CN107190103B - Multiplex PCR primer set, kit and method for simultaneous detection of three fish viruses - Google Patents

Abstract

The invention provides a multiplex PCR primer group, a kit and a detection method for simultaneously detecting eel herpes virus EHV, Japanese eel epidermal necrosis virus JEECV and eel polyoma virus AmPV, according to the obtained nucleic acid sequence, the invention designs specific primers for amplifying EHV, JEECV and AmPV, avoids forming stable primer dimer between the primers, analyzes the specificity of the primer amplification sequence, obtains a primer group which consists of 6 nucleic acid sequences and can simultaneously have high amplification sensitivity and specificity to EHV, JEECV and AmPV, and comprises the following steps: SEQ ID NO: 1-6. The invention can simultaneously detect and identify EHV, JEECV and AmPV in the same PCR reaction system, and has the advantages of simple operation, strong specificity and the like; the present invention can judge the result directly according to the difference of the amplification length, and is more efficient and practical.

Description

Multiplex PCR primer set, kit and method for simultaneous detection of three fish viruses

technical field

The invention belongs to the technical field of microorganism detection of aquatic diseases, in particular to a kind of eel herpesvirus (Eel herpesvirus, EHV), Japanese eel endothelial cell necrosis virus (Japanese eel endothelial cells-infecting virus, JEECV) and flower eel polyoma virus ( Anguilla marmorata polyoma-likevirus, AmPV) multiplex PCR primer set, kit and detection method for three DNA viruses.

Background technique

my country is one of the major eel farming countries in the world, and its output value occupies an important position in my country's aquatic product export trade. The main cultured species are Japanese eel (A.japonica), European eel (A. anguilla), American eel (A.rostrata), and flower eel (A.Marmorata). Due to large-scale intensive farming, outbreaks of major viral diseases are becoming more frequent, causing huge economic losses. Identifying the causative agent of the disease is an important prerequisite for effective disease prevention and control. We took the lead in establishing a variety of eel virus detection methods in China, and carried out epidemiological investigations on related viruses. Eel herpesvirus (EHV), Japanese eelendothelial cells-infecting virus (JEECV) and flower eel polyoma virus (Anguilla marmoratapolyoma-like virus, AmPV) belong to the same DNA virus. The pathogenicity of eels are all manifested as gill rot, bleeding and other symptoms, which are difficult to distinguish in clinical diagnosis. We have established PCR detection methods for these three viruses, which are time-consuming and labor-intensive.

EHV is a double-stranded DNA virus with an envelope, which has caused huge economic losses to European eel and Japanese eel breeders in many countries, and it is also an important factor causing the reduction of wild European eel. Our previous study showed that it can be detected in European eel, Japanese eel, American eel and flower eel. Clinical and pathological studies have shown that EHV can cause typical symptoms such as "rotten gills", "debonding", and "red head". However, the existing PCR tests are based on the DNA polymerase gene sequence of EHV to design primers. Because the DNA polymerase gene is highly conserved, eel iris virus may be detected during the test, which is false positive for EHV.

JEECV is a double-stranded circular DNA virus with a highly conserved LTLG gene homologous to the large T antigen gene of polyoma virus. It is the pathogenic cause of Viral endothelial cell necrosis of eel (VECNE) in Japanese eel. pathogen. Since the 1980s, VECNE has frequently erupted, causing huge economic losses to Japanese eel farmers. The typical symptoms of its disease include reddening of the eel's fin rays, swelling of the abdomen, and congestion of the gills and liver. We established the PCR detection method of JEECV for the first time in China and successfully applied it to the diagnosis of the disease.

AmPV, like JEECV, has the LTLG gene of polyoma virus. Sequence analysis showed that the sequence we amplified from the diseased eel was quite different from the AmPV isolated from Taiwan, China, with a homology of 94%. AmPV can cause typical symptoms such as gill rot and body surface hemorrhage.

Therefore, there is an urgent need to provide a multiplex PCR primer set for detecting these three viruses, invent a detection kit and optimize the experimental conditions, so as to realize the simultaneous detection of these three viruses.

