CN107190103B - Multiplex PCR primer group, kit and method for simultaneously detecting three fish viruses - Google Patents
Multiplex PCR primer group, kit and method for simultaneously detecting three fish viruses Download PDFInfo
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- C12Q2600/00—Oligonucleotides characterized by their use
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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
Technical Field
The invention belongs to the technical field of aquatic disease microorganism detection, and particularly relates to a multiplex PCR (polymerase chain reaction) primer group, a kit and a detection method capable of simultaneously detecting three DNA viruses, namely Eel Herpes Virus (EHV) and Japanese Eel epidermal necrosis virus (JEECV), and Anguilla Eel polyoma virus (AmpV).
Background
China is one of the main eel culture countries in the world, and the production value of the eel culture method plays an important role in the export trade of aquatic products in China. The bred varieties are mainly Japanese eels (A.japonica), European eels (A.anguilla), American eels (A.rostrata) and anguilla marmorata (A.marmorata). Due to large-scale intensive culture, major viral epidemic outbreaks are increasingly frequent, and huge economic losses are caused. The definition of pathogenic pathogens of diseases is an important prerequisite for effective development of disease prevention and control. A plurality of eel virus detection methods are established in China, and epidemiological investigation of related viruses is carried out. Eel Herpes Virus (EHV), Japanese Eel endothelial cell-infecting virus (JEECV) and Anguilla polyoma virus (AmPV) belong to the same DNA viruses, and the pathogenicity of the three viruses to eels is manifested by symptoms such as gill rot and bleeding, and are difficult to distinguish clinically. The PCR detection methods of the three viruses are respectively established, and the detection is time-consuming and labor-consuming; meanwhile, the mixed infection of the two is also found for many times.
EHV is a double-stranded DNA virus with envelope, which has caused great economic loss to many farmers of European eels and Japanese eels, and is also an important factor in the reduction of wild European eels. Our previous studies showed that it can be detected on European eel, Japanese eel, American eel and anguilla marmorata. Clinical and pathological studies have shown that EHV can cause classic symptoms of gill rot, de-sticking, red head, etc. However, in the existing PCR detection, primers are designed according to the DNA polymerase gene sequence of EHV, and because the DNA polymerase gene is highly conserved, eel iridovirus may be detected during detection, which is represented as EHV false positive.
JEECV is a double-stranded circular DNA virus, has an LTLG gene which is homologous with a large T antigen gene of polyoma virus and is highly conserved, and is a pathogenic agent of vascular endothelial necrosis of eel (VECNE) in Japan. Since the 80 s of the last century, VECNE has often exploded, causing great economic losses to farmers of anguilla japonica. Typical symptoms of the disease include red fin-ray, abdominal distension, gill and liver congestion of eel. We establish the JEECV PCR detection method for the first time in China and successfully apply to the diagnosis of diseases.
AmPV, like JEECV, has the LTLG gene of polyoma virus. Sequence analysis shows that the sequence amplified from the diseased anguilla marmorata is greatly different from AmPV isolated from Taiwan, and the homology is 94%. AmPV can cause typical symptoms of the anguilla marmorata gill rot, body surface bleeding and the like.
Therefore, it is highly desirable to provide a multiplex PCR primer set for detecting the three viruses, an invention of a detection kit and optimized experimental conditions, so as to achieve simultaneous detection of the three viruses.
Disclosure of Invention
The invention aims to provide a convenient and rapid multiplex PCR detection method with strong specificity and high accuracy, and can simultaneously detect Eel Herpes Virus (EHV), Japanese Eel epidermal necrosis virus (JEECV) and Anguilla polyoma virus (AmpV).
