and specific AEAdoV detection method. qPCR is a commonly used detection method for viral pathogens, with the advantages of strong specificity and high sensitivity. In view of the characteristics of AEAdoV's latent infection, the present invention provides a primer and a kit for quantitatively detecting AEAdoV using SYBR GreenI qPCR method, which has the advantages of strong specificity, high sensitivity and good repeatability. It provides a detection method for rapid and accurate quantitative detection of American eel adenomas in fish during the viral incubation period and in fish without clinical symptoms, which is conducive to early detection of viral infection and taking active prevention and control measures as soon as possible. Summary of the Invention

[0004] The present invention aims to provide a primer pair and a kit for quantitatively detecting American eel adenomas virus. The kit can be used for rapid and quantitative detection of American eel adenomas virus in eel tissue and cell samples, and has the characteristics of strong specificity, high sensitivity, good repeatability, etc.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A primer pair for quantitatively detecting American eel adenomatous virus, wherein the nucleotide sequence of the primer pair is:

[0007] Sequence AEAdoV-qF (SEQ ID NO. 1):

[0008] 5'-GACACTGATGCAGTTGACGCAC-3',

[0009] Sequence AEAdoV-qR (SEQ ID NO. 2):

[0010] 5'-CTGGCGTACTCTATCGAGCCT-3'.

[0011] The primer pair is used for preparing a reagent kit for detecting AEAdV.

[0012] A fluorescent quantitative PCR kit for detecting AEAdV, comprising the primer pair.

[0013] Further, the fluorescent quantitative PCR kit further comprises a positive control DNA and a negative control.

[0014] Further, the positive control DNA is a recombinant plasmid comprising the nucleotide sequence shown as SEQ ID NO. 3; and the negative control is sterilized ddH2O.

[0015] A fluorescent quantitative PCR method for detecting AEAdV for non-disease diagnosis and treatment purposes, which uses genomic DNA of a sample to be detected as a template and uses the primer pair to perform fluorescent quantitative PCR reaction.

[0016] Further, the reaction system of the fluorescent quantitative PCR is as follows: 2x SYBR Green I qPCR Mix 10 μl, 10 μM of the upstream and downstream primers each 0.4 μL, template 1 μL, and ddH2O 8.2 μL.

[0017] Further, the reaction conditions of the fluorescent quantitative PCR are as follows: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing for 34 s, and 40 cycles.

[0018] The present application has the following beneficial effects:

[0019] (1) The primer pair provided by the present application is obtained by optimization, and the target gene thereof is AEAdV superfamily 3 helicases (S3H) gene.

[0020] (2) Based on the primer pair, the present invention provides a kit for quantitatively detecting American eel adenomatous virus. The kit has high sensitivity and strong specificity, with no amplification of several common eel DNA viruses and negative controls; and good repeatability, with an intra-group coefficient of variation of less than 1% and an inter-group coefficient of variation of less than 2%.

[0021] (3) The kit for quantitatively detecting American eel adenomatous virus provided by the present invention can be used to quantitatively detect samples suspected of being infected with American eel adenomatous virus, and the positive detection rate reaches 97%. This provides a new detection method for quickly and accurately quantitatively detecting the amount of American eel adenomatous virus in samples, and can be applied to the diagnosis and epidemiological research of American eel adenomatous virus disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is the standard curve of the qPCR detection method of the present invention.

[0023] Figure 2 is the melting curve of the qPCR detection of the present invention, 10 8 ~10 1 The melting curves of the three-copy plasmids all had only one melting peak, with Tm values ​​ranging from 85.33 to 85.78°C.

[0024] Figure 3 is the sensitivity test result of qPCR of the present invention, where 1-9 are 10 8 ~10 0 10 copies of the recombinant plasmid were used as negative control.

[0025] Figure 4 Specific detection results of qPCR of the present invention, where 1 is AEAdoV, 2 is a positive control, 3-5 are WSSV, KHV, and RGV, respectively, and 6 is a negative control. DETAILED DESCRIPTION

[0026] The present invention is described in detail below with reference to the embodiments.