SUMMARY OF THE INVENTION

The object of the present invention is to provide a kind of convenience, high specificity, high accuracy, and can simultaneously detect eel herpesvirus (Eel herpesvirus, EHV), Japanese eel endothelial cells-infecting virus (Japanese eel endothelial cells-infecting virus, JEECV) and Multiplex PCR detection method for Anguilla marmorata polyoma-likevirus (AmPV).

For achieving the above object, the present invention provides a kind of multiplex PCR primer set and multiplex PCR detection kit that detect EHV simultaneously in same PCR reaction system, JEECV and AmPV, and realize by following technical scheme:

According to the obtained nucleic acid sequence, the present invention designs specific primers for amplifying EHV, JEECV and AmPV, avoids the formation of stable primer dimers between the primers, and analyzes the specificity of the amplified sequences of the primers to obtain The primer set consists of 6 nucleic acid sequences, which can simultaneously have high amplification sensitivity and specificity for EHV, JEECV and AmPV, including: SEQ ID NO: 1-6; specifically, can be divided into SEQ ID No. 1, Primer set 1 for detecting EHV consisting of the nucleotide sequence shown in SEQ ID No. 2, and primer set 1 for detecting JEECV consisting of the nucleotide sequence shown in SEQ ID No. 3 and SEQ ID No. 4 Set 2 and set 3 of primers for detecting AmPV consisting of the nucleotide sequences shown in SEQ ID No.5 and SEQ ID No.6.

The specificity detection results of primer pairs showed that the PCR reaction system had good specificity. The primer set 1 for detecting EHV has no non-specific amplification on the viruses with close homology such as Koi herpesvirus (KHV) and Eel iridovirus (EIV), and primer set 2 for detecting JEECV has no non-specific amplification on AmPV. Specific amplification, primer set 3 for detecting AmPV has no specific amplification for JEECV.

The results of the sensitivity test of primer pairs showed that the multiplex PCR detection method could detect the minimum 10 copies of EHV, JEECV and AmPV.

The multiplex PCR detection kit for EHV, JEECV and AmPV includes the primer set consisting of the above 6 nucleic acid sequences, PCR buffer, dNTP, Taq DNA polymerase, ddH ₂ O and positive control DNA plasmid. Among them, the positive control plasmid is the amplified sequence of EHV, JEECV and AmPV connected to the pMD19-T vector, and the concentration is 108 copies/μL. The amplified DNA sequence is as follows:

The insert sequence of EHV plasmid pMD19-EHV is SEQ ID No.7;

The insert sequence of JEECV plasmid pMD19-JEECV is SEQ ID No.8;

The insert sequence of AmPV plasmid pMD19-AmPV is SEQ ID No.9.

The method of multiplex PCR detection of EHV, JEECV and AmPV with the above detection primer set and detection kit is as follows:

Prepare the DNA template of the sample to be tested

Use tissue genomic DNA extraction kit or viral genomic DNA extraction kit to extract the genomic DNA in the sample to be tested.

PCR detection

Take 2 μL of the sample DNA extracted in the above method as a PCR template, and prepare the following PCR reaction system on ice:

Placed in a PCR machine for PCR amplification, and the amplification conditions are:

Pre-denaturation: 94°C for 3 minutes;

Cycling: denaturation at 94°C for 10 seconds, annealing at 60°C for 15 seconds, extension at 72°C for 45 seconds, 40 cycles;

Extension: 5 minutes at 72°C;

result judgment

Take 10μL of PCR product and use 1% agarose gel electrophoresis for electrophoresis. If the electrophoresis result has an amplification band at the 240bp position, it means that there is EHV in the disease material; if the electrophoresis result has an amplification band at the 505bp position, it means that the disease material With JEECV; if there is an amplification band at the 793bp position as a result of electrophoresis, it means that the disease material has AmPV. If multiple bands appear simultaneously in the electrophoresis result, it means that there is a mixed infection of the virus corresponding to the band size in the disease material. If there are no visible amplification bands in the above three positions, it means that the above three viruses are not present in the patient material.