In order to achieve the above purpose, the present invention provides a multiplex PCR primer set and a multiplex PCR detection kit for simultaneously detecting EHV, JEECV and AmPV in the same PCR reaction system, and the multiplex PCR detection kit is realized by the following technical scheme:
according to the obtained nucleic acid sequence, the invention designs specific primers for amplifying EHV, JEECV and AmPV, avoids forming stable primer dimers among the primers, analyzes the specificity of the primer amplification sequence, obtains a primer group which consists of 6 nucleic acid sequences and can have high amplification sensitivity and specificity to EHV, JEECV and AmPV, and comprises the following components: SEQ ID NO: 1 to 6; specifically, the primer set can be divided into a primer set 1 for detecting EHV, which consists of nucleotide sequences shown in SEQ ID No.1 and SEQ ID No.2, a primer set 2 for detecting JEECV, which consists of nucleotide sequences shown in SEQ ID No.3 and SEQ ID No.4, and a primer set 3 for detecting AmPV, which consists of nucleotide sequences shown in SEQ ID No.5 and SEQ ID No. 6.
The specificity detection result of the primer pair shows that the PCR reaction system has good specificity. The primer group 1 for detecting EHV has no non-specific amplification to the viruses with close homology, such as Koi Herpesvirus (KHV) and Eel Iridovirus (EIV), the primer group 2 for detecting JEECV has no specific amplification to AmPV, and the primer group 3 for detecting AmPV has no specific amplification to JEECV.
The sensitivity test result of the primer pair shows that the multiplex PCR detection method can detect EHV, JEECV and AmPV of the minimum 10 copies.
The EHV, JEECV and AmPV multiplex PCR detection kit comprises a primer group consisting of the 6 nucleic acid sequences, PCR buffer solution, dNTP, Taq DNA polymerase and ddH2O and positive control DNA plasmid. Wherein, the positive control plasmid is the amplified sequences of EHV, JEECV and AmPV connected with pMD19-T vector, the concentration is 108 copies/. mu.L, the amplified DNA sequence is as follows:
the insertion sequence of the EHV plasmid pMD19-EHV is SEQ ID No. 7;
the insertion sequence of the JEECV plasmid pMD19-JEECV is SEQ ID No. 8;
the insertion sequence of the AmPV plasmid pMD19-AmPV is SEQ ID No. 9.
The method for performing the multiple PCR detection of EHV, JEECV and AmPV by using the detection primer group and the detection kit comprises the following steps:
preparing DNA template of sample to be detected
And extracting the genome DNA in the sample to be detected by using a tissue genome DNA extraction kit or a virus genome DNA extraction kit.
PCR detection
Taking 2 μ L of the sample DNA extracted by the above method as a PCR template, preparing the following PCR reaction system on ice:
placing the mixture in a PCR instrument for PCR amplification, wherein the amplification conditions are as follows:
pre-denaturation: 3 minutes at 94 ℃;
and (3) circulation: denaturation at 94 ℃ for 10 seconds, annealing at 60 ℃ for 15 seconds, extension at 72 ℃ for 45 seconds, and 40 cycles;
extension: 5 minutes at 72 ℃;
determination of results
Taking 10 mu L of PCR product, carrying out electrophoresis by using 1% agarose gel electrophoresis, and if the electrophoresis result has an amplification band at the position of 240bp, indicating that the pathological material has EHV; if the electrophoresis result has an amplified band at the position of 505bp, the JEECV is shown in the pathological material; if the electrophoresis result shows that an amplified band exists at the position of 793bp, the disease has AmPV. If a plurality of bands appear simultaneously in the electrophoresis result, the mixed infection of the viruses corresponding to the band sizes in the pathological materials is shown. If no amplified band is visible at any of the three positions, it indicates that the three viruses are absent from the disease.
The invention also comprises application of the primer group in preparation of products for simultaneously detecting eel herpes virus EHV, Japanese eel epidermal necrosis virus JEECV and eel polyoma virus AmPV. The product comprises a kit for detecting three fish viruses, a gene probe, a gene chip and the like. The methods for preparing gene probe and gene chip can refer to the prior art.
The multiple PCR detection primer group, the detection kit and the detection method provided by the invention can be used for simultaneously detecting and identifying EHV, JEECV and AmPV in the same PCR reaction system, and have 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.