[0027] Virus strains, cell lines, and test materials used in the following examples:

[0028] American Eel Adomavirus (AEAdoV), Ranagryliovirus (RGV), Koi Herpes Virus (KHV), and Whitespot Syndrome Virus (WSSV) are stored at the Institute of Biotechnology, Fujian Academy of Agricultural Sciences. Twenty-five eel tissue samples suspected of being infected with AEAdoV were collected and stored at the Institute of Biotechnology, Fujian Academy of Agricultural Sciences.

[0029] Example 1: Primer design and screening for a kit for quantitative detection of American eel adenomas virus Based on the genomic sequence of the American eel adenomas virus, 10 pairs of eel herpes virus-specific primers were designed using DNASTAR lasergene V7.1 software for the superfamily 3 helicases (S3H) gene. These primers were used as alternative primers for the detection method of the present invention and were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

[0030] AEAdoV was inoculated into the eel ovary cell line (EO). After the cells developed typical lesions, the supernatant was collected and DNA was extracted. The DNA was used as a template for qPCR amplification using synthesized primers. Based on the amplification curve and Ct value, a primer pair AEAdoV-qF (SEQ ID NO.1) / AEAdoV-qR (SEQ ID NO.2) with good amplification efficiency and strong specificity was finally screened for the establishment of subsequent detection methods.

[0031] Table 1 Preferred primers for quantitative detection of American eel adenomatous virus

[0032]

[0033] Example 2: Kit for quantitative detection of American eel adenomatous virus

[0034] 1. Preparation of positive plasmid standards

[0035] The target fragment (SEQ ID NO. 3) was cloned into the pCE2-TA / Blunt plasmid. The resulting recombinant plasmid served as a positive control DNA (stored at the Institute of Biotechnology, Fujian Academy of Agricultural Sciences). Its concentration and purity were determined using an ultramicro-UV spectrophotometer. The number of plasmid copies per μL of extracted DNA was calculated using a DNA / RNA Copy Number Calculator (http: / / www.endmemo.com / bio / dnacopynum.php). The DNA was stored at -80°C until further use. The measured plasmid concentration was 135.5 ng / μL, and the calculated plasmid copy number was 3.1 × 10 10 copies / μL.

[0036] 2. Establish a standard curve

[0037] The positive plasmid DNA was diluted 10-fold to 10 8 ~10 1qPCR amplification of the AEAdoV-qF (SEQ ID NO. 1) / AEAdoV-qR (SEQ ID NO. 2) primer pair was performed at 8 concentrations per μL as template. Three replicates were performed at each concentration. Sterile ddH2O was used as a negative control instead of template to generate a standard curve. The reaction system and conditions were as follows: 20 μL reaction system: 10 μL 2× SYBR Green I qPCR Mix, 0.4 μL each of the upstream and downstream primers (10 μM / L), 1 μL template, and 8.2 μL ddH2O. Amplification conditions: 95°C initial denaturation for 30 s; 95°C denaturation for 5 s; 60°C annealing for 34 s, for 40 cycles. Result determination: If the Ct value of the sample to be tested is less than 35 under the conditions that the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, then it is judged as AEAdoV positive; if the Ct value of the sample to be tested is ≥35 under the conditions that the negative control has no Ct value and no amplification curve, the positive control has a Ct value less than 35 and a typical amplification curve appears, then it is judged as AEAdoV negative; if an amplification curve appears in the negative control, the experimental result is invalid.

[0038] The standard curve was drawn by using the positive plasmid copy number and Ct value ( Figure 1 ), the linear relationship between the positive plasmid copy number and the Ct value is y = -3.206x + 38.14, the correlation coefficient is 0.999, and the amplification efficiency is 105.067%, indicating that the Ct value of the qPCR has a good linear relationship with the copy number. Melting curve analysis shows that ( Figure 2 ), 10 8 ~10 1 The melting curves of the copy-positive plasmids all had only one melting peak, with Tm values ​​ranging from 85.326 to 85.784°C, indicating that the reaction products were all specifically amplified without nonspecific amplification or primer dimer generation.