The invention also includes the application of the primer set in preparing a product for simultaneous detection of eel herpes virus EHV, Japanese eel epidermal cell necrosis virus JEECV and flower eel polyoma virus AmPV. The products include kits for detecting three fish viruses, gene probes, gene chips and the like. Wherein, for the production methods of gene probes and gene chips, reference may be made to the prior art.

The multiplex PCR detection primer set, detection kit and detection method provided by the invention can simultaneously detect and identify EHV, JEECV and AmPV in the same PCR reaction system, and have the advantages of simple operation, strong specificity and the like; The difference in growth degree directly determines the result, which is more efficient and practical.

Description of drawings

FIG. 1 is an electropherogram of amplification specificity identification using primer set 1, primer set 2, and primer set 3. FIG. M: DL2000 standard molecular weight Marker; 1: EHV; 2: KHV; 3: EIV; 4: JEECV; 5: AmPV; 6: AmPV; 7: JEECV;

Fig. 2 is the electrophoresis image of identifying EHV, JEECV and AmPV using primer set and kit. M: DL2000 standard molecular weight Marker; 1: EHV; 2: JEECV; 3: AmPV;

Figure 3 is the electropherogram of the sensitivity of identifying EHV, JEECV and AmPV using primer set and kit. M: DL2000 standard molecular weight Marker; 1: 108 copies; 2: 107 copies; 3: 106 copies; 4: 105 copies; 5: 104 copies; 6: 103 copies; 7: 102 copies; 8: 100 copies; 9: Negative control;

Fig. 4 is the electrophoresis image of identifying EHV, JEECV, AmPV disease material using the above-mentioned primer set and kit. M: DL2000 standard molecular weight Marker; 1: Positive control; 2: Mixed infection of EHV and JEECV; 3: Mixed infection of EHV and AmPV; 4: EHV; 5: JEECV; 6: AmPV; 7: No disease detected; 8: negative control;

Detailed ways

In order to describe the technical content, structural features, achieved objects and effects of the present invention in detail, the following detailed description is given in conjunction with the embodiments and the accompanying drawings.

(1) Design and optimization of primers

The cells were isolated and identified 8 strains of EHV, and the open reading frame sequences of 6 genes such as EHV ORF8 and ORF95 were cloned, and their homology was analyzed. 250bp. Using the EHV DNA isolated and cultured from cells as a template, PCR amplification was carried out by a conventional PCR method, and the amplified products were analyzed by gel electrophoresis. Primer set 1 with high amplification efficiency was screened.

Three JEECV-positive disease materials were isolated and identified from the cells. The open reading frame sequence of JEECV LTLG was cloned, and its homology was analyzed. Two pairs of primers were designed in the conserved region, and the size of the amplified product was set at about 500bp. Using the genomic DNA extracted from JEECV positive disease materials as template, PCR amplification was carried out by conventional PCR method, and the amplified products were analyzed by gel electrophoresis. Primer set 2 with high amplification efficiency was screened.

Cells were isolated and identified 5 AmPV-positive disease materials, the open reading frame sequence of AmPV LTLG was cloned, and its homology was analyzed. Using the software Primer Primer 5.0, 2 pairs of primers were designed in the conserved region, and the size of the amplified product was set at About 750bp. Using the genomic DNA extracted from AmPV positive disease materials as template, PCR amplification was carried out by conventional PCR method, and the amplified products were analyzed by gel electrophoresis. Primer set 3 with high amplification efficiency was screened.

(2) Extraction of genomic DNA from disease materials

The material of suspected viral diseases of eel was collected, and the liver, spleen, kidney and other visceral tissues were collected, and the genomic DNA was extracted with a tissue DNA extraction kit after homogenization.

(3) Specificity of multiplex PCR detection

In order to detect the specificity of the amplification of the above-mentioned primer set, KHV and EIV with higher homology to EHV were used as templates to detect the specificity of the amplification of EHV of the primer set 1; because the relationship between JEECV and AmPV is close, and no other fish have been seen Therefore, the specificity of primer set 2 to amplify JEECV and the specificity of primer set 3 to amplify AmPV were detected by using the genomic DNA extracted from JEECV and AmPV positive patients as templates, respectively. The results showed that the primer set 1 for detecting EHV could only specifically amplify EHV, but could not amplify KHV and EIV; the primer set 2 for detecting JEECV could only specifically amplify JEECV but not AmPV; the primer set 3 for detecting AmPV only amplified It can specifically amplify AmPV, but not JEECV (as shown in Figure 1).