Drawings
FIG. 1 is an electrophoretogram for identifying the amplification specificity using primer set 1, primer set 2, and primer set 3. M: DL2000 standard molecular weight Marker; 1: an EHV; 2: KHV; 3: EIV; 4: JEECV; 5: AmPV; 6: AmPV; 7: JEECV;
FIG. 2 is an electrophoretogram for identifying EHV, JEECV, AmPV using a primer set and a kit. M: DL2000 standard molecular weight Marker; 1: an EHV; 2: JEECV; 3: AmPV;
FIG. 3 is a sensitive electrophoretogram for identifying EHV, JEECV, AmPV using the primer set and the 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 an electrophoretogram for identifying EHV, JEECV, and AmPV pathological materials by using the primer set and the kit. M: DL2000 standard molecular weight Marker; 1: a positive control; 2: mixed infection of EHV and JEECV; 3: mixed infection of EHV and AmPV; 4: an EHV; 5: JEECV; 6: AmPV; 7: the disease material is not detected; 8: negative control;
Detailed Description
In order to explain technical contents, structural features, and objects and effects of the present invention in detail, the following detailed description is given with reference to the accompanying drawings in conjunction with the embodiments.
(1) Design and optimization of primers
8 strains of EHV are separated and identified by cells, open reading frame sequences of 6 genes such as EHV ORF8, ORF95 and the like are cloned, the homology of the sequences is analyzed, 6 pairs of primers are designed in a conserved region, and the sizes of amplification products are all set to be about 250 bp. Taking EHV DNA of cell separation culture as a template, carrying out PCR amplification by a conventional PCR method, and carrying out gel electrophoresis analysis on an amplification product. The primer set 1 having high amplification efficiency was selected.
3 parts of JEECV positive pathological materials are separated and identified by cells, an open reading frame sequence of JEECV LTLG is cloned, the homology of the JEECV LTLG is analyzed, 2 pairs of primers are designed in a conserved region, and the size of an amplification product is set to be about 500 bp. Taking genome DNA extracted from the JEECV positive pathological material as a template, carrying out PCR amplification by a conventional PCR method, and carrying out gel electrophoresis analysis on an amplification product. The primer set 2 having high amplification efficiency was selected.
5 portions of AmpV positive pathological materials are separated and identified by cells, an open reading frame sequence of AmpV LTLG is cloned, the homology of the AmPV LTLG is analyzed, 2 pairs of primers are designed in a conservative area by using software Primer 5.0, and the size of an amplification product is set to be about 750 bp. And (3) taking the genome DNA extracted from the AmPV positive pathological material as a template, carrying out PCR amplification by using a conventional PCR method, and carrying out gel electrophoresis analysis on an amplification product. The primer set 3 having high amplification efficiency was selected.
(2) Extraction of genomic DNA from disease material
Collecting suspected viral disease material of eel, taking viscera tissues such as liver, spleen, kidney and the like, homogenizing, and extracting genome DNA by using a tissue DNA extraction kit.
(3) Specificity of multiplex PCR detection
In order to detect the specificity of the amplification of the primer group, KHV and EIV with higher homology with EHV are used as templates to detect the specificity of the EHV amplified by the primer group 1; because the relationship between JEECV and AmPV is close and no report of other fish polyoma viruses is found, the specificity of the primer group 2 for amplifying the JEECV and the specificity of the primer group 3 for amplifying the AmPV are detected by respectively taking the genomic DNA extracted from the positive pathological materials of the JEECV and the AmPV as templates. The result shows that the primer group 1 for detecting EHV can only specifically amplify EHV, but cannot amplify KHV and EIV; the primer group 2 for detecting the JEECV can only specifically amplify the JEECV and cannot amplify the AmPV; primer set 3 for detection of AmPV can only specifically amplify AmPV and cannot amplify JEECV (as shown in fig. 1).