[0039] 3. Sensitivity test: Using the gradient diluted positive plasmid DNA as template, the designed primers were used to perform qPCR amplification of AEAdoV-qF (SEQ ID NO.1) / AEAdoV-qR (SEQ ID NO.2), and the lowest copy number of Anguilla anguilla adenomatous virus that could be detected was analyzed. The results showed that the lowest copy number detected by qPCR could reach 10 1 ( Figure 3 ), with higher sensitivity.

[0040] 4. Specificity detection

[0041] Specific qPCR detection was performed using genomic DNA and positive plasmid DNA of AEAdoV, RGV, KHV and WSSV as templates. The results showed that amplification curves were generated for AEAdoV and the positive control, while no amplification signals were generated for RGV, KHV, WSSV and the negative control ( Figure 4 ).

[0042] 5. Repeatability test

[0043] Take 10 4 and 10 5 The positive plasmid with two concentrations of copies / μL was used as a template for qPCR repeatability testing. Three replicates were set for each concentration, and the intra-group repeatability of the method was analyzed based on the coefficient of variation of the Ct value. The experiment was repeated three times to analyze the inter-group repeatability.

[0044] Table 2 Repeatability of qPCR detection of American eel adenomatous virus

[0045]

[0046] As shown in Table 2, the intra-group coefficients of variation were all less than 1%, namely 0.08% and 0.47% respectively; the inter-group coefficients of variation were all less than 2%, namely 1.5% and 0.22% respectively, indicating that the method has good repeatability and can ensure the stability and reliability of the test results.

[0047] Example 3: Application of the Quantitative Detection Kit for Anguilla anguilla Adenoma Virus

[0048] 35 tissue samples of suspected American eel "hemorrhagic gill rot" diseased materials collected and stored in the laboratory were taken, DNA was extracted as a template, and the samples were tested using the American eel adenomatous virus quantitative detection kit provided by the present invention.

[0049] Table 3 Quantitative detection kit for American eel adenomatous virus in samples suspected of eel "hemorrhagic gill rot"

[0050]

[0051] As can be seen from Table 3, the American eel adenomatous virus quantitative detection kit provided by the present invention was used to detect 35 collected samples suspected of having "hemorrhagic gill rot" of American eel, and 34 positive samples were detected, with a positive detection rate of 97%.

[0052] The primer pair and kit for quantitative detection of American eel adenomas virus provided by the present invention have high sensitivity, strong specificity, good repeatability, and good application effect. They can be used for rapid and quantitative detection of American eel adenomas virus, and are also of great significance for the prevention and control of eel "hemorrhagic gill rot disease".

Claims

1. A primer pair for quantitative detection of American eel adenomatous virus, characterized in that: The sequence of the upstream primer AEAdoV-qF is SEQ ID NO.1, and the sequence of the downstream primer AEAdoV-qR is SEQ ID NO.

2.

2. Use of the primer pair as claimed in claim 1 in preparing a kit for detecting American eel adenomatous virus.

3. A fluorescent quantitative PCR kit for detecting American eel adenomatous virus, characterized in that: The fluorescent quantitative PCR kit comprises the primer pair according to claim 1.

4. The fluorescent quantitative PCR kit according to claim 3, characterized in that: The fluorescent quantitative PCR kit also contains positive control DNA and negative control.

5. The fluorescent quantitative PCR kit according to claim 4, characterized in that: The positive control DNA is a recombinant plasmid containing the nucleotide sequence shown in SEQ ID NO. 3; the negative control is sterilized ddH2O.

6. A fluorescent quantitative PCR method for detecting anguilla adenomatous virus for purposes other than disease diagnosis and treatment, characterized in that: The genomic DNA of the sample to be detected is used as a template and a fluorescent quantitative PCR reaction is performed using the primer pair as claimed in claim 1.

7. The fluorescent quantitative PCR method according to claim 6, characterized in that: The reaction system of the fluorescent quantitative PCR was as follows: 10 μL of 2×SYBR Green I qPCR Mix, 0.4 μL of each of 10 μM upstream and downstream primers, 1 μL of template, and 8.2 μL of ddH2O.

8. The fluorescent quantitative PCR method according to claim 6, wherein: The reaction conditions of the fluorescent quantitative PCR were as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing at 60°C for 34 s, and 40 cycles.

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