(4) Establishment of multiplex PCR detection method

Primer set 1, primer set 2, and primer set 3 were mixed in a ratio of 1:1:1, and the final concentration was 10 μM. PCR amplification was carried out at the annealing temperature of 55, 60, and 65, respectively, and then the amplification conditions were optimized to establish the following multiplex PCR detection method: 10X PCR buffer (Mg2+plus) 5μL, dNTP (2.5mM each) 4μL, Taq DNA Polymerase (5U/μL) 0.5 μL, primer set 2 μL, supplemented with ddH ₂ O to 50 μL. Placed in a PCR machine for PCR amplification, the amplification conditions are: pre-denaturation: 94 Pre-denaturation: 94°C for 3 minutes; cycle: 94°C denaturation for 10 seconds, 60°C annealing for 15 seconds, 72°C extension for 45 seconds, 40 cycles; Extension: 5 minutes at 72°C. 10 μL of PCR products were taken, subjected to 1% agarose gel electrophoresis, observed and photographed on a gel imaging system. Cell culture EHV, JEECV, and AmPV positive disease materials were detected by the detection method established in the present invention, and the results were able to efficiently detect the corresponding viruses, and no non-specific bands were found (as shown in Figure 2).

(5) Sensitivity of multiplex PCR

The EHV, JEECV, and AmPV sequences amplified with primer set 1, primer set 2, and primer set 3 were respectively connected to the cloning vector pMD19-T. After sequencing and verification, the DNA content was determined, and the copy number was calculated. The number was mixed 1:1:1 at a concentration of 108 copies/μL as a positive control for the kit. The positive control was serially diluted 10 times and detected by the established multiplex PCR detection method. The results showed that this method could specifically detect the lowest 10 copies of EHV, JEECV, and AmPV (as shown in Figure 3).

(6) Positive eel disease materials detected by multiplex PCR

In order to verify the validity of the above-mentioned multiplex PCR detection method, the disease materials with positive EHV, JEECV, AmPV, mixed infection and undetected disease materials were selected by conventional PCR method, and the disease materials were detected by the above-mentioned multiplex PCR detection method, respectively. EHV, JEECV, AmPV positive disease material, and EHV and JEECV, EHV and AmPV positive disease material mixed infection were detected, and the results were completely consistent with the results detected by conventional PCR method (as shown in Figure 4).

Although the above embodiments have been described, those skilled in the art can make additional changes and modifications to these embodiments once they know the basic inventive concept, so the above is only the implementation of the present invention For example, it does not limit the scope of patent protection of the present invention. Any equivalent structure or equivalent process transformation made by using the contents of the description and drawings of the present invention, or directly or indirectly used in other related technical fields, are similarly included in this document. The invention is within the scope of patent protection.

SEQUENCE LISTING

<110> Institute of Biotechnology, Fujian Academy of Agricultural Sciences

<120> Multiplex PCR primer set, kit and method for simultaneous detection of three fish viruses

<130> 2017

<160> 9

<170> PatentIn version 3.5

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Claims (3)

1. A multiplex PCR primer group for simultaneously detecting eel herpes virus EHV, Japanese eel epidermal necrosis virus JEECV and anguilla marmorata polyoma virus AmPV is characterized in that sequences of primers in the primer group are respectively SEQ ID NO: 1-6.

2. The primer group of claim 1, which is used for preparing a product for simultaneously detecting eel herpesvirus EHV, Japanese eel epidermal necrosis virus JEECV and eel polyoma virus AmPV.

3. A multiplex PCR detection kit for simultaneously detecting eel herpes virus EHV, Japanese eel epidermal necrosis virus JEECV and anguilla marmorata polyoma virus AmPV comprises: 10XPCR buffer solution, dNTPs mixed solution, primer group, Taq DNA polymerase and sterile deionized water, and can be used as the positive control DNA plasmid, characterized by that: the detection primer set is the multiplex PCR primer set according to claim 1.

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