(4) Establishment of multiplex PCR detection method
And (3) carrying out primer combination on the primer group 1, the primer group 2 and the primer group 3 according to the proportion of 1: 1: 1, to a final concentration of 10. mu.M. Respectively carrying out PCR amplification under the conditions of annealing temperatures of 55, 60 and 65, and then optimizing the amplification conditions to establish the following multiplex PCR detection method: 10XPCR buffer (Mg2+ plus) 5. mu. L, dNTP (2.5mM each) 4. mu.L, Taq DNA Polymerase (5U/. mu.L) 0.5. mu.L, primer set 2. mu.L, supplement ddH2O to 50. mu.L. Placing the mixture in a PCR instrument for PCR amplification, wherein the amplification conditions are as follows: pre-denaturation: 94, pre-denaturation: 3 minutes at 94 ℃; and (3) circulation: denaturation at 94 ℃ for 10 seconds, annealing at 60 ℃ for 15 seconds, extension at 72 ℃ for 45 seconds, and 40 cycles; extension: 5 minutes at 72 ℃. 10 μ L of the PCR product was electrophoresed through 1% agarose gel and photographed by observing on a gel imaging system. When the detection method established by the invention is used for detecting the EHV, JEECV and AmPV positive disease materials of cell culture, corresponding viruses can be efficiently detected in the result, and no non-specific band is found (as shown in figure 2).
(5) Sensitivity of multiplex PCR
Connecting EHV, JEECV and AmPV sequences amplified by the primer group 1, the primer group 2 and the primer group 3 to a cloning vector pMD19-T respectively, measuring the DNA content after sequencing verification, calculating the copy number, and then, adding three plasmids according to the copy number 1: 1: 1 mix at a concentration of 108 copies/. mu.L and serve as a positive control for the kit. The positive control was diluted 10-fold in a gradient and tested using the established multiplex PCR assay. The results show that the method can specifically detect EHV, JEECV and AmPV with the minimum of 10 copies (as shown in FIG. 3).
(6) Positive eel disease material for multiplex PCR detection
In order to verify the effectiveness of the multiplex PCR detection method, the conventional PCR method is selected to detect EHV, JEECV and AmPV positive pathological materials, mixed infection pathological materials and undetected pathological materials, the multiplex PCR detection method is used to detect the pathological materials, EHV, JEECV and AmPV positive pathological materials and EHV, JEECV, EHV and AmPV mixed infection positive pathological materials are respectively detected, and the result is completely consistent with the result detected by the conventional PCR method (as shown in figure 4).
Although the embodiments have been described, once the basic inventive concept is obtained, other variations and modifications of these embodiments can be made by those skilled in the art, so that the above embodiments are only examples of the present invention, and not intended to limit the scope of the present invention, and all equivalent structures or equivalent processes using the contents of the present specification and drawings, or any other related technical fields, which are directly or indirectly applied thereto, are included in the scope of the present invention.
SEQUENCE LISTING
<110> institute of biotechnology of academy of agricultural sciences of Fujian province
<120> multiple PCR primer group, kit and method for simultaneously detecting three fish viruses
<130>2017
<160>9
<170>PatentIn version 3.5
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ataggttcgt gctgtccttc aa 22
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gagatcggaa gatgtggagt ctatgaggtc cgccatcgtc cacgttctgg agtcagagtc 180
gccgtttgac aagcagttca agtttggatc ctacgtgact ctgattgtgc tgggttgttt 240
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cccaccagag gaaagacaaa gactggagca aaggctgaaa aggataaatg aggtatgggc 60
aaaatacctg gacatccatc agagacaaca gacgtggttt gacatatccc ccagtaaaag 120
ggggggcacg ctgactgtac ctgaatacct ggctaaatat tgcactacta tacaggatgg 180
tcagtttgtg cagactgtac tggtgcaggt gccctgggct aagctgggat cagtggtcac 240
agaactgaaa aaatacaaac atgtagatct aattgcaggc ggggaccccc gggaggaccc 300
cccaacaggc atagcagttg tgcaatttgt gcataggcaa aaggaatctg cattgaaggg 360
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caggcagtct gtgaacccaa acaccatagc aaactcattt aacaacttaa tgctgaacgc 360
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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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| CN108315487B (en) * | 2018-04-16 | 2021-06-22 | 福建省农业科学院生物技术研究所 | Primer group and kit for detecting eel herpesvirus and application of primer group and kit |
| CN110863067A (en) * | 2019-11-29 | 2020-03-06 | 厦门海关技术中心 | Primer pair and kit for detecting eel herpes virus |
| CN112852987A (en) * | 2021-03-16 | 2021-05-28 | 青岛农业大学 | PCR (polymerase chain reaction) specific primer and detection method for detecting eel dermatophytosis |
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